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Expression, activation, and regulation of matrix 2 (MMP-2) in the bovine corpus luteum

Bo Zhang University of New Hampshire, Durham

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Recommended Citation Zhang, Bo, "Expression, activation, and regulation of 2 (MMP-2) in the bovine corpus luteum" (2002). Doctoral Dissertations. 86. https://scholars.unh.edu/dissertation/86

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. EXPRESSION, ACTIVATION, AND REGULATION OF MATRIX

METALLOPROTEINASE 2 (MMP-2) IN THE BOVINE CORPUS LUTEUM

BY

BO ZHANG

B. S., Henan Normal University, 1994

M. S., Chinese Academy of Sciences, 1997

DISSERTATION

Submitted to the University of New Hampshire

in Partial Fulfillment of

the Requirements of the Degree of

Doctor of Philosophy

in

Animal Sciences

May, 2002

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This dissertation has been examined and approved.

Dissertation Director, D r^ lu l C.W. Tsang Associate Professor Animal and Nutritional Sciences University of New Hampshire

Dr. William A. Condon, Professor Animal and Nutritional Sciences University of New Hampshire

Dr. Stacia A. Sower, Professor Biochemistry and Molecular Biology University of New Hampshire

Dr. Marsha A. Moses, Associate Professor Department of Surgery Children’s Hospital Harvard Medical School

Dr. David H. Townson, Assistant Professor Animal and Nutritional Sciences University of New Hampshire

Afrit 2 ‘7/zoa'2- Date

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This dissertation is dedicated to

my wife Hongying Zhang

and

my son Nickolas J. Zhang

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

It is my sincerest wish to acknowledge several people without whom this work

would have been impossible.

First of all, I must express my deepest gratitude to Dr. Paul Tsang, my dissertation

director. I thank him not only for giving me the chance to pursue my Ph. D. degree in his

laboratory, but also mainly for his incredible support, continuing encouragement, great

understanding, and thoughtful consideration throughout my time here in New Hampshire.

He gave me the greatest possible freedom in my research. He also went into great depth

in helping me to prepare each of my presentations, correct each of my research reports,

and instruct me in all laboratory techniques. I am so fortunate to have had the opportunity

to work with him. The experiences that he and I shared here will benefit my entire life.

I extend my heartfelt thanks to my committee members: Dr. Bill Condon, Dr.

Stacia Sower, Dr. Marsha Moses, and Dr. Dave Townson, for their advice, time and

energy over the past years. Their guidance and foresight helped clarify my research

targets, articulate my vision, and inspire my thinking. Furthermore, their intellectual input

has brought an additional richness and depth to my research.

I also would like to thank Dr. Li Yan at Children’s Hospital, Harvard Medical

School for his cooperation in DNA sequencing and in vitro tube formation assay, and Drs.

Bo Rueda and Jim Pru at the Massachusetts General Hospital, Harvard Medical School,

and Dr. John Davis at the University of Nebraska for their collaboration on the luteal-

derived endothelial cell culture study.

iv

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I am profoundly grateful to all the faculty and staff members, and my fellow

graduate students in our department. Their help and support make me feel at home here. I

also would like to thank the graduate and undergraduate students in Dr. Tsang’s

laboratory for assisting me in my research. I especially thank Dominic Ambrosi for his

great work in the MMP-13 study.

I would like to recognize the financial support from the United States Department

of Agriculture, the Northeast Regional Project, and the American Cancer Society. I also

thank the Sigma Xi Society for a Grant-in-Aid research award to support my MMP-2 and

TIMP-2 study, and the University of New Hampshire Graduate School for supporting me

with a Summer Teaching Assistant Fellowship and a Dissertation Fellowship.

Lastly, I would like to acknowledge my parents, my parents-in-law, my wife

Hongying, and my son Nickolas for their understanding, encouragement, and love. Their

support gave me endless motivation to accomplish this dissertation.

v

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

DEDICATION...... iii

ACKNOWLEDGEMENTS...... iv

LIST OF TA BLES...... viii

LIST OF FIGURES...... ix

ABSTRACT...... xi

CHAPTER PAGE

CHAPTER I. MATRIX (MMPS) AND TISSUE INHIBITORS OF METALLOPROTEINASES (TIMPS) IN OVARIAN PHYSIOLOGY: A LITERATURE REVIEW...... 1

Introduction ...... 1 Characteristics and Properties of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Metalloprotcinases (TIMPs) ...... 3 MMPs/TIMPs in Follicular Development and Atresia ...... 28 MMPs/TIMPs in Ovulation ...... 34 MMPs/TIMPs in Corpus Luteum Formation, Maintenance, and Regression ...... 41 MMPs/TIMPs in Ovarian Angiogenesis ...... 45 Perspectives ...... 49

CHAPTER II. BOVINE MATRIX METALLOPROTEINASE 2 (MMP-2): MOLECULAR CLONING, EXPRESSION IN THE CORPUS LUTEUM, AND INVOLVEMENT IN ANGIOGENESIS...... 58

Abstract...... 58 Introduction ...... 59 Materials and Methods ...... 60 Results...... 65 Discussion ...... 67

CHAPTER m. BOVINE MEMBRANE TYPE I MATRIX METALLOPROTEINASE (MT1- MMP): MOLECULAR CLONING AND EXPRESSION IN THE CORPUS LUTEUM 80

Abstract...... 80 Introduction ...... 80 Materials and Methods ...... 82 Results...... 88

vi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Discussion ...... 90

CHAPTER IV. TEMPORAL AND SPATIAL EXPRESSION OF TISSUE INHIBITORS OF METALLOPROTEINASES 1 AND 2 (TIMP-1 AND -2) IN THE BOVINE CORPUS LUTEUM...... 104

Abstract...... 104 Introduction ...... 105 Materials and Methods ...... 106 Results...... 110 Discussion ...... Ill

CHAPTER V. REGULATION OF MATRIX METALLOPROTEINASE 2 (MMP-2) EXPRESSION IN LUTEAL AND ENDOTHELIAL CELLS BY CYTOKINES...... 124

Abstract...... 124 Introduction ...... 125 Materials and Methods ...... 127 Results...... 130 Discussion ...... 131

SUMMARY AND CONCLUSIONS...... 141

LIST OF REFERENCES...... 144

APPENDIX...... 181

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

TABLE PAGE

1-1. The MMP family...... 51

1 -2. Biochemical and molecular features of MMPs ...... 52

I -3. ECM and non-ECM substrates of MMPs ...... 54

1 -4. Biochemistry and molecular features of TIMPs ...... 57

viii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

FIGURE PAGE

1 -5. Domain organization of the MMPs ...... 53

2-1. Sequence and homology analysis of bovine MMP-2 ...... 72

2-2. Tissue distribution of bovine MMP-2 mRNA ...... 74

2-3. MMP-2 mRNA expression in the bovine CL ...... 75

2-4. MMP-2 expression in the bovine CL ...... 76

2-5. MMP-2 localization in the bovine CL ...... 77

2-6. MMP-2 activity in bovine capillary endothelial cells ...... 78

2-7. Effects of MMP-2 antibody on in vitro capillary tube formation of BCE cells ...... 79

3-1. Sequence and homology analysis of bovine MT1-MMP ...... 95

3-2. MT1-MMP mRNA expression in bovine tissues ...... 98

3-3. MT1-MMP mRNA expression in the bovine CL ...... 99

3-4. MT1-MMP protein expression in the bovine CL ...... 100

3-5. MMP-2 activity in the bovine C L...... 101

3-6. Cellular localization of MT1-MMP in the bovine CL ...... 103

4-1. TIMP-1 mRNA expression in the bovine CL ...... 117

4-2. TIMP-2 mRNA expression in the bovine CL ...... 118

4-3. Activities of tissue inhibitors of metalloproteinases (TIMPs) in the bovine C L ...... 119

4-4. TIMP-1 protein expression in the bovine CL ...... 120

4-5. TIMP-2 protein expression in the bovine CL ...... 121

4-6. Cellular localization of TIMP-l and -2 in the bovine C L ...... 122

5-1. Dose dependent responses of MMP-2 expression in luteal cells to TNFa ...... 136

5-2. Dose dependent responses of MMP-2 expression in luteal cells to TNFa ...... 137

ix

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5-3. Time dependent responses of MMP-2 expression in luteal cells to TNFa ...... 138

5-4. Time dependent responses of MMP-2 expression in luteal cells to TNFa ...... 139

5-5. Regulation of MMP-2 expression in luteal-derived endothelial cells to cytokines ...... 140

x

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

EXPRESSION, ACTIVATION, AND REGULATION OF MATRIX

METALLOPROTEINASE 2 (MMP-2) IN THE BOVINE CORPUS LUTEUM

by

Bo Zhang

University of New Hampshire, May, 2002

Matrix metalloproteinases (MMPs) have been postulated to be important for

angiogenesis and for tissue remodeling events associated with corpus luteum (CL)

development. The MMPs are mainly regulated at three levels, transcription, activation,

and inhibition by endogenous tissue inhibitors of metalloproteinases (TIMPs). Unlike

most MMPs, pro-MMP-2 activation is accomplished on the cell surface rather than

extracellularly. The objectives of the present study were to clone bovine cDNAs for

MMP-2 and its activator, membrane type 1- (MTl-)MMP, to investigate the expression

and localization of MMP-2, MT1-MMP, TIMP-1, and TIMP-2 in three ages of CL, and

to explore the regulation of MMP-2 expression in luteal and endothelial cells by

cytokines. Molecular cloning and sequence analysis demonstrated that the bovine MMP-

2 and MTl-MMP genes are highly similar to their homologs in other species. Although

the levels of MMP-2 and MTl-MMP transcripts were constant in the early (day 4), mid

(day 10), and late (day 16) stage CL, active MMP-2 and MTl-MMP were

xi

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significantly increased (p<0.05) from the early to the mid and late stages. In addition, the

patterns of TIMP-2 mRNA and protein expression were similar to MMP-2 and MTl-

MMP, being expressed at higher (p<0.05) levels in the mid and late stages than the early.

Immunohistochemistry demonstrated that MMP-2, MTl-MMP, and TIMP-2 were

localized in endothelial and large luteal cells. In contrast, TIMP-1 mRNA and protein

were highly expressed in the early and mid cycle CL, but decreased in the late stage.

TIMP-1 was detected in vascular smooth muscle cells and large luteal cells. In a luteal

cell culture system, TNFa stimulated MMP-2 expression in a dose and time dependent

manner. In luteal-derived endothelial cells, TNFa increased while IFNy inhibited MMP-2

expression. In the presence of both cytokines, IFNy attenuated the stimulatory effects of

TNFa alone, bringing MMP-2 expression down to control levels. In conclusion, the

coordinate expression of TIMP-2, and active MMP-2 and MTl-MMP suggests that the

MT1 -MMP/TIMP-2/pro-MMP-2 system may be spatially and temporally available for

pro-MMP-2 activation in the bovine CL. Furthermore, cytokines regulate MMP-2

expression in luteal and endothelial cells.

xii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

MATRIX METALLOPROTEINASES (MMPS) AND TISSUE INHIBITORS OF

METALLOPROTEINASES (TIMPS) IN OVARIAN PHYSIOLOGY:

A LITERATURE REVIEW

I. Introduction

II. Characteristics and Properties of Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of

Metalloproteinases (TIMPs)

III. MMPs/TIMPs in Follicular Development and Atresia

IV. MMPs/TIMPs in Ovulation

V. MMPs/TIMPs in Corpus Luteum Formation. Maintenance, and Regression

VI. MMPs/TIMPs in Ovan an Angiogenesis

VII. Perspectives

I. Introduction

The ovary, a primary female reproductive organ, has two principal functions. Ovaries release

mature egg(s) or oocyte(s) at appropriate intervals for fertilization and successful propagation of

the species (Findlay. 1991). In addition, they produce steroid hormones to regulate reproductive

cy clicity, to prepare the adult female for fertilization and implantation of the zygote, and to

support pregnancy. The follicle and corpus luteum. two structures found in the ovary, are largely

responsible for these functions.

Follicles consist of an inner avascular granulosa compartment harboring the oocyte, an

external vascularized thecal layer, and a in between to separate these two

compartments. The development of follicles from primordial to primary, then to secondary, and

-l-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. eventually the final Graafian stages involves not only the proliferation of granulosa and theca

cells, but also the breakdown and resynthesis of to accommodate the

enlargement and maturation of the follicle (Rodgers et al.. 1999: Rodgers et al., 2000). When

stimulated by the preovulatory surge of gonadotropins, the dominant Graafian follicle ruptures

and releases the mature oocyte into the oviduct. This event requires the breakdown of tissue

barriers, such as the basement membrane where granulosa cells rest, the theca externa,

fibers, tunica albuginea, and a second basement membrane beneath the surface epithelium, before

the ovarian surface is breached to release the egg (Espey. 1994a: Espey. 1994b). Following

ovulation, fibroblasts and endothelial cells of the thecal layer invade the granulosa layer. The

gonadotropin surge also initiates luteinization. a process by which the remaining ruptured follicle

is transformed into a new endocrine gland called the corpus luteum. which secretes progesterone

to support pregnancy (Niswender and Nett. 1994). If fertilization does not occur, the CL will

undergo a degenerative process, referred to as Iuteolysis or luteal regression, enabling a new

reproductive cycle to commence.

All these physiological events that occur during the course of a normal reproductive cycle

necessitate remodeling of the extracellular matrix (ECM). which require the participation of four

classes of proteolytic : serine , cysteine proteases, aspartic proteases, and

metalloproteases. Of these, a wealth of evidence suggests that matrix metalloproteinases (MMPs)

and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs). are known to

play important roles, not only in ECM remodeling in pathological situations such as rheumatoid

arthritis, wound healing (Ravanti and Kahari. 2000) and tumor invasion and metastasis (Kahari

and Saanalho-Kere. 1999: Stamenkovic. 2000). but in physiological processes such as embrvo

development, bone formation, implantation, and the ovarian events described above (Smith et al.,

1999: Vu and Werb. 2000). The present review focuses on the molecular and biochemical

characteristics of MMPs and TIMPs. and their roles in follicular development, ovulation, corpus

luteum development and regression, and angiogenesis in the ovary.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II. Characteristics and Properties of Matrix Metalloproteinases (MMPs) and Tissue

Inhibitors of Metalloproteinases (TIMPs)

The matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent

proteolytic enzymes that share a similar protein structure and collectively are able to digest all

known ECM macromolecules (Matrisian. 1990: Birkedal-Hansen et al., 1993). However,

individual MMP members have distinct protein structures and different preferences for a

particular group of matrix proteins (Massova et al.. 1998). The MMP family members are

products of different genes. So far. more than 25 members have been identified in this

family (see Table 1-1). The characteristic domains of MMPs include (see Figure 1-1): (1) the

signal peptide domain, which directs the enzyme into the rough endoplasmic reticulum during

synthesis: (2) the propeptide domain, which maintains enzyme latency until it is removed or

disrupted: (3) the catalytic domain, which contains the highly conserved Zn:‘ binding region

(HExGHxxGxxHS/T) and dictates enzyme activity: (4) the hemopexin domain, which is

responsible for the substrate specificity' of MMPs: and (5) a small hinge region, which acts as a

"hub", enabling the hemopexin region to present substrate to the active core of the catalytic

domain. For the subfamily of membrane-type MMPs. there is also a transmembrane domain,

which has a membrane-spanning segment of about 20 hydrophobic amino acids and an

intracellular domain of approximately 24 amino acids.

1. MMP family

Based on these structural and functional features. MMP family members can be classified

into different subfamilies (see Table 1-2): (I) , including MMP-1, MMP-8, and

MMP-13: (2) . including MMP-2 and MMP-9: (3) stromelysins, including

stromelysin-l. and -2: (4) membrane-type metalloproteinases (MT-MMPs), including MT1-6

MMP: and (5) other MMPs. which require further characterization. This last group includes

- J -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-19. MMP-18. and MMP-7 There are now more than twenty-five members in the MMP

family.

(1) Collagenases Collagenases are the first enzymes to be characterized in the MMP

family (Gross and Lapiere. 1962). Thus far. four members have been identified in this

subfamily. They are: collagenase I (MMP-1), collagenase 2 (MMP-8), collagenase 3

(MMP-13). and X enopus collagenase 4 (MMP-18). These collagenases cleave the triple helix of

type I. II. and III collagen exclusively at the Gly 77 ;-Leu/Ileu 776 peptide bond, which is located

very close to three-quarters of the distance from the amino terminus of the substrate molecule.

The N-terminal V* and C-terminal '/4-length fragments that are generated then undergo

spontaneous denaturation and become susceptible to further cleavage by other MMPs such as

gelatinases (Miller et al.. 1976: Hasty et al.. 1987: Dioszegi et al., 1995: Mitchell et al.. 1996:

Reboul et al.. 1996). However, each of these collagenases has various preferences for different

collagen types.

Although the first vertebrate collagenase activity was originally described by Gross et al.

(1962) in amphibian tissues, it took about one decade before human collagenase was purified in

skin (Bauer et al.. 1970) and fibroblasts (Stricklin et al., 1977). The cDNA o f human collagenase

was also cloned from fibroblasts (Goldberg et al.. 1986). This enzyme, bearing the designation

matrix metalloproteinase-1 (MMP-1), has served as the prototype for all other interstitial

collagenases. MMP-1 is secreted from cells as two latent forms, one with molecular weight of 52

kDa and the other 57 kDa. which depend on the status of the Asn-linked glycosylation (Dioszegi

et al.. 1995). After activation, the enzyme is processed to a 42 kDa form (Table 1-2). MMP-l

preferentially degrades type III collagen. It also cleaves type I, II, and X , type I gelatin,

the antiprotease pranti trypsin. and it is likely that other substrates exist for this enzyme as well

(W elgus et al.. 198 la: Welgus et al., 198 lb). A variety of cell types, including tumor cells,

endothelial cells, fibroblasts, keratinocytes, chondrocytes, osteoblasts, and hepocytes, have the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ability to produce MMP-1. indicating the importance of this MMP in both pathological and

physiological conditions (Birkedal-Hansen etal.. 1993; Birkedal-Hansen. 1995).

Collagenase-2 (MMP-8) is also called due to its original

identification in human neutrophils as a distinct protein (Macartney and Tschesche. 1983: Hasty-

et al.. 1984) and gene product (Hasty et al.. 1990) from MMP-1. However, the collagenase

activity in human neutrophil was first described in the late 1960s (Lazarus et al., 1968). One

feature unique to MMP-8 is that this enzyme is stored in specific granules in neutrophil

leukocytes (Murphy et al.. 1977) and released upon phagocytic stimulation (Oronsky et al., 1973).

In addition, although the function is still unclear, there is a high level o f glycosylation in MMP-8

(Birkedal-Hansen et al.. 1993). The reported molecular weight of MMP-8 varies depending on the

investigators. This may be due to method of determination (purified protein vs. deduced from

cDNA). In the purified protein, the calculated molecular weight may be affected by the level of

glycosylation on the enzyme. In general, it is believed that the latent form of MMP-8 is

approximately 75 kDa. but it is usually processed during extraction protocols to about 58 kDa

(this form still lacks catalytic activity). The active form of MMP-8 has a molecular mass of

around 40-42 kDa. In addition to its expression in polymorphonuclear leukocytes during their

maturation (Hasty et al.. 1987). MMP-8 is also produced in melanoma cells (Giambemardi et al..

1998). rheumatoid synovial fibroblasts (Hanemaaijer et al.. 1997), endothelial cells, and

chondrocytes (Cole et al.. 1996). Furthermore, its high expression in rat postpartum uterus

indicates that it may play a potential role during involution of this reproductive organ (Balbin et

al.. 1998). This enzyme displays a substrate specificity profile different from that of MMP-1

(Hasty et al.. 1987). The preferred substrate for MMP-8 is , not type in collagen,

which is digested at a higher rate by MMP-l (Hasty et al., 1987). Intriguingly, MMP-8 also

degrades collagen type Q at a much greater (~ 150 times) rate than MMP-1 (Knauper et al.,

1997b: Jeffery. 1998). indicating that it may be involved in diseases such as rheumatoid arthritis

and osteoarthritis, where type II collagen degradation plays a major role. Moreover, MMP-8 is

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. capable o f cleaving aggrecan (Fosang et al., 1996a; Arner et al., 1997), but at sites distinct from

the major enzyme called cartilage (Arner et al., 1997).

Collagenases 3. designated as MMP-13. was first sequenced from human breast tumor

(Freije et al., 1994). The latent and active forms of MMP-13 possess a molecular mass of about

52 and 42 kDa, respectively. Unlike MMP-1. which has a low activity on type D collagen, MMP-

13 cleaves type II collagen at a higher rate than types I and III. Similar to MMP-8, this efficient

catalytic action of MMP-13 on type II collagen signifies its importance in the physiology and

pathology of cartilage, where type II collagen is the predominant ECM protein. Additionally,

MMP-13 displays considerably greater gelatinolytic activity than collagenolytic activity (Welgus

et al.. 1985a; Mitchell et al.. 1996). Of particular interest MMP-13 has broad substrate

specificity. MMP-13 also catalyzes type IV. IX. X and XIV collagens, . fibrillin-1,

fibronectin. . aggrecan. and inhibitors of serine proteinases (Fosang et al., 1996a;

Knauper et al.. 1996a; Knauper et al.. 1997a; Ashworth et al.. 1999). Thus, in addition to its

original localization in breast carcinomas (Freije et al.. 1994; Heppner et al., 1996; Uria et al..

1997). MMP-13 is expressed in a variety of tumor types, including chondrosarcomas (Uria et al.,

1998). melanomas (Airola et al.. 1999). and squamous cell carcinomas (Johansson et al.. 1997;

Cazorla et al.. 1998). It is also associated with a number of diseases, where excessive collagen

degradation has been postulated to be causative, such as periodontitis (Uitto et al., 1998),

rheumatoid synovium (Stahle-Backdahl et al.. 1997), osteoarthritic cartilage (Mitchell et al..

1996; Shiopov et al.. 1997). and atherosclerosis (Sukhova et al.. 1999). Interestingly, this enzyme

is also highly expressed in the uterus (Welgus et al.. 1985b), suggesting its potential roles during

tissue remodeling of this reproductive organ.

Collagenase 4 (MMP-18) was recently identified in the African clawed toad, Xenopus laevis

(Stolow et al.. 1996). This is also the first sequenced MMP from an amphibian. The predicted

molecular masses of the latent and active forms of MMP-18 are 51 and 42 kDa, respectively

(Stolow et al., 1996). Collagenase 4 has the ability to degrade type I collagen (Stolow et al.,

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1996). rat type II and human type in collagens. a 1-antitrypsin, and a2-macroglobulin (Shi and

Sang. 1998). MMP-18 is transiently expressed in the region of branchial arches, thorax, and the

dorsal anterior axis during the late stages of Xenopus embryogenesis (Damjanovski et al., 1999:

Damjanovski et al.. 2000: Damjanovski et al.. 2001). The embryo lethality during the late stages

of embryogenesis caused by over-expression of MMP-18 further indicates the restricted temporal

and spatial expression of this enzyme (Damjanovski et al.. 2001). The homologs of collagenase 4

in other species have not been identified.

(21 Gelatinases There are two members in this group: A and .

Although a gelatinase activity, which was retrospectively determined to be gelatinase B. was

detected from rheumatoid synovial fluid in 1972 (Harris and Krane. 1972). it was about six years

later that gelatinase activity was first separated from collagenase and stromelysin (Sellers et al..

1978). Whereas collagenase was the first described MMP to degrade native collagen into

fiagments (Gross and Lapiere. 1962). gelatinases have activity against denatured collagens (or

collagen fiagments) (Sopata et al.. 1974). Based on their sizes and substrate specificities, these

two identified gelatinases are named 72-kDa gelatinase (. MMP-2) and 92-kDa

gelatinase (gelatinase B. MMP-9). respectively. The early experiments demonstrated that both

gelatinases were also able to digest soluble type IV collagen, producing characteristic V* N-

terminal and V* C-terminal fragments (Liotta et al.. 1979: Mainardi et al.. 1980: Fessler et al..

1984: Murphy et al.. 1989). Therefore, gelatinase A and B are also called 72-kDa and 92-kDa

type IV collagenase. respectively. However, it has been questioned whether MMP-2 and MMP-9

are true type IV collagenases in vivo since the full-length type IV collagen is not susceptible to

cleavage by either enzyme (Mackay et al.. 1990: Moll et al., 1990). Structurally, gelatinases are

characterized by three repeats of type II fibronectin-like gelatin binding regions, which enable

gelatinases to bind to denatured collagens (O'Farrell and Pourmotabbed, 1998).

The 72-kDa gelatinase A (MMP-2) is processed to an approximately 62 kDa active enzyme

via a membrane-dependent mechanism, while most other MMPs are activated extracellularly

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Brown et al., 1990: Strongin et al., 1993). In addition to gelatin and type IV collagen, MMP-2

possesses proteolytic actions on type I (Aimes and Quigley, 1995), type V (Okada et al., 1990),

type VII (Seltzer et al., 1989), and type X (Gadher et al., 1989; Welgus et al., 1990) collagens,

and other ECM components such as (Senior et al.. 1991), laminin (Giannelli et al., 1997),

(Imai et al.. 1995a), and aggrecan (Fosang et al.. 1992; Fosang et al., 1996b).

Surprisingly. MMP-2 also cleaves the non-ECM component amyloid protein precursor with a P*

secretase-like activity- (LePage et al.. 1995), indicating its involvement in Alzheimer's disease. It

is because of this wide range of substrate specificities that enables MMP-2 to play important roles

in a number of cellular processes, such as cell proliferation, differentiation, adhesion, and

migration. Most importantly. MMP-2 also has a significant role in tumor invasion and

angiogenesis. It binds integrin a ,p 3. facilitating the pericellular proteolysis during tumor invasion

and angiogenesis (Brooks et al.. 1996).

In addition to the cysteine-rich repeats of type II fibronectin-like inserts in the catalytic

domain. MMP-9 also has a unique proline-rich -like insert at the end of its hinge

region: the significance of which is still unclear (Wilhelm et al., 1989). The predicted molecular

weight of MMP-9 from its cDNA is about 76 kDa. However, the molecular weight determined by

biochemical analyses is about 92 kDa. This difference is due to N- and O-linked glycosylation on

this enzyme (Wilhelm et al.. 1989; Murphy and Crabbe. 1995). The active form of MMP-9 is

approximately 82 kDa. but progressive proteolytic and/or autolytic cleavage on the N- and C-

termini of the enzyme may process it into 65 kDa or even smaller forms (Fridman et al., 1995;

Murphy and Crabbe. 1995: Sang et al.. 1995). MMP-9 shares a similar range of substrates with

MMP-2 (Table 1-3). Distinct from MMP-2. MMP-9 is not able to hydrolyze type I collagen.

However, it has been reported that MMP-9 cleaves the N-terminai telopeptide of type I collagen

in an acidic environment (Okada et al.. 1995bb). Although MMP-9 degrades aggrecan, and link

protein, which stabilizes the interaction between aggrecans and hyaluronate in proteoglycan

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aggregates, its efficiency is much lower than MMP-2 (Fosang et al., 1992; Nguyen et al., 1993).

MMP-9 is also localized on the cell surface, where CD-44 serves as a docking protein to promote

tumor invasion and angiogenesis (Yu and Stamenkovic, 1999; Yu and Stamenkovic, 2000).

Accumulating evidence indicates that MMP-9 is a major MMP in a variety of normal

physiological processes such as implantation (Librach et al., 1991; Behrendtsen et al.. 1992), and

bone development (Reponen et al.. 1994; Blavier and Delaisse, 199S; Vu and Werb, 2000). Not

surprisingly, high levels of MMP-9 expression are also observed in pathological conditions,

including tissue injury- (Partridge et al.. 1993; La Fleur et al.. 1996; Pender et al.. 1997),

inflammation (Weeks et al.. 1993; Leppert et al.. 1995a; Leppert et al., 1995b; Opdenakker et al.,

2001), arthritis (Stein-Picarella et al.. 1994), and wound healing (Salo et al., 1994; Buisson et al..

1996). and tumor angiogenesis (Bergers et al.. 2000). Additionally, its expression has been

demonstrated in tumor cells from diverse sites of the body including colorectum (Pyke et al..

1993; Nielsen et al.. 1996). bone (Vu et al.. 1998), bone marrow (Ries et al.. 1994). brain (Rao et

al.. 1993). breast (Farias et al.. 2000). liver (Ashida et al.. 1996), lungs (Canete-Soler et al.. 1994;

Farias et al.. 2000). skin (Pyke et al.. 1992), and prostate (Dong et al.. 2001).

(3) Stromelvsins The stromelvsins include stromelysin-1 (MMP-3) and stromelvsin-2

(MMP-10). These two enzymes are classified into their own subgroup because they lack the

ability to cleave the triple helical regions of collagen, even though they share a similar domain

structure with the collagenases (Stemlicht and Werb. 2001). Furthermore, they are distinguished

from other MMPs by their amino acid sequences being noticeably similar to each other, but not to

other MMPs (Muller et al., 1988). Lastly, they possess a 9-amino acid residue insert in the hinge

region, which is absent from other MMPs.

The first neutral non-collagenolytic proteinase activity was noticed in human cartilage

(Sapolsky et al.. 1974) and in the conditioned medium from rabbit synovial fibroblasts (Werb and

Reynolds. 1974) at the same time by two independent research groups. The enzyme was later

named "stromelysin" because it was produced by stromal cells (Chin et al., 1985). Historically,

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stromelysin was also known as "collagenase activator protein” (Treadwell et al., 1986) and

"transin” (Matrisian et al.. 1986). cDNA cloning ultimately determined that the three proteins

were the same metalloproteinase, MMP-3 (stromelysin I) (Matrisian et al., 1985; Whitham et al..

1986; Fini et al., 1987; Wilhelm et al.. 1987; Saus et al.. 1988). The latent form o f MMP-3 has a

molecular mass of 56 kDa. while the active form is about 45 kDa (Nagase, 1995; Nagase, 1998).

Asn-linked glycosylation is also observed in MMP-3 (Wilhelm et al., 1987). However, not all the

secreted MMP-3 is glycosylated, for example. 80% of MMP-3 from fibroblasts is unglycosylated,

while the remaining ones are produced as a glycoprotein (Nagase. 1995). MMP-3 hydrolyzes

numerous ECM components including aggrecan (Wilhelm et al.. 1993). cartilage link protein

(Nguyen et al.. 1989). gelatin (Murphy et al.. 1992a). vitronectin (Imai et al.. 1995a). tenascin

(Imai et al.. 1994). fibronectin (Muir and Manthorpe. 1992). versican (Perides et al.. 1995).

clastin (Murphy et al.. 1992a). laminin (Bejarano et al.. 1988). perlecan (Whitelock et al.. 1996).

proteoglycan (Fosang et al.. 1991). and type II. IX. X. and XI collagens (Wu et al.. 1991).

However, it is unable to hydrolyze triple-helixal collagen peptide (Lauer-Fields et al.. 2000).

Stromelysin 2 (MMP-10) was the first MMP to be identified directly from rat cDNA clones

(Breathnach et al.. 1987). The deduced protein was also designated as transin 2. which has 71%

identity to MMP-3 (transin 1) at the amino acid level. The human MMP-10 was subsequently

cloned from human tumors (Muller et al.. 1988). MMP-10 is capable of activating pro-MMP-8

(Knauper et al.. 1994). There is limited information about the substrate specificity of MMP-10.

except its ability to effectively degrade cartilage link protein (Nguyen et al.. 1993) and gelatin

(Sanchez-Lopez et al.. 1993). In human endometrium. MMP-10 mRNA expression increases

during premenstrual and menstrual phases (Hampton and Salamonsen. 1994; Osteen et al., 1994).

The function of MMP-10 during tumor progression is still not clear.

(4) Membrane-type metalloproteinases (MT-MMPs) At present, there are six

membrane-type metalloproteinases: MTl-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP

(MMP-16), MT4-MMP (MMP-17). MT5-MMP (MMP-24), and MT6-MMP (MMP-26). MT-

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMPs contain a cleavage site for furin proteinases between the propeptide and catalytic domains,

providing the basis for furin enzyme-like dependent activation of latent MT-MMPs prior to

secretion.

The notion that pro-MMP-2 is activated on the cell membrane rather than in the extracellular

environment led to the discovery of MT-MMPs. MT1-MMP. the first member of the membrane-

type MMP subfamily, was cloned from a human placenta cDNA library (Sato et al.. 1994).

Distinct from all other MMPs that have been identified, this enzyme has a hydrophobic

transmembrane segment at the C-terminus following the hemopexin domain. Indeed. MT1-MMP

activates pro-MMP-2 (Sato et al.. 1994). Subsequent studies suggest that this activation process

also involves TIMP-2. which binds to MT1-MMP to form a MT1-MMP/TIMP-2 ”co-receptor”

for pro-MMP-2. The latter is then presented to an adjacent, active MT1-MMP. which cleaves the

propeptide domain of pro-MMP-2. initiating the activation process (Deryugina et al.. 1997: Butler

et al.. 1998: Cao et al.. 1998; Cao et al.. 1999: Murphy et al.. 1999b). In addition. MT1-MMP

converts latent MMP-13 (collagenase-3) to the active form (Knauper et al.. 1996c). Moreover.

MT1-MMP also functions as a proteolytic enzyme, capable of cleaving various ECM

components, including type I. II. and ID collagen, gelatin, fibronectin. laminin-1. vitronectin,

cartilage proteogly cans, and fibrillin-1 (Imai et al.. 19%: Pei and Weiss. 1996: d'Ortho et al..

1997; Ohuchi et al.. 1997; Aznavoorian et al.. 2001).

MT2-MMP was identified from a human lung cDNA library’ (Will and Hinzmann. 1995).

MT2-MMP is predominantly expressed in liver, placenta, intestine, colon, and testis (Will and

Hinzmann. 1995). MT2-MMP also activates pro-MMP-2. Different from MT1-MMP. however,

MT2-MMP activates pro-MMP-2 via a pathway independent of TIMP-2 (Morrison et al., 2001).

MT2-MMP also degrades laminin. fibronectin. and tenascin (d’Ortho et al.. 1997).

Having been identified at the same time as MT2-MMP. MT3-MMP was originally

designated as MT-MMP-2 (Takino et al.. 1995). MT3-MMP is primarily expressed in the brain.

MT3-MMP activates pro-MMP-2 and also hydrolyzes gelatin, casein, type III collagen, and

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fibronectin (Shimada et al.. 1999: Kang et al.. 2000), and can be detected in membrane-bound and

soluble forms (Matsumoto et al.. 1997: Shofuda et al.. 1997).

In addition to its original identification in breast carcinoma, MT4-MMP is also expressed in

normal tissues including brain, colon, ovary, and testis (Puente et al.. 1996). MT4-MMP is

structurally different from other MT-MMPs Instead of having a 20-amino acid tail after the

transmembrane domain (as observed in other MT-MMPs), MT4-MMP has a very short or no

cytoplasmic tail following the putative transmembrane domain(ltoh et al.. 1999). Further

sequence and biochemical analyses suggest that the C-terminus of MT4-MMP acts as a

glycosylphosphatidylinositol (GPI) anchoring signal, rather than as a transmcmbrane domain

(Itoh et al.. 1999). MT4-MMP is the first GPI-anchored proteinase in the MMP family. This

property enables it to be shed from plasma membranes by endogenous enzymes. MT4-MMP

converts membrane-bound pro-TNFa to its mature form (Wang et al.. 1999b: English et al..

2000). There are contradictory reports regarding its capability to activate pro-MMP-2

(Kolkenbrock et al.. 1999: Wang et al.. 1999b: English et al.. 2000). MT4-MMP is unable to

hydrolyze collagen types I. II. III. IV. and V. fibronectin. laminin or deconn, but degrades gelatin

and some synthetic MMP substrates (Kolkenbrock et al.. 1999: Wang et al.. 1999b).

MT5-MMP. originally identified from a human brain cDNA library, activates latent MMP-2

and is predominantly expressed in brain, kidney, pancreas, and lung (Llano et al., 1999: Pei.

1999a). It is highly expressed in brain tumors (Llano et al.. 1999). Interestingly, MT5-MMP is

also shed from the cell membrane by a furin type convertase. suggesting that it may function both

as a membrane-bound and soluble proteinase (Pei. 1999a: Wang and Pei. 2001). Proteoglycans

are the preferred substrates for MT5-MMP (Wang et al.. 1999a).

MT6-MMP was cloned from leukocytes (Pei. 1999b). Thus, it is also called leukolysin (Pei.

1999b). High levels of MT6-MMP expression was also observed in brain tumors (Velasco et al.,

2000). MT6-MMP is the second GPI-anchored enzyme in the MMP family, showing a similar C-

terminal sequence as MT4-MMP (Kojima et al.. 2000). MT6-MMP activates pro-MMP-2

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Velasco et al.. 2000). and cleaves type-IV collagen, gelatin, fibronectin and fibrin, but not

laminin-1 (Pei. 1999b: English et al., 2001: Kang et al., 2001). Cytokines may play an important

role in processing this enzyme from secretory vesicles and plasma membrane to the extracellular

milieu as a soluble, active protein (Kang et al.. 2001).

(51 Matrilvsins Currently, two members have been identified in this subfamily,

matrilysin I (MMP-7) and matrilysin 2 (MMP-26). They are the smallest MMPs since they lack

the hemopexin domain.

MMP-7 was first purified from the postpartum involuting rat uterus (Sellers and Woessner.

1980: Woessner and Taplin. 1988). Its cDNA was cloned and sequenced from a human tumor

library (Quantin et al.. 1989). This enzyme was also previously named as PUMP-l (putative

metalloproteinase-1). Due to its simple domain organization. MMP-7 is small in size, with a 28

kDa latent and 19 kDa active forms (Wilson and Matrisian. 1998). However, the minimal domain

structure docs not affect its broad substrate specificities at all. MMP-7 hydrolyzes type IV

collagen (Murphy et al.. 1991), gelatins (Quantin et al.. 1989). laminin (Imai et al.. 1995a),

fibronectin (Imai et al.. 1995b). elastin (Imai et al.. 1995b). vitronectin(Imai et al.. 1995a).

aggrecan (Fosang et al.. 1992). cartilage link protein (Nguyen et al.. 1993), proteoglycan (Imai et

al.. 1995b: Halpert et al.. 1996). fibulin (Sasaki et al.. 1996). and entactin (Sires et al.. 1993).

MMP-7 is involved in many normal tissue remodeling events by being expressed in the uterus

(Sellers and Woessner. 1980). hair follicles and sweat glands, small intestinal crypts, skin, and

airway epithelium (Giambemardi et al.. 1998: Wilson and Matrisian. 1998). Distinct from other

MMPs. expression of MMP-7 is restricted to glandular epithelium, and is secreted apically.

MMP-7 is also expressed by malignant epithelial cells in tumors of the breast (Heppner et al..

1996). prostate (Klein et al.. 1997). colon (Yoshimoto et al.. 1993), lung (Bolon et al.. 1997). and

gastrointestinal tract (Adachi et al.. 2001). This is unlike other MMPs, whose source is the

stromal cells that surround epithelial tumors, rather than the tumor cells themselves (Wilson and

Matrisian. 1996: Wilson etal., 1997).

- 1 3 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A novel metalloproteinase (MMP-26) has been recently identified independently by several

groups (de Coignac et al., 2000: Park et al., 2000; Uria and Lopez-Otin. 2000; Marchenko et al..

2001). Even though this enzyme has a high homology' (45% identity') with M M P-12 (macrophage

metalloproteinase). its protein structure is more related to MMP-7 (matrilysin) by having the

minimal domain organization, which consists of only the signal peptide, propeptide and catalytic

domains (de Coignac et al.. 2000; Marchenko et al.. 2001). Furthermore, MMP-26 also contains a

Thr(207) residue upstream of the zinc binding motif, which is a specific feature of all known

matrilysins (Abramson et al.. 1995; Uria and Lopez-Otin. 2000; Marchenko et al.. 2001).

Accordingly, the name matrilysin-2 has been suggested for this novel MMP (Uria and Lopez-

Otin. 2000: Marchenko et a l. 2001). The name "endometase" has also been used for this enzyme

as it is derived from a human endometrial tumor cDNA library (Park et al.. 2000). Distinct from

other MMPs. MMP-26 has a unique "cy steine switch" motif sequence PHCGVPD. which

replaces the highly conserved motif PRCGXXD. indicating its activation may be sensitive to pH

(Marchenko et al.. 2001). Of note. MMP-26 may not be a classical matrilysin in that there is low

sequence similarity between MMP-26 and MMP-7. Also, they are located on different

chromosomes and they have dissimilar predicted surface modeling. MMP-26 has a diverse array

of substrates, including gelatins, fibronectin, fibrogen. vitronectin. However. MMP-26 is unable

to degrade types I-IV collagen, tenascin C. and laminin V (Uria and Lopez-Otin. 2000;

Marchenko et al.. 2001). MMP-26 is highly expressed in the placenta (de Coignac et al.. 2000;

Park et al.. 2000). the uterus (Uria and Lopez-Otin. 2000) and a number of developing fetal

tissues (Marchenko et al.. 2001). Expression of MMP-26 in carcinomas of the endometrium, lung,

and prostate suggests its unique role in the progression of epithelial tumors (Uria and Lopez-Otin,

2000).

(6) Other MMPs Based on structure and substrate specificity, several MMPs do not fit into

any of the subgroups describe so far. although some investigators argue that matrilysin and

macrophage metalloelastase should be included in the group of stromelysins.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stromelysin-3 (MMP-11) was first cloned from a breast carcinoma cDNA library . Its

expression is restricted to the stromal cells surrounding the tumor cells (Basset et al.. 1990).

While stromelvsins 1 and 2 share a 76% identity to each other in amino acid sequence, MMP-11

only shows 39% and 36% sequence identity to other stromelvsins and other MMPs. respectively

(Sang and Douglas. 1996). Structurally. MMP-11 is also different from stromelvsins 1 and 2.

MMP-11 contains a 10-amino acid residue insert (RXK/RR) at the end of the propeptide domain,

w hich resembles a furin-like recognition site (Pei and Weiss. 1995). Indeed, intracellular

activation of pro-MMP-11 by Golgi-associated furin enzyme, which is different from the

extracellular activation of other MMPs. was observed (Pei and Weiss. 1995). The just described

characteristics distinguish MMP-11 (stromelysin-3) from the subfamily of stromelvsins.

Although MMP-11 cleaves some ECM proteins such as type IV collagen, laminin. fibronectin.

and aggrecan. its efficiency is much lower than stromelysin-1 (Murphy et al.. 1993b). However.

MMP-11 is critical for tumor progression, since MMP-11-deficient mice are resistant to

chemically-induced tumors (Noel et al.. 1996: Basset et al.. 1997). It has also been postulated that

expression of MMP-11 in adjacent cells is pivotal for the epithelial-to-mesenchvmal conversion

of tumor cells (Ahmad et al.. 1998). This tumor promoting capability of MMP-11 may result from

its ability to release mature or active growth factors from the corresponding latent forms

sequestered in the extracellular matrix (Basset et al.. 1997).

Macrophage metalloelastase (MMP-12) was first purified from the mouse as a 22 kDa

enzyme (Banda and Werb. 1981). which is processed from its 54 kDa latent form by conventional

propeptide removal and atypical processing at the C-terminus (Shapiro et al.. 1992). Cloning and

sequence analysis of mouse and human MMP-12 revealed that this enzyme has a domain

structure typical of collagenases. but it only shares 33-48% homology with other MMPs at the

amino acid level (Shapiro et al.. 1992: Shapiro et al.. 1993). Although MMP-12 is unable to

hydrolyze interstitial collagens. it cleaves a wide array of ECM components including elastin,

type IV collagen, type I gelatin, vitronectin, fibronectin. proteoglycans, laminin-1, and myelin

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. basic protein (Chandler et al., 1996; Gronski et al., 1997). MMP-12 has been related to a number

of macrophage-involved diseases such as pulmonary' emphysema (Hautamaki et al., 1997) and

Lewis lung carcinoma (Dong et al.. 1997).

Human MMP-19 was the first MMP identified by using the EST (expressed sequence tags)

database search, although its 5' end was determined by 5'-RACE (Cossins et al.. 1996). These

investigators called their enzyme MMP-18, without being aware that the then newly identified

Xenopus MMP was already given this designation. Two identical cDNAs. which were designated

as MMP-19. were later cloned from a human liver cDNA library and a library' constructed from

rheumatoid arthritis synovium (Kolb et al.. 1997; Pendas et al., 1997b). Since the latter research

was done on rheumatoid arthritis synovium inflammation, the enzyme was also called RASI-1

(Kolb et al.. 1997). MMP-19 lacks all the specific structural features of collagenases. gelatinases.

and membrane-type mctalloproteinases. For example. MMP-19 has a 16-acidic residue insert in

the hinge region rather than the 9-residue insertion rich in hydrophobic amino acids that is

specific for stromelvsins (Pendas et al.. 1997b). MMP-19 is a potent basement membrane enzyme

capable of hydrolyzing type IV collagen, laminin. fibronectin. type I gelatin, nidogen. tenascin.

aggrecan. and cartilage oligomeric matrix protein (COMP) (Stracke et al.. 2000a: Stracke et al..

2000b). MMP-19 is highly expressed in intestine, ovary, spleen, heart pancreas, lung, thymus,

testis, prostate, and placenta, indicating that it may plav a role in remodeling events in these

tissues (Cossins et al.. 1996: Pendas et al.. 1997b). Furthermore, the expression o f MMP-19 in

capillary endothelial cells of acutely but not chronically inflamed synovium, demonstrates that it

may be involved in angiogenesis (Kolb et al.. 1999). Interestingly. MMP-19 expression is

observed in tumor and endothelial cells of benign tumors, while it disappears as the tumor

progresses towards the invasive and neoplastic phenotypes (Djonov et al., 2001). This suggests

that MMP-19 may be important for tumor survival but not progression.

Enamelysin (MMP-20) was first cloned from a cDNA library of porcine enamel organ

(Bartlett et al.. 1996). Its human homolog was subsequently cloned from an odontoblastic cell

- 1 6-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cDNA library (Llano et al., 1997). The deduced MMP-20 protein consists of 483 amino acid

residues, bearing a 54 kDa molecular mass. Although MMP-20 has the characteristic domain

organization of MMPs. the specific structural features of collagenases, gelatinases. membrane-

type MMPs. and stromelvsins are absent in MMP-20. MMP-20 degrades amelogenin, casein,

aggrecan. and cartilage oligomeric matrix protein (COMP), but has weak action against gelatin

(Llano et al.. 1997; Stracke et al.. 2000a). The restricted expression of MMP-20 signifies its

importance in the processing of enamel matrix during tooth enamel formation (Grant et al.. 1999).

Moreover. MMP-20 expression is also detected in odontogenic tumors and tongue carcinomas

(Takata et al.. 2000; Vaananen et al.. 2001). (ntriguingly, MMP-20 is expressed in granulosa

cells, and is highly elevated by bradykinin treatment indicating its specialized role during

ovulation (Kimura et al.. 2001).

MMP-21 (XMMP) was first cloned from Xenopus laevis (Yang et al.. 1997). Different from

other MMPs. MMP-21 has a 37-amino acid-residue insert at the c-terminal end of the propeptide

domain. This insert, resembling a vitronectin segment, is followed by an RRKR motif, a furin­

like recognition site that is present in stromelysin-3 and MT-MMPs. In addition. MMP-21 lacks

the hinge region, which is typically present in other MMPs for connecting the catalytic and

hemopexin domains. Another specific feature of MMP-21 is that its C-terminal domain consists

of four repeats of vitronectin-like segments (Yang et al.. 1997). The expression of MMP-21 is

transiently induced at the gastrula stage, maintained at the neurula stage, but down-regulated in

the pre-tailbud embryo, suggesting its unique role during early embryo development of Xenopus

laevis. The human homolog of MMP-21. which shows 73% homology to XMMP at the amino

acid level, has been recently identified (Marchenko et al.. 2001). However, its substrate

specificity, and involvement in physiological and pathological processes require further

investigation.

MMP-22 (CMMP) was characterized in chicken embryo fibroblasts (Yang and Kurkinen,

1998). In the catalytic domain. MMP-22 has a unique cysteine residue in the sequence motif that

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. interacts with the structural (noncatalytic) zinc ion. A homologous cysteine residue is also present

in XMMP (Yang et al.. 1997) and MMP-19 (Cossins et al., 1996; Pendas et al., 1997b). The

recombinant MMP-22 enzyme digests casein and gelatin (Yang and Kurkinen, 1998), suggesting

that it possesses collagenoiytic and gelatinolytic activities. The presence of MMP-22 in

mammalian species is not known at this time.

MMP-23 was first cloned from human uterus and testis cDNA libraries, but it was named

MMP21/22 by the investigators (Gururajan et al.. 1998a; Gururajan et al.. 1998b). The identical

sequence was re-discovered from a human ovary cDNA library, bearing the name MMP-23

(Velasco et al.. 1999). The mouse and rat homologs were also reported at the same time[(Pei,

1999c); rat homolog with GenBank accession number ABO 10960 by Ohnishi et al.. 1999], In all

of these species, the domain organization of MMP-23 is strikingly different from all other MMPs

in that it lacks not only a recognizable signal peptide at the N-terminus and the cysteine switch

consensus PRCGVPD in the propeptide domain, but also the hemopexin-like sequence at the C-

terminal end. which is replaced by an Ig-like C2 type (or ILIR-Iike) domain (Pei. 1999c; Velasco

et al.. 1999) Although the cysteine switch motif is absent in MMP-23. the enzyme contains a

conserved furin recognition sequence (RRRR). which is also present in MMP-11 (stromelysin-3)

and MT-MMPs. suggesting it has an activation mechanism different from other MMPs (Pci.

1999c; Velasco et al.. 1999). Surprisingly, a cysteine array motif, which is located downstream of

the catalytic domain, is noted in mouse MMP-23 (Pei. 1999c). while it is not mentioned in the

human homolog (Velasco et al.. 1999). Thus, this enzyme is also named cysteine array matrix

metalloproteinase or CA-MMP (Pei. 1999c). Even though the typical signal peptide is absent.

MMP-23 contains a hydrophobic segment at the N-terminal end (Pei. 1999c). Studies

demonstrate that this hydrophobic region serves as a signal anchor to localize MMP-23, enabling

it to act as a type II transmembrane MMP [type I transmembrane has either a transmembrane

segment (M Tl. 2. 3. 5-MMPs) or a glycosylphosphatidylinositol region (MT4,6-MMPs) at the

C-terminus|, which couples enzyme secretion and activation by a single proteolytic cleavage (Pei

- 1 8-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al.. 2000). This novel mechanism may establish a new pathway for regulating MMP

trafficking. The recombinant MMP-23 degrades a common synthetic MMP substrate and gelatin

(Pei. 1999c: Velasco et al., 1999). In addition to its expression in adult tissues such as heart and

lung, MMP-23 is predominantly expressed in reproductive tissues including prostate, ovary, and

testis. However, its expression in placenta is relatively low or negative (Pei, 1999c: Velasco et al..

1999). The expression of MMP-23 in the ovary will be discussed in a later section.

MMP-27 is a newly identified member in the MMP family, and has a domain organization

characteristic of collagenases. However, although the sequence information has been reported in

the NCBI (GenBank accession number NM_022122 by de Coignac). there are no reports

regarding the biochemical and molecular properties, or tissue expression and cellular localization

of this enzyme.

MMP-28 (Epilysin) is a novel MMP member identified independently by two research

laboratories (Lohi et al.. 2001: Marchenko and Strongin. 2001). Since this novel is

prominently expressed in the epidermis, it is appropriately designated as "epilvsin”. MMP-28

contains the characteristic signal peptide, propeptide, catalytic, hinge, and hemopexin domains,

characteristic of most MMPs. It also has several peptide sequences typical of MMPs. such as the

conserv ed cysteine switch sequence PRCGVTD and the catalytic domain sequence HEIGH (Lohi

et al.. 2001: Marchenko and Strongin. 2001). Unlike other MMPs. however, a unique feature of

its catalytic domain is that it has threonine residues (HEIGHTLGLTH). Structurally. MMP-28 is

similar to MMP-11 (stromelysin-3) in that it has a furin recognition sequence (RRKKR)

following the cysteine switch sequence. But phylogenetic analysis on the catalytic domains of

MMP-28 and other MMPs demonstrates that this enzyme is most closely related to MMP-19.

w hich shows 46% identity to MMP-28 (Lohi et al.. 2001). Therefore, it has been postulated that

MMP-28 is the first member of the MMP-19 subgroup (Marchenko et al., 2001). The

recombinant MMP-28 protein has caseinolytic activity, which can be completely inhibited by

EDTA and a synthetic MMP inhibitor, batimastat (Lohi et al., 2001). In addition to its strong

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression in keratinocvtes, MMP-28 expression is also observed in a broad range of fetal tissues

(lung, brain, skeletal muscle, and kidney), adult tissues (kidney and pancreas), and a number of

carcinomas including colon adenocarcinoma, ovarian carcinoma, and pancreatic adenocarcinoma

(Marchenko et al.. 2001). This suggests that this novel MMP may have a significant role in

normal tissue remodeling and tumor progression.

2. Regulation of MMP activity

MMP activities are pivotal for normal physiological processes, while over-expression of

these enzymes typically results in a variety of diseases and pathological conditions. Therefore, the

temporal expression and spatial localization of MMPs need to be closely coordinated with

activity. This requires strict regulation of MMPs. which occurs at different levels.

(11 Transcriptional regulation As a general rule. MMPs arc not constitutively

expressed in most tissue types. Instead, the transcriptional expression of MMPs can be induced by

a variety of growth factors and cytokines, while their expression can also be suppressed, for

example, by transforming growth factor beta (TGFP) and glucocorticoids (Matrisian. 1990).

Other extracellular signals regulating MMP expression also include extracellular matrix proteins,

integrin-derived signals, and cell stress factors.

The regulation by the above extracellular signals is ultimately accomplished by acting on the

promoters of MMP genes (Borden and Heller. 1997). The most common promoter found in MMP

genes is the AP-l cis-element. which is usually localized at 65 to 79 base pairs upstream of the

transcriptional start site. MMP-1. -3. -7. -9. -10, -12. -13, and -19 are under the regulation of the

AP-l promoter (Gack et al.. 1994; Quinones et al.. 1994; Korzus et al.. 1997; Pendas et al.,

1997a; Chapman et al.. 1999; Mueller et al.. 2000; Campbell et al., 2001; Wu etal., 2001). Other

inducible cis-regulatory elements, such as Ets, SP1, SP3, and NF-k B, are also present in the

promoter region of most MMPs (Borden and Heller, 1997). Thus, transcriptional regulation of

- 2 0 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMPs is complex, since there are numerous signal transduction pathways for extracellular

signals, and one promoter may be affected by various signaling components. For example, the

AP-l cis-element is switched on when an AP-l transcription factor complex, which consists of

members of Jun and Fos proteins, is attached (Curran and Franza, 1988). c-Jun and c-Fos

components are under the regulation of at least three distinct signaling pathways, including

mitogen-activated protein kinases (MAPKs), stress-activated protein kinase/Jun N-terminal

kinases (SAPK/JNKs). and p38 (Heldin, 1995). Interactions between multiple signaling

components may. therefore, influence the expression pattern of MMPs (Korzus et al.. 1997;

Simon et al.. 2001).

Different from most other MMPs, MMP-2 is often constitutivelv expressed at the

transcriptional level. Gene structure analysis found that the MMP-2 promoter has high similarity

with the other two molecules that are involved in its activation process: TIMP-2 and MT1-MMP.

In vivo studies further demonstrated that MMP-2. MT1-MMP, and TIMP-2 are coordinated

expressed in a number of tissue types (Apte et al.. 1997: Chen and Wang, 1999; Kan war et al..

1999: Kurschat et al.. 1999: Longin et al.. 2001). suggesting that not all members of this protein

family are regulated at the same levels.

(2) Activation of latent MMPs As potent proteolytic enzymes on various ECM

components. MMPs are also tightly regulated at the activational level. MMPs are produced as

latent forms, and most are subsequently activated by extracellular proteolytic cleavage. The

latency of MMPs is maintained by the cysteine switch consensus sequence in the propeptide

domain. The conserved cysteine residue covalently interacts with the catalytic zinc ion in the

active core of the enzyme. The latter is thus prevented from interacting with a water molecule

(Van Wart and Birkedal-Hansen. 1990). Either proteolytic cleavage on the propeptide by

proteinases. or chemical modification on the SH group of cysteine residues, can bring about

disruption of the Zn2*-Cvs interaction, allowing the catalytic zinc ion to interact with H20

(Nagase. 1997). The activity o f MMPs is attained by this intramolecular reaction. The final step

- 2 1 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. may also involve autocatalytic removal of the propeptide. Chemical modification is achieved by

reagents such as 4-aminophenylmercuric acetate (APMA), HOCL. and denaturants. Also, the

propeptide of most MMPs is susceptible to cleavage by extracellular proteinases such as trypsin,

plasmin. chymotrypsin. and MMPs themselves (Nagase, 1997; Murphy et al., 1999b; Nagase and

Woessner, 1999). Therefore, these proteinases may serve as endogenous activators of MMPs.

Unlike other MMPs. MMP-11 (stromelysin-3) and all MT-MMPs have an additional 10-

amino acid residue furin-like enzyme recognition motif (RxK/RR) at the C-terminal end of the

propeptide domain (Pei and Weiss. 1995). The same insert is also observed in a novel member of

the MMP family. MMP-23. which does not possess the typical cysteine switch consensus

(PPCG[V/N1PD) in the propeptide domain (Pei. 1999c). This structural feature indicates that

these MMPs can possibly be activated intracellularly rather than extracellularly, by furin. a Golgi-

associated subtiiisin-like proteinase. Indeed, it has been demonstrated that furin is able to process

latent MMP-11 to its active form (Pei and Weiss. 1995). Furthermore, this processing can be

stopped by synthetic furin inhibitors (Santavicca et al.. 1996). The transmembrane-deleted MT-

MMP1 can also be activated intracellularly (Pei and Weiss. 1996). Intracellular processing of

MMP-23 is also observed (Pei et al.. 2000; Ohnishi et al.. 2001). even though furin is unable to

enhance MMP-23 processing in a transfection model (Ohnishi et al.. 2001).

Notably , although MMP-2 harbors the characteristic cysteine switch motif in its propeptide

domain, it is resistant to cleavage by many proteinases that are able to activate most other MMPs.

Instead, it is activated by membrane-type metalloproteinases (MT-MMPs) (Sato et al., 1994; Sato

and Seiki. 1996; Murphy et al.. 1999a; Seiki, 1999). Among them, MT1-MMP is the most

studied. Efficient activation of pro-MMP-2 by MT1-MMP requires TIMP-2, which forms a

trimeric complex by binding to MTI-MMP to form a "co-receptor*’ for pro-MMP-2. On the cell

surface, an adjacent active MTI-MMP then cleaves the propeptide of pro-MMP-2 to initiate the

activation process (Cao et al.. 1998; Zucker et al.. 1998; Deryugina et al., 2001). However,

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TIMP-2 may not be necessary for activation by other MT-MMPs. For example, MT2-MMP

activates pro-MMP-2 in a TIMP-2 independent manner (Morrison et al., 2001).

(3) Inhibition by endogenous inhibitors Once activated, the activity of MMPs in the

extracellular milieu is controlled by a group of endogenous inhibitors known as tissue inhibitors

of metalloproteinases (TIMPs), which belongs to a multigene family (Woessner and Nagase,

2000). Other proteinase inhibitors, such as a-macroglobulins, also inhibit MMP activity (Sottrup-

Jensen. 1989). There are two significant aspects regarding the roles of TIMPs and a2-

macroglobulin. On the one hand. a2-macroglobulin. which is abundantly present in all tissue

fluids and acts nonspecificallv against MMPs. may serve as a major inhibitor throughout the

entire body, whereas TIMPs may regulate MMP activity locally by their tissue or cell specific

expression. On the other hand, the inhibitory action of a2-macroglobulin on MMPs is irreversible

until the a2-macroglobulin/MMP complexes are eliminated via scavenger receptor-mediated

endocytosis. while the inhibition of MMPs by TIMPs is reversible (Stemlicht and Werb. 2001).

In addition to a2-macroglobulin and TIMPs. another group of inhibitors is emerging. Recent

studies suggest that ECM proteins may play dual roles during tissue remodeling. Besides their

functions as structural components for supporting cells and tissues and as targets of MMPs for

ECM turnover, they may also regulate MMP activities via their proteolytic products. For

example, the noncollagenous NC1 domain of type IV collagen possesses not only sequence

similarity with TIMPs but also inhibitory activity against MMPs (Netzer et al.. 1998), being

capable of inhibiting tumor progression and angiogenesis (Petitclerc et al.. 2000). The C-terminal

domain of procollagen C-terminal proteinase enhancer (CT-PCPE) also shows MMP inhibitory

activity by reverse zvmography (Mott et al., 2000). N-terminal sequencing and structural

comparison further demonstrate that this fiagment harbors six cysteine residues, which are

conserved in the N-terminal domain of TIMPs (Murphy and Willenbrock, 1995). Nevertheless,

although the inhibitory effects of the C- and N-terminal fragments of CT-PCPE on MMP-2 are

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. remarkably lower than TIMPs (Netzer et al.. 1998; Mott et al., 2000), they may present a new

group of inhibitors whereby they are released upon proteolytic cleavage of the full length

molecule.

3. TIMPs

TIMPs specifically and reversiblv inhibit the activity of MMPs. So far. four members of the

tissue inhibitors of metalloprotcinases (TIMPs) gene family are known; TIMP-1, -2. -3. and -4.

Table 1-4 summarizes some of the basic molecular and biochemical features of these inhibitors.

All TIMPs have 12 conserved cysteine residues, which form six disulfide bonds and 6-looped. 2-

domain structures. Loops 1-3. which are localized at the N-terminal domain, are highly

conserved. The importance of these loops is established by the fact that they are necessary and

sufficient for the inhibitory ability' of TIMPs against MMPs (Murphy and Willenbrock. 1995;

Woessner and Nagase. 2000). The three loops at the C-terminus domain (loops 4-6) may be

responsible for determination of preferred MMP targets of individual TIMPs (Willenbrock and

Murphy. 1994). Overall, the four TIMPs show 37-51% sequence identity (Woessner and Nagase.

2000). The ability of TIMPs to block the autocatalytic activation of latent MMPs and to limit the

degradative actions of activated MMPs is attained by their capability to bind both latent and

active MMPs with a 1:1 stoichiometric ratio (Bode and Maskos. 2001). The relatively high

variability in the C-terminal domains of TTMPs may contribute to determine their preferred MMP

targets (Brew et al.. 2000; Bode and Maskos. 2001). In addition, the differential regulation and

wide tissue localization of each individual TIMP are associated with their corresponding distinct

biological functions.

TIMP-1 is a 28.5 kDa N-linked glycoprotein (Gasson et al., 1985). Although it has been

generally recognized that TIMP-1 inhibits the activities of all known MMPs (Edwards. 2001), it

has no or very low inhibitory’ actions on MT-MMPs (Will et al., 1996). TIMP-1 preferentially

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. binds to MMP-9 by interacting strongly with the hemopexin domain of MMP-9 with its C-

terminai domain (Goldberg et al., 1989: Murphy and Willenbrock, 1995).

TIMP-2 is an unglycosylated protein with a molecular mass of about 2 1 kDa (De Clerck et

al.. 1989: Stetler-Stevenson et al.. 1989). Unlike other TIMPs, the C-terminal sequence of TIMP-

2 is extended and negatively charged (Murphy and Willenbrock. 1995). TIMP-2 readily forms

complexes with MMP-2 through its C-terminal domain (Goldberg et al., 1989: Kolkenbrock et

al.. 1994).

TIMP-3 is an N-glycosylated protein (Apte et al.. 1995). Its molecular mass ranges from 24

to 27 kDa depending on the degree of glycosyiation. It preferentially inhibits the activity’ of

MMP-1. -2. -3. -9. and -13 (Apte et al.. 1996: Knauper et al., 1997a: Butler et al., 1999). TIMP-3

is the only TIMP that is strongly associated with the ECM (Leco et al.. 1994; Yu et al.. 2000).

The biological importance of TIMP-3 is highlighted by the fact that mutation in this molecule

results in Sorsby's Fundus Dystrophy, an autosomal dominant disorder that is manifested by early

degeneration o f the retina (Apte et al.. 1995).

TIMP-4 has a molecular mass o f about 24 kDa (Greene et al.. 1996: Liu et al.. 1997). It is

not known whether this protein is glycosylated. TIMP-4 is the only member of the TIMP family

that was identified by using the expressed sequence tag approach (Greene et al.. 1996). The full-

length cDNA was cloned from a human heart cDNA library (Greene et al., 1996). The gene

structure of TIMP-4 was determined from genomic DNA (Olson et al.. 1998). Homology analysis

revealed that TIMP-4 is closer to TIMP-2 and TIMP-3 (51%) than to TIMP-1 (37%). In addition

to the original observation o f its abundant presence in human heart tissues (Greene et al., 1996),

TIMP-4 is also highly expressed in a broad range of rodent tissues, including brain, heart, ovary,

and skeletal muscles ((Leco et al.. 1997: Wu and Moses, 1998), suggesting its role as an

important tissue-specific regulator of ECM turnover. TIMP-4 significantly inhibits tumor growth

and metastasis of human breast cells probably via its potent inhibitory action on a number of

MMPs, including MMP-I, -2, -3, -7. -8, -9, -12, -13, and -14 (Bigg et al., 1997; Liu etal., 1997;

-25-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wang et al., 1997; Stratmann et al., 2001a; Stratmann et al., 2001b). This is achieved primarily

through TIMP-4"s reliance on residue 2 (Ser2) in its inhibitory domain (Stratmann et al., 2001a).

TIMP-4 binds to MMP-2 with high efficiency similar to TIMP-2. However, unlike TIMP-2,

which promotes activation of pro-MMP-2 by coupling MMP-2 and MTI-MMP to form a trimeric

complex on the cell membrane. TIMP-4 inhibits pro-MMP2 activation (Bigg et al., 2001;

Hernandez-Barrantes et al.. 2001). Therefore, the balance between TIMP-2 and TIMP-4 may be

part o f a mechanism that dictates the local regulation of MMP-2 activity'.

4. Non-conventional functions of MMPs and TIMPs

Accumulating evidence suggests that MMPs are multifunctional proteins. In addition to

facilitating ECM turnover. MMPs have broad substrate specificities on non-ECM proteins (Table

1-3).

One notable example of the novel functions of MMPs is their ability to regulate the activity

of enzymes, including themselves. MTI-MMP can activate pro-MMP-2 on the cell surface (Sato

et al.. 1994). while MMP-3 initiates the activation of pro-MMP-1. -3. -7. -8. -9 and -13 (Nagase.

1995; Nagase. 1998). In addition, proteolytic enzyme activity is mainly regulated by endogenous

inhibitors. For example, serine proteinase inhibitors (serpins) inhibit plasmin, which belongs to a

class of proteases that has a serine amino acid residue in their . These inhibitors, in turn,

are cleaved by MMP-9 (Liu et al.. 2000) and MMP-11 (Pei et al.. 1994), resulting in their

inactivation, which alters the balance between enzymes and their inhibitors.

Cleavage of ECM proteins by MMPs can regulate the availability of growth (actors and

cytokines, which are associated with matrix protein. For example, fibroblast growth factor (FGF)

is released when perlecan is cleaved by MMP-1 and MMP-3 (Whitelock et al., 1996), while

transforming growth factor (3 (TGFf3) becomes available when is degraded by MMP-2, -

3. and -7 (Imai et al., 1997). Furthermore, there are a number of growth factors and cytokines that

are synthesized as pro-forms, which are biologically inactive. Direct cleavage of these precursors

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by MMPs processes them into their active forms. For example, MMP activity is required for

release o f TN Fa from its cell membrane bound form (Gearing et al., 1994; McGeehan et al.,

1994; Lombard et al.. 1998), while removal o f the N-terminal sequence o f interleukin ip (ILip)

by MMP-2. -3, and -9 converts it to its biologically functional form (Schonbeck et al., 1998). On

the other hand, the biological activity of these cytokines can also be down-regulated by MMPs.

N-terminal proteolytic processing of monocyte chemoattractant protein 3 (MCP-3) by MMP-2

results in a MCP-3 antagonist which binds to MCP receptors, but does not induce signal

transduction (McQuibban et al.. 2000). Furthermore, MMPs act on growth factor receptors. For

example, cleavage of FGF receptor type I by MMP-2 releases its ectodomain from the cell

surface. This soluble FGF receptor then alters the availability of FGF to its cell membrane

receptors (Levi et al.. 1996).

Cell-cell and cell-matrix interactions are important for cell migration and invasion. MMPs

influence these processes by directly cleaving cell adhesion molecules, such as integrin P4 and E-

cadherin (Lochter et al.. 1997; von Bredow et al.. 1997). Proteolytic cleavage of E-cadherin by

MMP-3 and MMP-7 releases an 80 kDa fragment which stimulates cell migration and invasion

(Noe et al.. 2001). Keratinocyte migration, on the other hand, is induced by MMP-2 dependent

cleavage of laminin-5 (Giannelli et al.. 1997). Lastly, hydrolysis of cell adhesion molecule CD-44

and cell surface tissue transglutaminase (tTG) by MTI-MMP alters tumor cell adhesion and

migration (Belkin et al.. 2001; Kajita et al.. 2001).

Besides MMPs. the TIMPs are also a multifunctional group of proteins. In addition to

inhibiting MMP activity. TIMP-1 and -2 exhibit growth promoting ability on several different cell

lines (Docherty et al.. 1985; Gasson et al.. 1985; Stricklin and Welgus, 1986; Hayakawa et al..

1992; Hayakawa et al.. 1994; Nemeth et al.. 1996). Also, TIMP-1 is associated with

steroidogenesis in rat gonadal cells (Boujrad et al., 1995). Furthermore, the nuclear localization of

TIMP-1 shows a cell cycle dependent pattern (Zhao et al., 1998), indicating that this protein may

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulate the cell cy cle. In addition to its inhibitory activity', TIMP-2 facilitates pro-MMP-2

activation by assembling an MTI-MMP/TIMP-2/pro-MMP-2 tn-molecular complex on the cell

surface (Butler et al .. 1998). Lastly , a death domain is found in the N-terminus o f the TIMP-3

protein (Bond et al.. 2000). Indeed. TIMP-3 over-expression induces apoptosis in smooth muscle

cells (Baker et al.. 1998). This may be due to its ability to induce the type II apoptotic pathway

(Bond et al.. 2002).

Taken together. MMPs regulate cell proliferation, survival, adhesion, migration, and cell-cell

and cell-matrix interactions by their proteolytic actions on non-ECM proteins that include the

precursors and receptors of growth factors and cytokines, these hormones themselves, cell

adhesion molecules, and enzymes. As a group of proteins w ith small molecular masses. TIMPs

also affect cell growth and survival. Because they possess multiple functions, the roles of MMPs

and TIMPs in physiological and pathological processes are even more complex (Chambers and

Matrisian. 1997: Blavier et al.. 1999: McCawley and Matrisian. 2000).

III. MMPs/TIMPs in Follicular Development and Atresia

In all mammalian species, the follicle is a morphological and functional unit which sustains

oocyte growth and maturation. Follicular growth begins with primordial follicles which are

located in the inner part of the ovarian cortex. Entry of primordial follicles into the growth phase

is characterized bv conversion of the non-growing, flattened (squamous) pre-granulosa cells

surrounding the oocyte into a single layer of cuboidal granulosa cells. At this stage, it is termed a

primary follicle, which is also distinguished by the formation of a zona pellucida around the

oocyte (Cortvrindt and Smitz. 2001). Granulosa cells continue to proliferate resulting in the

oocyte being surrounded by two or three layers of these cells. At this stage, they are termed

secondary follicles. As secondary- follicles grow, stromal cells next to the basement membrane of

the follicle become aligned and form the theca cell layer. At the end of this stage, a vascular

network is also developed (will be discussed in section VI) in the theca layer. The blood supply

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. not only provides nutrients and oxygen for the growth of the follicle, but also exposes the follicle

to factors circulating in the blood stream, which influence follicle development. In the tertiary

follicle, the granulosa and theca cells undergo further development and a cell-free, fluid-filled

cavity called an "antrum'' (means cave in Greek) is formed. Therefore, the follicle at this stage is

also called the antral follicle. Based on the appearance and the size of the antrum, the follicles at

this stage can be further distinguished as "preantraT or "early antral"'. In the theca layer, the cells

next to the basement membrane become epitheliod in appearance and form a so-called theca

interna, while the peripheral cells, which form the theca externa, maintain their spindle shape.

The theca interna cells undergo cytodifferentiation and acquire organelles characteristic of

steroid-secreting cells such as mitochondria and smooth endoplasmic reticulum. As follicles

continue to grow and synthesize hormones, they will reach the final stage called the Graafian

follicle.

Taken together, follicular growth and development are characterized by proliferation of

granulosa cells, differentiation of theca cells from the ovarian stroma, and deposition of a

basement membrane separating the vascular theca from the avascular granulosa cell layer. As the

follicular membrane expands within the limits of the ovarian stroma, the surface area of the

follicle dramatically increases. For example, the bovine follicle increases approximately 360.000

fold from the primordial to the preovulatory stage. In primates, the mature follicle is almost 400

fold larger than the primordial follicle. Follicle development is accomplished in an extracellular

environment that consists of collagen, laminin. and fibronectin. Thus, extensive tissue remodeling

is required to accommodate the rapid cellular proliferation as follicles develop. Indeed, the

composition of follicular basement membrane changes during bovine follicle development

(Rodgers et al.. 1998). In addition, modification of the surrounding ovarian stroma is also

necessary for the growing follicle to reach a place on the surface of the ovary where the oocyte

can be released at ovulation. Remodeling of the ECM during angiogenesis also occurs within the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. theca layer. These structural reorganization events in the ovarian ECM are thought to be

achieved at least in part, by the MMPs/TIMPs system.

MMPs appear to play a role in follicular development of cattle. In the bovine ovary, MMPs

constitute one family of the most relevant genes that are associated with differentiated follicles

bearing developmentally competent oocytes (Robert et al.. 2001). Both MMP-2 and MMP-9

activities were detected in bovine follicular fluid (Khandoker et al.. 2001). In vitro studies on

bovine follicles further indicate the expression of MMP-9 is related to follicle health, and thus can

be used as a marker to assess follicle development (McCaffery et al.. 2000). Of note, MMP-9

expression in the bovine follicle is augmented by ascorbic acid (Thomas et al.. 2001). which has

been postulated to be important for follicular health.

In developing rat follicles. MMP-2 and MMP-9 mRNAs are localized to the theca cells and

the surrounding stroma (Liu et al.. 1998; Curry et al.. 2001). Consistent with its mRNA

distribution. MMP-9 protein is restricted to theca and interstitial cells (Bagavandoss. 1998).

However. MMP-2 immunoreactivity is observed in both theca-interstitial and granulosa cells,

although the latter has a lower expression than the former (Bagavandoss. 1998). This discrepancy

may be due to the inability (low sensitivity) of in situ hybridization to detect the low copies of

MMP-2 mRNA in granulosa cells. Although MMP-2 mRNA is expressed in theca-interstitial

cells. MTI-MMP. the activator of pro-MMP-2. is detected in both granulosa cells and theca-

interstitial cells (Liu et al.. 1998). Similar to MMP-2. the expression of MTI-MMP is also up-

regulated by gonadotropins during follicle development (Liu et al.. 1998). MMP-13 (collagenase-

3) is also detected in theca-interstitial but not granulosa cells of rat growing follicles, suggesting a

potential role of this MMP in follicular development (Balbin et al., 1996). Indeed, significant

increases in MMP-13. MMP-2. and MMP-9 mRNA, and the corresponding collagenolytic and

gelatinolytic activities, are observed during folliculogenesis induced by gonadotropin (Cooke et

al.. 1999). In the intact mouse follicle culture modeL MMP-2 activity is up-regulated by ascorbic

acid (Murray et al.. 2001). suggesting that the promoting effects of this vitamin on follicle

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. integrity and survival may be mediated by regulating MMP activity. During gonadotropin-primed

follicular development in rats. MMP-23 expression is switched from granulosa cells to theca-

extema and the ovarian epithelium (Ohnishi et al., 2001). FSH, via the cAMP signal transduction

pathway, reduced MMP-23 expression in granulosa cells, while the expression of MMP-23 is

increased in theca-interstitial cells regardless of the presence of LH (Ohnishi et al.. 2001). This

indicates that MMP-23 is not only involved in follicle development but is also regulated in a cell

specific manner.

Besides MMPs. TIMP-1 mRNAs are also localized to theca and stroma during follicle

development in the rat (Curry et al.. 2001). TIMP-1 protein is localized in the thecal cells of

follicles, and blood vessels of the ovary (Bagavandoss. 1998). The co-localization of MMPs and

TIMPs indicates that the tissue remodeling events during follicular development are strictly

regulated.

Among species with long reproductive cycles. TIMP-1 is also detected in ovine (Smith and

Moor. 1991; Smith et al.. 1993; Smith et al.. 1994a; Mclntush et al.. 1996). bovine (Smith et al..

1996). porcine (Smith et al.. 1994b; Driancourt et al.. 1998; Shores and Hunter. 2000). primate

(Chaffin and StoufFer. 1999). and human (Curry et al.. 1988) follicles. TIMP-1 mRNA is

increased in bovine (Smith et al.. 1996) and ovine (Smith et al.. 1994a) preovulatory follicles,

while ovine TIMP-1 protein in the ovary' (Mclntush et al.. 1996) is only observed after the

gonadotropin surge. As suggested in a Sertoli cell model. TIMP-1 expression may be stimulated

by FSH via a cAMP. PKA-dependent pathway. Furthermore, the PKC pathway also mediates

signals that up-regulate TIMP-1 production (Ulisse et al.. 1994).

TIMP-2 mRNA expression is observed in bovine (Smith et al., 1996), and ovine (Smith et

al.. 1995) follicles. In the latter. TIMP-2 is primarily expressed in theca cells, but its expression

was not affected by the gonadotropin surge (Smith et al.. 1995). However, after the gonadotropin

surge, a remarkable increase in TIMP-2 expression is induced in the bovine ovary (Smith et al.,

1996). TIMP-2 is also present in theca and granulosa cells, and follicular fluid of large and small

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pig follicles (Shores and Hunter. 2000). TIMP-2 mRNA is expressed in the theca and stromal

compartments during rat folliculogenesis (Curry et al.. 2001). Although TIMP-2 expression can

be up-regulated by FSH via a cAMP. PKA-dependent pathway, it is resistant to stimulation

through the PKC pathway (Ulisse et al.. 1994).

The localization of TIMP-3 mRNA exhibits a different pattern from that for TIMP-1 and -2.

Although TIMP-3 mRNA is detected in theca and stroma of developing rat follicles, its

expression in granulosa cells is only observed in certain follicles, but not in the follicles that are

adjacent to them (Curry et al.. 2001). In the mouse ovary. TIMP-3 mRNA is also expressed in the

theca and granulosa cells of small and large follicles (Inderdeo et al.. 1996). Similarly. TIMP-3 is

observed in pig small and large follicles (Shores and Hunter. 2000). The study of TIMP-1. -2. and

-3 production in pig follicles shows that the expression of these TIMPs is regulated by factors

such as follicular size and the phase of follicular development (Shores and Hunter. 2000).

Although TIMP-4 is abundantly expressed in ovarian tissue (Leco et al.. 1997). its

localization in the follicular compartments has not been elucidated. Reverse zvmography

demonstrates that this inhibitor may be present in equine follicular fluid (Riley et al.. 2001).

Because of their specialized ovarian structure and ovulation pattern, the equine gonads are a

good model to study the tissue remodeling events in follicle growth and development. Different

from the ovaries of other mammalian species, in which follicles can ovulate from any site on the

ovarian surface, the follicles in equine ovaries migrate from the stroma to the ovulatory fossa,

where ovulation occurs. Both MMP-2 and MMP-9 are present in equine follicular fluid. While

MMP-2 is the predominant geiatinase. its level does not vary significantly during follicle

development (Riley et al.. 2001). However. MMP-9 increases dramatically, when follicles grow'

from < 10mm to 1 l-20mm in diameter. Both these gelatinases are localized to the granulosa,

theca. and stromal cells. Furthermore, all four tissue inhibitors of metalloproteinases (TIMPs 1-4)

are also present in follicular fluid, but their levels do not change significantly during follicle

development (Riley et al.. 2001). Similar to the localization of MMP-2 and MMP-9, all these

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TIMPs are immunolocalized to follicular granulosa and theca cells and the surrounding stromal

cells. The co-localization of MMPs and TIMPs suggests that MMP activity is locally regulated

during the tissue remodeling that occurs when follicles develop and migrate to the ovulation fossa

in mares. Furthermore, accumulating evidence suggests that one of the roles of reiaxin during

ovarian follicle development and ovulation is to regulate proteolytic enzyme activity. In an in

vitro equine ovarian stromal cell (EOSC) culture sy stem, reiaxin increases both latent and active

MMP-2. latent MMP-9. TIMP-1, and TIMP-2 (Song et al.. 1999). This suggests that reiaxin may

contribute to equine follicle growth and migration, and facilitate ovulation by modulating the

degradation of ECM in ovarian stromal tissue. The growth factor. TGFp. also plays an important

role, as it significantly stimulates the activity of MMP-2 and MMP-9 and TIMP-1 production by

EOSC (Song et al.. 1999). The PKC signal transduction pathway may be involved in the

stimulation of MMP-2. MMP-9. TIMP-2. and TIMP-3. since PMA (phorbol 12-mvristate 13-

acctate) increases the production of these enzymes and inhibitors (Song et al.. 1999).

Follicles also undergo atresia, a process that involves apoptosis. Abnormal proteolytic

degradation of the ECM may initiate an apoptotic signal or mediate the apoptosis process. In

human ovary, low levels of MMP-1 in the apical wall of the follicle are associated with atresia.

The level of stromelysin-1 (MMP-3) is inversely related to follicle size (Bogusiewicz et al..

2000). In the mouse model, although stromelysin-3 (MMP-11) is coordinatelv expressed with

apoptotic follicles, the stromeiysin-3-deficient mouse shows no difference in the apoptotic rates

of follicles from the wild type (Haggiund et al.. 2001). Therefore, stromelysin-3 may not be

sufficient for inducing or completing follicle atresia, but it may be involved in this process. In

non-atretic follicles, activity is high in granulosa and thecal cells and low

in follicular fluid. Atresia is associated with declining collagenase activity in both cell types and

increasing activity in follicular fluid (Garcia et al.. 1997). GelaUnase activities are also correlated

with follicular atresia. Specifically, active MMP-2 and pro-MMP-9 are present only in atretic

follicular fluid (Khandoker et al., 2001).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The physiological roles of these metalloproteinases. therefore, may include their direct

actions on ECM components for modulating the local extracellular milieu, and their indirect

effects on cell proliferation by processing growth factors from their latent forms and/or modifying

their receptors.

IV. MMPs/TIMPs in Ovulation

Although a variety of hypotheses has been proposed for the mechanism of ovulation, most of

them, if not all. include a coordinated series of proteolytic degradative events which result in the

breakdown of the follicle wall. As early as about one century ago (1916). Schochet pointed out

the importance of proteolytic enzymes in the process of ovulation by degrading the ECM at the

apex of a Graafian follicle. Numerous proteolytic enzymes were thereafter identified and

characterized during the ovulation process (Espey. 1974; Parr. 1975; Tsafriri. 1995).

Besides oocyte maturation, deterioration of the follicle wall and follicle apex leads to

ovulation (Thibauit and Levasseur. 1988; Murdoch and McCormick. 1992). The apical wall of the

preovulatory follicle, from the innermost to the outermost side, is composed of granulosa cells,

basement membrane, theca interna, theca externa, tunica albuginea, a second basement

membrane, and epithelium. During ovulation, which is initiated by a surge of the pituitary

gonadotropin, luteinizing hormone (LH). all these layers of ECM and connective tissues have to

be compromised for follicle rupture and expulsion of the oocyte. These events begin with

alterations in the vasculature of the follicle, which results in the formation of an avascular area

known as the macula pellucida or stigma (Parr. 1975). Then, the tunica albuginea is the first place

w here disintegration occurs, followed by the degradation of ECM in the epithelium. As a result,

fibroblasts and epithelial cells are no longer in contact, and ultimately this leads to the loss of

integrity of the whole surface epithelium. In some species such as mouse and rat, the outer layer

of basement membrane, which lies beneath the surface epithelium, undergoes edema, which then

causes fragmentation of the collagen matrix. The blood capillaries in the theca layer subsequently

-34-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. disintegrate and the blood seeps into the interstitium. As the time of ovulation approaches, the

follicle expands and the number of layers of fibrils decreases over the apex, resulting in the

deterioration of the basal lamina localized between granulosa and theca layers (Martin and

Miller-Walker. 1983). Collectively, follicle wall breaching and the loss of apical integrity

eventually lead to leakage of follicular fluid and release of the oocyte.

One of the major breakthroughs in the early studies of ovulation was accomplished with the

demonstration that antral pressure does not increase prior to follicle rupture (Bronson et al.,

1979). This observation, therefore, shifted the research focus to the deterioration and weakening

of the follicle wall around the stigma area, which appears to be a necessary prerequisite to the

ovulatory process (Parr. 1975: Martin and Miller-Walker. 1983). The degradation of the follicle

wall is reflected by the changes in collagen content during follicle development, which increases

with increasing follicle volume (Morales et al.. 1983). However, right before ovulation, the

collagen content decreases dramatically (Martin and Miller-Walker. 1983: Morales et al.. 1983).

All these matrix degradation events during ovulation may be a consequence of the proteolytic

action of MMPs. For example, collagenase and gelatinase activities are associated with the apex

of preovulatory follicles (Fukumoto et al.. 1981: Murdoch and McCormick. 1992: Curry and

Osteen. 2001).

Collagenase was the first proteolytic enzyme identified in the ovulation process.

Collagenolytic activity is first detected in the rabbit and sow Graafian follicles during ovulation

(Espey and Rondell. 1968). Higher collagenolytic activity is localized in the follicular apex area

than the basal wall of the ovulatory follicles, suggesting degradation of extracellular matrix

proteins may dictate the site o f follicle rupture (Murdoch and McCormick. 1992). In the rat ovary,

collagenase activity is correlated with the ovulatory process (Curry et al.. 1985: Palotie et al.,

1987: Woessner et al.. 1989: Hirsch et al., 1993). An ovulatory dose o f hCG increases interstitial

collagenase (MMP-1) mRNA about twenty-five times in rat follicles, where it is predominantly

localized in both granulosa cells of preovulatory follicles and the residual ovarian tissues (Reich

-35-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al.. 1991). A continuous increase of neutral coliagenase activity is observed in the follicular

wall during the ovulatory process in humans (Yajima et al., 1980). Further investigation shows

that granulosa cells are the major cellular source of coliagenase. which is produced at maximal

levels at ovulation and decreases abruptly thereafter (Fukumoto et al., 1981).

In the domestic fowl, coliagenase activity is detected in the ovarian follicle wall (Fujii et al..

1981). Although there are no significant changes of coliagenase activity observed up to the point

of ovulation (Tojo et al.. 1982). local administration of proteolytic enzymes induced the rupture

of the ovarian follicle (Nakajo et al.. 1973). LH and progesterone increased the total activity' of

neutral and acid proteases and coliagenase. which are primarily concentrated in the stigma region

(Ogawa and Goto. 1984). Retrospectively, the coliagenase activity described at this stage is

interstitial coliagenase.

During ovulation of rabbit follicles. MMP-1 is expressed in theca intema. theca externa,

interstitial cells and germinal epithelium. In addition. MMP-1 is also induced in the capillary'

lumina around the apex of preovulatory follicles, and increases in granulosa and theca intema

cells that are around the orifice of the ruptured follicles (Tadakuma et al.. 1993).

The ovulatory process is regulated by multiple factors. Gonadotropin induces an increase in

collagenolytic activity (Curry et al.. 1985; Reich et al.. 1985; Hirsch et al.. 1993). which is

reflected, in part by the increase in interstitial coliagenase expression at the transcriptional level

(Reich et al.. 1991). Coliagenase 3 (MMP-13) is also highly expressed in thecal cells/stroma of

rat antral follicles (Baibin et al.. 1996). suggesting a plausible role for it during ovulation. It is up-

regulated by LH via a tyrosine kinase -dependent pathway during ovulation in rats (Komar et al.,

2001). Besides gonadotropins, a number of paracrine factors have regulatory effects on

coliagenase activity'. Relaxin stimulates the release of coliagenase activities from rat granulosa

cells (Too et al.. 1984). Prolactin, on the other hand, is unable to promote collagenolytic activities

(Hirsch et al.. 1993). Indomethacin (an inhibitor of cyclooxygenase) blocks the stimulatory

effects of hCG on coliagenase mRNA expression and collagenolytic activity (Reich et al., 1985;

-36-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reich et al.. 1991). In the ewe. indomethacin negates the stimulated collagenolysis in follicular

tissue by gonadotropin (Murdoch et al., 1986), suggesting that prostaglandins are involved in

enhancing coliagenase activity. Prostaglandins have been shown to be stimulatory for proteolytic

enzyme activity during ovulation in rabbits (Miyazaki et al.. 1991). Furthermore, coliagenase is

also up-regulated by TNFa. which accumulates in the oocyte-cumulus cell complex (Johnson et

al.. 1999). implying that multiple ceil types are involved in the regulation of coliagenase activity

during ovulation. In non-human primates, progesterone is indispensable for the stimulatory

effects of gonadotropin on interstitial coliagenase expression (Chaffin and Stouffer. 1999).

However, progesterone, prostaglandin E2. prostaglandin F^. and 17p-estradiol have no effect on

coliagenase activity in the human follicle wall (Norstrom and Tjugum. 1986). but relaxin and

oxytocin increase coliagenase activity (Norstrom and Tjugum. 1986). Notably, oxytocin

decreases collagen synthesis (Tjugum et al.. 1986). Collectively, these results suggest that the

regulation of coliagenase activity is diverse and may be species dependent.

Type IV collagenolytic activity' was first detected in human follicular fluid (Puistola et al..

1986). This activity' increases as ovulation approaches, and decreases after follicle rupture

(Puistola et al.. 1986). In an in vitro culture model o f rat follicles, type IV coliagenase activity' is

stimulated by gonadotropin, showing a similar pattern of changes as type I collagenolytic activity

(Palotie et al.. 1987). Retrospectively, these type IV collagenolytic activities are attributed to

gelatinases (A and B). as confirmed by gelatin zymography (Curry et al.. 1992). The efficient

degradation of the major component of the basement membrane, collagen type IV. by these

activities may be pivotal for ovulation. Furthermore, type IV collagenolytic activity is also

present within the follicular fluid and increases toward ovulation (Puistola et al.. 1986). While

interstitial coliagenase increases at 3-6 hours after hCG administration (Reich et al., 1985; Reich

et al.. 1991). type IV coliagenase expression, on the other hand, peaks at 9 hours after hCG

treatment (Reich et al.. 1991; Curry et al.. 1992). Increased expression of MMP-9 in the late stage

-37-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of the ovulatory process is also observed in macaque (Chaffin and Stouffer, 1999). Furthermore,

interstitial coliagenase is expressed in both granulosa and residual cells, while type IV

coliagenase is restricted to residual (thecal-interstitial) cells (Reich et al., 1991). The

aforementioned type IV coliagenase activity is predominantly due to gelatinase B (MMP-9),

which is highly expressed in the theca-interstitial compartment (Hurwitz et al., 1993; Curry et al.,

2001). Taken together, these observations indicate that interstitial coliagenase is expressed at the

early stages of ovulation to loosen the connective tissues between the two basement membranes,

while residual cells produce gelatinase B (type IV coliagenase activity) at the final stages of

ovulation to tear the major boundaries, the two layers of basal lamina, for oocyte extrusion. The

abrupt decrease of this enzyme after the rupture of follicles further suggests its unique role during

ovulation (Puistola et al.. 1986). Other factors, such as interleukin-1 (IL-1), stimulate gelatinase B

production by theca-interstitial cells (Hurwitz et al.. 1993). However, transforming growth factor-

beta 1 (TGF-pi) attenuates the stimulatory action of IL-1 (Hurwitz et al.. 1993), suggestinga

complex intraovarian regulatory loop for fine-tuning this MMP activity.

The importance of gelatinase A in the ovulatory' process was recognized in the ewe. when

immunization with the N-terminal peptide of inhibin a43 (aN) reduced the fertility. Specifically,

latent and active MMP-2 levels increase as ovulation approaches (Russell et al.. 1995: Gottsch et

al.. 2000). while immunization with aN significantly reduces gelatinase A activity (Russell et al..

1995). The increase in MMP-2 activity at ovulation, however, is abolished by administration of

TNFa antiserum (Gottsch et al.. 2000). When combined with the in vitro data that shows the

stimulatory effects of TNFa on MMP-2 activity (Gottsch et al.. 2000), these results indicate that

MMP-2. which is regulated by inhibin and TNFa. is important for the tissue remodeling process

during ovulation. In rats, relaxin stimulates the secretion of MMP-2 from theca-interstitial cells

but not granulosa cells. This autocrine or paracrine action of relaxin on MMP-2 activity may

contribute to its ability to facilitate ovulation (Hwang et al., 1996). Furthermore, MMP-2 and its

-38-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell surface activator, MT1-MMP. are up-regulated in theca-interstitial cells of large preovulatory

follicles after hCG stimulation (Liu et al.. 1998). This indicates that the MT1-MMP/MMP-2

activation system may be associated with the tissue destruction events during ovulation. In the

macaque. MMP-2 expression is also up-regulated via a progesterone-independent pathway during

the ovulatory process (Chaffin and Stouffer, 1999).

In addition to the two major MMP subgroups- collagenases and gelatinases, other MMPs

may also participate in the tissue destruction process during ovulation. For example, gonadotropin

induces increased expression of matrilvsin in the macaque ovary (Chaffin and Stouffer, 1999).

Although stromelvsin-3 (MMP-11) is constitutively expressed during ovulation, maximal levels

of MMP-19 in granulosa and theca-interstitial cells of large preovulatory’ and ovulating follicles

are induced by hCG in the mouse ovary’ (Hagglund et al.. 1999). In the porcine ovary, bradykinin

up-regulates expression of MMP-3. MMP-20. and MT1-MMP in granulosa cells (Kimura et al..

2001). ADAMTS-I (a disintegrin and metalloproteinase with thrombospondin-like motifs), which

is a novel MMP-related proteolytic enzyme and which appears to be the regulatory target of

progesterone, is induced in granulosa cells of mouse preovulatory follicles by LH (Espey et al..

2000; Robkeretal . 2000).

Besides MMPs. the expression patterns of metalloproteinase inhibitors are also altered. A

metalloproteinase inhibitor activity was first identified in human follicular fluid (Curry' et al..

1988). Based on the characterized biochemical features, this metalloproteinase inhibitor activity

is attributed to TIMP-1. Additionally, a-macroglobulins (a 1-macroglobulin. a2-macroglobulin.

and a l inhibitor3). a group of plasma proteins with broad inhibitor activity, are also

present in the follicular fluid of human and rat follicles (Curry et al., 1989; Curry et al., 1990: Zhu

and Woessner, 1991). In the rat ovary, the activities o f these a-macroglobulins increase as

ovulation approaches (Zhu and Woessner, 1991). The absence o f a2-macroglobulin mRNA in

granulosa cells suggests that this serum-derived inhibitor is transported by the blood stream from

-39-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. other sources in the body (Curry et al.. 1990), while detection of TIMP-1 mRNA in granulosa

cells indicates that this inhibitor may serve as a local regulator of proteolysis during ovulation

(Curry et al., 1990; Hagglund et al., 1999; Curry et al., 2001). In addition to granulosa cells,

TIMP-l is also localized to theca-interstitial cells in the mouse ovary (Hagglund et al., 1999).

suggesting that there is probably a species difference in the tissue distribution of this endogenous

MMP inhibitor.

Similar to MMPs. the TIMPs are also regulated. TIMP-1 expression is dramatically

increased in rat (Curry et al., 1989: Mann et al.. 1991) and mouse (Hagglund et al., 1999) ovaries

after hCG administration. In rat granulosa cells, a LH-cAMP-dependent protein kinase-A

pathway or a cAMP-independent protein kinase-C pathway augments TIMP-1 expression (Mann

et al.. 1991: Mann et al.. 1993), while neither prostaglandin nor estrogen affects the basal or LH-

augmented TIMP-1 expression (Reich et al.. 1991: Mann et al.. 1993). In addition, calcium is also

involved in modulating TIMP-1 expression, since the ionophore. A23187. decreases granulosa

cell-derived TIMP-l activity (Cannon et al.. 1997). Although aN is involved in the regulation of

MMP-2 during ovulation (Russell et al.. 1995). the paracrine effects of this factor has not been

determined for TIMP-1 (Dharetal.. 1998). In non-human primates, the stimulatory'effects on

TIMP-1 and TIMP-2 expression by gonadotropin are dependent on progesterone (Chaffin and

Stouffer. 1999).

Proteolytic enzymes other than members of the MMP family, such as plasminogen and

plasminogen activator, are also found in the follicle wall (Beers, 1975: Shimada et al., 1983:

Hsueh et al.. 1988: Liu et al.. 1991: Chun et al.. 1992: Hagglund et al., 1996: Liu, 1999).

Inhibitors of plasminogen and plasmin significantly inhibit ovulation efficiency (Yoshimura and

Wallach. 1987). Notably, the fertility in mice with a deficiency of either tPA or uPA is normal,

while ovulation is reduced in mice lacking both PAs (Leonardsson et al., 1995). The plasmin

inhibitor is effective only when it is administrated at the early stage (Woessner et al., 1989), while

full inhibitory capability of the coliagenase inhibitor on ovulation is observed when it is

-40-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. administrated as late as 7 hours after LH (Butler et al., 1991). These findings suggest that plasmin

may serve as the activator for collagenases during ovulation.

All in all. ovulation is a complex physiological process involving a number of proteolytic

enzymes and their corresponding inhibitors. MMPs and TIMPs undoubtedly play an important

role in the proteolytic events during ovulation. Ovarian paracrine and autocrine factors, as well as

gonadotropins, constitute a complex loop for regulating MMP activity. Further investigations on

the temporal and spatial expression patterns, and regulatory mechanisms of proteolytic enzymes

and endogenous inhibitors, are necessary for advancing our understanding of this exquisitely

controlled tissue remodeling process.

V. MMPs/TIMPs in CL Formation. Maintenance, and Regression

The corpus luteum (CL) is a transient endocrine gland that secretes progesterone to establish

and maintain pregnancy (Niswender and Nett 1994). The CL also plays a central role in survival

of the embryo, regulation of cyclicity, and controlling ovulation and length of the reproductive

cycle. The CL develops from the remnants of the follicle following ovulation (Niswender and

Nett. 1994). Dramatic structural and functional changes are associated with the development,

maintenance and regression o f the CL (Nisw ender and Nett. 1994). At the time of ovulation, the

basement membrane breaks down, and the follicle wall folds toward the cavity of the ruptured

follicle (Pederson. 1951). During this process, theca interna cells, fibroblasts and endothelial cells

undergo mitosis, and migrate into the central region of the postovulatory follicle, contributing

towards the formation of the CL (Parry et al.. 1980: Cavender and Murdoch, 1988; O'Shea et al..

1989: Zheng et al.. 1994). In addition, disruption o f the basement membrane facilitates invasion

of capillary vessels from the theca intema into the granulosa layer (Cavender and Murdoch,

1988). The latter, therefore, undergoes a striking transition from an avascular to a vascular

compartment (Pederson, 1951: Ford et al., 1982: Wiltbank et al., 1988). This neovascularization

provides a blood supply for the differentiated theca-derived small and granulosa-derived large

-41 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. luteal cells (Dharmarajan et al., 1985: Wiltbank et al., 1988: Wiltbank et al., 1989), and is

associated with increased progestin production by the CL (Smith et al., 1994c). During the mid­

cycle phase of luteal development, neovascularization is slowed down and the integrity of the CL

is maintained to provide a relatively steady environment for continued steroidogenesis (Jabionka-

Shariff et al.. 1993). Regression of the CL at the end of the cycle is also an example of a

remarkable tissue reorganization process (Nett et al.. 1976: Niswender and Nett, 1994: Murdoch.

1996).

These remodeling events in the CL require the participation of MMPs and TIMPs. TIMP-1 is

one of the first members in this identified to be closely associated with luteal

physiology. In mouse adult tissues, the highest level of TIMP-1 was detected in the ovary', where

the expression is predominantly restricted to the CL (Nomura et al.. 1989: Edwards et al.. 1992).

indicating a significant role of this MMP inhibitor in luteal physiology. HMP-1 immunoreactivc

staining is detected in rat (Bagavandoss. 1998). porcine (Tanaka et al.. 1992), and ovine

(Mclntush and Smith. 1998) luteal capillaries. In addition, abundant expression of this inhibitor is

predominantly localized in large luteal cells of species with relatively long luteal phases, such as

sheep (Mclntush and Smith. 1998). and pig (Tanaka et al.. 1992). More specifically. TIMP-1 is

packed in the secretory' granules of large luteal cells (Mclntush et al.. 1996). While TIMP-1

mRNA does not vary in the ovine CL over the estrous cycle (Smith et al.. 1994a), its levels are

maximal in the early stage and decline with age o f bovine (Freudenstein et al.. 1990: Smith et al..

1996) and porcine (Pitzel et al.. 2000) CL. Although TIMP-1 is most abundantly expressed in the

CL (Tanaka et al.. 1992) and thus the major secreted protein product of the sheep ovary (Smith

and Moor. 1991: Smith et al.. 1993). circulating concentrations of this protein remain at a

consistent level over the estrous cycle (Mclntush et al.. 1997). In the bovine CL, immunoreactive

TIMP-l-like protein is unchanged over the estrous cycle (Goldberg et al.. 1996). The expression

of TIMP-1 is also hormonally regulated (O'Sullivan et al., 1997). Triiodothyronine (T3) and

-42-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. follicle stimulating hormone (FSH), alone and in combination, stimulate TIMP-1 production in

human luteinized granulosa cells (Goldman et al., 1997).

The 5' non-coding region of bovine TIMP-1 is homologous to the protein coding region of

the bovine StAR gene (Hartung et al., 1995), suggesting a possible role of TIMP-1 in steroid

production. Indeed. TIMP-1 facilitates steroidogenesis in Leydig, granulosa, and theca cells in

vitro (Boujrad et al.. 1995; Nothnick et al.. 1997; Shores and Hunter, 2000). In addition, TIMP-1

deficient mice have lower serum testosterone concentration in males (Nothnick et al., 1998), but

normal serum 17p-estradiol and progesterone levels in females (Nothnick et al., 1997).

Collectively, these data suggest that TIMP-1 may act as a co-regulator of steroidogenesis.

However, the underlying mechanism of TIMP-1 in steroidogenesis and whether it is involved in

luteal steroid production require further investigation.

There are two species of TIMP-2 mRNA; one migrates at 3.5 kb. while the other migrates at

Ikb. Both mRNA species are detected in rat (Nothnick et al.. 1995; Simpson et al.. 2001) CL.

While a single Ikb species is detected in ovine CL (Smith et al.. 1995), the 3.5 kb species is the

only TIMP-2 mRNA in human CL (Duncan et al.. 1998). In the bovine CL. TIMP-2 transcripts

and immunoreactive TIMP-2-like protein increase over the estrous cycle (Smith et al., 1996;

Goldberg et al.. 1996). while ovine luteal TIMP-2 mRNA expression in the early luteal phase is

greater than the mid- and late stages (Smith et al.. 1995). In contrast TIMP-2 mRNA expression

does not vary in the human CL during the menstrual cycle (Duncan et al., 1998). It has been

shown that the large luteal cells of ovine CL are the cellular sources of TIMP-2 (Smith et al.,

1995). However. TIMP-2 is localized to the theca-lutein cells and the surrounding connective

tissue stroma in the human CL (Duncan et al., 1998). These data suggest that species differences

may exist regarding the temporal and spatial expression patterns of TIMP-2.

The gelatinases are detected in the conditioned medium of luteal cells from the early bovine

CL (Tsang et al., 1995) and luteinized bovine granulosa cells (Zhao and Luck, 1996). The early

stage rat CL possesses higher levels of coliagenase and gelatinase activities than any other stage

-43-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Nothnick et al.. 1996). MMPs play important roles in CL development and function. During CL

formation, ewes immunized with the N-terminal peptide of inhibin a43-subunit (aN) manifested

incomplete infolding of surrounding tissues and theca/vessel invaginations. The reduced fertility

that resulted was attributed to decreased levels of MMP-2 (Russell et al.. 1995). Furthermore,

immunization with MMP-2 antibody caused incomplete CL formation in ewes (Gottsch et al..

2001). In human (luteinized) granulosa cells, levels of gelatinases are reduced while there is a

concurrent increase in TIMP-l production following exposure to hCG (Aston et al.. 1996).

suggesting one of the actions of gonadotropin (LH) in the CL is possibly to stabilize ECM

remodeling at the early stages of CL development.

CL maintenance also includes active tissue remodeling events. MMP-2 and MMP-9

activities are present in the bovine CL throughout the estrous cycle (Goldberg et al.. 1996).

Higher levels of MMP-2. MMP-9. and MMP-1 gene expression are observed in the mid stage

porcine CL than in the early stage (Pitzel et al.. 2000). In the rat. MT1-MMP is constitutivelv

expressed in CL throughout pseudopregnancy (Liu et al.. 1999). While MMP-2 is mainly

expressed during luteal development. MMP-13 (coliagenase 3) is only expressed in the regressing

CL (Liu et al.. 1999). The MMP-2 protein is localized in endothelial and large luteal cells of the

developing rat CL (Bagavandoss. 1998). Interestingly. MMP-9 is localized on the plasma

membrane of luteal and interstitial cells in rat CL (Bagavandoss. 1998). indicating that this

enzyme may perform pericellular proteolysis through its cell surface receptor, such as CD44 (Yu

and Stamenkovic. 1999). Besides MMPs. other proteolytic enzymes, such as proteoglycanase, are

also associated with luteal maintenance (Nothnick et al.. 1996).

Extensive tissue deterioration occurs during CL regression (Niswender and Nett, 1994).

During structural regression induced by PRL in the hypophysectomized rat both latent and

active forms of MMP-2 are increased (Endo et al.. 1993). Similarly, enhanced MMP-2 activity

and mRNA expression are also observed in GnRH agonist (GnRHa) (Goto et al., 1999) and GH

(Kiya et al., 1999) induced structural regression. During rat luteal regression induced by

-44-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cloprostenol (a PGF:a analog), MMP-13 is induced only after progesterone levels decreases (Liu

et al.. 1999), indicating that functional regression may initiate physiological signal(s) to bring

about MMP expression, which are involved in structural regression. Indeed, the rise in MMP-2

activity’ is coincident with the nadir in progesterone during the early onset of luteolysis in ewes

(Towle et al.. 2002). In the human CL. an increase in MMP-2 activity may be associated with

luteolysis (Duncan et al.. 1998), since MMP-2 expression is decreased during luteal rescue

(Duncan et al.. 1998) or following treatment with hCG in luteinized human granulosa cells

(Stamouli et al.. 1996). In contrast. MMP-1 (coliagenase 1) expression is not changed during

luteal rescue (Duncan et al.. 1998). During luteal regression of the pseudopregnant rat

coliagenase mRNA levels are increased (Nothnick et al.. 1996). Recently, this coliagenase was

most likely determined to be collagenase-3 (Liu et al.. 1999).

TIMP-l expression is decreased during PGF;a-induced luteal regression in the cow (Juengel

et al.. 1994) and the ewe (Towle et al.. 2002). Interestingly, in the ewe. this occurs before the

decline of progesterone and the rise in MMP-2 (Towle et al.. 2002). During porcine luteal

regression induced by PGF:a. the serum concentration of TIMP-1 is also significantly decreased

(Mclntush et al.. 1997). A significant fall in TIMP-l expression is also observed in marmoset CL

during PGF:a and GnRHa induced luteolysis (Duncan et al.. 1996a). In these species, the reduced

level of TIMP-1 may result in enhanced MMP activity, which favors ECM degradation during

luteal regression. However, in the rat TIMP-1 levels are increased during luteal regression (Liu et

al.. 1999). Furthermore, during rescue of the human CL, TIMP-l and TIMP-2 expression do not

vary (Duncan et al.. 1996b: Duncan et al., 1998). Therefore, there may be species differences

regarding the expression of TIMP-1 during luteal regression.

VI. MMPs/TIMPs in Ovarian Angiogenesis

-45-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Follicle development is accompanied by blood vessel recruitment (Cavender and Murdoch.

1988; Fraser and Lunn, 2000; Mattioli et al., 2001; WulfFet al., 2001). The rapid and

hypertrophic growth of the CL, which requires an abundant supply of oxygen and nutrients,

depends on concomitant vascular growth or angiogenesis (Reynolds et al., 2000). Angiogenesis.

the formation of new capillaries from preexisting vessels, is critical for follicle development and

CL formation. Angiogenesis is exquisitely orchestrated by the cell-cell and cell-matrix

interactions in the capillary. The process of angiogenesis can be divided into the following steps:

(1) production of angiogenic factors from the demand tissue; (2) spatiotemporal regulation of

endothelial cell gene expression by these angiogenic factors; (3) fragmentation of existing

capillary basement membrane; (4) migration of endothelial cells; (5) proliferation o f endothelial

cells: and (6) recruitment of pericytes and formation of capillary lumen (Moses, 1997; Klagsbrun

and Moses. 1999).

MMPs are involved in various steps of the angiogenic process. MMP-deficient mice

demonstrate delayed or reduced angiogenic events during normal development and tumor

progression (Itoh et al.. 1998; Vu et al.. 1998). Furthermore, both endogenous and synthetic MMP

inhibitors block angiogenesis in vivo and in vitro (Knauper et al.. 1996b; Hiraoka et al.. 1998).

Thus, it seems likely that MMPs also play central roles in ovarian angiogenesis.

The interest in ovarian angiogenesis originates in the finding that this organ is a rich source

of angiogenic factors (Gospodarowicz and Thakral. 1978; Frederick et al.. 1984; Makris et al..

1984). Follicular fluid initiates angiogenesis (Frederick et al., 1984; Frederick et al., 1985),

through its stimulatory effects, in part on endothelial cell proliferation (Rose and Koos, 1988). In

addition, both intact follicles and dispersed granulosa cells produce a vascular chemoattractic

factor (Rone et al.. 1993). Intense expression of vascular endothelial growth factor (VEGF)

mRNA and protein is confined to granulosa and theca cells at the late stage of follicle

development consistent with the establishment of a vasculature around the follicle wall at this

time (Ravindranath et al.. 1992; Kamat et al., 1995). The importance o f VEGF in follicle

-46-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. development is further established in the human ovary, where it is only expressed in healthy

follicles, but not atretic ones (Gordon et al., 1996; Yamamoto et al., 1997). Furthermore,

inhibition of ovarian angiogenesis by administration of a VEGF antibody in the late follicular

phase of rhesus monkeys delays follicle development (Zimmermann et al., 2001). The expression

of angiogenin, another potent angiogenic factor, is also closely correlated to follicle development

(Lee et al.. 1999). With the exception that TIMP-1 is localized in blood vessels surrounding

developing follicles (Bagavandoss. 1998). little is known about the expression and localization of

MMPs and TIMPs in capillaries around follicles. As in the case of tumor angiogenesis, however,

MMPs and TIMPs produced by theca and interstitial cells may facilitate blood vessel recruitment

during follicle development.

CL development is associated with extensive angiogenesis (Basset 1943: Meyer and

McGeachic. 1988: Tsukada et al.. 1996). Right before ovulation, the basement membrane that

separates the granulosa and theca layers is fragmented. At the same time, capillary basement

membrane in the theca intema compartment is also broken down (Matsushima et al.. 1996:

Tsukada et al.. 1996). Immediately following ovulation, endothelial cells migrate into the

previously avascular granulosa layer to form new blood vessel sprouts. Accompanying this

process, there are a series of changes in the microvascular extracellular matrix, including

fibronectin. collagen type IV. and laminin (Matsushima et al.. 1996: Amselgruber et al., 1999).

However, there are contradictory reports regarding whether endothelial cells or pericytes lead the

outgrowth of the newly formed vessels (Matsushima et al.. 1996: Redmer and Reynolds. 1996:

Amselgruber et al.. 1999: Reynolds et al.. 2000). This discrepancy may be due to technical limits

of the research methods applied in the studies. Pericytes play important roles during capillary

formation and vasculature survival (Nehls et al.. 1992: Hirschi and D'Amore, 1996). Further

investigation on the cellular angiogenic events during CL formation may provide valuable

information regarding the interactions between endothelial cells and pericytes.

-47-

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The luteal angiogenic process is so intense that endothelial cells make up 85% of the

proliferative cell population during CL development (Reynolds et al.. 1994). By mid cycle,

endothelial cells are the most abundant cell type in the bovine CL, and constitute about 50% of

total number o f cells (Lei et al.. 1991; Zheng et al., 1993; Zheng et al., 1994). At this stage, the

CL is so vascularized that the majority of steroidogenic ceils are in contact with one or more

capillaries, and the blood flow in this gland is among the greatest of any tissue in the body

(Reynolds et al.. 1992; Redmer and Reynolds. 1996).

Consistent with this rapid development of the vasculature, various angiogenic factors are

identified in the CL. including FGF (fibroblast growth factor), NGF (nerve growth factor), EGF

(epidermal growth factor). IGF (insulin-like growth factor). TGF (transforming growth factor).

VEGF (vascular endothelial growth factor), and angiopoeitins (Gospodarowicz et al.. 1977a;

Gospodarowicz et al.. 1977b; Gospodarowicz and Thakral. 1978; Gospodarowicz et al.. 1985;

Phillips et al.. 1990; Reynolds et al.. 1992; Kamat et al.. 1995; Redmer and Reynolds. 1996;

Laitinen et al.. 1997; Abulafia and Sherer. 2000). Among these factors, heparin-binding factors,

such as FGFs and VEGFs. have been postulated to be key regulators (Redmer and Reynolds.

1996: Reynolds et al.. 2000). VEGF is expressed in the primate and human CL from early to late

stages, but is absent in regressing tissues (Ravindranath et al.. 1992; Gordon et al., 1996). In

addition, total angiogenic activity is present in the bovine CL throughout the estrous cycle, but

there is a slight increase w ith age (Redmer et al.. 1988; Reynolds et al.. 2000). Therefore, it may

not be appropriate to view the mid-stage CL as non-angiogenic (Goede et al., 1998). Indeed, in

the human CL. although the total volume of luteal cells decreases in the late stage, the vascular

volume progressively grows until the late regressing stage (Gaytan et al., 1999). This is consistent

with the observation in the bovine CL. where blood vessel regression lasts several weeks after

luteolysis (Augustin et al.. 1995). It has been postulated that the vasculature in the early stages of

luteolysis may serve as a route for degradation products destined for breakdown and excretion

(Redmer et al., 1988). Besides the endometrium, the CL also exhibits blood vessel regression.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Endothelial cell detachment and vessel occlusion are two major phenomena during luteal blood

vessel regression (Modlich et al.. 1996). These phenotypic changes of luteal endothelial cells may

be mainly related to changes in their cell surface adhesion molecules (Augustin et al.. 1995).

MMPs and TIMPs may participate in the regulation of the luteal angiogenic process. In the

rat (Bagavandoss. 1998) and sheep (Mclntush et al.. 1996). TIMP-1 is localized in luteal

capillaries, suggesting that this endogenous MMP inhibitor may participate in maintaining the

integrity of the capillary. Administration of a MMP-2 antibody in ewe results in abnormal luteal

vasculature and incomplete CL formation (Gottsch et al.. 2001). implicating the critical role of

this enzyme in luteal angiogenesis.

Ovarian angiogenesis shares similarities with tumor angiogenesis. For example, in both

processes, angiogenesis is view'ed as a response to nutrient deficiency, and the same regulators

and angiogenic factors, such as hypoxia-inducible factor 1 (HIF-1) and VEGF. are involved

(Waleh et al.. 1995; Necman et al.. 1997). Therefore, elucidation o f the molecular mechanisms of

ovarian angiogenesis and the normal capillary degeneration process during luteolysis. especially

those that involve MMPs and TIMPs. may provide insights toward a better understanding of

tumor angiogenesis.

VII. Perspectives

Ovarian physiological events, such as follicle development, ovulation, corpus Iuteum

development and angiogenesis. require the participation of proteolytic enzymes and their

endogenous inhibitors, such as MMPs and TIMPs. respectively, to orchestrate the associated

dynamic tissue remodeling that occurs By modify ing the extracellular milieu. MMPs and TIMPs

modulate the cellular or biochemical properties of cells, facilitating proliferation, growth, or

differentiation. Beyond this. MMPs also affect other physiological events by liberating growth

factors and cytokines from their biologically inactive forms, or modifying the receptors for

growth factors or cytokines. In addition. TIMPs may directly modulate cellular behaviors, such as

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. steroidogenesis and proliferation. However, a great deal of work remains before the physiological

functions of MMPs and TIMPs in the reproductive system are fully elucidated. From the above, it

is clear that the ovary is an excellent research model for studying the roles of MMPs and TIMPs

during normal tissue remodeling and angiogenesis. The information obtained will perhaps prove

valuable in designing therapeutic strategies for physiological processes associated with infertility

or for pathological conditions, such as cancer, retinopathies, and rheumatoid arthritis.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-1 The MMP family

MMP Trivial name Other names EC Subfamilies number number MMP-l Coliagenase 1 Interstitial coliagenase 3.4.24.7 Collagenases MMP-2 Gelatinase A 72 kDa gelatinase 3.4.24.24 Gelatinases type IV coliagenase MMP-3 Stromelvsin-1 Transin. proteoglycanase 3.4.24.17 Stromelvsins MMP-4* Procollagen peptidase =MMP-3 - MMP-5* ^-coliagenase -MMP-2 - MMP-6* Acid metalloproteinase =MMP-3 - MMP-7 Matrilvsin Pump-1 3.4.24.23 Matrilvsins MMP-8 Collagenase-2 Neutrophil coliagenase 3.4.24.34 MMP-9 Gelatinase B 92 kDa gelatinase 3.4.24.35 Gelatinases type IV coliagenase MMP-10 Stromelvsin-2 Transin 2 3.4.24.22 Stromelvsins MMP-11 Stromelvsin-3 Furin motif Others MMP-12 Metalloelastase 3.4.24.65 Others MMP-13 Collagenase-3 Rodent interstitial Collagenases coliagenase MMP-14 Membrane-type I MT1-MMP. MT-MMP1 MT-MMPs metalloproteinase MMP-15 Membrane-ripe 2 MT2-MMP. MT-MMP2 MT-MMPs metalloproteinase MMP-16 Membrane-ripe 3 MT3-MMP. MT-MMP3 MT-MMPs metalloproteinase MMP-17 Membrane-ripe 4 MT4-MMP. MI -MMP4 MT-MMPs metalloproteinase MMP-18 Coilagenase-4 Xenopus coliagenase Collagenases MMP-19 No trivial name RASI-1. Stromelvsin-4 Others MMP-20 Enameivsin Others MMP-21 XMMP Others MMP-22 CMMP Chicken metalloproteinase Others MMP-23 No trivial name CA-MMP Others MMP-24 Membrane-ripe 5 MTS-MMP. MT-MMP5 MT-MMPs i i metalloproteinase MMP-25 Membrane-type 6 MT6-MMP. MT-MMP6. MT-MMPs metalloproteinase Leukolvsin MMP-26 Matrilvsin-2 Endometase Others MMP-27 No trivial name Human homolog of Others CMMP MMP-28 Epilysin Others

Note: I. Asterisks denote names that are not longer used. 2. Adapted from Stemlicht and Werb (2001) Annu Rev Cell Dev Biol 17:463.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-2 Biochemical and molecular features of MMPs

Subfamilies Trivial name MMP Molecular Chromosome localization number mass (kDa) D/O L A Human Mouse Coliagenase-1 MMP-1 52 42 Ilq22-q23 B Collagenase-2 MMP-8 85 64 Uq21-q22 B Collagenases Collagenase-3 MMP-13 52 42 llq22.3 9(A1-A2) B Collagenase-4 MMP-18 53 42 B Gelatinases Gelatinase A MMP-2 72 66 16q 13 8(42.9) C Gelatinase B MMP-9 92 84 20ql 1.2-q 13.1 2(H1-H2) D Stromelvsins Stromelvsin-1 MMP-3 57 45 1 lq23 9(1.0) B Stromelvsin-2 MMP-10 54 44 Ilq22.3-q23 B MT-MMP1 MMP-14 66 54 14q 11-q 12 14(12.5) F MT-MMP2 MMP-15 72 60 16ql3-q21 8(45.5) F MT-MMP3 MMP-16 64 53 8q21 4(3.6) F MT-MMPs MT-MMP4 MMP-17 57 53 12q24.3 5(1.0) G MT-MMP5 MMP-24 73 62 20q 11,2-q 12 2(1.0) F MT-MMP6 MMP-25 63 62 I6p 13.3 G Matrilysins Matrilvsin-1 MMP-7 28 19 Ilq21-q22 9(1.0) A 1 Matrilvsin-2 MMP-26 29 19 llpl5 A Stromelvsin-3 MMP-11 64 46 22q 11.2 10(40.9) E Metalloelastase MMP-12 54 22 1 Iq22.2-q22.3 9(1.0) B RASI-1 MMP-19 54 45 12q 14 10(71.0) B Other MMPs Enamelvsin MMP-20 54 22 Uq22.3 9(1.0) B XMMP MMP-21 70 53 H CMMP MMP-22 51 43 1 lq24 B CA-MMP MMP-23 44 31 lp36.3 1 MMP-27 B Epilysin MMP-28 59 45 17ql 1.2 E

D/O = domain organization. Lettters denote arrangement of domains described in Figure 1-1.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1-1 Domain Organization of the MMPs

Signal Peptide Propeptide

Catalytic domain | | Furin-like recognition site

Hemopexin-like domain | Fibronectin type II-like insert

Transmembrane domain [] Collagen type V-like insert

111 Glycophosphatidyl inositol-anchoring domain Cytoplasmic tail

Cysteine/proline rich IL-1 receptor like sequence ^ Hinge region

| Vitronectin-like sequence Note: Letters (A-I) denote domain organization

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-3 ECM and non-ECM substrates of MMPs

MMP Non-ECM substrates subfamilies MMPs ECM Substrates Substrates Notes Collagen I, II, III. VII. VIII. MCP-1, MCP-3, MCP-4, X. XI: gelatin L SDF. Pro-ILip, ILip, Hormones entactin/nidogen. Pro-TNFa MMP-1 fibronectin. laminin. Pro-MMP-1, Pro-MMP-2 Enzvmes perlecan vitronectin, IGF-BP-2, IGF-BP-3, a l- aggrecan cartilage link proteinase inhibitor, a l- Inhibitors protein, myelin basic antichymotrypsin a2- protein, tenascin decorin macroglobuiin Collagen I. II. Ill: gelatin, MCP-1. Pro-TNFa Hormones MMP-8 entactin aggrecan. tenascin L-selection CAM Pro-MMP-8 Enzymes IGF-BP, a 1-proteinase Inhibitors Collagenases inhibitor. a2- macroglobulin. Collagen I. II. III. VI. IX. MCP-3. SDF. Pro-TNFa Hormones X. XIV; gelatin I. MMP-13 fibronectin. vitronectin, Pro-MMP-13. Pro-MMP-9 Enzvmes aggrecan. osteonectin a 1 -antichymotrypsin. Inhibitors o2-macroglobulin MMP-18 Collagen type 1.11. Ill: gelatin Collagen I. III. IV. V. VII. Pro-TGFpi. 2; Pro-TNFa. Hormones X. XI: gelatin I. fibrillin. Pro-ILip. Big endothelin- BM-40. neurocan. 1. SDF. MCP-3. Substance MMP-2 fibronectin. elastin. P vitronectin, aggrecan. FGFR-1 Receptors cartilage link protein, myelin basic protein, Galectin-3 CAM osteonectin, tenascin. laminin-5. decorin. Pro-MMP-1. Pro-MMP-2. Enzymes breyican Pro-MMP-13 a 1-proteinase inhibitor. Inhibitors a2-macroglobulin IGFBP-3. IGFBP-5. Pregnancy zone protein Gelatinases Collagen IV. V. XL XIV; Pro-IL-8. SDF, GROa. Hormones gelatin 1. decorin. elastin. PF-4. Pro-TNFa. Pro- fibrillin, laminin. TGFpl, Pro-ILip, MMP-9 vitronectin, aggrecan. CTAP-m/NAP-2 cartilage link protein, Cell-surface IL-2Ra. Receptors myelin basic protein, FGF-R1 osteonectin. Galec tin-3 CAM Pro-MMP-2, Pro-MMP-9, Enzymes Pro-MMP-13, Plasminogen al-proteinase inhibitor, Inhibitors a2-macroglobulin

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Collagen III, IV. V. VII. MCP-1, MCP-2, MCP-3, IX. X, XI; gelatin L MCP-4, SDF, Pro-TNFa, Hormones decorin. elastin. Pro-HB-EGF, Pro-ILip entactin/nidogen. fibrillin, L-selection, E-cadherin CAM MMP-3 fibronectin. laminin. Pro-MMP-l. Pro-MMP-3 Enzymes Stromelvsins vitronectin, aggrecan. Pro-MMP-7. Pro-MMP-8 cartilage link protein, Pro-MMP-9. Pro-MMP-13 myelin basic protein, Plasminogen osteonectin, tenascin a 1-proteinase inhibitor. Inhibitors a2-macroglobulin, IGFBP-3,al- antichymotrypsin Collagen III. IV. V; gelatin Pro-MMP-1. Enzymes MMP-10 I. elastin. fibronectin. Pro-MMP-8. aggrecan. cartilage link Pro-MMP-10 protein Collagen types I. II. Ill: MCP-3, SDF. Pro-TNFa Hormones gelatin, fibronectin. Cell-surface CD44. Cell- CAM vitronectin, aggrecan surface (tTG) MMP-14 Pro-MMP-2. Pro-MMP-13 Enzymes a 1-proteinase inhibitor. Inhibitors a2-macroglobulin Pro-TNFa Hormones MT-MMPs MMP-15 Proteoglycan Cell-surface bound tTG

I Pro-MMP-2 Enzvmes Collagen type III. Pro-TNFa Hormones MMP-16 fibronectin Cell-surface bound tTG CAM i i Pro-MMP-2 Enzvmes MMP-17 Gelatin, fibrin/fibrinogen Pro-TNFa Hormones Pro-MMP-2 Enzvmes Fibronectin. proteoglycans, Pro-MMP-2 Enzymes MMP-24 gelatin Collagen type IV. gelatin, Pro-MMP-2 Enzymes MMP-25 laminin-1, fibronectin. Pro-MMP-9 proteoglycans (DSPG. a 1-proteinase inhibitor Inhibitors CSPG). fibrin/fibrinogen Collagen I. IV. decorin. Pro-a-definsin, Cell Hormones elastin. entactin/nidogen. surface bound Fas-Ligand, fibrillin, fibronectin. Pro-TNFa Matriiysins MMP-7 laminin. decorin E-cadherin. P4 integrin CAM vitronectin, aggrecan. Pro-MMP-l. Pro-MMP-2 Enzymes cartilage link protein, Pro-MMP-7, Pro-MMP-9 myelin basic protein, Plasminogen osteonectin, tenascin a 1-proteinase inhibitor, Inhibitors a2-macroglobulin MMP-26 Collagen type IV. gelatin. Pro-MMP-9 Enzymes fibronectin. fibrin/fibrinogen IGFBP-1. a 1 -proteinase Inhibitors inhibitor

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-11 Fibronectin, laminin, IGFBP-l Inhibitors aggrecan a 1-proteinase inhibitor a2-macroglobulin MMP-12 Fibronectin, elastin, Pro-TNFa Hormones laminin. proteoglycan, Plasminogen Enzymes Gbrin/Gbrinogen, myelin a 1-proteinase inhibitor Inhibitors basic protein MMP-19 Collagen type IV, gelatin, laminin, Gbronectin tenascin. Other MMPs entactin, aggrecan, COMP, Gbrin/Gbrinogen MMP-20 Amelogenin. COMP, aggrecan MMP-21 no substrates defined MMP-22 no substrates defined MMP-23 Gelatin MMP-27 no substrates defined MMP-28 no substrates deGned

1. Abbreviations: CAM: cell adhesion molecules: GROa: growth related oncogene a ; PF-4: platelet factor 4; CTAP-III/NAP-2: connective tissue activating protein III/ neutrophil-activating peptide-2: SDF: Stromal cell-derived factor: COMP: cartilage oligomeric matrix protein: Cell-surface (tTG): cell surface tissue transglutaminase: TNFa: Tumor necrosis factor a: TGFf): Transforming growth factor (3: IL: Interleukin: HBEGF: Heparin-binding epidermal growth factor. FGFR: Fibroblast growth factor receptor. ILR: Interleukin receptor. MCP: Monocyte chemoattractant protein.

2. Adapted from McCawley and Matrisian (2001) Curr Opin Cell Biol 13: 534

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1-4 Biochemistry and molecular features of TIMPs

TIMPs Molecular Glycosvlation Cellular Chromosome mRNA size weight localization of localization (kDa) protein TIMP-l 29 Glvcosvlated diffusible Xpl 1.3-11.23 0.9 kb TIMP-2 21 Unglycosylated diffusible I7q25 3.5 kb and 1 kb TIMP-3 24-27 glycosylated ECM bound 22ql2.1-13.2 4.5.2.8, and 2.4 kb TIMP-4 23-24 unresolved diffusible 3p25 1.2 kb

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II

BOVINE MATRIX METALLOPROTEINASE-2 (MMP-2): MOLECULAR CLONING,

EXPRESSION IN THE CORPUS LUTEUM, AND INVOLVEMENT IN ANGIOGENESIS

Abstract

Angiogenesis and other tissue remodeling events in the corpus luteum (CL) are mediated

by matrix metalloproteinases (MMPs). Previously, we cloned the bovine homolog of membrane-

type I metalloproteinase (MT1-MMP). and showed that active MT1-MMP is correlated to MMP-

2 activity in the CL during the estrous cycle. The aims of the present study' were to clone the

bovine MMP-2 gene, to investigate its temporal and spatial expression in three ages of CL

obtained over the bovine estrous cycle, and to determine its potential roles in endothelial cells

during capillary tube formation. The bovine MMP-2 cDNA. which was isolated from a UNI-ZAP

II capillary endothelial cell cDNA library-, encoded a protein of 661 amino acids. Luteal tissues

were collected from non-lactating dairy cows on days 4. 10. and 16 (n=3/dav: day 0 = estrus) of

the estrous cycle. Northern blotting and Western blotting revealed that the levels of MMP-2

mRNA (3. Ikb) and immunoreactive pro-MMP-2 protein (68 kDa) did not differ (P>0.05) in any

age of CL examined. In addition to large luteal cells. MMP-2 was localized in endothelial cells in

ail ages of CL by immunohistochemistry. The potential roles of MMP-2 in angiogenesis were

evaluated using bovine capillary endothelial (BCE) ceils in a Matrigel capillary' tube-formation

assay. Tube formation by BCE cells was remarkably suppressed by a neutralizing antibody

against MMP-2. Taken together, these data suggested that the activity of MMP-2 and its

localization in endothelial cells may be critical for CL development e.g., by regulating capillary

tube formation.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Introduction

The formation and development of the corpus luteum (CL) include extensive matrix

remodeling events. After ovulation, the basement membrane degrades, and the follicle wall folds

toward the cavity of the collapsed follicle (Niswender and Nett, 1994: Smith et al.. 1994).

Fibroblasts and luteinized theca intema cells proliferate and migrate into the cavity of the

ruptured follicle, where they mix with luteinized granulosa cells (Pederson, 1951). In addition,

endothelial cells from capillaries in the theca intema compartment invade the granulosa layer to

form new blood vessels (Plendl. 2000). This process of new capillary formation is termed

angiogenesis (Moses. 1997). These tissue remodeling events likely require the participation of a

large family of zinc and calcium dependent proteolytic enzymes, matrix metalloproteinases

(MMPs). which collectively degrade all known components of the extracellular matrix (ECM)

(Massova et al.. 1998: Smith et al.. 1999: Curry and Osteen. 2001).

Among the more than 25 members of the MMP family, only a few have been studied in

the CL. The patterns of expression and activities of MMP-2 and MMP-9 have been investigated

in bovine (Tsang et al.. 1995: Goldberg et al.. 1996: Zhang et al.. 2002). ovine (Russell et al..

1995: Towle et al.. 2002). porcine (Pitzcl et al.. 2000). rat (Nothnick et al.. 19%: Bagavandoss.

1998: Liu et al.. 1999). and human (Duncan et al.. 1998) CL. Furthermore. MMP-1 (collagenase-

1) expression was detected in rat (Nothnick et al.. 1996) and human (Duncan et al.. 1998) CL,

while MMP-13 expression was found in bovine (Zhang and Tsang: unpublished data), ovine

(Ricke et al.. 2002). and rat (Liu et al.. 1999) CL. Within this select group of enzymes, the action

of MMP-2 is most notable because its reduced levels impair CL formation (Russell et al., 1995).

In part this may be attributed to the broad substrate specificity of MMP-2, especially its efficient

degradative action on type IV collagen, the major component of the basement membrane (Yu et

al.. 1998). Additionally, administration of a MMP-2 antibody blocks vasculature formation

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. during CL development, supporting the potential role of this enzyme in blood vessel recruitment

(Gottsch et al.. 2001).

Another distinguishing feature of MMP-2 is its activation process. Most MMPs are

secreted as zymogens and then cleaved extracelluiarly into their active forms by serine

proteinases (Murphy et al.. 1999). However. pro-MMP-2 is activated, instead, on the cell surface

by membrane-type 1 metalloproteinase (MT1-MMP) (Murphy et al.. 1999). Previously, we

cloned the bovine MT1-MMP gene, and demonstrated that the active form of MT1-MMP is

correlated to MMP-2 activity in three ages of CL obtained over the estrous cycle (Zhang et al..

2002).

However, much remains to be learned about MMP-2. especially in domestic ruminants,

before \vc can elucidate its role or function in the CL. Therefore, in the present study, we have

cloned the bovine MMP-2 cDNA. and profiled its temporal and spatial expression patterns in CL

obtained during the estrous cycle. The role of MMP-2 during angiogenesis was studied by using

an in vitro capillary tube formation assay.

Materials and Methods

1. Molecular Cloning and Sequence Analysis

A bovine UNI-ZAP II cDNA library was constructed using RNA isolated from BCE cells

of the adrenal cortex with Oligo(dT) primers. The primers for cloning bovine MMP-2 were

designed based on the conserved catalytic region of known MMP-2 sequences of other species.

The cDNA encoding the catalytic domain of bMMP-2 was directly amplified from this cDNA

library using the polymerase chain reaction (PCR). Successive rounds of PCR were performed

using new primers designed based on the obtained sequence data to clone the rest of bMMP-2

cDNA. PCR products were cloned into PCR TA-cloning vector (Invitrogen, Carlsbad. CA) for

subsequent DNA sequencing.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sequencing of cDNA was performed using PCR-based automated sequencing methods

(Biopolvmer Facility and DNA Sequencing Core Facility, Children's Hospital, Boston, MA).

Protein database search was assessed with advanced BLAST service provided by the National

Center for Biotechnology Information (http://www.ncbi.nim.nih.gov/) with default settings. DNA

and protein sequence analysis was conducted using the MacVector 6.0 DNA sequence analysis

software package (Accelrvs Inc. San Diego. CA). Multiple alignment analysis was performed

using Genetyx Sequence Analysis Software (Software Development Company, Tokyo, Japan).

2. Tissue Distribution of Bovine MMP-2

For Northern blot analysis, a cDNA fragment consisting of mostly the 5'-untranslated

region (UTR) of bMMP-2 was used to generate cDNA probes, which were random primer-

labeled with a 3:P-dCTP using the Ready Prime cDNA Labeling Kit (Amersham. Piscatawav.

NJ). Poly(A)' RNA from different bovine tissues was purified using a modified guanidinium

thiocvanate method followed by poly (A)' RNA selection with two rounds of oligo(dT)-cellulose

columns. After separation with 1.0% (w/v) agarose electrophoresis containing formaldehyde.

RNA was transferred to nylon membranes (Schleicher & Schuell. Keene, NH). A Northern blot

of 2.5 |ig polv(A)*-RNA was hybridized overnight at 65° C with the radiolabeled probes at a

concentration of 2x 107 cpm/10 ml. The blot was washed to a final stringency of 0. lxSSC and

0 1% SDS (w/v) at 65° C. Hybridized probes were detected with autoradiography.

3. Animals and Tissue Collection

Ail animal procedures in the present study were performed according to protocols

approved by the Institutional Animal Care and Use Committee (IACUC) at the University of New

Hampshire. Corpora lutea were collected from regularly cycling, non-lactating dairy cows housed

at the University of New Hampshire Dairy- Teaching and Research Center. Luteal tissues were

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. removed on days 4. 10. and 16 of the estrous cycles (day 0 = estrus: n=3/day). For day 4 C L the

ovary was removed by colpotomv from animals under an epidural anesthesia [2% mepivacaine

hydrochloride (w/v); 0.01 mL/kg body weight; Upjohn. Kalamazoo, NO], The CL tissues were

then dissected from the ovarian stroma. The day 10 and 16 CL tissues were removed from the

ovary by enucleation.

4. Expression of MMP-2 in the Bovine CL during the Estrous Cvde

Total RNA was extracted from luteal tissues using TRIZOL (GIBCO-BRL, Carlsbad,

CA) according to the manufacturer's instructions. Twenty micrograms of total RNA were

fractionated on 1.0% (w/v) agarose gels containing formaldehyde and were transferred onto

Hybond™-N* nylon membranes (Amersham Pharmacia Biotech. Piscataway. NJ). Membranes

were first incubated in pre-hybridization buffer [50% (v/v) formamide. 5xSSC. 0.2% (w/v) SDS.

and 2% (w/v) blocking reagent| for two hours at 42°C. Hybridization was carried out in the pre-

hybridization buffer containing MMP-2 cDNA probes at 50°C overnight. These cDNA probes

were labeled with digoxigenin (DIG)-dUTP using the DIG DNA random-primed Labeling Kit

(Roche Molecular Biochemicals. Indianapolis. IN). The blots were then washed twice at room

temperature in 2xSSC with 0.1% (w/v) SDS. followed by higher stringency washes in 0.2xSSC

with 0.1% (w/v) SDS at 65°C. Hybridized probes were detected using an anti-DIG antibody

conjugated to alkaline phosphatase (used at 1:5000; Roche Molecular Biochemicals. Indianapolis,

IN) in 1% (w/v) blocking reagent. The signals were detected by CSPD (Roche Molecular

Biochemicals. Indianapolis. IN), a chemiluminescent substrate for alkaline phosphatase. After

visualization, the membranes were subsequently stripped according to manufacturer's

instructions, and re-hybridized with cyclophilin cDNA probes.

5. Western Blot Analysis

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Total protein was extracted from luteal tissues as previously described (Zhang et al.,

2002). Equal amounts of protein were subjected to SDS-PAGE on 10% (w/v) polyacrylamide

gels, and were transferred to Protran® nitrocellulose membranes (Schleicher & Schuell, Keene,

NH). Nonspecific binding sites were blocked by incubating the membranes with 5% (w/v) nonfat

dry milk in TBST buffer [lOmM Tris-HCl, l50mM NaCI. 0.05% (v/v) Triton X-100, pH 8.0j.

Membranes were then incubated with rabbit anti-human MMP-2 antibody (used at 1:5000;

Chemicon. Temecula. CA). After extensive washes with TBST buffer (five times, 15 minutes per

wash). HRP-conjugated anti-rabbit-IgG (1:10.000) was applied onto membranes as a secondary

antibody. The signals were detected with the SuperSignal* West Pico Chemiluminescent

Substrate (Pierce. Rockford. 1L) according to the manufacturer's instructions.

6. lmmunohistochemistrv

Frozen sections (6pm) were prepared from luteal tissues collected at three stages of the

estrous cycle using a Crvotome (Shandon. Pittsburgh. PA). Sections were fixed in cold acetone

for 5 minutes. The endogenous peroxidase activity' was quenched by incubating sections in 0.03%

(v/v) H20 : for 30 minutes at room temperature. Nonspecific sites were blocked by 10% (w/v)

BSA before incubating the slides with a mouse anti-human MMP-2 antibody (1:250; Oncogene

Research Products. San Diego. CA). Following incubation with the biotin-conjugated goat anti­

mouse IgG secondary antibody (1:2000; Pierce. Rockford. IL), MMP-2 was visualized using

VECTASTAIN® Elite ABC Kits (Vector Laboratories. Burlingame. CA). Sections were

subsequently counterstained with Gill's 2 hematoxylin (Shandon, Pittsburg, PA). For each tissue

sample, a consecutive section placed on the same slide was used as a negative control, with BSA

substituting for the primary antibody. For each tissue (3 corpora lutea for each age), 10 to 20

sections were stained. A minimum of 20 areas per section was examined.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7. BCE Cell Culture and Gelatin Zvmogrrahic Analysis

SDS substrate zymography electrophoresis was performed based on a previously

described method with modifications (Tsang et al., 1995; Zhang et al., 2002). Medium

conditioned by BCE cells were mixed with non-reducing SDS sample buffer before fractionation

in 10% (w/v) polyacrylamide gels containing 0.1% (w/v) gelatin (Bio-Rad, Hercules, CA). After

electrophoresis, gels were washed twice with 2.5% (v/v) Triton X-100 (15 minutes per wash),

followed by incubation with substrate buffer [50 mM Tris-HCl. 5 mM CaCL, 1 pM ZnCI;, and

0.02% (w/v) NaN 3. pH7.6| at 37°C for 24 hours. The gels were stained with 0.1% (w/v)

Coomassic Brilliant Blue R-250 (BioRad. Hercules..CA). and the gelatinolytic activities were

shown as clear zones against a blue background.

To verify- that bMMP-2 is a bona fide metal-dependent proteinase, zymogram gels were

incubated with substrate buffer containing a MMP-specific inhibitor 1.10 phenanthroline (10

mM; Sigma. St. Louis. MO). The latent and active forms of MMP-2 were distinguished by

incubating BCE conditioned medium with ImM 4-aminophenylmercuric acetate (APMA)

(Sigma. St. Louis. MO), an organomercurial activator of MMPs. at 25°C for 30 minutes before

zymographic analysis.

8. In Vitro Capillary Tube Formation Assay

The in vitro capillary tube formation assay was carried out as previously described

(Kubota et al.. 1988). Cell culture surfaces were pre-coated with Matrigel (Becton Dickinson.

Waltham. MA) at a density of 100 pl/cm2 growth area. After incubating at 37°C for 30 minutes,

culture surfaces were washed with PBS. BCE cells were plated at 25.000 cells/ cm2 growth area

in growth medium that contains 5% (v/v) fetal bovine serum (FBS) and 1% (v/v) glutamine

penicillin-streptomycin (GIBCO-BRL, Carlsbad. CA).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To assess the potential function of MMP-2 in angiogenesis, the capillary tube formation

assay was carried out under conditions that MMP-2 activity was neutralized by an anti-MMP-2

antibody (1:100) (A lbinietal.. 1991; Pauly etal.. 1994; Kenagy et al., 1997), which was kindly

provided by Dr. William Stetler-Stevenson at NDi. In the negative control, normal mouse serum

was added in the same dilution.

9. Data Analysis

Densitometric intensities of bands on Northern and Western blots were determined by

UN-SCAN-IT gel™ automated digitizing system (Silk Scientific, Orem, UT). Data were

analyzed by ANOVA. followed by Tukev's test for multiple comparisons.

Results

I. Cloning and Sequence Analysis of Bovine MMP-2

Bovine MMP-2 was PCR-cloned from a cDNA library' constructed using mRNA isolated

from BCE cells of the adrenal cortex. Compiled cDNA sequences of bovine MMP-2 were aligned

using MacVector sequence analysis software to generate a cDNA of 2169 basepairs (bp),

including an open reading frame (ORF) of 1986 bp. a 5’-untranslated region (UTR) of 181 bp.

and a partial 3'-UTR of 70 bp (Figure I A; GenBank accession number AF290428).

The predicted bovine MMP-2 protein is composed of 661 amino acids with a calculated

molecular mass of about 74 kDa. From the amino terminus, bovine MMP-2 is composed of the

hydrophobic signal domain (M1 to \ M). the pro-domain (A31 to N 110), the catalytic domain (Ym

to G447). the hinge region (A448 to L469). and the hemopexin domain (C470 to C661) (Figure IB). The

catalytic domain contains a conserved sequence HEXGHXXGXXHS/T, which functions as the

for the catalytic zinc atom (HEFGHAMGLEHS in the bovine MMP-2). In addition,

the catalytic domain of bovine MMP-2 is interrupted by three fibronectin type Q-like repeats,

which function as binding sites for gelatin and serve as a hallm ark o f gelatinases. Multiple

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sequence alignment analysis demonstrated that bovine MMP-2 shares 94.7%, 94.1%, 93.5%,

93%. and 95.9% sequence identity to its human, rat mouse, rabbit and pig homologs,

respectively (Figure 1C).

2. Tissue Distribution of Bovine MMP-2

Expression of bMMP-2 in various bovine tissues was analyzed by Northern blotting

(Figure 2A). Bovine MMP-2 is encoded by a single mRNA species of approximately 3.1 kb. The

density of Northern blots was evaluated by scanning densitometry analysis, and relative

expression levels of MMP-2 mRNA in different tissues are presented as percentage of density

compared to that in the lung, w hich had the strongest expression and was assigned as 100%

(Figure 2B). A moderate expression of MMP-2 was also detected in the kidney (50.0%), heart

(30.2%) and spleen (15.0%). However, there were extremely low and non-detectable levels of

MMP-2 mRNA in the brain (6.7%) and in the liver (0%). respectively.

3. Expression and Localization of MMP-2 in the Bovine CL

The pattern of MMP-2 expression in the early, mid-, and late stage CL during the estrous

cycle was investigated at the mRNA level by Northern blot analysis (Figure 3A). and at the

protein level by Western blot analysis (Figure 4A). Northern blot analysis detected a single 3.1 kb

MMP-2 transcript in all CL tissues. No apparent differences in the levels of bMMP-2 mRNA

w ere observed among these three ages of CL (p>0.05). Western blot analysis detected an

approximately 69 kDa protein band, which corresponds to the latent form of MMP-2. in all three

ages of CL (Figure 4A). Densitometric analysis demonstrated that the level of the latent MMP-2

did not change (p>0.05) during the estrous cycle (Fig 4B).

The spatial pattern of MMP-2 expression in the bovine CL was studied by

immunohistochemistry. In early (d4), mid (dlO). and late (dl6) cycle CL (Figure 5A-C,

respectively), positive staining for MMP-2 was localized in endothelial and large luteal cells.

- 6 6 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Large (steroidogenic) luteal cells were identified based on cell size and presence of lipid droplets.

Endothelial cells were identified by staining a consecutive section with an antibody for Factor

Xm. an endothelial cell marker (Figure 5E). No staining was observed when the primary

antibody was omitted (Figure 5D and 5F).

4. Detection of MMP-2 Activity in Bovine Capillary Endothelial (BCE1 Cells

MMP-2 activity' in BCE cells was studied using gelatin zymography A 68 kDa

geiatinolytic activity, which corresponds to the protein size of MMP-2. was readily detected in

serum-free medium conditioned by BCE cells (Figure 6. lane 1). Under the cell culture

conditions, the majority of gelatinase activity expressed by BCE cells is in the latent form. This

was validated by incubation of BCE conditioned medium with 1.0 mM APMA. which resulted in

a conversion of the 68 kDa proMMP-2 to the 62 kDa active forms (Figure 6. lane 2).The detected

geiatinolytic activity was from an MMP because its activity was completely inhibited by an

MMP-spccific inhibitor. I. 10-phenanthroline (Figure 6. lane 3).

5. MMP-2 is Required for In vitro Capillary Tube Formation

The role of MMP-2 during angiogenesis was studied by using an in vitro capillary tube

formation system. As shown in Figure 7 A. BCE cells formed extensive capillary-like structures

w hen they were plated on Matrigel. The most extensive tube formation was observed at 8 to 12

hours after cell plating and was dependent on the presence of 5% serum (data not shown). When

incubated with a monoclonal MMP-2 antibody at 1:100 dilution, BCE cells showed reduced

degree of in vitro capillary tube formation (Figure 7B).

Discussion

In addition to their involvement in pathological extracellular degradation events such as

tumor invasion and metastasis, matrix metalloproteinases (MMPs) have been shown to be

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. important in normal tissue remodeling processes that relate to female physiology-, including

follicle development ovulation, CL development and regression, and implantation (Smith et al..

1999: Curry and Osteen. 2001). However, information regarding detailed expression profiles of

these enzymes and their physiological functions, especially in species with long luteal phases, is

limited. In the present study, we reported the full length cDNA sequence of bovine MMP-2. its

expression and localization in the bovine CL. and its involvement in capillary formation. The

open reading frame of the bovine MMP-2 gene encodes a protein consisting of 661 amino acids,

showing a high degree of identity to its human, rat. mouse, rabbit, and pig homologs. This

indicates that this enzyme is highly conserved among mammals. Bovine MMP-2 possesses the

classical domain organization of gelatinases. including the three head-to-tail repeats of fibronectin

type II-like inserts. These inserts function as binding sites for gelatin and are critical for the

proteinase activity on ECM substrates such as casein, gelatin, and type IV collagen (Murphy et

al.. 1994; Shipley et al.. 1996). The tissue distribution pattern of bovine MMP-2 is not only-

similar to that observed in mouse (Gack et al.. 1994). but is also correlated to its membrane-

associated activator. MT1-MMP (Zhang et al.. 2002). Thus, in cattle, the coordinated expression

of these two MMPs in multiple tissues suggests that MMP-2 is activated similarly in a variety of

cell types.

Only a few studies have reported the activity and expression patterns of MMP-2 in CL

obtained during the estrous cycle or undergoing regression. The MMP-2 transcript and protein are

highly expressed during the early stages of rat CL development (Liu et al.. 1999), while

significant increases are observed in the late rather than the early and mid stages in the human CL

(Duncan et al.. 1998). In the present study. MMP-2 transcript and pro-MMP-2 protein were

constitutively expressed in early, mid. and late stages of the bovine CL. Furthermore, enhanced

MMP-2 expression is associated with luteoivsis in sheep (Towle et al., 2002), human (Duncan et

al.. 1998). and rat (Endo et al.. 1993). Eight hours after a systemic infusion o f the luteolysin,

PGFio, MMP-2 activity is significantly increased (Towle et al., 2002). PGF2a also induces high

- 6 8 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. production of MMP-2 by porcine luteal cells (Pitzel et al., 2000). When human chronic

gonadotropin (hCG) is given to rescue the human CL from regressing, MMP-2 expression is

reduced (Duncan et al.. 1998). Collectively, these data indicate that there are species differences

in the expression patterns of MMP-2 in the CL. Furthermore, the degradation of the ECM

mediated by MMP-2 may be important for the involution of the CL.

The temporal expression of MMP-2 follows a pattern similar to its endogenous activator.

MT1-MMP. While the latent forms of MMP-2 and MT1-MMP remain unchanged during the

bovine estrous cycle, the activities of both enzymes paralleled each other, being low in the early

stage CL. but significantly enhanced in the mid and late stages (Zhang et al.. 2002). Previously,

zymography revealed the presence of active MMP-2. which was validated by incubation with

APMA to distinguish between latent and active forms of MMP-2 (Zhang et al.. 2002). Thus, our

inability to detect the active form of MMP-2 in immunoblots in the present study was most likely

the result of using a heterologous antibody. Besides MMP-2. MT1-MMP also activates MMP-13

(collagenase 3). MMP-13 was expressed at a constant level in the bovine CL during the estrous

cycle (Zhang and Tsang: unpublished data). However, in the rat CL. which predominantly

expresses MMP-2 at the early stage. MT1-MMP is constitutively expressed in all stages

examined, while MMP-13 is highly expressed in the late stage (Liu et al.. 1999). Following

PGF;a-induced luteoiysis in sheep. MMP-2 and MMP-13 mRNA are increased by 6 hours, while

MT1-MMP mRNA is increased by 15 minutes, and remains elevated through 48 hours (Ricke et

al.. 2002). Thus, there is also species variation in the interplay between MMP-2 and MMP-13.

and their activator in vivo. MT1-MMP.

Although MMP-2 transcripts are localized in human theca-lutein cells (Duncan et al.,

1998). MMP-2 mRNA and activity has been predominantly observed in large luteal cells of a

variety of species. Rat luteinized granulosa cells express MMP-2 mRNA (Curry et al., 2001),

while immunoreactive MMP-2 is detected in luteal cells of developing rat CL (Bagavandoss,

1998). In addition. MMP-2 mRNA and activity are detected in porcine large luteal cell cultures

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Pitzel et al., 2000). In the present study, immunohistochemistry demonstrated that large

(steroidogenic) luteal cells expressed MMP-2, which is consistent with our previous observation

that MMP-2 activity is present in conditioned medium of bovine luteal cell cultures (Tsang et al.,

1995). The co-localization of MMP-2 and MT1-MMP in large luteal cells suggests that activation

of pro-MMP-2 is locally regulated. For example. MMP-2. per se. binds to cell membranes via the

integrin OvP3 (Brooks et al.. 1996). promoting localized pericellular proteolysis to accommodate

the increasing size of large luteal cells during CL development (Schwall et al.. 1986; Zheng et al..

1994). As proposed by Smith et al. (Smith et al.. 1999). the molecular regulation of the

luteinization process and the changes in the biochemical properties of luteal cells are possibly

attributed to dynamic interactions between the ECM and the cell.

Another example of cell-cell and cell-matrix interactions is angiogenesis. a hallmark of

CL development (Basset. 1943: Reynolds et al.. 2000). Angiogenesis is a multi-step process that

involves the interplay between angiogenic factors, endothelial cell migration and proliferation,

and ECM remodeling, which ultimately lead to recruitment of pericytes and formation of

capillary lumen (Moses. 1997: Klagsbrun and Moses. 1999). As a highly vascularized gland, the

CL is a rich source of angiogenic factors (Reynolds et al.. 2000). Over the course of CL

development. 85% of proliferative cells are endothelial cells (Reynolds et al.. 1994), which are

the most abundant cell type in the mid cycle CL (Lei et al.. 1991: Zheng et al.. 1993).

Extracellular proteolysis, which requires the participation of MMPs, is an integral part of

the angiogenic process (Moses. 1997). Besides the large luteal cells. MMP-2 was also localized in

endothelial cells of the bovine CL. In the rat CL MMP-2 is also detected in the same cellular

compartments (Bagavandoss. 1998). The in vivo cellular localization of MMP-2 is supported by a

recent in vitro study, showing that MMP-2 was the predominant gelatinase in CL-derived

endothelial cells (Zhang et al.. unpublished data). The present study also showed that an MMP-2

antibody inhibits capillary' tube formation by endothelial cells, an indispensable step in

angiogenesis. Taken together, these data suggest that MMP-2 activity may be necessary for

- 7 0 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. neovascularization (Hiraoka et al., 1998). In ewes, administration of a MMP-2 antibody results in

incomplete CL formation, whereby the build up of normal vasculature is blocked (Gottsch et al..

2001). In the same animal model, immunization of the N-terminal peptide of the inhibin (143-

subunit (aN) also causes incomplete CL development concurrent with reduced MMP-2 activity

(Russell et al.. 1995). In addition. MMP-2 activity is critical for angiogenic processes in other

tissues. For example, the MMP-2 gene knockout mouse shows remarkable resistance to tumor-

induced angiogenesis (Itoh et al.. 1998). while corneal angiogenesis is diminished in MMP-2-

deficient mice (Kato et al.. 2001).

MMP-2 may play multiple roles during angiogenesis. The cleavage of plasminogen

generates a 38 kDa internal fragment which is called angiostatin and has inhibitory action on

angiogenesis (O'Reilly et al.. 1999). MMP-2 also cleaves big ET-1 into an active and potent

vasoconstrictor. ET-l (Fernandez-Patron et al.. 1999). Moreover, the cleavage of calcitonin gene-

related peptide (CGRP). a potent vasodilator, by MMP-2. remarkably reduces its vasodilatory

potency (Femandez-Patron et al.. 2000). In addition to its degradation on ECM components,

therefore. MMP-2 may regulate angiogenesis and vascular function via diverse pathways.

Considering our previous observation that active MT1-MMP is correlated to MMP-2

activity (Zhang et al.. 2002). the constitutive expression of MMP-2 transcript during the estrous

cycle suggests that MMP-2 activ ity is predominantly regulated at the level of activation in the

bovine CL. In addition, expression of MMP-2 in large luteal cells may facilitate pericellular

proteolysis of the ECM during CL development. Furthermore, localization of MMP-2 in

endothelial cells and inhibition of capillary tube formation by a MMP-2 antibody indicate that

this enzyme may be critical for angiogenesis in the CL.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sequence and homology analysis of bovine MMP-2

q -191

gcacqaggcqgqctgggggccgggccacgctctgctgagccgggcaaagccgaggagaccgaataqaatagcccctrggagcgcagcgec -91

gcgcgggggageaggegccagecaggcggcqacgcggccacacgcaccgagcctgccacceccgggcgacgcgcggggcecgggagcgca -1 atgaccgaggcgcgagtgLcccggggcgcgctggccgcccttctgcgggcgctctgcgccctgggctgcctgttgggccgtgccgccgcc 90

MTEARVSRGALAALLF. AI.CALGC1.1. 3RAAA

gcgccgt cgeecatcatcaaaittcccggcgatgtcgcecccaaaacggacaaagagt tggctgtgcaatacctaaacaccttctacggc 160

APS? I I KFPGDVAFK7DKELAVQYLM7FYG tgcceeaaggagagctgtaacttgtttgtgctgaaggacaccctgaagaagatgcagaagttcttegggttaccccagacaggtgaactg 2^0

CPKESCNLFVLKDTLKKMQKFFGLPQTGEL gaceagagcaccatugagaccatgcggaagccgcgctgtggcaaccccgacgtggccaactacaacttcttcccccgaaagcccaagtgg 260

^GSTOETMRKPRCGNPDVANYNFFPRKPKW

gacaagaaccaqatcacatacaggatcattggctacacacctgacctggacceccagacagtggatgatgccttcgctcgtgcettccaa 450

SKNSITYRIIGYTPDLDPQTVDGAFARAFC

gtctggagcgatgtgactccgetacggtcttctcggatccatgatggagaggetgacatcatgateaactttggccgctgggagcatgga 54 0

VWSGV?PLRFSRIHDGEADIM:NFGRWEHG

qatgggtacccttttgatggcaaagacgqgctcetggctcatgccttcgccccgggccctggagttgggggagattcccactttgatgac 630

OGYPFDGKOJGLLAHAFAPGPGVGGDSHFDD gatgagctgcggaccctgggagaaggacaagtggtccgtgtgaagtacgggaatgctgacggggaatattgcaagttccccttccggttc ?20

0ELS7LGEGCVVRVKYGSADGEYCKFPFRF

aac^gcaagqagta eaccagctgcacaqacacaggccgcagcgarggcttcetctggtgttecaccacatacaactttgacaaggacgqc 610

N'GKEYTSCTDTGRSOGF^WCSTTYSFDKGG aagtatggctretgecercatgaagcectgttcaecatgggcggcaacgccgacggacaqccctgcaagttcccgttccgcttecaqggc 90 0

KYGFZPHEALFTMGGNADGGPCKFFFRFCo aegcctcaeqaeagctgcaccacggaqgqccgcacqgdcgqctdcegctqqtqtgqcaccaccgaqgactaccaccgcqacaaqqaqtac

TS:0SC7TEGRTrGYRWCGT7EDY7R2KEY

gqcttctgcccggagaccgccargrccactgtgggcgggaactcggaaggtgccccatgtgtcctccccttcacettectgggcaacaag 1060

■3FCPE7AMS7VGGM5EGAPCVLFF7FLGNK

racgagagccgcaccagcgctgqcegcagtgatgggaagttgtggtgtgcgaccacctccaactacgatgatgaccgcaagtggggcttc 1170

HES07SAGRSDGKLWCAT7SMYDD0P. KWGF

tgccccgaccaacggtacagcctgttcctggtggcagcccatgagtttggccatgcaatggggctggagcacccacaggaccctgqagcc 12 60

CPGQGYSLFLVAAHEFGHAMGLEHSGDPGA

etgatggegcccactratacccacaccaaqaacttccqcctgtcceatqatgacatccagggcatccaagaacccratggggcctcccct 125C

1.MAF I Y7Y7KNFRL.3KZDIQGI 3ELYGASP

gacat tgatactqgcaccggccccaccccaaccctgggccccgtcactcctgagctctgcaaacaggacatcgtcctcgacggcatrtct 1440

2:G7G7GP7?7«G?VTPELCKQD: v FDG: s

cagatccgtggggagatettccrctccaaggacccatteatetggcgaacagcgacaccacgtgacaagcccacagggcccctgctggta 1530

:: r g s : f f f k 3 r f : wr7 V 7 P R d k ? 7 G pllv

gccacatt rrggcrrgagctgccggaaaagatcgatgctgtgtargaagacccacaggaggagaaggctgtgttctttgcagggaacgaa 1620

A7FWFE1.FEKIDAVYEDPQEEKAVFFAGNE tactgggtetattcagccageaccccggaqcgagggtaccccaagccactgaccaccccggggcteccccctggtgtccaqaaggtgqac 1“10

Y W V : £ A S 7 1 E R G Y ? K ? L 7 £ L G L P r G V Q K V 2 gctgcccctaactggagcaagaacaagaagacgcacatcrregccggagacaaactctggagatacaatgaggtgaagaagaaaatggat 1600

AAFSM5KNKK7YIFAGDKFWRYMEVKKKMG cetggcctccccaagcccaccgccgatgcctggaacgccatrcctgataaccrggacgctgtggtggacctgcagggcgggggtcacagc IS 90

PGFrKLIADAWNAIPDNLDAVVDLQGGGHS cacctccrcaagggcgcctatcacctgaagttggagaaccaaagtctgaagagcgtgaagttcggaagcatcaaatccgatcggctgggc 1960

Y FFKGAYY LKLENQSLKSVKFGS IKSDWLG cgetgagctggct z cgcctceeccagggcctgccecteeatcacctgctgcacaccagggcctgagcaccagggaaggacccgggtgggc 2G~C

661 gtggcagccetcagttctgtaattaatcagcattctcacccccacctggtaatttaagaaaccctagagtggctctgccctgtgctcaag 2160 taaaactga 2163

- 7 2 -

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B Signal Pro Cat I I I ♦> vt WV> V\V> » wW v>V> v> V>

Fibrontctin type Zn~-binding site IMik* inserts (HEFGHAMGLEHS)

Species signal propeptide catalytic hinge hemopexin overall

Bovine 100 100 100 100 100 100

Human 70 98 96 86 97 94.7

Mouse 73 98 95 82 97 94.1

Rat 63 98 95 82 97 93.5

Rabbit 67 99 95 77 93 93

Pig 83 100 96 95 96 95.9

Figure 2-1. Sequence analysis of bovine MMP-2. A) Nucleotide sequence o f the bovine MMP-2 cDNA and its deduced amino acid sequence. The protein sequence is shown in uppercase letters and the DNA sequence in lowercase. While the numbers on the left are for the amino acid sequence, the ones on the right are for the cDNA sequence. B) Schematic diagram showing the domain organization of bovine MMP-2. Signal, Pro, Cat, Hinge, and Hex represent the signal peptide, propeptide, catalytic, hinge, and hemopexin/vitronectin domains, respectively. The fibronectin type II-like inserts and the Zn2* binding consensus site in the catalytic domain are also indicated. C) Multiple sequence alignment of bovine MMP-2 and its homologs from other species. The numbers in the boxes indicate the percentages of amino acid sequence identity in corresponding domains. The overall homology percentages for the entire molecule are shown on the right GenBank accession numbers of the sequences analyzed are bovine MMP-2 AAG28169, human MMP-2 A28153, mouse MMP-2 A42496, rat MMP-2 S34780, rabbit MMP-2 S70365, and pig MMP-2 AAK97133.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tissue distribution of bovine MMP-2 mRNA

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Figure 2-2. Tissue distribution of bovine MMP-2 mRNA expression. A) Northern blot analysis of MMP-2 mRNA expression in various bovine tissues, including the brain, heart, kidney, liver, lung, and the spleen. A single transcript o f approximately 3.1 kb is marked by the arrow on the left. B). Relative expression levels of MMP-2 mRNA in different bovine tissues. Densitometric intensities of MMP-2 Northern blots were determined by UN-SCAN-IT digitizing systems (Salk Scientific, Orem, UT), and are presented in percentages as compared to that in the lung, which was assigned as 100%.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 mRNA expression in the bovine CL

Early Mid- Lata Agas of CL

Figure 2-3. MMP-2 mRNA expression in luteal tissues. A). Northern blotting detected a 3.1 kb MMP-2 mRNA species in the early (E; day 4), mid- (M; day 10), and late (L; day 16) stage CL (upper panel). The same membranes were stripped and hybridized with a cyclophilin probe as a control for RNA loading (lower panel). B). Relative levels of MMP-2 mRNA are presented as ratios of the densitometric value of MMP-2 to cyclophilin. Dissimilar letters denote significance at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 protein expression in the bovine CL

Pro-MMP-2 Active MMP-2

Esrly Mid- Late Ages of CL

Figure 2-4. Representative Western blot of MMP-2 in bovine CL obtained over the estrous cycle. A) This particular MMP-2 antibody detected the pro- (-69 kDa) but not the active form of MMP- 2 in the early (E: day 4), mid (M; day 10), and late (L; day 16) stage CL. Molecular masses (kDa) of prestained protein standards (Bio-Rad Laboratories, Hercules, CA) are shown on the left. HT denotes conditioned medium of HT1080 cells. B) Densitometric analysis showed that the levels of pro-MMP-2 did not vary (p>0.05) among different stages of CL. Dissimilar letters denote significance at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 localization in the bovine CL

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i _** * B n # ’ • 1 * 3 8 Figure 2-5. Immunohistological staining of MMP-2 in the early, mid-, and late stage CL. MMP-2 protein expression was localized in endothelial (black arrows) and large luteal cells (white arrow's) o f the early (A; day 4), mid (B; day 10), and late (C; day 16) stages of CL. Endothelial cells were identified (black arrows) based on staining with Factor V K (E). No staining was observed in the negative controls (D and F, for MMP-2 and Factor VIII, respectively), in which the primary antibody was replaced by 10% BSA. Magnification is at 400x.

- 7 7

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 activity in bovine capillary endothelial cells

(kDa) 1 2 3 150 100 75 Pro-MMP-2 Active MMP-2 50

Figure 2-6. Gelatin zymographic analysis of medium conditioned by BCE cells. A major 68 and a faint 62 kDa band, which correspond to pro and active MMP-2, respectively, were observed in medium conditioned by BCE cells (lane 1). Incubation with APMA converted the 68 kDa pro- MMP-2 to its 62 kDa active form (lane 2). All zones of clearance disappeared following incubation with 1, 10-phenanthroline. The molecular masses (kDa) of Perfect Protein™ Marker (Novagen, Madison, WT) are indicated on the left.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Effects of MMP-2 antibody on in vitro capillary tube formation of BCE cells

Figure 2-7. Effects of a neutralizing antibody against MMP-2 on in vitro capillary formation of BCE cells. Phase contrast photography of capillary-like tube formation of BCE cells cultured on Matrigel under control conditions (A), or in the presence of a neutralizing anti-MMP-2 antibody (1:100; B).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III

BOVINE MEMBRANE-TYPE I MATRIX METALLOPROTEINASE (MT1-MMP):

MOLECULAR CLONING AND EXPRESSION IN THE CORPUS LUTEUM

Abstract

Matrix metalloproteinase-2 (MMP-2) is produced as a zymogen, which is subsequently,

activated by membrane-type 1 metal loproteinase (MT1-MMP). The objectives of the present

study were to clone bovine MTl-MMP and to investigate its expression in the corpus luteum

(CL). CL were harvested from nonlactating dairy cows on day 4. 10. and 16 of the estrous cycle

(day 0=estrus; n=3 for each age). The bovine MTl-MMP cDNA contained an open reading frame

of 1749 base pairs, which encoded a predicted protein of 582 amino acids. Northern blotting

revealed no differences (p>0.05) in MTl-MMP mRNA levels between any ages of CL. Western

blotting demonstrated that two species, the latent form (-63 kDa) and the active form (-60 kDa)

of MTl-MMP. were present in CL throughout the estrous cycle. Active MTl-MMP was lower

(p<0.05) in the early CL than the mid- and late stages, where MMP-2 activity’, as revealed by

gelatin zymography. was also elevated. Furthermore, immunohistochemistry revealed that MTl-

MMP was localized in endothelial, large luteal, and fibroblast cells of the CL at different stages.

Taken together, the differential expression and localization of MTl-MMP in the CL suggest that

it may have multiple functions throughout the course of the estrous cycle, including activation of

pro-MMP-2.

Introduction

The corpus luteum (CL) is a dynamic, transient endocrine gland, which develops from

the postovulatory’ follicle (Niswender and Nett, 1994). In addition to functional changes, the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. development, maintenance and regression of the CL are associated with structural events such as

tissue remodeling and angiogenesis (Smith et al.. 1999; Reynolds et al., 2000). It is believed that

this tissue remodeling process includes breakdown and resynthesis of extracellular matrix (ECM)

components (Luck and Zhao. 1995; Smith et al.. 1999). which require the participation of matrix

metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) (Hulboy et al.,

1997; Mclntush and Smith. 1998; Smith et al.. 1999). Although the literature is still emerging, a

few of the secreted MMPs and TIMPs have been investigated in CL and follicles of species with

long luteal phases, including the bovine (Juengel et al.. 1994; Tsang et al., 1995; Goldberg et al..

1996; Smith et al.. 1996; Zhang and Tsang, 2000b). ovine (Murdoch et al.. 1986; Smith et al..

1994; Smith et al.. 1995). porcine (Pitzel et al.. 2000). human (Duncan et al.. 1998), and macaque

(Chaffin and Stouffer. 1999).

Matrix metalloproteinases are secreted as latent proenzymes, which are proteolvtically

cleaved to their active forms (Birkedal-Hansen. 1995; Massova et al.. 1998). It is proposed that

some serine proteinascs. such as trypsin, neutrophil elastase and plasmin. can initiate the

extracellular activation process of pro-MMPs by a “cysteine switch" mechanism (Van Wart and

Birkedal-Hansen. 1990). Unlike other secreted MMPs. however. pro-MMP-2 is inefficiently

activated by this proteolytic activation mechanism, because its sequence is not susceptible to

cleavage by serine proteinases (Collier et al.. 1988). Instead. pro-MMP-2 activation is localized

on the ceil surface (Murphy et al.. 1992b). where membrane-type 1 matrix metalloproteinase

(MTl-MMP) has been identified as its activator in placenta and several different kinds of tumor

cells (Sato et al.. 1994; Sato et al.. 1997a).

Currently, six different membrane-type MMPs (MT1-MT6 MMP) have been identified

(Ravanti and Kahari. 2000; Vu and Werb. 2000) with MTl-MMP being the most well studied.

Similar to other non-membrane type MMPs, MTl-MMP is characterized by having a signal

peptide at the amino terminus, followed by a pro-peptide, catalytic domain, hinge region, and

hemopexin-like domain at the C terminus (Sato et al., 1994; Massova et al., 1998). In contrast to

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. secreted MMPs, MTl-MMP contains a distinct transmembrane domain, which has about 25

hydrophobic amino acid residues, enabling it to be expressed on the cell surface (Massova et al.,

1998). The human MTl-MMP nucleic acid and protein sequence was the first to be deduced

(Sato et al.. 1994). and subsequently, rat (Okada et al.. 1995a), mouse (Apte et al., 1997), and

rabbit (Sato et al.. 1997b) MTl-MMPs were cloned and sequenced.

MTl-MMP possesses a variety of functions. As a matrix-degrading enzyme, MTl-MMP

has collagenolytic and geiatinolytic activities during connective tissue remodeling (Imai et al..

1996: Ohuchi et al.. 1997). With the exception o f MT4-MMP. the most well known role o f MT-

MMPs. including MTl-MMP. is their ability to activate pro-MMP-2. a key enzyme associated

with a variety of pathological and physiological processes. Thus, it is not surprising that MTl-

MMP expression is correlated with wound healing (Ravanti and Kahari. 2000) and a variety of

disease states including tumor invasion and metastasis (Polette and Birembaut. 1998). and

rheumatoid arthritis (Yamanaka et al.. 2000). Interestingly. MTl-MMP expression is also

associated with several physiological processes, including bone resorption, implantation, and

mammary gland development (Vu and Werb. 2000). In rats and mice. MTl-MMP mRNA is

expressed during follicular development, ovulation, and formation and regression of the CL (Liu

et al.. 1998: Goto et al.. 1999: Hagglund et al.. 1999: Liu et al.. 1999).

In order to establish the presence and activity of MTl-MMP in a domestic ruminant, the

objectives of the present study were to clone and sequence the bovine specific MTl-MMP, and

determine the pattern of mRNA and protein expression in relation to MMP-2 activity in early,

mid. and late stage CL obtained throughout the estrous cycle.

Materials and Methods

1. Molecular Cloning and Sequencing of Bovine MTl-MMP cDNA

A bovine UNI-ZAP II cDNA library was constructed using RNA isolated from bovine

capillary endothelial cells of adrenal cortex with Oligo(dT) primers. The catalytic domain of

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bovine MTl-MMP cDNA was amplified from this library using PCR primers designed according

to the conserved regions of MTl-MMP of other species (Massova et al., 1998). The remaining

cDNA was amplified using primer-walking. PCR products were subsequently cloned into the

PCRTA-cloning vector (Invitrogen. Carlsbad, CA) for DNA sequencing. cDNA sequencing was

performed with PCR-based automated sequencing methods (Biopolymer Facility and DNA

Sequencing Core Facility. Children's Hospital, Boston. MA). Protein database searches were

assessed by using the BLAST service of the National Center for Biotechnology Information

(Bethesda. MD). DNA and protein sequence analyses were conducted using the Mac Vector 6.0

DNA sequence analysis software package (Kodak. Rochester, NY).

2. Animals and Tissue Collection

Corpora lutea were collected from regularly cycling, nonlactating dairy cows that were

housed at the University of New Hampshire Dairy Teaching and Research Center. Luteal tissues

were removed on days 4. 10. and 16 of the estrous cycle (day 0 = estrus: n=3 per day). For day 4

CL. the ovary was removed by coipotomy after the cow received an epidural anesthetic [2% (v/v)

mepivacaine hydrochloride: 0.01 mLAg BW: (Upjohn. Kalamazoo. MI)], and the CL was then

dissected from the ovarian stroma. The day 10 and 16 CL were removed from the ovary by

enucleation. All animal experimentation protocols were approved by the Institutional Animal

Care and Use Committee (IACUC) at the University of New Hampshire.

3. Tissue Distribution of MTl-MMP

In order to determine the tissue distribution of MTl-MMP. poly(A)+ RNA from bovine

liver, brain, kidney, lungs, heart and spleen was purified using a modified guanidinium

thiocyanate method (Chomczvnski and Sacchi. 1987) followed by poly(A)+ RNA selection with

two rounds of oligo(dT)-cellulose columns. Poiy(A)+ RNA (2.5pg) was fractionated on 1%

formaldehyde agarose gels before transfer onto nylon membranes (Schleicher & SchuelL Keene,

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. NH). A 700bp cDNA probe, corresponding to nucleotides 900 to 1600 of bovine MTl-MMP

coding sequence, was labeled with 32p-dCTP using the Rediprime™ II Random Prime Labeling

System (Amersham Pharmacia Biotech, Piscataway, NJ). The blots were then hybridized

overnight at 65°C with radiolabelled probes at a concentration of 2x 107 cpm/lOml, followed by

washing with lxSSC (150mM sodium chloride. 15mM sodium citrate, pH 7.0) at room

temperature and 0. lxSSC at 65°C.

4. Northern Blotting of Luteal Tissues

For analysis of luteal tissues, the same cDNA probe described above was labeled with

digoxigenin (DIG)-dUTP using the DIG DNA Labeling Kit (Roche Molecular Biochemicals.

Indianapolis. IN). Total RNA was isolated from luteal tissues using the guanidine thiocyanate-

phenol acid extraction protocol as described by Chomczynski and Sacchi (Chomczynski and

Sacchi. 1987). Following quantification by absorbance at 260nm. 20pg of total RNA was

fractionated on 1 % (w/v) agarose gels containing formaldehyde and transferred onto Hybond™-

N+ nylon membranes (Amersham Pharmacia Biotech. Piscataway. NJ) by capillary blotting using

20xSSC (3M sodium chloride. 300mM sodium citrate. pH 7.0). After an overnight transfer,

nucleic acids were cross-linked with a UV Crosslinker (Hoefer. San Francisco. CA).

Prehybridization was performed at 50°C in standard hybridization buffer containing 50% (v/v)

formamide. 5xSSC. 0.2% (w/v) SDS. and 2% (w/v) blocking reagent (Roche Molecular

Biochemicals. Indianapolis. IN) for 2 hours. The membranes were then hybridized in the same

buffer containing DIG-labeled MTl-MMP cDNA probe overnight at 65°C. The blots were

washed twice at room temperature in 2xSSC in 0.1% (w/v) SDS, followed by higher stringency

washes with 0.1 xSSC in 0.1% (w/v) SDS at 65°C. The membranes were then equilibrated in

washing buffer [0.1M maleic acid. 0.15M NaCL 0.3% (v/v) Tween-20, pH 7.5] for 1 minute,

followed by a 30-minute incubation in 1% (w/v) blocking solution (in washing buffer). The

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. membranes were then incubated in the same blocking solution containing an anti-DIG antibody

conjugated to alkaline phosphatase (1:5000; Roche Molecular Biochemicals, Indianapolis, IN).

After two rinses with washing buffer, the blots were incubated with CSPD (Roche Molecular

Biochemicals. Indianapolis. IN), a chemiluminescent substrate for alkaline phosphatase. Signals

were visualized after development of Kodak XAR-5 films by a Konica Medical Film Processor

(Tokyo. Japan).

S. Western Blot Analysis

Finely minced luteal tissue was homogenized in an extraction buffer [50mM Tris-HCl.

150mM NaCl. 0.02% (w/v) sodium azide. lOmM EDTA. 1% (v/v) Triton X-100. lOpg/ml

aprotinin. lpg/ml pepstatin A. Ipg/ml aminoethyl benzenesulphonyl fluoridej described by Lehti

et al. (Lehti et al.. 1998) with minimal modification (pH 7.5). A ratio o f 0.125g luteal tissue and

1.0 mi extraction buffer was maintained. Following extraction, the samples were centrifuged at

800g for 10 minutes at 4°C to enable collection of the supernatant fraction.

To assess the protein expression pattern of MTl-MMP in different ages of CL. equivalent

amounts o f tissue extracts were subjected to SDS-PAGE on 10% (w/v) polyacrylamide gels.

Fractionated proteins were then electrophoreticallv transferred to Protran nitrocellulose

membranes (Schleicher & Schuell. Keene. NH). Nonspecific binding sites were blocked with 5%

nonfat powdered dry milk in TBST buffer [0.01M Tris-HCl. 0.15M NaCl. 0.05% (v/v) Triton X-

100. pH 8.0] for 2 hours. A rabbit anti human MTl-MMP polyclonal antibody (1:500: Chemicon

International Inc. Temecula. CA). which recognizes the conserved hinge region of the bovine

molecule, was diluted in 5% nonfat dry milk in TBST. applied onto the membranes, and

incubated overnight at room temperature. The membranes were then washed five times, 15

minutes each, with TBST before a 1 hour incubation with anti-rabbit IgG conjugated to

horseradish peroxidase (1:15.000: Pierce. Rockford, IL) at room temperature. After another five

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. washes as described above, the blots were developed writh the use of SuperSignal9 West Pico

Chemiluminescent Substrate (Pierce. Rockford, IL) according to the manufacturer’s instructions.

The protein bands were finally visualized after exposure to Kodak XAR-5 film (Kodak,

Rochester. NY). Low range Prestained SDS-PAGE Standard (Bio-Rad Laboratories, Hercules,

CA) was run in adjacent lanes.

6. Gelatin Zvmographv

Gelatinolytic activity in luteal tissue samples was detected using a modified protocol that

was previously described (Goldberg et al.. 1996). Briefly, equivalent amounts of tissue extracts

were electrophoresed on 10% polyacry lamide gels impregnated with 0.5mg/ml gelatin. After

electrophoresis. SDS was removed from gels by two 15-minute washes with 2.5% (v/v) Triton X-

100. and then incubated overnight at 37°C in substrate buffer (50mM Tris-HCl. 5mM CaCL.

50mM NaCl. pH 8.0). The gels were stained with 0.1% (w/v) Coomassie Brilliant Blue R-250

(Bio-Rad Laboratories. Hercules. CA) for 30 minutes, and destained with distilled H;0.

Gelatinolytic activity was revealed as clear bands against a blue-stained background. Perfect

Protein™ Markers (Novagen. Madison. WT) and a positive control, the conditioned medium of

HT1080 cells, were run in adjacent lanes.

In order to verify' that the gelatinolytic activities detected were metalloproteinases, gels

were incubated with substrate buffer containing lOmM 1.10-phenanthroline (Sigma, St Louis.

MO), a specific MMP inhibitor and a zinc ion chelator. By interfering with the zinc-containing

active site of MMPs. zones of clearance are prevented from forming. Furthermore, in order to

distinguish between the latent and active forms of MMPs. samples and conditioned medium of

HT1080 cells were incubated with 2mM p-aminophenylmercuric acetate (APMA; Sigma, St

Louis. MO) tor 2 hours at 37°C prior to zymography. This results in the cleavage of latent MMPs

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to their "active'1 lower molecular weight forms, which may undergo further processing, i.e.,

autolvtic cleavage.

7. Immunohistochemistrv

Tissue sections (6pm) were cut onto Superffost slides (Fisher Scientific, West Chester,

PA), and air-dried at room temperature for 30 min. Sections were then fixed in cold acetone for

10 min. and washed with phosphate-buffered saline (PBS. pH 7.4). Endogenous peroxidase

activity was quenched by incubating sections with 0.3% (v/v) hydrogen peroxide for 30 min.

Nonspecific binding was blocked for 30 min by 5% (w/v) BSA. Sections were subsequently

incubated at room temperature for 1 hour with a 1:250 dilution of a polyclonal rabbit anti-MTl-

MMP (Chcmicon International Inc. Temecula. CA). After washing, slides were incubated with

biotin conjugated goat anti-rabbit IgG (1:200; Vector Laboratories. Burlingame. CA). before

visualization with the VECTASTAIN'8 Elite ABC Kit (Vector Laboratories. Burlingame. CA)

according to the manufacturer's instructions. For each tissue sample, an adjacent section placed

on the same slide was used as a negative control, with BSA substituting for the primary antibody.

After completion of the color reaction, the slides were counter-stained with Gill 2 Hematoxylin

(Shandon. Pittsburgh. PA). For each tissue (3 corpora lutea for each age). 10 to 20 sections were

stained. A minimum of 20 areas per section was examined.

8. Densitometry

Northern blots. Western blots, and zymograms were analyzed with UN-SCAN-IT gel™

automated digitizing system (Silk Scientific Inc. Orem. UT). For Northern blots, densities of

MT1-MMP and 28S rRNA were used to calculate the MTI-MMP/28S rRNA density ratio for

each sample. Similarly, for zymography. levels of latent and active MMP2. and the HT1080

positive control were used to calculate the MMP2/HT1080 ratio for each sample.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9. Statistics

Each sample was run in triplicate. Quantitative values were expressed as means ± SEM.

All data were analyzed by ANOVA. followed by Tukev's test of pairwise comparisons to

determine differences between the three age groups of CL. A value of p<0.05 was considered

significant.

Results

1. Sequence Analysis of Bovine MT1-MMP

The bovine MT1-MMP cDNA contained an open reading frame of 1749 bp (GenBank

accession number AF290429: July 27th. 2000). which encoded a protein of 582 amino acids

(Figure IA). All three characteristic regions of the MT-MMP subfamily, the 11-amino-acid

insertion (IS-I) between the propeptide and catalytic domains, the 8-amino-acid (IS-II) insertion

tn the catalytic domain, and the 75-amino-acid (IS-UI) at the C terminus, were also present at

these positions in bovine MT1-MMP (Figure IB). Alignment of the predicted protein sequences

revealed significant homology between the bovine MT1-MMP protein and other species, with rat

being the highest (95.9%). followed by human (95.7%). mouse (95.0%). and rabbit (92.3%)

(Figure 1C).

2. Tissue Distribution of MT1-MMP mRNA

Northern blotting revealed a 3.5 kb transcript in a variety of bovine tissues, with the

strongest signal in the lungs, followed by spleen, kidney, heart and liver. The signal in the brain

was undetectable (Figure 2).

3. Expression of MT1-MMP in Bovine Corpus Luteum

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A single 3.5 kb transcript was detected by Northern blotting in luteal tissues collected at

early, mid-, and late stages of the estrous cycle (Figure 3A). However, the levels of MT1-MMP

mRNA. expressed as a ratio of the MT1-MMP band density for each CL sample to its

corresponding 28S rRNA band density, were similar (p>0.05) among the three stages (Figure

3B).

MTl-MMP was studied in bovine CL by Western blot analysis. In all samples, two bands

with a relative molecular mass of -63 kDa, consistent with the latent form of MTl-MMP, and

-60 kDa. consistent with the active form of MTl-MMP. were observed (Figure 4A). The level of

active MTl-MMP protein in the d4. early CL was significantly lower (p<0.05) than mid- and late

stages (Figure 4B).

4. Expression of MTl-MMP is Correlated to Pro-MMP-2 Activation

MMP-2 activity in the bovine CL during the estrous cycle was determined by gelatin

zymography (Figure 5 A). Visual observation revealed an -68 (pro-MMP-2) and -62 kDa (active

MMP-2) species in all luteal samples. While the intensity- of the -68 kDa species did not vary, the

intensity of the -62 kDa species in the d4. early CL. appeared to be less than the other two older

stages. Incubation of samples with 1. 10-phenanthroline prevented the appearance of gelatinolytic

zones of clearance (data not shown), indicating that these clear zones on the zymograms reflected

MMP activities. Lastly, the decrease in band intensity- of the -68 kDa band after treatment of

luteal samples and conditioned medium of HT 1080 cells with APMA for two hours at 37°C

suggested that this enzyme species was the latent form of MMP-2 (Figure 5C).

Densitometric analysis revealed that the levels of the -68 kDa pro-MMP-2 were similar

(p>0.05; data not shown) among the three stages of CL. However, the level of the -62 kDa active

form of MMP-2 increased during the progression of the estrous cycle, being significantly greater

(p<0.05) in dlO and d!6 CL than in the early stage (Figure 5B).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. Cellular Distribution of MTl-MMP

Immunohistochemistry was used to investigate the cellular localization of MTl-MMP in

the bovine CL. Overall, the cellular distribution of MTl-MMP varied throughout the life span of

the CL. In the early (Figure 6A) and mid-cycle (Figure 6B) CL. MTl-MMP was localized in

endothelial ceils, and on the cell membrane and cytoplasm of large luteal cells, consistent with

cellular processing of the MTl-MMP protein in cytoplasmic organelles before becoming resident

on the cell membrane. In the late stage CL (Figure 6C). the expression of MTl-MMP was

predominantly observed in fibroblasts, which were identified based on cell shape. However,

endothelial and large luteal cells were weakly stained in the late stage CL. No staining was

observed when the primary antibody was omitted (Figure 6D).

Discussion

To date. MTl-MMP nucleotide and predicted protein sequences are known for the human

(Sato et al.. 1994). mouse (Apte et al.. 1997). rat (Okada et al.. 1995a). and rabbit (Sato et ai..

1997b). while there are no reports, to our knowledge, regarding its sequence and expression in

agriculturally important species such as domestic ruminants. In the present study, we report the

bovine-specific MTl-MMP complementary nucleotide sequence and demonstrate its expression

and localization in the bovine CL. The bovine MTl-MMP cDNA consisted of 1746 bp

nucleotides, which encode a 582 amino-acid protein. Sequence alignment indicated that the

bovine MTl-MMP was highly similar (>92%) to the rat human, mouse, and rabbit homologs. In

addition, bovine MTl-MMP shares the characteristics typical of other membrane-type

metalloproteinases. e.g.. the 11 amino acids insertion (IS-l) between the propeptide and the

catalytic domains that serves as a putative convertase recognition site, the RXKR motif (IS-II) in

the catalytic domain, and the transmembrane segment (IS-III) (Murphy et al., 1999a). These

results strongly suggest that this molecule is highly conserved among the mammals investigated

thus far. Furthermore, as in the human (Sato et al., 1994) and the rabbit (Sato et al., 1997b),

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bovine MTl-MMP had broad tissue distribution, suggesting multiple sites of action in these

species.

Since information regarding MTl-MMP and ovarian function is limited, the remainder of

the present study focused on the bovine CL. Previously. MTl-MMP transcripts have been shown

to be present in mouse ovaries (Hagglund et al., 1999) and in developing and regressing rat CL

(Goto et al.. 1999; Liu et al.. 1999). Here, we found that MTl-MMP mRNA was constitutivelv

expressed in all three stages of luteal development, however, the level of active MTl-MMP

protein was greater in the mid- and late stage bovine CL than that in the early stage. A major

function of MTl-MMP is to activate latent MMP-2. In contrast to other MMPs. the activation

process of pro-MMP-2 is unusual in that it is not through the extracellular proteolytic cleavage by

serine proteinases (Murphy et al.. 1999b). but rather the activation of pro-MMP-2 is localized on

the cell membrane (Murphy et al.. 1992b). A ternary molecular complex model is hypothesized in

which TIMP-2 binds to MTl-MMP forming a cell surface "co-receptor" for pro-MMP-2 (Butler

et al.. 1998; Zucker et al.. 1998). This cell surface bound pro-MMP-2 is subsequently activated by

the adjacent active MTl-MMP (Zucker et al.. 1998; Stetler-Stevenson. 1999). Indeed, in rats (Liu

et al.. 1998). MTl-MMP and MMP-2 mRNA are coordinately expressed and localized during

follicular development and ovulation. In the bovine CL. we demonstrated that active MMP-2

enzyme levels correlated to that of active MTl-MMP. Thus, together with our observation that

TIMP-2 was also coordinately expressed (Zhang and Tsang, 2001), we suggest that in the bovine

CL. as is the case in other tissues. MTl-MMP is an endogenous activator of pro-MMP-2.

The cellular localization of MTl-MMP in luteal tissue was also investigated. The

formation and development of the CL are associated with tissue remodeling and angiogenesis

(Smith et al.. 1999; Reynolds et al.. 2000). Angiogenesis is the formation of new capillaries from

pre-existing vessels. This complex, multi factor-regulated process requires angiogenic factors to

stimulate production of MMPs, which degrade the basement membrane surrounding endothelial

cells (Moses. 1997; Klagsbrun and Moses. 1999). This alteration o f the ECM leads to endothelial

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell proliferation and migration. As vessels extend, additional MMPs are required to break down

the ECM. accommodating the growth of sprouting vessels (Klagsbrun and Moses. 1999). MTl-

MMP might mediate these processes because angiogenesis is impaired in MTl-MMP deficient

mice (Zhou et al.. 2000). and it is highly associated with malignant tumor angiogenesis (Hiraoka

et al.. 1998). In the present study. MTl-MMP was localized in endothelial cells of the young

bovine CL. where it might directly digest ECM proteins (Imai et al.. 1996; Ohuchi et al., 1997;

Butler et al.. 1998). Indeed, co-incubation of purified MTl-MMP and collagen types I. IL and m

before SDS-PAGE (Ohuchi et al.. 1997) yielded cleavage products, while very weak gelatinolytic

activity by MTl-MMP mutants lacking a transmembrane domain was observed using

zymography (Imai et al.. 1996). This may explain the lack of a readily detectable band in

zymograms of our luteal tissue samples. Besides MTl-MMP. MMP-2 is also required for

angiogenesis (Moses. 1997). In an in vivo tumor system. MMP-2 activity is necessary for the

switch to the angiogenic phenotype during tumor development (Fang et al.. 2000). Consistent

with these findings is the fact that injection of ewes with a MMP-2 antibody results in defects of

the CL vasculature (Gottsch et al.. 2001). Together with our preliminary finding that MMP-2 is

localized in bovine luteal endothelial cells (Zhang and Tsang, 2001). MTl-MMP might mediate

the angiogenic process by activating pro-MMP-2. which in turn digests collagen type IV. a major

component of the basement membrane (Moses. 1997). Thus, following ovulation, when the

follicle is transformed to become the richly vascularized CL. MTl-MMP might have a dual role

as a proteolytic enzyme and an activator of pro-MMP-2.

As the CL develops, ongoing angiogenesis is necessary to establish and maintain the

mature vascular network (Dickson et al.. 2001). In fact, the density of the vasculature is highest in

mid-cycle cow (Zheng et al.. 1993) and mature human (Gaytan et al., 1999) CL. correlating with

the high metabolic rate, and the high rate of blood flow and progesterone production by the CL

(Bruce and Moor. 1976; Zheng et al., 1993). Progesterone either decreases (Jaggers et al., 1996;

Zhang et al.. 2000c) or increases (Chaffin and StoufFer. 1999; Luo and Liao, 2001) MMP activity

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and angiogenesis. In part this may be attributed to cell type or species differences. In the present

study', the active form of both MTl-MMP and MMP-2 enzymes increased in mid- and late stage

CL. consistent with the need for MMP production and activity for vascular maintenance.

In addition to endothelial cells, MTl-MMP was prominently expressed on the

membranes of mid-cycle large luteal cells. Accompanying the development of the luteal

vasculature, there is also a rapid increase in luteal weight and size (Zheng et al., 1994), although

this increase is primarily attributed to the proliferation of non-luteal cells such as fibroblasts and

endothelial cells (Farin et al.. 1986: Lei et al.. 1991), and small luteal cells (Zheng et al., 1994).

The sizes of large luteal cells also enlarge from the early to mid-cycle stage (Schwall et al.. 1986:

Lei et al.. 1991). To accommodate this growth. MTl-MMP might act directly or indirectly

through activation of locally produced pro-MMP-2 to commence the pericellular degradation of

the ECM (Hiraoka et al.. 1998). The activated MMP-2 may then bind to luteal cell membranes

through an integrin such as OvP3 (Brooks et al.. 1996). Thus, in large luteal cells. MTl-MMP and

its associated actions might facilitate cell-matrix and cell-cell interactions, which ultimately could

regulate steroidogenesis (Smith et al.. 1999).

At the end of estrous cycle, luteolysis ensues, whereby luteal progesterone production

(Niswender and Nett. 1994). size and weight (Niswender and Nett. 1994: Zheng et al.. 1994)

decrease dramatically. There is also apoptosis of luteal cells (Juengel et al.. 1993). In the present

study. MTl-MMP was localized in fibroblasts of the late stage, day 16 CL. At this time, these

cells also undergo proliferation (Lei et al.. 1991). This switch in cellular distribution of MTl-

MMP. when compared to the young and mid-cvcie CL. might occur in anticipation of the

functional and structural changes that are soon to follow. Indeed, fibroblast MTl-MMP might

participate in the luteolvtic process by degrading connective tissue ECM as part of the structural

demise of the CL.

Although the literature base is relatively small, there are reports of differences or

consistent trends in expression patterns of MMPs and TIMPs in species with long luteal phases.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Similar to the present study, high MMP-2 activity is observed in the late stage human CL

(Duncan et al.. 1998). and in mid and late stages of the porcine CL (Pitzel et al., 2000). When

MTl-MMP is low. MMP-9 (Goldberg et al., 1996) and TIMP-1 mRNA (Smith et al., 1996) are

increased early in the cycle, while TIMP-2 protein (Goldberg et al.. 19%; Zhang and Tsang,

2001) and mRNA (Smith et al.. 1996; Zhang and Tsang, 2001). along with MTl-MMP (present

study), are increased during the mid-luteal phase in the bovine. In the human CL, MMP-9 peaks

in early and late stages, but TIMP-1 and -2 do not change over the luteal phase (Duncan et al..

1998). In contrast TIMP-1 and -2 mRNA levels decline with age of the porcine CL. while

MMP-l and -9 are highest in the late phase (Pitzel et al.. 2000). However, in the sheep CL.

TtMP-2 mRNA in the early phase is greater than the late phase (Smith et al.. 1995). but TIMP-1

mRNA does not vary over the cycle (Smith et al.. 1994). Collectively, among the non-laboratory

mammals, a number of MMPs. including MTl-MMP in the bovine, and TIMPs. may work in

concert to regulate remodeling of the vasculature and the ECM during the life span of the CL.

In summary , the present study reports the cDNA and predicted protein sequences of

bovine MTl-MMP and the pattern of mRNA and protein expression in CL harvested over the

bovine estrous cycle. Active MTl-MMP and active MMP-2 protein levels were coordinately

increased in mid- and late stage CL. supporting the role of MTl-MMP as an activator of pro-

MMP-2 in vivo. The localization of MTl-MMP in endothelial cells, in large luteal cells, and in

late cycle fibroblasts was associated with the angiogenesis and structural remodeling that occur

over the life span of the CL. Collectively, these data indicate that MTl-MMP might act on the

ECM to modulate the local environment, which in turn may influence vascular development and

the function of luteal cells, e.g.. hormone biosynthesis, by affecting intercellular and cell-matrix

communication.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sequence and homology analysis of bovine MTl-MMP

ggcgtgggccccgccgcggagcccgcttggcccggctgcccggacagtcgcggacc 60 atgtctcccgccccacgacccgcctgcagtctgctgctccccgtgctcacgctcgcctcc 120 1 MSPAPRPACSLLLPVLTLAS gcgctcgcctccctcagctctgcccagagcagcttcagccccgaagcctggctgcagcag 180 21 ALASLSSAQSSFSPEAWLQQ catggctacctgccccctggagacctgcggacccacacacagcgctcaccccagtccctc 240 41 HGYLPPGDLRTHTQRSPQSL tcagctgccattgctgccatgcagaggttctatggtctgcgagtgacaggcaaggctgat 300 61 SAAIAAMQRFYGLRVTGKAD gcagacaccatgaaggccatgaggcgcccccgctgtggtgttccagacaagtttggggct 360 81 ADTMKAMRRPRCGVPDKFGA gaaatcaaggccaatgttcgaaggaagcgctatgccatccagggactcaaatggcagcat 420 101 EIKANVRRKRYAIQGLKWQH aatgagatcaccttctgcatccagaactacacccccaatgtgggcgagtatgccacattt 480 121 NEITFCIQNYTPNVGEYATF gaggccatccgcaaggcattccgagcgcgggagagtgcaacgccactgcgctcccgagag 540 141 E A I RKAFRVWESATPLRFRE gtaccccatgcccacacccgtgagggccacgagaagcaggccgacaccatgatcttcttc 600 161 VPYAYIREGHEKQADIMIFF gctgaaggcttccatggtgacagcacgcctttcgacggcgagggtggctccctggcccat 660 181 AEGFHGDSTPFDGEGGFLAH gcctacttcccaggccccaacaccggaggggacacccactttgactctgccgagcctcgg 720 201 AYFPGPNIGGDTHFDSAEPW actgtccggaacgaggacccaaacgggaatgacatctttctggtggctgtgcacgagccg 780 221 TVRNEDLNGNDI FLVAVHEL ggccatgccctgggccttgagcattccaacgatccctcggccatcatggcaccctttcac 840 241 GHALGLEHSNDPSAIMAPFY cagtggatggacacagagaactccgtgctgcctgatgacgaccgccggggcatccagcaa 900 261 QWMDTENFVLPDDDRRGIQQ etctatgggagtaagtcaggatcccccactaagatgccccctcaacccaggaccacctcc 960 281 LYGSKSGSPTKMPPQPRTTS aggccttccgtccccgataagcccaaaaatcccacctacgggcccaacatctgtgatggg 1020 301 RPSVPDKPKNPTYGPNICDG aactttgacaccgtggccatgcttcgaggggagatgtttgtcttcaaggagcgttggttc 1080 321 NFDTVAMLRGEMFVFKERWF tggcgcgtaaggaaaaaccaggtgatggacggataccccatgcccatcggccagttctgg 1140 341 WRVRKNQVMDGYPMPIGQFW cggggcctgcccgcatccatcaacactgcctatgagaggaaggatggcaagtttgtcttc 1200 361 RGLPASINTAYERKDGKFVF ttcaaaagagacaagcactgggtgtttgacgaagcttccctggagcctggctaccccaag 1260 381 FKGDKHWVFDEASLEPGYPK cacattaaggagctgggccgaggactgcctactgacaggattgatgctgctctcttctgg 1320 401 HIKELGRGLPTDRIDAALFW atgcccaatggaaagacetatttcttccgaggaaacaagtactaccgtttcaacgaggag 1380 421 MPNGKTYFFRGNKYYRFNEE ctcaggatagtggaaagcgaataccccaaaaacatcaaggtctgggaaggaatccctgag 1440 441 LRIVESEYPKNIKVWEGIPE tctcccagagggtcattcatgggcagtgacgaagtcttcacttacttctacaaggggaac 1500 461 SPRGSFMGSDEVFTYFYKGN aagtactggaaattcaacaaccagaagctgaaggttgagccgggctaccccaagtcagcc 1560 481 KYWKFNNQKLKVEPGYPKSA ctgcgggactggatgggctgcccatcatcggggggccagccggatgaggggactgaggag 1620 501 LRDWMGCPSSGGQPDEGTEE gagacggaggtgatcatcatcgaggtggacgaggaaggcagcggggcagtgagcgcggcc 1680 521 ETEVI I IEVDEEGSGAVSAA gccgtggtgctccccgtgctgctgctgctcctggtgctggcggtgggcctcgccgtcttc 1740 541 AVVLPVLLLLLVLAVGLAVF ctcttcagacgrcacgggacccccaagcgactgctctactgccagcgctccctgctggac 1800 561 FFRRHGTPKRLLYCQRSLLD aag gtctga 1810 581 K 'v •

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B Signal paptid* Pro-paptidn 9ovin* 1 MSPB»R»BCSLLLlVLTLR**LA*LSSBQ-SBPSPII«LggB01l»ieBLR*IT6RSlQS 5i Is 1 MSmntPSnCLLLFIXTLOTALASUiSAQasarsnaKLQQTOTLRNCZJtTBTQRaPQS 60 i NnkmmiumnsTALMumQaiRnrunggTeTLmsLMiTsurQS 60 Babbit i MBPBmnmxiLPU.niOTAtMbOMQaiiPimMLQgsaTLPPiBiKmgHPQi so Rat i M uuirasum inm uium Qtim iiM iaQTCnfKOLKnflUffli co ••••••• •••• ••• •••« •• •••••••*• •••••••••••••••••• ______Pro-poptldn ______Bovin* so T.tBBTBRMaprwuiwtawtaRnTitRMiRnirawBipafg sg n iiiim iBiogtmQ in Ioann si tiBBTBBiqgwigwaiBUMansaiiapRcowpiag M M iiaiittRiQaLiig 120 Mouan 61 LSaRXRMOCrre&avtORRD! QGLKMQ 120 Rabbit 61 imiMMOWT' QOUNQ 120 Hat 6i iiRBrBBMCpptcigvpqtRPiowaisapRcgvppiaqi QGUWQ 120 3ta®ra*a******!•« ItSa-Ste-flri'-L: . . C a t a l y t i c (XS-t) Bovin* 120 nmTTcigpTT>nvcn R»rKRiRKB»«wi«R»piRWKvwMm>ssiiKOi>PiMxr no Ioann 121 imiCTCIflPTTRKVBITRTTIRlRKRWVISBTWJtreVPyRyilBQgmORP DCF 110 Moon* Rabbit. 121 RRRiTrciqwrr gKvsRiAtrRAiRKAiRviiRaaTyiRrRrviiJHmncRrKoanxMir no Rat 121 PIIITrCiaRTrPKVgBlAiriRXRRRrRVWIBRTRWRIVlTOniBWHIRaROOqi no ••••••••••••a •••••• ••••••••••••••*•••••• '**•«• • •••»•••• ______C a t a l y t i c ______(X *- 1 1 ) ______Bovin* n o raie>iCD6TPW>qM6«Juuurrtc»inacPTi>i>«Rmftv a o puwaiPtn.vBvi» 23* ■uaan n i nKaPMDSTPVDau8ruuouryfannaaDTiyBs»sp«ivniDura»>iPtvRvn 2«o Moua* 111 rRB69M0ST»VMB06rXJUUim6»IIXQGDTI»3RB9VTVqpBBXJK»Xn.VRVn240 Rabbit n i rRiaPMP«T?yDCMOPiJuuurppQPwiaaPTi»psaBPiiTvi —omGW)irtvRviE 240 R at 111 rBSSPRCBSTVVDCiaGPIJUUUrrPSPinQaOTHVOSRSPIRVgMSOLIiaiDXPtVRVHS 240

Catalytic . _ Bing* Bovin* 240 UaUim fll WRBaniRWTgnCTIIIPVllOPDBRCXgQETtgtBCRPTKHtRQRBTT 200 Ioann 241 T<8 I RT.gLri 3SPPSRneHrt fllBOTlOTVLROODRROIQQlTqgHCrynOOfqPRTT 300 Moua* 241 UaniLClflCCMRna»fTQIBI>TmWt>PCOaROIQQLTCSK36i>TK>OPQRRTT 300 R a b b it 241 LCHALCLCTSRDPSJUMRPTTqiBPTMyLLPDOtlRCTaOLTCSQSCgPTRCIXlIPGQP 300 Rat 241 W B18 HBIII)PiaiMR>r »giM>t l llPVlJOOOW>CI001t CHtt86 PTm»QHttT 300

B in g * Hnaop*vin Bovin* 300 ■uaan 301 Moua* 301 SRMVVeiCVRMPRysyRlCMHfMVMSiRBBIWVKBMVVRVMBQVICCyVMfXOQP 360 Rabbit 301 agLLrRispgRPTTOPKiaxaciPTVRvrRanu’vrRRiBifiiRVRiaiQVicqiPMPiaQL 360 Rat 301 «PSVR)KPniFTT6PNXC0flWVDfVMILRSBIWn(BMBIiatViaaiQVICeYmPX0QP 360 Hnaop*xin Bovin* 360 419 I uaan 361 IbgLRAJXIITATRRRDCRrViniBDtlWVIDRBSUFU im i RBLBRgT.rrPKiaJUar 420 Moua* 361 Rabbit 361 HGLPaamTATlRKDCK3VTmg)KIBVrP«aallPOTPiaiI KILgRCLPTOKXDAaU 420 Rat 361 BR61>»IXllTAt«RIDttRfVPH60IHIIV»OlBSlJIPCXHaHKBlCR61HPKX0»Rir 420 B«*op*xin Bovin* 420 RMF»mXXir BQnCXTRr)l«BlRIVlSITTKinXVWBCIFB3FR6CT»ICSP»VTTTrTKS479 loaan 421 WHUILl t»PBBWCHRWI« ll«RVPSITPiaiXrV6HBIPRai»MHPIVITTrTKS 410 Mou*« 421 1 P»ORITFFaa«ITtBTIBR>RRVP81 TTI0»XKW«aiFli rBfl4WieroiVTTYrTXa 410 Rabbit 421 ■Q»BO«TTrrR

Bovin* 410 ■naan 411 Mona* 491 Rabbit 491 Rat 491 • itiininttiiM liM tltiiiiiK A ; Tranaaaahran* dcaain (X9-XIX) Bovin* S 40 (loot) 592 Rnaan 540 (95.71 ) 592 Moua* 540 (951) 592 Rabbit 540 (92. 3*) 592 Rat 540 BKVVKPVBUXHrian (95. 9) 592

(XS-XIXI

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c

Species Signal Pro Cat Hinge Hex TM Overall

Bovine 100 100 100 100 100 100 100

Human 75 96 98 92 98 98 95.7

Mouse 72 91 98 97 97 97 95

Rabbit 72 97 96 50 96 96 92.3

Rat 69 95 98 97 97 97 95.9

Figure 3-1. Sequence and homology analysis of bovine MTl-MMP. A) Nucleotide sequence of bovine MTl-MMP cDNA and its deduced amino acid sequence. The protein sequence is shown in uppercase letters and the DNA sequence in lowercase. While the numbers on the left are for the amino acid sequence, the ones on the right are for the cDNA sequence. B) Alignment of predicted MTl-MMP amino acid sequences of bovine, human, mouse, rat, and rabbit. The asterisks indicate the amino acid residues that are conserved among the five MTl-MMPs. The consensus sequences of the convertase recognition site (IS-I), the catalytic domain insertion (IS-II), and transmembrane domain (IS-III) are shaded. The signal peptide, propeptide, catalytic, hinge, hemopexin-like, and transmembrane domains are also indicated. There was a 95.7%, 95%, 92.3%, and 95.9% homology of bovine MTl-MMP to human, mouse, rabbit, and rat MTl-MMP, respectively. C) Homology analysis of bovine MTl-MMP domains with other species. The signal peptide (Signal), propeptide (Pro), catalytic (Cat), hinge (Hinge), hemopexin (Hex), and transmembrane (TM) domains are indicated. Species names appear on the left. The whole molecule (Overall) and individual domains of MTl-MMPs are compared. The numbers represent percentages. The identity scores are determined by setting the bovine MTl-MMP as 100 percent.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MTl-MMP mRNA expression in bovine tissues

e t e£ u 00 n V e 3 2 9i > s "EL CD s J (A

28S —

18S —

Figure 3-2. Tissue distribution of bovine MTl-MMP mRNA. For Northern analysis, mRNA was extracted from bovine brain, heart, kidney, liver, lung, and spleen before probing with bovine MTl-MMP. The positions of 28S and 18S rRNA are indicated on the left. A single 3. kb transcript was detected (arrow).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MTl-MMP mRNA expression in the bovine CL

M

, ;V ‘ -A^1, •• V* '-i T. p l S s 28S rRNA — ‘ ^ >' Vr - - '< ■

18S rRNA — STTWa?-'.-

28S rRNA

B

| 0.4 1 0.3

Early Mid Late

Figure 3-3. Expression of MTl-MMP mRNA in different ages of bovine CL. A) The upper panel shows that a single 3.5 kb MTl-MMP transcript (arrow) was detected in early (E; day 4), mid- (M: day 10), and late (L; day 16) cycle CL. Corresponding positions of 28S and 18S rRNA are indicated on the left. The lower panel shows ethidiutn bromide staining of 28S rRNA. B) The levels of MTl-MMP mRNA, expressed as the densitometric ratio of MTl-MMP to 28S rRNA, were not different (p>0.05) among any age of CL. Dissimilar letters denote significant difference at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MTl-MMP protein expression in the bovine CL

M

^ Latent MT1 -MMP ■M i Jffig — +------Active MTl-MMP

51.8

B 25 I b b A 20

I 15

h 10 BS

l.iiEarly Mid Late

Figure 3-4. Expression of MTl-MMP protein in different ages of bovine CL. A) In early (E; day 4). mid (M; day 10), and late (L; day 16) cycle CL, the -63 kDa latent and the -60 kDa active MTl-MMP species were observed. B) Densitometric analysis (average total pixelsxlO5) showed that the active form of MTl-MMP in early (day 4) CL was significantly lower (p<0.05) than in mid (day 10) and late (day 16) CL. Dissimilar letters denote significant difference at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 activity in the bovine CL

MW HT E M L

75 kDa Pro-MMP-2 Active MMP-2 50 kDa

35 kDa

B

•c 2 0.6

Early Mid Late

1 2 3 4 5 6 7 8 9

75 kDa Pro-MMP-2 Active MMP-2 50 kDa 35 kDa

20 kDa

Samples: MW HT HT E E M M L L

APMA: -

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3-5. Gelatin zymographic analysis of MMP-2 activity in bovine CL. A) Perfect Protein™ Markers (Novagen, Madison, WI) were loaded in lane 1 (MW). The positive control, PMA (Phorbol 12-myristate 13-acetate)-stimulated HT1080 conditioned medium (HT), was loaded in lane 2. Representative early (E; day 4), mid (M; day 10), and late (L; day 16) luteal samples were loaded in lanes 3 to 5, respectively. The arrows on the right indicate the pro- and active forms of MMP-2 in the positive control and CL samples. The relative molecular masses of protein markers are indicated on the left. B) The active form of MMP-2, expressed as the densitometric ratio of CL samples to HT 1080 conditioned medium, was significantly lower (p<0.05) in early (day 4) cycle CL than in mid (day 10) and late (day 16) stages. Dissimilar letters denote significant difference at p<0.05. C) Treatment with APMA. Samples of HT1080 conditioned medium (HT) and representative early (E; day 4), mid (M; day 10), and late (L; day 16) CL extracts were incubated at 37°C with (+) or without (-) 2mM APMA before zymographic analysis. Perfect Protein™ Markers were loaded in lane I. The numbers on the left indicate relative molecular mass (kDa). Arrows indicate the pro- and active forms of MMP-2.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cellular localization of MTl-MMP in the bovine CL

W 0 m m * r m s t . % * • 9 jfo* t36 • **V«iP»3fr £*-.* fE&J* * ? ' t n ^ SMwsp*

j . V . ».t

Figure 3-6. MTl-MMP immunohistochemistry in the bovine CL. A) In the early (day 4) cycle CL, positive staining is observed in endothelial (black arrows), and the cell membrane and cytoplasm of large luteal (white arrows) cells (x400). B) In the mid (day 10) cycle CL, the staining is localized on large luteal cell plasma membranes and cytoplasm (white arrows), and endothelial cells (black arrows; x400). C) In the late (day 16) stage CL, there was predominant staining in fibroblasts (white arrowheads; x400), and weak staining in endothelial (black arrows) and large luteal (white arrows) cells. D) Negative control of a representative section of mid (day 10) cycle CL. No positive staining was observed (x400).

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER IV

TEMPORAL AND SPATIAL EXPRESSION OF TISSUE INHIBITORS OF

MET AL LOPROTEINASES I AND 2 (TIMP-l AND -2) IN THE

BOVINE CORPUS LUTEUM

Abstract

The matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue

inhibitors of metalloproteinases (TIMPs), may mediate the dramatic structural and

functional changes in the corpus luteum (CL) over the course of its life span. Besides

regulating MMP activity, TIMPs are involved with a variety of cellular processes,

including cell proliferation and steroidogenesis. In order to gain a greater understanding

of the role of TIMPs in luteal function, we investigated the temporal and spatial

expression of TIMP-1 and TIMP-2 in the bovine CL. Luteal tissues were collected from

early (4 days old, estrus=day 0), mid (10-11 days old) and late (16 days old) phases (n=3

for each phase) of the estrous cycle. Northern blotting revealed that the TIMP-1 transcript

(0.9kb) was expressed at a higher (p<0.05) level in early and mid cycle CL than in the

late stage. On the other hand, two TIMP-2 mRNA species, one major I kb species and

one minor 3.5kb species, were significantly increased (p<0.0S) in the mid and late cycle

CL than in the early. Reverse zymography demonstrated four metalloproteinase

inhibitory protein bands with relative molecular masses that are consistent with these

reported for TIMP-1 to -4. TIMP-1 was the major species in the bovine CL. Western

blotting showed no differences in TIMP-1 (29 kDa) protein levels between early and mid

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phases, while its levels decreased (p<0.05) from the mid to late stage CL. Conversely,

TIMP-2 (22 kDa) protein was low in the early CL, but significantly increased (p<0.05) in

the mid and late stages. Immunohistochemistry revealed that both TIMP-1 and -2 were

localized in large luteal cells from all three ages of CL. Furthermore, TIMP-1 was also

localized in vascular smooth muscle cells, but TIMP-2 was restricted to the endothelial

cells in the capillary compartment. Overall, these results suggested that the variations in

TIMP-l and -2 expression may suggest diverse roles for these multifunctional MMP

inhibitors in the physiology of the CL during the estrous cycle.

Introduction

The corpus luteum (CL) is a transient, dynamic endocrine gland, which develops from

the postovulatory follicle (Niswender and Nett. 1994). Dramatic structural and functional changes

are associated with the development, maintenance and regression of the CL (Niswender and Nett

1994). These remodeling events require the participation of matrix metalloproteinases (MMPs). a

large family of zinc and calcium dependent proteolytic enzymes that collectively digest all known

macromolecules constituting the extracellular matrix (Matrisian. 1990: Massova et al.. 1998).

Generally, the catalytic activity of the MMPs are highly regulated at three levels, gene

expression, proteolytic activation of proenzymes, and inhibition by binding of endogenous tissue

inhibitors of metalloproteinases (TIMPs) to the catalytic domain (Matrisian, 1990: Woessner and

Nagase. 2000).

So far. four TIMPs. including TIMP-1 (Carmichael et al.. 1986), TIMP-2 (Stetler-

Stevenson et al.. 1989). TIMP-3 (Pavloff et al.. 1992). and TTMP-4 (Greene et al., 1996), have

been identified. TIMP-1. a glycoprotein with a molecular mass of approximately 29kDa, is the

first member of this family to be cloned (Carmichael et al.. 1986). TIMP-1 can bind the active

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. forms of all known MMPs (Woessner and Nagase, 2000). In addition, TTMP-1 also binds the

latent form of MMP-9 (Itoh et al., 1995). Besides its capacity to inactivate the activity of MMPs,

TIMP-1 also possesses mitogenic activity to a variety of cell types, such as gingival fibroblasts

and erythroid precursor cells (Docherty et al.. 1985: Hayakawa et al.. 1992). In addition, TIMP-l

may regulate steroidogenesis (Boujrad et al.. 1995). In CL from a variety of species, protein and

messenger RNA expression of TIMP-l have been observed, including cow (Freudenstein et al..

1990; Goldberg et al.. 1996). sheep (Smith et al.. 1994), rat (Nothnick et al.. 1995), mouse

(Indcrdeo et al.. 1996). monkey (Duncan et al.. 1996a). and human (Duncan et al.. 1996b).

Different from TIMP-1. TIMP-2 is an unglvcosylated protein with an approximate

molecular mass o f 22 kDa (Stetler-Stevenson et al.. 1989: Goldberg et al.. 1989). TIMP-2 binds

most active MMPs and inhibits their proteolytic activity' (Woessner and Nagase. 2000). Among

all members of the MMP family. TIMP-2 preferentially binds to MMP-2 (Goldberg et al.. 1989;

Olson et al.. 1997). Paradoxically, however. TIMP-2 is also involved in pro-MMP-2 activation by

forming a MTl-MMP/TIMP-2/pro-MMP-2 tri-molecular complex on the cell membrane

(Deryugina et al.. 1997; Cao et al.. 1998; Butler et al.. 1998). Similar to TIMP-1. TIMP-2

possesses potent stimulatory activity on proliferation of a variety of ceil types (Hayakawa et al..

1994). TIMP-2 mRNA expression was observed in the sheep (Smith et al.. 1995). cow (Smith et

al.. 1996). human (Duncan et al.. 1998). rat (Simpson et al.. 2001). and mouse (Inderdo et al..

1996) CL. while a TIMP-2-like protein was detected in the cow CL (Goldberg et al., 1996).

Because the variety and intensity of remodeling events that occur over the life span of the

CL suggest a temporal and spatial interplay between MMPs and TIMPs. we presently

investigated the expression patterns and cellular distribution of TIMP-1 and TIMP-2 in the early,

mid. and late developmental stages of bovine CL obtained over the estrous cycle.

Materials and Methods

1. Animal Model and Tissue Collection

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Corpora lutea were collected from cyclic, nonlactating dairy cows, which were housed at

the University of New Hampshire Dairy Teaching and Research Center. CL were removed on day

4. 10. and 16 of the estrous cycle (day 0 = estnis; n=3 per day). For day 4 CL, the ovary was

removed by an ecraseur after the cow received an epidural anesthetic [2% (w/v) mepivacaine

hydrochloride: 0.01 mL/kg BW: Upjohn, Kalamazoo. MI], and the CL was then dissected from

ovarian stroma. The day 10 and 16 CL were enucleated from the ovarian stroma. All animal

experimentation protocols in the present study were approved by the Institutional Animal Care

and Use Committee (IACUC) at the University of New Hampshire.

2. Northern Blotting

TIMP-l and TIMP-2 mRNA expression in the CL were studied by Northern blotting as

previously described (Zhang et al.. 2002). Briefly, total RNA was extracted from luteal tissues

using TRIZOL (GIBCO-BRL. Carlsbad. CA) according to the manufacturer's instructions.

Twenty micrograms of total RNA were fractionated on 1.0% (w/v) agarose gels containing

formaldehyde and were transferred onto Hybond™-N~ nylon membranes (Amersham Pharmacia

Biotech. Piscataway. NJ). Membranes were first incubated in pre-hybridization buffer [50% (v/v)

formamide. 5xSSC. 0.2% (w/v) SDS. and 2% (w/v) blocking reagent| for two hours at 42°C.

Hybridization was carried out in hybridization buffer containing TIMP-1 (Waterhouse et al..

1990). TIMP-2 (Stetler-Stevenson et al.. 1989). or cvclophilin (a generous gift from Dr. Robert

Thompson. University of Michigan) cDNA probes at 50°C overnight. These cDNA probes were

labeled with digoxigenin (DIG)-dUTP using the DIG DNA random-primed Labeling Kit (Roche

Molecular Biochemicals. Indianapolis. IN). The blots were then washed twice at room

temperature in 2xSSC with 0.1% (w/v) SDS. followed by higher stringency washes in 0.2xSSC

with 0.1% SDS at 65°C. Hybridized probes were detected using an anti-DIG antibody

conjugated to alkaline phosphatase (used at 1:5000: Roche Molecular Biochemicals, Indianapolis,

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IN) in L% (w/v) blocking reagent. The signals were detected by CSPD (Roche Molecular

Biochemicals, Indianapolis. IN), a chemiluminescent substrate for alkaline phosphatase. The blots

were visualized by developing Kodak XAR-5 films with a Konica Medical Film Processor

(Tokyo. Japan).

3. Reverse Zvmoeraphv

In order to simultaneously identify- total TIMP proteins in the bovine CL. reverse

zymographic analysis was performed. Equivalent aliquots (15pl) of CL tissue extracts were

mixed with loading buffer [2% (w/v) SDS. 10% (v/v) glycerol. 0.1% (w/v) bromophenol blue.

50mM Tris-HCl. pH6.8) and applied to 12% (w/v) polyacrylamide gels containing 0.1% (w/v)

SDS and 0.5 mg/ml gelatin. After electrophoresis, gels were rinsed twice with 2.5% (v/v) Triton

X-100. followed by incubation with conditioned medium of fibrosarcoma HT1080 cells for 4

hours. After rinsing, gels were incubated overnight at 37°C in substrate buffer (50mM Tris-HCl.

0.2M NaCl. 5mM CaCU. pH8.0). Subsequently, gels were stained for 30 minutes with 0.1% (w/v)

Coomassie Blue G-250. and destained with a solution of 30% (v/v) methanol and 10% (v/v)

glacial acetic acid. TTMPs were visualized as blue bands on a clear background. Prestained SDS-

PAGE standards (Bio-Rad Laboratories. Hercules. CA) were run along side samples as molecular

weight references.

4. Western Blotting Analysis

To investigate the protein expression of TIMP-1 and TIMP-2 in different ages of CL.

equivalent amounts of tissue extracts were subjected to SDS gel electrophoresis on 12% (w/v)

polyacrylamide gels under reducing conditions before transfer onto nitrocellulose membranes

(Schleicher & SchuelL Keene. NH) Prestained SDS-PAGE standards (Bio-Rad Laboratories,

Hercules. CA) were also loaded on the gel in an adjacent lane. Nonspecific binding sites were

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. blocked with 5% (w/v) nonfat powdered milk in TBST [0.01M Tris-HCl, 0.15M NaCl, and

0.05% (v/v) Tween-20. pH8.0] for 2 hours. A mouse anti human TIMP-1 monoclonal antibody

(1:100; Oncogene Research Products, Cambridge, MA) in 5% nonfat powdered milk in TBST

was added onto the membrane, and allowed to incubate overnight at room temperature. The

membranes were then washed five times with TBST (each time for 15 minutes), and then

incubated for 1 hour with goat anti-mouse IgG conjugated to horseradish peroxidase (1:15.000.

Pierce. Rockford. IL) at room temperature. After five. 15-minute washes, the blots were

developed using the SuperSignal® West Pico Chemiluminescent Substrate (Pierce. IL).

according to the manufacturer's instructions. The protein bands were finally visualized after

development of Kodak XAR film (Konica Medical Film Processor. Tokyo. Japan).

S. Immunohistochemistrv

Frozen tissue sections (6pm) were cut onto Superfrost*/Plus slides (Fisher Scientific.

Pittsburg. PA), and air-dried at room temperature for 30 min. Sections were fixed in cold acetone

for 10 min. and then washed with phosphate-buffered saline (PBS. pH7.4). Endogenous

peroxidase activity was quenched by incubating with 0.3% (v/v) hydrogen peroxide for 30 min.

Nonspecific binding was blocked for 30 min by 5% (w/v) BSA. Sections were subsequently

incubated at room temperature for 1 hour with a 1:40 dilution of mouse anti human TIMP-1

monoclonal antibody (Oncogene Research Products. Cambridge. CA) or with a 1:50 dilution of

mouse anti human TIMP-2 monoclonal antibody (Oncogene Research Products, Cambridge, CA).

After washing, slides were incubated with goat anti-mouse IgG followed by VECTASTAIN*

Elite ABC Kit (Vector Laboratories. CA), according to the manufacturer's instructions.

To further validate the localization of TIMP-1 in vascular smooth muscle cells (VSMC),

consecutive sections were also stained with an antibody against a-actm. a cellular marker for

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VSMC. For every tissue, an adjacent section placed on the same slide was used as a negative

control, where BSA substituted for the primary antibody.

For each tissue (3 corpora lutea for each age). 10 to 20 sections were stained. A minimum

of 20 areas per section was examined.

6. Data analysis

Intensities of Northern and Western blots were determined by UN-SCAN-IT gel™

automated digitizing system (Silk Scientific. Orem. UT). The data were analyzed by ANOVA.

followed by Tukey's test for multiple comparisons.

Results

1. TIMP-1 and TIMP-2 mRNA Expression in the Bovine CL during the Estrous Cvde

Northern blotting demonstrated that a single TTMP-1 transcript (0.9kb) was present in all

three stages of CL (Figure 4-1 A). The 18S rRNA was used to normalize sample loading. The

dcnsitometric ratio of TIMP-1 mRNA to 18S rRNA was high in the early and mid- cycle CL. but

decreased significantly (p<0.05) in the late stage (Figure 4-IB).

Two TIMP-2 mRNA species, a major one at lkb and a minor one at 3.5 kb. were present

in all ages of CL (Figure 4-2A). In contrast to the expression pattern of TIMP-1 mRNA. both

species of TIMP-2 mRNA. shown as the densitometric ratio of TIMP-2 mRNA to a house­

keeping gene cyclophilin. were low in the early stage, but increased significantly (p<0.05) in the

mid and late cycle CL (Figure 4-2B).

2. Mctalloproteinase Inhibitor Activities in the Bovine CL

Reverse zymography revealed four protein bands in luteal tissues. These four protein

bands had relative molecular masses of ~30kDa. ~27kDa. - 24kDa, and ~22kDa. corresponding

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. those reported for TIMP-1. -3, -4. and -2, respectively (Figure 4-3). TIMP-1 was the predominant

TIMP present in the bovine CL.

3. TIMP-1 and TIMP-2 Protein Levels in the Bovine CL

Western blotting revealed an approximately 30kDa immunoreactive TIMP-1 protein in

all ages of CL examined (Figure 4-4A). The level of TIMP-1 protein was not different between

the early and mid-cycle CL (p=0.324). but it was significantly decreased (p<0.05) in the late stage

CL (Figure 4-4B). A 22 IcDa TIMP-2 immunoreactive protein was also observed in all three ages

of CL (Figure 4-5 A). The TIMP-2 protein level in the mid and late cycle CL was significantly

greater (p<0.05) than in the early stage (Figure 4-5B).

4. Cellular Localization of TIMP-1 and TIMP-2 Proteins in the Bovine CL

Immunohistochemistry demonstrated that TIMP-1 was present in both large luteal cells

and vascular smooth muscle cells (Figure 4-6 B, D. and F). The localization of TIMP-1 in VSMC

compartments was validated with staining for a-actin. a cellular marker for VSMC. Although

large luteal cells from all three stages of CL expressed TIMP-1. visual observations revealed that

the highest level of expression was in cells of the mid-cycle CL. TIMP-1 was expressed in

vascular smooth muscle ceils of the early and late, but not the mid cycle CL (Figure 4-6 B. D and

F). Vascular smooth muscle cells were identified by staining with a-actin (Figure 4-6 G). In all

ages of CL. TTMP-2 was consistently localized in the endothelial and large luteal ceils (Figure 4-6

A. C and E). No positive signals were observed in sections devoid of primary antibody (Figure 4-

6 H).

Discussion

Accumulating evidence supports the notion that matrix metalloproteinases (MMPs) play

important roles during CL development and demise. Once activated, these enzymes are strictly

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulated by a group of endogenous inhibitors, the TIMPs. Furthermore, the two most studied

members in this TIMP family. TIMP-1 and TIMP-2. possesses a variety of other functions.

Therefore, it is necessary to determine the temporal and spatial expression patterns of these two

inhibitors before their physiological roles in the CL can be elucidated.

In the present study. TIMP-1 mRNA was highly expressed in the early and mid cycle

bovine CL. but was decreased in the late stage. This finding was similar to previous observations

by others (Freudenstein et al.. 1990; Smith et al.. 1996). Likewise, in the porcine CL. TIMP-1

transcript was also highly expressed in the early stage, and is slightly reduced as the estrous cycle

progresses before it is significantly decreased in the regressing stage (Pitzel et al., 2000). In

contrast. TIMP-1 mRNA expression in the ovine (Smith et al.. 1994) and human (Duncan et al..

1998) CL does not change throughout the estrous or menstrual cycle, respectively. In the

pseudopregnant rat. yet a different pattern emerges, whereby the strongest TIMP-1 mRNA

expression is observed during CL formation and regression (Liu et al.. 1999). Clearly, there are

species differences with regard to the temporal expression of the TIMP-1 transcript in the CL.

Despite these differences, we chose to pursue our studies in cows. In particular, as a relevant

animal model for humans, cows have a relatively long cy cle length, during which a single ovum

is shed. Also, a single CL provides sufficient tissue for multiple analyses.

The expression of a TIMP-1 specific protein in three ages of CL was also demonstrated

in the present study. This is consistent with our previous study that showed the presence of a

TIMP-l-like protein in CL obtained over the estrous cycle (Goldberg et al.. 1996). Furthermore,

reverse zymographv showed that TIMP-1 was the predominant TIMP in the bovine CL, similar to

the finding in sheep (Smith et al.. 1993). Lastly, the patterns of TIMP-1 protein and mRNA

paralleled each other, being high in the early and mid stages, but decreased in the late cycle CL.

The high levels of TIMP-1 in the early and mid cycle CL may be needed to regulate the extensive

tissue remodeling events that occur during CL formation and development. The reduced TIMP-1

mRNA and protein expression in the late stage CL may portend the decline of this inhibitor

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. observed during luteolysis in bovine (Juengel et al., 1994), ovine (Towle et al., 2002), porcine

(Pitzel et al., 2000). and primate (Duncan et al.. 1996a) CL. Collectively, these data implicate

TIMP-1 might be an important player in the physiology of the CL.

The TIMP-1 protein was localized in large luteal cells of early, mid, and late stages of the

bovine CL. A similar observation was reported in the ovine CL. where TIMP-1 and oxytocin

were co-localized in secretory granules of large luteal cells (Mclntush et al., 1996). In addition.

TIMP-1 expression was also detected in isolated ovine (Smith et al., 1994) and porcine (Pitzel et

al.. 2000) large luteal cells, and luteinized human granulosa cells (Aston et al.. 1996). The

presence of TIMP-1 in steroidogenic cells may be associated with its ability to enhance steroid

production (Boujrad et al.. 1995). In part, this may be related to a 124 bp-nucleotide sequence

similarity between the protein coding region of the bovine steroidogenic acute regulatory (StAR)

gene and the 5' non-coding region of TIMP-1 (Hartung et al.. 1995). Additionally, although

female mice lacking the TIMP-1 gene do not show detectable differences in serum estradiol- 17p

concentrations, the TIMP-1 deficient male mice produce higher levels of total serum testosterone

than the wild type (Nothnick et al.. 1997: Nothnick et al.. 1998). Furthermore, cell culture studies

demonstrated that TIMP-1 increases estradiol-17p production by granulosa cells from both

TIMP-1 deficient and wild-type mice (Nothnick et al.. 1997). and enhances estradiol- I7p and

progesterone production from porcine thecal cells (Shores and Hunter, 2000). Therefore, the

strong expression of TTMP-1 in large luteal cells may be related to its collateral role in

steroidogenesis.

Because of the predominant expression of TIMP-1 in large luteal cells, this inhibitor is

used as a cellular marker for this cell type (Haworth et al.. 1998). However, other cell types in

the CL are also positive for TIMP-1 expression. For example, the present study showed that

TIMP-1 was also localized in the vascular smooth muscle cells (VSMC) of the bovine CL.

Although the cellular source was not specified, the capillary compartments in ovine (Mclntush et

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. al.. 1996) and rat (Bagavandoss, 1998) CL also stain positively for TIMP-1. The localization of

TIMP-1 in the vascular smooth muscle compartment supports its potential role during

angiogenesis and vascular maintenance. Indeed, over-expression of TIMP-1 in VSMC by

exogenous gene transfer reduces VSMC cell proliferation and migration (Forough et al., 1996;

George et al.. 1998). In addition, stimulation of TIMP-1 expression impairs angiogenesis in a

variety of tumor types (Martin et al.. 1999; Guedez et al.. 2001; Bloomston et al., 2002).

Therefore. TIMP-1 acts as a negative regulator of blood vessel formation (Moses et al.. 1990).

During early CL development, extensive angiogenesis occurs, and theca-derived luteal cells and

fibroblasts invade through the breached basement membrane into the cavity of the ruptured

follicle. These early events in the angiogenic process require net proteolytic/MMP activity. In the

last step of angiogenesis. recruitment and maintenance of pericytes (VSMC) are critical for

vascular maturation and survival (Nehls et al.. 1992; Hirschi and D'Amore. 1996). Thus, the high

level of TIMP-1 expression in VSMC in 4-day old CL may provide an environment where MMP

action is inhibited as the vasculature matures. As the CL ages, its structure remains relatively

stable. Although angiogenesis is ongoing, it is slowed-down during CL maintenance (Tsukada et

al.. 1996; Amselgruber et al.. 1999; Dickson et al.. 2001). This may be the reason for the absence

of TIMP-1 in the VSMC compartment at this stage of the bovine estrous cycle. In the 16-day old

CL. TIMP-1 was detected again in the VSMC compartment. This localized expression of TIMP-1

is consistent with the view of Redmer et al. (1988). who proposed that maintenance of the

v asculature may be necessary for the transport of degraded products during luteoiysis, which

ultimately results in a massive decrease in CL size and weight as regression ensues.

Although TIMP-1 was the most abundant TIMP in luteal tissues, the other three TIMPs

were also detected in the bovine CL by reverse zymography. Among them, we chose to focus our

attention on TIMP-2. It has been reported that TIMP-2 is involved in the pro-MMP-2 activation

process by binding a MT1-MMP to form a "co-receptor” for pro-MMP-2 on the cell surface

(Butler et al.. 1998). This bound pro-MMP-2 is then presented to an adjacent MTl-MMP for

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activation (Deryugina et al.. 1997; Butler et al., 1998). Although a transmembrane-deleted M Tl-

MMP activates pro-MMP-2 without the participation of TIMP-2 (Pei and Weiss, 1996), TIMP-2

deficient mice have a dramatically reduced ability to activate pro-MMP-2 (Wang et al.. 2000). In

the present study, both TIMP-2 mRNA and protein levels were low in the early CL, but

significantly increased in the mid and late stages. This expression pattern was similar to those for

active MT1-MMP and MMP-2 (Zhang et al.. 2002), suggesting that the MT1 -MMP/TIMP-2/pro-

MMP-2 tri-molecular system may be available for MMP-2 activation in vivo in the bovine CL.

The coordinate expression of these three molecules is also observed during embryo development

(Apte et al.. 1997). which supports the presence of this activation system in a variety of tissue

types.

In addition to the temporal correlation of TIMP-2 with active MT1-MMP and MMP-2

expression in the CL. TIMP-2 was also co-localized with these two molecules in endothelial and

large luteal cells. TIMP-2 has a variety of functions in endothelial cells. On the one hand, its

stoichiometrically correlated expression with MT1-MMP and pro-MMP-2 may facilitate the

activation process of pro-MMP-2. which is critical for the angiogenesis process. On the other

hand, inhibition of angiogenesis by TIMP-2 cannot be excluded, because over-expression of

TIMP-2 blocks vascular smooth muscle cell invasiveness (Cheng et al.. 1998) and reduces

angiogenic ability (Li et al.. 2001). The latter observation is due. in part to down-regulation of

vascular endothelial cell growth factor (VEGF) (Hajitou et al.. 2001). Furthermore, TIMP-2 also

possesses growth inhibitory ability on endothelial cells (Murphy et al.. 1993a).

The localization of TIMP-2 in large luteal cells may also contribute to the activation of

pro-MMP-2 in this cell type by assembling the trimeric complex on the cell membrane. The in

situ activated MMP-2 then binds onto integrin ou.p3, where localized pericellular proteolysis

ensues. This resulting degradation of the ECM is needed to accommodate the enlargement of

large luteal cells from the early to mid and late stages (Schwall et al., 1988; Zheng et al., 1994).

Although there is no direct evidence showing the involvement of TIMP-2 in steroidogenesis, the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dynamic interactions between large luteal cells and their local ECM niche may induce

biochemical changes related to the steroid biosynthetic process in this cell type (Smith et al.,

1999). For example, disruption o f the links between ECM. integrins, and the cytoskeleton

(Murdoch et al.. 1996) may perturb the intracellular transport of substrates, such as cholesterol,

for steroidogenesis (Niswender et al., 2000). However, additional in vitro and in vivo studies are

needed to elucidate the physiological roles of these TIMPs in this endocrine gland.

In summary, the present study demonstrated that the temporal and spatial expression of

TIMP-2 was correlated to the expression patterns of active MT1-MMP and MMP-2. This

suggested that these three molecules might be coordinateiy expressed in the bovine CL to

facilitate the activation of pro-MMP-2 in vivo, and in turn coordinate physiological processes

such as enlargement of large luteal cells and luteal angiogenesis. The expression pattern of TIMP

1 was temporally and spatially distinct from TIMP-2. The predominant expression of TIMP-1 in

the CL suggested that it might be a key regulator in CL physiology.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TIMP-1 mRNA in the bovine CL

E M L

TIM P-1

18S rR N A

Early Mid Late Ages of CL

Figure 4-1. TIMP-1 mRNA expression in the bovine CL. A) Northern blotting of TIMP-1 mRNA in early (E), mid (M), and late (L) stage CL is shown in the upper panel. The arrow indicates the 0.9kb TIMP-1 transcript The ethidium bromide stained 18S rRNAs in corresponding luteal samples are shown in the lower panel. B) Changes in TIMP-1 mRNA, shown as a densitometric ratio of TIMP-1 mRNA to 18S rRNA, are illustrated. Dissimilar letters denote significant difference at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TIMP-2 mRNA in the bovine CL

TIMP-2

cyclophilin

B

□ 3.5kb u 0 l kb 3 ■ > .a 2.5< Z E o M ev a 0.5-

Mid LateEarly Ages of CL

Figure 4-2. TIMP-2 mRNA expression in the bovine CL. A) The upper panel shows Northern blotting of TIMP-2 mRNA in early (E), mid (M), and late (L) bovine CL. The arrows indicate the 3.5 and 1 kb species. The lower panel is the same membrane hybridized with a human cyclophilin probe. B) Densitometric ratios of the two TIMP-2 mRNA bands to cyclophilin in corresponding stages are shown. Dissimilar letters denote significant difference at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Activities of tissue inhibitors of metalloproteinases (TEMPs) in the bovine CL

STD 30 kDa 27 kDa

24 kDa 22 kDa

Figure 4-3. Reverse zymographic analysis of tissue inhibitors of metalloproteinases (TIMPs). Inhibitor activities in the early (E), mid (M), and late (L) stages of bovine CL are shown. Lane 1 indicates the Prestained SDS-PAGE standards (STD; Bio-Rad Laboratories, Hercules, CA). Their corresponding molecular masses (kDa) are indicated on the left. Four bands possessing MMP inhibitory activities (indicated by arrows on the right) were observed in the luteal samples. These bands correspond to TIMP-1 (-30kDa), TIMP-3 (~27kDa), TIMP-4 (~24kDa). and TIMP-2 (~22kDa), respectively.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TIMP-1 protein expression in the bovine CL

30kDa

B

8

7 6

5

4

3

2

1 0 Early Mid Late Ages of CL

Figure 4-4. TIMP-1 protein expression in the bovine CL during the estrous cycle. A) A representative Western blot o f TIMP-1 in early (E), mid (M), and late (L) CL collected over the estrous cycle. Conditioned medium of HT1080 cells (HT) was ioaded in the first lane and was used as a positive control. The arrow indicates the 30 kDa immunoreactive TIMP-1 protein. B) TIMP-1 protein levels in different stages are presented as a ratio of band intensity in luteal samples to that in HT1080 conditioned medium. Dissimilar letters denote significant difference at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TIMP-2 protein expression in the bovine CL

TIM P-2

o i------,------, Early Mid Late Ages of CL

Figure 4-5. TIMP-2 protein expression in the bovine CL throughout the estrous cycle. A) A representative Western blot of TIMP-2 in the bovine CL. Molecular masses (kDa) of protein standards are shown on the left. As indicated by the arrow, a single 22 kDa TIMP-2 immunoreactive protein band is present in early (E), mid (M), and late (L) stages of CL. B) Densitometric analysis of TIMP-2 protein expression among different stages of CL. Dissimilar letters denote significant difference at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cellular localization of TIMP-1 and -2 in the bovine CL

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4-6. Immunohistochemistry of TIMP-2 (A, C, E; x400) and TIMP-1 (B, D, F; x200) in the early (A and B), mid (C and D), and late (E and F) stages of CL. Vascular smooth muscle cells were identified by staining with a-actin (G; x200). Positive staining (red color) is indicated by white arrows, black arrows, and white triangles in large luteal cells, endothelial ceils, and vascular smooth muscle cells, respectively. In a representative negative control (H; x400), no positive staining was observed.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V

REGULATION OF MATRIX METALLOPROTEINASE 2 (MMP-2) EXPRESSION IN

ENDOTHELIAL AND LUTEAL CELLS BY CYTOKINES

Abstract

Our previous studies showed that MMP-2 activity varies in day 4, day 10. and day 16

bovine CL obtained over the estrous cycle. We also observed that MMP-2 is localized in

endothelial and large luteal cells. Although cytokines regulate progesterone and prostaglandin

production by the corpus luteum (CL), little is known about their effects on MMPs in the ovary.

Thus, the aim of the present study was to investigate cytokine regulation cf MMP-2 expression by

endothelial and luteal cells. In experiment I. luteal ceils were isolated from mid cycle bovine CL

(n=3). Dissociated cells were then incubated with I. 10. or 100 ng/ml tumor necrosis factor

(TNFa) for 12 hours, or with 100 ng/ml TNFa for 6. 12. 24. and 48 hours. Zymography revealed

that the two highest doses of TNFa increased (p<0.05) the level of pro-MMP-2 above the 1 ng/ml

treatment and control groups. In addition. pro-MMP-2 also increased in a time-dependent

manner, being the highest at 48 hours. Using concentrated medium. Western blotting showed that

active MMP-2 was present in cells treated with the 100 ng/ml dose of TNFa. Furthermore, the

level of active MMP-2 was the highest (p<0.05) at 48 hours. In experiment Q. endothelial cells

were isolated from cow CL during early pregnancy. Luteal-derived endothelial cells were treated

with either 100 ng/ml TNFa. 200 lU/ml interferon y (IFNy), or a combination of these two

cytokines, for 24 hours. Zymographic analysis revealed that TNFa stimulated, while IFNy

inhibited latent MMP-2 expression. Furthermore, when combined, IFNy attenuated the

stimulatory effect by TNFa, and the level of latent MMP-2 being not different from controls.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Together, these data suggest that MMP-2 expression in the CL may be regulated, in part, by

cytokines.

Introduction

Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent

proteolytic enzymes that have been shown to be important in ovarian physiology, including

follicular development, follicle atresia, ovulation, corpus luteum development, and ovarian

angiogenesis (Smith et al.. 1999). Among the more than 25 members that have been identified in

this family, matrix metalloproteinase 2 (MMP-2: geiatinase A) is one of the most well studied.

This may be due to its broad substrate specificity' on ECM proteins. In addition to its efficient

degradation on gelatins. MMP-2 also cleaves type I (Aimes and Quigley. 1995). type V (Okada et

al.. 1990). type VII (Seltzer et al.. 1989). and type X (Gadher et al.. 1989: Welgus et al.. 1990)

collagens. and other ECM components such as elastin (Senior et al.. 1991). laminin (Imai et al..

1995b: Giannelli et al.. 1997). vitronectin (Giannelli et al.. 1997). and aggrecan (Fosang et al..

1992). In particular. MMP-2 efficiently hydrolyzes soluble collagen type IV. which is one of the

major barriers encountered during structural remodeling in a variety of tissues. Furthermore, this

proteolytic enzyme affects cell behaviors by regulating the availability of growth factors,

cytokines, and their receptors (McCawley and Matrisian. 2001). For example, proteolytic

cleavage of biologically inactive pro- transforming growth factor (TGF) p 1 and P2 (Imai et al..

1997). pro-tumor necrosis factor a (TNFa) (McGeehan et al.. 1994). and pro-interleukin ip

(ILip) (Schonbeck et al.. 1998) by MMP-2 yields the active molecules. Also, N-terminal

processing of chemokines. such as monocyte chemoattractant protein 3 (MCP-3) and stromal cell

derived factor (SDF) la and ip results in loss of biological activity (McQuibban et al., 2000:

McQuibban et ai.. 2001). Furthermore. MMP-2 acts on the Val368-Met369 peptide bond of the

fibroblast growth factor receptor type 1 (FGFR1) ectodomain to release a soluble extracellular

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. domain, which modulate the mitogenic activities of FGF (Levi et al., 1996). Together, these

properties of MMP-2 establish its complex but critical roles in tumor invasion and metastasis.

Likewise, it may also be necessary for normal physiological processes, such as ovulation, and CL

development.

MMP-2 has been identified in the bovine (Tsang et al., 1995; Goldberg et al.. 1996:

Zhang et al.. 2002). ovine (Russell et al.. 1995: Towle et al., 2002), porcine (Pitzel et al., 2000).

rat (Nothnick et al.. 1996; Bagavandoss. 1998; Liu et al., 1999), and human (Duncan et al., 1998)

CL. In ewes. MMP-2 activity is critical for CL development and luteal neovascularization

(Russell et al.. 1995; Gottsch et al.. 2001). Increased MMP-2 expression or activity is also

associated w ith luteal regression in a variety species, including rat (Endo et al.. 1992). sheep

(Reike ct al.. 2002: Towle et al.. 2002), and human (Duncan et al.. 1998). Although luteinizing

hormone (LH) has no effect on MMP-2 expression by luteal cells isolated from 4 day old bovine

CL (Tsang et al.. 1995). its expression in vivo is augmented by prolactin and PGF;a in rat (Endo

et al.. 1992) and sheep (Towle et al.. 2002) CL. respectively.

The regulation of MMP-2 expression, which is unique and complex, occurs at the

activational or translational level. Recently, we showed that MMP-2. and membrane type 1 MMP

(MT1-MMP) and tissue inhibitor of metalloproteinases 2 (TIMP-2), the two key regulators that

activate it. are co-Iocaiized in endothelial and large luteal cells of the ovine CL (Zhang and

Tsang. 2001: Zhang et al.. 2002). MMP-2 can also be regulated at the transcriptional level.

Because it lacks well characterized regulatory' elements in the promoter region, MMP-2 has been

long viewed intractable to either stimulatory or inhibitory reagents (Mauviel. 1993). In part, this

may be the reason for the paucity of in vitro studies on the regulation of MMP-2 expression in the

ovary'. However, a recent study demonstrated that the MMP-2 promoter has a GC-rich region,

even though it lacks a typical TATA box (Qin et al.. 1998). Further sequence analysis revealed

that a number of cis-acting regulatory elements, includingp53, AP-l, AP-2. Spl, Ets-l, C/EBP,

CREB. and PEA3. are present in the up-stream regulatory region of the MMP-2 gene (Bian and

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sun. 1997; Qin et al.. 1998). Therefore, these elements could be involved in the regulation of

MMP-2 by growth factors and cytokines. Notably, the CL is a potentially rich source of a variety'

of cytokines (Pate. 199S: Terranova and Rice, 1997; Pate and Landis Keyes, 2001). Furthermore,

cytokines participate in CL physiology, by being involved in steroidogenesis, apoptosis.

angiogenesis. and luteolysis (Pate and Landis Keyes. 2001).

Among the cytokines identified in the CL. TNFa and interferon y (IFNy) have been

implicated in regulating endothelial and luteal cell physiology. Towards this end. the aim of the

current study was to investigate regulation of MMP-2 by TNFa and IFNy in luteal and

endothelial cells isolated from bovine CL.

Materials and Methods

1. Isolation and culture of luteal cells

Corpora lutea were collected from regularly cycling, nonlactating dairy cows on day 10

of the estrous cycle (day 0= estrus; n=3). After removing connective tissues. CL were dissociated

as previously described (Tsang et al.. 1995). Briefly, luteal tissues were minced before placing

into a spinner flask containing 25ml of Ham's F-12. with 1% bovine serum albumin (BSA). and

Type I collagenase (Worthington Biochemical. Lakewood. NJ; 2000 units/g tissue) for one hour

at 37°C. During this period, tissues were triturated every 10 minutes. After one hour, dissociated

cells were centrifuged sequentially at I90g, 1 lOg, and 80g to remove collagenase, connective

tissues and other tissue debris. Cell viability and number were determined by trypan blue

exclusion and counting on a hemacytometer, respectively.

Luteal cells (1 x 106) were seeded in T25 flasks containing 4 ml Ham's F-12 culture

medium, supplemented with insulin/selenium/transfening (ITS; Upstate Biotechnology. Lake

Placid. NY) and gentamicin (30pg/ml; GIBCO-BRL. Paisley. PA). After an overnight incubation,

unattached cells were removed by rinsing with fresh Ham's F-12 medium. Then, for experiment

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. luteal cells were treated with 1. 10, or 100 ng/ml TNFa (n=3 per dose; R&D Systems,

Minneapolis. MN) for 12 hours. For experiment n. luteal cells were treated with TNFa

(lOOng/ml) and incubated for 6, 12. 24. or 48 hours. Cell number was determined before

treatment and at the end of each time point.

2. Endothelial cell isolation and culture

Endothelial cells from early pregnant cow CL were provided by Dr. Bo Rueda at the

Massachuessets General Hospital and Dr. John Davis at the University of Nebraska. The

isolation, purification, and characterization of these luteal-derived endothelial cells were

commercially accomplished by BioWhittaker Molecular Applications (Walkersville. MD).

Endothelial cells were then cultured in 24 well plates (5000 cells/cm:) in EGM-2 MV medium

(BioWhittaker. Walkersville. MD) containing 3% fetal bovine serum (FBS) and a cocktail of

growth factors (BioWhittaker. Walkersville. MD) based on the manufacturer's recommendations.

After the cells attained 90% confluency. the spent medium was replaced with serum-free EGM-2

MV before they were treated with lOOng/ml TNFa (Upstate Biotechnology. Lake Placid. NY).

200 units/mi IFNy (a generous gift from Dr. Dale Godson), or a combination of both for 24 hours

(n=3 w ells per treatment). Cell numbers were determined at the end of the treatment period.

3. Gelatin rvmogranhv

MMP-2 activity was determined by zymographic analysis on 10% acrylamide SDS gels

containing 0.5mg/ml gelatin (Zhang et al.. 2002). Conditioned medium of luteal and endothelial

cell cultures was normalized to cell number. Equivalent aliquots of conditioned medium were

loaded into the gels. Perfect Protein™ Markers (Novagen. Madison, WI) and a positive control,

the conditioned medium of HT 1080 cells, were run in adjacent lanes.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Following electrophoresis, SDS was removed by rinsing twice with 2.5% (v/v) Triton X-

100 (15 minutes per wash). The gel was then incubated in substrate buffer (0.05M Tris-HCl.

5mM CaCk 0.05M NaCl. pH 8.0) at 37°C for 18 hours, followed by staining with 0.1% (w/v)

Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Hercules, CA) for 30 minutes.

Gelatinolytic activities were identified as clear zones against a blue background after destaining

with ddH;0.

4. Western blotting

Conditioned medium from luteal cell cultures was concentrated 20 times by using

Microcon* YM-3 Centrifugal Filter Devices (Millipore. Bedford. MA). Aliquots of concentrated

medium equivalent to 10.000 cells were used for Western blot analysis as previously described

(Zhang et al.. 2002). Briefly, media were separated on 12% acryiamide SDS gels under reducing

condition, w hich was accomplished by including lOmM (final concentration) dithiothreitol

(Sigma. St Louis. MO) in the loading buffer and boiling the samples for 3 minutes prior

electrophoresis. Proteins were then electrophoretically transferred onto Protran® nitrocellulose

membranes (Schleicher & Schuell. Keene. NH). Nonspecific binding sites on the membranes

were blocked by incubation with 5% (w/v) nonfat dry milk in TBST buffer ( lOmM Tris. 150mM

NaCl. 0.05% Tween-20. pH 8.0) for 2 hours at room temperature. A mouse anti human MMP-2

monoclonal antibody ( lug/ml in 5% nonfat dry milk: Oncogene Research Products. San Diego.

CA) was incubated with membranes overnight at 4°C. After four washes with TBST buffer (15

minutes per wash), a horse radish peroxidase (HRP) conjugated goat anti mouse IgG antibody

(1:10.000: Pierce. Rockford. IL) was applied onto membranes for 1 hour. Immunoreactive protein

bands were visualized with SuperSignal® West Pico chemiluminescent substrate (Pierce,

Rockford, EL) according to manufacturer's instructions.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5. Statistical analysis

Densitometric intensities of bands on zymograms and Western blot membranes were

analyzed by UN-SCAN-IT gel™ automated digitizing system (Silk Scientific, Orem, UT). The

data were analyzed by ANOVA. The Dunnet test was used for comparisons between treatment

and control groups, while the Bonferroni test was used for comparisons among different

treatments (Zar. 1999). A value of p<0.05 was considered to be significantly different.

Results

1. Dose dependent responses of MMP-2 expression to TNFa in luteal cells.

Zymographic analysis of luteal cell conditioned media revealed two major gelatinolvtic

species with relative molecular masses of 88 and 68 kDa. which correspond to MMP-2 and

MMP-9 family members, respectively (Figure 5-1 A). While latent MMP-2 and MMP-9 were the

major forms observ ed. less intense bands of active MMP-2 and MMP-9 were also detected.

Densitometric analysis demonstrated that the level of latent MMP-2 increased coordinatelv with

TNFa concentration. Compared to the control group, latent MMP-2 was significantly increased

(p<0.05) in cells treated with 10 ng/ml and 100 ng/ml TNFa (Figure 5-1B). Interestingly, latent

MMP-9 also increased in a similar manner (data analysis not shown).

Western blotting of concentrated luteal cell conditioned medium demonstrated that active

MMP-2 was only detected in the 100 ng/ml TNFa treatment group, while latent MMP-2 was

present in all samples (Figure 5-2A). Furthermore, levels of active and latent MMP-2 were the

greatest (p<0.05) in the 100 ng/ml of TNFa treatment group, while no differences were observed

among all other groups.

2. Time dependent responses of MMP-2 expression to TNFa in luteal frih.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The stimulatory effect of TNFa ( lOOng/ml) on MMP-2 expression in luteal cells was shown

to be time dependent. Gelatin zvmography revealed that latent MMP-2 was increased with length

of incubation time (Figure 5-3 A). While no significant differences were detected between the 6-

and 12-hour groups (Figure 5-3 B). latent MMP-2 at 24- and 48-hours was greater than the earlier

time points. Furthermore, when compared to their contemporaneous control group, latent MMP-2

expression in the presence of TNFa was significantly greater (p<0.05) only at the 24 and 48-hour

time points (Figure 5-3B).

Western blotting confirmed the pattern of change observed in zymograms, whereby latent

MMP-2 increased with incubation time. While latent MMP-2 was lowest and greatest at the 6-

and 48-hour time points, respectively, there was no difference between the 12- and 24- hour time

points. Also, latent MMP-2 in cells treated with TNFa was greater in contemporaneous controls

at all time points except at 6-hours. Furthermore, active MMP-2 was detected in cells treated

w ith 100 ng/ml TN Fa at all time points (Figure 5-4A). Notably, active MMP-2 was significantly-

increased only at the 48-hour time point (Figure 5-4B).

3. Differential regulation of MMP-2 expression bvTNFa and IFNv in luteal-derived

endothelial cells.

In luteal-derived endothelial cells, zvmography demonstrated that MMP-2 was the

predominant gelatinase. since MMP-9 was undetectable. Furthermore, latent MMP-2 was the

major form, while minor active MMP-2 was observed in all groups (Figure 5-5A). Treatment

with TNFa significantly increased (p<0.05). while IFNy inhibited (p<0.05) MMP-2 expression.

When combined. IFNy attenuated the stimulatory effect of TNFa alone, and the level of latent

MMP-2 expression was similar to the control group (p>0.05) (Figure 5-5B).

Discussion

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMP-2 is critical for ovulation, luteal angiogenesis, and CL development (Gottsch et al.,

2000; Gottsch et al., 2001). Unlike other MMPs, the activation of pro-MMP-2 is accomplished on

the cell surface by the coordinate interactions between MT1-MMP and TIMP-2 (Nagase, 1997).

The regulation of MMP-2 gene expression is also unique among MMPs due to its atypical

promoter organization (Qin et al.. 1999). Little is known regarding the regulation of this enzyme

in the ovary. LH has no effects on MMP-2 expression by luteal cells from 4 day old bovine CL

(Tsang et al.. 1995). while estradiol up-regulates MMP-2 production by human granulosa-lutein

cells (Puistola et al.. 1995). In equine ovarian stromal cells, transforming growth factor p (TGFP)

stimulates MMP-2 activity (Song et al.. 1999). In addition, a number of cytokines exhibit

differential regulatory effects on MMP-2 expression. For example. TNFa up-regulates MMP-2

activity in ovine follicular explants, while incubation with TNFa antiserum reduces MMP-2

activity (Gottsch et al.. 2000). Furthermore, intrafollicular administration of TNFa antiserum

blocks ovulation. Although the un-ruptured follicles, nonetheless, underwent luteinization. a fully

formed CL with accompanying vasculature was not observed (Gottsch et al.. 2000). These data

suggest that MMP-2. under the regulation of TNFa. may be critical for CL formation and

angiogenesis.

We previously reported that MMP-2 is localized to large luteal cells and endothelial cells

in the bovine CL (Zhang and Tsang, 2001). In the present study, we investigated the regulation of

MMP-2 expression in these two cell types by TNFa and/or IFNy. In bovine luteal cells. TNFa

stimulated MMP-2 expression in a dose and time dependent manner. Similarly, TNFa also

stimulates MMP-2 expression in porcine large luteal cells (Pitzel et al.. 2000). The CL produces

TNFa. Macrophages are the major sources of TNFa in the porcine CL (Zhao et al., 1998), while

large luteal cells also secrete TNFa at a significantly higher level than endothelial and small

luteal cells (Zhao et al.. 1998). In both porcine and bovine CL, the large luteal cells are positive

for TNFa receptor type I (TNFR I) mRNA expression (Richards and Almond, 1994; Sakumoto et

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. al.. 2000a). Considering these data, we postulate that MMP-2 production may be regulated by

TNFa in a paracrine and/or autocrine manner.

Of interest the present study demonstrated that only the highest dose of TNFa (100

ng/ml) stimulated pro-MMP-2 activation, even though the level of pro-MMP-2 increased with

increasing concentration of TNFa. This indicated that TNFa may have multiple cellular signal

transduction pathways to regulate MMP-2 expression and activity. On the one hand, TNFa may

work through transcriptional activators, such as p53 and/or NFicB, to stimulate MMP-2 gene

expression (Marti et al.. 1993: Bian and Sun. 1997). which results in the increase of pro-MMP-2.

On the other hand. TNFa may modulate activation of MMP-2 via a nongenomic pathway. For

instance, efficient activation of MMP-2 is accomplished on the cell membrane by assembling a

trimeric complex comprising MT1-MMP. TIMP-2. and pro-MMP-2 (Cao et al.. 1998; Butler et

al.. 1998). In the bovine CL. we reported the coordinate expression of MT1-MMP and TIMP-2

w ith MMP-2 activity (Zhang and Tsang, 2001: Zhang et al.. 2002). Since this activation is

performed on the cell membrane, it can be greatly affected by factors that modulate membrane

trafficking, including fluidity, stability, and other biophysical features (Lehti et al.. 2002). Indeed.

ConA stimulates pro-MMP-2 activation by shifting endogenous MT1-MMP from the intracellular

compartment to the cell surface (Jiang et al.. 2001). Also, binding of TNFa to its membrane

receptor (TNFR I) induces activation of sphingomyelinases, which hydrolyze N-acylsphingosin-

1-phosphorylcholine. a phospholipid preferentially found in the plasma membrane of mammalian

cells, to ceremide (Kronke. 1999). The latter can act as a second messenger to trigger a variety of

cellular functions. Notably, conversion of sphingomyelin, a sphingolipid situated predominantly

in the outer part of the cell membrane, to ceremide. a second messenger, may affect the

biophysical structure of plasma membranes. In addition, TNFa also increases PGF;a production

by activating phospholipase A2 enzymes, which liberate arachidonic acid (ACA) from membrane

phospholipids for the synthesis of eicosanoids (Townson and Pate, 1996; Sakumoto et al., 2000b)

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Therefore, one of the cellular functions of TNFa might be through

sphingomyelinase/sphingomyelin and/or PLA2/ACA pathway(s) to affect the oligomerization of

MT1-MMP on the cell surface, which then modulates pro-MMP-2 activation (Lehti et al.. 2002).

In the present study, induction of pro-MMP-2 activation by 100 ng/ml TNFa was observed as

early as 6 hours after treatment, indicating that it is likely acting via a nongenomic pathway rather

than being regulated at the transcriptional level. By using inhibitors against different second

messengers, studies are underway to further pinpoint the detailed cellular signal transduction

events triggered by TNFa for MMP-2 expression and activation.

Similar to luteal cells. MMP-2 expression in endothelial cells was also enhanced by

TNFa treatment. Luteal-derived endothelial cells have greater levels of TNFRI mRNA than luteal

cells (Sakumoto et al.. 2000a). suggesting that endothelial cells are also a major target of TNFa.

Indeed, injection of the rabbit CL with TNFa induced blood vessel regression, a process

characterized by strictures, obstructions, and ragged surfaces on the vessels (Nariai et al.. 1995).

This is similar to the vessel regression observed during luteolysis in cows (Modlich et al., 19%).

Therefore, one action of increased TNFa during the late luteal phase and in the regressed CL may

be to stimulate MMP-2 expression and activation in endothelial cells. The increased MMP-2

activity allows endothelial cells to degrade the surrounding ECM proteins, to detach from the

underlying basement membrane (Modlich et al.. 1996), and to initiate blood vessel regression.

In contrast to TNFa. IFNy inhibited MMP-2 expression by luteal-derived endothelial

cells. In addition, co-treatment with TN Fa and IFNy resulted in MMP-2 expression that is similar

to controls, indicating possible interactions between the second messengers induced by these two

cytokines. IFNy has anti-proliferative effects on luteal-derived microvascular endothelial cells

(Fenyves et al.. 1994). IFNy also up-regulates endothelial cell expression o f E-cadherin (Fenyves

et al.. 1993), a cell adhesion molecule. This is consistent with its ability to increase intercellular

junctions in an in vitro cell culture system (Ricken et al., 1996). Taken together, IFNy may play

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. an important role in vasculature maintenance by down-regulating MMP-2 expression, inhibiting

endothelial cell proliferation, and reinforcing intercellular junctions between endothelial cells.

These biological functions of IFNy on luteal-derived endothelial cells may contribute to its

luteotrophic effects (Prakash et al.. 1997).

Although the physiological roles of IFNy and TNFa on luteal physiology are very

complex, they have been postulated to play important roles in luteolysis (Pate, 1994). IFNy

mRNA levels decline in late diestrus and early luteolysis (Petroff et al.. 1999), although its

receptor in the CL remains to be elucidated. TNFa mRNA levels in the CL do not vary

throughout the estrous cycle (Petroff et al.. 1999; Sakumoto et al.. 2000a). However, the TNFa

protein significantly increases from the mid to late stage (Shaw and Britt. 1995: Sakumoto et al..

2000a). Although the concentration of TNFa receptors (TNFR I) is significantly lower in the late

stage than in others, high affinity binding sites for TNFa are detected in the regressed CL

(Sakumoto et al.. 2000a). Collectively, decreased IFNy expression, increased TNFa level, and

appearance of high affinity TNFa receptor may act in concert to increase MMP-2 expression and

activation, which we have observed in the regressing bovine CL (Zhang et al.. unpublished

observation).

In summary, the present study demonstrated the stimulatory effects of TNFa on MMP-2

expression and activation in luteal cells and luteal-derived endothelial cells. In contrast, IFNy

attenuated MMP-2 expression in endothelial cells. The regulatory effects of these cytokines on

MMP expression may contribute to their roles in modulating CL physiology, i.e., angiogenesis,

vascular maintenance, and luteolysis.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dose dependent responses of MMP-2 expression in luteal cells to TNFa

TNFa (12h)

HT M C 1 10 100 (ng/ml) lOOkDa 7SkDa Pro-MMP-2 Active MMP-2 50kDa

3SkDa

2SkDa

2.5 o s 2 0 1 E 2 * 5 c s 0.5

Control 1no/ml 10no/ml IQOna/ml TNFa

Figure 5-1. Zymographic analysis of medium conditioned by bovine luteal cells treated with different concentrations of TNFa for 12 hours. A) In this representative zymogram, Perfect Protein™ Marker (Novagen, Madison, WI) molecular weight standards are listed on the left and shown in lane 2 (M). In lane 1, HT stands for the conditioned medium of HT1080 cells. In lane 3, C denotes the control culture medium from luteal cells in the absence of treatment. In lanes 4-6, the concentrations of TNFa are indicated on the top. B) Densitometric analysis of pro-MMP-2. Dissimilar letters denote significance at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dose dependent responses of MMP-2 expression in luteal cells to TNFa T N Fa (I2h)

HT C I 10 100 (ng/ml) U3kDa 92kDa Pro-MMP-2 Active MMP-2 S2kDa

35kDa

A'afUCi 28.9kDa

20kDa

B

□ latent MMP-2 B active MMP-2

6

5

2 4 o a a rE-i !•a a • 2 -E-

1 a ’ a' a ’ 0 Control 1 no/ml 10no/ml 100no/ml TNFa

Figure 5-2. Immunoreactive MMP-2 in luteal cells treated with different concentrations of TNFa. A) Representative Western blot of latent and active MMP-2. Molecular masses of Prestained protein standards (Bio-Rad Laboratories, Hercules, CA) are listed on the left. In lane 1, HT is the conditioned medium of HT1080 ceils. In lane 2, C represents the control group. Lanes 3-5 are samples from luteal cells treated with 1,10, or 100 ng/ml of TNFa for 12 hours. The latent and active MMP-2 bands are indicated on the right. B) Densitometric analysis of latent (O) and active MMP-2 among different groups. Dissimilar letters denote significance at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Time dependent responses of MMP-2 expression in luteal cells to TNFa

12h 24h

TNFa lOOkDa 75kDa ■Pro-MMP-2 SOkDa ■Active MMP-2

35kDa 25kDa

B □ control ■ treatment

2.5

• 1.5

&

Figure 5-3. Time dependent responses of MMP-2 expression in bovine luteal cells treated with TNFa (100 ng/ml). A) Representative zymogram of luteal cells incubated in the absence (-) and presence (+) of TNFa. Perfect Protein™ Marker (Novagen, Madison, WI) molecular weight standards are indicated on the left and shown in lane 1 (M). In lane 10, conditioned medium from HT1080 cells (HT) were loaded. Samples incubated for 6-, 12-, 24-, or 48-hours were loaded in lanes 2-9. Arrows indicate pro- and active MMP-2. B) Densitometric analysis of latent MMP-2. Dissimilar letters denote significance (p<0.05) among different incubation times following TNFa treatment. Asterisks represent significant difference (p<0.05) between respective control and TNFa treatment groups at each time point.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Time dependent responses of MMP-2 expression in luteal cells to TNFa

6h 12h 24h 48h A h t 1----- 1 1----- 1 1----- 1 1----- 1 TNFa(100ng/ml) - - + - + - + - + U3kDa — 92kDa — Pro-MMP-2 52kDa — Active MMP-2

35kDa — 28.9kDa—

20kDa —

B

Latent MMP-2 Active MMP-2 □ control a treatment □ control a treatment i

6 0.5 III 6h 12h 24h 12h 24h Time Time

Figure 5-4. Time course responses of latent and active MMP-2 in bovine luteal cells treated with TNFa. A) Representative Western blot of conditioned medium from luteal cells in the absence (-) and presence (+) of TNFa. Molecular masses of Prestained protein standards (Bio-Rad Laboratories, Hercules, CA) are indicated on the left. In lane I, FIT represents the conditioned medium of HT1080 cells. Samples incubated for 6-, 12-, 24-, or 48-hour were loaded in lanes 2-9. Latent and active MMP-2 bands are indicated by arrows on the right. B) Statistical analysis of active and latent MMP-2 levels. Dissimilar letters denote significance (p<0.05) among different incubation times following TNFa treatment. Asterisks represent significant difference (p<0.05) between respective control and TNFa treatment groups at each time point.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Regulation of MMP-2 expression in luteal-derived endothelial cells by cytokines

A M HT Control TNFa IFNy TNFa+IFNy 100 ld)a

75 kDa

50 kDa

35 kDa

25 kDa

B 2.5

0 2 1 0 1.5 1 E 2 1 *e 5 <3 0.5

0 control TNFa IFNy TNFa* IFNy

Figure 5-5. MMP-2 expression in luteal-derived endothelial cells in response to TN Fa and IFNy, alone and in combination. A) Representative zymogram of conditioned medium of luteal-derived endothelial cells. Molecular masses of Perfect Protein™ Markers (Novagen, Madison, WI) are indicated on the left and shown in lane 1 (M). In lane 2, HT represents HT1080 conditioned medium. In lanes 3-6, control and cytokine treatments, TNFa (lOOng/ml) and IFNy (200IU/mI), alone or in combination, are shown. B) Densitometric analysis of MMP-2 levels. Dissimilar letters denote significance at p<0.05.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUMMARY AND CONCLUSIONS

In the present studies, we cloned the full length cDNAs of the bovine matrix

metalloproteinase-2 (MMP-2) and membrane type 1 metalloproteinase (MT1-MMP) genes.

Multiple alignment analysis indicated that the predicted proteins for these two molecules have a

high level of sequence identity with their homologs from other mammalian species, suggesting

that they have been conserved through evolution. Although the transcript levels of both molecules

were unchanged in three ages of bovine corpus luteum (CL) obtained over the estrous cycle, their

protein levels varied. While latent MT1-MMP was higher (p<0.05) in the early CL than the other

two stages, the active MT1-MMP was significantly increased from the early stage to the mid and

late cycle CL. In spite of the fact that the level of pro-MMP-2 remained constant in all ages of CL

evaluated, the level of active MMP-2 in the mid and late stages was significantly higher than that

in the early CL. In addition, MT1-MMP and MMP-2 were localized to endothelial and large

luteal cells. Furthermore. MT1-MMP expression was also detected in the fibroblast-like cells of

the late cycle CL. Thus, the correlation between active MTI-MMP and active MMP-2 throughout

the estrous cycle and their co-localization in the same cellular compartments suggests that MTI-

MMP may serve as the in vivo activator for pro-MMP-2.

Recent studies report that the activation process of pro-MMP-2 by MTI-MMP is

regulated by an endogenous tissue inhibitor of metalloproteinases 2 (TIMP-2). This inhibitor

bridges these two MMPs by binding MTI-MMP on the cell surface via its N-terminus and pro-

MMP-2 at its C-terminal domain. This assembled trimeric complex presents pro-MMP-2 to an

adjacent, active MTI-MMP, which cleaves the N-terminal pro-peptide domain of pro-MMP-2,

initiating its activation. In order to determine whether TIMP-2 is coordinately expressed with

MTI-MMP and MMP-2. we investigated the temporal and spatial expression of TIMP-1 and

TIMP-2 in the bovine CL. TIMP-1 mRNA and protein were expressed at higher levels in the

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. early and mid stages than the late cycle CL. However. TIMP-2 mRNA and protein expression

was low in early CL, but significantly increased in the mid and late stages, corresponding to the

changes observed for active MTI-MMP and MMP-2 proteins. Furthermore, TIMP-2 was also

localized in endothelial and large luteal cells in bovine CL, while TIMP-1 was localized in large

luteal cells, and in vascular smooth muscle cells in the early and late stage CL. These data

indicated that the MTl-MMP/TlMP-2/pro-MMP-2 system might be available for pro-MMP-2

activation in the bovine CL. In addition, the localization of TIMP-1 in the vascular smooth

muscle cell compartment suggested that it might have a role in luteal angiogenesis and vascular

maintenance.

Besides its distinct activation mechanism. MMP-2 also possesses a different promoter

organization from other MMPs. The last part of our experiments was to initiate studies on the

regulation o f MMP-2 in luteal and endothelial cells by using in vitro cell culture systems. TNFa

stimulated MMP-2 expression and activation in luteal cells in a time and dose dependent manner.

Although TNFa increased MMP-2 expression in luteal-derived endothelial cells. IFNy inhibited

its expression. In the presence of both cytokines. IFNy attenuated the stimulatory effects of

TNFa. reducing MMP-2 expression to control levels.

Collectively, the in vivo data suggest that the MTI -MMP/TIMP-2/pro-MMP-2 system

might be available in the CL for pro-MMP-2 activation. The localization of these three molecules

in the same cellular compartments further support their coordinated actions during turnover of the

CL extracellular matrix, such as during enlargement of large luteal cells and luteal angiogenesis.

Furthermore, localization of TIMP-1 in vascular smooth muscle cells suggests that it is another

player participating in the regulation of angiogenesis and vascular maintenance. Lastly, the in

vitro study using luteal and endothelial cells indicated that the cytokines, TNFa and IFNy, might

serve as local regulators of MMP-2 expression and activation.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The data provided by the present study on the expression, activation, and regulation of

MMP-2 will enable us to conduct future experiments to continue elucidating the roles of this and

other MMP enzymes in angiogenesis and the physiology of the corpus luteum.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX

APPROVAL OF ANIMAL PROTOCOLS BY THE UNH INSTITUTIONAL

ANIMAL CARE AND USE COMMITTEE

U niversity o f N e w H a m psh ir e Office of Sponsored Research Service Building 51 College Road Durham, New Ham pa hire 03824*3585 (6031862-3564 FAX

LAST NAME Tsang FIRST NAME Paul

DEPT Animal and Nutritional Sciences - Kendall Hail NEXT REVIEW 12/17/2002 DATE 991202 OFF-CAMPUS IACUC # ADDRESS (If appiicabla) REVIEW LEVEL 3

DATE OF NOTICE 11/19/2001

PROJECT Association of Fertility with Temporal Changes in Ovarian Function of Domestic Ruminants TITLE

The Institutional Animal Care and Use Committee (IACUC) has reviewed and approved your request for a time extension for this protocol. Approval is granted until the ‘Next Review Date' indicated above. You will be asked to submit a project report with regard to the involvement of animals before that date. If your project is still active, you may apply for extension of IACUC approval through this office.

The appropriate use and care of animals in your project is an ongoing process for which you hold primary responsibility. Changes in your protocol must be submitted to the IACUC for review and approval prior to their implementation. If you have questions or concerns about your project or this approval, please feel free to contact the Regulatory Compliance Office at 862-2003 or 862-3536.

P lease note: Use of animals in research and instruction is approved contingent upon participation in the UNH Occupational Health Program for persons handling animals. Participation is mandatory for all principal investigators and their affiliated personnel, employees of the University and students alike. A Medical History Questionnaire accompanies this approval; please copy and distribute to ail listed project staff who have not completed this form already. Completed questionnaires should be sent to Dr. Gladi Porsche, UNH Health Services.

Please refer to the IACUC # above in ail correspondence related to this project The IACUC wishes you success with your research.

FoMhe IACUC, r f i 1- John A. Utvaitis, Ph.D.

cc: Rle ORIQAPPt 12/17/1999

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.