UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

“Role of Matrix in acrolein-induced Mucin 5 (subtype A and C) increase”

A dissertation submitted to the

Division of Research and Advance Studies University of Cincinnati

in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY

In the Department of Environmental Health of College of Medicine February 14th, 2006

by

HITESH DESHMUKH M.B., B.S. UNIVERSITY OF MUMBAI, 2001

DISSERTATION COMMITTEE: George D Leikauf Ph.D. (Chair) Michael T Borchers Ph.D. Jeffrey A Whitsett M.D. Thomas R Korfhagen M.D., Ph.D.

ABSTRACT:

Chronic obstructive pulmonary disease (COPD) is major public health problem worldwide induced by cigarette smoking. The pathogeneses of COPD is characterized by progressive difficulty in breathing and excessive mucus production. Previous studies have demonstrated that acrolein, a constituent of cigarette smoke increased mucin 5

(subtype A and C) (MUC5AC) production in the airways, however, the mechanism is unclear. The overall hypothesis of this dissertation is that acrolein increases MUC5AC production through activation of matrix metalloproteinases (MMPs). In airway epithelial cells, acrolein increased MUC5AC transcripts independent of oxidative stress, via a ligand-dependent, epidermal growth factor receptor (EGFR) and mitogen activated protein kinase (MAPK) activation. Acrolein increased MMP9 and MMP14 activity in vitro and in vivo. Small interfering (si) RNA to MMP9, MMP14 and a disintegrin and domain protein (ADAM) 17 inhibited the acrolein-induced increase in

MUC5AC transcripts in vitro. Acrolein-induced increase in MUC5AC transcripts and

(-/-) (+/+) protein was lower in -targeted MMP9 mice (Mmp9 ) than in Mmp9 mice.

Acrolein increased MMP9 and MMP14 transcripts via an EGFR-MAPK dependent mechanism and decreased the transcript level of tissue inhibitor of metalloproteinase protein (TIMP) 3, an endogenous inhibitor of ADAM17, MMP9 and MMP14 in vitro and in vivo. Thus, acrolein activates MMP9 to increase MUC5AC transcripts and protein via an initial EGFR ligand dependent mechanism. Chronic exposure to acrolein increases the protein levels and activity of MMP9 and MMP14, and decreases the transcript level of

TIMP3 to initiate a positive feedback loop leading to persistent MUC5AC production.

ACKNOWLEDGEMENTS:

“Shree Ganeshayah Namah”

I would like to express my deep appreciation to Dr George Leikauf for his advice, support and friendship, and for encouraging me to strive for excellence in all aspects of my academic and professional development.

I would like to express my gratitude to other members of my dissertation committee, Dr

Michael Borchers, Dr Thomas Korfhagen and Dr Jeffrey Whitsett, for their advice and support throughout the course of my graduate studies.

I am especially indebted to Dr Michael Borchers, Dr Scott Wesselkamper and Lisa Case who were called upon very frequently to rescue my experiments and for their unwavering support and guidance. I am grateful to Colin Shaver and John Mason for their invaluable help in my research.

To my brother, Ashish and my friends, Purvi Chawla, Rucha Sane, Kiran Khadke and

Mihir Mahajan, I remain forever thankful for helping me tide over my doubts and instilling confidence, without which this work could never be completed.

And to my parents, Suryakant and Bhavana Deshmukh, my deepest appreciation for imparting to me the love of learning and for the constant encouragement and support for this endeavor and making this worthwhile.

TABLE OF CONTENTS

TABLE OF CONTENTS i

LIST OF FIGURES iv

LIST OF TABLES vi

CHAPTER 1 Chronic Obstructive Pulmonary Diseases Background 1─1 Etiology and Pathogenesis 1─2 Mucus overproduction in COPD 1─4 Structure of Mucins 1─5 Mucin gene family 1─6 MUC5AC regulation 1─7 MUC5AC overproduction and Epidermal Growth Factor Receptor 1─7 Matrix Metalloproteinases 9 1─9 Transcriptional regulation 1─10 Activation of pro-MMP9 1─13 Pericellular localization of MMP activity 1─14 Matrix Metalloproteinase 14 1─15 Transcriptional regulation 1─16 Activation of pro-MMP14 1─16 Endogenous MMP inhibitors 1─17 Regulation of Cell Signaling by MMPs 1─19 Cell surface proteolysis and cellular signals 1─19 Regulation of paracrine signaling 1─20 Acrolein Sources of human exposure 1─22 Health effects of acrolein 1─24 Toxicokinetics of acrolein 1─27 In-vitro toxicity studies 1─28 Acrolein and lipid peroxidation 1─29 Focus of Dissertation 1─30 Specific Aims 1─31

i CHAPTER 2: Metalloproteinases mediate acrolein-induced Mucin 5 AC expression Abstract 2─1 Introduction 2─2 Methods and Materials 2─3 Results 2─5 Discussion 2─8 References 2─10 Supplementary Materials 2─11

CHAPTER 3: An autocrine feedback loop regulating matrix metalloproteinase (MMP) 9 leads to persistent mucus production in the airways Abstract 3─1 Introduction 3─2 Methods and Materials 3─6 Results 3─13 Discussion 3─15 Figure Legends 3─20 References 3─24 Tables 3─33 Figures 3─36

CHAPTER 4: Matrix metalloproteinase 14 mediates acrolein-induced mucin 5 (subtype A and C) increase Abstract 4─1 Introduction 4─2 Methods and Materials 4─5 Results 4─10 Discussion 4─12 Figure Legends 4─16 References 4─20 Tables 4─28 Figures 4─40

CHAPTER 5 Discussion Acrolein and mucus production 5─1 Acrolein and oxidative stress 5─2 MUC5AC overproduction and Epidermal Growth Factor Receptor 5─3 Matrix metalloproteinase 9 and MUC5AC production 5─5 Matrix metalloproteinase 14 and MUC5AC production 5─9 Tissue inhibitor of metalloproteinase proteins (TIMP)s 5─11 and MUC5AC production Summary 5─13 Significance to human health 5─15

ii

BIBLIOGRAPHY 6─1

iii LIST OF FIGURES

1. Acrolein-induced increase in MUC5AC transcripts in NCI-H292 cells is

independent of oxidative stress. 2-5

2. Acrolein-induced increase in MUC5AC transcripts involves phosphorylation of the

epidermal growth factor receptor and mitogen activated protein kinase. 2-5

3. Acrolein-induced increase in MUC5AC transcripts involves a tissue inhibitor of

metalloproteinase protein (TIMP) 3 sensitive metalloproteinase. 2-6

4. Acrolein-induced increase in MUC5AC transcripts is mediated by a disintegrin

and metalloproteinase domain protein (ADAM) 17 and matrix metalloproteinase

(MMP) 9. 2-7

5. Acrolein increases the release and subsequent activation of pro-MMP9. 2-7

6. Acrolein increases MMP9 transcript and decreases TIMP3 transcripts in NCI-

H292 cells and normal bronchial epithelial (NHBE) cells. 2-8

7. Acrolein activates pro-MMP9 in cell free system. 3-36

8. Acrolein increases MMP9 transcripts and decreases TIMP3 transcripts in the

lungs of acrolein-exposed mice. 3-37

9. Acrolein increases immunostaining with anti-MMP9 antibodies in the airways of

acrolein-exposed mice. 3-39

10. Acrolein increases MMP9 protein and activity in the lungs of acrolein-exposed

mice. 3-40

11. MMP9 mediates acrolein-induced increase in MUC5AC in lungs. 3-42

12. Acrolein-induced increase in MMP9 transcripts is mediated by metalloproteinase

mediated EGFR-ligand dependent mechanism by activating MAPK3/2. 3-45

13. Mechanism of acrolein-induced increase in MUC5AC. 3-49

iv 14. MMP14 transcripts increase in acrolein-treated NCI-H292 cells and NHBE cells

and increases MMP14 transcripts and protein in the lungs of acrolein-exposed

mice. 4-28

15. Acrolein increases MMP14 activity in acrolein treated NCI-H292 cells and in the

lungs of acrolein-exposed mice. 4-31

16. Furin mediates acrolein-induced increase in MMP14 activity. 4-33

17. Acrolein-induced increase in MMP14 transcripts is mediated by

metalloproteinase mediated EGFR-ligand dependent mechanism by activating

MAPK3/2 and MAPK8. 4-34

18. Small interfering (si) RNA against MMP14 decreased MMP14 transcripts and

protein. 4-37

19. Acrolein-induced increase in MUC5AC transcripts is mediated by MMP14. 4-38

20. Role of MMP14 in acrolein-induced increase in MUC5AC. 4-40

21. Immunostaining with anti-MMP14 antibodies increased in the airways of human

subjects with COPD. 4-41

22. Cysteine switch mechanism for activation of pro-MMP9. 5-7

23. Mechanism of MMP9 and MMP14 mediated increase in MUC5AC after acrolein

treatment. 5-14

v

LIST OF TABLES

1. Tissue inhibitors of metalloproteinase proteins (TIMP) s. 1-18

2. Sources of acrolein. 1-23

3. Effects of inhaled acrolein on laboratory animals. 1-27

4. Primers and probes used in real time polymerase chain reaction. 3-33

vi CHAPTER 1

Introduction

CHRONIC OBSTRUCTIVE PULMONARY DISEASE:

Background: Chronic obstructive pulmonary disease (COPD) is a major and growing global health problem. COPD was the sixth most common cause of death worldwide in

1990, the fifth most common cause of death in 2001, and the Global Burden of Disease

Study predicted that it would become the third most common cause of death by 2020

(Lopez 1998). In the United States alone, COPD has already risen to the fourth leading cause of death, and is the only common cause of death whose prevalence has increased over the past 20 years (Pauwels 2004). Moreover, it is an increasingly common cause of chronic disability, and is predicted to become the fifth most common cause of disability in the world by 2020 (Leikauf 2002). COPD is also an increasingly common cause of hospital admissions and loss of time from work, resulting in a major health care expenditure of over $14 billion/yr (Barnes 2004). The total (both direct and indirect) costs of COPD estimated by the NHLBI were in excess of $34 billion/yr in 2001 (Petty

2003). Yet among common diseases, COPD has been relatively neglected, with less investment in research on its underlying cellular and molecular mechanisms, and no therapies that have been shown to slow the relentless progression of the disease (Gross

1999).

Etiology and pathogenesis of COPD: COPD is caused by long-term exposure to inhaled irritant gases and particles contained in cigarette smoke. In developed countries, smoking accounts for more than 90% of COPD cases (Markewitz 1999). In developing countries, the inhalation of smoke from biomass fuels is also a leading cause of COPD, particularly among women who cook in poorly ventilated homes. The progressive

1-1 reduction in the forced expiratory volume in 1.0 sec (FEV1) and increasing severity of disease over time results in progressive shortness of breath on exertion that slowly advances to respiratory failure (Barnes 2000, Petty 2003, Pauwels 2004). COPD is a collective term describing two separate lung diseases, emphysema and chronic bronchitis (Pauwels 2001). It is marked initially by dyspnea and occasional cough, which becomes more pronounced as the disease progresses. Death and disability from COPD are related to an accelerated decline in lung function over time, with a loss of more than

50 ml per year in FEV1, as compared with a normal loss of 20 ml per year (Fletcher

1977). The cellular and molecular events leading to COPD are multifactorial, involving macrophages, neutrophilic airway inflammation, proteinase-antiproteinase imbalance, oxidative stress, and recurrent infection (Hogg 2004). Histopathological studies reveal that inflammation occurs widely in the peripheral airways (bronchioles) and lung parenchyma. The bronchioles are obstructed by fibrosis and infiltration of macrophages and T lymphocytes. Destruction of lung parenchyma is also accompanied by an increased in macrophages and T lymphocytes, which are predominantly CD8+ (cytotoxic)

T cells (Finkelstein 1995, Barnes 2000). Bronchial-biopsy results are similar to the histopathological findings and include excessive macrophages, CD8+ T cells, and neutrophils (O'Shaughnessy 1997).

The proteinase-anti-proteinase imbalance hypothesis is thought to play a key role in cigarette smoke induced chronic lung disease (Cawston 2003). It proposes that an anti- proteinase protects the normal lung from locally elaborated proteinases, and abnormal increase in proteinases and/or reduction in pulmonary anti-proteinases, like alpha-1 antitrypsin, contribute to the pathogenesis of COPD. In the past, studies examining proteinases have focused on the role of the serine proteinases (such as neutrophil elastase and proteinase 3) and the cathepsin family. Increasing evidence supports a

1-2 central role for matrix metalloproteinases (MMP)s in cigarette smoke induced COPD.

Concentrations of MMP1, MMP8, and MMP9 increase in bronchoalveolar lavage (BAL) fluid from COPD patients as compared to controls (Finlay 1997, Betsuyaku 1999).

Macrophages derived from COPD patients have increased MMP9 transcripts and secrete more MMP9 when stimulated with cigarette smoke extract (Russell 2002).

MMP2 and MMP9 protein (measured by immunohistochemistry) increased in the lungs of COPD patients (Segura-Valdez 2000). In mice exposed to cigarette smoke,

Hautamaki et al (1997) demonstrated that macrophage metalloelastase (MMP12) was necessary for the full development of emphysema.

MUCUS OVERPRODUCTION IN COPD: Three major mechanisms have been

implicated in the accelerated loss in FEV1. There is a loss of elasticity and the destruction of the alveolar attachments of airways within the lung as a result of emphysema, which results in a loss of support and closure of small airways during expiration (Hogg 1994). The second is the narrowing of small airways as a result of inflammation and scarring (Hogg 1968), and the third is the blocking of the lumen of small airways with mucous secretions (Lange 1990). Small airways (<2-3 mm diameter) normally contribute 20-25 % of total airway resistance (Hogg 1968), which can be increased markedly when the airway lumen is decreased by accumulation of excess mucus resulting in a ventilation-perfusion mismatch and insufficient oxygenation of blood. Studies have found positive associations between mucus production and decline in lung function, hospitalization and death (Vestbo 1996). Moreover, excess mucus in the airways also contributes to infections which can further exacerbate COPD and contribute to morbidity and mortality in patients with COPD (Wilson 1998).

1-3 Mucus is a viscoelastic gel that lines the respiratory epithelium and protects against infectious and environmental agents. Mucus consists mainly of water (95%) combined with salts, lipids, and various proteins, including mucin glycoproteins (Reid 1986).

Consistency of mucus varies throughout the airways to enable efficient mucociliary clearance (Spicer 1971). In the distal airways, mucus is enriched with lower molecular weight and lower viscosity proteins (including surfactant and Clara cell secretory proteins). The secretions within submucosal glands also vary. The distal tubule regions contain serous cells, which produce lower molecular weight and less viscous secretions, whereas the proximal tubule regions contain more mucous cells and produce secretions of higher molecular weight and greater viscosity (Leikauf 2002). The mucin glycoprotiens provides the viscoelastic properties of mucous and serous secretions.

Thus, though mucin glycoproteins are an important component of host defense, they represent an important cause of airway obstruction when present in excess.

Structure of mucins: Mucins are large molecules (> 100 kDa) that are composed of

50-85% carbohydrates (Widdicombe 1989). Each mucin protein contains an apomucin core (typically 80% of the coding sequence) that is enriched in hydroxyamino acids, serine, and theronine (Carlson 1968). These amino acids contain O-glycosylation sites for oligosaccharides. The length and number of O-glycosylation domains vary between mucins, and each domain contains repeated series of sequences (variable-number tandem repeats) (Porchet 1991). Each mucin transcript is capable of producing multiple peptides via a wide variety of post-translational modifications. Mucin oligosaccharides are joined to the protein core through an initial α-O-glycosidic linkage of N- acetylgalactosamine (GalNAc) to the hydroxyl region of serine or theronine (Seregni et al

.1997). Various branching oligosaccharide chains contain fucose, galactose, N- acetylglucosamine, and N-acetylneuraminic acid. Peripheral to the core of these five

1-4 monosaccharides are sulfate or neuramic acids that produce a polyanionic (ie, acidic) molecule. Small amounts of mannose also are found in mucins. Mucin O-glycosylation is accomplished by one of six uridine diphosphate-GalNAc-polypeptide-α-N-GalNAc (Hennebicq 1998). Because one apomucin sequence can lead to numerous lengths of transcript, each transcript can vary in stability, and each peptide can have various combinations of several hundred different carbohydrate chains, this complex assembly scheme allows for wide mucin heterogeneity between and within individuals. This mechanism provides for huge diversity in an extracellular protein that the cell needs to manage a continuously altering environment (Leikauf 2002).

Mucin gene family: Mucins are encoded by at least 20 , including a cluster localized to human 11p15 (Van klinken 1995), of which at least 9 genes are expressed in the lungs (MUC 1, MUC2, MUC 3A, 3B, 4, 5AC, 5B, 6-9, 11-13, 15-20)

(Reviewed by Leikauf 2002). Mucins found in tracheobronchial secretions include

MUC2, MUC5AC, MUC5B, MUC7, and MUC8 (Reviewed by Rose 2001). MUC5AC and

MUC5B appear most frequently in studies of human secretions (Rose 1989). MUC5AC, due to its large size, was initially described as two genes. Two large partial clones (then named MUC5A and MUC5C) were subsequently found to be parts of the same gene.

MUC5AC colocalizes with glycoprotein-positive specialized epithelial (goblet) cells and submucosal gland mucous cells and is highly inducible by various irritants (Borchers

1998). In contrast to MUC5AC, MUC5B is limited mainly to mucous cells in submucosal glands (Desseyn 1998) and is constitutively expressed (Borchers 1998).

Mucus overproduction in the airways: Mucus overproduction is a complex pathological process involving increase in the number of mucus producing cells

(hyperplasia), degranulation of mucus-secreting cells, and increased transcription of

1-5 mucin genes. Chronic exposure to tobacco smoke results in an increase in goblet cell number in humans and experimental animals (Reid 1950). Degranulation of mucus producing cells mediated by leukotrienes (Henderson 1996), mast cell chymase

(Sommerhoff 1989) and neutrophil elastase (Sommerhoff 1990) contribute to mucus overproduction. However, excess mucus produced by the increased number of goblet cells in the peripheral airways is a major contributing factor in the pathophysiology of

COPD (Hogg 1968) and there is consistent association between increased goblet cell number and increased MUC5AC expression in the lungs (Li 1998; Guzman 1996).

MUC5AC regulation: MUC5AC expression is increased by irritants and mediators including cigarette smoke, bacterial products like lipopolysaccharide (LPS), oxidative stress, cytokines including tumor necrosis factor (TNF)α, interleukin (IL)-4, IL-5, IL-9, IL-

13, and eicosanoids (prostaglandin E2, 12- and 15-hydroxyeicosatetraenoic acid)

(Leikauf 2002). Cigarette smoke (Takeyama 2001), activated eosinophils (Burgel 2001),

IL-13 (Kim 2002), neutrophil elastase (Kohri 2002), Pseudomonas aeruginosa (Kohri

2002), and phorbol 12-myristate 13-acetate (PMA) (Shao 2003) increase MUC5AC in airway epithelial cells (NCI-H292) by activating the epidermal growth factor receptor

(EGFR) and the mitogen activated protein kinase (MAPK) cascade. Inhibition of EGFR activity decreases the levels of mucin in response to various stimuli and reduced the goblet cell metaplasia (Takeyama 1999).

MUC5AC overproduction and epidermal growth factor receptor: The EGFR family of receptor tyrosine kinases in mammals contains four members: EGFR (ErbB1), ErbB2,

ErbB3, and ErbB4 (Lemmon 1994). Of these, EGFR, an 1186-amino acid residue transmembrane glycoprotein, is the prototypical member and is expressed in many cell types (Schlessinger 1988). Structurally, EGFR consists of an extracellular ligand binding

1-6 domain, a single alpha-helical transmembrane domain, an intracellular tyrosine kinase domain, and a COOH-terminal region that contains auto-phosphorylation sites (King

1989). Upon binding of specific polypeptide ligands including EGF, transforming growth factor (TGF)-α, betacellulin, heparin-binding EGF (Hb-EGF), epiregulin, and amphiregulin, EGFR undergoes homo- or hetero-dimerization and activation of its intrinsic tyrosine kinase activity (Schlessinger 1988). These events lead to the auto- phosphorylation of multiple tyrosine residues in the COOH terminal tail of the molecule that serve as binding sites for cytosolic signaling proteins containing Src homology 2

(SH2) domains and phosphotyrosine-binding domains. Activated EGFR, in turn, activates the Ras/MAPK pathway through initial tyrosine phosphorylation of the adaptor protein SHC, subsequent formation of an SHC-Grb2-Sos complex and Ras-mediated induction of Raf function. This prototypical-signaling pathway couples EGFR stimulation to gene transcription and mitogenesis.

In addition to its cognate ligands, the EGFR is activated by stimuli that do not directly interact with the EGFR ectodomain, including GPCR ligands, other receptor tyrosine kinase (RTK) agonists, cytokines, chemokines, and cell adhesion elements. EGFR can also be activated by non-physiological stimuli such as UV- and gamma-radiation, osmotic shock, membrane depolarization, heavy metal ions, and radical-generating agents, such as H2O2 (Prenzel 1999). EGFR ligands are synthesized as glycosylated membrane bound precursors (Massauge 1993), which are cleaved by proteinases to release functional ligands (Thorne 1994). Although the identity of proteinases commonly responsible for EGFR transactivation in MUC5AC expression has yet to be fully established, various MMPs are implicated in the ectodomain shedding of pro-EGFR ligands (Dempsey 2002). A related disintegrin and metalloproteinase domain protein

1-7 (ADAM), ADAM17, can also mediate EGFR activation in NCI-H292 cells following PMA or cigarette smoke treatment (Shao 2003, 2004).

MATRIX METALLOPROTIENASES

Background: Matrix metalloproteinases belong to the metzicin super family of metalloproteinases. They depend on the presence of a metal ion, usually Zn+2 for (i.e. ability to cleave internal peptide bonds) activity. More than 25 vertebrate MMPs and 22 human homologues have been identified to date (Reviewed by

Nagase & Woessner 1999). Though individual MMPs have distinct substrate specificities, nevertheless, together they can cleave numerous extracellular substrates, including virtually all ECM proteins (reviewed in Sternlicht 2001). All MMPs have an N- terminal signal sequence (or “pre” domain) that is removed after it directs their synthesis to the endoplasmic reticulum. Most MMPs are secreted; however, six display transmembrane domains and are expressed as cell surface . The protein also contains a propeptide “pro” domain that maintains latency until it is removed or disrupted, and a catalytic domain that contains the conserved zinc-binding region. In addition to their conserved zinc-binding motif (usually HEF/LGHS/ALGLXHS), and “Met turn” (usually ALMYP) (Bode 1993), the MMPs share added stretches of , giving them a fairly conserved overall structure (Stocker 1995). This chapter focuses principally on MMP9 and MMP14.

Matrix metalloproteinase 9: MMP9 is structurally the most complex members of the

MMP family (Cuzner and Opdenakker, 1999). MMP9 has a gelatin binding domain, composed of three fibronectin-repeats, inserted between the active-site domain

(Allan 1995) and the Zn2+-binding domain, and S/T/P-rich type V domain in a suggested hinge region. The Zn2+-binding domain of human MMP9 contains the

1-8 conserved sequence AHEXGHXXGXXH, in which the three histidines are responsible for the coordination of the catalytic Zn2+- ion. Together with the active domain, the Zn2+- binding domain forms the and is essential for the enzymatic activity. In the human proenzyme, the fourth ligand of the Zn2+ is C86 of the conserved sequence

PRCGXPD in the prodomain. This prodomain is removed by various types of proteolysis or is distorted by substrate binding (Bannikov 2002) to yield the active enzyme through the cysteine-switch mechanism (Van Wart and Birkedal-Hansen, 1990). The function of the hemopexin-domain is less clear. It was suggested to play a role in the substrate specificity and for various TIMPs. The fibronectin type II repeats are responsible for binding to gelatin, laminin, and type I and IV. The human and murine forms of

MMP9 have significant homology and the gene sequences and distribution of 13 exons and introns are similar (Masure 1993). Human MMP9 is localized to chromosome 20 q12-q13, and the mouse MMP9 has been localized to chromosome 2 (Leco 1997).

Once transcribed, MMP9 undergoes various post-translational modifications. MMP9 is heavily glycosylated (Rudd 1999) and contains three possible attachment sites for N- linked glycans, one of which is situated in the prodomain. The two other sites are located in the active domain. The collagen type V-like domain of MMP9 contains repeats of T/SXXP, which are attachment sites for the multiple O-linked glycans (Van den Steen 1998). These O-linked glycans give MMP9 a “bottle-brush” structure, as described for many glycoproteins, including mucins. In many cell types, MMP9 is produced as a mixture of monomers and homodimers. In addition, neutrophils produce a third form, a covalent complex of MMP9 with neutrophil B-associated lipocalin (NGAL) (Kjeldsen 1993).

1-9 Regulation of matrix metalloproteinase 9: To accomplish its normal functions, MMP9 must be present in the right cell type and pericellular location, at the right time, and in the right amount, and it must be activated or inhibited appropriately. MMP9 activity is tightly regulated at the transcriptional and post-transcriptional levels and also controlled at the protein level via its activators, inhibitors and its cell surface localization.

Transcriptional Regulation of MMP9: The biological function of MMP9 is largely regulated by its differential patterns of expression. In addition, expression of MMP9 is restricted spatially. Normal MMP9 expression is limited to osteoclasts, macrophages, trophoblast cells, hippocampal neurons, and migrating keratinocytes at the margins of healing wounds (Mohan 1998, Munaut 1999). MMP9 expression is regulated by numerous stimulatory and suppressive factors including several cytokines and growth factors, including IL-1α (Fabunmi 1996), IL-2 (Montgomery 1993), IL-8 (Van den Steen

2000), interferon (IFN) γ (Hujanen 1994), epidermal growth factor (EGF) (Harvey 1995;

Miyagi 1995), basic fibroblast growth factor (FGF) (Weston and Weeks, 1996), and transforming growth factor (TGF) β (Fini 1995). These stimuli induce the expression and/or activation of c-fos and c-jun proto-oncogene products, which heterodimerize and bind to various transcription factor binding sites in the MMP9 promoter.

More than 50 regions responsible for cell-specific expression in vivo have been identified in Mmp9 using mice carrying β-galactosidase reporter transgenes under the control of various portions of the MMP9 promoter (Mohan 1998). The MMP9 promoter is in a 2-kb

5' flanking region that contains activator protein (AP)-1, AP-2 (Sato 1993; Campbell

2001), SP-1 (Himelstein 1997), nuclear factor (NF)-κB (Han 2001) and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets)-binding sites (Sato and Seiki,

1993) and TGF-ß–inhibitory element (Huhtala 1991). AP1 sites give MMP9 the ability to

1-10 be induced by phorbol ester and act synergistically with adjacent Ets-binding sites and targeted disruption of the murine Ets2 transcription factor results in early embryonic lethality and deficient MMP9 expression (Yamamoto 1998). In addition, a functional p53- has been identified in the MMP9 promoter (Bian and Sun 1997).

Expression of MMP9 is also regulated by cell adhesion molecules (CAM) (Edvardsen

1993), components of (ECM) including laminin (Corcoran 1995), tenascin, and fibronectin (Tremble 1994). Cell-cell contacts also increased the expression of MMP9 in fibroblasts (Seagin 1996). MMP9 expression is also stimulated by transformation with variety of oncogenic viruses including SV40 in embryonic fibroblasts (Wilhelm 1989) and tumor promoters like PMA in corneal epithelial cells (Fini and Girard, 1990), skin keratinocytes and HT1080 cells (Wilhelm 1989; Huhtala 1991).

Basal and inducible levels of MMP expression are also influenced by genetic variations that may, in turn, influence the development or progression of several diseases.

Common bi-allelic single-nucleotide polymorphisms (SNPs) that influence the rate of transcription have been identified within several MMP promoters (Ye 2000). A SNP in the MMP9 promoter contains either a cytidine or thymidine 1562 bp upstream of the

MMP9 transcription start site (Zhang 1999). This SNP is associated with coronary artery disease progression (Ye 2000) and upper lung dominant emphysema (Ito 2005) despite their modest influence on gene transcription.

Signaling pathways for expression of MMP9: Different signaling cascades are involved in MMP regulation, depending on the stimulus and the cell type. Growth factors including EGF and TGF-α, increased MMP9 transcript via activation of EGFR and

MAPK3/2 (McCawley 1999). All three MAPK signaling pathways are involved in regulating MMP9 expression. MAPK3/2 (ERK1/2) and MAPK8 (JNK) activity was

1-11 essential for MMP9 expression in oncogenic transformed rat embryo cells and SCC cells, which display constitutive activation of both MAPK3/2 and/or MAPK8 (Gum

1997;Himelstein 1997; Simon 1999). Increase in MMP9 expression by IL-1 and TNF-α is mediated through activation of the transcription factors NF-κB and AP-1 via

JNK/SAPK or p38 MAPK pathways (Baud and Karin, 2001). Besides IL-1β and TNF-α, other cytokines like IL-17 have also increase MMP9 expression via MAPK signaling

(Jovanovic 2000). PMA induced MMP9 expression in SCC cells, required stimulation of the MAPK14 (p38) pathway (Simon 1998; Simon 2001). Thus, MMP9 expression is driven by the three MAPK signaling cascades. Despite numerous advances, the cross- talk between the many signaling pathways, nuclear factors, and gene regulatory elements that regulate MMP9 expression are barely understood. Little is known about

MMP9 transcript stablization, however MMP1 and MMP3 transcripts are stabilized by phorbol esters and EGF, whereas MMP13 transcripts are stabilized by PDGF and glucocorticoids and destabilized by TGF-β (Delany 1995, Vincenti 2001).

Although most MMPs are constitutively secreted once they become translated, MMP9 is synthesized by differentiating granulocytes in the bone marrow, stored in the specific and gelatinase (tertiary) granules of circulating neutrophils, respectively, and released following neutrophil activation by inflammatory mediators (Sternlicht and Werb 2002).

Activation of Latent MMP9: MMP9 is first synthesized as inactive proenzyme, pro-

MMP9. Latency is maintained by an unpaired cysteine sulfhydryl group near the C- terminal end of the propeptide domain. Proteolytic removal of the propeptide domain or by ectopic perturbation of the cysteine-zinc interaction (VanWart and Birkedal-Hansen

1990) opens the cysteine-to-zinc switch to a water molecule. The extracellular activation of MMP9 can be initiated by other already activated MMPs including MMP2 (Fridman

1-12 1995), MMP3 (Ogata 1992), MMP7 (Imai 1997; Sang 1995), MMP10 (Nakamura 1998),

MMP13 (Knauper 1997) or by several serine proteinases including neutrophil elastase

(Ferry 1997), tissue kallikrein (Desrivieres 1993) and mast cell chymase (Fang 1996) that can cleave peptide bonds within MMP9 prodomain.

Pericellular localization of proteolytic activity: Many of the extracellular signaling events that regulate cell behavior occur at or near the cell membrane, and many cellular signals are created or canceled via pericellular proteolysis (Werb 1997, Werb & Yan

1998). The processing of pericellular proteins by proteolysis is an ideal means of regulating extracellular signal transduction. Although pericellular proteolysis may sometimes reflect the exclusive expression of a critical substrate at or near the cell surface, there are specific mechanisms that confine or concentrate proteinases in the immediate pericellular microenvironment. The expression of membrane-bound MMPs, the binding of MMPs to cell surface receptors, the presence of cell surface receptors for

MMP-activating enzymes such as urokinase plasminogen activator (uPA), plasmin, thrombin, and elastase; and the concentration of MMPs on pericellular ECM molecules serve to localize MMPs to the cell surface and to specific cell surface sub domains.

These localization mechanisms often enhance MMP activation, limit the access of MMP inhibitors, concentrate MMPs within the vicinity of their targets, and limit the extent of proteolysis to discrete pericellular regions.

Latent pro-MMP9 binds with high affinity to type IV collagen α2 chains on the surface of several cell types, thus concentrating it at the cell-ECM interface in direct contact with its target (Olson 1998, Toth 1999). Activated MMP9 binds to the cell surface hyaluronan receptor CD44 (Bourguignon 1998) and localization of MMP9 to the cell surface by

CD44 promotes tumor cell invasion and angiogenesis and mediates the activation of

1-13 latent TGF-β by MMP9 (Yu & Stamenkovic 2000). Moreover, cell surface heparan sulfates act as docking molecules for tissue inhibitor of metalloproteinase protein (TIMP)

3. Thus there exist localized pericellular feed-back loop which can direct the appropriate expression, activation, and physical placement of MMP9.

Matrix metalloproteinase 14: MMP14 is a prototypical member of membrane tethered group of MMP gene family (MT-MMP). Unlike other MMPs, which are secreted into the pericellular space, MT-MMP are membrane bound by at least three distinct anchoring mechanisms, including type I transmembrane domain for MMP14. MMP14 expression is associated with a variety of cellular and developmental processes, as well as multiple pathophysiological conditions (reviewed by Yana and Seiki 2002). MMP14 displays a broad spectrum of activity against ECM components such as type I and II collagen, fibronectin, vitronectin, laminin, fibrin and (d’Ortho 1997) and MMP14's collagenolytic activity is critical during development, as mice deficient in MMP14 suffer severe complications in remodeling of skeletal and extraskeletal connective tissues resulting in earlier death (Holmbeck 1999). So far, MMP14 is the only MMP that has been found to be essential for survival.

MMP14 structure: MMP14 has a signal peptide directing its trafficking to the cell membrane, a prodomain which keeps the enzyme inactive and a catalytic domain with a conserved HEXXHXXGXXH motif for Zn+2 binding, a hinge region which connects catalytic domain with the c-terminal hemopexin like domain and a transmembrane hydrophobic sequence followed by a short cytoplasmic tail (Sato 1994). In addition,

MMP14 contains an RXK/RR consensus sequence which is recognized by pro-protein convertase (Takino 1995)

1-14 Regulation of matrix metalloproteinase 14: MMP14 activity is tightly regulated at the transcriptional and post-transcriptional levels and also controlled at the protein level via its activators, inhibitors and its cell surface localization. MMP14 is mainly expressed in cells of mesenchymal origin including fibroblasts, muscular cells, and osteoblasts in developing embryo (Kinoh 1996). In contrast to MMP9, MMP14 is expressed in most of the tissues with high expression in the lungs, kidney and the intestine (Takino 1995). In lungs, MMP14 is expressed in surface epithelial cells (Fukuda 1998), type II cells

(Kunugi 2001) and endothelial cells (Hiraoka 1998). MMP14 expression is regulated by numerous stimulatory and suppressive factors including several cytokines and growth factors, IL-2 (Galvez 2004) and IL-8, growth factors including EGF (Kheradmand 2002), fibroblast growth factor (FGF) 1 (Udaykumar 2004), vascular endothelial growth factor

(VEGF) (Munaut 2003), insulin like growth factor (IGF)-1 (Zhang 2003) induced MMP14 expression in prostrate carcinoma cell lines. These stimuli induce the expression and/or activation of c-fos and c-jun proto-oncogene products, which heterodimerize and bind to various transcription factor binding sites in the MMP14 promoter. Not much is known about the signal transduction pathways involved in increased MMP14 expression.

Inhibition of MAPK3/2 decreased MMP14 expression in fibrosarcoma cells (Tanimura .

2003). MAPK3/2 but not MAPK8 or MAPK14 regulate increased MMP14 expression in rat endothelial cells (Boyd 2005).

Activation of MMP14: Because of its critical role in physiology and pathology, MMP14 activity on the cell surface is subject to multiple control mechanisms. Like other MMPs,

MMP14 is produced as an inactive zymogens (60 kDa) containing a secretory signal sequence and a propeptide whose proteolytic cleavage is required for MMP14. A basic tetrapeptidic sequence RXRR is inserted between the propeptide and the catalytic domain. This furin recognition motif is cleaved by proprotein convertases in the trans

1-15 Golgi network during the trafficking of MT-MMPs from the endoplasmic reticulum to the plasma membrane (Rozanov 2001). Activation of MMP14 by a furin-independent alternative pathway has been reported in some types of cells. This alternative activation pathway depends on autoproteolytic activation or on the action of non-furin proprotein convertases, or on other proteinases located at the plasma membrane (Sato 1996; Cao

1995; Okumura 1997).

Since MMP14 is expressed on the cell surface as an active form, inhibition is one of the critical steps to regulate its activity. Active MMP14 (56 kDa) undergoes further processing to a 44-45 kDa species by MMP2 or MMP14 itself (Lehti 1998; Stanton 1998;

Toth 2002). This removes the catalytic domain of MMP14 making it inactive, and is a mechanism of downregulation. Thus, MMP14 can be ‘functionally active’ and as a result undergoes further processing to an ‘functionally inactive’ form. MMP14 is internalized by clathrin-dependent and caveolae-dependent pathways (Jiang 2001). Internalization removes MMP14 from the cell surface and is thus a mechanism of downregulation.

Clathrin-dependent internalization of MMP14 is mediated through the C-terminal cytoplasmic tail (Uekita 2001). MMP14 is also inhibited by endogenous inhibitors

TIMP2, 3, and 4, but not by TIMP1 (Will 1996; Bigg 2001). A GPI-anchored glycoprotein, reversion-inducing-cysteine rich protein with Kazal motifs (RECK) is a potent inhibitor of MMP14 (Oh 2001).

Regulation of cell signaling by MMPs: In addition to cleaving most of the structural

ECM molecules, MMPs can also cleave several circulating, cell surface and pericellular proteins, which enables them to regulate cell behavior. These mechanisms include the alteration of cell-matrix and cell-cell interactions; the release, activation, or inactivation of autocrine or paracrine signaling molecules; and the potential activation or inactivation of

1-16 cell surface receptors. Extracellular matrices are not just passive cellular scaffolds; they influence cell behavior by sequestering signaling molecules, such as growth factors and growth factor-binding proteins, and by acting as ligands for cellular adhesion receptors, such as integrins, that transduce signals to the cell interior (Streuli 1999). ECM regulates basic processes such as cell shape, movement, growth, differentiation, and survival by controlling cell adhesion and the cytoskeletal machinery (Lukashev & Werb

1998). Thus various MMPs can also influence these same processes by altering the composition and structural organization of the ECM, thereby altering matrix-derived signals. Moreover, proteolytic ECM remodeling results in the release of modular breakdown products with biologic activity.

ECM molecules also act as binding reservoirs for various growth factors and cytokines that are released once the ECM molecules are degraded. For example, the small collagen-associated proteoglycan decorin acts as a depot for TGF-β, and its degradation by various MMPs makes the otherwise sequestered TGF-β available to carry out its biologic functions (Imai 1997). One such function is to inhibit the expression of several

MMP genes. Thus MMP-mediated activation and release of TGF-β may act as a negative feed-back mechanism to limit MMP expression and further TGF-β release.

Endogenous Metalloproteinase Inhibitors: TIMPs represent a family of at least four

20–29-kDa secreted proteins (TIMPs 1–4) that reversibly inhibit the MMPs in a 1:1 stoichiometric fashion (Strenlicht & Werb 1999) (Table 1). They share a conserved gene structure, and 12 similarly separated cysteine residues. These invariant cysteines form six intrachain disulfide bridges to yield a conserved six-loop, two-domain structure.

Though TIMP 1 and 2 contain only the first three loops, they retain their inhibitory activity, thus indicating that portions of the N-terminal domain interact with the MMP

1-17 catalytic site (Murphy & Willenbrock 1995). Individual TIMPs differ in their ability to inhibit various MMPs (reviewed in Woessner & Nagase 2000). TIMP1 inhibits MMP9

(Sellers 1979), but not MMP14. TIMP3 binds with MMP9 and inhibits it. TIMP2 and

TIMP3 inhibit MMP14, whereas TIMP1 does not. Likewise, TIMP1 is a relatively poor inhibitor MMP16, and TIMP3 appears to be a more potent inhibitor of MMP9 than other

TIMPs. TIMP3 is also unique in its ability to inhibit ADAM10 and 17(Kashiwagi 2001). In addition, the TIMPs differ in terms of their gene regulation and tissue-specific patterns of (Edwards 2001). TIMP3 also has the unique ability to bind via its C- terminal domain to heparin sulfates within the ECM, thereby concentrating it to specific regions within tissues and basement membranes (Langton et al. 1998).

Table 1: Tissue inhibitor of metalloproteinase proteins (TIMP)s

TIMP1 TIMP2 TIMP3 TIMP4

Mol Wt (kDa) 28.5 21 24-27 24

Chromosomal Xp11.23-11.4 17q2.2-2.5 22q12.1-13.2 3p25 location

Promoter TATA box-less TATA box, AP-1 TATA box, 6 AP-1 TATA box less, AP-1, PEA3 sites no AP-1

Glycosylation yes no highly no

Solublility yes yes ECM associated yes

Regulation Transcriptional Constitutive Transcriptional Transcriptional

MMP14 inhibition no yes yes yes

ADAM17 no no yes no inhibition

Trimolecular MMP9/TIMP1 MMP14/TIMP2 No complex with No complex with complex /MMP3 /pro-MMP2 MMP2 or MMP14 MMP2 or MMP14 formation

1-18 TIMPs are not the only endogenous MMP inhibitors. Indeed, α2-macroglobulin is a major endogenous inhibitor of the MMP9 (Sottrup-Jensen and Birkedal-Hansen 1989), and its importance may have been overlooked. α2-macroglobulin is an abundant plasma protein and thus represents the major inhibitor of MMPs in tissue fluids, whereas

TIMPs may act locally. Moreover, because α2-macroglobulin/MMP complexes are removed by scavenger receptor-mediated endocytosis, α2-macroglobulin plays an important role in the irreversible clearance of MMP9, whereas TIMPs inhibit MMP9 in a reversible manner.

Cell surface proteolysis and cellular signals: MMPs can potentially influence cell behavior by cleaving cell-cell adhesion proteins, by releasing bioactive cell surface molecules, or by cleaving cell surface molecules that transduce signals from the extracellular environment. MMP14 modulates the bioavailability of TGFβ by releasing active TGFβ from cell surface complexes involving αvβ8 integrin (Mu 2002). A variety of growth factors, growth factor-binding proteins, growth factor receptors are MMP substrates. The degradation of insulin-like growth factor-binding protein (IGF-BP) by several soluble MMPs releases IGFs (Manes 1999). Cleavage of perlecan releases fibroblast growth factors (Whitelock 1996). HB-EGF shedding is mediated by a specific proteolytic cleavage at a juxtamembrane site through the action of MMP3 (Suzuki 1997) and MMP7 (Yu 2002).

Several growth factor receptors are released by MMPs, for example, the release of soluble FGFR-1 is mediated by MMP2 (Levi 1996). MMP14 by mediating the activation of MMP13 and MMP2 (Sternlicht and Werb 2002) can regulate a wide range of cellular processes. Monocyte chemoattractant protein-3 (MCP-3) is inactivated by MMP14

(McQuibban 2002). Pro-TNFα can be proteolytically activated by MMP14 (d’Ortho

1-19 1997). Cell surface adhesion receptor CD44 (Kajita 2001), the αv chain of the αvβ3 integrin (Deryugina 2002), and proteoglycans like syndecan-1 (Endo 2003) are also processed by MMP14. Low density lipoprotein receptor (LDLR)-related protein (LRP), a cell surface-associated endocytic receptor, is implicated in the internalization and degradation of multiple ligands such as thrombospondins, α2-macroglobulin- complexes, urokinase- and tissue-type plasminogen activators, MMP2, MMP9 and

MMP13 (Rozanov 2004). The cleavage of LRP by MMP14 leads to the control of the bioavailability and fate of many ligands and soluble MMPs in cancer progression

(Strickland 2002). MMP2, cleaves cell surface FGF receptor 1 releasing a soluble receptor fragment that retains its ability to bind FGF (Levi 1996). MMP9 cleaves IL-2 receptor α (IL2Rα) on T cells and significantly down regulates their proliferative response to IL-2 in culture (Sheu 2001).

Regulation of paracrine signals: By releasing several cell surface and matrix-bound growth factors and cleaving cell surface receptors, MMPs also regulate the availability of paracrine signals either by inactivating them directly or by inactivating their soluble binding proteins. For example, MMP9 can cleave plasminogen to generate the angiogenesis inhibitor angiostatin (Patterson and Sang 1997). Many of these MMP- mediated mechanisms contribute to a variety of biologic processes, but it is still largely unclear which mechanisms are crucial.

1-20

ACROLEIN

Background: Acrolein (H2C=CH-CHO) is also known as acrylaldehyde, acrylic aldehyde, allyl aldehyde, ethylene aldehyde, 2-propenal, and prop-2-en-1-al (Izard and

Liebermann, 1978). Acrolein (specific gravity = 0.9 g/ml; boiling point = 52.5°C) is a colorless to yellowish flammable liquid at room temperature. Acrolein is highly volatile liquid (vapor pressure = 274 mm Hg @ 25°C) with a disagreeable odor.

Sources of human exposure

Occupational exposure: Exposure to acrolein in occupational settings occurs as the result of its varied uses and its formation during the combustion and pyrolysis of materials such as wood, petrochemical fuels, and plastics. Some of these occupations include those involved in the production of acrylates, methionine, perfumes, plastics, refrigerants, rubber, or textile resins (Ghilarducci and Tjeerdema 1995). Firefighters are at risk to exposure to acrolein when battling house fires and wild fires (Ghilarducci and

Tjeerdema 1995; Gochfeld 1995; Lees 1995; Materna 1992). The concentrations of acrolein measured in a NIOSH house fire study ranged from not detected to 3.2 ppm, with half of the exposures exceeding the 0.3 ppm short-term exposure limit.

Outdoor exposure: Production of acrolein in the air occurs through photochemical reactions of volatile organic compounds (VOC), released from stationary and mobile sources, including solvent and fuel vapors, automobile exhaust (Ghilarducci and

Tjeerdema 1995; Liu 1999) and from facilities involved in the manufacture or use of products containing acrolein; like glycerine, methionine, glutaraldehyde and other

1-21 organic chemicals (Arntz 2002; Etzkorn 2002). Exposure of the general population occurs primarily through inhalation and the mean ambient acrolein concentration is 6.2 ppb (Destaillats 2002). Acrolein has been detected in exhaust gases from both gasoline engines (0.01-10 ppm) and diesel engines (0.05-4.5 ppm) (Nishikawa 1987).

Indoor exposure: Concentrations in indoor air may exceed outdoor levels 2 to 20 fold times (Evans 2000) mainly through the exposure to environmental tobacco smoke

(ETS). More than 30 million nonsmokers in the United States were exposed to acrolein concentrations in indoor air ranging from 0.8-1.5 ppm (Nazaroff and Singer, 2004). ETS is enriched in acrolein (as high as 100-700 µg acrolein/ cigarette) and other low molecular weight aldehydes due to altered combustion chemistry of a smoldering cigarette. Levels between 0.1-10 ppm were detected in bars and restaurants. In homes where wood stoves were used, concentrations from 0.2-3 ppm have been reported

(Highsmith and Zweidinger, 1988). Acrolein is also generated by pyrolysis of vegetable oils or organic chemcials. Levels as high as 24 ppm were detected 15 cm above heated cooking oil (Fortes 1994). Burning of polyethylene foam can generate 2-23 ppm of acrolein (Boettner 1980).

Endogenous acrolein: Acrolein is generated endogenously during the process of lipid peroxidation (Esterbauer 1991). Acrolein has been identified as both a product and initiator of lipid peroxidation (Adams and Klaidman, 1993, Uchida 1999). This oxidation process occurs in all cells, and is enhanced in many toxic and pathophysiological situations, hence there is a potential for virtually continuous exposure to acrolein.

Acrolein is a byproduct in the metabolism of cyclophosphamide, an anticancer agent

(Alarcon 1976). Acrolein is also generated from theronine by neutrophil myeloperoxidase at sites of inflammation (Anderson 1997).

1-22

Table 1: Sources of acrolein

Source Limit Reference

Auto Gas exhaust Not detected to 27.2 Gasoline engine ppm Lipari 1982; Nishikawa 1987

Diesel engine 0.05-0.3 ppm Seizinger 1972,

Tobacco Smoke Cigarette smoke 3-220 ug/cigarette Dong 2000; Hoffman 1975 Marijuana smoke 92-145 ug/cigarette Hoffman 1975; Horton 1975 Smoking areas of Bars/Restaurant 0.1-10 ppm Nazaroff and Singer 2004

Smoke Wood 50 ppm Einhorn 1975 Cotton 60 ppm Kerosine < 1 ppm

Wood burning fireplaces 0.2-3 ppm Lipari 1984,

Pyrolysis Pyrolysis of polyethylene 2-23 ppm Boettner 1980 Pyrolysis of cooking oil 2-24 ppm

Burning candle 0.1-0.5 ppm Lau 1997

Health effects of acrolein

Human studies:

Acute Exposures: Exposure to 5.5 ppm of acrolein resulted in painful eye and nose irritation after 20 seconds, and 22 ppm was immediately intolerable (Esterbauer 1991).

Human subjects exposed to 0.6 ppm acrolein for 40 minutes developed nasal and throat irritation (Weber-Tschopp 1977). A 27-month-old boy exposed to probable high levels of acrolein (and other chemicals) from burning vegetable oil for one hour, developed diffuse brochiectasis in a few months (Mahut 1993). In summary, based upon the available

1-23 human data, levels as low as 0.09 ppm for 5 minutes may elicit subjective complaints of eye irritation with increasing concentrations leading to more extensive eye, nose and respiratory symptoms. No chronic studies of humans exposed to acrolein are reported in the literature.

Animal studies

Acute exposures: Fischer 344 rats exposed 100–40,000 ppm of acrolein of for short periods of time (

1986). Rabbits exposed to 375-489 ppm of acrolein developed edema, necrosis of the lung parenchyma, and damage to the bronchial linings of the large airways within 3 days of exposure (Beeley 1986). Acute inflammatory reactions were found in conjunction with areas of necrosis.

Sprague-Dawley rats exposed to 81 ppm of acrolein for 1 hour developed clinical signs of sensory irritation, respiratory distress and hypoactivity. There was congestion, mottled discoloration of the lungs, intra-alveolar hemorrhage and clear fluid in the trachea. Acute exposure of mice, rats, and guinea pigs to concentrations of 0.3–17 ppm acrolein for several minutes induced vasodilation (Morris 1999), as well as an increase in airflow resistance and a reflex decrease in respiratory rate by activation of the sensory nerve endings in the nasal mucosa (Alarie 1973; Buckley 1984; Kane and Alarie 1977;

Leikauf 1989; Morris 1999, 2003; Murphy 1963; Nielsen 1984; Steinhagen and Barrow

1984). Since acrolein exposure in the workplace is usually concurrent with other chemicals, particularly aldehydes, several studies have examined the effects of acrolein

1-24 exposure with pre-exposure and co-exposure to other chemicals. In Fischer 344 rats exposed to 15 ppm formaldehyde 6 hr/day, for 9 days followed by exposure to acrolein for 10 minutes on the 10th day. RD50 (concentration of acrolein which reduced the respiratory rate by 50%) increased to 29.6 ppm in pre-exposed animals compared to 6 ppm in the controls. This suggests that pre-exposure to lower concentrations of sensory irritants desensitize animals to sensory irritation effects of acrolein. Bronchial hyper- reactivity increased in guinea pigs exposed to 0.31-1.26 ppm of acrolein for 2 hour

(Leikauf 1989, 1991).

Chronic Exposures: Chronic exposure of guinea pigs, rats, and mice to acrolein causes pulmonary inflammation, decreases in respiratory rate, and nasal lesions, effects also seen upon acute exposure. The effects of inhaled acrolein on laboratory animals are shown in Table 2. Fischer 344 rats were exposed to acrolein at 0, 0.4, 1.4, or 4.0 ppm for 6 hr/day, 5 days/week for 62 exposure days to examine the lung function and pathology (Kutzman 1981, 1985). Exposure to 4.0 ppm of acrolein resulted in mortality due to severe acute bronchopneumonia. Lung hydroxyproline per mg protein (as an index of lung collagen) was increased 113 and 137% above controls (p<0.05) in the 1.4 and 4.0 ppm (3.2 and 9.2 mg/m3) groups, respectively. The surviving animals demonstrated bronchiolar epithelial necrosis and sloughing, bronchiolar edema with macrophages, and focal pulmonary edema. Rats exposed to 0.4 and 1.4 ppm of acrolein did not exhibit pulmonary lesions attributable to acrolein exposure. No adverse histopathology was noted in other tissues examined.

Male Wistar rats (5-6/group) were exposed 6 hr/day, for 3 consecutive days, in a nose- only exposure chamber to acrolein at concentrations of 0, 0.25, 0.67, or 1.4 ppm

(Cassee 1996). After one 6-hour exposure, no treatment-related histopathological

1-25 lesions were found in any of the treatment groups. After 3 days, slight to moderate effects were noted in acrolein exposed animals There was disarrangement, necrosis, thickening and desquamation in the respiratory/transitional epithelium, rhinitis or in the severe cases, single cell necrosis or atrophy of the olfactory epithelium.

White albino male mice exposed to 25-50 ppm for 6 hr/day, 5 days/week for 2 weeks.

There was no mortality when the animals were exposed to 25 ppm but the mortality increased significantly (91% mortality) when mice were exposed to 50 ppm (Philippin

1970). Mucus hypersecretion and increased mucin 5 (subtype A and C) (MUC5AC) transcripts was observed in the lungs of Sprague-Dawley rats exposed to 3 ppm acrolein for 6 hr/day, 5 days/week for 2 weeks (Borchers 1997). FVB/NJ male mice exposed to

3.0 ppm acrolein for 6 hr/day, 5 days/week for 3 weeks had a significant and persistent increase in macrophages in bronchoalveolar lavage fluid (Borchers 1999).

1-26 Table 3: Effect of inhaled acrolein on laboratory animals:

Species Exposure Concentration Principal findings References duration ppm

Rats Wistar 6 h/day for 3 0, 0.25, 0.67, Nasal necrosis of respiratory epithelium and increased proliferation Cassee (1996) days and 1.4 up to 0.67 ppm; 1.4 ppm group not evaluated.

S-D 6 h/day 0, 0.1, 1.0, No effect on macrophage killing of inhaled K. pneumonia. Sherwood (1986) 5 days/wk for 3 and 3 wks

F-344 6 h/day for 62 0, 0.4, 1.4, 1. High mortality at 4 ppm. Kutzman (1981); days and 4 2. Increase in lung collagen at 1.4 and 4 ppm (p<0.05). Kutzman (1985). 3. Elastin content in 4 ppm group twice controls. Costa (1986) 4. Bronchial necrosis and pulmonary edema at 4 ppm.

Bouley .(1975, SPF-OFA 77 days 0, 0.55 1. Decrease in alveolar macrophage. 1976) 2. No effects on reproductive potential.

Mouse Swiss- 6 h/day for 1. Lesions of moderate severity in respiratory epithelium except for Webster 5 days 1.7 severe squamous metaplasia. Buckley (1984) 2. Ulceration and necrosis in olfactory epithelium with squamous metaplasia. 3. Incomplete recovery after 72 hours.

FVB/NJ 6 h/day 3 Acrolein-induced excessive macrophage accumulation associated Borchers (1999) 5 days/wk for 3 with mucus hypersecretion. wks

CDI 3 h/day for 5 days 0.1 Decreased (p<0.01) in percent killing of S. zooepidemicus and Aranyi (1986) K. pneumonia.

Toxicokinetics of acrolein

Absorption and distribution: Respiratory studies in dogs indicate that inhaled acrolein is retained at rates of 75-80% in the upper respiratory tract (URT) with a lesser rate of retention (65-70%) for the lower respiratory tract. At inhaled concentrations of 176-264 ppm, 80-85% was retained in the total respiratory track at varying ventilation rates, suggesting little distribution elsewhere (Egle 1972). Studies with radiolabeled [2, 3-14C] acrolein in rats (Parent 1996, 1998) indicate little systemic distribution of acrolein.

Metabolism and excretion: Acrolein reacts directly with protein and non-protein sulphydryl groups mainly at the cell surfaces, but when absorbed into the cells it can react with primary and secondary amines found in proteins (Ghilarducci and Tjeerdema,

1995). In proteins, it preferentially attacks free sulphydryl (SH) groups of cysteine, amino groups of lysine and histidine residues (Esterbauer 1991). Acrolein binds to serum albumin and low-density lipoproteins including ApoA1, invitro (Uchida 1998).

Conjugation of the carbon of acrolein with sulphydryl groups is rapid (rate constant 220

M-1sec-1) and essentially irreversible (Esterbauer 1976), and leads to thiazolidine derivatives and a decrease in glutathione (GSH) stores without an increase in oxidized

GSH (GSSG). This pathway results in an acrolein-GS adduct, GS-propionaldehyde which is then further metabolized by both high- and low-affinity forms of mitochondrial and cytosolic aldehyde and alcohol dehydrogenase (Mitchell and Peterson, 1989); one resultant product has been identified as 3-hydroxypropyl-mercapturic acid (Kaye and

Young 1970). This product has been isolated from urine of rats after subcutaneous injection of acrolein (Kaye 1973) and after inhalation and intraperitoneal (ip) injection of

Wistar rats (Linhart 1996). The reduction of the acrolein-GSH adduct by alcohol dehydrogenase to 3-hydroxypropyl-mercapturic acid was postulated as a potentially important pathway (Mitchell and Peterson, 1989).

1-28

In Vitro Toxicity studies: Concentrations of acrolein used in the invitro experiments in current study were 0.03-0.1 µM. A number of in vitro studies have demonstrated that acrolein has the potential to perturb the environments of human and laboratory animal cells in which GSH plays an important role. GSH depletion in isolated rat hepatocytes incubated with 0.25-0.5 µM acrolein caused lipid peroxidation and impaired integrity of cell membranes (Zitting and Heinonen 1980). GSH protects cells by removing reactive metabolites such as electrophilic carbonium ions. Thus, GSH depletion deprives the cell of its natural defense against ubiquitous reactive metabolites and leaves the thiol groups in critical proteins vulnerable to attack by oxidation, cross-linking, and the formation of mixed disulfides or covalent adducts.

In vitro concentrations of 25 to 100 µM are lethal to pulmonary artery endothelial cells

(Kachel and Martin, 1994, Patel and Block, 1993), bronchiolar epithelial cells (Grafström

1988), and both bronchial (Krokan 1985) and cardiac fibroblasts (Torasson 1989).

Acrolein at concentrations of 5-50 µM resulted in disruption of actin cytoskeletal fibers in cultured pulmonary artery endothelial cells (Joseph 1994). Survival of human alveolar macrophages was significantly decreased following 24 hours exposure to acrolein concentrations of 25 µM or greater (Li 1997). Acrolein (3 µM) decreased the colony forming efficiency 50% in human bronchial epithelial cells (Grafström 1988) and in cultured human bronchial fibroblasts (Krokan 1985). Acrolein inhibited the release of the cytokines IL-1, TNF-α, and IL-12, and induced apoptosis and necrosis in human alveolar macrophages by inhibiting the phosphorylation of NF-κB (Li 1997, 1998). Acrolein also inhibits the in vitro synthesis of prostaglandin E2 in rat resting and zymosan-stimulated alveolar macrophages in a dose dependent manner (Grundfest 1982).

1-29 Acrolein and lipid peroxidation: Lipid peroxidation is implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging. Lipid peroxidation proceeds by a free-radical chain reaction mechanism and yields reactive aldehydes as including acrolein, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) and as end products (Uchida 1999). There is increasing evidence that aldehydes generated endogenously during lipid peroxidation contribute to the pathophysiologic effects associated with oxidative stress in cells and tissues.

Acrolein is the most electrophilic of all α, β-unsaturated aldehydes and shows the highest reactivity with nucleophiles such as proteins (Esterbauer 1991). Acrolein-lysine adducts were detected in atherosclerotic plaques in the aorta, where it was with macrophage-derived foam cells (Uchida 1998). The role of acrolein as an endogenous cytotoxic compound in pathogenesis of inflammation needs further investigation.

1-30 FOCUS OF THE DISSERTATION:

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity (>14 million cases) and mortality (>110,000 deaths/yr) in the United States (Anderson 2001).

COPD is marked by excessive mucus production, chronic cough, shortness of breath, and labored breathing, and results in an irreversible loss of lung function (Markewitz

1999). The etiology of COPD has been studied extensively, and it is clearly linked to cigarette smoking and other environmental exposures (Davis 1989). However, questions remain about the pathophysiological mechanisms controlling this complex condition, and despite numerous clinical trials, current therapy is limited mainly to supportive measures. COPD involves disruption of the alveolar (emphysema) and alteration of the airway (bronchitis) architecture. The latter involves a phenotypic shift in the epithelium with decreases in ciliated and Clara cells accompanied by an increase in mucus producing cells (Reid 1950). This shift is mediated by metalloproteinase mobilization of EGF family ligands that bind to and activate receptor-type protein tyrosine kinases, including EGFR (Takeyama 1999). EGFR, in turn, activates mitogen-activated protein kinase (MAPK) signaling that mediates cell proliferation, , and in the airway epithelium, mucin gene expression. Matrix metalloproteinases play a critical role in pathogenesis of COPD (Atkinson 2003). However, the role of MMPs in mucus overproduction is unclear. Various inflammatory mediators including, chemokines like

IL-8, cytokines like TNF-α, reactive oxygen are present in high concentration in the sputum of COPD patients (Keatings 1996), particularly during exacerbations (Aaron

2001). These agents are also known to increase transcription of multiple MMPs, in various cell lines (Opdenakker 2002). Previously Borchers et al. reported a critical role for MMP12 in acrolein-induced MUC5AC expression (1999). Excessive macrophage accumulation was observed in FBV/NJ control mice as compared to Mmp12-/- knock out mice. Macrophage accumulation correlated with level of MUC5AC transcripts in the

1-31 lungs. Kim et al. demonstrated that mice exposed to lipopolysaccaharide (LPS) demonstrated mucus metaplasia (2004). Pretreatment with an MMP inhibitor effective against several MMPs, decreased the LPS-induced mucin production. This study seeks to identify the specific MMPs involved in irritant-induced MUC5AC increase and investigate the interactions between MMPs and EGFR mediated pathways to orchestrate a persistent increase in MUC5AC production.

HYPOTHESIS:

MMPs mediate increased MUC5AC in the lungs airways after acrolein exposure.

SPECIFIC AIM 1:

Determine the identity of various MMPs involved in acrolein-induced increase in

MUC5AC. (Covered in manuscripts II and III).

SPECIFIC AIM 2:

Determine the signal transduction pathway involved in acrolein-induced MUC5AC increase. (Covered in manuscript I).

1-32 CHAPTER 2

Manuscript I

Metalloproteinases mediate Mucin 5, subtype A and C (MUC5AC) expression by epidermal growth factor receptor activation.

ABSTRACT

Chronic obstructive pulmonary disease (COPD) is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke increases MUC5AC, a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses ≥ 10 µM, higher than those sufficient (0.03 µM) to increase MUC5AC mRNA suggesting that

MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor

(EGFR) and mitogen activated protein kinase 3/2 [MAPK 3/2(ERK1/2)]. Pretreating the cells with an EGFR neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small interfering RNA (siRNA) directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase.

Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloprotienase-3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Taken together these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.

2-1 Metalloproteinases Mediate Mucin 5AC Expression by Epidermal Growth Factor Receptor Activation

Hitesh S. Deshmukh, Lisa M. Case, Scott C. Wesselkamper, Michael T. Borchers, Linda D. Martin, Howard G. Shertzer, Jay A. Nadel, and George D. Leikauf

Departments of Environmental Health and Pulmonary and Critical Care Medicine, University of Cincinnati, Cincinnati, Ohio; Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina; Cardiovascular Research Institute and Departments of Medicine and Physiology, University of California–San Francisco, San Francisco, California

Chronic obstructive pulmonary disease is marked by alveolar at serine and theronine residues (5). Mucins are encoded by at enlargement and excess production of airway mucus. Acrolein, a least 15 genes, including a cluster localized to human chromo- component of cigarette smoke, increases mucin 5AC (MUC5AC), a some 11p15 (6), with at least nine genes expressed in the lungs prevalent airway mucin in NCI-H292 cells by transcriptional activa- (5). Mucin 5AC (MUC5AC) constitutes the majority of mucin tion, but the signal transduction pathways involved in acrolein- glycoproteins in the airway secretions of humans (7) and is highly induced MUC5AC expression are unknown. Acrolein depleted inducible (8–10). Specialized epithelial (goblet) cells are the cellular glutathione at doses of 10 ␮M or greater, higher than those ␮ major source of MUC5AC in the airways. sufficient (0.03 M) to increase MUC5AC mRNA, suggesting that Cigarette smoking is the most common cause of chronic ob- MUC5AC expression was independent of oxidative stress. In con- structive pulmonary disease (3). In humans, chronic exposure to trast, acrolein increased MUC5AC mRNA levels by phosphorylating tobacco smoke results in an increase in the number of goblet cells epidermal growth factor receptor (EGFR) and mitogen-activated ϭ protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with because of hyperplasia and metaplasia (11). Acrolein (CH2 an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, CHCHO) is a potent irritant aldehyde present in tobacco smoke decreased the acrolein-induced MUC5AC mRNA increase. Small, and is a constituent of wood smoke, diesel exhaust, and photo- interfering RNA directed against ADAM17 or MMP9 inhibited the chemical smog (12). Acrolein reacts rapidly with cellular nucleo- acrolein-induced MUC5AC mRNA increase. Acrolein increased the philes (e.g., sulphydryl-containing cysteines and peptides) and release and subsequent activation of pro-MMP9. Acrolein increased depletes cellular thiols, including reduced glutathione (GSH), MMP9 and decreased tissue inhibitor of metalloproteinase 3 thereby inducing oxidative stress in primary airway epithelial (TIMP3), an endogenous inhibitor of ADAM17, transcripts. To- cells (13). Acrolein increases matrix metalloproteinase 12– gether, these data suggest that acrolein induces MUC5AC expres- dependent mucus metaplasia in mice (14) and MUC5AC mRNA sion via an initial ligand-dependent activation of EGFR mediated in NCI-H292 cells (9). Increased MUC5AC expression can result by ADAM17 and MMP9. In addition, a prolonged effect of acrolein from increased transcription of MUC5AC (8) and stabilization may be mediated by altering MMP9 and TIMP3 transcription. of mRNA (9). However, the mechanism by which acrolein in- Keywords: bronchitis; emphysema; human bronchial epithelial cells; creases MUC5AC expression remains unknown. mucin Multiple agents, including cigarette smoke (15), activated eosinophils (16), interleukin-13 (17), neutrophil elastase (18), Chronic obstructive pulmonary disease affects more than 16 Pseudomonas aeruginosa (19), and phorbol 12-myristate 13- million people, and is the fourth leading cause of death in the acetate (PMA) (20) increase MUC5AC in airway epithelial cells United States (1). It is characterized by airflow obstruction, (NCI-H292) by activating the epidermal growth factor receptor chronic bronchitis, and emphysema (2). Chronic obstructive pul- (EGFR)/mitogen-activated protein kinase (MAPK) cascade. In- monary disease is marked by pathologic abnormalities in the hibition of EGFR activity decreases mucin production and re- submucosal glands and surface epithelium, which lead to exces- duces goblet cell metaplasia in response to various stimuli (21). sive airway mucus production (3). Mucus is a viscoelastic gel Binding of the EGFR ligands to EGFR results in receptor dimer- that lines the respiratory tract epithelium and protects against ization and subsequent autophosphorylation of specific tyrosine infectious and environmental agents. Mucus consists of water residues in the cytoplasmic domains of the receptors (22). EGFR (95%) combined with salts, lipids, and various proteins, including is also phosphorylated by treatment with ionizing radiation (23) mucin glycoproteins (4, 5). Mucins are large, heterogeneous mol- in the absence of ligand binding to EGFR. Ͼ ecules ( 20,000 kD), consisting of a protein backbone (apo- Endogenous EGFR ligands are synthesized as glycosylated mucin) to which multiple carbohydrate side chains are attached membrane-bound precursors (24), which are cleaved by protein- ases to release functional ligands (25). Pro-EGFR ligands are cleaved by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase domain proteins (ADAMs) (26–28). Cig- (Received in original form August 3, 2004; accepted in final form November 3, 2004) arette smoke increases the expression and release of EGFR Supported by the National Institutes of Health grants ES10562, ES06096, ligands, such as transforming growth factor ␣, amphiregulin, and ES07250, HL65612, and AI46556. This work is in partial fulfillment of doctoral diphtheria toxin receptor (DTR), also known as heparin binding degree requirements at the University of Cincinnati (H.S.D.). growth factor (HB-EGF), in airway epithelial cells (29). Ciga- Correspondence and requests for reprints should be addressed to George D. rette smoke (30) and PMA (20) increased MUC5AC production Leikauf, Ph.D., University of Cincinnati, P.O. Box 670056, Cincinnati, OH 45267- in NCI-H292 cells via transforming growth factor-␣–dependent 0056. E-mail: [email protected] EGFR activation mediated by ADAM17. In addition to ADAM17, This article has an online supplement, which is accessible from this issue’s table other members of the ADAM and MMP protein subfamilies of contents at www.atsjournals.org are involved in release of the EGFR ligands in response to Am J Respir Crit Care Med Vol 171. pp 305–314, 2005 Originally Published in Press as DOI: 10.1164/rccm.200408-1003OC on November 5, 2004 treatment with various agents (31). MMP2 and MMP9 mediate Internet address: www.atsjournals.org EGFR activation in mouse pituitary gonadotrope cells (␣T3-1)

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(32). MMPs and ADAMs are regulated at the level of transcrip- MUC5AC:3Ј: TCA CAG CCG GGT ACG CGT TGG CAC AAG tion (33) by activation of the precursor zymogens (34) and by TGG, and 5Ј: TGC TAT TAT GCC CTG TGT AGC CAG GAC action of endogenous inhibitors, tissue inhibitors of metallopro- TGC (37) teinase proteins (TIMPs). An increase in the levels of TIMP3 ADAM17:3Ј: AAT GAG AGC AAA GAA TCA AGC CCT GTC and TIMP1 can inhibit the activity of ADAM17 (35) and MMP9 TC-3Ј, and 5Ј: AAG CTT GAT TCT TTG CTC TCA CCT GTC (36), respectively. TC (38) The present study was designed to determine the mechanism MMP9:3Ј: GGA GAC CTG AGA ACC AAT CTC, and 5Ј:TCC of MUC5AC expression by acrolein. First, we examined whether AAT AGG TGA TGT TGT CGT (39) acrolein increases MUC5AC mRNA by depleting GSH (i.e., TIMP1:3Ј: GGC CAT CGC CGC AGA TCC, and 5Ј: GCT GGG inducing oxidative stress). Second, we examined the role of TGG TAA CTC TTT ATT TCA EGFR/MAPK cascade in acrolein-induced MUC5AC mRNA TIMP3:3Ј: CTG ACA GGT CGC GTC TAT GA, and 5Ј: GGC increase. We determined the role of EGFR ligands in MUC5AC GTA GTG TTT GGA CTG GT (40) expression by acrolein. The role of ADAM17 or MMP9 was The PCR protocol used was as follows: (1) 15 minutes, 95ЊC(2); examined by using specific small, interfering RNA (siRNA) se- Њ Њ quences and by measuring the MMP9 activity in conditioned n cycles 30 seconds, 95 C(3); 30 seconds, 57 C(4); and 30 seconds, 72ЊC; after cycling, the sample was heated (10 minutes, 72ЊC) and medium after acrolein treatment. We also determined whether cooled (4ЊC; total number of cycles: n ϭ 30 for MUC5AC,nϭ 18 for acrolein alters the expression of ADAM17 and MMP9 or their ␤-actin,nϭ 27 for TIMP1 and TIMP3,nϭ 30 for ADAM17, and respective endogenous inhibitors, TIMP3 and TIMP1. n ϭ 34 for MMP9).

METHODS Quantitation of PCR Products PCR products were quantitated by densitometry. DNA (10 ␮l) was Cell Culture and Acrolein Exposure electrophoresed on a 2% agarose gel containing 0.5 ␮g/ml of ethidium NCI-H292 cells (Cat. No. CRL-1848; American Type Culture Collec- bromide in Tris-acetate– ethylenediaminetetraacetic acid buffer (Cat. tion, Manassas, VA) were grown in 75-cm2 plastic tissue culture flasks No. BP1355; Fisher Biotech, Fair Lawn, NJ). After electrophoresis, (Cat. No. 3376; Corning, Corning, NY). NCI-H292 cells were main- DNA was by scanned by a Typhoon 8600 phosphor imager (Amersham tained in RPMI 1640 medium (Cat. No. 30-2001; ATCC), supplemented Biosciences, Piscataway, NJ) and analyzed by an image quant software with 10% fetal calf serum (Cat. No. 30-2020; ATCC), penicillin (100 program (Amersham Biosciences). For each RT-PCR, a serial dilution U/ml), and streptomycin (100 ␮g/ml; both from Sigma, St. Louis, MO; (0.5–0.032 ␮g) of total mRNA from the NCI-H292 or NHBE cells was 37ЊC, pH 7.4). The cells were seeded at a density of 5,000 cells/cm2 and amplified and included on each gel to obtain an internally consistent passaged at an approximately 90% confluence. For acrolein treatment, reference curve. Each sample was analyzed in the linear portion of the 2 NCI-H292 cells were seeded (5,000 cells/cm ) into 30-mm six-well plates curve. The relative amount of mRNA was determined by comparing (Cat. No. 3506; Corning). Once confluent, the cells were incubated the total intensity of each sample against the standard curve. Each Њ (37 C, pH 7.4) for 24 hours in serum-free medium (RPMI 1640). In a sample was analyzed in duplicate, and MUC5AC, TIMP1, TIMP3, few studies, normal human bronchial epithelial (NHBE) cells (Cat. No. ADAM17, and MMP9 mRNA levels were expressed as fold increase 2 CC2540; Cambrex Biosciences, Baltimore, MD) were cultured in 75-cm or decrease according to control levels after each was normalized to plastic tissue culture flasks and maintained in bronchial epithelial cell ␤-actin. growth medium (Cat. No. CC3170, Cambrex Biosciences). For acrolein 2 treatment, NHBE cells were seeded (5,000 cells/cm ) into 30-mm six- Acrolein Induces Oxidative Stress well plates. Once confluent, the cells were incubated (37ЊC, pH 7.4) for 24 hours in bronchial epithelial cell basal medium. NCI-H292 cells were exposed to increasing concentrations of acrolein ␮ Њ The cells were treated with 0.03 ␮M acrolein (Cat. No. 36520; Alfa (0.01–100 M) for 4 hours (37 C, pH 7.4). After exposure, the reagents Aesar, Ward Hill, MA), 25 ng/ml epidermal growth factor (EGF) (Cat. were removed, the cells were washed with PBS, and reduced GSH was No. 9908; Cell Signaling Technologies, Beverly, MA) in phosphate- measured as previously described (41). Briefly, the cells were lysed buffered saline (PBS; Cat. No. 14287–080; Invitrogen, Carlsbad, CA) with ice-cold homogenization solution containing 154 mM KCl, 5 mM for 4 hours (37ЊC, pH 7.4). After exposure, the solution was removed, diethylenetriaminepentaacetic acid, and 0.1 M (K3[PO]4) buffer (pH 6.8) and the cells were washed with PBS and lysed by Trizol reagent (Cat. using a homogenizer (Tekmar, Cincinnati, OH) at maximum speed. An No. 15596–026; Invitrogen). Total RNA was isolated by isopropanol/ aliquot was removed for protein determination using the bicinchoninic chloroform (Cat. No A416-4; Fischer, St. Louis, MO) precipitation and acid method (Pierce, Rockford, IL). An equal volume of solution con- suspended in RNAase-free water. taining 40 mM HCl, 10 mM diethylenetriaminepentaacetic acid, 20 mM ascorbic acid, and 10% trichloroacetic acid was added to the homogenate. Reverse Transcription and Polymerase Chain Reaction The suspension was centrifuged at 12,000 ϫ g for 10 minutes. The super- ␮ Total RNA from each sample was reverse transcribed into cDNA using natant solution was centrifuged through a 0.45- m micro-centrifuge filter the following reaction mixture: 2.5 ␮g total RNA from each sample in (Cat. No. 78976; Millipore, Billerica, MA), and GSH was measured 10 ␮l RNAse-free water, 1 ␮l of oligo dT-15 (Cat. No. C1101; Promega, by fluorescent spectrophotometry using o-phthalaldehyde (Cat. No. Madison, WI), 1 ␮l of 10 mM deoxynucleotide triphosphate (Cat. No. ICN216717; Fischer). 10297; Invitrogen). The reaction mixture was incubated (65ЊC, 5 min- utes) and then chilled (4ЊC). First strand buffer (5ϫ,4␮l), 2 ␮l 0.1 M Role of EGFR Activation in Acrolein-induced MUC5AC dithiothreitol (DTT), 1 ␮l SuperScript II (Cat. No. 180640; Invitrogen), mRNA Increase ␮ and 1 l RNase inhibitor (Cat. No. N2111; Promega) were then added NCI-H292 cells were pretreated (1 hour, 37ЊC) with 0.25 ␮M AG1478, Њ to the reaction and further incubated (42 C, 1 hour). The reaction was an EGFR kinase inhibitor (Cat. No. 658552; Calbiochem, San Diego, Њ Њ terminated by heating the mixture (70 C, 5 minutes) and stored at 4 C. CA). Cells were then treated with 0.03 ␮M acrolein or EGF (25 ng/ml) ␮ cDNA (2 l) was used in the subsequent polymerase chain reaction in PBS. To measure MUC5AC mRNA level, cells were washed with (PCR) using hot-start polymerase (Hotstar Taq, Cat. No. 203205; Qia- ice-cold PBS after 4 hours and RNA was isolated. To determine EGFR ␮ gen, Valencia, CA) in a 50- l reaction mixture containing the following activation, the cells were washed with ice-cold PBS after 1 hour and ␮ ϫ ␮ ␮ components: 5 l of PCR buffer (10 ), 10 l of Q solution, 200 M lysed with ice-cold radioimmunoprecipitation assay (RPA) lysis solution ␮ of each deoxynucleotide triphosphate, 0.2 M of each primer, and 0.625 (Cat. No. 20–188; Upstate, Waltham, MA) containing the following: ␮ l of Hotstar Taq polymerase. Primers used for PCR were from Sigma- 0.05 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.25% deoxycholic acid, 1% Genosys (Austin, TX). The sequence of primers used were as follows: NP-40, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsul-

␤-actin:3Ј: GGG GTC TAC ATG GCA ACT GTG AGG AGG phonylfluoride, 1 mM Na3VO4, 1 mM NaF, 1 mg/ml aprotinin, 1 mg/ml GGA, and 5Ј: AAA CCT GCC AAA TAT GAT GAC ATC AAG leupeptin, and 1 mg/ml pepstatin. Cell lysates were centrifuged (12,000 ϫ AAG g, 5 minutes, 4ЊC) and precleared by incubating (1 hour, 4ЊC) with

2-3 Deshmukh, Case, Wesselkamper, et al.: Acrolein-induced Mucin Production 307 washed 60 ␮l protein G agarose bead slurry (Cat. No.16–266; Upstate). 0.03 ␮M acrolein or 25 ng/ml EGF in PBS. RNA was isolated and the Protein concentration was determined by the bicinchoninic acid method, level of MUC5AC mRNA was determined. and cell lysates were diluted to 1 ␮g/␮l. Cell lysate (500 ␮l) was incu- bated (1 hour, 4ЊC) with 2 ␮g of anti-EGFR antibody (LA-22; Cat. No. Role of ADAM17 and MMP9 in Acrolein-induced 05–104; Upstate) and 60 ␮l protein G agarose bead slurry. Agarose Mucin 5AC mRNA Increase ϫ beads were collected by pulsing (12,000 g, 5 seconds), and the super- To determine the role of ADAM17 in acrolein-induced MUC5AC natant was drained. The beads were washed three times with ice-cold mRNA increase, 21-nt siRNA sequences of ADAM17; sense 5Ј-AAGC ␮ ϫ cell lysis solution. The agarose beads were resuspended in 50 l2 TTGATTCTTTGCTCTCA-3Ј antisense, 5Ј-AATGAGAGCAAA sodium dodecyl sulfate (SDS) sample buffer (Cat. No. LC2676; Invitro- GAATCAAGC-3Ј (20) were used. The 21-nt siRNA was prepared in vitro gen). The beads were boiled (5 minutes) and the supernatant was by a Silencer siRNA construction kit (Cat. No. 1620; Ambion, Austin, resolved by SDS polyacrylamide gel electrophoresis using 4 to 12% Tris- TX) according to the manufacturer’s instructions. To determine the glycine gels (Cat. No. EC6028; Invitrogen). The protein was transferred role of MMP9 in acrolein-induced MUC5AC mRNA increase, the electrophoretically to polyvinyldifluoride membrane (Cat. No. LC2005; siRNA was synthesized chemically (Ambion) from sequences described Invitrogen), which was incubated with 5% fat-free skimmed milk in previously (42). The sense siRNA sequence was 5Ј-CAUCACCUAU Tris-buffered saline (TBS) containing 0.05% Tween 20 (room tempera- UGGAUCCAAdTdT-3Ј. The antisense siRNA was 5Ј- UUGGAUCCA ture, 1 hour) (Cat. No. P7949, Sigma, St. Louis, MO) and incubated AUAGGUGAUGdTdT-3Ј. The sequences were annealed according to Њ ␮ overnight at 4 C with 2 g/ml antiphosphotyrosine antibodies (Cat. the manufacturer’s instructions. ADAM17 and MMP9 siRNA (0.03 ␮M) No. 05–321; Upstate). The membrane was washed twice with TBS were transfected into 40% confluent NCI-H292 cells using the Silencer containing 0.05% Tween 20 and then incubated (1 hour, room tempera- siRNA transfection kit (Cat. No. 1630; Ambion) according to the manu- ture) with 1:5000 rabbit anti-goat IgG horse radish peroxidase–linked facturer’s instructions. As a control, cells were transfected with scram- secondary antibody (Cat. No.12–349; Upstate). The membrane was bled siRNA (Cat. No. 4800; Ambion). Gene silencing was confirmed washed twice with TBS and bound antibodies were visualized using 48 hours later by RT-PCR. an enhanced chemiluminescent kit (Cat. No. RPN 2108; Amersham Biosciences) according to the manufacturer’s instructions. The mem- Gelatin Zymography brane was stripped using a stripping solution containing 2% SDS, 16 mM Cells were treated (4 hours, 37ЊC) with 0.03 ␮M acrolein or 5 ␮M Tris-HCl (pH 6.7) at 60ЊC for 1 hour. The membrane was incubated Њ H2O2 (Cat. No. 202460250; Arcos Organics) in PBS. The medium was overnight at 4 C with 1:1000 anti-EGFR antibody (Cat. No. 05–321; collected, centrifuged (12,000 ϫ g, 5 minutes, room temperature) to Upstate) using an enhanced chemiluminescent kit. remove cell debris, and concentrated 20-fold using concentration de- vices (Cat. No. 42416; Millipore). In some experiments, after acrolein Role of MAPK Activation in Acrolein-induced MUC5AC or H2O2 (4 hours, 37ЊC) treatment, the reagents were removed and mRNA Increase fresh serum-free RPMI 1640 medium was added to the cells. The cells NCI-H292 cells were pretreated (1 hour, 37ЊC) with 10 ␮M PD98059, were incubated (37ЊC) for an additional 20 hours. The medium was a MAP2K (MEK) inhibitor (Cat. No. 51300; Calbiochem). Cells were collected, centrifuged (12,000 ϫ g, 5 minutes, room temperature) to then treated with acrolein (0.03 ␮M) or EGF (25 ng/ml) in PBS. To remove cell debris, and concentrated 20-fold using concentration de- measure MUC5AC mRNA levels, the cells were washed with ice-cold vices. Protein concentration was determined by the bicinchoninic acid PBS after 4 hours and RNA was isolated. To determine MAPK activa- method. Samples containing 15 ␮g of protein were mixed with 2ϫ SDS tion, the cells were washed with ice-cold PBS after 1 hour and lysed sample buffer. with ice-cold RPA lysis solution. Cell lysates were centrifuged, and Protein was resolved by SDS polyacrylamide gel electrophoresis protein concentration was determined by the bicinchoninic acid method. using 10% Tris- glycine gels containing 0.1% gelatin as a substrate (Cat. Cell lysates containing equal amounts of proteins were then mixed with No. EC6175; Invitrogen). Gels were washed two times in zymogram 2ϫ SDS sample buffer and boiled. The protein was resolved by SDS renaturing solution (Cat. No. LC2670; Invitrogen; 30 minutes, room Њ polyacrylamide gel electrophoresis and transferred electrophoretically to temperature). Gels were preincubated (30 minutes, 37 C) in zymogram polyvinylidene fluoride membrane, which was incubated with 5% fat-free developing solution (Cat. No. LC2671; Invitrogen) and subsequently Њ skimmed milk in TBS containing 0.05% Tween 20 (1 hour) (Cat No. incubated (18 hours, 37 C) in zymogram developing solution. Gels were P7949, Sigma) and incubated overnight at 4ЊC with 1:1,000 anti–phospho- stained in 0.5% Coomassie blue R-250 (Cat. No. 24567; Sigma) in 40% MAPK3/2 (ERK1/2), anti–phospho-MAPK8 (JNK), or anti–phospho- methanol, 10% acetic acid (1 hour, room temperature), and destained MAPK14 (p38; Cat. No. 9910; Cell Signaling Technologies). The mem- in 40% methanol, 10% acetic acid (1 hour, room temperature) with a rinse and two changes of destaining solution to visualize digested bands brane was washed and then incubated at room temperature for 1hour with in the gelatin matrix. Gels were photographed using a digital camera. 1: 5,000 goat anti-rabbit IgG horse radish peroxidase–linked secondary antibody (Cat. No. 9211; Cell Signaling Technologies). Bound antibody Western Blotting for MMP9 Protein was visualized using an enhanced chemiluminescent kit. The membrane Њ ␮ ␮ was stripped and then incubated overnight at 4ЊC with 1:1,000 anti- Cells were treated (4 hours, 37 C) with 0.03 M acrolein or 5 MH2O2 MAPK3/2 (ERK1/2), anti-MAPK8 (JNK), or anti-MAPK14 (p38) anti- in PBS. After 4 hours, the reagents were removed and fresh serum- bodies (Cat. No. 9911; Cell Signaling Technologies) and visualized using free RPMI 1640 medium was added to the cells. The cells were incu- Њ an enhanced chemiluminescent kit. bated (37 C) for an additional 20 hours. The medium was collected, centrifuged (12,000 ϫ g, 5 minutes, room temperature) to remove cell Mechanism of Epithelial Growth Factor Receptor debris, and concentrated 20-fold using concentration devices. The pro- tein concentration was determined by bicinchoninic acid method. Sam- Activation by Acrolein ples containing 15 ␮g of protein were mixed with 2ϫ SDS sample buffer NCI-H292 cells were pretreated (1 hour, 37ЊC) with 10 ␮g/ml of LA-1, containing 2.5% betamercaptoethanol (Cat. No. 516732; Sigma) and a neutralizing antibody to the EGFR (Cat. No. 05–101; Upstate) and boiled (5 minutes). Protein was resolved by SDS polyacrylamide gel treated (4 hours, 37ЊC) with 0.03 ␮M acrolein or 25 ng/ml EGF in PBS. electrophoresis using 4 to 12% Tris-glycine gels and transferred electro- RNA was isolated and the level of MUC5AC mRNA was determined phoretically to polyvinylidene fluoride membrane, which was incubated as before. To determine whether acrolein-induced MUC5AC mRNA with 5% fat-free skimmed milk in TBS containing 0.05% Tween 20 increase involves a metalloproteinase, cells were pretreated (1 hour, (1 hour, room temperature) and incubated (overnight, 4ЊC) with 1:100 37ЊC) with 10 ␮M GM 6001, a broad-spectrum metalloproteinase inhibi- anti-MMP9 antibody (Cat. No. SA-106; Biomol International, Plymouth tor (Cat. No. 364205; Calbiochem) and treated with 0.03 ␮M acrolein Meeting, PA). The membrane was washed twice with TBS containing or 25 ng/ml EGF in PBS. RNA was isolated and the level of MUC5AC 0.05% Tween 20 and then incubated (room temperature, 1hour) with mRNA was determined. To further determine the identity of metallo- 1:1,000 goat anti-rabbit IgG horse radish peroxidase–linked secondary proteinase involved in acrolein-induced MUC5AC mRNA increase, antibody (Cat. No. A6154; Sigma). The membrane was washed twice cells were pretreated (1 hour, 37ЊC) with recombinant 2 ␮g/ml TIMP3 with TBS. Bound antibody was visualized using an enhanced chemilumi- (Cat. No. PF095; Calbiochem). Cells were treated (4 hours, 37ЊC) with nescent kit.

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Effect of Acrolein on ADAM17, MMP9, TIMP1, and TIMP3 mRNA Expression Metalloproteinases are regulated principally at the levels of transcrip- tion and by the activity of their endogenous inhibitors, TIMPs. To determine the role of TIMPs in acrolein-induced MUC5AC mRNA increase, NCI-H292 or NHBE cells were treated (4 hours, 37ЊC) with 0.03 ␮M acrolein or 25 ng/ml EGF in PBS. After exposure, the solution was removed, and the cells were washed with PBS and lysed by Trizol reagent. Total RNA was isolated and suspended in RNAase-free water. The level of ADAM17, MMP9, TIMP1, and TIMP3 transcript was measured by RT-PCR and quantitated as described before.

Statistical Analysis For analysis of the results of mRNA measurements of MUC5AC, two- way analysis of variance for repeated measurements was used to deter- mine statistically significant differences among group, followed by a Student-Newman-Keuls test for multiple comparisons. A probability of less than 0.05 was accepted as a statistically significant difference. All data are expressed as mean Ϯ SEM.

RESULTS MUC5AC mRNA Increase by Acrolein Is Independent of Oxidative Stress Previous studies have indicated that acrolein depletes GSH at concentrations 3 ␮M or greater and protein thiols at 10 ␮Mor greater in primary bronchial epithelial cells (13). In NCI-H292 cells, the threshold concentration of acrolein was 10 ␮M or more, with 30 ␮M significantly decreasing GSH levels (Figure 1). This was in excess of concentration of acrolein (0.03 ␮M) that induced MUC5AC mRNA increase in NCI-H292 cells (Figure 2B), sug- gesting that acrolein-induced MUC5AC expression may be inde- pendent of oxidative stress.

MUC5AC mRNA Increase by Acrolein Involves Phosphorylation of EGFR and MAPK2/3 Acrolein at a concentration of 0.03 ␮M increased the tyrosine phosphorylation of the EGFR in NCI-H292 cells (Figure 2A). The increase in tyrosine phosphorylation was reduced by

Figure 2. Acrolein increases mucin 5AC (MUC5AC) mRNA by phosphor- ylating epidermal growth factor receptor (EGFR). Confluent NCI-H292 cells were pretreated (1 hour, 37ЊC) with 0.25 ␮M AG1478, an EGFR kinase inhibitor, and treated with 0.03 ␮M acrolein or 25 ng/ml EGF or PBS. (A) EGFR phosphorylation increased after acrolein or EGF treat- ment (1 hour, 37ЊC). Pretreatment with AG1478 decreased the acrolein- or EGF-induced EGFR phosphorylation. This is a representative blot of tests done in duplicate on three separate occasions. (B) MUC5AC mRNA levels increased after acrolein or EGF treatment (4 hours, 37ЊC). Pretreat- ment with AG1478 diminished these responses. The results are expressed as fold increase in the levels of MUC5AC mRNA of the acrolein- or EGF- treated cells (with or without inhibitor) as compared with the PBS-treated cells after each was normalized to ␤-actin. Values are mean Ϯ SE (n ϭ 9). *Significantly different from PBS-treated samples, †significantly different from the acrolein-treated samples, or ‡significantly different from EGF- Figure 1. Acrolein reduces glutathione (GSH) levels in human bronchial treated samples (p Ͻ 0.05). (C) RT-PCR results demonstrating the inhibition epithelial cells at concentrations above 10 ␮M. Confluent NCI-H292 of acrolein- or EGF-induced increase in MUC5AC mRNA by pretreatment cells were treated (4 hours, 37ЊC) with increasing doses of acrolein with AG1478. This is a representative gel of tests done in duplicate on (0.03–100 ␮M). Oxidative stress was assessed by measuring the GSH nine separate occasions. IP ϭ immunoprecipitation; WB ϭ western blot. levels (o-opthalaldehyde fluorescence). Control (phosphate-buffered sa- line [PBS] alone) levels were 77 Ϯ 7 nmol GSH/mg protein. Values are mean Ϯ SEM (n ϭ 8). *Significantly different from the PBS-treated samples (p Ͻ 0.05).

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pretreating the cells with AG1478, an EGFR tyrosine kinase PD98059, decreased the MAPK3/2 phosphorylation induced by inhibitor (Figure 2A). Pretreatment with AG1478 decreased the acrolein or EGF (Figure 3A). Pretreating the cells also decreased levels of MUC5AC mRNA induced by acrolein or EGF (Figures the levels of MUC5AC mRNA induced by acrolein or EGF 2B and 2C). EGFR phosphorylation leads to activation of (Figure 3B). Thus, acrolein-induced MUC5AC expression in MAP2K (MEK), which in turn phosphorylates and activates NCI-H292 cells is mediated by EGFR phosphorylation and MAPK. Acrolein (0.03 ␮M) increased the phosphorylation of MAPK3/2 phosphorylation. the MAPK3/2 (ERK1/2) and MAPK8 (JNK) but had no effect on MAPK14 (p38). Pretreating the cells with a MAP2K inhibitor, MUC5AC mRNA Increase by Acrolein Involves an Endogenous EGFR Ligand and Is Dependent on a TIMP3-sensitive Metalloproteinase Pretreatment with a neutralizing antibody to EGFR (LA-1) de- creased the acrolein or exogenous EGF-induced MUC5AC mRNA increase (Figure 4A). Pretreatment with a broad-spectrum metallo- proteinase inhibitor (GM6001) decreased the acrolein-induced but not the exogenous EGF-induced MUC5AC mRNA increase (Figure 4B). Pretreatment with TIMP3, an inhibitor of ADAM17, partially inhibited the increase in acrolein-induced MUC5AC mRNA levels (Figure 4C). Thus, acrolein-induced MUC5AC ex- pression in NCI-H292 cells involves an EGFR ligand–dependent mechanism mediated, in part, by a TIMP3-sensitive metallopro- teinase.

Figure 4. Acrolein increases MUC5AC mRNA by an EGFR ligand–dependent mechanism mediated by a metalloproteinase. Confluent NCI-H292 cells were pretreated (1 hour, 37ЊC) with 10 ␮g/ml LA-1, an EGFR-neutraliz- ing antibody; 10 ␮M GM6001, a broad-spectrum metalloproteinase inhibitor; or 2 ␮g/ml recombinant tissue inhibitor of metalloproteinase protein 3 (TIMP3) and then ex- posed (4 hours, 37ЊC) to 0.03 ␮M acrolein or 25 ng/ml EGF or PBS. (A) Pretreatment with LA-1 inhib- ited the acrolein- or EGF-induced increase in MUC5AC mRNA. (B) Pretreatment with GM6001, in- hibited the acrolein-induced but Figure 3. Acrolein increases MUC5AC mRNA by activating mitogen- not the EGF-induced increase in activated protein kinase 3/2 (MAPK3/2 [ERK1/2]) and MAPK8 (JNK). MUC5AC mRNA. (C) Pretreat- Њ ␮ Confluent NCI-H292 cells were pretreated (1 hour, 37 C) with 5 M ment with TIMP3 partially inhib- ␮ PD98059, a MAP2K (MEK) inhibitor and then treated with 0.03 M ited the acrolein-induced increase acrolein or 25 ng/ml EGF or PBS. (A) Immunoblot demonstrating an in MUC5AC mRNA. Values are increase in MAPK3/2 and MAPK8 phosphorylation after treatment with mean Ϯ SE (n ϭ 9). *Significantly Њ acrolein or EGF (1 hour, 37 C). Pretreatment with PD98059, a MAP2K different from PBS-treated samples, inhibitor, decreases the acrolein- or EGF-induced MAPK3/2 and MAPK8 †significantly different from acro- phosphorylation. This is a representative blot of tests done in duplicate lein-treated samples, ‡significantly on three separate occasions. (B) MUC5AC mRNA levels increased after different from EGF-treated samples Њ acrolein or EGF treatment (4 hours, 37 C). Pretreatment with PD98059 (p Ͻ 0.05, n ϭ 9). diminished these responses. The results are expressed as fold increase in the levels of MUC5AC mRNA of the acrolein- or EGF-treated cells (with or without inhibitor) as compared with the PBS-treated cells after each was normalized to ␤-actin. Values are mean Ϯ SE (n ϭ 8). *Signifi- cantly different from PBS-treated samples, †significantly different from the acrolein-treated samples, or ‡significantly different from EGF-treated samples (p Ͻ 0.05).

2-6 310 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 171 2005 2-7 Acrolein-induced MUC5AC mRNA Increase Is Mediated (Figure 6A). An additional gelatinase with a molecular weight by ADAM17 and MMP9 of approximately 82 kD, representing the active form of MMP9, was observed in acrolein- or H2O2-treated samples, which was ADAM17 or MMP9 siRNAs inhibited the increase in acrolein- absent in the control samples (Figure 6A). Extending the incuba- induced MUC5AC mRNA levels (Figure 5A). Cells cotransfected tion period to 24 hours also increased MMP2 and MMP9 activity with ADAM17 and MMP9 siRNA showed an approximately 80% in the conditioned medium after acrolein or H2O2 treatment inhibition of acrolein-induced MUC5AC mRNA level (Figure (Figure 6B). Western blots demonstrated protein levels of pro- 5B). The EGF-induced increase of MUC5AC mRNA levels was MMP9 (92 kD) and MMP9 (82 kD) in the conditioned medium not inhibited (Figures 5A and 5B). Cells transfected with non- increased following acrolein or H2O2 treatment as compared sense (scrambled) siRNA had MUC5AC mRNA levels compa- with the PBS-treated (control) samples (Figure 6C). rable to the control and responded appropriately to acrolein Acrolein Increases MMP9 mRNA and Decreases TIMP3 mRNA (Figures 5A and 5B). Thus, acrolein-induced MUC5AC expres- in NCI-H292 and Normal Human Bronchial Epithelial Cells sion in NCI-H292 cells is mediated by ADAM17 and MMP9. When the acrolein treatment was extended to 24 hours, the Acrolein Increases Release and Subsequent Activation MMP9 mRNA level in NCI-H292 cells increased (Figure 7). of Pro-MMP9 Moreover, MMP9 mRNA levels increased in NHBE cells after Two with a molecular weight of approximately 90 kD (pro-MMP9) and approximately 70 kD (pro-MMP2) increased

in the conditioned medium after acrolein or H2O2 treatment

Figure 5. Acrolein increases MUC5AC mRNA by an EGFR ligand–dependent mechanism mediated by a disintegrin and metallo- proteinase domain pro- tein 17 (ADAM17) and matrix metalloproteinase 9 (MMP9). (A) NCI-H292 cells were transfected with small interfering RNAs (siRNA) directed against ADAM17 (0.03 ␮M), MMP9 (0.03 ␮M), or non- sense (scrambled) siRNA (0.05 ␮M) as control. After 48 hours, cells were exposed to 0.03 ␮M acrolein or 25 ng/ml EGF or PBS (4 hours, 37ЊC). The siRNA against ADAM17 or MMP9 partially inhibited acrolein- induced MUC5AC mRNA Figure 6. Acrolein stimulates MMP9 activity in conditioned cell medium increase (Group 3) but via protein release. Confluent NCI-H292 cells were treated (4 hours, not EGF-induced MUCA5C 37ЊC) with 0.03 ␮M acrolein or 5 ␮MH2O2.(A) Zymogram performed mRNA increase (Group 5). with the conditioned medium collected immediately after acrolein or NCI-H292 cells not trans- ;kD, MMP9 90 ف) H2O2 (4 hours, 37ЊC) treatment. Gelatinase activity -kD, MMP2) is increased in the conditioned medium from acrolein 70 ف fected with siRNA for ADAM17 or MMP9 (positive or H2O2-treated samples as compared with the PBS-treated (control) control; Groups 2 and 4) samples. This is a representative zymogram of test done in duplicate responded to acrolein or on three separate occasions. (B) Zymogram performed with the condi- EGF treatment by an in- Њ tioned medium collected 20 hours after acrolein or H2O2 (4 hours, 37 C) crease in MUC5AC mRNA. treatment. Acrolein or H2O2 was added to PBS, incubated for 4 hours. (B) NCI-H292 cells were PBS was removed, complete medium (RPMI 1640) was added, and cells ,kD 90 ف) cotransfected with siRNA were incubated for an additional 20 hours. Gelatinase activity kD, MMP2) is increased in the conditioned medium from 70 ف ;against both ADAM17 MMP9 ␮ ␮ (0.03 M) and MMP9 (0.03 M) or non-sense (scrambled) siRNA acrolein- or H2O2-treated samples as compared with the PBS-treated (0.05 ␮M) as control. After 48 hours, cells were exposed to 0.03 ␮M (control) samples. This is a representative zymogram of test done in acrolein or 25 ng/ml EGF or PBS (4 hours, 37ЊC). Cells transfected with duplicate on three separate occasions. (C) Western blot performed with

siRNA against ADAM17 or MMP9 show an inhibition of acrolein-induced the conditioned medium collected 20 hours after acrolein or H2O2 (4 MUC5AC mRNA increase of approximately 80%, but not an EGF- hours, 37ЊC) treatment. MMP9 (pro-MMP9 [92 kD] and active MMP9 induced MUCA5C mRNA increase. NCI-H292 cells transfected with [82 kD]) protein levels are increased in the conditioned medium from

scrambled (control) siRNA did not show any decrease in acrolein or acrolein- or H2O2-treated samples as compared with the PBS-treated EGF-induced MUC5AC mRNA increase. Values are mean Ϯ SE (n ϭ (control) samples. These immunoreactive proteins migrated with the 6). *Significantly different from PBS-treated samples or †significantly 92-kD purified pro-MMP9 (last lane). This is representative blot of tests different from the acrolein-treated samples (p Ͻ 0.05). done in duplicate on three separate occasions.

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Figure 7. Acrolein increases dependent on EGFR phosphorylation, because pretreating the MMP9 and decreases TIMP3 cells with an EGFR kinase inhibitor, AG1478, inhibited this transcript level, leaving the effect (Figures 2A and 2B). Previously, Takeuchi and coworkers ADAM17 and TIMP1 transcript (51) demonstrated that acrolein induced EGFR phosphorylation level unchanged in NCI-H292 at a concentration of 50 ␮M and, importantly, this concentration or normal human bronchial epi- of acrolein led to apoptosis in keratinocytes. We found that thelial (NHBE) cells. Confluent acrolein depleted GSH and induced oxidative stress at concen- cells were treated (4 hours, ␮ 37ЊC) with 0.03 ␮M acrolein or trations greater than 10 M (Figure 1). Oxidative stress in the PBS. Level of ADAM17, MMP9, form of H2O2 phosphorylates EGFR but the pattern of tyrosine TIMP1, and TIMP3 mRNA was phosphorylation is different from that of ligand-induced EGFR measured by RT-PCR. The re- phosphorylation (52). Importantly, this type of EGFR phosphor- sults are expressed as fold in- ylation does not lead to phosphorylation (activation) of down- crease or decrease in the mRNA stream MAPKs (53). We found that acrolein led to phosphorylation levels of various genes as com- of MAPKs, including MAPK3/2 (ERK1/2) and MAPK8 (JNK; pared with the PBS-treated Figure 3A), and acrolein-induced MUC5AC mRNA increase is samples after normalizing to dependent on MAP2K phosphorylation, as indicated by PD98059 ␤-actin. Values are mean Ϯ SE (n ϭ 6). *Significantly different from inhibition (Figure 3B). Thus, acrolein increases MUC5AC expres- PBS-treated samples (p Ͻ 0.05, n ϭ 6). sion by phosphorylating EGFR and phosphorylating MAP2K, which in turn phosphorylates MAPK3/2 and MAPK8, independent of oxidative stress (Figure 8). Acrolein-induced MUC5AC expression is dependent on acrolein treatment for 24 hours. Acrolein also decreased TIMP3 EGFR ligand release mediated by metalloproteinase. EGFR mRNA in NHBE cells and NCI-H292 cells (Figure 7). The level ligands are synthesized as glycosylated membrane-bound precur- of TIMP1 mRNA and ADAM17 mRNA remained unchanged sors (24). Various EGFR ligands, such as transforming growth (Figure 7). factor-␣, amphiregulin, and diphtheria toxin receptor, are ex- pressed in NCI-H292 cells (29). Shao and coworkers showed DISCUSSION that transforming growth factor-␣ mediated MUC5AC increase by PMA (20) and cigarette smoke (30). After pretreating the Acrolein is a constituent of cigarette smoke, wood smoke, diesel NCI-H292 cells with an EGFR-neutralizing antibody (LA-1), exhaust, and cooking fumes (43). Acrolein levels are higher in we found that MUC5AC mRNA increase by acrolein depends secondhand as compared with mainstream cigarette smoke, be- on binding of an EGFR ligand to the receptor (Figure 4A). cause of lower combustion temperatures of smoldering cigarettes Endogenous EGFR ligands are released by proteolytic cleavage (44). Acrolein can penetrate the upper respiratory passages (45) of membrane-bound proforms mediated by metalloproteinases and react with macromolecules as highly reactive zwitterions such as MMPs and ADAMs (26, 27). By pretreating the NCI- ϩ ϭ Ϫ ␣ ␤ ( CH2CH CHO ) through electron rearrangement of an - H292 cells with a broad-spectrum metalloproteinase inhibitor unsaturated bond (46). Previously, Borchers and coworkers (47) (GM6001), we found that MUC5AC mRNA increase after acro- reported that rats exposed to acrolein develop mucus metaplasia lein treatment depends on a metalloproteinase (Figure 4B). The and that mucus hypersecretion in the airways was preceded by present study did not measure the release of EGFR ligands in an increase in MUC5AC mRNA level (47). Acrolein increased the medium. However, Richter and coworkers (29) previously MUC5AC mRNA in NCI-H292 cells at concentrations between have demonstrated that EGFR ligands, including transforming 0.001 and 0.03 ␮M (9). growth factor-␣, amphiregulin, and diphtheria toxin receptor, Acrolein-induced MUC5AC expression is independent of ox- are released from NCI-H292 cells into the cell culture medium idative stress. Acrolein reacts directly with protein and nonpro- on treatment with cigarette smoke. tein sulfhydryl groups, and with primary and secondary amines Acrolein-induced MUC5AC expression involves ADAM17 found in proteins and nucleic acids (48). The conjugation of the and MMP9. We sought to determine the identity of metallopro- acrolein with sulfhydryl groups is rapid and essentially irrevers- teinases involved in acrolein-induced MUC5AC expression. Pre- ible (49), and leads to a decrease in cellular GSH stores. Acrolein viously, Shao and colleagues found that ADAM17 mediated treatment also decreased the availability of precursor amino EGFR activation in NCI-H292 cells on treatment with PMA acids used in GSH and protein synthesis in pulmonary endothe- (20) or cigarette smoke (30). We found that pretreating the cells lial cells (50). In NHBE cells, Grafstrom and colleagues (13) with TIMP3, an inhibitor of ADAM17, inhibited partially the found that acrolein induced oxidative stress by depleting GSH. acrolein-induced MUC5AC mRNA increase (Figure 4C). We We found that acrolein decreased the level of GSH in a concen- used siRNA directed against ADAM17 in NCI-H292 cells and tration-dependent manner in NCI-H292 cells. However, the confirmed the role of ADAM17 in acrolein-induced MUC5AC threshold concentration of acrolein that decreased GSH was 10 expression (Figure 5). Because the inhibition of acrolein-induced to 30 ␮M (Figure 1). In contrast, the threshold dose of acrolein MUC5AC mRNA increase by TIMP3 pretreatment or sufficient to increase MUC5AC mRNA was 0.03 ␮M or less, ADAM17 siRNA was partial, there was a possibility that more approximately a 300- to 1,000-fold lower concentration (Fig- than one metalloproteinase may be involved in acrolein-induced ure 2). Because oxidative stress occurred at concentrations of MUC5AC mRNA increase. Various MMPs, including MMP2 10 ␮M or greater, oxidative stress seems unlikely to be involved and MMP9, are expressed in airway epithelial cells (53). In mouse in MUC5AC expression by acrolein. pituitary gonadotrope (␣T3-1) cells, MMP2 and MMP9 can me- Acrolein increases MUC5AC expression by phosphorylating diate EGFR transactivation (32). We used siRNA directed EGFR and by activating downstream MAPK signaling. Multiple against MMP9 in NCI-H292 cells and found inhibition of the agents, including cigarette smoke (15), interleukin-13 (16), and MUC5AC mRNA increase by acrolein (Figure 5A). Thus, in PMA (20), induce MUC5AC production in airway epithelial addition to ADAM17, MMP9 may also be involved in MUC5AC cells by phosphorylating EGFR. We found that the acrolein- mRNA increase by acrolein (Figure 8). These results complement induced MUC5AC mRNA increase was accompanied by and the previous observations by Shao and coworkers (20, 30) that

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Figure 8. Mechanism of acro- lein-induced MUC5AC mRNA increase. Acrolein induces pro- teinases, ADAM17, and MMP9 to cleave the cell-surface EGFR ligands. This effect can be blocked by a broad-spectrum metallopro- teinase inhibitor, GM6001, or siRNA directed against ADAM17 or MMP9. The endogenous EGFR ligands bind to EGFR (inhibited by neutralizing antibody, LA-1) and phosphorylate the tyrosine residues (inhibited by AG1478). EGFR phosphorylation leads to MAP2K (MEK) phosphorylation (inhibited by PD98059), which in turn phosphorylates and activates MAPK3/2 (ERK1/2) and MAPK8 (JNK; not shown). These events are accompanied with an in- crease in the levels of MUC5AC mRNA. With prolonged acrolein exposure, MMP9 transcript lev- els increase and TIMP3 transcript levels de crease, consistent with continued activation of EGFR/ MAPK signaling through ADAM17 and MMP9. Thus, acrolein initiates MUC5AC expression rapidly through EGFR- mediated signaling followed by alterations in metalloproteinases that could prolong MUC5AC production. MMP9 could thereby induce mucus hypersecretion along with its known effects on emphysema, hallmarks of chronic obstructive pulmonary disease.

ADAM17 is involved in MUC5AC mRNA increase by PMA CD-44 is increased on bronchial epithelial cells in areas of damage or cigarette smoke. Moreover, cells cotransfected with siRNA (61). CD-44 coimmunoprecipitates with EGFR (62) and recruits against ADAM17 and MMP9 showed a greater inhibition of active MMP7 and pro–DTR to form a complex on the cell surface MUC5AC mRNA increase by acrolein compared with transfect- (63). Further studies are necessary to determine the mechanism ing the cells with siRNA against either ADAM17 or MMP9 by which MMP9 can cleave pro-EGFR ligands. alone (Figure 5B). With prolonged treatment, acrolein alters MMP9 and TIMP3 Acrolein increases MMP9 protein and gelatinase activity in transcript levels. MMPs are tightly regulated at the transcrip- the cell culture medium. We observed two gelatinases, pro-MMP9 tional and post-transcriptional level and are also controlled at (92 kD) and pro-MMP2 (66 kD), in the cell culture medium after the protein level via their activators, inhibitors, and cell-surface acrolein treatment (Figure 6A). MMP9 is synthesized as pro- localization (54). MMP9 is regulated at the level of transcription MMP9 (92 kD), which is kept inactive by interaction between by several cytokines and growth factors, including EGF (34). The cysteine-sulphydryl groups in the propeptide domain and the transcript level of MMP9 is elevated in interleukin-13–induced zinc ion bound to the catalytic domain (34). Activation to MMP9 emphysema (64). We found that acrolein increased MMP9 (82 kD) requires proteolytic removal of the prodomain (54) out- mRNA (Figure 7) in NCI-H292 cells. Because metalloprotei- side the cell by other proteinases. We found that control samples nases are often increased in tumor cell lines (65), we used NHBE had only the proform (inactive) of MMP9, whereas the acrolein- cells to confirm increase in MMP9 mRNA after acrolein treat- or H2O2-treated samples showed the proform and the activated ment (Figure 7). We also observed increased MMP9 gelatinolytic form of MMP9 (Figure 6A). We also found that protein level activity in NCI-H292 cells after 4 hours (Figure 6A) and 24 of pro-MMP9 in acrolein- or H2O2-treated samples was greater hours (Figure 6B). TIMP1 is an endogenous inhibitor of MMP9 than the control samples (Figure 6C). Thus, acrolein, in addition (36). TIMP1 binds MMP9 in a 1:1 stoichoimetric fashion and to increasing the MMP9 protein and gelatinase activity in the keeps it inactive (66). We found that acrolein had no effect on cell culture medium, can also activate pro-MMP9. Extracellular TIMP1 mRNA in both NCI-H292 cells and NHBE cells (Figure activation of pro-MMP9 can be initiated by a proteinase cascade 7). TIMP3, a natural inhibitor of ADAM17 (35), is also regulated involving already activated MMPs (34), including MMP2 (55). at the transcriptional level (67). We found that acrolein decreased We found that acrolein treatment increased the MMP2 activity TIMP3 mRNA (Figure 7) in NCI-H292 and NHBE cells but had in the cell culture medium (Figure 6A). MMP2 could activate no effect on ADAM17 mRNA (Figure 7). Thus, in addition to pro-MMP9 in NCI-H292 cells after acrolein treatment; however, rapid ADAM17- and MMP9-mediated ligand-dependent activa- further studies are necessary to confirm the role of MMP2 in tion of EGFR, acrolein increases MMP9 activity and alters tran- activating pro-MMP9 after acrolein treatment. scription of proteins critical to this pathway, which in turn may The mechanism by which MMP9 cleaves cell-surface pro-EGFR prolong the effect on MUC5AC production (Figure 8). ligands is unknown. Surface-bound MMP9 may be responsible for In summary, acrolein rapidly induces MUC5AC expression processing pro-EGFR ligands. MMP9 binds with high affinity to through an EGFR-MAPK pathway mediated by metalloprotei- various substrates, including hylaluronan receptor (CD-44) (56, nases ADAM17 and MMP9. In addition, acrolein can produce a 57), ␣-2 chain of collagen IV (58), intracellular adhesion molecule prolonged increase in MUC5AC expression through an increase (59), and docking proteins such as ␤-integrin (60). In the airways, in MMP9 (transcript and activity) and a decrease in TIMP3

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(transcript). Together, these interactions would be consistent 16. Burgel PR, Lazarus SC, Tam DC, Ueki IF, Atabai K, Birch M, Nadel with extended mucin production following exposure to this irri- JA. Human eosinophils induce mucin production in airway epithelial cells via epidermal growth factor receptor activation. J Immunol tant, a component of cigarette smoke. Samples of lung tissues 2001;167:5948–5954. from patients with cigarette smoke–related emphysema show an 17. Shim JJ, Dabbagh K, Ueki IF, Dao-Pick T, Burgel PR, Takeyama K, increase in MMP9 (68). Thus, in addition to playing an important Tam DC, Nadel JA. IL-13 induces mucin production by stimulating role in alveolar enlargement and matrix degradation, MMP9 epidermal growth factor receptors and by activating neutrophils. Am may also be involved in mucus hypersecretion. J Physiol Lung Cell Mol Physiol 2001;280:L134–L140. 18. Kohri K, Ueki IF, Nadel JA. Neutrophil elastase induces mucin produc- Conflict of Interest Statement : H.S.D. does not have a financial relationship with tion by ligand-dependent epidermal growth factor receptor activation. a commercial entity that has an interest in the subject of this manuscript; L.M.C. Am J Physiol Lung Cell Mol Physiol 2002;283:L531–L540. does not have a financial relationship with a commercial entity that has an interest 19. Kohri K, Ueki IF, Shim JJ, Burgel PR, Oh YM, Tam DC, Dao-Pick T, in the subject of this manuscript; S.C.W. does not have a financial relationship Nadel JA. Pseudomonas aeruginosa induces MUC5AC production with a commercial entity that has an interest in the subject of this manuscript; via epidermal growth factor receptor. Eur Respir J 2002;20:1263–1270. M.T.B. does not have a financial relationship with a commercial entity that has 20. Shao MX, Ueki IF, Nadel JA. 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2-11 ONLINE DATA SUPPLEMENT

LEGEND

Fig. E1: Standard curve used for quantitating MUC5AC mRNA.

PCR products obtained from multiple dilutions of total RNA by reverse transcription

PCR. PCR products were quantitated by densitometry. DNA (10 µl) was electrophoresed on a 2% agarose gel containing 0.5 µg/ml of ethidium bromide in Tris- acetate-EDTA buffer (Cat. No. BP1355-1, Fisher Biotech, Fair Lawn, NJ). After electrophoresis, DNA was by scanned by Typhoon 8600 phosphor imager (Amersham biosciences, Piscataway, NJ) and analyzed by Imagequant software program

(Amersham Biosciences, Piscataway, NJ) to obtain a reference curve. Each sample was analyzed in the linear portion of the curve. The relative amount of mRNA was determined by comparing the total intensity of each sample against the standard curve.

Fig. E2: siRNA efficiently knocks down ADAM17 and MMP9 expression. NCI-H292 cells were transfected with increasing concentrations of siRNA directed against ADAM17 or MMP9. RNA was isolated after 48 h and the level of ADAM17 and MMP-9 mRNA was determined by RT-PCR. The results are expressed as fold change in the level of

ADAM17 and MMP9 mRNA as compared to the control (un-transfected cells) after each was normalized to β-actin. Values are mean ± standard error (n = 6). * indicates significant difference from the control (non transfected) cells. From these finding, a final concentration of 0.03 µM of ADAM17 and MMP9 siRNA was used for gene silencing.

2-12 FIGURES

Figure E1

2-13 Figure E2

2-14 CHAPTER 3

Manuscript II

An autocrine feedback loop regulating Matrix Metalloproteinase (MMP) 9 leads to persistent mucus production in the airways.

Abstract

Chronic obstructive pulmonary disease (COPD), a major public health problem worldwide, is characterized by progressive difficulty in breathing and mucus overproduction. Acrolein, a constituent of cigarette smoke increased mucin 5 (subtype A and C) production in the airways, however, the mechanism of acrolein-induced MUC5AC increase remains unclear. Acrolein- induced increase in MUC5AC transcripts and protein is associated with increased MMP9 transcripts, protein and metalloproteinase activity in the lungs of Mmp9 (+/+) mice. Acrolein- induced increase in MUC5AC transcripts and protein was lower in gene targeted MMP9 mice

(Mmp9 (-/-)). Acrolein decreased the transcript level of endogenous MMP inhibitor, tissue inhibitor of metalloproteinase protein (TIMP) 3 in vivo. In addition acrolein increased the transcript level and activity of MMP9 in airway epithelial cells. Moreover acrolein directly increased pro-MMP9 activation in vitro. Increase in MMP9 transcripts after acrolein treatment was inhibited by pretreatment with a neutralizing antibody against EGFR or EGFR kinase inhibitor. Pretreatment with a mitogen activated protein kinase (MAPK) 3/2 inhibitor, but not

MAPK8 or MAPK14 inhibitor diminished the acrolein-induced increase in MMP9 transcripts. A metalloproteinase inhibitor diminished the acrolein-induced increase in MMP9 transcripts. Thus acrolein rapidly activates pro-MMP9 to increase MUC5AC transcripts via an EGFR-ligand dependent mechanism. Chronic exposure to acrolein, initiates a feedback loop to increase

MMP9 transcripts and simultaneously decrease TIMP3 transcripts to cause a persistent increase in mucus production.

3-1 Introduction:

Chronic obstructive pulmonary disease (COPD) is a major and growing global health problem.

Predicted to be the third most common cause of death by 2020 (Lopez 1998), COPD is caused by long-term exposure to inhaled noxious gases, with tobacco smoking accounting for > 90 percent of new cases. Death and disability from COPD are related to an accelerated decline in lung function (Fletcher 1977), that is a result of remodeling of the airway and alveolar architecture. Small airways (<2-3 mm diameter) normally contribute less than 20% of total airway resistance (Hogg 1968), but this increases markedly in COPD due to narrowing of the airway lumen and mucus hypersecretion. Mucus plugging in the small airways leads to ventilation-perfusion mismatch and insufficient oxygenation of blood, and excessive mucus production is associated with decline in lung function, hospitalization and death (Vestbo 1996).

The pathogenesis of COPD involves a balance between proteinases and proteinase inhibitors

(Hogg 2004). Matrix metalloproteinases (MMPs) is a major family of zinc-dependent neutral proteinases. Studies of human lung biopsy samples and induced sputum show increases in many proteinases, including MMP1 (Imai 2001), MMP2 (Ohnishi 1998), MMP8 (Betsuyaku

1999), MMP9 (Finlay 1997), and MMP14 (MT1-MMP) (Ohnishi 1998) in smoking-related COPD.

Increased gelatinolytic activity associated with MMP2 and MMP9 has been identified in BAL fluid from COPD patients (Finlay 1997 and Betsuyaku 1999).

Mucus, a viscoelastic gel composed of water (95%) and mucins, lines the respiratory epithelium and protects against infectious and environmental agents (Reid 1986). Mucins are a large heterogeneous glycoproteins, consisting of a protein backbone (apomucin) to which multiple carbohydrate side chains are attached by glycosyltransferases at serine and theronine residues that are contained in variable number of tandem repeat regions (Leikauf 2002). Mucins are

3-2 encoded by at least 15 genes, many localized in a gene cluster on human chromosome 11p15

(Leikauf 2002). Biochemical analysis indicates that mucin 5 (subtype A and C) (MUC5AC) and mucin 5B (MUC5B) constitute the majority of mucin glycoproteins in the airway secretions of humans (Rose 1989). Although MUC5B is primarily constitutively expressed, MUC5AC is highly inducible (Borchers 1998). Specialized epithelial (goblet) cells are the major source of

MUC5AC in the airways. Goblet cells are typically limited in number and found mainly in the large airways of healthy individuals, but with tobacco smoke exposure the number of goblet cells, especially in the small diameter airways, increases markedly (Reid 1950, Hogg 1968).

Cigarette smoke consists of many irritants, but one of the most prevalent and potent is acrolein

(3-228µg acrolein/cigarette). Notably, second-hand tobacco smoke contains high levels of acrolein because aldehydes are enriched in side stream smoke due to poor combustion of smoldering cigarettes. In indoor air, smoking one cigarette per m3 of room-space in 10-13min can lead to acrolein concentrations of 2.0-3.7ppm (450-840µg/m3) (Jermini 1976). Acrolein is also a constituent of wood smoke, diesel exhaust, and photochemical smog, and can be generated from biomass fuels and by cooking with oils (Leikauf 2002). Once inhaled, acrolein can penetrate the upper respiratory passages and deposit throughout the lower respiratory tract.

+ - Because it forms a highly reactive zwitterion ( CH2CH=CHO ) through electron rearrangement of the α-β unsaturated bond, inhaled acrolein readily reacts with various molecules on the airways surface and thus it nearly completely retained in the respiratory epithelium (Egle 1972).

Exposure of laboratory animals to acrolein can lead to decreased lung function (Costa 1976), increased goblet cell number in the small airways (Borchers 1998), and increased steady state transcript levels of MUC5AC in human airway epithelial cells (Borchers 1999).

Surprisingly, the role of MMPs in mucus overproduction has not been clearly elucidated. There have been few studies investigating the role of metalloproteinases in mucus overproduction.

3-3 Previously Borchers et al. reported a critical role for a monocyte/macrophage metalloelastase,

MMP12, in acrolein-induced MUC5AC expression (1999). Excessive macrophage accumulation was observed in Mmp12 (+/+) strain-matched control mice as compared to Mmp12 (-/-) gene- targeted mice, and macrophage accumulation correlated with level of MUC5AC transcripts in the lungs. Mice exposed to lipopolysaccharide (LPS) develop mucus metaplasia and pretreatment with an MMP inhibitor decreased the LPS-induced mucin production (Kim 2004).

Various inflammatory mediators including, chemokines like interleukin (IL)-8, cytokines like tumor necrosis factor (TNF)-α, reactive oxygen are present in high concentration in the sputum of COPD patients (Keatings 1996), particularly during exacerbations (Aaron 2001). These agents increase transcription of various MMPs, including MMP2 and MMP9 in different cell lines

(Opdenakker 2002).

Using human airway epithelial cells in vitro, we demonstrated that acrolein increased MUC5AC transcripts through an epidermal growth factor receptor-mitogen activated protein kinase

(EGFR-MAPK) pathway (Deshmukh 2005). This response is mediated by ectodomain shedding of EGFR-ligands that is initiated by metalloproteinases, a disintegrin and metalloproteinase domain containing proteins (ADAM) 17 and MMP9. This lead us to propose that acrolein could produce a prolonged increase in MUC5AC expression, via an autocrine feedback loop. This loop involves activation of MMPs that release EGFR-ligands, that bind to and active EGFR and

MAPK signaling. Once activated, EGFR-MAPK signaling mediated an increase in MMP9 transcript levels and decrease in an inhibitor of ADAM17, tissue inhibitor of metalloproteinase protein (TIMP) 3 transcript levels. In the present study, we sought to better understand this mechanism and confirm these observations in vivo. To accomplish this we exposed mice to acrolein and measured MMPs and TIMPs transcripts, and MMP9 protein, and activity in Mmp9

(+/+) (strained-matched controls). To determine whether MMP9 mediated mucin expression, we measured MUC5AC levels in Mmp9 (-/-) gene-targeted mice. We then treated human airway

3-4 epithelial cells with acrolein and assessed the role of EGFR/MAPK signaling in the accumulation of MMP9 transcripts.

3-5 Methods:

Experimental design: To determine whether acrolein-exposure alters the steady state transcript levels of various murine MMPs) and their inhibitors, TIMPs in mouse lung, Mmp9 (+/+) (male, 6-8 wk; Jackson Laboratories, Bar Harbor, ME) were exposed to acrolein (Cat. No. 36520, Alfa

Aesar, Ward Hill, MA) (2.0 ppm x 6 h/d x 5 d/wk x 4 wk) as previously described (Borchers

1998). To confirm the role of MMP9 in MUC5AC increase after acrolein exposure in vivo, wild type Mmp9(+/+) mice and gene targeted MMP9 deficient, Mmp9(-/-) mice (FVB.Cg-Mmp9tm1Tvu

Stock No. 004104, donated to Jackson Laboratories by Zena Werb) (male, 6-8 wk) were exposed to acrolein (2.0 ppm x 6 h/d x 5 d/wk x 4 wk). These mice were generated by

Coussens et al. (2000) using a targeting vector containing a neomycin resistance gene driven by the mouse phosphoglycerate kinase promoter to disrupt a most of exon 1 and all of intron 2 of the Mmp9 gene. The construct was electroporated into 129S-derived ZW4 embryonic stem

(ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts, and the resulting chimeric males were mated with Swiss Black females. Progeny animals were mated to Black Swiss mice for an unknown number of generations before being mated with FVB/N animals for 5 generation. The strain-matched control Mmp9(+/+) mice were FVB/NJ strain (Stock

No. 001800 Jackson Laboratories). After the exposures, the animals were killed (n = 6/group), and the trachea and lung were removed.

Tissue preparation: Mmp9(+/+) or Mmp9(-/-) mice were killed immediately after exposure by an intraperitoneal injection of pentobarbital sodium (50 mg/kg; Nembutol, Abbott Laboratories,

Chicago, IL) and severing of the posterior abdominal aorta. The chest cavity was opened and the right inferior lobe and the left lobe were clamped, excised, frozen in liquid nitrogen and stored (-70°C) for later mRNA analysis, western blot, zymography and MMP9 metalloproteinase activity assay. To obtain tissue for immunohistochemsitry, a cannula was inserted in the middle

of the trachea and the lung was instilled (pressure: 30 cm H2O) with 10% phosphate-buffered

3-6 Formalin (Cat. No. SF100, Fisher, Fair Lawn, NJ). The trachea was ligated, and the inflated lung was immersed in fixative (24 h, 4°C). Fixed tissues were dissected after 24 h, and the trachea and midlobe sections of the left lung were washed with phosphate-buffered saline (PBS)

(Cat. No. 14287–080, Invitrogen, Carlsbad, CA), dehydrated through a series of graded ethanol solutions (30-70%), and processed into paraffin blocks (Hypercenter XP, Shandon, Ramsey,

MN).

Quantitation of transcript levels: To determine whether acrolein-exposure alters the transcript levels of various murine MMPs, TIMPs and MUC5AC, total RNA from each mouse was reverse transcribed into cDNA as previously described (Deshmukh 2005). cDNA (2 µL) was used in the subsequent PCR reaction using Taqman universal master mix (2x, 12.5 µL) (Cat. No. 4304437,

Applied Biosystems, Foster City, CA), in a 25 µL reaction mixture containing 0.2 µM forward and reverse primers (Sigma Genosys, Austin TX) and 0.1µM Taqman sequence specific FAM-

TAMRA probes (Synthegen, Houston, TX). The sequences of the specific primers and probes used for PCR are in table 1. PCR was performed using Storm 7600 as follows: 95°C for 15 minutes followed by 40 cycles of 95°C for 15 s and 60°C for 1 minute. To determine whether acrolein-treatment alters the transcript level of MMP9 in NCI-H292 cells, total RNA was reverse transcribed into cDNA and (2 µl) used in the subsequent PCR reaction using SYBR™Green

Quantitech real time PCR master mix (2x, 12.5 µL) (Cat. No. 204143, Qiagen, Valencia, CA) in a 25 µL reaction mixture containing 0.2 µM forward and reverse primers (MMP9: Cat. No.

PPH00152A, RPL32: Cat. No. PPH02371A, Super Array, Frederick, MD). PCR was performed using Storm 7600 (Applied Biosystems) as follows: 95°C for 15 minutes followed by 40 cycles of

95°C for 30 s, 57°C for 30 s and 72°C for 30 s, followed by a disassociation stage (72°C, 10 minutes). For each RT-PCR, a serial dilution (0.5-0.032 µg) of total mRNA was amplified to obtain a standard curve. The relative amount of transcript was determined by comparing each sample against the standard curve. Each sample was analyzed in quadruplicate, normalized to

3-7 GAPDH for murine transcripts or ribosomal protein L32 (RPL32) for human transcripts, and results were expressed as fold increase or decrease with respect to the control.

MMP9 immunohistochemsitry: For immunohistochemical detection of MMP9, paraffin sections

(5 µm) were treated (30 min, 95°C) with citrate buffer (10 mM Citric acid, 0.05% Tween 20, pH

6.0), rinsed (2X) in PBS and incubated (30 min, 25°C) with serum blocking solution (2% goat serum, 1% BSA, 0.1 % Triton X-100 and 0.5 % Tween 20). Sections were incubated (30 min,

25°C) with anti-MMP9 (1:100) antibody (Cat. No. IM37L, Calbiochem, San Diego, CA) in antibody dilution buffer (1% BSA). Endogenous peroxidase activity was quenched (15 min,

25°C) with 3% H2O2 in methanol. The section was incubated (30 min, 25°C) with horse radish peroxidase (HRP) -labeled goat anti-mouse secondary antibody (1:5000) (Cat. No. K5355,

Dako Cytomation, Fort Collins, CO) in antibody dilution buffer, rinsed (2X, PBS) and incubated

(10 min, 25°C) with chromogen, 3, 3’diamino benzidine tetrachloride (DAB) (0.05%) (Cat. No.

K5355, Dako Cytomation) in PBS and counterstained (1 min, 25°C) with hematoxylin. The sections were visualized under Spot 2000 microscope (40X objective) and the images were captured by a cooled CCD camera with Metamorph™ (Meta Imaging, Molecular devices,

Downington, PA).

MMP9, MUC5AC and GAPDH western blot: To determine whether acrolein alters the protein level of murine MMP9 and MUC5AC, frozen lung tissue was homogenized (Tekmar, Cincinnati,

OH) at maximum speed and lysed with ice-cold tissue protein extraction reagent (TPER) (Cat.

No. 78510, Pierce, Rockford, IL) containing 1X protease inhibitor cocktail (Cat. No. 78410,

Pierce). The whole lung homogenates were centrifuged (12,000 g, 5 min, 4°C) and supernatant was used to determine the protein concentration using the bicinchoninic acid (BCA) method.

Lung protein (50 µg) was mixed with 2X SDS sample buffer (Cat. No. 516732, Sigma, St. Louis,

MO) and boiled (5 min). Protein was resolved by SDS polyacrylamide gel electrophoresis using

3-8 4-12 % Tris-Glycine gels (Cat. No. EC6028, Invitrogen, Carlsbad, CA) and transferred electrophoretically to polyvinylidene dichloride (PVDF) membrane (Cat. No. LC2005; Invitrogen), which was incubated with 5% fat-free skimmed milk in tris-buffered saline (TBS) containing

0.05% Tween 20 (1 h, 25°C) and incubated (overnight, 4°C) with anti-MMP9 antibody (1: 1000)

(Cat. No. AB16306, Abcam, Cambridge, MA) or anti-MUC5AC antibody (1:100) antibody (Cat.

No. MS-145-P, Clone 45M1, Neomarkers, Fremont, CA). The membrane was washed twice with TBS containing 0.05% Tween 20 (0.5% TBS-T) and then incubated (1 h, 25°C) with 1:

4000 goat anti-rabbit IgG HRP linked secondary antibody (Cat. No. 7074, Cell Signaling

Technology, Waltham, MA) for MMP9 or 1:4000 goat-anti-mouse HRP linked secondary antibody (Cat. No. SC-2005, Santa Cruz Biotechnology, Santa Cruz, CA) for MUC5AC. The membrane was washed twice with 0.5% TBS-T and bound antibody was visualized using enhanced chemiluminescent kit (Cat. No. RPN2108, Amersham Biosciences, Piscataway, NJ).

The membrane was stripped using a stripping solution containing 2 % SDS, 16 mM Tris-HCl

(pH 6.7) at 60°C for 1 h. The membrane was incubated (overnight, 4°C) with 1:1000 anti-

GAPDH antibody (Cat. No. AB9485, Abcam) for loading control. The membrane was washed twice with 0.5% TBS-T and then incubated (1 h, 25°C) with 1: 4000 goat anti-rabbit IgG HRP linked secondary antibody, washed twice with 0.5% TBS-T and bound antibody was visualized using enhanced chemiluminescent kit.

Gelatin zymography: To determine if acrolein alters the metalloproteinase activity in mouse lung after exposure to acrolein, lung protein (50 µg) in 2x Tris-Glycine gel loading buffer was electrophoresed on a 10 % Tris-Glycine gel containing 0.1 % gelatin (Cat. No. EC6175,

Invitrogen). Gels were washed two times (30 min, room temperature) in zymogram renaturing solution (Cat. No. LC2670, Invitrogen) and then preincubated (30 min, room temperature) in zymogram developing solution (Cat. No. LC2671, Invitrogen) and subsequently incubated (12 h,

37°C) in zymogram developing solution. Gels were stained in 0.5% Coomassie blue R-250 in

3-9 40% methanol, 10% acetic acid (1 h, 25°C), and destained in 40% methanol, 10% acetic acid (1 h, 25°C) with a rinse and two changes of destaining solution to visualize digested bands in the gelatin matrix. Gel was photographed using a digital camera.

MUC5AC ELISA: Level of murine MUC5AC protein in the lungs was determined by ELISA using a protocol described before (Takeyama 2000). Briefly, 50 µl of total lung homogenates containing 100 µg protein or (100 – 0. 01 µg/ml) standard (Bovine mucus; Cat. No. M3895,

Sigma) was incubated (2 h, 37°C) with bicarbonate-carbonate buffer (50 µl) in a 96-well plate

(Maxisorp Nunc; Fisher Scientific). Plates were washed three times with 0.5% TBS-T and blocked with 2% bovine serum albumin, fraction V (Sigma), (1 h, room temperature). Plates were again washed three times with 0.5 % TBS-T and then incubated (overnight, 4°C) with 100

µl of (1:100) MUC5AC monoclonal antibodies (clone 45 1, NeoMarkers) diluted with 0.5% TBS-

T. The wells were washed three times with 0.5 % TBS-T and incubated (1 h, 37°C) with 100 µl of (1:2000) with goat-anti-rabbit IgG-HRP linked antibody (Santa Cruz Biotechnologies). The wells were washed three times with 0.5% TBS-T and incubated with (1 h, room temperature)

( with o-phenylenediamine Sigma) and stopped with 1 N H2SO4. Absorbance was read at

492 nm. The relative amount of MUC5AC protein was determined by comparing each sample against the standard curve. Each sample was analyzed in duplicate in the linear portion of the standard curve.

Cell culture, inhibitor pretreatment and in vitro acrolein treatment: NCI-H292 cells (ATCC, Cat.

No. CRL-1848, Manassas, VA) were grown in 75-cm2 plastic tissue culture flasks (Corning, Cat.

No. 3376, Corning, NY) and maintained in RPMI 1640 medium (ATCC, Cat. No. 30-2001), supplemented with 10% fetal calf serum (ATCC, Cat. No. 30-2020), penicillin (100 U/ml), and streptomycin (100 µg/ml; both from Sigma, St. Louis, MO) (37°C, pH 7.4). For acrolein treatment, NCI-H292 cells were seeded (5,000 cells/cm2) into 30 mm six-well plates (Corning,

3-10 Cat. No. 3506, Corning, NY). Once confluent, the cells were incubated (37°C, pH 7.4) for 24 h in serum-free medium (RPMI 1640). To determine if acrolein treatment increases the steady state transcript level of human MMP9, NCI-H292 cells were treated (4 h, 37°C) with increasing concentrations of acrolein (0.01-10 µM) or PBS. To determine the role of epidermal growth factor receptor (EGFR) in acrolein-induced increase in human MMP9 transcript level, confluent

NCI-H292 cells were pre-treated (1 h, 37°C) with an EGFR kinase inhibitor, AG1478 (0.025 µM)

(Cat. No. 658552, Calbiochem, San Diego, CA) or a neutralizing antibody to EGFR, LA-1 (10

µg/ml) (Cat. No. 05–101, Upstate, Charlottesville, VA). To determine the role of various mitogen activated protein kinase (MAPK) activation in acrolein-induced increase in hMMP9 transcript level, NCI-H292 cells were pre-treated (1 h, 37°C) with MAPK kinase (MAP2K) inhibitor,

PD98059, (5 µM) (Cat. No. 51300, Calbiochem) or c-jun N-terminal kinase (JNK) inhibitor,

SP600125, (5 µM) (Cat. No. 420119, Calbiochem) or p38 MAPK inhibitor, ML3403 (5 µM) (Cat.

No. 506121, Calbiochem) and then incubated (4 h, 37°C) with acrolein (0.1 µM). After treatment, the cells were washed with PBS, lysed by Trizol reagent and total RNA was isolated by isopropanol/chloroform precipitation and suspended in RNAase free water.

MMP9 activity assay: Lungs from acrolein-exposed Mmp9(+/+) mice or acrolein-treated NCI-

H292 cells were homogenized using extraction buffer (50 mM Tris-HCl pH 7.6, 1.5 mM NaCl,

0.5 mM CaCl2,1 µM ZnCl2, 0.01% Brij 35 and 0.25% Triton™ X-100). The samples (100 µl) or standards (MMP9, 0.4-0.001 µg/µl) were then incubated (overnight, 4°C) in 96 well micro-titre plate coated with anti-MMP9. The wells were washed (2X, Wash solution containing 0.01 M

Sodium Phosphate pH 7.0 and 0.05% Tween™ 20) and incubated (3 h, 37°C) with detection- enzyme (urokinase) and substrate (S-2444™) in reaction buffer (50 mM Tris-HCl pH 7.6, 1.5 mM NaCl, 0.5 mM CaCl2, 1 µM ZnCl2, Heparin, 1 unit/ml and 0.01% Brij™ 35) and read spectrophotometrically (405 nm). The relative amount of MMP9 activity was determined by comparing each sample against the standard curve. Each sample was analyzed in the linear

3-11 portion of the curve in duplicate and results were expressed as fold change in MMP9 metalloproteinase activity.

Direct activation of pro-MMP9: To determine if acrolein directly activates pro-MMP9, 20 ng purified human pro-MMP9 (Cat. No. PF038, Calbiochem) was incubated (0-4 h, 37°C) with (0.1-

10 µM) acrolein or (1 mM) 4-aminophenylmercuric acetate (APMA) in 50 µl of 50 mM Tris-HCl,

® pH 7.6, 1.5 mM NaCl, 0.5 mM CaCl2, 1 µM ZnCl2 and 0.01% Brij -35. An aliquot (10 µl) of the acrolein or APMA treated pro-MMP9 was used subsequently for gelatin zymography as described before.

3-12 Results

Acrolein increased MMP9 activity in vitro. Incubation of pro-MMP9 with organomercurial compound, APMA, removes ~8-9 kDa of N-terminal prodomain of pro-MMP9 to generate ~ 84 kDa active isoform (Shapiro 1995). Acrolein increased the formation of ~86 kDa and ~84 kDa isoforms, representing active MMP9 in a concentration dependent fashion (≥ 0.3 µM) (Fig 1A and 1B) as early as 30 min (Fig 1C). Additional isoforms corresponding to molecular weight

~150-200 kDa were observed on the zymogram, which is consistent with presence of dimers of pro-MMP9 and active MMP9 (Min 2002) in purified MMP9 preparations. Thus acrolein (≥ 0.3

µM) can directly activate pro-MMP9.

Acrolein altered the transcript level of MMP9s and TIMP3: Mmp9(+/+) mice exposed to acrolein had increased steady state transcript levels of MMP9. Transcript levels of MMP2, MMP3,

MMP7, MMP12 and a distegrin and metalloproteinase domain protein (ADAM) 17 were unchanged (Fig 1A). Steady state transcript levels of TIMP3, an inhibitor of ADAM17 decreased in the lung of acrolein exposed Mmp9 (+/+) mice, whereas TIMP1, TIMP2 and TIMP4 transcript levels were unchanged (Fig 1B). Thus acrolein increased MMP9 transcript levels and decreased transcript levels of TIMP3, an inhibitor of MMP9 and ADAM17.

MMP9 protein and activity increased in Mmp9 (+/+) mice lung exposed to acrolein: The increase in steady state transcript levels of MMP9 induced by repetitive acrolein exposure was accompanied by an increase in the protein levels of MMP9. The airways from Mmp9 (+/+) mice exposed to acrolein had increased MMP9 immunostaining (Fig 2, Panel A) compared to the airways from control (unexposed) Mmp9 (+/+) mice (Fig 2, Panel B). No immunostaining for

MMP9 was observed in the airways of Mmp9 (-/+) mice (negative control) (Fig 2, Panels C and

D). Similarly, MMP9 protein in the whole lung homogenates in acrolein-exposed Mmp9 (+/+) mice

3-13 also increased as compared to control (unexposed) Mmp9 (+/+) mice (Fig 3A). Gelatinase activity

(~103 KDa: pro-MMP9, ~86 KDa: MMP9) increased in the whole lung homogenates from acrolein-exposed Mmp9 (+/+) mice (Fig 3B). MMP9 activity also increased significantly in the lung of Mmp9 (+/+) mice after acrolein exposure as compared to controls (Fig 3C). Thus acrolein increased MMP9 protein and activity in airways of mice exposed to acrolein.

MMP9 mediates increased MUC5AC in the lungs after acrolein exposure. The steady state transcript (Fig 4A) and protein levels of MUC5AC by ELISA (Fig 4B) and western blot (Fig 4C) increased significantly in acrolein-exposed Mmp9 (+/+) lung and acrolein-exposed Mmp9 (-/-) mice as compared to the unexposed littermates. However, the increase in MUC5AC transcripts and protein was significantly lower in acrolein-exposed Mmp9 (-/-) mice than Mmp9 (+/+) mice (Fig 4A,

4B and 4C). Thus, mice deficient in MMP9 responded less, supporting our hypothesis that acrolein-induced increases in MUC5AC are mediated by MMP9.

MMP9 transcripts levels increased after acrolein treatment through EGFR/MAPK3/2 signaling.

Acrolein increased the MMP9 transcripts in a concentration dependent manner in NCI-H292 cells, with 0.1 µM significantly increasing the level of MMP9 transcripts (Fig 5A). Increased

MMP9 transcript levels after acrolein treatment were mediated by epidermal growth factor receptor (EGFR). Pretreatment with AG1478, an EGFR kinase inhibitor or LA-1, a neutralizing antibody against EGFR reduced the acrolein-induced increase in MMP9 transcript (Fig 5B).

EGFR activation leads to activation of downstream mitogen activated protein kinase (MAPK).

Pretreating the cells with a MAP2K inhibitor, PD98059, reduced the acrolein-induced increase in

MMP9 transcripts (Fig 5C). Pretreatment with a C-jun N-terminal kinase (JNK) inhibitor or a p38

MAP kinase inhibitor had no effect on acrolein-induced increase in MMP9 transcripts (Fig 5C).

Thus, increase in MMP9 transcripts in acrolein-treated airway epithelial cells is mediated by

EGFR-MAPK3/2 signal transduction pathway.

3-14 Discussion:

Acrolein initiates mucus overproduction in vivo. Animals exposed repeatedly to acrolein develop histological changes including epithelial damage, mucus metaplasia and bronchiolitis, accompanied by excessive macrophage accumulation in the airways (Feron 1978, Borchers

1998). Acrolein exposure induced mucous cell metaplasia in airways, and increased MUC5AC transcripts in the lung of Sprague-Dawley rats (Borchers 1998) and FVB/NJ mice (Borchers

1999). However, the mechanism of acrolein-induced increase in MUC5AC is unclear.

MMPs are a family of neutral proteinases that are minimally composed of a prodomain that requires cleavage for activation of a zinc-binding catalytic domain. Though MMPs are involved in pathogenesis of emphysema, the role of MMP in mucus overproduction in COPD is unclear.

Previously we reported a critical role for MMP12 in acrolein-induced MUC5AC expression

(Borchers 1998). Acrolein increased MUC5AC transcripts and macrophage accumulation in lungs of wild type littermate controls, Mmp12 +/+ as compared to Mmp12 -/- mice. Similarly, LPS induced mucus metaplasia in rat lung, and this response is accompanied by EGFR phosphorylation (Kim 2004). Moreover, pretreatment with an MMP inhibitor decreased the LPS- induced mucin production in rats (Kim 2004). However, because this inhibitor is effective against several MMPs, it was important to determine which MMPs and TIMPs were involved in the processes that lead to increased MUC5AC transcript levels.

We sought to determine the identity of MMP involved is acrolein-induced MUCA5C increase.

Transcript levels of MMP2, MMP3, MMP7, MMP12 and MMP13 were unaltered in mouse lung after acrolein-exposure; however transcript levels of MMP9 increased significantly. MMP9 protein level increased in the lung after acrolein exposure. In the lung, bronchial epithelial cells

(Yao 1999), Clara cells (Betsuyaku 2000), alveolar type II cells (Pardo 1999), fibroblasts

(Wilhelm 1989), smooth muscle cells (Kenagy 1994), and endothelial cells (Schnaper 1993)

3-15 produce MMP9. Immunostaining for MMP9 increased in the mouse airways exposed to acrolein as compared to airways in unexposed animals (Fig 2). The intensity of immunostaining was greatest in the extracellular matrix below the surface epithelial cells and in epithelial cells. This supports the role of the epithelial cells as a source of the secreted MMP9. In addition, it is likely in the airway epithelial cell receives the greatest dose of acrolein and this may be sufficient to initiate the response, which would be consistent with our previous in vitro studies. To confirm the role of MMP9 in acrolein-induced MUC5AC increase in vivo, gene-targeted Mmp9 (-/-) and strain matched control Mmp9 (+/+) mice were exposed to acrolein. Acrolein increased MUC5AC transcripts and protein levels in the lungs of both Mmp9 (-/-) and the wild type Mmp9 (+/+) mice as compared to the control (Fig 4). However, the increase in MUC5AC transcript and protein was significantly lower in the lungs of acrolein exposed Mmp9 (-/-) mice than Mmp9 (+/+) (Fig 4). This finding supports a role in vivo for MMP9, and suggests that other MMPs or ADAMs may play a role in the response. Likely other candidates are MMP12 and ADAM17, and possibly other

MMPs activated in the lung during injury and remodeling.

Due to wide-ranging biological consequences, MMP9 activity is tightly controlled. MMP9 is synthesized as an inactive pro-enzyme (proMMP9) and is activated by proteolytic removal of an amino-terminal prodomain upon secretion into the extracellular space. The prodomain keeps the enzyme latent through the interaction of a cysteine residue with a zinc ion in the active site.

Oxidants and other compounds interact with the protective cysteine. This reduces latency by altering protein conformation that permits entrance of water essential for catalysis and accelerates the autocatalytic loss of the propeptide domain. This process is called the "cysteine switch" mechanism (Van Wart 1990). Because of its ability to covalently modify macromolecules and disrupt critical cellular functions and cause mutations (Izard 1978,

Esterbauer 1991, Cohen 1992), acrolein is considered an important mediator of cell damage.

Acrolein-protein adducts has been found to accumulate in ischemic tissue (Uchida 1999) and in

3-16 atherosclerotic lesions (Shao 2005). Acrolein induced the release of pro-MMP9 in the cell culture supernatant from NCI-H292 cells exposed to acrolein (Deshmukh 2005). Acrolein directly activated pro-MMP9 in a concentration dependent fashion (≥ 0.3 µM) (Fig 1A and 1B) as early as 30 min (Fig 1C). MMP9 activity increased in the acrolein treated NCI-H292 cells (Fig

5D) as well as acrolein-exposed mouse lung (Fig 4A). Thus acrolein can potentially disrupt the interactions between the cysteine residues and the zinc ion in the active site and thus directly activate MMP9 in absence of other active MMPs, including MMP2.

Enzymatically active MMPs can potentially influence cell behavior by cleaving cell-cell adhesion proteins, by releasing bioactive cell surface molecules, or by cleaving cell surface molecules that transduce signals from the extracellular environment. A variety of growth factors, growth factor-binding proteins, growth factor receptors are MMP substrates. Heparin-binding EGF (HB-

EGF) shedding is mediated by a specific proteolytic cleavage at a juxtamembrane site through the action of MMP3 (Suzuki 1997) and MMP7 (Yu 2002). MMP2 and MMP9 can cleave cell surface bound pro-EGFR ligands and transactivated EGFR in pituitary gonadotrophic cells

(Roelle 2004). Previously small interfering (si) RNA directed against MMP9 inhibited the acrolein-induced increase in MUC5AC transcripts in airway epithelial cells via ligand-dependent

EGFR activation (Deshmukh 2005).

MMP9 activity is also regulated at the transcriptional and post-transcriptional levels and also controlled at the protein level via its inhibitors. MMP9 gene expression is regulated by numerous growth factors, including epidermal growth factor (EGF) (Harvey 1995; Miyagi 1995).

Because, acrolein at lower concentrations (0.3 µM) increased phosphorylation of EGFR in NCI-

H292 cells (Deshmukh 2005), we next investigated the role of EGFR in acrolein-induced increases in MMP9 transcripts. Pretreatment with an EGFR kinase inhibitor, AG1478 or an

EGFR neutralizing antibody, LA-1, decreased the acrolein-induced increase in MMP9

3-17 transcripts, suggesting EGFR activation is involved in acrolein-induced MMP9 transcript increase. Phosphorylation of EGFR leads to activation of downstream MAP kinases. Increase in MMP9 expression by IL-1 and TNF-α is mediated through activation of the transcription factors NF-κB and AP-1 via MAPK8 or MAPK14 pathways (Baud and Karin, 2001). Besides IL-

1β and TNF-α, other cytokines like IL-17 have also increase MMP9 expression via MAPK signaling (Jovanovic 2000). PMA induced MMP9 expression in squamous cell carcinoma

(SCC) cells, required stimulation of the MAPK14 (p38) pathway (Simon 1998; Simon 2001).

Pretreatment with MEK inhibitor, PD98059, but not the JNK inhibitor, SP600125 or p38 inhibitor,

ML3403, decreased the acrolein-induced increase in MMP9 transcripts, suggesting that MAPK

3/2 and not MAPK8 or MAPK14 are involved in the response initiated by acrolein. Thus acrolein-induced increase in MM9 (Fig 6) and MUC5AC transcripts (Deshmukh 2005) is mediated by signaling through similar EGFR-MAPK pathway.

MMP9 activity is also regulated by the endogenous inhibitors, TIMPs (Murphy and Docherty,

1992). TIMP1 (Yao 1999), TIMP2, TIMP3 and TIMP4 (Thomas 2000) are expressed in bronchial epithelium. TIMP2 and to a lesser extent TIMP1 and TIMP3 bind with MMP9 and inhibit its activity (Olson 1997). TIMP3 binds with high affinity to ADAM17 and inhibits it (Amour

1998). In contrast to TIMP2, which is constitutively expressed (Gomez 1997), TIMP1, TIMP3, and TIMP4 are highly inducible by extra cellular signals like growth factors, phorbol esters, matrix proteins, inflammatory cytokines and transforming oncogenes (Baker 2002). Acrolein decreased the transcript levels of TIMP3 in the acrolein-exposed mouse lung, whereas the transcript levels of TIMP1, TIMP2 and TIMP4 were unaltered (Fig 2B). The decrease in TIMP3 could explain how ADAM17 activity may be involved in the increase in MUC5AC even though

ADAM17 transcript levels did not change. ADAM17 is important in cigarette smoke induced

MUC5AC expression (Shao 2004). Although the activity of ADAM17 was not measured in this experiment, it is also likely to have contributed to shedding of pro-EGFR ligands and increased

3-18 MUC5AC transcript levels. Even tough ADAM17 transcript levels were unchanged, transcript levels of TIMP3, an inhibitor of ADAM17 (Amour 1998) decreased invitro in NCI-H292 cells treated with acrolein (Deshmukh 2005) and in vivo in lung of acrolein-exposed mice. In addition activation of MAPK3/2 (ERK 1/2) is consistent with increased ADAM17 activity. Activation of

MAPK3/2 results in phosphorylation of a theronine (Thr-735) residue in ADAM17, which stimulated the trafficking of ADAM17 from the endoplasmic reticulum to the cell surface (Soond

2005). Because TIMP3 and MMP9 are produced by the epithelial cell, the decrease in TIMP3 and the increase in MMP9 are consistent with autocrine induction.

Autocrine loops are established when soluble ligands secreted by cells bind to and stimulate receptors on their own surfaces (Sporn and Roberts 1992). Acrolein, by disrupting the cysteine residues in the pro-domain of MMP9 can directly activate pro-MMP9 to increase MMP9 activity.

MMP9 proteolytically processes various pro-EGFR ligands (Roelle 2003) on the cell surface which can then bind to EGF receptor on the same cell or the adjacent cell. EGFR-MAPK activation increased the transcription of MUC5AC (Takeyama 2000). Moreover, acrolein increased the MMP9 transcripts through EGFR-MAPK signaling (Fig 6) and stimulated the release of pro-MMP9 in the conditioned medium (Deshmukh 2005) which can be subsequently activated by acrolein. Thus acrolein increased MMP9 transcripts, protein, pro-MMP9 release and pro-MMP9 activation and increased MMP9 activity to release more pro-EGFR ligands and establish an autocrine positive feedback loop.

In summary, acrolein, a major component of cigarette smoke, increased MMP9 activity to increase mucin transcription. In addition, chronic exposure initiates an autocrine feedback loop that further stimulates transcription of MMP9 and simultaneously represses transcription of

TIMP3. A combination of these responses leads to persistent mucus production (Fig 7).

3-19 Figure Legends:

Figure 1. Acrolein increased matrix metalloproteinase 9 activity in vitro. Recombinant human pro-MMP9 (100 ng) was incubated (37°C, 30 min-4h) with (0.1-10 µM) or 1 mM 4-aminophenyl mercuric acetate (APMA) in 50 µl reaction buffer (50 mM Tris-HCl pH 7.6, 1.5 mM NaCl, 0.5 mM

CaCl2, 1 µM ZnCl2, and 0.01% Brij™ 35). Pro-MMP9 activation was determined by gelatin zymography. (A) Acrolein increased the activation of pro-MMP9 (92 kDa) to MMP9 (~84, 86 kDa) in vitro in a concentration dependent manner. Each lane was loaded with pro-MMP9 incubated (37°C, 4h) with acrolein (0.1-10 µM) or 1 mM 4-aminophenly mercuric acetate

(APMA) and is representative of each group (n=3/treatment). (B) Acrolein increased the activation of pro-MMP9 (92 kDa) to MMP9 (~84, 86 kDa) as early as 30 min. Each lane was loaded with pro-MMP9 incubated (37°C, 30 min - 4h) with acrolein (0.3 µM) and is representative of each group (n=3/treatment).

Figure 2. Matrix Metalloproteinases (MMP), a disintegrin and metalloproteinase domain proteins (ADAM) 17 and tissue inhibitor of metalloproteinase proteins (TIMP) transcript levels are altered in MMP9(+/+) mouse lung after acrolein exposure. MMP9 (+/+) mice were exposed to acrolein (2.0 ppm x 6h/d x 5d/wk x 4wks) or filtered air (control) and the lungs were removed for mRNA isolation. Transcript level of various MMPs, TIMPs and ADAM17 was determined by quantitative real-time PCR (qRT-PCR). (A) Transcript levels of MMP9 and MMP14 increased following acrolein exposure, whereas transcript levels of MMP2, 3, 7, and 13 were unchanged.

ADAM17 transcript level was also unchanged. (B) Transcript level of TIMP3 was decreased, whereas TIMP1, 2, and 4 were unchanged. The results are expressed as fold change in the level of transcript after normalizing to β-actin. (Values are mean ± SEM. * Significantly different from control. n=6, ANOVA, Student-Newman-Keul’s test).

3-20 Figure 3. Immunohistochemical staining for Matrix Metalloproteinase (MMP) 9 in the airway epithelium increased in the lung of Mmp9(+/+) mice exposed to acrolein. Mmp9 (+/+) mice were exposed to acrolein (2.0 ppm x 6h/d x 5d/wk x 4wks) or filtered air (control). After 4wks, the lungs were removed and fixed. Serial sections of paraffin embedded lung tissue were immunostained with anti-MMP9 antibody (Ab) or anti-immunoglobulin (Ig) G Ab (isotype control). Immunostaining with anti-MMP9 Ab increased in the respiratory epithelium of MMP9

(+/+) mice (D) after acrolein exposure as compared to control mice (B). No immunostaining was observed in the respiratory epithelium with anti-IgG Ab in the control (A) or acrolein-exposed

MMP9 (+/+) mice (C). (Original magnification 400 x)

Figure 4. Matrix metalloproteinase 9 protein and activity increased in MMP9(+/+) mouse lung following acrolein exposure. MMP9 (+/+) or MMP9 (-/-) mice were exposed to acrolein (2.0 ppm x

6h/d x 5d/wk x 4wks) or filtered air (control). After 4wks, the lungs were removed and whole lung homogenate was used for western blotting, zymograms and MMP9 activity assay. (A)

Protein levels of MMP9 as determined by Western Blot increased in mouse lung exposed to acrolein, whereas GAPDH protein levels remained changed (loading control). Each lane was loaded with protein (50 µg) obtained from an individual mouse that is representative of each group (n=5/treatment). (B) Gelatinase activity (~ 103 kDa and ~ 94 kDa: MMP9) increased in the mouse lung exposed to acrolein. Each lane was loaded with protein (25 µg) obtained from an individual mouse that is representative of each group (n=5/treatment). (C) MMP9 activity levels increased in mouse lung exposed to acrolein. Active MMP9 in lung homogenate was captured with anti-MMP9 antibody which was then used to activate a modified murokinase detection enzyme to cleave a chromomeric peptide substrate and the resultant color was read spectrophotometrically at 405 nm. In all experiments a serial dilution of known sample of active

MMP9 was run simultaneously to obtain a reference curve. Each sample was analyzed in the linear portion of the curve and the relative amount of active MMP9 was determined by

3-21 comparing OD405 of each sample against the standard curve. The results are expressed as fold increase in the MMP9 activity as compared to the control. (Values are mean ± SEM. *

Significantly different from control. n=6, ANOVA, Student-Newman-Keul’s test).

Figure 5. Matrix metalloproteinase 9 mediates acrolein-induced increase in mucin 5 (subtype

A&C) (MUC5AC) in Mmp9(+/+) mouse lung. Mmp9 (+/+) mice were exposed to acrolein (2.0 ppm x

6h/d x 5d/wk x 4wks) or filtered air (control) and whole lung homogenates were used to determine MUC5AC protein levels by Western blot and ELISA. (A) RNA was isolated to determine transcript levels of MMP9 by quantitative real-time PCR (qRT-PCR). The results are expressed as fold change in the level of MMP9 transcripts after normalizing to GAPDH.

Transcript levels of MUC5AC increased more in the lung of Mmp9(+/+) mouse than Mmp9(-/-) mouse following acrolein exposure. (B) Protein levels of MUC5AC as determined by ELISA increased more in the lung of Mmp9(+/+) mouse than Mmp9(-/-) mouse following acrolein exposure

(n=5). For ELISA a serial dilution of known sample of MUC5AC was run to obtain a reference curve. Each sample was analyzed in the linear portion of the curve. (C) Protein levels of

MUC5AC as determined by Western Blot increased more in MMP9(+/+) control mice than Mmp9(-

/-) mouse lung follow acrolein exposure whereas GAPDH protein levels as determined by WB remained unchanged (loading control). Each lane was loaded with protein (50 µg) obtained from an individual mouse that is representative of each group (n=5/treatment). The results are expressed as fold increase in the MUC5AC protein as compared to the control. (Values are mean ± SEM, * significantly different from the control, ANOVA, Student-Newman-Keul’s test, n=5)

Figure 6. Acrolein-induced increases in transcript levels of MMP9 are mediated by epidermal growth factor receptor (EGFR) and mitogen activated protein kinase (MAPK) signaling in human airway epithelial cells. Confluent NCI-H292 cells were pretreated (37°C, 1 h) with 0.250 µM

3-22 AG1478, an EGFR kinase inhibitor, or 5 µM, PD98059, a MAPK3/2 inhibitor or 5 µM,

SP600125, a JNK inhibitor or 5 µM ML3403, a p38 inhibitor and then incubated (37°C, 4h) with

0.01 µM acrolein or vehicle. RNA was isolated and the level of MMP9 transcript was determined by quantitative real time PCR (qRT-PCR). The results are expressed as fold change in the level of MMP9 transcripts after normalizing to RPL32. (A) Acrolein increased

MMP9 transcript levels in a dose-dependent manner in NCI-H292 cells as compared to the control (vehicle treated cells). (B) Acrolein-induced increases in transcript levels of MMP9 are diminished in cells treated with an EGFR neutralizing antibody (LA-1) or an EGFR kinase inhibitor (AG1478). (C) Acrolein-induced increases in transcript levels of MMP9 are diminished in cells treated with a MAPK3/2 inhibitor (PD98059), whereas transcript levels for MMP9 were not diminished in cells treated with a JNK inhibitor (SP600125) or a p38 kinase inhibitor

(ML3403). (Values are mean ± SEM. * Significantly different from control. † Significantly different from the acrolein treated samples (without inhibitor) =6, ANOVA, Student-Newman-

Keul’s test, n=6-9).

Figure 7: Autocrine feedback loop regulating Matrix Metalloproteinase (MMP) 9 leads to persistent mucin 5 (subtype A and C) (MUC5AC) increase. Acrolein-induced increase in

MUC5AC is mediated by MMP9. Acrolein increased MMP9 transcripts and decreased tissue inhibitor of metalloproteinase proteins (TIMP) 3transcripts. Acrolein-induced increase in MMP9 transcripts is mediated by a metalloproteinase (inhibited by GM6001). Inhibition of EGFR (by neutralizing antibody, LA-1, or EGFR kinase inhibitor, AG1478) decreased the acrolein-induced increase in MMP9 transcripts. Inhibition of mitogen activated protein kinase (MAPK) 3/2, (by

PD98059) but not MAPK8 (by SP600125) or MAPK14 (by ML3403) diminished the acrolein- induced increase in MMP9 transcripts. Acrolein increased MMP9 activity in the lungs and protein in the airways. Thus chronic exposure to acrolein initiates an autocrine feedback loop

3-23 that stimulates transcription of MMP9, and simultaneously represses transcription of TIMP3. A combination of these responses leads to persistent mucus production.

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3-33 Table 1:

Murine MUC5AC:

Forward primer: 5’-AAAGACACCAGTAGTCACTCAGCAA-3’

Reverse primer: 5’-CTGGGAAGTCAGTGTCAAACCA-3’

Probe: FAM-TCACACACAACCACTCAACCAGTGACCA-TAMRA

Murine MMP2:

Forward primer: 5’-AACTACGATGATGACCGGAAGTG-3’

Reverse primer: 5’-TGGCATGGCCGAACTCA-3’

Probe: FAM-TCTGTCCTGACCAAGGATATATAGCCTATTCCTCG-TAMRA

Murine MMP3:

Forward primer: 5’-GGAAATCAGTTCTGGGCTATACGA-3’

Reverse primer: 5’-TAGAAATGGCAGCATCGATCTTC-3’

Probe: FAM-AGGTTATCCTAAAAGCATTCACACCCTGGGTCT-TAMRA

Murine MMP7:

Forward primer: 5’-GCAGAATACTCACTAATGCCAAACA-3”

Reverse primer: 5’-CCGAGGTAAGTCTGAAGTATAGGATACA-3’

Probe: FAM-CCAAAATGGCATTCCAGAATTGTCACCTAC-TAMRA

Murine MMP9:

Forward primer: 5’-CGAACTTCGACACTGACAAGAAGT-3’

Reverse primer: 5’-GCACGCTGGAATGATCTAAGC-3’

Probe: FAM-TCTGTCCAGACCAAGGGTACAGCCTGTTC-TAMRA

3-34 Murine MMP12:

Forward primer: 5’-GAAACCCCCATCCTTGACAA-3’

Reverse primer: 5’-TTCCACCAGAAGAACCAGTCTTTAA-3’

Probe: FAM-AGTCCACCATCAACTTTCTGTCACCAAAGC-TAMRA

Murine TIMP1:

Forward primer: 5’-CATGGAAAGCCTCTGTGGATATG-3’

Reverse primer: 5’-AAGCTGCAGGCACTGATGTG-3’

Probe: FAM-CTCATCACGGGCCGCCTAAGGAAC-TAMRA

Murine TIMP2:

Forward primer: 5’-CCAGAAGAAGAGCCTGAACCA-3’

Reverse primer: 5’-GTCCATCCAGAGGCACTCATC-3’

Probe: FAM-ACTCGCTGTCCCATGATCCCTTGC-TAMRA

Murine TIMP3:

Forward primer: 5’-GGCCTCAATTACCGCTACCA-3’

Reverse primer: 5’-CTGATAGCCAGGGTACCCAAAA-3’

Probe: FAM-TGCTACTACTTGCCTTGTTTTGTGACCTCCA-TAMRA

Murine TIMP4:

Forward primer: 5’-TGCAGAGGGAGAGCCTGAA-3’

Reverse primer: 5’-GGTACATGGCACTGCATAGCA-3’

Probe: FAM-CCACCAGAACTGTGGCTGCCAAATC-TAMRA

Murine ADAM17:

3-35 Forward primer: 5’-GAAGAAGTGCCAGGAGGCGATT-3’

Reverse primer: 5’-CGGGCACTCACTGCTATTACCT-3’

Probe: FAM-ATGCTACTTGCAAAGGCGTGTCCTACTGC-TAMRA

Murine GAPDH:

Forward primer: 5’-GTCGTGGATCTGACGTGCC-3’

Reverse primer: 5’-TGCCTGCTTCACCACCTTCT-3’

Probe: FAM-CCTGGAGAAACCTGCCAAGTATGATGACA-TAMRA

3-36 Figures:

Figure 1

3-37 Figure 2

6 A * 5

l)

o

ntr 4 o

3

ript levels

nc

ange over c h

Tras 2

(fold c 1

0 MMP2 MMP3 MMP7 MMP9 MMP12 ADAM17

3-38 Figure 2

B

control) 1.0

ed to

ipt levels r c *

as compar ans 0.5 Tr ge

chan old

f (

0.0 TIMP1 TIMP2 TIMP3 TIMP4

3-39 Figure 3:

3-40 Figure 4

Figure 4

3-41

C * 5

rol) 4 ty i

er cont 3 ne MMP9 activ i 2 d change ov mur ol (f 1

0 Control Exposed

A

5 ) *

ls e 4

cript lev † ans r 3 *

2 Figure 5

hange as compared to controls 1 murine MUC5AC t (fold c

3-42 0 Mmp9(+/+) Mmp9(-/-)

Figure 5

3-43 5 B * ) s

rol 4 by ELISA cont o

3

ared t † *

2 C5AC protein levels

1 d change as comp ne MU i ol (f mur

0 Mmp9(+/+) Mmp9(-/-)

Figure 5

3-44

A *

l)

o 6 s ntr l o e * v

e *

4 ancript l r mpared to c

t

9 P

Figure 6 2 *

human MM d change as co

ol (f

0 3-45 0.01 µM 0.03 µM 0.06 µM0.1 µM Acrolein

Figure 6

3-46 Figure 6 MAPK3/2 Ac EGF rolein

R human MMP9 transcript level inhibitor (fold change as compared to control) in 0 2 4 6 hi bi B tor ++ - -- + - ++ - - * † - * + † + * -

3-47

C

rol) 6 * * cont o * pared t

anscript level 4 r 9 t

2 human MMP old change as com (f

0 Acrolein --+++ JNK inhibitor ++- - - p38 inhibitor - + - - +

Figure 6

3-48

D *

rol) 6 cont o ipt level r c

4 mpared t † † * * 2 human MMP9 trans old change as co (f

0 Acrolein --+++ LA-1 ++- - - GM6001 - + - - +

Figure 7

3-49

Supplementary figures:

3-50 Figure S1

600 B * Control A

IS Acrolein

L 500 E by s l

e 400 v e n l ei mucus) 300

of † prot ml C *

(ng/ 200

100 murine MUC5A

0 Mmp9(+/+) Mmp9(-/-)

Figure S1: Matrix metalloproteinase 9 mediates acrolein-induced increase in mucin 5 (subtype

A&C) (MUC5AC) in Mmp9(+/+) mouse lung. Mmp9 (+/+) mice were exposed to acrolein (2.0 ppm x

6h/d x 5d/wk x 4wks) or filtered air (control). and whole lung homogenates were used to determine MUC5AC protein levels by ELISA. Serial dilution of known sample of bovine mucus

(100 – 0. 01 µg/ml) was run to obtain a reference curve. Each sample was analyzed in the linear portion of the curve. MUC5AC protein increased in the lung of Mmp9(+/+) mouse as compared to Mmp9(-/-) mouse following acrolein exposure (n=5). (Values are mean ± SEM, * significantly different from the control, ANOVA, Student-Newman-Keul’s test, n=5)

3-51 CHAPTER 4

Manuscript III:

Matrix metalloproteinase (MMP) 14 mediates acrolein-induced increase in mucus production in the airways.

Abstract:

Chronic obstructive pulmonary disease (COPD) is major public health problem worldwide induced by cigarette smoking and is characterized by progressive difficulty and an increase in mucus production. Acrolein, a constituent of cigarette smoke increased mucin 5 (subtype A and C) (MUC5AC) production in the airways, however, the mechanism is unclear. Acrolein increased MMP14 activity in airway epithelial cells.

Repeated exposure to acrolein increased the level of MMP14 activity and protein in the lungs. Decreasing the MMP14 protein and activity in vitro by small interfering (si) RNA to MMP14 diminished the acrolein-induced increase in MUC5AC transcripts. Acrolein increased MMP14 transcripts in airway epithelial cells in a concentration dependent fashion. Increase in MMP14 transcripts after acrolein treatment was inhibited by pretreatment with a neutralizing antibody against EGFR or EGFR kinase inhibitor.

Pretreatment with a mitogen activated protein kinase (MAPK) 3/2 inhibitor, but not

MAPK8 or MAPK14 inhibitor diminished the acrolein-induced increase in MMP14 transcripts. Repeated exposure to acrolein, thus initiates a feedback loop that to increase MMP14 protein and activity to cause a persistent increase in mucus production.

4-1 Introduction:

Chronic obstructive pulmonary disease (COPD) is a major and growing global health problem. Predicted to be the third most common cause of death by 2020 (Lopez 1998),

COPD is caused by long-term exposure to inhaled noxious gases, with tobacco smoking accounting for > 90 percent of new cases (Markewitz 1999). Death and disability from

COPD are related to an accelerated decline in lung function (Fletcher 1977), that is a result of remodeling of the airway and alveolar architecture. Small airways (<2-3 mm diameter) normally contribute less than 20% of total airway resistance (Hogg 1968), but this increases markedly in COPD due to narrowing of the airway lumen and mucus hypersecretion. Mucus plugging in the small airways leads to ventilation-perfusion mismatch and insufficient oxygenation of blood, and excessive mucus production is associated with decline in lung function, hospitalization and death (Vestbo 1996).

Mucus, a viscoelastic gel composed of water (95%) and mucins, lines the respiratory epithelium and protects against infectious and environmental agents (Reid 1986).

Mucins are a large heterogeneous glycoproteins, consisting of a protein backbone

(apomucin) to which multiple carbohydrate side chains are attached by glycosyltransferases at serine and theronine residues that are contained in variable number of tandem repeat regions (Leikauf 2002). Mucins are encoded by at least 15 genes, many localized in a gene cluster on human chromosome 11p15 (Leikauf 2002).

Biochemical analysis indicates that mucin 5 (subtype A and C) (MUC5AC) and mucin 5B

(MUC5B) constitute the majority of mucin glycoprotiens in the airway secretions of humans (Rose 1989). Although MUC5B is primarily constitutively expressed, MUC5AC is highly inducible (Borchers 1998). Specialized epithelial (goblet) cells are the major source of MUC5AC in the airways. Goblet cells are typically limited in number and found

4-2 mainly in the large airways of healthy individuals, but with tobacco smoke exposure the number of goblet cells, especially in the small diameter airways, increases markedly

(Reid 1950, Hogg 1968). Recent studies suggest that this shift is mediated by metalloproteinase mediated mobilization of epidermal growth factor (EGF) family ligands that bind to and activate receptor-type protein tyrosine kinases, including epidermal growth factor receptor (EGFR) (Takeyama 2000, Burgel 2001, Shao 2004). EGFR phosphorylation, in turn, activates mitogen-activated protein kinase (MAPK) signaling

(Schlesinger 1998) that mediates cell proliferation, wound healing, and in the airway epithelium, mucin gene expression (Takeyama 2000). Importantly, irritants in cigarette smoke, especially acrolein, can trigger these events (Borchers 1999, Deshmukh 2005).

Matrix metalloproteinases (MMPs), a major family of zinc-dependent neutral proteinases plays a critical in pathogenesis of emphysema in COPD (Hogg 2004). Less is known about the role of MMPs in mucus overproduction in COPD. Previously Borchers et al. reported a critical role for a monocyte/macrophage metalloelastase, MMP12, in acrolein- induced MUC5AC expression (1999). Excessive macrophage accumulation was observed in Mmp12 (+/+) strain-matched control mice as compared to Mmp12 (-/-) gene- targeted mice, and macrophage accumulation correlated with level of MUC5AC transcripts in the lungs. Similarly, acrolein increased MUC5AC transcripts and protein in lungs of wild type littermate controls, Mmp9 +/+ as compared to Mmp9 -/- mice (Manuscript

2: Fig 4).

In the current study we demonstrate that MMP14 mediates acrolein-induced increase in

MUC5AC transcripts. Acrolein increased MMP14 activity in vitro in NCI-H292 cells and in the lungs of FVB/NJ mouse exposed to acrolein. MMP14 transcripts and protein increased after acrolein-treatment in vitro and in vivo via signaling through an epidermal

4-3 growth factor receptor (EGFR) and mitogen activated protein kinase (MAPK) pathway.

Furthermore, immunostaining for MMP14 increased in the airways of human subjects with COPD. Small interfering (si) RNA against MMP14 decreased the acrolein-induced increase in MUC5AC transcripts in NCI-H292 cells. Thus MMP14 mediates acrolein- induced increase in MUC5AC transcripts.

4-4 Methods:

Acrolein exposures: FVB/NJ mice (male, 6-8 wk; Jackson Laboratories, Bar Harbor, ME) were exposed to acrolein (Cat. No. 36520, Alfa Aesar, Ward Hill, MA) (2.0 ppm x 6 h/d x

5 d/wk x 4 wk) as previously described (Borchers 1998). The mice were killed immediately after exposure by an intraperitoneal injection of pentobarbital sodium

(50 mg/kg; Nembutol, Abbott Laboratories, Chicago, IL) and severing of the posterior abdominal aorta. The chest cavity was opened and the lungs were excised, frozen in liquid nitrogen and stored (-70°C) for later mRNA analysis, western blot, and MMP14 metalloproteinase activity assay. To obtain tissue for immunohistochemsitry, a cannula was inserted in the middle of the trachea and the lung was instilled (pressure: 30 cm

H2O) with 10% phosphate-buffered Formalin (Cat. No. SF100, Fisher, Fair Lawn, NJ).

The trachea was ligated, and the inflated lung was immersed in fixative (24 h, 4°C).

Fixed tissues were dissected after 24 h, and washed with phosphate-buffered saline

(PBS) (Cat. No. 14287–080, Invitrogen, Carlsbad, CA), dehydrated through a series of graded ethanol solutions (30-70%), and processed into paraffin blocks (Hypercenter XP,

Shandon, Ramsey, MN).

Quantitation of transcript levels: To determine whether acrolein-exposure alters the transcript levels of MMP14, total RNA from each mouse or NCI-H292 or NHBE cells was reverse transcribed into cDNA as previously described (Deshmukh 2005). cDNA (2 µL) was used in the subsequent PCR reaction using Taqman universal master mix (2x, 12.5

µL) (Cat. No. 4304437, Applied Biosystems, Foster City, CA), in a 25 µL reaction mixture containing 0.2 µM forward and reverse primers (Sigma Genosys, Austin TX) and 0.1µM

Taqman sequence specific FAM-TAMRA probes (Synthegen, Houston, TX). The sequences of the specific primers and probes used for PCR are in table 1. PCR was performed using Storm 7600 as follows: 95°C for 15 minutes followed by 40 cycles of

4-5 95°C for 15 s and 60°C for 1 minute. For each RT-PCR, a serial dilution (0.5-0.032 µg) of total mRNA was amplified to obtain a standard curve. The relative amount of transcript was determined by comparing each sample against the standard curve. Each sample was analyzed in quadruplicate, normalized to GAPDH for murine transcripts or ribosomal protein L32 (RPL32) for human transcripts, and results were expressed as fold increase or decrease with respect to the control.

MMP14 and GAPDH western blot: To determine whether acrolein alters the protein level of murine MMP14 and MUC5AC, frozen lung tissue was homogenized (Tekmar,

Cincinnati, OH) at maximum speed and lysed with ice-cold tissue protein extraction reagent (TPER) (Cat. No. 78510, Pierce, Rockford, IL) containing 1X protease inhibitor cocktail (Cat. No. 78410, Pierce). The whole lung homogenates were centrifuged

(12,000 g, 5 min, 4°C) and supernatant was used to determine the protein concentration using the bicinchoninic acid (BCA) method. Lung protein (50 µg) was mixed with 2X

SDS sample buffer (Cat. No. 516732, Sigma, St. Louis, MO) and boiled (5 min). Protein was resolved by SDS polyacrylamide gel electrophoresis using 4-12 % Tris-Glycine gels

(Cat. No. EC6028, Invitrogen, Carlsbad, CA) and transferred electrophoretically to polyvinylidene dichloride (PVDF) membrane (Cat. No. LC2005; Invitrogen), which was incubated with 5% fat-free skimmed milk in tris-buffered saline (TBS) containing 0.05%

Tween 20 (1 h, 25°C) and incubated (overnight, 4°C) with rabbit anti-MMP14 antibody

(1:1000) (Cat. No. AB8102, Chemicon). The membrane was washed twice with TBS containing 0.05% Tween 20 (0.5% TBS-T) and then incubated (1 h, 25°C) with 1: 4000 goat anti-rabbit IgG HRP linked secondary antibody (Cat. No. 7074, Cell Signaling

Technology, Waltham, MA). The membrane was washed twice with 0.5% TBS-T and bound antibody was visualized using enhanced chemiluminescent kit (Cat. No.

RPN2108, Amersham Biosciences, Piscataway, NJ). The membrane was stripped using

4-6 a stripping solution containing 2 % SDS, 16 mM Tris-HCl (pH 6.7) (1 h, 60°C ) and then incubated (overnight, 4°C) with 1:1000 anti-GAPDH antibody (Cat. No. AB9485, Abcam) for loading control. The membrane was washed twice with 0.5% TBS-T and then incubated (1 h, 25°C) with 1: 4000 goat anti-rabbit IgG HRP linked secondary antibody, washed twice with 0.5% TBS-T and bound antibody was visualized using enhanced chemiluminescent kit.

MMP14 immunohistochemsitry: For immunohistochemical detection of MMP14, paraffin sections (5 µm) were treated (30 min, 95°C) with citrate buffer (10 mM Citric acid, 0.05%

Tween 20, pH 6.0), rinsed (2X) in PBS and incubated (30 min, 25°C) with serum blocking solution (2% goat serum, 1% BSA, 0.1 % Triton X-100 and 0.5 % Tween 20).

Sections were incubated (overnight, 4°C) with anti-MMP14 (1:200) antibody (Cat. No.

MAB3317, Chemicon, Temecula, San Diego, CA) in antibody dilution buffer (1% BSA).

Endogenous peroxidase activity was quenched (15 min, 25°C) with 3% H2O2 in methanol. The section was incubated (30 min, 25°C) with horse radish peroxidase

(HRP) -labeled goat anti-mouse secondary antibody (1:5000) (Cat. No. K5355, Dako

Cytomation, Fort Collins, CO) in antibody dilution buffer, rinsed (2X, PBS) and incubated

(10 min, 25°C) with chromogen, Nova Red (0.05%) (Cat. No. SK48000, Vector

Laboartories, Burlingame, CA) in PBS and counterstained (1 min, 25°C) with hematoxylin. The sections were visualized under Spot 2000 microscope (40X objective) and the images were captured by a cooled CCD camera with Metamorph™ (Meta

Imaging, Molecular devices, Downington, PA).

Cell culture, inhibitor pretreatment and in vitro acrolein treatment: NCI-H292 cells

(ATCC, Cat. No. CRL-1848, Manassas, VA) were grown in 75-cm2 plastic tissue culture flasks (Corning, Cat. No. 3376, Corning, NY) and maintained in RPMI 1640 medium

4-7 (ATCC, Cat. No. 30-2001), supplemented with 10% fetal calf serum (ATCC, Cat. No. 30-

2020), penicillin (100 U/ml), and streptomycin (100 µg/ml; both from Sigma, St. Louis,

MO) (37°C, pH 7.4). In a few studies, normal human bronchial epithelial (NHBE) cells

(Cat. No. CC2540, Cambrex Biosciences, Baltimore, MD) were cultured in 75-cm2 plastic tissue culture flasks and maintained in bronchial epithelial cell growth medium (Cat. No.

CC3170, 3171 and CC-4175, Cambrex Biosciences, Baltimore, MD). For acrolein treatment, NCI-H292 or NHBE cells were seeded (5,000 cells/cm2) into 30 mm six-well plates (Corning, Cat. No. 3506, Corning, NY). Once confluent, the cells were incubated

(37°C, pH 7.4) for 24 h in serum-free medium (RPMI 1640) for NCI-H292 cells or bronchial epithelial cell basal medium (Cat. No. CC3171, Cambrex) for NHBE cells. To determine if acrolein treatment increases the steady state transcript level of human

MMP14, NCI-H292 cells were treated (4 h, 37°C) with increasing concentrations of acrolein (0.01-10 µM) or PBS. To determine the role of epidermal growth factor receptor

(EGFR) in acrolein-induced increase in human MMP14 transcript level, confluent NCI-

H292 cells were pre-treated (1 h, 37°C) with an EGFR kinase inhibitor, AG1478 (0.025

µM) (Cat. No. 658552, Calbiochem, San Diego, CA) or a neutralizing antibody to EGFR,

LA-1 (10 µg/ml) (Cat. No. 05–101, Upstate, Charlottesville, VA). To determine the role of various mitogen activated protein kinase (MAPK) activation in acrolein-induced increase in hMMP9 transcript level, NCI-H292 cells were pre-treated (1 h, 37°C) with MAPK kinase (MAP2K) inhibitor, PD98059, (5 µM) (Cat. No. 51300, Calbiochem) or c-jun N- terminal kinase (JNK) inhibitor, SP600125, (5 µM) (Cat. No. 420119, Calbiochem) or p38

MAPK inhibitor, ML3403 (5 µM) (Cat. No. 506121, Calbiochem) and then incubated (4 h,

37°C) with acrolein (0.1 µM). After treatment, the cells were washed with PBS, lysed by

Trizol reagent and total RNA was isolated by isopropanol/chloroform precipitation and suspended in RNAase free water.

4-8 MMP14 activity assay: Ground lung from acrolein-exposed FVB/NJ mice or NCI-H292 cells were incubated (15 min, 4°C) with MMP14 extraction buffer (50 mM Tris-HCl pH

7.6, 1.5 mM NaCl, 0.5 mM CaCl2,1 µM ZnCl2,0.01% Brij 35 and 0.25% Triton™ X-100) and centrifuged (2000 x g, 10 min, 4°C). The supernatant (100 µl) or standards

(MMP14, 0.4-0.001 µg/µl) were then incubated (overnight, 4°C) in 96 well micro-titre plate coated with anti-MMP14. The wells were washed (2X, Wash solution containing

0.01 M Sodium Phosphate pH 7.0 and 0.05% Tween™ 20) and incubated (3 h, 37°C) with detection-enzyme (urokinase) and substrate (S-2444™) in reaction buffer (50 mM

Tris-HCl pH 7.6, 1.5 mM NaCl, 0.5 mM CaCl2, 1 µM ZnCl2, Heparin, 1 unit/ml and 0.01%

Brij™ 35) and read spectrophotometrically (405 nm). The relative amount of MMP14 activity was determined by comparing each sample against the standard curve. Each sample was analyzed in the linear portion of the curve in duplicate and results were expressed as fold change in MMP14 metalloproteinase activity. To determine the role of furin convertase in increased MMP14 activity after acrolein treatment, confluent NCI-

H292 cells were pre-treated (1 h, 37°C) with an furin inhibitor, hexa-D-arginine, (0.05

µM) (Cat. No. 344931, Calbiochem) and then incubated (4 h, 37°C) with acrolein (0.1

µM). After treatment, the cells were washed with PBS and MMP14 activity was determined.

4-9 Results

Acrolein increased MMP14 transcript and protein in the lungs: MMP14 transcripts increased in acrolein-treated normal bronchial epithelial (NHBE) cells (Fig 1A); furthermore acrolein increased MMP14 transcripts in airway epithelial (NCI-H292) cells in a concentration-dependent manner (Fig 1B). Repetitive acrolein exposure increased murine MMP14 transcript levels in FVB/NJ mouse lung (Fig 1C). MMP14 protein in the whole lung homogenates in acrolein-exposed FVB/NJ mice increased as compared to control (unexposed) mice (Fig 1D). Thus MMP14 transcripts and protein increased after acrolein treatment.

Acrolein increased MMP14 activity in the lungs. MMP14 activity increased in acrolein- treated NCI-H292 cells after acrolein exposure (8.78 ± 0.24 ng/ml) as compared to the control (2.87 ± 0.24 ng/ml) (Fig 2A). MMP14 activity also increased in acrolein-exposed

FVB/NJ lung (1.6 ± 0.43 ng/ml) as compared to control (0.52 ± 0.043 ng/ml) (Fig 2B).

Pretreatment with a furin inhibitor, hexa-ariginine diminished the acrolein-induced increase in MMP14 activity (Fig 2C).

MMP14 mediates increased MUC5AC after acrolein exposure. To determine the role of

MMP14 in acrolein-induced MUC5AC increase, we transfected NCI-H292 cells with siRNA directed against MMP14. MMP14 siRNA decreased MMP14 transcript, protein levels and activity (Fig 4A). NCI-H292 cells transfected with MMP14 siRNA had reduced levels of constitutive MUC5AC transcripts and demonstrated no increase in MUC5AC transcripts after acrolein treatment (Fig 4B). In contrast, non transfected cells and cells transfected with scrambled siRNA demonstrated normal response to acrolein (Fig 4B).

Thus, NCI-H292 cells transfected with MMP14 siRNA responded less to acrolein

4-10 treatment, supporting our hypothesis that acrolein-induced increases in MUC5AC are mediated by MMP14.

MMP14 transcripts levels increased after acrolein treatment through epidermal growth factor receptor (EGFR)/ mitogen activated protein kinase (MAPK) 3/2 signaling.

Increased MMP14 transcript levels after acrolein treatment were mediated by epidermal

EGFR. Pretreatment with AG1478, an EGFR kinase inhibitor (Fig 3A) or LA-1 (Fig 3C), a neutralizing antibody against EGFR diminished the acrolein-induced increase in

MMP14 transcript. EGFR activation leads to activation of MAP kinases (Schlessinger

1998). Pretreating the cells with a MAP2K inhibitor, PD98059, (Fig 3A) or C-jun N- terminal kinase (JNK) inhibitor, SP600125 (Fig 3B) or a metalloproteinase inhibitor,

GM6001 (Fig 3C) diminished the acrolein-induced increase in MMP14 transcripts.

Pretreatment with a p38 MAP kinase inhibitor had no effect on acrolein-induced increase in MMP14 transcripts (Fig 3B). Thus, increase in MMP14 transcripts in acrolein-treated airway epithelial cells is mediated by a metalloproteinase mediated EGFR ligand- dependent mechanism with signaling through MAPK3/2 and MAKP8.

4-11 Discussion:

Matrix metalloproteinases are involved in mucus overproduction in COPD. LPS-induced mucus metaplasia in rat lung, and pretreatment with an MMP inhibitor decreased the

LPS-induced mucin production in rats (Kim 2004). However, because this inhibitor is effective against several MMPs, it was important to determine the identity of specific

MMPs involved in the processes that lead to increased MUC5AC transcript levels.

Previously we reported a role for MMP9 (Manuscript in preparation) and MMP12 in acrolein-induced MUC5AC expression (Borchers 1998). MMP14 activity increased in the acrolein-exposed mouse lung (Fig 2A) as well as acrolein treated NCI-H292 cells

(Fig 2B). Unlike MMP9 and MMP12, MMP14 is associated with the cell surface through a type 1 transmembrane domain (Takino 1995). MMP14 activity is tightly controlled at the transcriptional and post-translational levels. MMP14 is produced as a latent pro- peptide. The proform keeps the enzyme latent through the interaction of a cysteine residue with a zinc ion in the active site. MMP14 contains an RXK/RR furin-like enzyme recognition motif between the propeptide and catalytic domains, which is activated by intracellular subtilisin-type serine proteinases including furin before MMP14 reaches the cell surface (Pei 1995). Pretreatment with a furin inhibitor diminshed the acrolein- induced increase in MMP14 activity in NCI-H292 cells (Fig 2C). In addition, MMP14 can be processesed autocatalytically (Hernandez-Barrantes 2000; Lehti 1998) to enzymatically active MMP14. Both inhaled acrolein as well as endogenously generated acrolein reacts directly with protein and non-protein sulphydryl groups mainly at the cell surfaces and with primary and secondary amines found in the intracellular proteins

(Ghilarducci and Tjeerdema, 1995). Conjugation of the carbon of acrolein with sulphydryl groups is rapid and essentially irreversible (Esterbauer 1976). In lungs,

MMP14 is expressed in surface epithelial cells (Fukuda 1996) and in type II cells (Kunugi

2001). Acrolein is likely to react with cysteine residues in the pro-domain to perturb the

4-12 cysteine switch and activate MMP14. Acrolein can also cross link MMP14 leading to

MMP14 dimer or oligomer formation, producing autocatalysis and self-activation or interfere with internalization of MMP14, thus increasing the amount of active MMP14 on the cell surface.

Additionally, MMP14 activity is regulated at the transcriptional level. Various growth factors including EGF induce MMP14 expression. Egfr (-/-) mice have low expression of

MMP14 in the lungs (Kheradmand 2002). MMP14 transcripts increased in the lung of

FVB/NJ mice exposed to acrolein. Acrolein treatment increased the transcript levels of

MMP14 in NCI-H292 cells and NHBE cells. The signal transduction pathways involved in increased MMP14 expression are unclear. Inhibition of MAPK3/2 decreased MMP14 expression in fibrosarcoma cells (Tanimura 2003). MAPK3/2 but not JNK or p38 increased MMP14 expression in rat endothelial cells (Boyd 2005). Moreover, constitutively active MEK increased MMP14 expression in MDK cells (Munshi 2004). We observed that acrolein-induced increase in MMP14 transcripts was diminished by pretreating the cells with a neutralizing antibody against EGFR or an EGFR kinase inhibitor (Fig 3A and 3C). Pretreatment with MEK inhibitor, PD98059 (Fig 3A), and the

JNK inhibitor, SP600125, but not the p38 inhibitor, ML3403 (Fig 3B), decreased the acrolein-induced increase in MMP9 transcripts, suggesting that MAPK 3/2 (ERK1/2) and

JNK (MAPK8), but not p38 (MAPK14) are involved in the response initiated by acrolein.

Further more pretreatment with metalloproteinase inhibitor, GM6001 diminished the acrolein-induced increase in MMP14 transcripts (Fig 3C). Together these results indicate that acrolein-induced increase in MMP14 transcripts involves a metalloproteinase mediated, EGFR ligand dependent mechanism with signaling through

MAPK3/2 and MAPK8.

4-13 The tissue inhibitor of metalloproteinase proteins (TIMPs) represent a family of at least four 20–29-kDa secreted proteins (TIMPs 1–4) that reversibly inhibit the MMPs in a 1:1 stoichiometric fashion (Strenlicht & Werb 1999). TIMP1 (Yao 1999), TIMP2, TIMP3 and

TIMP4 (Thomas 2000) are expressed in bronchial epithelium. TIMP2 (Zucker 1998) and

TIMP3 (Will 1996), but not TIMP1 (Kinoshita 1996) inhibit MMP14 activity. TIMP3 also has the unique ability to bind via its C-terminal domain to heparin sulfates proteoglycans within the ECM, thereby concentrating it to specific regions within tissues and basement membranes (Langton 1998). Unlike other TIMPs, TIMP3 is subject to a high degree of transcriptional regulation (Yeow 2001). Transcript levels of TIMP3 are decreased in the lungs of FVB/NJ mice after acrolein exposure (Manuscript in preparation). Because

TIMP3 and MMP14 are produced by the airway epithelial cells, the decrease in TIMP3 and the increase in MMP14 are thus consistent with increased MMP14 activity.

Epithelial cell repair and mucin synthesis are regulated by EGFR (Nadel 2001). Various agents including cigarette smoke (Takeyama 2001), activated eosinophils (Burgel 2001),

IL-13 (Kim 2002) and neutrophil elastase (Kohri 2002) increase MUC5AC in airway epithelial cells (NCI-H292) by activating EGFR and the MAPK cascade. Previously we demonstrated that acrolein increased the transcript levels of MUC5AC in NCI-H292 cells

(Borchers 1999) and normal bronchial epithelial (NHBE) cells (Deshmukh 2005) through

EGFR-MAPK pathway that is initiated by ectodomain shedding of EGFR-ligands. This process is dependent on activation of metalloproteinases, including a disintegrin and metalloproteinase domain containing proteins (ADAM) 17 and MMP9. However, the inhibition of increase in MUC5AC transcripts after acrolein treatment was incomplete in

NCI-H292 cells transfected with siRNA against ADAM17 and MMP9 (Deshmukh 2005), indicating a role for yet another metalloproteinase in acrolein-induced MUC5AC increase. We used siRNA to confirm the role of MMP14 in acrolein-induced MUC5AC

4-14 increase. siRNA directed against MMP14 efficiently decreased the transcripts, protein level by western blot and MMP14 activity. NCI-H292 cells transfected with siRNA had lower constitutive levels of MUC5AC transcripts as compared to untransfected cells or cells transfected with scrambled siRNA. There was no significant increase in the transcript levels of MUC5AC in NCI-H292 cells transfected with MMP14 siRNA after acrolein treatment. Untransfected cells responded appropriately to acrolein treatment.

EGF regulates epithelial differentiation in NCI-H292 cells cultured at air liquid interface.

Removal of EGF from culture medium decreased goblet cell differentiation in NCI-H292 cells cultured at air liquid interface (Atherton 2003). It is likely that decrease in the constitutive levels of MUC5AC transcripts by MMP14 siRNA involves disruption of the basal shedding of pro-EGFR ligands. Given the ability of MMP14 to activate pro-MMP2

(Sato 1993) and MMP13 (Cowell 1998) and to directly cleave a range of extracellular matrix molecules (Pei and Weiss) and growth factors including TGF-β which also regulates MUC5AC expression (Wang 2002; Shao 2005), it is not surprising that MMP14 is critical in acrolein-induced MUC5AC increase.

In summary, acrolein, a major component of cigarette smoke, increases MMP14 activity to increase mucin transcription. In addition, chronic exposure initiates an autocrine feedback loop that further stimulates transcription of MMP14 and simultaneously represses transcription of TIMP3. A combination of these responses leads to persistent mucus production.

4-15 Figure Legends

Figure 1. Acrolein increased matrix metalloproteinase (MMP) 14 transcripts and protein levels. FVB/NJ mice were exposed to acrolein (2.0 ppm x 6h/d x 5d/wk x 4wks) or filtered air (control). (A) MMP14 transcript levels were measured by quantitative real- time PCR (qRT-PCR). The results are expressed as fold change in the level of transcript after normalizing to GAPDH. MMP14 transcripts increased in the acrolein-exposed

FVB/NJ mouse lung as compared to the control. (B) MMP14 protein level by western blot increased in acrolein-exposed FVB/NJ mouse lung after acrolein exposure. Each lane was loaded with protein (60 µg) obtained from an individual mouse that is representative of each group (n=5/treatment). (C) Confluent serum starved NCI-H292 cells or normal bronchial epithelial (NHBE) cells were treated (4 h, 37 °C) with 0.001-

0.03 µM acrolein. Level of MMP14 transcript was determined by quantitative real time

PCR (qRT-PCR). The results are expressed as fold change in the level of MMP14 transcripts after normalizing to RPL32. MMP14 transcripts increased in normal bronchial epithelial (NHBE) cells after acrolein treatment. (D) MMP14 transcripts increased in a concentration-dependent manner in NCI-H292 cells after acrolein treatment. (Values are mean ± SEM. * significantly different from control. ANOVA, Student-Newman-Keul’s test, n=5-9).

Figure 2. Acrolein increased MMP14 activity. (A) FVB/NJ mice were exposed to acrolein (2.0 ppm x 6h/d x 5d/wk x 4wks) or filtered air (control). MMP14 activity was determined by capturing active MMP14 with anti-MMP14 antibody which was then used to activate a modified pro-urokinase detection enzyme to cleave a chromomeric peptide substrate and the resultant color was read spectrophotometrically at 405 nm. In all experiments a serial dilution of known sample of active MMP14 was run simultaneously to obtain a reference curve. Each sample was analyzed in the linear portion of the curve

4-16 and the relative amount of active MMP14 was determined by comparing OD405 of each sample against the standard curve. MMP14 activity increased in the FVB/NJ mouse lung after acrolein exposure. (B) Confluent serum starved NCI-H292 cells were treated

(4 h, 37 °C) with 0.3 µM acrolein. MMP14 activity was determined as described before.

Acrolein increased MMP14 activity in NCI-H292 cells. (C) Confluent serum starved

NCI-H292 cells were pretreated with furin inhibitor, hexa-D-arginine (0.625 µM) and then incubated (4 h, 37 °C) with 0.3 µM acrolein. MMP14 activity was determined as described before. Pretreatment with hexa-D-arginine decreased the acrolein-induced increase in MMP14 activity. (Values are mean ± SEM. * Significantly different from control, ANOVA, Student-Newman-Keul’s test, n=5-10).

Figure 3. Acrolein-induced increases in transcript levels of matrix metalloproteinase

(MMP) 14 are mediated by epidermal growth factor receptor (EGFR) and mitogen activated protein kinase (MAPK) signaling in human airway epithelial (NCI-H292) cells.

(A) Acrolein-induced increases in transcript levels of MMP14 are diminished in cells treated with an EGFR neutralizing antibody (LA-1) or an EGFR kinase inhibitor

(AG1478). (B) Acrolein-induced increases in transcript levels of MMP14 are diminished in cells treated with a MAPK3/2 inhibitor (PD98059), whereas transcript levels for

MMP14 were not diminished in cells treated with a c-jun N-terminal kinase (JNK) inhibitor (SP600125) or a p38 kinase inhibitor (ML3403). (C) Acrolein-induced increases in the transcript levels of MMP14 are increased in transforming growth factor (TGF)-α over expressing mice as compared to the wild type littermate controls. Confluent NCI-

H292 cells were pretreated (37°C, 1 h) with 0.250 µM AG1478, an EGFR kinase inhibitor, or 5 µM, PD98059, a MAPK3/2 inhibitor or 5 µM, SP600125, a JNK inhibitor or

5 µM ML3403, a p38 inhibitor and then incubated (37°C, 4h) with 0.01 µM acrolein or vehicle. RNA was isolated and the level of MMP14 transcript was determined by

4-17 quantitative real time PCR (qRT-PCR). The results are expressed as fold change in the level of MMP9 transcripts after normalizing to RPL32. Mice expressing TGF-α and their wild type littermate controls, were exposed to acrolein (2.0 ppm x 6h/d x 5d/wk x 4wks) or filtered air (control) and the lungs were removed for mRNA isolation. Transcript level of MMP14 was determined by quantitative real-time PCR (qRT-PCR). The results are expressed as fold change in the level of transcript after normalizing to GAPDH. (Values are mean ± SEM. * significantly different from control. † significantly different from the acrolein treated samples (without inhibitor) =6, ANOVA, Student-Newman-Keul’s test, n=6-9).

Figure 4. Matrix metalloproteinase (MMP) 14 mediates acrolein-induced increases in mucin 5 (subtype A and C) transcripts in human airway epithelial (NCI-H292) cells. (A)

MMP14 transcripts level, protein and metalloproteinase activity are diminished in NCI-

H292 cells transfected with small interfering (si) RNA directed against MMP14 as compared to cells transfected with scrambled siRNA (negative control) or untransfected cells. (B) Acrolein-induced increases in MUC5AC transcripts are diminished in NCI-

H292 cells transfected with siRNA against MMP14 as compared to the cells transfected with scrambled (non-sense) siRNA. 40 % confluent NCI-H292 cells were transfected with siRNA against MMP14 or scrambled siRNA (negative control) or not-transfected with siRNA and then incubated (37°C, 36 h). To determine if siRNA diminished the transcript levels, protein and activity of MMP14, RNA and protein was isolated and the level of MMP14 transcript was determined by quantitative real-time PCR (qRT-PCR) and western blotting. MMP14 activity was determined by capturing active MMP14 in NCI-

H292 cells with anti-MMP14 antibody which was then used to activate a modified pro- urokinase detection enzyme to cleave a chromomeric peptide substrate and the resultant color was read spectrophotometrically at 405 nm. In all experiments a serial

4-18 dilution of known sample of active MMP14 was run simultaneously to obtain a reference curve. Each sample was analyzed in the linear portion of the curve and the relative

amount of active MMP14 was determined by comparing OD405 of each sample against the standard curve. To determine if siRNA diminished the acrolein-induced increases in

MUC5AC transcripts, 40 % confluent NCI-H292 cells were transfected with siRNA against MMP14 or scrambled siRNA (negative control) or not-transfected with siRNA and then incubated (37°C, 36 h) and then treated with 0.03 µM acrolein (37°C, 4 h).

RNA was isolated and the level of MUC5AC transcript was determined by quantitative real-time PCR (qRT-PCR). The results are expressed as fold change in the level of

MMP14 or MUC5AC transcripts after normalizing to RPL32. (Values are mean ± SEM. * significantly different from control. † Significantly different from the acrolein treated samples (without MMP14 siRNA), ANOVA, Student-Newman-Keul’s test, n=6-9).

Figure 5: Matrix Metalloproteinase (MMP) 14 mediates acrolein-induced increase in mucin 5 (subtype A and C) (MUC5AC). Acrolein increase MMP14 activity to increase

MUC5AC transcripts via an epidermal growth factor receptor (EGFR) ligand dependent mechanism. Acrolein increased MMP14 transcripts in vitro. Acrolein-induced increase in MMP9 transcripts is mediated by a metalloproteinase (inhibited by GM6001).

Inhibition of EGFR (by neutralizing antibody, LA-1, or EGFR kinase inhibitor, AG1478) decreased the acrolein-induced increase in MMP9 transcripts. Inhibition of mitogen activated protein kinase (MAPK) 3/2, (by PD98059) but not MAPK8 (by SP600125) or

MAPK14 (by ML3403) diminished the acrolein-induced increase in MMP14 transcripts.

Acrolein increased MMP14 activity in the lungs and protein in the airways.

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4-27 Figures:

Figure 1

A * 3 ntrol) o s t p anscri r 2 14 t mapred to the c o c ne MMP 1 ange as muri h (fold c

0 Control Acrolein

4-28

Figure 1

4 C Contr ol Acrolein )

rol * 3 cont

o

pts * i r

c

ans 2

MMP14 tr 1

d change as compared t

ol (f

0 NCI-H292 NHBE

4-29

Figure 1

5 D

* 4

ntrol) * o 3

2 *

old change over c f

( human MMP14 transcripts 1

0 Control 0.01 0.03 0.1 0.3

Acrolein (µM)

4-30 Figure 2

3 A * rol)

y he cont t i t v i o 2 P14 act M

rine M 1 u m (fold change as comapred t

0 Control Acrolein

4-31

Figure 2

4 B * rol)

3 he cont t o ty vi ti c

a 2 MMP14

1 (fold change as comapred t

0 Control Acrolein

4-32 Figure 2

4 C *

3 control) o vity i † 14 act 2 *

† † human MMP 1 * old change as comapred t (f

0 Acrolein - - + + Furin - + - + Inhibitor

4-33 Figure 3

A *

3 rol) cont level o † † * 2 * mpared t

1 human MMP14 transcript old change as co (f

0 Acrolein --+++ EGFR inhibitor ++- - - MEK inhibitor - + - - +

4-34 Figure 3

B * * 3 rol) cont level o †* *

2 anscript pared t r 14 t

1 human MMP old change as com (f

0 Acrolein --+++ JNK inhibitor ++- - - p38 inhibitor - + - - +

4-35 Figure 3

C *

3 rol) cont level o † † 2

mpared t * *

1 human MMP14 transcript old change as co (f

0 Acrolein --+++ LA-1 ++- - - GM6001 - + - - +

4-36 Figure 4

1.2 A )

rol 1.0 s cont o 0.8 anscript r

14 t 0.6 * P *

an MM 0.4 * hum 0.2 (fold change as compared t

0.0 Control 0.015 0.03 0.06

MMP14 siRNA (µM)

4-37 Figure 4

B * 3

rol) * s cont o

2 pared t

1 †

human MUC5AC transcript * old change as com (f

0

MMP14 siRNA - + - Scrambled siRNA - - +

4-38 Figure 5:

4-39 Table 1

Murine MMP14:

Forward primer: 5’-AGGAGACAGAGGTGATCATCATTG-3’

Reverse primer: 5’-GTCCCATGGCGTCTGAAGA-3’

Probe: FAM-CCTGCCGGTACTACTGCTGCTCCTG-TAMRA

Murine GAPDH:

Forward primer: 5’-GTCGTGGATCTGACGTGCC-3’

Reverse primer: 5’-TGCCTGCTTCACCACCTTCT-3’

Probe: FAM-CCTGGAGAAACCTGCCAAGTATGATGACA-TAMRA

Human MMP14:

Cat. No. Hs00237119.m1

Applied Biosystems, Foster City, CA

Human RPL32

Cat. No. Hs00851655.g1

Applied Biosystems, Foster City, CA

4-40 Supplementary Figures:

Figure S1

Fig S1: Lung specimens were obtained from human subjects undergoing lung transplant surgery for treatment of COPD under institutional review board-approved protocols at the Washington University Medical Center and immunostaining with anti-MMP14 was performed as described in materials and methods. Immunostaining with anti-MMP14 antibody (Ab) increased in the lungs from COPD subjects (C and D) as compared to healthy subjects (A and B).

4-41 CHAPTER 5

DISCUSSION

Acrolein and Mucus production: Cigarette smoking is associated with an increased risk of developing COPD. It contains numerous irritants but none stronger than acrolein and acrolein concentration are increased in environmental tobacco smoke due to altered combustion of aldehydes at lower temperatures. More than 30 million nonsmokers in the United States are exposed to acrolein concentrations in indoor air ranging from 0.8-

1.5 ppm and levels between 0.1-10 ppm have been detected in bars and restaurants

(Jermini 1976, Nazaroff and Singer, 2004). Acrolein is a constituent of wood smoke, diesel exhaust, and photochemical smog, and can be generated from biomass fuels and by cooking with oils (Nishikawa 1987, Boetnner 1980). Acrolein and other 2-alkenals are also generated endogenously by the oxidation of membrane fatty acids during oxidative stress (Esterbauer 1991, Uchida 2000, Shacter 2000, Furuhata 2002) or the oxidation of alpha-amino acids including theronine by myeloperoxidase during inflammation

(Vasilyev 2005). 2-Alkenals contain two electrophilic reaction centers, the partially positive carbon 1 or 3, which attack nucleophiles, especially thiol containing proteins.

Among all the unsaturated 2-alkenals, acrolein is by far the strongest electrophile

(Esterbauer 1991) and the most irritating (i.e. concentrations as low as 0.06 ppm can cause eye irritation within 5 minutes) (Darley 1960, Steinhagen 1984). Acrolein is thus an important mediator of cell damage because of its ability to covalently modify macromolecules, which disrupt critical cellular functions and cause mutations (Izard

1978, Esterbauer 1991, Cohen 1992). Acrolein-protein adducts has been found to accumulate in ischemic tissue (Uchida 1999) and in atherosclerotic lesions (Shao 2005).

5-1 Animals exposed repeatedly to acrolein develop histological changes including epithelial damage, mucus metaplasia and bronchiolitis, accompanied by excessive macrophage accumulation in the airways (Feron 1978, Borchers 1998). Acrolein exposure induced mucus cell metaplasia in airways, and increased MUC5AC transcripts in the lung of

Sprague-Dawley rats (Borchers 1998) and FVB/NJ mice (Borchers 1999). Acrolein at lower concentrations (0.03-0.1µM) increased the transcript levels of MUC5AC in NCI-

H292 cells (Borchers 1999) and normal bronchial epithelial (NHBE) cells (Manuscript 1;

Fig 6). However, the mechanism of acrolein-induced increase in MUC5AC is unclear.

Acrolein and oxidative stress: Both inhaled acrolein as well as endogenously generated acrolein can react directly with protein and non-protein sulphydryl groups mainly at the cell surfaces and with primary and secondary amines found in the intracellular proteins (Ghilarducci and Tjeerdema, 1995). In proteins, it preferentially attacks free sulphydryl (SH) groups of cysteine, amino groups of lysine and histidine residues (Esterbauer 1991). Conjugation of the carbon of acrolein with sulphydryl groups is rapid (rate constant 220 M-1sec-1) and essentially irreversible (Esterbauer.

1976). Acrolein decreased the availability of precursor amino acids used in glutathione

(GSH) synthesis in pulmonary endothelial cells (Takeuchi 2001). In vitro concentrations of 25-100 µM acrolein deplete cellular GSH and are lethal to pulmonary artery endothelial cells (Kachel and Martin, 1994, Patel and Block, 1993), bronchiolar epithelial cells (Grafström 1988), and both bronchial (Krokan 1985) and cardiac fibroblasts

(Torasson 1989).

Acrolein decreased the level of GSH in a concentration dependent manner in NCI-H292 cells. However, the threshold concentration of acrolein that decreased GSH was 10-30

µM (Manuscript 1: Fig 1). In contrast, the concentration of acrolein sufficient to cause

5-2 significant increase in MUC5AC transcripts in NCI-H292 cells was 0.03 µM, about a 300-

1000 lower concentration (Manuscript 1: Fig 2). Since oxidative stress occurred at concentrations ≥ 10 uM, oxidative stress seems unlikely to be involved in MUC5AC expression by acrolein.

MUC5AC production and EGFR: MUC5AC production is increased by irritants and mediators including cigarette smoke, bacterial products like LPS, oxidative stress, cytokines (TNFα, IL-4, IL-5, IL-9, and IL-13), and eicosanoids (prostaglandin E2, 12- and

15-hydroxyeicosatetraenoic acid) (Leikauf 2002). Many of these agonists including cigarette smoke (Takeyama 2001), activated eosinophils (Burgel 2001), IL-13 (Kim

2002), neutrophil elastase (Kohri 2002), Pseudomonas aeruginosa (Kohri 2002), and

PMA (Shao 2003) increase MUC5AC in airway epithelial cells (NCI-H292) by activating

EGFR. Inhibition of EGFR activity decreases the levels of mucin in response to various stimuli and reduced the goblet cell metaplasia (Takeyama 1999). In addition to its cognate ligands, the EGFR is activated by stimuli that do not directly interact with the

EGFR ectodomain, including GPCR ligands, other receptor tyrosine kinase (RTK) agonists, cytokines, chemokines, and cell adhesion elements. EGFR can also be activated by non-physiological stimuli such as UV- and gamma-radiation, osmotic shock, membrane depolarization, heavy metal ions, and radical-generating agents, such as

H2O2 (Prenzel 1999). Previously, acrolein has been found to activate EGFR in keratinocytes (Takeuchi 2001) and neutrophils (Finkelstein 2001), albeit at high concentrations (>10-50 µM) that are sufficient to induce apoptosis or cell death in human bronchial epithelial (NHBE) cells (Nardini 2002). In contrast, Acrolein phosphorylated

EGFR at concentrations ≤ 0.1 µM in NCI-H292 cells, a lower concentration than 10-30

µM, which depleted GSH (Manuscript 1: Fig 2A). Oxidative stress in form of H2O2 phosphorylates EGFR but the pattern of tyrosine phosphorylation is different from that of

5-3 ligand-induced EGFR phosphorylation (Ravid 2002). Importantly, this type of EGFR phosphorylation does not lead to phosphorylation (activation) of downstream MAPKs (Xu

2002). We found that acrolein led to phosphorylation of MAPKs including MAPK3/2

(ERK1/2) and MAPK8 (JNK) (Manuscript 1: Fig 3A), and acrolein-induced increase in

MUC5AC transcripts is dependent on MAP2K phosphorylation (Fig. 3B). Thus acrolein increases MUC5AC transcripts by phosphorylating EGFR and phosphorylating MAP2K, which in turn phosphorylates MAPK3/2 and MAPK8, independent of oxidative stress.

Various EGFR ligands like TGF α, amphiregulin Hb-EGF are expressed in NCI-H292 cells (Shao 2004). Pretreating the NCI-H292 cells with an EGFR neutralizing antibody

(LA-1) diminished the acrolein-induced increase in MUC5AC transcripts suggesting that ligand-dependent EGFR activation is essential in acrolein-induced MUC5AC increase

(Manuscript 1: Fig 4A). EGFR ligands are synthesized as glycosylated membrane bound precursors (Massauge 1993), which are cleaved by proteinases to release functional ligands (Thorne 1994). Although the identity of proteinases commonly responsible for EGFR transactivation in MUC5AC expression has yet to be fully established, various MMPs are implicated in the ectodomain shedding of pro-EGFR ligands (Dempsey 2002). Pretreating the NCI-H292 cells with a broad-spectrum metalloproteinase inhibitor (GM6001) diminished the acrolein-induced increase in

MUC5AC transcripts, indicating that acrolein-induced MUC5AC increase is dependent on a metalloproteinase (Manuscript 1: Fig 4B).

Matrix metalloproteinases 9 and MUC5AC production: Matrix metalloproteinases

(MMPs) are a family of neutral proteinases that are minimally composed of a prodomain that requires cleavage for activation of a zinc-binding catalytic domain. MMPs play an important role in pathogenesis of COPD (Atkinson 2003). However, much of the

5-4 published literature pertains to emphysema with little information linking the mucus overproduction with MMPs. Previously Borchers et al. reported a critical role for MMP12 in acrolein-induced MUC5AC expression (1998). Acrolein increased MUC5AC transcripts and macrophage accumulation in lungs of wild type littermate controls,

Mmp12 +/+ as compared to Mmp12 -/- mice. Similarly, LPS induced mucus metaplasia in rat lung, and this response is accompanied by EGFR phosphorylation (Kim 2004).

Moreover, pretreatment with an MMP inhibitor decreased the LPS-induced mucin production in rats (Kim 2004). However, because this inhibitor is effective against several MMPs, it was important to determine the identity of MMPs involved in the process that lead to increased MUC5AC transcript levels.

MMP2 and MMP9 can mediate EGFR transactivation in pituitary gonatrope cells (Roelle

2004). Transcript levels of MMP9 increased in NCI-H292 cells and NHBE cells treated with 0.03 µM acrolein (Manuscript 1: Fig 7). Acrolein increased the transcript levels of

MMP9 in vivo in the lung of FVB/NJ mice exposed to acrolein. MMP9 protein level increased in vivo after acrolein exposure (Manuscript 2: Fig 2A and Fig 3B).

Immunostaining for MMP9 increased in the mouse airways exposed to acrolein as compared to airways in unexposed animals (Manuscript 2: Fig 4).

Acrolein increased MMP9 activity: Acrolein increased the release and subsequent activation of pro-MMP9. Gelatinases with molecular weight of ~90 kDa (pro-MMP9) and

~70 kDa (pro-MMP2) increased in the conditioned medium of NCI-H292 cells after acrolein or H2O2 treatment (Manuscript 1: Fig 6A). An additional gelatinase with molecular weight ~82 kDa representing the active form of MMP9 was observed in acrolein or H2O2-treated samples, which was less in the control samples. Moreover, western blots demonstrated protein levels of pro-MMP9 (92 kDa) and MMP9 (82 kDa) in

5-5 the conditioned medium increased following acrolein or H2O2 treatment as compared to the phosphate-buffered saline (PBS) treated (control) samples (Manuscript 1: Fig 6B).

Acrolein directly activated pro-MMP9 in a concentration dependent fashion (≥ 0.3 µM)

(Manuscript 2: Fig 1A and 1B) as early as 30 min (Fig 1C) in a cell free system. Due to wide-ranging biological consequences, MMP9 activity is tightly controlled at the transcriptional and protein levels. MMP9 is synthesized as an inactive pro-enzyme

(proMMP9) and is activated by proteolytic removal of an amino-terminal domain upon secretion into the extracellular space. The proform keeps the enzyme latent through the interaction of a cysteine residue with a zinc ion in the active site. Oxidants and other compounds interact with the protective cysteine. This reduces latency by altering protein conformation that permits entrance of water essential for catalysis and accelerates the autocatalytic loss of the propeptide domain. This process is called the "cysteine switch" mechanism (Van Wart 1990). Because of its ability to covalently modify macromolecules and disrupt critical cellular functions and cause mutations (Izard 1978,

Esterbauer 1991, Cohen 1992), acrolein is considered an important mediator of cell damage. Acrolein-protein adducts has been found to accumulate in ischemic tissue

(Uchida 1999) and in atherosclerotic lesions (Shao et al 2005). Thus acrolein can potentially disrupt the interactions between the cysteine residues and the zinc ion in the active site and thus directly activate MMP9 in absence of other active MMPs, including

MMP2.

5-6 Figure D1:

Figure D1: The 'cysteine-switch' mechanism regulating the pro-MMP9 activation.

The sulphydryl group of a conserved cysteine (C) at the carboxyl terminus of the pro- domain acts as a fourth inactivating ligand for the catalytic zinc atom in the active site; this results in the exclusion of water and keeps the MMP9 latent. Acrolein can covalently modify the sulphydryl group of a conserved cysteine (C) at the carboxyl terminus of the pro-domain and thus disrupt the cysteine-zinc pairing to allow the entry of water molecules into the catalytic site.

Active MMP9 can cleave cell surface bound pro-EGFR ligands (Roelle 2004) and could thus increase the level of MUC5AC transcripts after acrolein treatment. Gene silencing was used to determine the role of MMP9 in the acrolein-induced MUC5AC increase.

The siRNA against MMP9 effectively knocked down the MMP9 transcript levels [–3.5 +

0.9-fold 48h after transfection, respectively (Manuscript 1: Supplemental data Fig. 2E).

Lowering the level of MMP9 with siRNA inhibited the increase in acrolein-induced

MUC5AC mRNA (Manuscript 1: Fig 5A). To confirm the role of MMP9 in acrolein-

5-7 induced MUC5AC increase in vivo, gene-targeted Mmp9 (-/-) and strain matched control

Mmp9 (+/+) mice were exposed to acrolein. Acrolein increased MUC5AC transcripts and protein levels in the lungs of both Mmp9 (-/-) and the wild type Mmp9 (+/+) mice as compared to the control (Manuscript 2: Fig 4). Thus, in summary acrolein rapidly activated MMP9, which can process membrane, bound pro-EGFR ligands to activate

EGFR and downstream MAP kinases to increase MUC5AC transcripts.

Mechanism of acrolein-induced increase in MMP9 transcripts: MMP9 activity is also regulated at the transcriptional and post-transcriptional levels and also controlled at the protein level via its inhibitors. MMP9 gene expression is regulated by numerous stimulatory and suppressive factors including several cytokines and growth factors, including IL-1α (Fabunmi 1996), IL-2 (Montgomery 1993), IL-8 (Van den Steen 2000), interferon (IFN) γ (Hujanen 1994), EGF (Harvey 1995; Miyagi 1995), basic fibroblast growth factor (FGF) (Weston and Weeks, 1996), and TGF-β (Fini 1995). Because, acrolein at lower concentrations (0.3 µM) increased phosphorylation of EGFR in NCI-

H292 cells (Manuscript 1: Fig 2A), the role of EGFR in acrolein-induced increases in

MMP9 transcripts was investigated. Pretreatment with an EGFR kinase inhibitor,

AG1478 or an EGFR neutralizing antibody, LA-1, decreased the acrolein-induced increase in MMP9 transcripts (Manuscript 2: Fig 5B and 5D), suggesting EGFR activation is involved in acrolein-induced MMP9 transcript increase. Phosphorylation of

EGFR leads to activation of downstream MAP kinases. Increase in MMP9 expression by IL-1 and TNF-α h is mediated through activation of the transcription factors NF-κB and AP-1 via JNK/SAPK or p38 MAPK pathways (Baud and Karin, 2001). Besides IL-1β and TNF-α, other cytokines like IL-17 have also increase MMP9 expression via MAPK signaling (Jovanovic 2000). PMA induced MMP9 expression in SCC cells, required stimulation of the MAPK14 (p38) pathway (Simon 1998; Simon 2001). Thus MMP9

5-8 expression is driven by the three MAPK signaling cascades. Pretreatment with MEK inhibitor, PD98059, but not the JNK inhibitor, SP600125 or p38 inhibitor, ML3403, decreased the acrolein-induced increase in MMP9 transcripts, suggesting that MAPK 3/2

(ERK1/2), and not JNK (MAPK8) or p38 (MAPK14) are involved in the response initiated by acrolein (Manuscript 2: Fig 5B and 5C).

The inhibition of increase in MUC5AC transcripts after acrolein treatment in NCI-H292 cells transfected with MMP9 siRNA was not complete (Manuscript 1: Fig 5B). Similarly, there was an increase in the MUC5AC transcript and protein level in the lungs of acrolein-exposed Mmp9 -/- mice albeit it was significantly lower than the increase in

MUC5AC transcript and protein level in the wild type littermates, Mmp9 +/+ mice

(Manuscript 2: Fig 4). These finding suggested a possibility that, another metalloproteinase may be involved in acrolein-induced increase in MUC5AC transcripts.

ADAM17 is important in processing of pro-EGFR ligands (Sunnarborg 2002). Increase in MUC5AC in NCI-H292 cells after treatment with cigarette smoke or PMA is mediated by ADAM17 (Shao 2004 and 2005). siRNA against ADAM17 was used to confirm the role of ADAM17 in acrolein-induced increase in MUC5AC transcripts. Increase in acrolein-induced increase in MUC5AC transcripts was diminished in cells transfected with ADAM17 siRNA (Manuscript 1: Fig 5A). Moreover, cells co-transfected with siRNA against ADAM17 and MMP9 showed a greater inhibition of MUC5AC transcript increase by acrolein compared with transfecting the cells with siRNA against either ADAM17 or

MMP9 alone (Manuscript 1: Fig 5B). Although the activity of ADAM17 was not measured in this study, it is also likely to have contributed to shedding of pro-EGFR ligands and increased MUC5AC transcript levels. Even tough ADAM17 transcript levels were unchanged, transcript levels of TIMP3, an inhibitor of ADAM17 (Amour 1998) decreased in vitro in NCI-H292 cells treated with acrolein (Manuscript 1: Fig7) and in

5-9 vivo in lung of acrolein-exposed mice. In addition activation of MAPK3/2 (ERK 1/2) is consistent with increased ADAM17 activity. Activation of MAPK3/2 results in phosphorylation of a theronine (Thr-735) residue in ADAM17, which stimulated the trafficking of ADAM17 from the endoplasmic reticulum to the cell surface (Soond 2005).

Matrix metalloproteinase 14 and MUC5AC production: However, the inhibition of

MUC5AC transcript increase by acrolein in cells co-transfected with siRNA against

MMP9 and ADAM17 was not complete (Manuscript 1: Fig 5B), suggesting that yet another MMP may be involved in mediating acrolein-induced MUC5AC increase.

MMP14 is a membrane tethered member of MMP family which associates with the cell surface through a type 1 transmembrane domain (Takino 1995). MMP14 is important in lung development as evidenced by defect in formation of alveolar septae (Apte 2005) in

Mmp14-/- mice. MMP14 can activate pro-MMP2 (Strongin 1995) and pro-MMP13

(Knauper 1996) which in turn, can activate pro-MMP9 (Leeman 2002). Transcript levels of MMP14 increased in NCI-H292 cells and NHBE cells treated with 0.03 µM acrolein

(Manuscript 3: Fig 1C and 1D). Acrolein increased the transcript and protein levels of

MMP14 in vivo in the lung of FVB/NJ mice exposed to acrolein (Manuscript 3: Fig 1A).

Immunostaining for MMP14 increased in the airways of human subjects with COPD

(Manuscript 3: Fig 5).

siRNA was used again to confirm the role of MMP14 in acrolein-induced MUC5AC increase. siRNA directed against MMP14 efficiently decreased the transcripts and protein level of MMP14 in vitro (Manuscript 3: Fig 4A and 4B). NCI-H292 cells transfected with siRNA had lower levels of MUC5AC transcripts as compared to untransfected cells or cells transfected with scrambled siRNA (Manuscript 3: Fig 4C).

There was no significant increase in the transcript levels of MUC5AC in NCI-H292 cells

5-10 transfected with MMP14 siRNA after acrolein treatment. Untransfected cells responded appropriately to acrolein treatment. These results indicate that MMP14 plays a critical role in acrolein-induced MUC5AC increase. EGF regulates epithelial differentiation in

NCI-H292 cells cultured at air liquid interface. Removal of EGF from culture medium decreased goblet cell differentiation in NCI-H292 cells cultured at air liquid interface

(Atherton 2003). It is likely that decrease in the constitutive levels of MUC5AC transcripts by MMP14 siRNA involves disruption of the basal shedding of pro-EGFR ligands. MMP14 can modulate the bioavailability of TGFβ by releasing active TGFβ from cell surface complexes involving αvβ8 integrin (Mu 2002), which in turn can regulate MUC5AC expression (Wang 2002; Shao 2005).

Acrolein increased MMP14 activity: Acrolein increased the activity of MMP14 in NCI-

H292 cells exposed to acrolein (Manuscript 3: Fig 2) and in the lungs of FVB/NJ mice exposed to acrolein (Manuscript 3: Fig 2A). Unlike other secreted MMPs, MMP14 contains an RXK/RR furin-like enzyme recognition motif between the propeptide and catalytic domains, which can be activated by intracellular subtilisin-type serine proteinases before MMP14 reaches the cell surface (Pei 1995). Pretreatment with a furin inhibitor decreased partially the acrolein-induced increase in MMP14 activity

(Manuscript 3: Fig 2C), suggesting the presence of an alternate mechanism for increased MMP14 activity after acrolein treatment. MMP14 has a unique regulatory mechanism in which the active enzyme undergoes a series of processing steps, either autocatalytic (Hernandez-Barrantes 2000, Lehti 1998) or mediated by other proteinases

(Pei 2001) initially to an enzymatically active ~56 kDa species and ultimately to an inactive membrane-tethered ~44-kDa species lacking the entire catalytic domain, thereby regulating the activity and nature of the MMP14s at the cell surface and at the pericellular space. Acrolein by modifying the cysteine residues near the transmembrane domain or

5-11 in the catalytic domain could potentially interfere with the autocatalytic processing and thus increase the amount of active MMP14 present on the cell surface and thus potentially increase MMP14 activity.

Mechanism of acrolein-induced increase in MMP14 transcripts: MMP14 activity is also regulated at the transcriptional and post-transcriptional levels and also controlled at the protein level via its inhibitors (Zucker 2003). Acrolein treatment increased the transcript levels of MMP14 in NCI-H292 cells and NHBE cells. Growth factors including

EGF increase MMP14 transcripts (Kheradmand 2002). Mice lacking Egfr had reduced expression of Mmp14 in the lungs (Kherdamand 2002). MMP14 transcripts increased in the lung of FVB/NJ mice exposed to acrolein (Manuscript 3: Fig 1). Immunostaining for

MMP14 increased in the airways of human subjects with COPD (Manuscript 3: Fig S1).

In lungs, MMP14 is expressed in surface epithelial cells (Fukuda 1996) and in type II cells (Kunugi 2001) epithelium and in lung adenocarcinoma cells (Sato 1994). Not much is known about the signal transduction pathways involved in increased MMP14 expression. Inhibition of MAPK3/2 decreased MMP14 expression in fibrosarcoma cells

(Tanimura 2003). MAPK3/2 but not JNK or p38 regulate increased MMP14 expression in rat endothelial cells (Boyd 2005). Moreover, constitutively active MEK increased

MMP14 expression in MDK cells (Munshi 2004). Pretreatment with a neutralizing antibody against EGFR, LA-1 (Manuscript 3: Fig 3C) or an EGFR kinase inhibitor,

AG1478 (Manuscript 3: Fig 3A) diminished the acrolein-induced increase in MMP14 transcripts confirming the role of EGFR in acrolein-induced increase in MMP14 transcripts. Pretreatment with MEK inhibitor, PD98059, and the JNK inhibitor,

SP600125, but not the p38 inhibitor, ML3403, decreased the acrolein-induced increase in MMP9 transcripts (Manuscript 3: Fig 3A and 3B), suggesting that MAPK 3/2 (ERK1/2) and JNK (MAPK8), but not p38 (MAPK14) are involved in the response initiated by

5-12 acrolein. Further more pretreatment with metalloproteinase inhibitor, GM6001 diminished the acrolein-induced increase in MMP14 transcripts (Manuscript 3: Fig 3C). Together these results indicate that acrolein-induced increase in MMP14 transcripts involves a metalloproteinase mediated, EGFR ligand dependent mechanism with signaling through

MAPK3/2 and MAPK8.

Tissue inhibitor of metalloproteinase proteins (TIMPs) and MUC5AC production:

TIMPs represent a family of at least four 20–29-kDa secreted proteins (TIMPs 1–4) that reversibly inhibit the MMPs in a 1:1 stoichiometric fashion (Strenlicht & Werb 1999).

TIMP1 (Yao 1999), TIMP2, TIMP3 and TIMP4 (Thomas 2000) are expressed in bronchial epithelium. TIMP2 and to a lesser extent TIMP1 and TIMP3 bind with MMP9 and inhibit its activity (Olson 1997). TIMP2 (Zucker 1998) and TIMP3 (Will 1996), but not TIMP1 (Kinoshita 1996) inhibit MMP14 activity. TIMP3 also has the unique ability to bind via its C-terminal domain to heparin sulfates proteoglycans within the ECM, thereby concentrating it to specific regions within tissues and basement membranes (Langton

1998). Unlike other TIMPs, TIMP3 is subject to a high degree of transcriptional regulation (Edwards 2001). Transcript levels of TIMP3 are decreased in vitro in NCI-

H292 cells after acrolein treatment (Manuscript 1: Fig 6) and in the lungs of FVB/NJ mice after acrolein exposure (Manuscript 2: Fig 2B). Because TIMP3 and MMP14 are produced by the epithelial cell, the decrease in TIMP3 and the increase in MMP14 are thus consistent with autocrine induction.

Summary: Thus acrolein rapidly activates pro-MMP9 which can cleave cell surface bound pro-EGFR ligands to activate EGFR in a ligand-dependent manner and activate down stream MAPK3/2 to increase MUC5AC transcripts. Repeated exposures to acrolein increases MMP9 and MMP14 trancript, protein and activity and decrease the

5-13 transcript levels of TIMP3, an endogenous inhibitor of MMP9 and MMP14. This is consistent with increased MMP9 and MMP14 activity which can process more pro-EGFR ligands on the cell surface to establish a feedback loop to cause a persistent increase in

MUC5AC.

Figure D2:

Figure D2: Mechanism of acrolein-induced increase in mucin 5, subtype A and C

(MUC5AC): Acrolein-induced increase in MUC5AC is mediated by matrix metalloproteinase (MMP) 9 and MMP14. Acrolein activates pro-MMP9 and increases

MMP9 and MMP14 activity. MMP9 and MMP14 (inhibited by metalloproteinase inhibitor,

GM6001 or small interfering (si) RNA or tissue inhibitor of metalloproteinase protein

(TIMP) 3) can cleave cell membrane bound pro-epidermal growth factor receptor

(EGFR) ligands and phosphorylates EGFR (inhibited by neutralizing antibody or EGFR inhibitor, AG1478) in a ligand dependent fashion and activates down stream mitogen activated protein kinase (MAPK) 3/2 (Inhibited by PD98059). Prolonged treatment with

5-14 acrolein increases MMP9 and MMP14 transcripts via a ligand-dependent EGFR and

MAPK activation in vitro and repeated exposure to acrolein increased MMP9 and

MMP14 protein and activity in vivo. Acrolein decreases the transcript level of TIMP 3, an endogenous inhibitor of MMP14. Thus chronic exposure to acrolein initiates an autocrine feedback loop that stimulates transcription of MMP9 and MMP14, and simultaneously represses transcription of TIMP3. A combination of these responses leads to persistent mucus production.

Significance to human health: Epidemiological data suggest that mucus

overproduction is significantly associated with a more rapid decline in FEV1 and increased hospitalization of patients with COPD (Vestbo 1996). While quitting smoking is the preferred approach in treatment of mucus overproduction in COPD, this has proved difficult in the majority of patients. Current therapy for mucus overproduction includes facilitating clearance through use of bronchodilators, thiol containing drugs, reducing airway inflammation with glucocotricoids (Braga 1989). However, these approaches rely on non specific mechanisms and to date there are few reports of consistent therapeutic effects (Lundgren and Shelhamer 1990). It is more important to develop drugs that inhibit the mucus overproduction, but at the same time do not suppress normal mucus production or impair mucociliary clearance. Tetracyclines and hydroxamates, such as batimastat (BB-94) and the orally active marimastat (BB-2516), are currently used in clinical trials, but the musculoskeletal side effects of such drugs may be a problem in long-term use. This study identifies the specific MMPs involved in increased mucus production and demonstrates the mechanism by which inhaled irritants can activate signaling pathways that regulate mucin gene expression. Knowledge of the specific MMPs involved in increased mucus production and the specific signal transduction pathways involved in increased MMP transcripts will help in development of

5-15 specific transcription inhibitors and specific enzyme inhibitors, which are likely to be better tolerated in chronic therapy.

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