CITED2, AN AUTOREGULATED TRANSCRIPTIONAL
MODULATOR, IN TGF-β SIGNALING
by YU-TING CHOU
Submitted in partial fulfillment of the requirements For the degree of Doctor Philosophy
Adviser: Dr. Yu-Chung Yang
Department of Pharmacology CASE WESTERN RERERVE UNIVERSITY
August, 2006
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
______Yu-Ting Chou candidate for the Ph.D. degree *.
Monica M. Montano (signed)______(chair of the committee)
Edward Stavnezer ______
Paul N. MacDonald ______
Yu-Chung Yang ______
______
______
(date) ______4/3/2006
*We also certify that written approval has been obtained for any proprietary material contained therein.
To my parents and sister….
Table of Contents
List of Figures v
List of Abbreviation viii
Abstract x
Chapter I General Introduction 1-31
1.1 Cited2 and Cited family proteins
1.2 Knockout models of Cited members
1.3 Cited members and neoplastic transformation
1.4 TGF-β superfamily
1.5 TGF-β receptors
1.6 Smads as key effectors in TGF-β signaling
1.7 Smad interacting transcription factors, co-activators, and co-repressors
1.8 TGF-β and tumorigenesis
1.9 Matrix metalloproteinases
1.10 Posttranscriptional control in TGF-β signaling
1.11 Statement of purpose
Chapter II Cited2 modulates TGF-β mediated upregulation of 32-85
MMP9
i 2.1 Introduction
2.2 Materials and Methods
2.3 Results
2.3.1 Cited2 is a co-activator for Smad3
2.3.2 Cited2 interacts with Smads 2 and 3
2.3.3 Cited2 promotes Smad3/p300-mediated transcription
2.3.4 Cited2 enhances TGF-β-mediated expression of MMP9
2.3.5 Smad pathway is involved in upregulation of MMP9 by
TGF-β in MDA-MB-231 cells
2.3.6 Cited2 enhances TGF-β-mediated MMP9 promoter reporter
Activity
2.3.7 TGF-β regulates MMP9 expression by recruiting Cited2 and
Smad3 to the MMP9 promoter
2.3.8 Cited2 plays a role in TGF-β-mediated invasion of
MDA-MB-231 cells
2.4 Discussion
Chapter III Posttranscriptional control of Cited2 by TGF-β: 86-132
regulation via Smads and Cited2 coding region
ii 3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.3.1 TGF-β downregulates Cited2 in MDA-MB-231 cells
3.3.2 Smad-dependent pathway is involved in TGF-β mediated
downregulation of Cited2
3.3.3 TGF-β downregulates Cited2 by posttranscriptional
regulation
3.3.4 TGF-β increases the turnover rate of Cited2 mRNA in
MDA-MB-231 cells
3.3.5 Untranslated regions of Cited2 mRNA are not responsible
for TGF-β mediated downregulation of Cited2
3.3.6 Cited2 coding sequence is necessary and sufficient
for TGF-β mediated downregulation of Cited2
3.3.7 Translation of Cited2 coding sequence is involved
in the downregulation of Cited2 by TGF-β
3.4 Discussion
Chapter IV Future direction and Summary 132-167
iii 4.1 Introduction
4.2 Cited2, Nodal, mammary gland development and
tumorigenesis
4.3 p38MAPK, cis-elements, and trans-acting factors involved
in TGF-β mediated downregulation of Cited2
4.4 Genes regulated by Cited2 in mouse embryo fibroblasts
Summary 168-178
Bibliography 179-200
iv List of Figures
Figure I-1 Alignment of amino acid sequences of Cited family proteins. 24
Figure I-2 TGF-β signaling from receptor to nucleus 26
Figure I-3 Different functional Smads in TGF-β signaling. 28
Figure I-4 The functional domains of Smads. 30
Figure II-1 Cited2 enhances Smad3-mediated transactivation. 63
Figure II-2 Cited2 interacts with Smad2 and Smad3. 65-66
Figure II-3 Cited2 enhances Smad3/p300-mediated transcription 69
through p300/Cited2/Smad3 interaction.
Figure II-4 Cited2 modulates TGF-β-mediated upregulation of MMP9. 71-73
Figure II-5 Smad pathway is involved in TGF-β-mediated 76
upregulation of MMP9.
Figure II-6 Cited2 enhances the expression of MMP9 promoter reporter. 78
Figure II-7 Cited2 and Smad3 are recruited to the MMP9 promoter. 80
Figure II-8 Cited2 enhances TGF-β-mediated invasion of MDA-MB-231 82
cells.
Figure III-1 TGF-β downregulates Cited2 in MDA-MB-231 cells. 110-111
Figure III-2 Smad pathway is involved in the downregulation of Cited2 113-115
v by TGF-β.
Figure III-3 TGF-β mediated Cited2 downregulation is 118
posttranscriptional.
Figure III-4 TGF-β stimulation accelerates turnover rate of Cited2 120
mRNA.
Figure III-5 5’ and 3’ untranslated region of Cited2 are not responsible 122
for TGF-β mediated downregulation of Cited2.
Figure III-6 Cited2 coding region is essential for its downregulation 124-125
by TGF-β.
Figure III-7 TGF-β mediated downregulation of Cited2 depends on 128-129
translation of Cited2 coding region.
Figure IV-1 Cited2 regulates Nodal expression in MCF-10A cells. 146
Figure IV-2 Human Nodal promoter. 148
Figure IV-3 Mammary gland structure and growth of MCF-10A 150
cells in three dimensional culture
Figure IV-4 Cited2 expression during ErbB2/neu-induced tumorigenesis. 152
Figure IV-5 ERK pathway is not involved in TGF-β mediated 154
downregulation of Cited2.
vi Figure IV-6 JNK pathway is not activated by TGF-β in MDA-MB-231 156
cells.
Figure IV-7 p38MAPK is required for Cited2 downregulation by 158
TGF-β.
Figure IV-8 PI-3 Kinase pathway is not essential for Cited2 160
downregulation by TGF-β.
Figure IV-9 Cited2 regulated genes. 162
Figure IV-10 Cited2 enhances migration of MEFs. 164
Figure IV-11 Expression of Cited2 affects cell growth in serum free 166
medium.
Figure IV-12 Proposed TGF-β mediated downregulation of Cited2 model. 175
Figure IV-13 Proposed self-balancing model of MMP9 upregulation by 177
TGF-β.
vii List of Abbreviation
AMH anti-Müllerian hormone
BMP Bone morphogenetic protein
CBP CREB-binding protein
Cited2 CBP/p300 interacting transactivator with E (Glutamic acid) and D (Aspartic acid) rich C-terminal domain, 2
CRD Coding region instability determinant
CRD-BP Coding region instability determinant-binding protein
ECM Extracellular matrix
EMT epithelial to mesenchymal transition
ERα Estrogen receptor alpha
FSH Follicle stimulating hormone
GAPDH Glyceraldehyde -3-phosphate dehydrogenase
HIF-1 Hypoxia inducible factor-1
MEF Mouse embryo fibroblast
MMP Matrix metalloproteinase
SBE Smad-binding element
TFAP2 Transcription factor AP2
viii
TβRI Transforming factor-β type I receptor
TβRII Transforming factor-β type II receptor
TGF- α Transforming factor-α
TGF- β Transforming factor-β
UTR Untranslated region
ix Cited2, an autoregulated transcriptional modulator, in TGF-β signaling
Abstract
by
YU-TING CHOU
Cited2 [CBP/p300 interacting transactivator with E (Glutamic acid) and D (Aspartic acid) rich C-terminal domain, 2] is a CBP/p300 binding transcription factor without
typical DNA binding domains. Cited2 interacts with LIM-homeobox gene 2 (Lhx2),
Transcription factor AP2 (TFAP2), and nuclear receptors to function as a transcriptional
co-activator. Cited2 is implicated in the control of cell growth and malignant
transformation in Rat1 cells. Cited1 enhances Smad mediated transcription, suggesting
that members of the Cited family may function as a co-activator in transforming growth
factor-β (TGF-β) signaling. We have explored the function of Cited2 in the TGF-β
signaling pathway. In promoter reporter assays, Cited2 enhances Smad3 mediated
transcription. This may occur through a direct physical association of Cited2 with Smads
2/3 and p300. We found that Cited2 modulates TGF-β mediated upregulation of Matrix
Metalloproteinase 9 (MMP9). In chromatin immunoprecipitation experiment, Cited2 and
x Smad3 are recruited to MMP9 promoter upon TGF-β stimulation. Knockdown of Cited2 in MDA-MB-231 cells attenuates TGF-β mediated cell migration, suggesting that Cited2 could play a role during tumor progression.
Cited2 is downregulated by TGF-β in MDA-MB-231 cells. We show that
Smad2/3/4 and p38MAPK pathways are involved in TGF-β mediated downregulation of
Cited2. Nuclear run-on analysis and Cited2 promoter/reporter assays demonstrate that downregulation of Cited2 by TGF-β is through posttranscriptional regulation. In transcriptional inhibitor assays, Cited2 mRNA turnover is increased under TGF-β stimulation. We examined the expression of recombinant Cited2 gene introduced into
MDA-MB-231 cells by stable transfection, and found that mRNA containing the Cited2 protein-coding region controlled by a heterologous promoter indeed responds to TGF-β mediated downregulation. Our findings support that TGF-β mediated downregulation of
Cited2 is under posttranscriptional control, through Smad and p38MAPK pathways, and requires the presence of its coding sequence.
The results of this thesis research provide supporting evidence of Cited2 as a transcription modulator in TGF-β signaling and also suggest a feedback mechanism that downregulation of Cited2 by TGF-β attenuates the expression of TGF-β responsive genes
xi such as MMP9 and MMP13. As MMP9 and MMP13 are highly involved in tumorigenesis, our research proposes that Cited2 could be a potential anticancer target.
xii CHAPTER I
General Introduction
Cited2 is a transcriptional modulator and plays significant roles during
development. Cited2 is implicated in the control of cell growth and malignant
transformation in Rat1 cells. TGF-β signaling is highly involved in development and
tumor progression. Both Cited1 and Cited2 are downregulated by TGF-β, and Cited1
functions as a Smad co-activator. We are interested in understanding the role of Cited2 in
TGF-β signaling and the mechanism of Cited2 downregulation by TGF-β.
Cited2 and Cited family proteins
Cited is a novel family of transcriptional co-activators without typical DNA binding domains. The Cited family includes four nuclear protein members, Cited1
(formerly called MSG1) (Shioda et al., 1996), Cited2 (formerly called MRG1)
(Dunwoodie et al., 1998; Shioda et al., 1997; Sun et al., 1998), Cited3 (Andrews et al.,
2000) and Cited4 (formerly called MRG2) (Braganca et al., 2002). Cited1 was first isolated from murine pigmented melanoma cells and was shown to be associated with melanogenesis (Shioda et al., 1996). Cited2 was later identified from a gene database
1 search by sequence homology to Cited1 and independently identified by our laboratory in
a differential display screen for genes that are induced by IL-9 in a mouse T helper cell
line (Shioda et al., 1997; Sun et al., 1998). Cited3 was detected in birds, frogs, and fish,
but not in mammals (Andrews et al., 2000; Ng et al., 2003). Cited4 was identified by
sequence homology to Cited2 and is highly expressed in mammary epithelial cells
(Braganca et al., 2002; Yahata et al., 2002). Members of the Cited family share several
conserved regions (Fig.I-1). The C-terminal conserved region (CR2) contains glutamic
acid and aspartic acid rich sequences with transactivation function. The predicted
secondary structure of the CR2 domain is composed of two amphipathic helices separated
by a strictly conserved proline residue (Yahata et al., 2002). Conserved region 1 (CR1) is
well conserved in Cited1, Cited2, and Cited3, but is poorly conserved in Cited4 (Fig.I-1).
In contrast, N-terminal conserved region (CR3) is shared by Cited2, Cited3 and Cited4, but not Cited1 (Fig.I-1). The predicted CR3 domain forms an independent helix structure
(Yahata et al., 2002). The well conserved CR2 domain binds directly to the CBP/p300 transcriptional integrators and activates transcription (Bhattacharya et al., 1999). Cited family members interact with several transcription factors. Cited1 was shown to function as a transcriptional co-activator of Smad4 in a manner dependent on its interaction with the CBP/p300 transcriptional co-activator (Shioda et al., 1998; Yahata et al., 2000).
2 Cited1 also binds estrogen receptor alpha (ERα) and stabilizes estrogen dependent
interaction between ERα and p300. Cited1 functions as a co-activator to enhance ERα
mediated TGF-α expression (Yahata et al., 2001). Cited2 interacts with nuclear receptor
PPARα and enhances PPARα mediated transcription (Tien et al., 2004). Cited2 also binds to the LIM domain of Lhx2 and functions as a co-activator to stimulate glycoprotein hormone alpha-subunit gene expression (Glenn and Maurer, 1999). Cited2 and Cited4 interact with TFAP2 and enhance TFAP2 mediated gene transcription
(Braganca et al., 2003; Braganca et al., 2002).
Basal level expression of Cited2 is regulated by Sp1/Sp3 and ETS transcription factors (Han et al., 2001). Cited2 is also induced by many cytokines and biological stimuli, including IL-1α, -2, -4, -6, -9, and -11, granulocyte/macrophage colony- stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum,
lipopolysaccharide and hypoxia in different cell types (Sun et al., 1998). In hypoxic cells,
Cited2 is induced through the HIF-1 pathway, but Cited2 expression also regulates HIF-1 mediated transactivation through a negative feedback mechanism (Bhattacharya et al.,
1999). Cited2 and HIF-1α share a common LPXL motif which interacts with the CH1 domain of p300 (Freedman et al., 2003). The formation of a Cited2-p300 complex is favored because the binding affinity of Cited2 for CH1 is higher in comparison with the
3 HIF-1α (Freedman et al., 2003). As more molecules of Cited2 accumulate in the cell
through HIF-1 mediated transcription of the Cited2 gene, p300 is sequestered by Cited2
resulting in fewer p300 molecules available to sustain HIF-1α mediated transcription.
Knockout models of Cited members
During mouse embryogenesis, Cited1 is predominantly expressed in the heart tube, limb bud and sclerotome, and Cited2 is expressed largely in the precardiac and anterior visceral mesoderm and neuroectoderm (Dunwoodie et al., 1998). Cited4 is highly expressed in hematopoietic tissues and endothelial cells (Yahata et al., 2002). In contrast,
Cited3 is strongly expressed during early stages of embryonic development in chick mesonephric tubules and eye, and dog pronephros (Andrews et al., 2000). Loss-of- function of Cited members during development has been studied through knockout mouse models. Cited1 null mutants exhibit growth restriction phenotype at 18.5 days postcoitum, and some of them die shortly after birth (Rodriguez et al., 2004). Cited1 is
essential for the formation of normal spongiotrophoblast layer during placental
development and subsequently for embryo viability (Rodriguez et al., 2004). Cited1
knockout mice, which survive after birth, show retarded mammary ductal growth at
4 puberty and dilated ductal structures with a lack of spatial restriction of the subtending branches (Howlin et al., 2006).
We and others generated Cited2 knockout mice and showed that Cited2 null embryos die during gestation with cardiac malformations, adrenal agenesis, abnormal cranial ganglia and exencephaly (Bamforth et al., 2001; Barbera et al., 2002; Yin et al.,
2002). The cardiac defects of Cited2 null embryos include atrial and ventricular septal defects, overriding aorta, double-outlet right ventricle, persistent truncus arteriosus and right-sided aortic arches. Expression of ErbB3, a marker of neural crest cells, was reduced and abnormally distributed in mid-embryogenesis suggesting that Cited2 may control neural crest cell migration (Bamforth et al., 2001). In addition, the phenotypes of
Cited2 null embryos include right isomerism, abnormal cardiac looping and hyposplenia
(Weninger et al., 2005). Nodal signaling determines left/right pattern during embryogenesis (Brennan et al., 2002). Cited2 null embryos lack expression of the Nodal target genes, Nodal and Lefty2, and Pitx2 in the left lateral plate mesoderm and Lefty1 in the presumptive floor plate (Bamforth et al., 2004). As Nodal belongs to TGF-β superfamliy and its expression is controlled by both Cited2 and Smad pathways (Besser,
2004), Cited2 may play roles in TGF-β/Nodal-Smad signaling.
5 Cited members and neoplastic transformation
Cited1 is highly expressed in several malignant melanomas, but not in benign nevi, suggesting that Cited1 is associated with melanoma progression, particularly UV- induced lesions (Sedghizadeh et al., 2005). Cited1 is also highly expressed in papillary
thyroid carcinoma (Prasad et al., 2004). Cited2 is strongly expressed in Ewing's sarcomas
(EWS) (Baer et al., 2004), Engelbreth-Holm-Swarm (EHS) tumors (Futaki et al., 2003),
and invasive breast cancer cell line MDA-MB-231 (Chapter II). Cited2 is induced by several cytokines such as IL-9 and IL-11 (Sun et al., 1998). Overexpression of Cited2
converts TS1 cells from IL-9-dependent to IL-9-independent growth, one of the features of cellular transformation (Sun et al., 1998). Cited2 regulates cell growth in mouse embryonic fibroblasts (Kranc et al., 2003). Cited2 null MEFs cease growth prematurely, which is associated with a reduction in growth fraction, senescent cellular morphology, and increased expression of cell proliferation inhibitors p16(INK4a), p19(ARF), and p15(INK4b) (Kranc et al., 2003). Overexpression of Cited2 in Rat1 cells causes loss of cell contact inhibition and leads to growth in soft agar and tumor formation in nude mice
(Han et al., 2001). As Cited2 is highly expressed in certain tumor cells and has transforming activity, it is possible that Cited2 plays a significant role in tumor progression.
6
TGF-β superfamily
Cited1 functions as a Smad4 co-activator to enhance TGF-β mediated
transcription (Shioda et al., 1998), suggesting that members of the Cited family could
play roles in TGF-β signaling. TGF-β belongs to the TGF-β superfamily, which is
composed of pleiotropic cytokines formed by dimeric proteins with conserved structures,
and has multiple functions. TGF-β superfamily includes over 60 proteins such as TGF-β,
Activin and Inhibin, Nodal, Myostatin, Bone Morphogenetic Protein (BMP), and Anti-
Müllerian Hormone (AMH). TGF-β is synthesized as a latent dimeric complex that is
secreted and subsequently cleaved to yield a smaller active dimer (Annes et al., 2003).
The precursor form of TGF-β can bind to various extracellular proteins enabling TGF-β
to be stored in the extracellular matrix (ECM), until activated by a physical process such
as tissue remodeling or wound healing (Annes et al., 2003). The ECM proteinases such as
matrix metalloproteinase MMP9 and MMP2 can cleave the latent form of TGF-β
complex and activate TGF-β (Yu and Stamenkovic, 2000). TGF-β was first identified as
a growth factor, which can promote TGF-α induced anchorage independent colony
formation in fibroblasts (Anzano et al., 1982). Later, TGF-β was found to inhibit or
stimulate cell proliferation, differentiation, motility, or death depending on the cell type
7 and the developmental state of a cell (Siegel and Massague, 2003). In addition, TGF-β is
involved in embryonic development and tissue repair (Kulkarni et al., 2002). TGF-β also
stimulates the production of matrix metalloproteinases, which facilitate remodeling of the
extracellular matrix and cell migration (Hagedorn et al., 2001). Inhibin was identified as a
cytokine that inhibits the secretion of follicle stimulating hormone (FSH) from the
pituitary gland, and later Activin, a related member, was found to stimulate FSH production (Bilezikjian et al., 2004). Inhibin is formed as a disulphide bond dimer of
inhibin-α and –β chains. In contrast, Activin is a inhibin-β homodimer (Bilezikjian et al.,
2004). Activin plays significant roles in the induction of dorsal mesoderm during early
embryogenesis and also regulates cell growth, differentiation and apoptosis of epithelial
and haematopoietic cells (McDowell and Gurdon, 1999; Shav-Tal and Zipori, 2002).
Antagonistic effects of Inhibin on Activin mediated release of FSH are through
competing with Activin for binding to betaglycan, an accessory protein of activin
receptors (Lewis et al., 2000). Nodal plays critical roles in the induction of dorsal
mesoderm, anterior patterning, and formation of left–right asymmetry during embryonic
development (Schier, 2003). Little is known about the role of Nodal in postnatal
development, except a recent study showing that Nodal is expressed in mouse mammary
gland (Kenney et al., 2004). Nodal enhances mammary duct branching and affects ductal
8 direction, suggesting that Nodal could function as a morphogen-like molecule during
mammary gland development (Kenney et al., 2004). Myostatin, also called GDF-8
(growth and differentiation factor-8), was first identified through sequence homology to
TGF-β superfamily cytokines (McPherron et al., 1997). Myostatin is specifically expressed in skeletal muscle. Myostatin negatively regulates skeletal muscle mass and determines both muscle fiber number and size, suggesting the role of Myostatin in muscle development and homeostasis (McPherron et al., 1997). BMP was identified as a cytokine that induces formation of bone and cartilage tissue (Urist et al., 1979). In addition, BMP induces epidermal formation (Munoz-Sanjuan et al., 2002) and directs the development of neural crest cells into neuronal phenotype (Christiansen et al., 2000).
BMP also induces somite development by inhibiting the process of myogenesis (Wan and
Cao, 2005). AMH induces Müllerian duct regression in the male fetus, the initial step of organogenesis of the male genital tract (Josso et al., 1998). In the absence of AMH, mullerian ducts of both sexes develop into uterus, Fallopian tubes, and the upper part of the vagina. AMH also inhibits the transcription of gonadal steroidogenic enzyme genes and blocks the differentiation and steroidogenesis of the immature ovary, the follicle of adult ovary and fetal and postnatal Leydig cells (Josso et al., 1998).
9 Although TGF-β superfamily proteins have divergent biological effects, they
transmit signals in a similar way through Smad transcription factor family into the nucleus. Based on the activated Smad signaling pathways, TGF-β superfamily cytokines
can be classified into Smad1/5/8-BMP, and Smad2/3-TGF-β subfamily. This thesis
focuses on how TGF-β regulates Cited2 expression, and roles of Cited2 in signaling of
TGF-β and a related factor, Nodal.
TGF-β receptors
Members of the TGF-β family bind to type II (TβRII) and type I (TβRI)
receptors, and both TβRII and TβRI are required for signal transduction. In addition,
some cell surface proteins, including betaglycan, endoglin, and the EGF-CFC family
proteins (also called Cripto) function as co-receptors to enhance the interaction between
receptors and cytokines of the TGF-β superfamily (Fonsatti et al., 2001; Gray et al.,
2002; Minchiotti, 2005). Both TβRII and TβRI contain serine/threonine kinase domains
in their intracellular portions. Upon binding of its ligands, TβRII recruits the TβRI into
an activated heterotetrameric receptor complex. TβRI contains a glycine and serine rich
region, known as the GS region, upstream of the serine/threonine kinase domain in the
cytoplasmic portion of the receptor. In the receptor complexes, TβRII kinases
10 transphosphorylate the Gly-Ser (GS) rich domain of TβRI (Fig.I-2). Following phosphorylation of the GS domain, TβRI kinases are activated and phosphorylate intracellular substrates, the Smad proteins. L45 loop, the nine amino acid sequence between kinase subdomains IV and V in TβRIs, interacts with residues in the L3 loop in the carboxy-terminal domains of the R-Smads and is crucial for signal specificity (Fig.I-
2) (Shi and Massague, 2003).
Smads as key effectors in TGF-β signaling
Smads function as signal transducers of the TGF-β family members. The family of Smad proteins consists of Smads with different functions. Smads 1, 2, 3, 5, and 8 act as substrates for TGF-β family receptors and they are collectively referred to as receptor- phosphorylated Smads (R-Smads). Smad4, also called Co-Smad, functions as a common partner for R-Smads and is required for the assembling of active transcriptional complexes (Liu et al., 1997). Smad6 and Smad7 are inhibitory Smads, also called I-
Smads, which interfere with Smad–receptor or Smad–Smad interactions (Hayashi et al.,
1997; Imamura et al., 1997) (Fig.I-3). Smads 1, 5, and 8 serve as substrates for the BMP and anti-Muellerian receptors, and Smads 2 and 3 for TGF-β, Activin, and Nodal receptors (Miyazawa et al., 2002). The structures of Smad proteins are highly conserved.
11 Both R-Smads and Co-Smad contain an N-terminal MH1 domain, which includes a
DNA-binding activity, and a C-terminal MH2 domain, which mediates the translocation into the nucleus and contains transactivation activity (Fig.I-4). In the basal state, MH1
and MH2 domains of R-Smads interact with each other and inhibit each other’s
functions: the MH2 domain represses MH1-mediated DNA binding, whereas the MH1
domain inhibits the transactivation function of MH2. The phosphorylation of the C-
terminal SSXS sequence of R-Smads by the TGF-β type I receptor relieves the two
domains from a mutually inhibitory interaction, which leads to the activation of R-Smad
(Derynck and Zhang, 2003). The activated R-Smads form complexes with the Co-Smad
through the MH2 domain once SSXS sequence is phosphorylated. Co-Smad is not
necessarily required for the nuclear translocation of R-Smads; however, Co-Smad is
important for the formation of functional transcriptional complexes (Fink et al., 2003).
Both Co-Smad and R-Smads, except Smad2, possess DNA binding activity and can
recruit different transcriptional cofactors. In the MH1 domain of R-Smads and Co-Smad,
an 11-residue β-hairpin protruding structure can directly interact with DNA at a 5’-
AGAC-3’ sequence known as a Smad-binding element (SBE) (Shi et al., 1998). In
contrast, Smad2 cannot bind DNA due to the presence of a unique 30-amino acid insert,
which lies just N-terminal to the β-hairpin. The affinity for interaction between Smads
12 and SBE is too low to support the binding of a Smad complex to a single SBE, thus high- affinity binding of the Smad complex usually occurs through the cooperation of Smads with different DNA-binding transcription factors (Shi and Massague, 2003).
Smad interacting transcription factors, co-activators, and co-repressors
Smad proteins interact with numerous proteins that can contribute to both DNA- binding and transcriptional activation. Smad binding transcription factors also act as specificity determinants due to their restricted expression in specific tissues or cell types.
For example, FoxH1 family members that interact with Smad2–Smad4 complexes are highly expressed during early vertebrate development (Attisano et al., 2001). Smad binding transcription factors also play roles in a synergistic or repressive manner with other signaling pathways. For example, Smad mediated transcription is enhanced by AP-
1 proteins in EGF signaling, but repressed by Stat1 in interferon γ signaling (Peron et al.,
2001; Ulloa et al., 1999). Therefore, Smad-interacting transcription factors determine the precise response to ligands in different cell types and distinct developmental stages to cooperate with other signaling pathways. The activated Smad complexes interact with
DNA binding transcription factors, and co-activators or co-repressors that can confer signaling direction, specificity, and magnitude.
13 DNA binding transcription factors such as FoxH1, Mixer, and OAZ interact with
Smads but have low transcriptional activity. They simply function as DNA binding
adaptors to enhance affinity between Smad complexes and DNA (Germain et al., 2000;
Hata et al., 2000). Some of Smad binding proteins are transcription factors or co- activators with their own ability to enhance transactivation such as JunB, TFE3,
CBFA/AML, LEF1/TCF, p300, and Cited1 (Hua et al., 1998; Miyazono et al., 2004;
Selvamurugan et al., 2004b; Shioda et al., 1998). Transcriptional co-activator CBP/p300
is essential for Smads mediated transcription as overexpression of E1A protein which
competes with Smads for binding to CBP/p300 blocks TGF-β mediated transcription
(Nishihara et al., 1998; Pouponnot et al., 1998; Shen et al., 1998; Topper et al., 1998).
CBP/p300 has intrinsic histone acetyltransferase (HAT) activity, which facilitates
transcription by decreasing chromosome condensation through histone acetylation and
increasing the accessibility of Smads with basal transcription machinery (Dennler et al.,
2005; Snowden and Perkins, 1998; van Grunsven et al., 2005). CBP/p300 may also
function as a transcription integrator to synergize functions between Smad proteins and
transcriptional co-activators such as SRC and members of the Cited family (Dennler et
al., 2005) (Chapter II). In addition to interacting with co-activators, Smad proteins also
bind co-repressors such as TGIF and Ski/SnoN, which negatively regulate Smad
14 mediated transcription (Luo et al., 1999; Stroschein et al., 1999; Sun et al., 1999; Wotton
et al., 1999; Xu et al., 2000). TGIF and Ski/SnoN recruit the N-CoR/mSin3/HDAC
repressors to the Smad complex and compete with p300 for binding to Smad proteins
(Luo et al., 1999; Nomura et al., 1999).
As Smad interacting transcription factors, co-activators, and co-repressors determine the specificity, magnitude, and duration of Smad mediated transcription, TGF-
β regulates the expression of certain Smad interacting proteins to amplify or attenuate
downstream signaling. For example, TGF-β stimulates junB expression through the Smad
pathway and the accumulated junB together with Smad proteins bind to MMP13
promoter to enhance MMP13 and MMP9 expression (Robinson et al., 2001;
Selvamurugan et al., 2004a; Selvamurugan et al., 2004b). In addition, Cited2 function as
a Smad coactivator to enhance MMP9 and MMP13 expression (Chapter II). Nonetheless,
Cited2 expression is downregulated by TGF-β through the Smad pathway (Chapter III).
The downregulation of Cited2 by TGF-β may represent a negative feedback control of
MMP9 upregulation. The balance between positive and negative feedback regulation
mediated by modulating the expression of Smad interacting factors determines the
specificity, magnitude and duration of TGF-β signaling (Chapter II).
15 TGF-β and tumorigenesis
TGF-β was originally identified as a growth factor which can induce anchorage-
independent growth of mouse fibroblasts (Anzano et al., 1982). Subsequent studies
indicated that TGF-β is a potent inhibitor for epithelial cell proliferation (Siegel and
Massague, 2003). TGF-β has been implicated both as a tumor suppressor and an
oncogene in mammary tumorigenesis (Roberts and Wakefield, 2003). Overexpression of
TGF-β in transgenic mouse models delays mammary gland development and further
reduces formation of carcinogen or oncogene- induced carcinomas (Pierce et al., 1995).
However, expression of TGF-β through an autocrine or paracrine mechanism is often
increased in the late-stage human breast cancer, particularly in association with invasion
and metastasis (Muraoka et al., 2002; Tobin et al., 2002). Overexpression of active TGF-
β1 or activated TβRI in the mammary gland of transgenic mice accelerates neu-induced tumor progression (Muraoka et al., 2003).
TGF-β induces epithelial to mesenchymal transition (EMT) in normal and transformed mammary epithelial cells, squamous carcinoma cells, ovarian adenosarcoma cells and melanoma cells (Brown et al., 2004). EMT is a process that epithelial cell layers
lose polarity and cell-cell contacts and undergo a dramatic remodeling of the
cytoskeleton. EMT is a feature of embryogenesis but later has been proposed to be
16 relevant in cancer as well. TGF-β induces EMT through downregulation of E-cadherin,
ZO-1, vinculin and keratin, and induces the expression of vimentin and N-cadherin, which in turn increases cell mobility (Zavadil and Bottinger, 2005). Several transcription factors such as Snail, Slug, Twist and Sip-1 that induce EMT during embryonic development also play significant roles in tumor progression (Barrallo-Gimeno and
Nieto, 2005; Kang and Massague, 2004; Yang et al., 2004). TGF-β has been shown to be an autocrine factor of breast tumor metastasis to promote cell growth and EMT (Janda et al., 2002). In addition, TGF-β induces changes in the microenvironment providing favorable conditions for cancer cell migration and endothelial capillary formation. For example, TGF-β induces expression of the matrix metalloproteinases, which enhance the migratory and invasive properties for cancer cells (Hagedorn et al., 2001).
.
Matrix metalloproteinases
The extracellular matrix (ECM) is composed of a complex mixture of insoluble molecules including gelatins, collagens, laminins, fibronectin, and heparan sulfate proteoglycans. Matrix metalloproteinases (MMPs) can degrade all the components of
ECM (Sternlicht and Werb, 2001). Matrix metalloproteinases can be divided into five classes based on their structure and substrate specificity. These are collagenases such as
17 MMP1 and MMP13, gelatinases such as MMP2 and MMP9, stromelysins such as MMP3 and MMP10, matrilysins such as MMP7 and MMP26 and membrane type MMPs (MT-
MMPs) (Sternlicht and Werb, 2001). Embryonic growth and tissue morphogenesis are fundamental events that require disruption of ECM barriers to allow cell migration.
MMPs play a significant role in matrix microenvironment remodeling to degrade structural components of ECM and basement membrane at different developmental stages. MMPs are also highly involved in tumor progression, including stimulation of cell migration, proliferation, and modulation of angiogenesis (Lynch and Matrisian, 2002).
Overproduction of MMP9 in nonmetastatic rat embryonic cells confers a metastatic phenotype (Bernhard et al., 1994). In glioma, expression of antisense or interference
RNA for MMP9 in human glioma cells inhibits their ability to form tumors in nude mice
(Lakka et al., 2005; Lakka et al., 2003). Absence of MMP9 has been shown to reduce the incidence and development of invasive squamous cell carcinoma (SCC) induced by the expression of HPV16 early region genes in basal keratinocytes (Coussens et al., 2000). In
MMP9 deficient mouse model, B16 melanoma cells derived tumors have less ability to metastasize into the lung (Itoh et al., 1999). Since MMP9 plays significant roles during tumor progression, studying the mechanism by which MMP9 is regulated may provide us insights in designing anti-tumor drugs (Chapter II).
18
Posttranscriptional control in TGF-β signaling
Expression of Cited1 and Cited2 is regulated by TGF-β in B16-F1 melanoma and
MDA-MB-231 breast cancer cells, respectively (Shioda et al., 1998) (Chapter III). In
addition to direct transcriptional regulation, TGF-β stimulation also modulates gene
expression posttranscriptionally by increasing the mRNA stability of ribonucleotide
reductase component R2 (Amara et al., 1993), elastin (Kucich et al., 2002), and receptor
for hyaluronan mediated mobility (RHAMM) (Amara et al., 1996), and destabilizing
CD40 mRNA (Nguyen et al., 1998). For example, the half-life of ribonucleotide
reductase R2 mRNA is significantly increased under TGF-β treatment (Amara et al.,
1993). The R2 message contains a sequence in the 3' UTR that binds a specific TGF-β responsive cytosolic p75 protein (Amara et al., 1995). Deletion of the cis-element in the
3’UTR prevents the binding of p75 to R2 transcript and causes destabilization of R2 mRNA. The sequence flanking the TGF-β responsive cis-element in the 3' UTR of R2
(AAU and GUGGUG) is an initial cleavage site in single-stranded RNA loops (Amara et al., 1995). TGF-β regulates cell proliferation by posttranscriptionally controlling expression of ribonucleotide reductase.
19 In addition to R2, elastin mRNA half-life is increased upon TGF-β stimulation. A
10-nucleotide sequence in exon 30 of elastin mRNA is essential for TGF-β mediated up- regulation of elastin (Zhang et al., 1999). Cytosolic 50-kDa protein (p50) interacts specifically with exon 30 of elastin mRNA. TGF-β treatment reduces mRNA-binding activity of p50, and stimulates elastin expression by stabilizing its mRNA, suggesting p50
is a negative regulator for elastin mRNA stabilization. The mechanism by which TGF-β
increases p75 binding to its cis-element in the 3' UTR of R2 or p50 dissociation from
exon 30 of elastin mRNA has not yet been defined. Overexpression of Smad7 blocks
TGF-β mediated up-regulation of elastin indicating that Smad pathway is involved
(Kucich et al., 2002); however, the mechanism of how Smads modulate elasin mRNA stability has not been characterized. It is hypothesized that Smad complexes may induce
expression of mRNA binding proteins or directly interact with mRNA binding proteins to
modify mRNA binding activity, which further affects turnover of elastin mRNA.
Statement of Purpose
Cited1 functions as a Smad4 co-activator and enhances TGF-β mediated
transcription, suggesting that other members of the Cited family may also play roles in
TGF-β signaling. Cited1 is downregulated by TGF-β in melanoma cell lines (Shioda et
20 al., 1998). Recent microarray analysis showed that Cited2 is downregulated by TGF-β in
MDA-MB-231 (breast cancer cells), MCF-10A (normal mammary epithelial cells), leiomyoma and myometrial smooth muscle cells (Chen et al., 2001; Luo et al., 2005).
Knockout mouse models indicated Cited2 plays significant roles in organ formation and determines left-right asymmetry during embryonic development; however, the function of Cited2 in mammary epithelial and breast cancer cells has not been studied. The
primary goal of my studies is to define the role of Cited2 in TGF-β signaling (Chapter II),
to reveal the mechanism of Cited2 downregulation by TGF-β (Chapter III), and to
understand the biological functions of Cited2 (Chapter II and Chapter IV).
Specific Aim 1: Characterize Cited2 function in TGF-β signaling pathway. Smads are the
key mediators in TGF-β signaling pathway. Smad binding proteins often determine the
magnitude, duration, and direction of TGF-β mediated gene expression. Since Cited1
enhances Smads mediated transcription, we hypothesize that Cited2 may also play a role
in TGF-β signaling. To test this hypothesis, we examine whether Cited2 modulates
Smads mediated transactivation through promoter/reporter assays. The physical
interaction between Cited2, Smads, and p300 are also tested. To further confirm the idea
that Cited2 functions as a co-activator for Smad mediated transcription, endogenous
21 TGF-β responsive genes regulated by Cited2 are identified through microarray analysis and further confirmed by siRNA experiments.
Specific Aim 2: Elucidate the mechanism of Cited2 downregulation by TGF-β. Through
microarray analysis, Cited2 was found to be downregulated by TGF-β in MDA-MB-231
cells. As Smads are key effectors in TGF-β signaling, we test whether the Smad pathway
is involved in TGF-β mediated downregulation. We block the Smad pathway by
overexpression of Smad7, or knockdown of Smad4 or Smad2/3 to examine the effect on
TGF-β mediated downregulation of Cited2 (Chapter III). TGF-β signaling modulates
gene expression through transcriptional control and/ or posttranscriptional mechanism.
To further characterize the mechanism of Cited2 downregulation by TGF-β, we use
nuclear run-on and Cited2 promoter/reporter assays to test whether Cited2
downregulation by TGF-β is at the transcriptional level. We further use actinomycin D
treatment to measure Cited2 mRNA half-life in the presence or absence of TGF-β to
examine posttranscriptional control. To further define the Cited2 downregulation
mechanism, expression of untranslated regions and coding region of Cited2 transcript are
tested for TGF-β mediated response (Chapter III).
22 Specific Aim 3: Reveal biological significance of Cited2. Cited2 has been shown to control the expression of proliferation inhibitors p16(INK4a), p19(ARF), and p15(INK4b). Since Cited2 is highly expressed in breast cancer but not in normal mammary epithelial cells, we hypothesize that Cited2 may regulate cell growth or tumor cell migration. To test this hypothesis, cell proliferation and matrix gel migration assays are performed to define the biological significance of Cited2 (Chapter II). Another approach to study the biological roles of Cited2 is through identification of genes regulated by Cited2 using microarray analysis (Chapter II and IV).
In short, we found that Cited2 functions as a Smad2/3 co-activator to enhance
TGF-β mediated transcription of MMP9. We also demonstrated that TGF-β mediated downregulation of Cited2 is through posttranscriptional control and both Smad pathway and coding region of Cited2 participate in the downregulation of Cited2. Interestingly, downregulation of Cited2 functions as a regulatory mechanism to modulate expression of late TGF-β responsive genes such as MMP9 and MMP13.
23 Figure I-1
24 Figure I-1 Alignment of amino acid sequences of Cited family proteins.
Amino acid sequences of Cited family proteins are aligned and analyzed with PRALINE
program. Three conserved regions of Cited family proteins are labeled. The color spectrum from dark blue to red represents the unconserved versus conserved amino acid.
25 Figure I-2
26 Figure I-2 TGF-β signaling from receptor to nucleus.
Upon ligands of the TGF-β superfamily binding to the TGF-β type II (blue) and I
receptors (green), Type II receptor kinases phosphorylate Type I receptors. Type I
receptors then transphosphorylate receptor Smads (R-Smads). Following phosphorylation, R-Smads form complexes with Smad4 (Co-Smad). The Smad complex is then translocated to the nucleus, where it interacts with Smad binding elements (SBE),
DNA binding transcription cofactors, and co-activators in a target gene-dependent manner (modified from Feng, et al. Annu Rev Cell Dev Biol. 2005;21:659-93.)
27 Figure I-3
28 Figure I-3 Different functional Smads in TGF-β signaling.
The family of Smad proteins consists of Smads with different functions. Receptor- phosphorylated Smads (R-Smads), which include Smads 1, 2, 3, 5, and 8, interact with type I receptors. Smad4 (Co-Smad) functions as a common partner for R-Smads.
Inhibitory Smads (I-Smads), which include Smad6 and Smad7, interfere with Smad– receptor or Smad–Smad interactions.
29 Figure I-4
30 Figure I-4 The functional domains of Smads.
Smad proteins are composed of MH1 domain, linker region, and MH2 domain.
31 CHAPTER II
Cited2 modulates TGF-β-mediated up-regulation of MMP9
Most of information in this chapter has been published in Chou et. al, Oncogen (2006)
Introduction
The Cited [CBP/p300-interacting transactivators with glutamic acid (E)/aspartic acid (D)-
rich C-terminal domain] gene family consists of four nuclear proteins--Cited1 (formerly
MSG1) (Dunwoodie et al., 1998; Shioda et al., 1996; Shioda et al., 1997), Cited2
(formerly MRG1/p35srj) (Bhattacharya et al., 1999; Dunwoodie et al., 1998; Leung et al.,
1999; Shioda et al., 1997; Sun et al., 1998), Cited3 (Andrews et al., 2000) and Cited4
(formerly MRG2) (Braganca et al., 2002). Members of this family function as transcriptional co-activators without classical DNA binding domains. Cited2 interacts with the LIM domain of Lhx2 to enhance Lhx2-dependent transcription of LH/FSH glycoprotein α-subunit genes (Glenn and Maurer, 1999). Cited2 and Cited4 interact with
TFAP2, thereby activating TFAP2-mediated transcription (Braganca et al., 2003;
Braganca et al., 2002). Transcriptional responses by Cited2 can be enhanced through its association with nuclear receptors, such as PPAR, to enhance transcription of target genes
(Tien et al., 2004). Cited2 expression is induced by many cytokines and biological
32 stimuli, including IL-1α, -2, -4, -6, -9, and -11, granulocyte/macrophage colony-
stimulating factor, interferon γ, platelet-derived growth factor, insulin, serum,
lipopolysaccharide and hypoxia in diverse cell types (Bhattacharya et al., 1999; Sun et
al., 1998).
Cited2 has been proposed to play an important function in regulating of cellular
responses to hypoxia. This is supported by the observation that hypoxia induces Cited2
expression. Elevated level of Cited2 then functions to downregulate HIF-1 driven transcription by competing with HIF-1α for binding to CBP/p300 (Bhattacharya et al.,
1999; Freedman et al., 2003; Yin et al., 2002). We and others have shown that disruption of the gene encoding Cited2 is embryonic lethal and causes defects in the development of heart and neural tube (Bamforth et al., 2001; Barbera et al., 2002; Yin et al., 2002).
TGF-βs are a highly conserved 25 kDa family of multifunctional autocrine, paracrine and endocrine regulators that signal through interaction with cell surface receptors (Attisano and Wrana, 2000; ten Dijke et al., 2000). Ligand-activated TGF-β receptors associate with and phosphorylate Smads 2 and 3, leading to nuclear translocation of these transcription factors, which serve as key intracellular mediators of
TGF-β signaling (Attisano and Wrana, 2000; ten Dijke and Hill, 2004). Receptor- activated Smad2 and Smad3, form hetero-complexes with Smad4, a common partner in
33 the assembly of transcriptional complexes. These complexes are translocated into the
nucleus and interact with a variety of transcription factors, such as AP-1, Sp1, FAST,
ATF-3, TFE-3, nuclear receptors, and E2F4/5, leading to activation or suppression of
transcription (Shi and Massague, 2003; ten Dijke et al., 2000; Zimmerman and Padgett,
2000). In addition to DNA binding transcription factors, the co-activator function of
CBP/p300 is essential for Smad-mediated transcription (Feng et al., 1998; Janknecht et
al., 1998; Nishihara et al., 1998; Topper et al., 1998). The profile of Smad-binding
cofactors during development or under various growth conditions determines cellular responses to TGF-β (Massague, 2000).
Cited1 interacts with Smad4 and enhances Smad4-mediated transcription in reporter
assays (Shioda et al., 1998; Yahata et al., 2000). Cited1 is downregulated by TGF-β in
melanoma cells (Shioda et al., 1998). Recently gene expression microarray analysis
revealed that Cited2 is regulated by TGF-β in a variety of cell lines (Chen et al., 2001;
Kang et al., 2003; Luo et al., 2005). These studies suggest that Cited2 may play a role in
modulating the TGF-β signaling network. To understand the function of Cited2 in TGF-
β-mediated signaling pathway and target gene expression, we performed microarray
analysis of gene expression modulated by TGF-β in MEFs following inducible
expression of Cited2, and found that Cited2 effectively regulates responses of TGF-β on
34 MMP9 expression. By knocking down Cited2 in MDA-MB-231 cells, we further discovered that Cited2 modulates TGF-β-mediated expression of MMP9 and may play an important role in tumorigenesis.
Materials and Methods
Reagents and antibodies
Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN).
Tetracycline and puromycin were obtained from Sigma (St. Louis, MO). Anti-MMP9 antibodies were ordered from Chemicon. Anti-Myc (A14), anti-Smad1/2/3 (SC7960), anti-Cited2 (MRG1, JA22) antibodies, and normal IgG1 (SC3877) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Flag (M2) and anti-β-actin antibodies were obtained from Sigma (St. Louis, MO). Anti-MMP13 (Ab-4) antibodies were purchased from Calbiochem (San Diego, CA).
Cell culture
MDA-MB-231 and MCF-7 cells were obtained from the American Type Culture
Collection. MCF-10A was provided by Dr. Amy Wilson-Delfosse (Case Western
Reserve University). Primary mouse embryo fibroblast (MEF) cultures were harvested and maintained as described (Yin et al., 2002). For generation of MEF cell lines, primary
35 MEFs were immortalized by retroviral infection with a retroviral vector containing
simian virus 40 large T antigen (Williams et al., 1988), provided by Dr. David Williams
(University of Cincinnati). Inducible Cited2 MEFs, Cited2-/-(LT+, pBPSTR1-Cited2),
were generated from a SV40 large T immortalized Cited2-/- MEF line infected with the
pBPSTR1 vector containing Cited2 cDNA. MEFs and HEK293T cells were maintained
in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal
bovine serum (HyClone). MDA-MB-231 and MCF-7 cells were maintained in minimum
essential medium supplemented with 1 nM insulin (Sigma) and 10% heat-inactivated
fetal bovine serum. MCF-10A cells were maintained in a 1:1 mixture of DMEM and
Ham's F-12 supplemented with 25% horse serum (Invitrogen), 10 µg/ml insulin,
0.5 µg/ml hydrocortisone (Sigma), and 20 ng/ml epidermal growth factor (Sigma).
Plasmids
Mouse Cited2 was cloned from mouse genomic DNA, followed by subcloning into
pcDNA3.1(-)B, pM, pVP-HA1, and PGEX4T1. pM was purchased from CLONTECH
and pVP16-HA1 was provided by Dr. Richard Baer (University of Texas Southwestern
Medical Center). pM was used to generate a fusion protein with the Gal4-DNA binding
domain. MH100, a Gal4 responsive luciferase reporter, was provided by Dr. Hung-Ying
Kao (Case Western Reserve University). Cited2 deletion mutants were PCR amplified
36 and cloned into pVP16-HA1 and pcDNA3.1(-)B. Rat Smad3 was subcloned into pM and
pcDNA3.1(-)B. Rat Smad3 mutant lacking MH2 domain was PCR amplified and
subcloned into pcDNA3.1(-)B. pRK5-Flag-Smad3 is a gift from Dr. Rick Derynck
(University of California, San Francisco). pSBE4-Luc was a gift from Dr. Bert Vogelstein
(Johns Hopkins University). ALK5*(T204D) and dominant negative Smad4/DPC4(1-
514) were provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center).
pBPSTR1 was obtained from Dr. Steven Reeves (Harvard University). Flag tagged
Smad7 with a consensus Kozak sequence was PCR amplified from Flag-CMV2 vector
and subcloned into pBABE-puro. A series of deletion mutants of the human MMP9
promoter reporter constructs were made and described previously (Ogawa et al., 2004).
Wild type, NF-κB, and AP-1 mutant of MMP9 promoter reporter were kind gifts from Dr.
Hiroshi Sato (Cancer Research Institute, Kanazawa University, Kanazawa, Japan) (Sato
and Seiki, 1993).
Luciferase assay, DNA transfection, and siRNA
For luciferase assays, MDA-MB-231 cells were transfected using Lipofectamine Plus reagent (Invitrogen) following the manufacturer’s instructions. HEK293T cells were transfected by the calcium phosphate method. Luciferase activity in cell lysates was determined by a dual luciferase reporter assay system (Promega) and normalized to sea
37 pansy luciferase activity of cotransfected pRL-CMV or pRL-SV40. Statistical
significance was determined by Student’s t-test. For siRNA knockdown experiment,
MDA-MB-231 cells were transfected with siRNA using Effectene transfection reagent
(Qiagen) for 60 hr following the manufacturer’s instructions. siRNAs for Cited2 and
ZNF76 (a negative control) were ordered from Dharmacon Research (Lafayette, CO).
The siRNA sequence for Cited2 is 5’-ugacggacuucgugugcaa-3’ and for ZNF76 5’-
ccagcgccaccaacuauaa-3’. The reconstitution of siRNA was performed following the
manufacturer’s instructions.
Purification of GST fusions, in vitro transcription/translation, and GST pull-down assay
Purification of GST-fusion proteins and GST pull-down assay have been described
previously (Chipuk et al., 2002). Briefly, cDNA from wild type of Cited2 (aa.1-269),
Cited2 C-terminal deletion mutant (aa. 1-199), wild type of Smad3 (aa.1-425), and
Smad3 deletion mutant (aa. 1-225) were subcloned into pcDNA3.1(-)B and in vitro transcribed and translated using TNT Coupled Reticulocyte Lysate Systems (Promega).
GST fusion proteins were immobilized on Glutathion-sepharose beads first. In vitro transcribed and translated peptides were incubated with 10 μg of GST-fusion protein in
PBS supplemented with 0.1% Triton X-100 and rotated overnight at 4°C. The beads were washed with the PBS/0.1% Triton X-100 buffer three times and resuspended in 50 μl of
38 2xSDS sample buffer. The pull-down products were heated at 95°C for 5 min and applied
to SDS-PAGE.
Retrovirus infection
pBPSTR1-Cited2 was generated by inserting Cited2 cDNA into multiple cloning sites of
pBPSTR1, a retroviral tet-off vector. pBABE-Smad7 was generated by inserting Smad7
cDNA into multiple cloning sites of pBABE-puro, a retrovirus vector. Phoenix packaging
cells were used to generate amphotropic retroviruses. Briefly, Phoenix cells were seeded
at a density of 3.5 x 106 cells per 10-cm dish, and transfected with 10 μg of retrovirus vector by the calcium phosphate method the following day. Forty-eight hours after transfection, virus-containing supernatant was collected. For generation of tet-off inducible Cited2 MEF lines, a Cited2-/- MEF line infected with retrovirus expressing
Cited2 was selected with 2.5 μg/ml of puromycin for one week. Individual clones were
picked and Cited2 protein and mRNA expression levels were monitored 24 hours after
culturing cells in 2 μg/ml of tetracycline. For generation of Smad7 overexpression cells,
MDA-MB-231 cells were infected with retrovirus expressing Flag-tagged Smad7,
followed by puromycin selection for one week. In order to avoid clonal variations, pools
of transfectants expressing Smad7 or vector alone were used and subjected to analysis
after puromycin selection and Western blot confirmation.
39 Western blot analysis
Cells were lysed into RIPA buffer (50 mM Tris-HCL, pH7.4, 150 mM NaCl, 1% NP-40,
1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20
μg/ml leupeptin). Lysates were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF-plus; Osmonics Inc.). Membranes were incubated with primary antibodies followed by incubation with secondary antibodies conjugated to horseradish peroxidase. Reacted secondary antibodies were detected using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
Northern blot analysis
Total RNA was isolated from cells using Trizol reagent (Invitrogen) following the manufacturer’s instructions. 10 μg per lane of total RNA was separated by 2.2 M formaldehyde agarose gel electrophoresis, and transferred to a nylon membrane (Magna;
Osmonics Inc.).
SYBER-Green real time PCR
RT-PCR primer pairs are: Mouse MMP9 (5’-tgtctggagattcgacttgaagtc-3’ and 5’- tgagttccagggcacacca-3’); Mouse β-actin (5’-accaactgggacgatatggagaaga-3’ and 5’- cgcacgatttccctctcagc-3’); Human Cited2 (5’-accatcaccctgcccacc-3’ and 5’- cgtagtgtatgtgctcgccca-3’); Human MMP9 (5’-tgacagcgacaagaagtg-3’ and 5’-
40 cagtgaagcggtacatagg-3’); Human MMP13 (5’-ttcctcttcttgagctggactca-3’ and 5’- tctgcaaactggaggtcttcct-3’); Human GAPDH (5’-gaaggtgaaggtcggagtc-3’ and 5’-
gaagatggtgatgggatttc-3’).
Total RNA was isolated by the Trizol (Invitrogen) method and reverse transcribed using the SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen). Real time PCR was performed with iQTM SYBER Green Supermix (BIO-Rad) in a MyiQ thermocycler (Bio-Rad) using a SyBr GreenTM detection protocol as outlined by the
manufacturer. Quantitations were normalized to endogenous GAPDH or β-actin. The
relative quantitation value for each target gene compared to the calibrator for that target is
expressed as 2-(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after
normalizing to GAPDH or β-actin).
Zymographic assay
Cited2-/-(LT+, pBPSTR1-Cited2) MEFs treated with tetracycline, or MDA-MB-231 cells
transfected with siRNA, were stimulated with or without TGF-β. After 24 hr, the
conditioned media were collected and mixed with 5X loading buffer (4% SDS, 20%
glycerol, 0.01% bromophenol blue, and 125 mM Tris-HCl, pH 6.8). The samples were
subjected to 7.5% SDS-PAGE containing 2 mg/ml gelatin (Sigma). After electrophoresis,
the gel was washed three times with 2.5% Triton X-100, briefly with distilled water, and
41
incubated with reaction buffer (50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 1 µM ZnCl2,
150 mM NaCl, 1% Triton X-100, and 0.002% sodium azide) for 16 hr at 37 °C. The gel was stained with 0.25% Coomassie Brilliant Blue R-250 and destained with 40%
methanol, 10% acetic acid as previously described (Heussen and Dowdle, 1980).
Microarray measurement
Cited2-/-(LT+, pBPSTR1-Cited2) MEFs were incubated with medium containing 2 μg/ml
tetracycline or solvent for 24 hours, followed by stimulation with 2.5 ng/ml TGF-β or
reconstitution buffer for another 4 hours. Total RNA was extracted by Trizol and high density oligodeoxynucleotide microarray analysis was performed in the Gene Expression
Analysis Core Facility of Case Western Reserve University.
Chromatin immunoprecipitation (ChIP)
Cells were incubated for 10 min at 37°C with medium containing 1% formaldehyde.
Formaldehyde cross-linking was stopped by adding glycine to a final concentration of
125 mM for 5 min at 37°C. Cells were then washed with ice-cold phosphate-buffered
saline containing protease inhibitors and 1 mM phenylmethylsulfonyl fluoride and
resuspended in SDS lysis buffer containing protease inhibitors for 5 min on ice. Samples
were sonicated to reduce the DNA length to 200-600 bp, cellular debris was removed by
centrifugation, and the supernatant was diluted 20-fold in dilution buffer supplemented
42 with protease inhibitors. Prior to chromatin immunoprecipitation, the samples were
precleared with protein A-agarose/carrier DNA/tRNA mixture. The supernatant was
recovered and used directly for immunoprecipitation experiments by incubation with
appropriate antibodies overnight at 4 °C. Immune complexes were mixed with protein A-
agarose/carrier DNA/tRNA mixture followed by incubation for 1 hr at 4 °C. After
immunoprecipitation, beads were collected and sequentially washed twice with 1 ml each
of the following buffers: low salt wash buffer, high salt wash buffer, LiCl wash buffer,
and TE buffer. The immunocomplexes were eluted twice by adding a 250 µl aliquot of a
freshly prepared solution of 1% SDS/0.1 M NaHCO3. 20 µl of 5 M NaCl and 1 µl of 10
mg/ml RNase were added to the samples and the cross-linking reaction was reversed by 6
hr incubation at 65 °C. The samples were then digested with proteinase K at 42 °C for 1
hr, and DNA was recovered by phenol/chloroform extraction and precipitated with two volumes of ethanol and 5 µl of 10 mg/ml glycogen. The input lysates were processed as above. DNA was resuspended in water and analyzed by quantitative real time PCR. The primer sequences of the human MMP9 promoter used are: 5’-agagaggaggaggtggtgtaagc-
3’ and 5’-ttggtgagggcagaggtgtc -3’.
[3H]Thymidine incorporation assay
43 MDA-MB-231 cells were transfected with siRNA. After 60 hr, cells were washed with
PBS and plated into 12-well plates at a density of 2×104 cells per well. The following day, cells were stimulated with or without 2.5 ng/ml of TGF-β for 33 hr. Cells were labeled with 4 μCi of [3H]thymidine for 3 hr and fixed with 10% trichloroacetic acid
(TCA) for 30 min at 25°C, followed by two washes with 10% TCA. DNA was solubilized by incubation in 500 μl of 0.2 N NaOH for 30 min, and radioactivity was counted using 400 μl of solubilized DNA in 4 ml scintillation fluid.
Matrigel invasion assay
Invasion through BM Matrigel was performed as previously described with minor modifications (Melchiori et al., 1987). MDA-MB-231 cells were transfected with siRNA.
After 60 hr, cells were stimulated with or without 2.5 ng/ml of TGF-β for another 24 hr.
Cells were detached and resuspended in MEM containing 0.1% BSA with antibiotics, with or without TGF-β in the absence of serum. A total of 1×105 cells were added to the upper well of a 24-well matrigel invasion chamber (BD Biosciences) and incubated at
37°C for 20 hr. The lower chamber contained MEM with 5% FBS. Cells that traversed through the filter were fixed, stained with 0.5% (W/V) crystal violet and counted in 5 random fields per insert. Each assay was repeated five times and performed in triplicate.
44 RESULTS
Cited2 is a co-activator for Smad3- Cellular responses to TGF-β are largely mediated by Smads, which serve as both transcription factors and transcriptional co- regulators. Much of the function of Smads is either mediated or modulated through association with a number of other transcription factors such as junB, ATF-3, and SnoN, whose expression may also be regulated by TGF-β (Jonk et al., 1998; Kang et al., 2003;
Stroschein et al., 1999). In line with those observations, TGF-β controls the expression of
Cited2, as demonstrated in MDA-MB-231 and MCF-10A cells (Chen et al., 2001).
Cited1, another member of Cited family, is a Smad4 co-activator (Shioda et al., 1998;
Yahata et al., 2000). We thus investigated the role Cited2 may play in TGF-β signaling pathway. MDA-MB-231 cells were cotransfected with Smad3 and/or Cited2 plus SBE4-
Luc, a TGF-β responsive reporter with four Smad binding elements (SBE) in the promoter region to which Smad3 binds. Smad3 increased SBE4-Luc activity without stimulation of TGF-β (Fig.II-1), consistent with other reports. Cotransfection of cells with Cited2 further enhanced Smad3-mediated transcription, although Cited2 alone had no effect on SBE4-Luc activity (Fig.II-1). In the presence of TGF-β, activated endogenous receptor Smads translocate to the nucleus and bind SBE, thereby activating transcription of SBE4-Luc reporter. Cited2 alone enhanced TGF-β-dependent activation
45 of SBE4-Luc reporter, implying that activated endogenous receptor Smads may mediate
this response. Consistent with this notion, under TGF-β stimulation, Smad3-mediated
transcription was further enhanced in the presence of Cited2 (Fig.II-1). These
experiments suggest that Cited2 could function as a co-activator to enhance Smad3-
mediated transcription.
Cited2 interacts with Smads 2 and 3- We tested whether Cited2 enhances Smad3-
mediated transcription by interacting with Smads, using a mammalian two-hybrid system.
Gal4-Smad3 fusion protein-mediated transcription of Gal4-binding site containing reporters, which was further enhanced by the coexpression of VP16 fused Cited2
(Cited2/VP16) (Fig.II-2A). These data suggest that Cited2 interacts with Smad3. We constructed Cited2 truncation mutants to map the Smad3 interacting region in Cited2.
There are three conserved regions (CR) among different members of the Cited family
(Fig.II-2B). Construct aa.1-199 of Cited2/VP16 which includes the CR1 and CR3, but not
CR2 domain of Cited2, did not interact with Gal4-Smad3 (Fig.II-2C). In contrast, construct aa.124-269 of Cited2/VP16 containing the CR2, but not CR1 and CR3 domain of Cited2, interacted with Gal4-Smad3 (Fig.II-2C), suggesting that the C-terminal region of Cited2 is essential for the interaction with Smad3. Equivalent expression levels of various Cited2 constructs were detected in Figure II-2C.
46 To further confirm the interaction between Cited2 and Smad3, GST pull-down
assay was performed. Full-length Cited2 and Cited2 mutant without the C-terminal
region were in vitro transcribed and translated (Fig.II-2D). As shown in Fig.II-2D, wild-
type Cited2, but not Cited2 C-terminal deletion mutant (Cited2 aa.1-199), interacted with
GST-Smad3, which further confirms the C-terminal region of Cited2 is necessary for
interaction with Smad3. Since the MH2 domain of Smad3 is responsible for interaction
with various transcription factors, we tested whether Cited2 interacts with Smad3 mutant
(Smad3 aa.1-225), which contains the MH1 domain and the linker region but not the
MH2 domain of Smad3. In vitro transcribed/translated full-length Smad3, but not Smad3 mutant lacking the MH2 domain, interacted with GST-Cited2 (Fig.II-2D), supporting that
MH2 domain of Smad3 interacts with Cited2. We tested whether other Smads interact with Cited2. As shown in Fig.II-2E, Cited2 interacted with GST-Smad2 and GST-Smad3, but not GST-Smad4 fusion protein. These experiments support our hypothesis that Cited2 interacts with Smads 2 and 3.
Cited2 promotes Smad3/p300-mediated transcription-Transcriptional co-activator p300 interacts with Smad3 and Cited2 through the C-terminus and the CH1 domain of p300, respectively (Bhattacharya et al., 1999; Nishihara et al., 1999; Nishihara et al.,
1998). We thus speculated that p300 may modulate the interaction between Cited2 and
47 Smad3. Since p300 interacts with only the phosphorylated Smad3 (Shen et al., 1998), we
cotransfected constitutively activated form of TGF-β receptor 1 (ALK5*) with Smad3,
Cited2 and/or p300 in HEK293T cells. Cotransfection with p300 increased the level of
Cited2 co-immunoprecipitated with Flag-tagged Smad3 (Fig.II-3A), indicating that p300
enhances the interaction between Cited2 and Smad3. It has been shown that p300 is
important for Smad-mediated transcription (Feng et al., 1998; Janknecht et al., 1998;
Nishihara et al., 1998; Topper et al., 1998). Transfection of HEK293T cells with p300 alone modestly enhanced Smad3-mediated transcription of SBE4-Luc (Fig.II-3B).
However, cotransfection of both Cited2 and p300 significantly promoted Smad3- mediated transcription of SBE4-Luc (Fig.II-3B).
We further asked whether p300 or Cited2 enhances Gal4-Smad3-mediated transcription by tethering Smad3 to Gal4 DNA binding domain. Interestingly, cotransfection of cells with both p300 and Cited2 significantly enhanced Gal4-Smad3- mediated transcription even in the absence of TGF-β (Fig.II-3C). In contrast, cotransfection of cells with p300 and a Cited2 mutant encoding aa.1-199 did not enhance
Smad3-mediated transcription (Fig.II-3C). These data support the conclusion that Cited2 enhances Smad3/p300-mediated transcription.
48 Cited2 enhances TGF-β-mediated expression of MMP9- In order to understand
the function of Cited2 in TGF-β signaling, we adopted a novel approach by manipulating
tetracycline controlled expression of Cited2 in Cited2 null MEFs, followed by microarray
analysis to search for TGF-β responsive genes regulated by Cited2. Cited2-/- MEF cells
were immortalized with retrovirus expressing SV40 large T antigen (LT), followed by stable transfection of a tet-off vector (pBPSTR1) containing the Cited2 coding sequence
to generate Cited2-/-(LT+, pBPSTR1-Cited2). Cited2 mRNA and protein were not
expressed in tetracycline treated Cited2-/-(LT+, pBPSTR1-Cited2), but detected in the
absence of tetracycline (Fig.II-4A, II-4B). We performed high density
oligodeoxynucleotide microarray analysis of RNAs from Cited2-/-(LT+, pBPSTR1-
Cited2) in the presence or absence of tetracycline for 24 hours and TGF-β stimulation for
another 4 hours. Matrix metalloproteinase 9 (MMP9) was one of the genes highly
stimulated by TGF-β in Cited2-/-(LT+, pBPSTR1-Cited2) in the absence of tetracycline.
By real-time analysis, expression of Cited2 significantly enhanced TGF-β-mediated
expression of MMP9 (Fig. 4C). The protein expression of MMP9 in conditioned medium
was confirmed by Western blot analysis (Fig.II-4D). We also performed gelatin
zymographic assays to measure changes in MMP9 (gelatinase B, 95-105 kDa) activity
following TGF-β stimulation in the presence or absence of Cited2 (Fig.II-4E). The basal
49 expression of MMP9 was similar in Cited2 on and off cells; however, under TGF-β stimulation, Cited2 strongly enhanced TGF-β-mediated expression of MMP9 as measured in the gelatin containing gel (Fig.II-4E). These data support the conclusion that
Cited2 modulates TGF-β-mediated MMP9 expression in Cited2 inducible MEFs.
Chen et al. showed that Cited2 is one of TGF-β responsive genes in MDA-MB-
231 breast cancer cells (Chen et al., 2001). We found that TGF-β downregulated Cited2 four hours following TGF-β treatment in MDA-MB-231 cell, which express high levels of this transcriptional co-activator (Fig.II-4F). Since Cited2 regulates TGF-β-mediated
MMP9 expression in MEFs, we asked whether Cited2 also modulates MMP9 expression in MDA-MB-231 cells. Knockdown of Cited2 using siRNA specific for Cited2 in MDA-
MB-231 cells resulted in decreased expression of Cited2 (Fig.II-4G, lane 3), but the expression level was not affected by si-Control (Fig.II-4G, lane 5). By treatment of
MDA-MB-231 cells with TGF-β for 24 hours, we observed TGF-β-mediated MMP9
mRNA expression was significantly attenuated among cells in which Cited2 was knocked
down (Fig.II-4H and II-4I). Zymographic assays showed that MMP9 protein was induced
by TGF-β in MDA-MB-231 cells, and the stimulated expression was reduced in cells
transfected with si-Cited2 (Fig.II-4J). These data support our model that Cited2
modulates TGF-β-mediated MMP9 expression in both MEFs and MDA-MB-231 cells.
50 Smad pathway is involved in up-regulation of MMP9 by TGF-β in MDA-MB-231
cells- Smad proteins are key mediators in TGF-β signaling, and Smad2/3 is involved in
up-regulation of MMP13 by TGF-β in MDA-MB-231 cells (Selvamurugan et al., 2004a;
Selvamurugan et al., 2004b). We tested whether Smad pathway plays a role in TGF-β-
mediated up-regulation of MMP9 in MDA-MB-231 cells. To block Smad pathway in
MDA-MB-231 cells, we overexpressed Smad7, an inhibitory Smad. Overexpression of
Smad7 attenuated phosphorylation of Smad2 (Fig.II-5A) and up-regulation of MMP9
mRNA (Fig.II-5B, II-5C) by TGF-β. Zymographic assay further demonstrated that
protein expression of MMP9 induced by TGF-β was reduced in cells with overexpression
of Smad7 (Fig.II-5D). These data support the conclusion that the Smad pathway is
involved in TGF-β-mediated up-regulation of MMP9, a target gene for Cited2, in MDA-
MB-231 cells.
Cited2 enhances TGF-β-mediated MMP9 promoter reporter activity- To localize
TGF-β responsive region in the MMP9 promoter, we designed a series of MMP9 promoter - luciferase reporter constructs, with point mutations or deletions, which were then used to study regulation of transcriptional control of MMP9 by TGF-β in MDA-
MB-231 cells. Deletions upstream of -151 bp in the MMP9 promoter region maintained
TGF-β-mediated response (Fig.II-6A). The proximal (-73/-79) AP-1 site (TGAGTCA) is
51 well conserved among human, rat, and mouse MMP9 promoters, and has high sequence homology to the TGF-β responsive elements, TGACTCA and TGAGTTCA, in human
MMP13 and rat PAI-1 promoters, respectively (Guo et al., 2005; Uria et al., 1998).
Given that both proximal AP-1 and NF-κB cis-elements are essential for mitogen- induced MMP9 up-regulation (Ma et al., 2004), proximal AP-1 and NFκB mutants of
MMP9 promoter were further tested for TGF-β-mediated response. MMP9 promoter with a specific mutation in the proximal AP-1 site not only decreased the basal level expression but also abolished TGF-β-mediated up-regulation of MMP9 transcription
(Fig.II-6A). Cotransfection of Cited2 was able to enhance TGF-β-mediated expression of wild type but not MMP9 promoter mutated at the AP-1 site (Fig.II-6B). Smad7 and dominant negative Smad4 have been shown to inhibit Smad pathway by interfering with phosphorylation and translocation of endogenous Smads, respectively (Hayashi et al.,
1997; Zhang et al., 1997). Coexpression of Smad7 or dominant negative Smad4 attenuated Cited2-mediated expression of MMP9 promoter reporter, suggesting that endogenous Smads are required for functional interaction with Cited2 to enhance MMP9 expression (Fig.II-6B). These data further confirm that Cited2 functions as a co-activator to enhance TGF-β-mediated up-regulation of MMP9.
52 TGF-β regulates MMP9 expression by recruiting Cited2 and Smad3 to the MMP9 promote- Our findings suggest that Cited2 enhances TGF-β-mediated expression of
MMP9. To further understand how Cited2 regulates the expression of endogenous genes
upon TGF-β stimulation, we performed real-time PCR to monitor the expression of
MMP9 and Cited2 mRNA (Fig.II-7A). During the first 4 hours of TGF-β stimulation,
MMP9 mRNA expression was not significantly enhanced, while Cited2 was downregulated by TGF-β. Interestingly, as Cited2 mRNA expression started to resume at
12 to 24 hours after stimulation, MMP9 expression was strongly enhanced (Fig.II-7A).
As it has been reported before that Smad3 is recruited to the MMP13 promoter upon
TGF-β stimulation (Selvamurugan et al., 2004b), we tested whether Smads and Cited2 are recruited to the promoter region of MMP9. Chromatin immunoprecipitation with the
MMP9 promoter was performed using antibodies against Cited2 and Smads. 4 hours after
TGF-β stimulation, occupancy of Smad2/3 on MMP9 promoter was increased (Fig.II-
7B). Interestingly, 24 hours after stimulation, occupancy of Cited2 was sharply increased on the MMP9 promoter (Fig.II-7B), during which an increased Smad2/3 on the promoter was also observed. These data, therefore, would support our hypothesis that Cited2 functions as a transcriptional modulator of Smad2/3 by simultaneously binding to the
MMP9 promoter to enhance TGF-β-mediated MMP9 expression.
53 Cited2 plays a role in TGF-β-mediated invasion of MDA-MB-231 cells- Cited2 is expressed at a higher level in MDA-MB-231 cells, an invasive breast cancer cell line, than in non-invasive lines, such as MCF-10A and MCF-7 cells (Fig.II-4F). The MDA-
MB-231 cell line is one of the breast cancer cell lines that are resistant to TGF-β- mediated growth arrest (Bandyopadhyay et al., 1999; Chen et al., 2001; Tobin et al.,
2002). Since Cited2 activates cell growth (Kranc et al., 2003), we knocked down Cited2 by siRNA and tested whether resistance of MDA-MB-231 cells to TGF-β-mediated growth arrest is due to high expression of Cited2 in MDA-MB-231 cells. Knockdown of
Cited2 by si-Cited2 did not significantly affect the growth rate of MDA-MB-231 under
TGF-β stimulation (Fig.II-8A). Since TGF-β enhances the invasion of MDA-MB-231 cells in the matrigel assay (Ilunga et al., 2004), and Cited2 modulates expression of
MMP9 (Fig. 4), which has been suggested to play important roles in the cancer cell invasion (Coussens et al., 2000; Hiratsuka et al., 2002; Itoh et al., 1999; Javelaud et al.,
2005; Santibanez et al., 2002), we tested whether Cited2 is involved in the invasion of
MDA-MB-231 cells. Consistent with the data from others, we found that TGF-β enhanced MDA-MB-231 invasion (Fig.II-8B). Interestingly, knockdown of Cited2 through si-Cited2 significantly attenuated TGF-β-mediated invasion of MDA-MB-231
54 cells (Fig.II-8B). These experiments imply that by modulating the expression of MMP9,
Cited2 could play a significant role during tumor invasion.
Discussion
Smads are key mediators in the TGF-β signaling pathway. Smad binding cofactors determine the magnitude, duration, and direction (activation or suppression) of Smad driven transcription during TGF-β stimulation (Attisano and Wrana, 2000; ten Dijke and
Hill, 2004). Transfection studies support that Cited1 functions as a Smad co-activator
(Shioda et al., 1998; Yahata et al., 2000). However, a direct demonstration that Cited1 regulates endogenous genes was never performed. In this study, we have provided evidence supporting that Cited2 interacts with Smad3 and functions as a Smad3 co- activator in TGF-β signaling. Cited2 is recruited to the endogenous MMP9 promoter along with Smads to enhance TGF-β-mediated MMP9 expression. Members of the Cited family modulate transcription mediated by DNA binding transcription factors such as
Lhx2 (Glenn and Maurer, 1999), TFAP2 (Braganca et al., 2003; Braganca et al., 2002), the nuclear receptor PPAR (Tien et al., 2004), and HIF-1α (Bhattacharya et al., 1999;
Fox et al., 2004; Freedman et al., 2003) through interaction with CBP/p300. The role for
CBP/p300 as an essential co-activator in Smad driven gene expression has been well
55 documented by observations that overexpression of E1A antagonizes the function of
CBP/p300, which leads to attenuation of Smad driven transcription (Feng et al., 1998;
Janknecht et al., 1998; Nishihara et al., 1998; Topper et al., 1998). However,
coexpression of CBP/p300 has modest effects on Smad-mediated transcription based on
several promoter/reporter assays (Feng et al., 1998; Warner et al., 2004). The effects of
p300 on Smad driven transcription may rely on the recruitment of other co-activators to
the Smad/p300 complex (Dennler et al., 2005). We show that p300 enhances the
interaction between Cited2 and Smad3 (Fig.II-3A). Coexpression of Cited2 and p300
enhances Smad3-mediated transcription (Fig.II-3B, II-3C), which corroborates the notion
that Cited proteins represent a novel family of transcription modulators to regulate the activities of various CBP/p300 complexes. It is interesting that overexpression of p300 without Cited2 and in the absence of TGF-β decreased Gal4-Smad3-mediated transcription (Fig.II-3C). Without TGF-β receptor-mediated phosphorylation, wild type
Smad3 does not interact with p300 (Shen et al., 1998). Overexpression of p300 may
interact with other endogenous transcription factor complexes such as Cited2 and TFAP2,
to decrease the endogenous pool of free Cited2, and in turn affect Gal4-Smad3-mediated
transcription. These data suggest other transcription factors including Cited2 are involved
in Smad3/p300-mediated transcription.
56 Our microarray analysis with tet-off inducible Cited2 MEF and siRNA-mediated
knockdown experiments in MDA-MB-231 cells show that Cited2 modulates TGF-β-
mediated expression of MMP9. MMPs are matrix metalloproteinases capable of
degrading extracellular matrix (ECM), and strict regulation of MMP synthesis is critical
for the maintenance of proper ECM expression (Sternlicht and Werb, 2001). In disease
states such as cancer, there is often high MMP activity at the tumor-stroma interface
(Lynch and Matrisian, 2002). Excessive deposition of ECM has been detected in
Engelbreth-Holm-Swarm (EHS) tumors and differentiated F9 embryonic carcinoma (F9-
PE). Interestingly, Cited1 and Cited2 are highly expressed in EHS tumors and up-
regulated during the differentiation of F9-PE, suggesting that Cited1 and Cited2 may be involved in the regulation of ECM (Futaki et al., 2003). MMP9 is activated through both proteolytic and non-proteolytic mechanisms. Full-length MMP9 is the major form of
MMP9 in cancer tissues (Fridman et al., 2003) and in MDA-MB-231, an invasive breast cancer cell line. Several studies have suggested that without the participation of proteolytic enzymes and removal of the inhibitory peptide, full-length MMP9 can be activated by binding to the substrates to cause conformational changes or oxidative modification of the cysteine side-chain thiol groups, in turn allowing zinc ion to become available for the catalytic function (Bannikov et al., 2002; Gu et al., 2002; Okamoto et
57 al., 2001). TGF-β is a potent growth inhibitor of normal epithelial cells, but also fosters tumor formation during later stages of tumorigenesis (Wakefield and Roberts, 2002).
TGF-β enhances the expression of MMP9, which plays significant roles during the dissemination and growth of cancer cells (Coussens et al., 2000; Hiratsuka et al., 2002;
Itoh et al., 1999).
While Cited2 is a transforming gene, as we previously showed, and overexpression of Cited2 in Rat1 cells causes tumor formation in nude mice (Sun et al.,
1998), the mechanism involved in tumor formation was unclear. Our current demonstration that Cited2 modulates TGF-β-mediated up-regulation of MMP9 and enhances in vitro tumor cell invasion may in part explain why overexpression of Cited2 promotes tumor formation in nude mice. In addition to MMP9, Cited2 also modulates
MMP13 expression in MDA-MB-231 cells (Supplementary information). Yokota et al. showed that, in a human chondrocyte cell line C-28/I2, MMP1 and MMP13 are downregulated by TGF-β, possibly through Cited2 (Yokota et al., 2003). In addition, they showed that Cited2 is up-regulated by TGF-β (Yokota et al., 2003), which is different from our study and those of others (Chen et al., 2001; Kang et al., 2003; Luo et al., 2005;
Tardif et al., 2001). These discrepancies could be due to the use of different cell lines.
Although Cited2 overexpression enhances TGF-β-mediated expression of MMP9 in
58 Cited2 inducible MEFs, MMP9 up-regulation by TGF-β is not totally abolished in the
Cited2 null background, suggesting that other transcription factors or cofactors may also be involved in MMP9 up-regulation by TGF-β.
Smad proteins are the main trans-acting factors involved in TGF-β stimulated
MMP13 expression in MDA-MB-231 cells (Selvamurugan et al., 2004a; Selvamurugan et al., 2004b). It has been reported that the AP-1 site in the MMP13 promoter region is essential for TGF-β stimulated MMP13 expression (Selvamurugan et al., 2004b; Uria et al., 1998). Here we demonstrated that mutation of the proximal AP-1 site in the MMP9 promoter abolishes TGF-β-mediated response. In addition to Smad binding elements,
Smads are recruited to the promoter through AP-1 complexes (Yamamura et al., 2000;
Zhang et al., 1998). Although Zhang et al. have showed that Smad3 directly binds to AP-
1 sequences in MMP1 promoter (Zhang et al., 1998), it is also suggested that Smad2/3 may interact with transcription factors, such as junB and Cbfa1/runx2, rather than directly binding to the DNA for the MMP13 promoter activity (Selvamurugan et al., 2004b).
Whether Smad3 and Cited2 directly bind to the MMP9 promoter or are recruited through other DNA binding transcription factors requires further investigation. We have observed more Cited2 are recruited to the MMP9 promoter in the absence of TGF-β than 4 hours after stimulation. Since Cited2 interacts with transcription factors such as TFAP2 and
59 Lhx2 other than Smads, we do not rule out the possibility that in the absence of TGF-β,
TFAP2 may also recruit Cited2 to the MMP9 promoter.
TGF-β activates Smads 2 and 3 by rapid (i.e, 15 min) yet transient phosphorylation (Selvamurugan et al., 2004a). It is difficult to reconcile that MMP9 and
MMP13 expression stimulated by TGF-β does not significantly occur till 24 h following stimulation (Selvamurugan et al., 2002). Our data here imply that Cited2 plays an essential role in regulating TGF-β-mediated MMP9 expression. Cited2 is recruited to the
MMP9 promoter and the kinetics of Cited2 expression correlates with the mRNA levels of MMP9 during TGF-β stimulation. These data suggest that by modulating the expression of Cited2, TGF-β creates a temporal and specific control to regulate the expression of late TGF-β responsive genes such as MMP9. Several regulatory mechanisms that create temporal controls in TGF-β signaling network have been demonstrated. SnoN, a Smad binding repressor, is rapidly degraded in response to TGF-
β. At later stages of TGF-β stimulation, TGF-β induces SnoN expression, resulting in termination of Smad-mediated transactivation (Stroschein et al., 1999; Sun et al., 1999).
Smad7, an antagonistic Smad induced by TGF-β, interferes with the binding between
Smad2/3 and TGF-β receptor, to attenuate TGF-β-mediated responses (Hayashi et al.,
1997). We also show that Cited2 is a Smad3/p300 co-activator, but is modulated by
60 TGF-β in MDA-MB-231 cells. As Cited2 expression resumes at later time points of
TGF-β stimulation, Cited2 enhances Smad2/3-mediated up-regulation of MMP9.
Interestingly, Cited1, another member of the Cited family and a Smad4 interacting co- activator, is also downregulated by TGF-β in B16-F1 melanoma (Shioda et al., 1998).
Whether such a temporal control identified in this study reflects a unique property also shared by other Cited family members to modulate TGF-β responses will require further
investigation.
In conclusion, the present data provides strong evidence for the involvement of
Cited2 in TGF-β signaling. Cited2 functions as a Smad2/3 binding co-activator and is
important for Smad3/p300-mediated transcription. In MEFs and MDA-MB-231 cells,
Cited2 modulates TGF-β-mediated up-regulation of MMP9. Knockdown of Cited2 in
MDA-MB-231 cells attenuates TGF-β-mediated invasion of MDA-MB-231 cells.
Unraveling the role of Cited2, a novel modulator in TGF-β signaling, could contribute to
the understanding of effects of TGF-β signaling on development and tumorigenesis.
Acknowledgments
We are grateful to Dr. David Donner for critical reading of manuscript; and Drs. Edward
Stavnezer, Paul MacDonald, and Monica Montano for helpful discussion and advice.
61 This work was supported by grants from National Institute of Health (RO1 CA78433 to
YCY).
62 Figure II-1
63
Figure II-1 Cited2 enhances Smad3-mediated transactivation.
1×105 MDA-MB-231 cells were plated and transfected with 300 ng of pSBE4-Luc together with 300 ng of pRK5-Flag-Smad3, pcDNA3.1-Cited2 or both. 24 hr after transfection, cells were treated with or without 2.5 ng/ml of TGF-β for another 24 hr,
followed by luciferase assay. Results are representative of three individual experiments.
*, p<0.05 comparing the indicated two bars. +, p<0.05 comparing the indicated two bars.
64
Figure II-2
A
B
C
65
D
E
66
Figure II-2 Cited2 interacts with Smad2 and Smad3.
(A) 4×104 HEK293T cells were cotransfected with 100 ng of MH100 reporter (Gal4 responsive luciferase reporter), pM-Smad3 (Gal4-Smad3), pVP16-HA1 vector containing
full-length Cited2 in-frame with VP16-HA1 (Cited2/VP16), and empty pVP16-HA1
vector (100ng each) in different combination for 48 hr, followed by luciferase assay. (B)
Schematic representation of Cited2 shows different domains of Cited2 and the deletion
constructs used in the experiment. CR represents the conserved region among different
members of the Cited family. (C) 4×104 HEK293T cells were cotransfected with MH100
reporter (100 ng), pM-Smad3 (100 ng), and pVP16-HA1 vector containing different portions of Cited2 (100 ng each) for 48 hr, followed by luciferase assay. *, p<0.01 comparing to VP16 alone. Cell lystes transfected with different Cited2 deletion VP16-HA fusion construct were subjected to Western blot analysis with antibodies against HA. (D)
(Left) Equal volumes of GST and GST-Smad3 bound to glutathione sepharose were mixed with in vitro transcribed and translated Myc tagged wild type Cited2 (WT) (aa. 1-
269) and Cited2 mutant (aa.1-199). GST-pull-down assay was performed as described in
Materials and Methods. 50% input and pull-down products were separated by SDS-
PAGE and detected by Western blot analysis with antibodies specific for Myc. (Right)
Equal volumes of GST and GST-Cited2 bound to glutathione sepharose were mixed with 67
in vitro transcribed and translated wild type Myc tagged Smad3 (WT) (aa. 1-425) and
Myc tagged Smad3 mutant without MH2 domain (∆MH2) (aa. 1-225). 50% input and
pull-down products were separated by SDS-PAGE and detected by Western blot analysis
with antibodies specific for Myc. (E) Top panel shows the binding of Myc tagged full-
length Cited2 to GST-Smad2, GST-Smad3, and GST-Smad4. 50% input and pull-down products were separated by SDS-PAGE and detected by Western blot analysis with antibodies specific for Myc. Bottom panel is coomassie blue stained gel showing relative amounts of GST, GST-Smad2, GST-Smad3, and GST-Smad4.
68
Figure II-3
A
B
C
69
Figure II-3 Cited2 enhances Smad3/p300-mediated transcription through
p300/Cited2/Smad3 interaction.
(A) 3×106 HEK293T cells were transfected with pRK5-Flag-Smad3, ALK5*, pcDNA3.1-
Cited2, and HA-tagged p300 expression vector (5 μg each) in different combination.
After 48 hr, cell lysates were immunoprecipitated (IP) for Flag-Smad3 and associated
Cited2 was determined by Western blotting (WB). The input represents 10% of the lysate. ALK5* represents a constitutive active TGF-β type I receptor (T204D). (B) 4×104
HEK293T cells were cotransfected with 300 ng of pSBE4-Luc together with pRK5-Flag-
Smad3 (300 ng), ALK5* (300 ng), pcDNA3.1-Cited2 (300 ng), and HA-tagged p300
expression vectors (300 ng) in different combination. 48 hr after transfection, cells were
lysed and subjected to luciferase assay. *, p<0.01 comparing the two indicated bars. **,
p<0.01 comparing the two indicated bars. (C) 4×104 HEK293T cells were cotransfected with 100 ng of MH100 reporter (Gal4 responsive luciferase reporter), 100 ng of pM-
Smad3 (Gal4-Smad3), HA-p300 (100 ng), and pcDNA 3.1-Cited2 or Cited2aa.1-199 expression vectors (100 ng each) in different combination for 48 hours, followed by luciferase assay. The empty vector was cotransfected to maintain an equal amount of
DNA in each experiment. Results are representative of three different experiments.
70
Figure II-4
A
B
C
D
71
E
F
G
72
H
I
J
73
Figure II-4 Cited2 modulates TGF-β-mediated up-regulation of MMP9.
(A) Cited2 inducible MEFs, Cited2-/-(LT+, pBPSTR1-Cited2), were generated by
retroviral infection of Cited2-/- MEFs with pBPSTR1-Cited2. Tetracycline-mediated downregulation of Cited2 was monitored by Western blot analysis using anti-Cited2
antibody. (B) Cited2-/-(LT+, pBPSTR1-Cited2) MEFs from (A) were cultured with or
without tetracycline for 24 hr, followed by adding 2.5 ng/ml of TGF-β or reconstitution
buffer to the media for another 4 hr. Total RNA was isolated and subjected to Northern
analysis with specific probes for Cited2 and GAPDH. (C) Total RNA from (B) was
subjected to real-time PCR with specific primers for mouse MMP9 and normalized with
mouse β-actin. *, P< 0.01 comparing –Tet (Cited2 on) to +Tet (Cited2 off) in the
presence of TGF-β. (D) Cited2-/-(LT+, pBPSTR1-Cited2) MEFs were treated with
tetracycline for 24 hr, followed by stimulation with 2.5 ng/ml of TGF-β for another 24 hr
in absence of serum. The serum free conditioned media were collected and condensed
with centricon 3, followed by Western blot analysis with specific antibodies against
MMP9. The relative intensity of specific bands was quantified with densitometry. (E)
Cited2-/-(LT+, pBPSTR1-Cited2) MEFs were treated with tetracycline for 24 hr, followed
by stimulation with 2.5 ng/ml of TGF-β for another 24 hr in the absence or presence of
serum. The conditioned media were collected, followed by gelatin zymographic assay. 74
(F) MCF-10A, MDA-MB-231, and MCF-7 cells were treated with or without 2.5 ng/ml
of TGF-β for 4 hr. Total RNA was isolated, followed by Northern analysis with specific
probes for Cited2 and GAPDH. (G) MDA-MB-231 cells were transfected with si-Cited2
(lane 3, 4) or si-Control (lane 5, 6; negative control) for 60 hr, followed by TGF-β
stimulation for another 4 hr (lane 2, 4, 6). Total RNA was extracted by Trizol, and
Northern analysis was performed. (H) MDA-MB-231 cells were transfected with si-
Cited2 or si-Control for 60 hr, followed by TGF-β stimulation for another 24 hr. Total
RNA was extracted, followed by quantitative real time PCR with specific primers for
human MMP9 and normalized with human GAPDH. (I) The RT-PCR products of RNA
samples from (H) were loaded onto a 12% polyacrylamide gel to demonstrate the relative
mRNA levels of MMP9, Cited2, and GAPDH. (J) Serum free conditioned media from
(H) were collected and subjected to gelatin zymographic assay. Results are representative of four individual experiments.
75
Figure II-5
A
B
C
D
76
Figure II-5 Smad pathway is involved in TGF-β-mediated up-regulation of MMP9.
(A) MDA-MB-231 cells were transfected with Flag-tagged Smad7 expression vector by retroviral infection as described in Materials and Methods. MDA-MB-231 cells transfected with pBABE-Smad7 or pBABE vector alone were treated with or without 2.5 ng/ml of TGF-β for 24 hr. Total cell lysates from pools of cells transfected with pBABE-
Smad7 or pBABE were then harvested and subjected to Western blot analysis with specific antibodies against phospho-Smad2 (p-Smad2), Flag, and β-actin. (B) Total RNA from MDA-MB-231 cells transfected with pBABE-Smad7 or pBABE was isolated and
subjected to quantitative real time PCR analysis with specific primers for MMP9 and
normalized with GAPDH. (C) RT-PCR products of RNA samples from (B) were loaded
onto a 12% polyacrylamide gel to demonstrate the relative mRNA levels of MMP9 and
GAPDH. (D) Serum free conditioned media from (B) were collected and subjected to
gelatin zymographic assay. Different pools of cells transfected with pBABE-Smad7 were
used and showed the similar results. Results are representative of three different
experiments.
77
Figure II-6
A
B
78
Figure II-6 Cited2 enhances the expression of MMP9 promoter reporter.
(A) Schematic representation of mutant constructs in the human MMP9 promoter. The
promoter was divided into A-F subregions. 5×104 MDA-MB-231 cells were plated and
transfected with 300 ng of various deletion constructs of MMP9 promoter reporters as indicated. 24 hr after transfection, cells were treated with or without 2.5 ng/ml of TGF-β for another 24 hr, followed by luciferase assay and normalized to sea pansy luciferase activity of cotransfected pRL-SV40. (B) 5×104 MDA-MB-231 cells were plated and
cotransfected with 300 ng of wild type MMP9 wild type (black bars) or AP-1 mutant
(white bars) promoter reporter and together with pcDNA3.1-Cited2 (100 ng), Flag-
CMV2-Smad7 (100 ng), and 100 ng of dominant negative Smad4 expression vector
(DN-Smad4) in different combination. 24 hr after transfection, cells were treated with or
without 2.5 ng/ml of TGF-β for another 24 hr, followed by luciferase assay and
normalized to sea pansy luciferase activity of cotransfected pRL-SV40. Results are
representative of two independent experiments. *, p<0.05 comparing to wild type MMP9
promoter in the presence of TGF-β. **, p<0.05 comparing to wild type MMP9 promoter
with Cited2 in the presence of TGF-β.
79
Figure II-7
A
B
80
Figure II-7 Cited2 and Smad3 are recruited to the MMP9 promoter.
(A) MDA-MB-231 cells were stimulated with 2.5 ng/ml of TGF-β for indicated time points. After TGF-β stimulation, total RNA was subjected to real-time PCR with specific primers for human MMP9 and normalized with human GAPDH. (B) MDA-MB-231 cells were stimulated with 1 ng/ml of TGF-β for indicated time points, followed by chromatin immunoprecipitation with antibodies against Smad2/3, Cited2 or normal IgG. The precipitated DNA was subjected to quantitative real time PCR with primers that amplify a 196 bp product (-195~+1) covering the TGF-β responsive region in the MMP9 promoter and normalized with the input products. Results are representative of two different experiments. *, p<0.05 comparing to zero time point of TGF-β stimulation. **, p<0.05 comparing to zero time point of TGF-β stimulation.
81
Figure II-7
A
B
82
Figure II-8 Cited2 enhances TGF-β-mediated invasion of MDA-MB-231 cells.
(A) MDA-MB-231 cells were transfected with si-Cited2 or si-Control for 60 hr, followed
by TGF-β stimulation. 33 hr after TGF-β stimulation, cells were pulse labeled with
[3H]Thymidine to measure the growth rate. (B) MDA-MB-231 cells were transfected with si-Cited2 or si-Control for 60 hr, followed by TGF-β stimulation for another 24 hr.
Cells were detached and resuspended in MEM containing 0.1% BSA with antibiotics, with or without TGF-β in the absence of serum. A total of 1×105 cells were added to the
upper well of a 24-well matrigel invasion chamber (BD Biosciences) and incubated at
37°C for 20 hr. The lower chamber contained MEM with 5% FBS. Number of migrated
cells through matrigel was quantified according to in vitro invasion assay described in
Materials and Methods. Results are representative of five individual experiments. *,
p<0.5 comparing to si-Control in the presence of TGF-β.
83
Supplemental Data
A
B
84
Supplemental Data
Cited2 modulates TGF-β-mediated up-regulation of MMP13
(A) MDA-MB-231 cells were transfected with si-Cited2 or si-Control for 60 hr, followed
by TGF-β stimulation for another 24 hr. Total RNA was extracted, followed by
quantitative real time PCR with specific primers for human MMP13 and normalized with
human GAPDH. (B) Total cell lysates from (A) were harvested and subjected to Western
blot analysis with specific antibodies against MMP13, Cited2 and β-actin.
85
CHAPTER III
Posttranscriptional control of Cited2 by TGF-β:
regulation via Smads and Cited2 coding region
Most information in this chapter has been submitted to
Journal of Biological Chemistry (2006)
Introduction
Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting
transactivators with glutamic acid (E) and aspartic acid (D)-rich tail] is one of the
founding members of transcriptional activators, previously named melanocyte-specific gene-related gene (MRG)1/p35srj (Bhattacharya et al., 1999; Dunwoodie et al., 1998;
Shioda et al., 1997; Sun et al., 1998). The members in this family function as
transcriptional modulators through interaction with the p300/cAMP response element-
binding protein (CBP) complex (Braganca et al., 2002; Glenn and Maurer, 1999; Yahata
et al., 2000). Cited2 interacts with Lhx2, to enhance the recruitment of CBP/p300 and the
TATA-binding protein leading to transcription of glycoprotein hormone α subunit genes
(Glenn and Maurer, 1999). On the other hand, Cited2 competes with HIF-1α for binding
to the CH1 domain of p300, and functions as a negative modulator in hypoxia signaling
86
pathway (Bhattacharya et al., 1999). In addition, Cited2 interacts with TFAP2 (Braganca
et al., 2003), and nuclear receptors such as ER (Yahata et al., 2001) and PPAR (Tien et
al., 2004) to function as a transcriptional co-activator.
Cited2-/- mouse embryos die during mid-gestation with profound developmental
abnormalities, including cardiac malformations, exencephaly and adrenal agenesis
(Bamforth et al., 2001; Barbera et al., 2002; Yin et al., 2002). Cited2 acts upstream of
Nodal, a member in TGF-β superfamily, and is required for normal establishment of the
left-right axis (Bamforth et al., 2004; Weninger et al., 2005). Overexpression of Cited2 in
Rat1 cells results in loss of cell contact inhibition, anchorage-independent growth in soft
agar, and tumor formation in nude mice, suggesting that Cited2 is a transforming gene
(Sun et al., 1998).
Cited2 is induced by many cytokines and biological stimuli, including IL-1α, -2, -4,
-6, -9, and -11, granulocyte/macrophage colony-stimulating factor, interferon γ, platelet- derived growth factor, insulin, serum, lipopolysaccharide (Sun et al., 1998) and hypoxia
(Bhattacharya et al., 1999) in diverse cell types. Cited1 (MSG1) is downregulated by
TGF-β in B16-F1 melanoma (Shioda et al., 1998), and functions as a Smad4 interacting co-activator (Shioda et al., 1998; Yahata et al., 2000); however, the mechanism involved in the downregulation of Cited1 is not clear. It is well established that the effects of TGF- 87
β are mediated through its interaction with cell surface receptors. Smad transcription
factors are intracellular key mediators of TGF-β signaling. Receptor-activated Smad2 and
Smad3 are phosphorylated by the activated TGF-β receptor complex, forming hetero-
complexes with Smad4, a common partner in the assembly of transcriptional complexes
(Shi and Massague, 2003). These complexes then translocate into the nucleus, and
interact with a variety of transcription factors such as p300 and E2F4/5 leading to
synergistic transcriptional activation or suppression of target genes (ten Dijke and Hill,
2004; Zimmerman and Padgett, 2000). Smad7 is an inhibitory Smad induced by TGF-β and is capable of inhibiting phosphorylation of Smad2 and Smad3 by competitive interaction with the TGF-β receptor complex (Hayashi et al., 1997). In addition to direct transcriptional regulation, TGF-β stimulation also modulates gene expression posttranscriptionally by increasing the mRNA stability of ribonucleotide reductase component R2 (Amara et al., 1993), elastin (Kucich et al., 2002), and receptor for hyaluronan mediated mobility (RHAMM) (Amara et al., 1996), and destabilizing CD40 mRNA (Nguyen et al., 1998).
We have shown that basal expression of Cited2 in fibroblasts is regulated by
Sp1/Sp3 and Ets transcription factors (Han et al., 2001). Cited2 is regulated by different cytokines and stimuli; however, the mechanisms that control Cited2 gene expression are 88
only partially understood (Sun et al., 1998). Recently, through microarray analysis,
Cited2 was reported to be downregulated by TGF-β in different cell lines (Chen et al.,
2001; Kang et al., 2003; Luo et al., 2005). In the current study, we confirmed that Cited2
is downregulated in MDA-MB-231 cells, in part through the Smad pathway. Based on
nuclear run-on analysis and Cited2 promoter/reporter assay, Cited2 transcription was not
affected by TGF-β. We showed that this TGF-β mediated downregulation occurs by a
posttranscriptional mechanism involving an increase in the turnover rate of Cited2
mRNA. By stable expression of Cited2 coding sequence under the control of a
heterologous promoter, we found that the expression of endogenous and exogenous
Cited2 mRNA is downregulated by TGF-β, suggesting that Cited2 coding sequence is
sufficient for proper regulation. As it has been shown previously that both TGF-β and
Cited2 play important roles during embryonic development and tumorigenesis,
unraveling the mechanism of how Cited2 is regulated by TGF-β may contribute to understanding the effects of TGF-β signaling on development and tumor progression.
Materials and Methods
Reagents and antibodies- Recombinant human TGF-β1 was purchased from R&D
Systems (Minneapolis, MN). Recombinant human BMP2 was ordered from PeproTech 89
Inc. (Rocky Hill, NJ). Actinomycin D, puromycin, and cycloheximide were obtained from Sigma (St. Louis, MO). Anti-Cited2 (MRG1, JA22) antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Smad2, anti-phospho-
Smad3, anti-phospho-Smad1/5 antibodies were ordered from Cell Signaling Technology
(Beverly, MA). Anti-Flag (M2) and anti-β-actin antibodies were obtained from Sigma
(St. Louis, MO).
Cells- MDA-MB-231 cells were obtained from the American Type Culture
Collection. MDA-MB-231 cells were maintained in minimum essential medium supplemented with 1 nM insulin (Sigma) and 10% heat-inactivated fetal bovine serum
(HyClone).
Plasmids- Cited2 promoter-luciferase construct, -2700/+833 pXP2, was derived from -2700/+121 pXP2 (Han et al., 2001) by PCR amplification from genomic subclone
pUCHi to include the entire 5’UTR of Cited2. CMV-luciferase plasmid was obtained by
sucloning luciferase into Flag-CMV2 plasmid. Human 3’UTR of Cited2 was obtained by
PCR amplification from human genomic DNA and subcloned downstream of luciferase
in the CMV-luciferase plasmid. Human Smad7 was provided by Dr. Yan Chen (Indiana
University), followed by subcloning into Flag-CMV2 plasmid. Flag tagged Smad7 with a
consensus Kozak sequence was PCR amplified from Flag-CMV2 vector and subcloned 90
into pBABE-puro. Mouse Cited2 was cloned from mouse genomic DNA, followed by subcloning into Flag-CMV2 vector. Flag-CMV2-Cited2 was generated by subcloning
Cited2 coding sequence into Xba I and BamH I sites of Flag-CMV2 plasmid. pBABE-
Stop-Cited2 was created by cutting Flag-CMV2-Cited2 with Hind III and Xba I and filling in with Klenow fragment to create a TAG stop codon between Flag and Cited2 coding sequence, followed by subcloning the mutant into pBABE-puro.
DNA transfection, luciferase assay and, siRNA- For luciferase assay,
MDA-MB-231 cells were transfected using Lipofectamine Plus reagent (Invitrogen) following manufacturer’s instructions. Luciferase activity in the cell lysate was determined by a dual luciferase reporter assay system (Promega) and normalized to sea pansy luciferase activity of cotransfected pRL-CMV. For siRNA knockdown experiment,
MDA-MB-231 cells were transfected wth siRNA using Effectene transfection reagent
(Qiagen) for 60 hr following the manufacturer’s instructions. si-Smad2 (M-003561-00)), si-Smad3 (M-020067-00), si-Smad4 (M-003902-01), si-Cited2, and si-Control (ZNF76) are ordered from Dharmacon Research (Lafayette, CO). The siRNA sequence for Cited2 is 5’-ugacggacuucgugugcaa-3’ and for ZNF76 5’-ccagcgccaccaacuauaa-3’. The reconstitution of siRNA was performed following the manufacturer’s instructions.
91
Retrovirus infection- pBABE-Cited2 and pBABE-Smad7 were generated by inserting corresponding cDNAs into multiple cloning sites of pBABE-puro, a retrovirus vector. Phoenix packaging cells were used to generate amphotropic retroviruses. Briefly,
Phoenix cells were seeded at a density of 3.5 x 106 cells per 10-cm dish, and transfected with 10 μg of retrovirus vector by the calcium phosphate method the following day.
Forty-eight hours after transfection, virus-containing supernatant was collected. For virus infection, one day before infection, 1 x 105 cells were seeded on a 6-cm dish. Cells were incubated with virus for 24 hours, followed by antibiotic selection with 0.8 μg/ml of puromycin for MDA-MB-231 cells. Individual clones were picked after puromycin selection. The mRNA and protein expression levels in the individual clones were monitored by Northern and Western blot analysis. Stable transfectants were pooled after selection. For generation of tet-off inducible Cited2 MEF lines, a Cited2-/- MEF line infected with retrovirus expressing Cited2 was selected with 2.5 μg/ml of puromycin for one week. Individual clones were picked and Cited2 protein and mRNA expression levels were monitored 24 hours after culturing cells in 2 μg/ml of tetracycline.
Western blot analysis- Cells were lysed in RIPA buffer (50 mM Tris-HCl, pH7.4,
150 mM NaCl, 1% NP-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride,
20 μg/ml aprotinin, and 20 μg/ml leupeptin). Lysates were separated by SDS-PAGE and 92
transferred to polyvinylidene difluoride membranes (PVDF-plus; Osmonics Inc.).
Membranes were incubated with primary antibodies followed by incubation with
secondary antibodies conjugated to horseradish peroxidase. Reacted secondary antibodies
were detected using an enhanced chemiluminescence detection system (Amersham
Pharmacia Biotech).
Northern blot analysis- Total RNA was isolated from cells using Trizol reagent
(Invitrogen) following manufacturer’s instructions. 10 μg per lane of total RNA was
separated by 2.2 M formaldehyde agarose gel electrophoresis, and transferred to a nylon
membrane (Magna; Osmonics Inc.).
Nuclear run-on assay- Nuclei from approximately 3 × 107 cells were collected
and nuclear run-on assay was performed as described previously (Greenberg and Ziff,
1984). Single-stranded DNA complementary to Cited2 and junB mRNA or alkaline
denatured double-stranded cDNA of Cited2, junB, GAPDH, and 36B4, and empty
pUC19 vector were slot-blotted onto a nylon membrane (Magna; Osmonics Inc.) for
hybridization with 32P-labled run-on products. Single-stranded DNA complementary to
Cited2 or junB mRNA was prepared by asymmetric PCR amplification according to standard procedures (Gyllensten and Erlich, 1988). The membrane was then washed and exposed to x-ray film. 93
SYBER-Green real time PCR- RT PCR primer pairs are: Human Cited2 (5’-
accatcaccctgcccacc-3’ and 5’-cgtagtgtatgtgctcgccca-3’); Human GAPDH (5’-
gaaggtgaaggtcggagtc-3’ and 5’-gaagatggtgatgggatttc-3’); Human Smad2 (5’-
aggagcaggcggaggagag-3’ and 5’-gtattacagttttgagtggtgatggc-3’); Human Smad3 (5’-
aatggtgcgagaaggcgg-3 and 5’-gacctggggatggtgatgc-3’); Human Smad4 (5’-
ctacgaacgagttgtatcacctgg-3’ and 5’-acgatggctgtccctcaaagt-3’). Total RNA was isolated by
the Trizol (Invitrogen) method and reverse transcribed by SuperScriptTM First-Strand
Synthesis System for RT-PCR (Invitrogen). Real time PCR was performed with iQTM
SYBER Green Supermix (BIO-Rad) in a MyiQ thermocycler (Bio-Rad) using
SyberGreenTM detection protocol outlined by the manufacturer. All quantitations were
normalized to an endogenous control GAPDH. The relative quantitation value for each
target gene compared to the calibrator for that target is expressed as 2-(Ct-Cc) (Ct and Cc
are the mean threshold cycle differences after normalization to GAPDH).
RESULTS
TGF-β downregulates Cited2 in MDA-MB-231 cells- Chen et al. (Chen et al.,
2001) showed previously by microarray analysis that Cited2 is one of the genes
downregulated by TGF-β in both MCF-10A and MDA-MB-231 cells. We performed 94
Northern analysis to confirm the microarray data. Cited2 was downregulated by TGF-β in both cell lines (Fig. III-1A). Since the basal expression of Cited2 was high in MDA-
MB-231 cells, and the downregulation by TGF-β was easily detected, we used MDA-
MB-231 cells to study TGF-β mediated Cited2 downregulation in the following experiments. TGF-β downregulated Cited2 expression in MDA-MB-231 cells in a dose dependent manner (Fig. III-1B) and 2.5 ng/ml of TGF-β was used throughout the study.
We performed the time course experiment to measure the TGF-β effect on Cited2 mRNA
and protein levels. Both Cited2 mRNA (Fig. III-1C) and protein (Fig. III-1D) levels were
downregulated by TGF-β. These experiments confirm that Cited2 is a TGF-β responsive
gene. We also observed that 24 hours after TGF-β stimulation, phosphorylated Smad2 (p-
Smad2) and Smad3 (p-Smad3) significantly decreased (Fig. III-1D) and Cited2 mRNA
and protein levels started to recover at the same time (Fig. III-1C, III-1D), suggesting that
Smad pathway may be involved in the downregulation of Cited2.
Smad-dependent pathway is involved in TGF-β mediated downregulation of
Cited2- Smad2, Smad3, and Smad4 are key mediators in TGF-β signaling pathway.
However, certain TGF-β responsive genes, such as fibronectin and novH, are either
upregulated or downregulated through Smad-independent pathways (Hocevar et al.,
1999; Lafont et al., 2002). In order to evaluate whether the Smad pathway is involved in 95
the downregulation of Cited2 by TGF-β, an expression plasmid for Smad7, an inhibitory
Smad, was introduced into MDA-MB-231 cells through retrovirus mediated gene transfer
(Fig. III-2A). Overexpression of Smad7 in cells decreased TGF-β mediated
phosphorylation of Smad2 (Fig. III-2A) and attenuated downregulation of Cited2 mRNA
(Fig. III-2B) and protein levels (Fig. III-2A) by TGF-β, supporting that the Smad
pathway is involved in the downregulation of Cited2. To further test which Smads are
involved in the downregulation of Cited2 by TGF-β, a short interference RNA specific
for Smad2 (si-Smad2), Smad3 (si-Smad3), or Smad4 (si-Smad4) were used in MDA-
MB-231 cells. Transfection with si-Smad4, but not si-Smad2 or si-Smad3, significantly
attenuated TGF-β mediated downregulation of Cited2 mRNA (Fig. III-2C). Receptor
Smads, Smad2 and Smad3, may work individually with Smad4 or in cooperation with
each other. Cotransfection with both si-Smad2 and si-Smad3 effectively decreased TGF-
β mediated downregulation of Cited2 mRNA (Fig. III-2D), suggesting that receptor
Smad is required for the downregulation of Cited2, and Smad2 or Smad3 may
compensate each other in individual knockdown experiments. Efficiency of knocking
down specific Smad2, Smad3, or Smad4 by siRNA was further confirmed in Figure 2E.
Taken together, these data support the conclusion that Smad4 and receptor Smad, Smad2
or Smad3, are involved in TGF-β mediated downregulation of Cited2. In addition to 96
TGF-β, BMP family also executes signaling through Smad4 but in combination with
different receptor Smads, Smad1/5/8 (Ogawa et al., 2004). We further tested whether
BMP signaling downregulates Cited2. Unlike TGF-β, BMP2 did not significantly
downregulate Cited2 (Fig. III-2F), although BMP2 mediated phosphorylation of Smad1/5
was detected in MDA-MB-231 cells (Fig. III-2G). These results suggest that specific
receptor Smads are involved in the downregulation of Cited2.
TGF-β downregulates Cited2 by posttranscriptional regulation- To determine
whether changes in the Cited2 mRNA level under TGF-β stimulation are due to changes
in the rate of transcription, nuclear run-on assays were performed using nuclei isolated
from either TGF-β treated or untreated MDA-MB-231 cells. Run-on transcripts were
labeled in vitro with [32P] UTP and hybridized to denatured double-stranded or antisense
single-stranded DNA of junB and Cited2 coding sequences. In cells stimulated by TGF-
β, the transcription of junB, a TGF-β responsive gene, was enhanced, while Cited2 transcription was not affected (Fig. III-3A), indicating that TGF-β mediated
downregulation of Cited2 is not through the transcriptional control. To further confirm
nuclear run-on analysis, serial deletion mutants of Cited2 promoter/reporter were tested
by luciferase assay (Fig. III-3B). Consistent with nuclear run-on analysis, reporter
activity of Cited2 promoter-luciferase constructs was not affected by TGF-β stimulation 97
in MDA-MB-231 cells (Fig. III-3C). These experiments suggest that TGF-β mediated downregulation of Cited2 is most likely through the posttranscriptional control.
TGF-β increases the turnover rate of Cited2 mRNA in MDA-MB-231 cells- To
determine if a change in the stability of Cited2 mRNA could account for the decreased
expression seen under TGF-β stimulation, we measured the degradation rate of Cited2
mRNA using transcriptional inhibitors. Actinomycin D inhibits transcription by forming
a stable complex with double-stranded DNA, thus inhibiting DNA-RNA primed RNA
synthesis. RNA was isolated at different times after the addition of actinomycin D to
either untreated or TGF-β pretreated cells, and Cited2 mRNA levels were measured by
Northern blotting (Fig. III-4A). These data were quantified by scanning densitometry and
normalized to the signal of GAPDH, which remained constant during actinomycin D
treatment, and plotted against time in Fig. 4B. The plot was used to derive half-life of
Cited2 mRNA of 81 min in untreated cells and 34 min in TGF-β treated cells. These data
support the conclusion that TGF-β stimulation increases the degradation of Cited2
mRNA.
Untranslated regions of Cited2 mRNA are not responsible for TGF-β mediated downregulation of Cited2- Untranslated regions (UTR) are known to play crucial roles in the posttranscriptional regulation of gene expression including 98
modulation of mRNA stability (Bashirullah et al., 2001; Yang et al., 1996; Yang and
Yang, 1994). We tested whether 5’UTR of Cited2 is responsible for TGF-β mediated downregulation, using a Cited2 promoter reporter plasmid containing intact 5’UTR, -
2700/+833-Luc (Fig. III-3B). Through luciferase reporter assay, we did not observe significant TGF-β mediated changes in the luciferase activity (Fig. III-5A). Since TGF-β induced ribonucleotide reductase R2 mRNA stabilization is through its 3’UTR (Amara et al., 1995), we cloned 3’UTR of Cited2 downstream of the luciferase coding sequence to test whether 3’UTR includes cis-elements responsible for TGF-β mediated downregulation (Fig. III-3B). By both luciferase reporter assay (Fig. III-5B) and Northern analysis (Fig. III-5C), we did not observe any TGF-β mediated changes in luciferase mRNA expression. These data suggest that untranslated regions of Cited2 are not
sufficient for its decreased mRNA stability under TGF-β stimulation.
Cited2 coding sequence is necessary and sufficient for TGF-β mediated
downregulation of Cited2- Since 5’ and 3’ UTR of Cited2 transcript are not involved in
TGF-β mediated mRNA instability, we next tested whether the coding region is involved.
To do this, we expressed Cited2 coding sequence under the control of a viral promoter/enhancer (pBABE-Cited2) in MDA-MB-231 cells by stable transfection. In pBABE, a mutagenised gag cassette lacking the translation initiation codon was 99
incorporated upstream of the Cited2 coding sequence (Morgenstern and Land, 1990) to distinguish exogenous from endogenous Cited2 mRNA by probing Northern blot with a gag-specific sequence (Fig. III-6A). Stable transfectants were isolated by selection with
puromycin, and RNA was isolated from transfectants stimulated by TGF-β. By Northern
analysis with the probe specific for gag, which recognizes exogenous Cited2, and the probe for coding region of Cited2, we found that both exogenous and endogenous Cited2 mRNAs were downregulated in response to TGF-β stimulation (Fig. III-6B). In tranfectants expressing pBABE vector alone, the gag tagged transcript was not
downregulated by TGF-β (Fig. III-6C), suggesting that TGF-β mediated downregulation
of exogenous Cited2 transcript was not due to transcriptional regulation of the MLV-
LTR. This result strongly supports our earlier data that TGF-β mediated downregulation of Cited2 occurs at a posttranscriptional level, and further reveals that the coding sequence of Cited2 is necessary and sufficient for TGF-β mediated downregulation.
Interestingly, Cited1, another member of the Cited family, is also downregulated by
TGF-β in B16-F1 melanoma (Shioda et al., 1998). Since Cited proteins are highly
homologous in the C-terminus, we tested whether the C-terminal conserved region
(aa.200-269) is essential for TGF-β mediated downregulation of Cited2. Exogenous
Cited2 transcript from cells expressing Cited2 aa.1-199 mutant (Fig. III-6D) lost TGF-β 100
mediated downregulation of Cited2 mRNA (Fig. III-6E). Basal expression of both
mRNA and protein from Cited2 C-terminal deletion mutant are much higher than wild
type Cited2. Deletion of Cited2 C-terminus increased the half-life of Cited2 mRNA (Fig.
III-6F, III-G), suggesting that C-terminus of Cited2 sequence affects its stability. These
data support the conclusion that C-terminus of Cited2 coding sequence is necessary for its downregulation.
Translation of Cited2 coding sequence is involved in the downregulation of
Cited2 by TGF-β- The coding sequence of c-myc and its coupled translation are essential
for the downregulation of c-myc mRNA during myogenesis (Wisdom and Lee, 1990;
Yeilding et al., 1998). To test whether translation of the Cited2 coding sequence per se is
involved in its mRNA downregulation by TGF-β, we examined the effects of translation
inhibitors, cycloheximide and puromycin, on Cited2 mRNA stability. Previous studies
have demonstrated that these two agents inhibit protein synthesis by different
mechanisms (Edwards and Mahadevan, 1992; Hua and Hod, 1992). Cycloheximide
blocks translation elongation through direct interaction with the 60S subunit of ribosomes
and results in polysome aggregation, whereas puromycin is an aminoacyl transfer RNA analog leading to polysome dissociation. As shown in Figure III-7A and III-7B, both cycloheximide and puromycin treatment superinduced the expression of Cited2 mRNA in 101
unstimulated cells, and attenuated downregulation of Cited2 in TGF-β stimulated cells.
These data suggest that protein translation process is involved in the regulation of Cited2
expression. Protein synthesis inhibitors could potentially block translation of the Cited2
transcript or inhibit synthesis of short-lived trans factors involved in the regulation of
mRNA stability. To distinguish these two possibilities, we blocked translation of Cited2
by the insertion of a termination codon between Cited2 coding region and the upstream
Flag sequence to generate pBABE-Stop-Cited2 mutant. Flag tagged Cited2 protein
expression was not detected in cells transfected with pBABE-Stop-Cited2 (Fig. III-7C).
The exogenous Cited2 transcript was downregulated by TGF-β in MDA-MB-231 cells
transfected with pBABE-Cited2, but not in cells tranfected with pBABE-Stop-Cited2
(Fig. III-7D). These data support the conclusion that Cited2 downregulation by TGF-β is
coupled to its translation.
DISCUSSION
Microarray analysis identified novel TGF-β responsive genes; however, only a few
of these genes have been further characterized to elucidate the mechanism of TGF-β
mediated downregulation. In this study, we show that TGF-β downregulates both Cited2
mRNA and protein expression in MDA-MB-231 cells. The decrease in Cited2 mRNA 102 under TGF-β stimulation is not accompanied by a corresponding decrease in the rate of transcriptional initiation or elongation, as measured by nuclear run-on analysis and promoter/reporter assays. Instead, the downregulation correlates with an increase in the turnover rate of Cited2 mRNA as measured by actinomycin D chase experiments, supporting that TGF-β mediated regulation occurs at the posttranscriptional level through changes in mRNA stability. Previous studies have suggested the involvement of cis elements such as SBE (Smad binding element) or TIE (TGF-β inhibitory element) and trans factors such as Smad, E2F or ATF-3 in TGF-β mediated transcriptional regulation
(Chen et al., 2001; Chen et al., 2002a; Jonk et al., 1998; Kang et al., 2003).
Posttranscriptional regulation under TGF-β stimulation, however, is far less understood.
In spite of that, there is evidence that TGF-β treatment of mammalian cells significantly increases the half-life of ribonucleotide reductase R2 through its 3’UTR (Amara et al.,
1993). In the case of Cited2, 3’UTR does not respond to TGF-β mediated regulation, when linked downstream of CMV driven luciferase reporter (Fig. III-5B, III-5C). Even though typical AU rich destabilization sequences are not observed in 3’UTR of Cited2, we detect Mos polyadenylation response element (Mos-PRE) in 3’UTR. Mos polyadenylation sites in the 3’UTR of Mos mRNA is responsible for cytoplasmic polyadenylation and translational activation of Mos mRNA during progesterone 103 stimulated xenopus oocyte maturation (Charlesworth et al., 2002). Progesterone induces
Cited2 mRNA expression in mouse uterus (Jeong et al., 2005). It will be interesting to test whether the Mos-PRE in 3’UTR of Cited2 is necessary for progesterone induced mRNA expression of Cited2.
To further elucidate the mechanism of TGF-β mediated downregulation of Cited2, we have analyzed the mRNA expression of recombinant Cited2 gene in MDA-MB-231 cells. Consistent with the notion that the regulation occurs at the posttranscriptional level, we observed correct regulation of exogenous Cited2 mRNA driven by a viral promoter,
MLV-LTR, suggesting that the protein coding sequence is sufficient to confer Cited2 downregulation by TGF-β. To our knowledge, this is the first demonstration that the mRNA stability of a TGF-β downregulated gene is controlled by its coding sequence.
Coding sequences are necessary for the mRNA stability of IL-11, c-myc, c-Fos, elastin,
LH receptor, albumin, and yeast MAT1α (Kash and Menon, 1999; Parker and Jacobson,
1990; Shyu et al., 1989; Wisdom and Lee, 1990; Yang et al., 1996; Yang and Yang,
1994; Zhang et al., 1999). The stability of these and many other labile mRNAs has been shown to be coupled to translation (Beauchamp et al., 1992; Shyu et al., 1989; Wisdom and Lee, 1990; Zhang et al., 1999), and polysome-associated endonucleases may be involved in this process (Lee et al., 1998; Yang et al., 2004; Zhang et al., 1999). We have 104
previously shown that phorbal ester induced IL-11 mRNA stabilization is in part through
the IL-11 coding sequence (Yang et al., 1996; Yang and Yang, 1994). Wisdom et al.
reported that c-myc mRNA is downregulated during myogenic differentiation, and the
downregulation is mediated by accelerated mRNA decay (Wisdom and Lee, 1990).
Stable expression of exogenous c-myc driven by metallothionein promoter is also
downregulated as the endogenous c-myc mRNA by a differentiation agent, HMBA, in
mouse erythroleukemia cells (Lachman et al., 1986). A C-terminal 249-nucleotide coding
region instability determinant (CRD) is identified to be responsible for the c-myc mRNA
instability (Bernstein et al., 1992). A CRD-binding protein (CRD-BP) binds to this region
and appears to protect the c-myc mRNA from endonuclease cleavage (Bernstein et al.,
1992). It is proposed that during myoblast differentiation, RNA binding activity of CRD-
BP is decreased, which in turn accelerates the degradation rate of c-myc mRNA (Lemm
and Ross, 2002). We have observed increased stability of Cited2 mRNA once the C-
terminus of Cited2 coding region was removed (Fig. III-6D-G), suggesting that C-
terminus is essential for the downregulation and may function as a CRD of Cited2.
Whether there are trans factors binding to CRD of Cited2 to protect the degradation of
Cited2 transcript requires further study. Like Cited2 downregulation by TGF-β, translation of c-myc mRNA is also necessary for its downregulation during myogenesis 105
(Wisdom and Lee, 1990; Yeilding and Lee, 1997). Translation pausing occurs in the
CRD of c-myc mRNA due to rare codons, such as arginine (CGA) and threonine (ACA),
and generates a ribosome-deficient region downstream of the pausing site (Lemm and
Ross, 2002). The ribosome–deficient region of c-myc is exposed to endonuclease attack
unless it is shielded by the CRD-BP (Lemm and Ross, 2002). Whether Cited2 downregulation involves translation pausing in the Cited2 coding sequence deserves further investigation.
By overexpression of Smad7, an inhibitory Smad, in MDA-MB-231 cells, we showed that downregulation of Cited2 by TGF-β is attenuated (Fig. III-2A, III-2B).
Consistent with this result, when Smad4 was knocked down in MDA-MB-231 cells,
TGF-β mediated effect on Cited2 downregulation was attenuated (Fig. III-2C),
supporting that the Smad pathway is involved in the regulation of Cited2 expression.
Smad2 and Smad3 may work independently or in cooperation with each other to mediate
gene expression. In this work, we further reveal only knockdown of both Smad2 and
Smad3, but not single receptor Smad, effectively attenuated TGF-β mediated
downregulation of Cited2, suggesting that common Smad, Smad4, may work with either
Smad2 or Smad3 for the downregulation of Cited2. BMP2 also executes signaling
through Sma4, but BMP2 activates different receptor Smads, Smad1/5/8. Unlike TGF-β, 106
BMP2 does not downregulates Cited2, suggesting that downregulation of Cited2 is
mediated by specific receptor Smads. Since individual receptor Smads interact with
different transcription factors and co-activators (Miyazawa et al., 2002), it is possible that
Smad2/3 interacting proteins may determine the expression of Cited2. While Smad pathway was inhibited through overexpression of Smad7 or si-Smads, we observed increased basal expression of Cited2. Since MDA-MB-231 is a cell line with high TGF-β autocrine activity (Bandyopadhyay et al., 1999; Martinez-Carpio et al., 1999), it is likely that increased basal expression of Cited2 is due to the attenuation of TGF-β autocrine by blockade of the Smad pathway through Smad7 and si-Smads. TGF-β also induces the stabilization of elastin mRNA in lung fibroblasts through the Smad pathway and elastin coding region, although the detailed mechanism involved is not clear (Kucich et al.,
2002; Zhang et al., 1999). It is possible that the Smad pathway may change Cited2 mRNA stability through affecting the activity or expression of mRNA binding proteins, thereby, increasing the turnover rate of the Cited2 transcript.
It is interesting that Cited1, another member of the Cited family, is also downregulated by TGF-β in B16-F1 melanoma (Shioda et al., 1998). The mechanism of
Cited1 downregulation by TGF-β is unclear. In this study, we found that Cited2 is regulated by TGF-β through posttranscriptional control in MDA-MB-231 cells. Members 107 of the Cited family share C-terminal conserved region. Interestingly, deleting C-terminal conserved region of Cited2 resulted in the loss of Cited2 downregulation by TGF-β, suggesting that TGF-β may downregulate Cited1 by a similar mechanism as Cited2.
TGF-β modulates downstream signaling through regulating expression of many transcription factors such as junB, ATF3, and Id1 (Kang et al., 2003; Selvamurugan et al.,
2004b). Both Cited1 and Cited2 interact with CBP/p300 and function as a transcriptional co-activator. Cited1 interacts with Smad4 and enhances TGF-β mediated transcription
(Shioda et al., 1998), suggesting that members of Cited family may play roles in TGF-β signaling. Recently, we found that Cited2 enhances TGF-β mediated MMP9 expression in MDA-MB-231 cells, and promotes tumor cell migration in matrigel assays
(unpublished data). In addition, Cited2 enhances NGAL(24p3) expression, which is further confirmed by the fact that knockdown of Cited2 in MDA-MB-231 cells decreases
NGAL(24p3) expression (supplemental data). Both Cited2 and NGAL(24p3) expression are downregulated by TGF-β in MDA-MB-231 cells, suggesting that TGF-β modulates
NGAL(24p3) expression in part through regulating Cited2 levels. NGAL(24p3) forms complexes with MMP9 and protects MMP9 from auto-degradation (Yan et al., 2001), which in turn facilitates breast cancer progression (Fernandez et al., 2005). We have shown that Cited2 is a transforming gene and overexpression of Cited2 in Rat1 cells leads 108
to tumor formation in nude mice (Sun et al., 1998). Since TGF-β plays important roles
during tumorigenesis (Dumont and Arteaga, 2000), our current hypothesis is that through
downregulation of Cited2, TGF-β modulates MMP9 and NGAL(24p3) levels, which may
further create a specific and temporal control on downstream events during tumor
progression.
In conclusion, we demonstrate that Cited2 is downregulated by TGF-β in MDA-
MB-231 cells through the Smad pathway. The downregulation of Cited2 by TGF-β is
posttranscriptional in part through the accelerated decay of Cited2 mRNA. TGF-β
regulated Cited2 mRNA stability is coupled to its translation. We reveal that C-terminus
of Cited2 coding sequence is necessary for TGF-β mediated downregulation. Future
studies will attempt to localize and define the elements in the coding sequence targeting
Cited2 mRNA downregulation and identify trans factors that mediate its downregulation
under TGF-β stimulation.
Acknowledgments
We are grateful to Drs. Edward Stavnezer, Paul MacDonald, and Monica Montano for
helpful discussion and advice. This work was supported by grants from National Institute of Health (RO1 HL48819 and RO1 CA78433 to YCY). 109
Figure III-1
A
B
C
110
D
111
Figure III-1 TGF-β downregulates Cited2 in MDA-MB-231 cells.
(A) MCF-10A and MDA-MB-231 were treated with or without 2.5 ng/ml of TGF-β for 4
hr. Total RNA was isolated, followed by Northern analysis with specific probes for
Cited2 and GAPDH. (B) MDA-MB-231 cells were treated with indicated concentrations
of TGF-β for 4 hr. Total RNA was subjected to Northern analysis with specific probes for
Cited2 and GAPDH. The relative intensity of specific Cited2 bands was quantified with
densitometry and normalized with GAPDH. (C) MDA-MB-231 cells were incubated with
2.5 ng/ml of TGF-β for the indicated time. Total RNA was subjected to Northern analysis
with specific probes for Cited2 and GAPDH. The levels of Cited2 were normalized with
the amount of GAPDH mRNA. (D) MDA-MB-231 cells were incubated with 2.5 ng/ml of TGF-β for the indicated time. Total cell lysate was subjected to Western analysis with specific antibodies for phospho-Smad2 (p-Smad2), Phospho-Smad3 (p-Smad3), Cited2, and GAPDH. Results are representative of four individual experiments.
112
Figure III-2
A B
C
D
113
E
F
G
114
H
I
115
Figure III-2 Smad pathway is involved in the downregulation of Cited2 by TGF-β.
(A) MDA-MB-231 cells over-expressing Smad7 (MDA-MB-231 pBABE-Flag-Smad7) were generated as described in Experimental Procedures. Cells were treated with
2.5ng/ml of TGF-β for 4 hr and total cell lysates were subjected to Western blot analysis with specific antibodies against Flag, phospho-Smad2, Cited2 and β-actin. The relative intensity of specific Cited2 bands was quantified with densitometry and normalized with
β-actin. (B) MDA-MB-231 stable transfectants with pBABE or pBABE-Flag-Smad7 were incubated with 2.5 ng/ml of TGF-β for 4 hr, followed by Northern analysis with specific probes for Cited2 and GAPDH. The relative intensity of specific Cited2 bands was quantified with densitometry and normalized with GAPDH. (C) MDA-MB-231 cells were transfected with si-Smad2, si-Smad3, si-Smad4 or si-Control for 60 hr, followed by
TGF-β stimulation for 4hr. Total RNA was isolated and subjected to Northern analysis
(top) as described in (B) and quantitative real time PCR analysis (bottom) with specific
primers for Cited2. (D) MDA-MB-231 cells were transfected with si-Control or both si-
Smad2 and si-Smad3 for 60 hr, followed by TGF-β stimulation for 4hr. Total RNA was
isolated and subjected to Northern analysis (top) and real time analysis (bottom) as
described in (C). (E) Total RNA from (C) was subjected to quantitative real time PCR
analysis with specific primers for Smad2, Smad3, and Smad4. (F) MDA-MB-231 cells 116 were treated with indicated concentrations of TGF-β or BMP2 for 4 hr. Total RNA was isolated and subjected to Northern analysis with specific probes for Cited2 and GAPDH.
(G) MDA-MB-231 cells were treated with 1 ng/ml TGF-β or 100 ng/ml of BMP2 for 30 min. Total cell lysates were isolated and subjected to Western blot analysis with specific antibodies against phospho-Smad1/Smad5 (p-Smad1/5), p-Smad2, and β-actin.
117
Figure III-3
A.
B
C
118
Figure III-3 TGF-β mediated Cited2 downregulation is posttranscriptional.
(A) 3×107 MDA-MB-231 cells were stimulated with TGF-β (2.5 ng/ml) for two hours
and the nuclei were collected for the nuclear run-on assay. Membrane 1 and 2 were slot-
blotted with alkaline denatured double-stranded cDNA of Cited2, junB and GAPDH.
Membrane 3 and 4 were slot-blotted with single-stranded DNA complementary to Cited2
and junB mRNA, double-stranded cDNA of 36B4, and empty pUC19 vector. 36B4 is
human acidic ribosomal phosphoprotein PO. Membrane 2 and 4 were hybridized with
32P-labled run-on products from TGF-β stimulated MDA-MB-231 cells. (B) Schematic
expression of mouse Cited2 genomic DNA and different luciferase reporters containing
Cited2 promoter, protein coding, 5’ or 3’ untranslated region (UTR). There are two exons
in Cited2 genomic sequence. The protein coding sequence is included in exon 2. (C)
MDA-MB-231 cells were transfected with serial deletion mutants of Cited2 promoter-
luciferase constructs as described (Han et al., 2001). 24 hr after transfection, cells were
treated with or without TGF-β for 12 hr, followed by luciferase assay. The luciferase
activities presented were normalized to sea pansy luciferase activity of cotransfected
pRL-CMV. Results are representative of three independent experiments.
119
Figure III-4
A
B
120
Figure III-4 TGF-β stimulation accelerates turnover rate of Cited2 mRNA.
(A) MDA-MB-231 cells were preincubated with or without TGF-β (2.5 ng/ml) for 2 hr, followed by the addition of actinomycin D (10 μg/ml) and total cellular RNA was isolated at various time points. RNA samples were then analyzed by Northern blotting as described previously. (B) The autoradiographic signals shown above were quantitated by scanning densitometry and plotted versus time. Half-lives of Cited2 mRNA were normalized according to the signal of GAPDH. Results are representative of four separate experiments.
121
Figure III-5
A
B
C
122
Figure III-5 5’ and 3’ untranslated region of Cited2 are not responsible for TGF-β
mediated downregulation of Cited2.
(A) MDA-MB-231 cells were transfected with Cited2 promoter-luciferase construct (-
2700/+833 pXP2), which contain the entire 5’ UTR of Cited2 or reporter pXP2 alone. 24
hr after transfection, cells were treated with or without TGF-β for 10 hr, followed by
luciferase assay. The luciferase activities presented were normalized to sea pansy
luciferase activity of cotransfected pRL-CMV. (B) MDA-MB-231 cells were transfected
with CMV-luciferase plasmid fused with 3’UTR of Cited2 (CMV-Luc-3’UTR pA) or
CMV-luciferase plasmid with 3’UTR of human growth hormone (CMV-Luc-hGH pA).
24 hr after transfection, cells were treated with or without TGF-β for 10 hr, followed by
luciferase assay. The luciferase activities presented were normalized to sea pansy
luciferase activity of cotransfected pRL-CMV. (C) MDA-MB-231 cells were transfected
with CMV-luciferase plasmid fused with 3’UTR of Cited2 and β-galactosidase plasmid
as the control for transfection efficiency. 24 hr after transfection, cells were treated with
or without TGF-β for 4 hr, and total RNA samples were isolated, followed by Northern
analysis with specific probes for luciferase, Cited2, and, β-galactosidase. Results are
representative of four independent experiments.
123
Figure III-6
A
B
C
124
D E
F
G
125
Figure III-6 Cited2 coding region is essential for its downregulation by TGF-β.
(A) Schematic expression of pBABE-Cited2 plasmid shows that transcription starts from
5’LTR containing an untranslated gag sequence followed by the mouse Cited2 coding region. (B) MDA-MB-231 stable transfectants with pBABE-Cited2 were treated with 2.5 ng/ml of TGF-β for indicated times, and total RNA samples were isolated, followed by
Northern analysis. Specific probe for gag recognizes exogenous mouse Cited2 mRNA fused with gag sequence (left panel). Specific probe for Cited2 coding region recognizes both exogenous mouse Cited2 (right panel upper band) and endogenous human Cited2 mRNA (right panel lower band). (C) MDA-MB-231 stable transfectants with pBABE vector alone were treated with 2.5 ng/ml of TGF-β for indicated times, followed by the same procedure described in (B). Since expression of exogenous Cited2 is higher than endogenous Cited2, it is hard to observe endogenous Cited2 expression in (B). The film in (C) was exposed longer than (B) to demonstrate endogenous Cited2, but not gag- tagged vector, was downregulated by TGF-β. (D) Total cell lysates from MDA-MB-231 stable transfectants with pBABE-Cited2 (full length, aa. 1-269) and pBABE-Cited2 aa.1-
199 were subjected to Western blot analysis with specific antibodies against Flag and β- actin. (E) MDA-MB-231 stable transfectants with pBABE-Cited2 (full length, aa.1-269) and pBABE-Cited2 aa.1-199 were treated with or without 2.5 ng/ml of TGF-β for 4 hr. 126
Total RNA samples from MDA-MB-231 stable transfectants were isolated and subjected
to Northern analysis with specific probes for gag and GAPDH. Results are representative
of four different experiments. (F) MDA-MB-231 stable transfectants with pBABE-Cited2
(full length, aa.1-269) and pBABE-Cited2 aa.1-199 were treated with 10 μg/ml of actinomycin D for indicated times. RNA samples were then analyzed by Northern analysis. (G) The autoradiographic signals shown above were quantitated by scanning densitometry and plotted versus time. Results are representative of three separate experiments.
127
Figure 7
A
B
128
C
D
129
Figure III-7 TGF-β mediated downregulation of Cited2 depends on translation of
Cited2 coding region.
(A) MDA-MB-231 cells were pretreated with 10 μg/ml of cycloheximide or 12.5 μg/ml of puromycin for 90 min, followed by 2.5 ng/ml of TGF-β stimulation for another 2 hrs.
Total RNA was isolated and Northern analysis was performed. (B) MDA-MB-231 cells were pretreated with different concentrations of cycloheximide or puromycin as indicated for 90 min, followed by 2.5 ng/ml of TGF-β for another 2 hrs. Total RNA was isolated
and quantitative real time PCR was performed with specific primers for Cited2 and
normalized with GAPDH. (C) Total cell lysates from MDA-MB-231 cells transfected
with pBABE, pBABE-Cited2, and pBABE-Stop-Cited2 were subjected to Western blot
analysis with specific antibodies against Flag. (D) MDA-MB-231 stable transfectants with pBABE-Cited2 and pBABE-Stop-Cited2 were treated with 2.5 ng/ml of TGF-β for
4hr. Total RNA samples from MDA-MB-231 stable transfectants were isolated and
subjected to Northern analysis with specific probes for gag and GAPDH. Results are
representative of three separate experiments.
130
Supplemental Data
A
B
C
131
Supplemental Data
Cited2 and TGF-β modulate NGAL(24p3) expression.
(A) Tet-off Cited2 inducible MEFs were treated with or without 2 μg/ml of tetracycline
for 24 hr. Total RNA was isolated and subjected to Northern analysis with specific
probes for Cited2, NGAL(24p3), and GAPDH. (B) MDA-MB-231 cells were transfected
with si-Control and si-Cited2 for 60 hr. Total RNA was isolated and subjected to
quantitative real time PCR with specific primers for NGAL(24p3) and normalized with
GAPDH. (C) MDA-MB-231 cells were treated with 2.5 ng/ml of TGF-β for indicated
times. Total RNA was isolated and subjected to real time PCR analysis with specific
primers for NGAL(24p3), Cited2, and GAPDH as described in (B).
132
CHAPTER IV
Future Studies
Introduction
This chapter focuses on addressing the function of Cited2 and its regulation based on the preliminary data. As Cited2 is expressed in both normal mammary epithelial and breast cancer cells, and its expression is increased during tumor progression in MMTV- neu model, we first ask whether Cited2 plays a role in mammary gland development and breast cancer progression. We have shown that both Smad pathway and the coding region of Cited2 are required for TGF-β mediated downregulation of Cited2 through posttranscriptional control. Since MAPK pathways are highly involved in TGF-β signaling, in the second part of this chapter, we will discuss the involvement of MAPKs, cis-elements and trans- factors in TGF-β mediated downregulation of Cited2. In the final part of this chapter, we will focus on Cited2 responsive genes identified from microarray study and discuss the roles of Cited2 in gene expression and biological responses.
Results and Discussion
Cited2, Nodal, mammary gland development and tumorigenesis 133
Cited2 null embryos show abnormal heart looping and right atrial and pulmonary
isomerism with features of the left-right-patterning defect (Bamforth et al., 2004). Loss of
Cited2 causes deceased expression of Nodal, Lefty2 and Pitx2 in the lateral mesoderm and of Lefty1 in the presumptive floor plate (Bamforth et al., 2004). The mechanism of
Cited2 mediated Nodal expression has not been explored yet. Nodal belongs to TGF-β
superfamily and is an important regulator of embryonic development. Nodal induces
mesoderm and endorderm, patterns the nervous system, and determines left-right
asymmetry (Brennan et al., 2002). Nodal signaling involves the activation of the type I
receptors ALK4/5/7 and the subsequent phosphorylation and activation of the effectors
Smad2/3. Activated Smad2/3 interact with Smad4 to form complexes and bind to the
promoter regions of Nodal, Lefty1, and Lefty2 to turn on the expression of these genes in
embryonic stem cells (Besser, 2004). Expression of Nodal creates a positive feedback via
the activation of Smad2/3 to induce its own expression (Besser, 2004). Little is known
about the role of Nodal in postnatal development. However, Nodal has been recently
found to be expressed in mouse mammary gland, suggesting that Nodal may play a role
in adult tissues (Kenney et al., 2004). Since Cited2 null mouse embryos show deficient
Nodal expression (Bamforth et al., 2004), we further examine whether Cited2 regulates
Nodal expression in mammary epithelial cells. Cited1 null mice have retarded mammary 134 ductal growth at puberty and dilated ductal structures with a lack of spatial restriction of the subtending branches (Howlin et al., 2006). Nodal stimulates mammary ducutal growth and branching (Kenney et al., 2004). We have demonstrated that expression of
Cited2 is higher in invasion breast cancer MDA-MB-231 cells than normal mammary epithelial MCF-10A and non invasive breast cancer MCF-7 cells (Chapter II). It will be interesting to examine the role of Cited2 during mammary gland development and breast cancer progression.
Cited2 regulates Nodal expression in MCF-10A cells- MCF-10A is a normal mammary epithelial cell line. Since Nodal expression is detected in the mammary gland, we tested whether Nodal is expressed in MCF-10A cells, and whether Cited2 regulates Nodal expression in cells. We detected Nodal expression in MCF-10A cells through real time
PCR analysis. Knockdown of Cited2 using siRNA specific to Cited2 (si-Cited2) in MCF-
10A cells significantly downregulated Nodal mRNA expression (Fig.IV-1A), suggesting that Cited2 regulates Nodal expression in MCF-10A cells. The decreased Cited2 mRNA levels in MCF-10A transfected with si-Cited2 but not si-Control demonstrated that si-
Cited2 specifically knocked down Cited2 in MCF-10A cells (Fig.IV-1B). Both TGF-β and Nodal activate downstream signaling through phosphorylation of Smads 2 and 3. We 135
further tested whether TGF-β activates Nodal expression in MCF-10A cells. TGF-β
enhanced Nodal expression in cells transfected with si-Control, but not in cells
transfected with si-Cited2 (Fg.IV-1A), suggesting that Cited2 also modulates Smad
mediated upregulation of Nodal in MCF-10A cells.
Identification of Cited2 responsive elements in Nodal promoter- Both Smad2/3 and
FoxH1 are essential for Nodal expression during embryonic development (Besser, 2004).
In the human Nodal promoter, one classical FoxH1 binding site and two putative Smad binding elements are detected 4 kb upstream of translation start site (Fig.IV-2). FoxH1 interacts with Smad2/3 to enhance Smad DNA binding activity (Attisano et al., 2001).
Cited1 has been reported to enhance FoxH1/ Smads mediated transcription (Shioda et al.,
1998). Since Cited2 interacts with Smad2/3 (Chapter II), we hypothesize that Cited2 interacts with the FoxH1/Smads complex in the Nodal promoter to promote Nodal transcription. To test this hypothesis, Nodal promoter region containing FoxH1 and putative SBEs will be cloned into the promoterless pGL3-basic luciferase vector. Serial deletion mutants of the Nodal promoter will be constructed to map the Cited2 responsive elements. Direct binding of Cited2 and Smad2/3 to the Nodal promoter will be tested through chromatin immunoprecipitation. 136
Cited2 mediated morphogenesis and oncogenesis of MCF-10A mammary epithelial cells-
Debnath et al. have developed an in vitro system to test the biological significance of genes involved in mammary gland morphogenesis and oncogenesis (Fig.IV-3) (Debnath et al., 2003). MCF-10A mammary epithelial cells can grow on a reconstituted basement membrane and form polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Overexpression of oncogenes in MCF-
10A cells such as ErbB2 and HPV E7 has been show to result in excessive proliferative activity with irregular acinar structures once cells are cultured on basement membrane gels (Fig.IV-3) (Debnath et al., 2003). Since Cited2 regulates ErbB3 and MMP9 expression (Bamforth et al., 2001) (Chapter II), which are involved in the motility and invasion in breast cancer metastasis, we will overexpress Cited2 in MCF-10A cells, followed by 3D culture assay to test the effects of overexpressed Cited2 on acinar structures in MCF-10A cells. Since Cited2 also controls expression of Nodal, which stimulates mammary duct growth and branching, we will also knockdown Cited2 in
MCF-10A cells in the 3D culture system to study the effect of Cited2 on acini morphogenesis.
137
Tissue specific Cited2 overexpression in mouse mammary gland and conditional knockout of Cited2 in mouse mammary gland- Cited1 null mice show irregular mammary gland development (Prasad et al., 2004). To further test whether Cited2 plays a role in mammary gland development, Cited2 expression levels in mouse mammary gland during different developmental stages will be examined by immunohistochemistry or in situ hybridization. Since Cited2 knockout mice are embryonic lethal, using the conditional knockout approach to observe the role of Cited2 in vivo during mammary gland development is required. Constuction of Cited2flox/flox mice is currently underway in our
laboratory. To generate tissue-specific Cited2 knockout mice in mammary gland,
Cited2flox/flox mice will be bred with WAP-Cre or MMTV-Cre mice (less specific). Since
WAP-Cre is expressed in alveolar epithelial cells of mammary tissue during lactation but
MMTV-Cre is confined to striated ductal cells of the salivary gland and mammary
epithelial cells in virgin and lactating mice (Wagner et al., 1997), different promoter
driven Cre systems will generate varied phenotypes of Cited2 conditional knockout.
Cited2 is highly expressed in certain breast cancer cells such as MDA-MB-231 and
MDA-MB-436 cells (Chapter II) (GDS820/207980_s_at), and its expression correlates
with tumor progression in MMTV-neu mouse model (Fig.IV-4) (Landis et al., 2006)
(GDS1222/101973_at). To further test the possibility that Cited2 enhances mammary 138 tumor progression, MMTV-LTR or WAP promoter driven Cited2 transgenic mouse will be generated and studied.
p38MAPK, cis-elements, and trans-acting factors involved in TGF-β mediated downregulation of Cited2
Cited2 is downregulated by TGF-β in MDA-MB-231 cells (Chapter III). We have demonstrated that Smad pathway is involved in the downregulation of Cited2 by TGF-β.
Since MAPK pathways are also crucial for TGF-β mediated response (Derynck and
Zhang, 2003), we further tested whether MAPKs are involved in TGF-β mediated downregulation of Cited2 by different MAPK inhibitors. In Chapter III, we have demonstrated that stable expression of exogenous Cited2 coding region is sufficient for
TGF-β mediated downregulation of Cited2 in MDA-MB-231 cells. Although, we further showed that the C-terminal conserved region of Cited2 is required for Cited2 downregulation, detailed mapping of the C-terminus of Cited2 responsible for downregulation has not been performed. Our testing hypothesis is that C-terminus of
Cited2 functions a coding region instability determinant (CRD), and CRD binding protein
(CRD-BP) binds to CRD of Cited2 mRNA to protect its degradation from polysomes
139
associated ribonucleases. It will be interesting to identify mRNA binding proteins which protect Cited2 mRNA from degradation.
p38MAPK pathway regulates TGF-β mediated downregulation of Cited2—In addition to
Smad pathway, MAPK pathways are highly involved in TGF-β signaling. We tested
whether ERK pathway plays any role in the downregulation of Cited2 by TGF-β. As
shown in Figure IV-5A, 50 μM of PD98059 decreased phosphorylated levels of p44/42
MAPK; however, blockade of ERK did not attenuate Cited2 downregulation by TGF-
β (Fig.IV-5B). When pretreatment of MDA-MB-231 cells with 50 μM JNK specific
inhibitor JNK iII, Cited2 was still downregulated by TGF-β (Fig.IV-6A), although the
same concentration of JNK iII inhibited autophosphorylation of JNK stimulated by UV
(Fig.IV-6B). Phosphorylated form of JNK was not detected in the presence or absence of
TGF-β (Fig.IV-6B), indicating that JNK pathway is not activated in MDA-MB-231 cells
by TGF-β stimulation. We also tested whether p38MAPK pathway is involved in the
downregulation of Cited2. Unlike ERK and JNK, phosphorylated level of p38 was
significantly increased under TGF-β stimulation (Fig.IV-7A). SB203580, a specific p38
inhibitor, acts by binding to the ATP-binding site of p38 molecule, to inhibit the kinase
activity (Tong et al., 1997). Interestingly, pretreatment of cells with a p38MAPK specific 140
inhibitor, SB203580, abrogated TGF-β mediated downregulation of Cited2 (Fig.IV-7B),
suggesting that p38MAPK is involved in the downregulation of Cited2. PI-3 Kinase
pathway is also involved in certain TGF-β regulatory events. We examined whether PI-3
Kinase specific inhibitor, Ly294002, can block TGF-β mediated downregulation of
Cited2. Pretreatment of cells with Ly294002 did not inhibit TGF-β mediated
downregulation of Cited2. These data demonstrate that in addition to Smad dependent
pathway (Chapter III), p38MAPK pathway is also involved in TGF-β mediated
downregulation of Cited2.
Mapping cis-elements in Cited2 coding region involved in Cited2 downregulation by
TGF-β- We have demonstrated that Cited2 coding region is sufficient for TGF-β
mediated downregulation in MDA-MB-231 cells (Chapter III). Deletion of the C-
terminal conserved region of Cited2 increases mRNA half-life and attenuates TGF-β mediated downregulation of Cited2, suggesting that the C-terminus of Cited2 functions as a coding region instability determinant (CRD). Serial Cited2 deletion mutants on the C- terminal region will be constructed to further map the cis-elements responsible for Cited2 downregulation by TGF-β. We have also shown that TGF-β mediated downregulation of
Cited2 is coupled to its translation. We further hypothesize that translation pausing on 141
Cited2 CRD results in mRNA degradation by polysome associated endonucleases. In
vitro translation of β-globin chimeric transcript containing Cited2 CRD or GAPDH
coding region will be used to test this hypothesis. It is expected that translation efficiency
for chimeric transcript with Cited2 CRD is lower than the transcript with GAPDH coding
region. Detail mapping of translation inefficiency sequence in the Cited2 CRD will be
performed through in vitro translation assay. Ribosome toeprinting assays (translation-
coupled primer extension) will be used to further map the location of pause sites on the
Cited2 CRD. The principle of toeprinting is that primer extension of reverse transcription
from the target transcript will continue untill polymerase encounters the stalled ribosome,
and the size of the extension product indicates the pause site (Hartz et al., 1988; Schaefer
et al., 1989).
Determinine trans-acting factors involved in Cited2 downregulation by TGF-β- Since we have found that Cited2 C-terminus functions as a CRD, it is possible that there are trans- factors which bind Cited2 CRD to protect mRNA from digestion by endonucleases. TGF-
β stimulation may decrease the mRNA binding activity of CRD binding protein, which in turn decreases Cited2 mRNA level. To search for Cited2 CRD binding proteins, RNA gel shift mobility assay will be performed to identify proteins binding to Cited2 CRD region. 142
For UV cross linking assay, cytoplasmic extract from cells treated with or without TGF-β
will be mixed with p32 labeled CRD of Cited2, followed by UV-cross linking and gel
separation. The cDNA sequence of the Cited2 CRD binding protein will be obtained by
purification of the protein, microsequencing, and RT-PCR. UV cross linking assay will be used to test whether the mRNA binding activity of CRD binding protein correlates with TGF-β stimulation.
Genes regulated by Cited2 in mouse embryo fibroblasts
We have performed microarray study and identified several genes that are regulated by Cited2 in MEFs. We found that Cited2 modulates TGF-β mediated MMP9
and NGAL(24p3) expression (Chapter II). NGAL(24p3) forms complexes with MMP9
and protects MMP9 from autodegradation (Yan et al., 2001). Overexpression of
NGAL(24p3) in MCF-7 breast cancer cells enhances tumor growth in nude mice, and
expression of NGAL(24p3) correlates with disease state in breast cancer progression
(Fernandez et al., 2005). Through regulating MMP9 and NGAL expression, Cited2 could play a significant role during tumor progression.
In addition, we observed that Cited2 enhances tensin2 expression (Fig.IV-9).
Tensin2 is a focal adhesion molecule containing actin-binding domain, the Src homology 143
2 (SH2) domain, and the phosphotyrosine binding (PTB) domain (Chen et al., 2002b).
Overexpression of Tensin2 in NIH3T3 cells promotes cell migration on fibronectin (Chen et al., 2002b). Although expression of Cited2 enhances cell migiration (Fig.IV-10), further experiments are required to determine whether Tensin2 is involved in Cited2 mediated cell migration.
Cited2 expression also enhances IGFBP3 expression (Fig.IV-9). IGFBP-3 inhibits insulin-like growth factors (IGFs) mediated mitogenic effect by competing with IGFs to
IGF receptors (Baxter, 2001). Recently, it has also been demonstrated that IGFBP3 might induce antiapoptotic effects depending on the cellular environment (Fang et al., 2004).
We did not observe significant apoptotic effect on MEFs with overexpression of Cited2 in the medium containing 10% serum, but we did observe that once in the serum free condition, expression of Cited2 in MEFs accelerated cell death (Fig.IV-11). Whether
IGFBP3 mediates cell death in MEFs with overexpression of Cited2 in low serum condition requires further investigation.
Sprr1A expression was enhanced in the presence of Cited2 (Fig.IV-9). Sprr1A
(Small Proline-Rich protein) is one of the major structural proteins of cornified cell envelope (CE), a structure synthesized at late stages of keratinocyte differentiation
(Baxter, 2001). Neuronal Sprr1A has also been shown to promote axonal outgrowth 144
(Bonilla et al., 2002). In addition, expression of Sprr1A correlates with mammary duct
growth and has been suggested to play a role during mammary gland development
(Morris et al., 2006). Cited2 also enhances expression of Tapsin and TLE2 (Fig.IV-9).
Tapasin interacts with MHC class I molecules and enhances peptide presentation on class
I molecules (Wright et al., 2004). TLE2 (called Groucho in Drosophila) belongs to TLE
family, which constitutes a family of transcription corepressors (Chen and Courey, 2000).
TLE family proteins are involved in various developmental processes, including lateral
inhibition, segmentation, sex determination, dorsal/ventral pattern formation, terminal pattern formation, and eye development (Chen and Courey, 2000). As Cited2 null embryos show numerous phenotypes such as neural tube defects and heart malformation,
whether the deregulated expression of Sprr1A, Tapsin2 and TLE3 correlates with Cited2
mediated phenotypes requires further investigation.
145
Figure IV-1
A
B
146
Figure IV-1 Cited2 regulates Nodal expression in MCF-10A cells.
MCF-10A cells were transfected with si-Cited2 or si-Control for 48 hr, followed by TGF-
β (2.5 ng/ml) stimulation for another 24 hr. Total RNA was extracted, followed by quantitative real time PCR with specific primers for human Nodal (A), human Cited2 (B) and normalized with human GAPDH.
147
Figure IV-2
148
Figure IV-2 Human Nodal promoter.
One classical FoxH1 binding site and two putative Smad binding elements are detected 4 kb upstream of translation start site.
149
Figure IV-3
150
Figure IV-3 Mammary gland structure and growth of MCF-10A acini in three dimensional cultures.
(A) Lobular structure of mammary gand. (B) Growth of normal or oncogene overexpressed MCF-10A acini in three dimentianl cultures (adopted from Debnath et al.
Methods. 2003 Jul;30(3):256-68).
151
Figure IV-4
152
Figure IV-4 Cited2 expression during ErbB2/neu-induced tumorigenesis.
Microarray analysis from Lendis et al. shows that Cited2 expression correlates with
tumor progression in MMTV-neu model. Activated neu allele is under the control of
mammary tumor virus promoter to enable mammary specific expression. To identify
early events of erbB2/neu-induced tumorigenesis, microarray analysis are performed with
RNA from tumors and preneoplastic mammary glands, which are compared with age- matched, wild-type control mammary glands (Landis et al., 2006).
.
153
Figure IV-5
A
B
154
Figure IV-5 ERK pathway is not involved in TGF-β mediated downregulation of
Cited2.
(A) MDA-MB-231 cells were pretreated with 50 μM of PD98059 or DMSO for 1 hour, followed by incubation of cells with 2.5 ng/ml of TGF-β for another 4 hours. The cell
lysates were subjected to Western blot analysis with antibodies against phosphorylated
forms of p42/44 MAPK and β-actin. (B) Total RNA from (A) was isolated and subjected
to Northern analysis with specific probes for Cited2 and GAPDH.
155
Figure IV-6
A
B
156
Figure IV-6 JNK pathway is not activated by TGF-β in MDA-MB-231 cells.
(A) MDA-MB-231 cells were pretreated with 50 μM of JNKiII or DMSO for 1 hour followed by incubation with 2.5 ng/ml of TGF-β for another 4 hours. Total RNA was isolated by Trizol and subjected to Northern analysis with specific probes for Cited2 and
GAPDH. (B) MDA-MB-231 cells were pretreated with 50 μM of JNKiII or DMSO for 1 hour followed by incubation with 2.5 ng/ml of TGF-β for 4 hours or UV stimulation for
30 minutes. Total cell lysates were subjected to Western blot analysis with antibodies against phosphorylated forms of JNK and β-actin.
157
Figure IV-7
A
B
158
Figure IV-7 p38MAPK is required for Cited2 downregulation by TGF-β.
(A) MDA-MB-231 cells were pretreated with 50 μM of SB203589 or DMSO for 1 hour, followed by incubation of cells with 2.5 ng/ml of TGF-β for another 4 hours. Total cell
lysates were harvested and subjected to Western blot analysis with antibodies against
phospho-p38MAPK and β-actin. (B) Total RNA from (A) was isolated by Trizol and subjected to Northern analysis with specific probes for Cited2 and GAPDH.
159
Figure IV-8
A
B
160
Figure IV-8 PI-3 Kinase pathway is not essential for Cited2 downregulation by
TGF-β.
(A) MDA-MB-231 cells were pretreated with 50 μM of Ly294002 or DMSO 1 hour followed by incubation with 2.5 ng/ml of TGF-β for another 4 hours. Total RNA was isolated by Trizol and subjected to Northern analysis with specific probes for Cited2 and
GAPDH. (B) MDA-MB-231 cells were pretreated with 50μM of Ly294002 or DMSO for
1 hour followed by incubation with 2.5 ng/ml of TGF-β for another 4 hours. Total cell lysates were subjected to Western blot analysis with antibodies against phosphorylated forms of AKT and β-actin.
161
Figure IV-9
A
B Cited2 inducible MEFs Cited2 on Cited2 off Tapasin (TAPBP) 2 1 TLE2 2 1 Tensin2 2.2 1 NGAL(24p3) 2.5 1 Sprr1A 3 1 IGFBP3 5 1
162
Figure IV-9 Cited2 regulated genes.
(A) Tet-off inducible Cited2 MEFs were generated by retrovirus infection of Cited2-/-
MEFs with pBPSTR1-Cited2. Tet-off inducible Cited2 MEFs were stimulated with or without tetracycline for 24 hours, followed by adding 2.5 ng/ml of TGF-β or reconstitution buffer for another 4 hours. Cited2, NGAL(24p3), IGFBP3, Sprr1A and
GAPDH mRNA expression levels were monitored by Northern analysis. (B) Total RNA from (A) were isolated and subjected to real time RT- PCR analysis with specific primers for EGLN3, Tapasin, TLE2 and Tensin2 and normalized with β-actin. Relative expression levels of NGAL(24p3), IGFBP3, and Sprr1A were quantified by densitometry and normalized to GAPDH levels.
163
Figure IV-10
A
-Tet (Cited2 on) +Tet (Cited2 off)
B
164
Figure IV-10 Cited2 enhances migration of MEFs.
(A) Tet-off inducible Cited2 MEFs were stimulated with or without tetracycline for 24
hours. Cells were trypsinized and seeded in serum-free medium containing 0.1% bovine
serum albumin onto the upper surface of the filters and allowed to migrate toward 10% of
FBS. After 8 hour incubation period, the cells that had migrated to the underside of the filter were fixed, stained with 0.5% crystal violet. (B) Cells that had migrated to the underside of the filter were counted by brightfield microscopy at ×200 in five random
fields.
165
Figure IV-11
-Tet (Cited2 on) +Tet (Cited2 off)
166
Figure IV-11 Expression of Cited2 affects cell growth in serum free medium.
Tet-off inducible Cited2 MEFs were stimulated with or without tetracycline in 10% FBS.
24 hours after incubation, cells were washed with PBS twice and regrow in serum free
medium for another 24 hours.
167
Summary
TGF-β signaling plays an important role during embryonic development and
tumor progression. In this thesis, we have revealed that Cited2 functions as a Smad
interacting transcriptional modulator. As Cited2 interacts with Smads 2 and 3, and p300,
Cited2 functions a transcriptional co-activator in the Smads/p300 complex to enhance
TGF-β mediated transcription of genes such as MMP9 and MMP13. Cited2 is highly
expressed in invasive breast cancer MDA-MB-231, but not in normal epithelial MCF-
10A and non-invasive MCF-7 cells. Knockdown of Cited2 in MDA-MB-231 cells
attenuates cell migration in matrigel assays. We have previously demonstrated that
overexpression of Cited2 in Rat1 cells causes anchorage independent cell growth in soft
agar and promotes tumor formation in nude mice. In addition, Cited2 expression highly
correlates with tumor progression in MMTV-neu transgenic model. These data suggest
that Cited2 could play a significant role during breast cancer progression. As MMP9 and
MMP13 are also highly involved in tumor progression, including stimulation of cell
migration, proliferation, and modulation of angiogenesis, this thesis provides in vitro mechanism for roles of Cited2 in tumorigenesis.
From knockout mouse studies, Cited2 regulates Nodal signaling and controls left/right pattering during embryonic development. As Nodal belongs to the TGF-β 168 superfamily and signals through Smad2 and 3 proteins, the role of Cited2 in TGF-
β/Nodal signaling becomes more intriguing. Nodal is expressed in mouse mammary gland and Nodal stimulation enhances mammary duct branching and affects ductal direction, which suggesting that Nodal could function as a morphogen-like molecule during mammary gland development. We also revealed that Cited2 regulates Nodal expression in normal mammary epithelial MCF-10A cells. The role of Cited2 in mammary gland development will need to be further investigated.
Although Cited2 is a transcriptional co-activator in TGF-β signaling, Cited2 expression is regulated by TGF-β. Cited2 is downregulated by TGF-β through posttranscriptional control. Knockdown of Smad4 or overexpression of Smad7 attenuates
TGF-β mediated downregulation of Cited2, suggesting that Smad pathway is involved in the downregulation. We further revealed it is Smad2/3-TGF-β, but not Smad1/5/8-BMP signaling, that downregulates Cited2. In addition, by using MAPK inhibitors, we demonstrated that p38MAPK is required for the downregulation of Cited2 by TGF-β.
TGF-β stimulation increases Cited2 mRNA turnover rate. Stable transfectants expressing exogenous Cited2 mRNA driven by a viral promoter, MLV-LTR, respond to TGF-β mediated downregulation, suggesting that coding region of Cited2 is essential for the downregulation. We also found that C-terminal conserved region of Cited2 is required for 169
Cited2 downregulation by TGF-β and functions as a coding region instability determinant
(CRD). By using protein synthesis inhibitors and inserting a translation termination
codon upstream of the Cited2 coding sequence, we found that Cited2 downregulation by
TGF-β is coupled to its own translation. c-Myc is downregulated during myoblast differentiation or by treatment of HMBA, a differentiation agent. A C-terminal 249- nucleotide coding region instability determinant (CRD) is identified to be responsible for the c-Myc mRNA instability (Bernstein et al., 1992). Based on c-Myc downregulation model, we hypothesize that Cited2 mRNA is protected by CRD binding protein (CRD-
BP), which interacts with the C-terminus of Cited2 mRNA to prevent degradation from polysome-associated endonucleases during translation process (Fig.S-1). TGF-β stimulation activates both Smad and p38MAPK pathways to affect expression or mRNA binding activity of CRD-BP; thereby, Cited2 mRNA turnover is increased (Fig.S-1). To solve the puzzle of Cited2 downregulation by TGF-β, several questions remain to be answered. Whether there are mRNA binding proteins which interact with the C-terminus of Cited2 to stabilize Cited2 mRNA needs to be further investigated. How TGF-β signaling through Smad and p38MAP pathways affects Cited2 mRNA stability and what is the connection between CRD-BP and Smad or p38MAPK pathway will require further study. 170
It is interesting that Cited2 as a transcription co-activator for Smad signaling is
regulated by TGF-β pathway. TGF-β pathway contains several regulatory feedback
mechanisms. For example, SnoN, a Smad binding co-repressor, is rapidly degraded in response to TGF-β. At later stages of TGF-β stimulation, TGF-β induces SnoN expression, resulting in termination of Smad-mediated transactivation (Stroschein et al.,
1999; Sun et al., 1999). Several TGF-β-Smad target genes are themselves Smad- interacting factors and generate so called “self-enabling” TGF-β transcriptional responses
(Kang et al., 2003). Such self-enabling responses provide a mechanism of maintaining specific Smad responses over extended periods. For example, TGF-β mediated repression
of Id1 is through both Smad3/4 and ATF-3 binding to the Id1 promoter (Kang et al.,
2003). A TGF-β activated Smad3/4 complex binds to the Id1 promoter, which initiates a burst of transcriptional activation. At the meantime, other Smad3/4 complexes bind to
ATF-3 promoter to induce expression of ATF-3, which is a transcriptional repressor. As it accumulates, ATF-3 becomes recruited by Smad3/4 complex to Id1 promoter, causing
Id1 repression (Kang et al., 2003). By requiring the sequential activation of two
transcription factors-Smad and ATF3-and coupling the activation of one to that of the
other, this mechanism provides a tight transcriptional control. Another example of “self- enabling” TGF-β regulation is MMP13 upregulation through interaction between Smad3 171
and junB. Under TGF-β stimulation, junB expression is transiently enhanced through
Smad complexes binding to the junB promoter. The accumulated junB together with
Smad3 become recruited to MMP13 promoter and enhance MMP13 expression.
Similarly, TGF-β upregulates MMP9 through the Smad pathway (Chapter II) and overexpression of junB enhances MMP9 expression (Robinson et al., 2001). However, this MMP13 upregulation model is not supported by the data that junB expression and
Smad3 phosphorylation are transient TGF-β responses, which happens in one hour.
However, significant upregulation of both MMP9 and MMP13 do not occur untill 24 hours after TGF-β stimulation (Selvamurugan et al., 2002; Selvamurugan et al., 2004a;
Selvamurugan et al., 2004b). In this thesis, we reported that Cited2 enhances TGF-β mediated upregulation of MMP13 and MMP9. Cited2 is downregulated by TGF-β and resumes 24 hours later. We also show that 24 hour after TGF-β stimulation Cited2 is significantly recruited to the MMP9 promoter, which causes enhanced MMP9 expression.
Since we have shown that Cited2 interacts with Smad2/3 and enhances Smad2/3 mediated transactivation, Cited2 may modulate Smad-JunB mediated “self-enabling”
MMP9 and MMP13 upregulation in MDA-MB-231 cells.
MDA-MB-231 is a cell line with high TGF-β autocine responses
(Bandyopadhyay et al., 1999) and phosphorylated Smads 2 and 3 are detected in cells 172
without treatment of exogenous TGF-β (Chapter III). We propose a “self-balancing”
model that under autocrine signaling, low levels of TGF-β may induce Cited2 through the
binding of the Smad complex to the MMP9 promoter, which causes basal expression of
MMP9 (Fig.S-2). Upon exogenous treatment of TGF-β, Cited2 is downregulated through
the Smad pathway while junB is upregulated (Fig.S-2). The downregulation of Cited2
attenuates the transactivation of the Smad-junB complexes on the MMP9 promoter
(Fig.S-2). Twenty four hours after stimulation, Cited2 level starts to recover and interacts
with the Smad-junB complex on the MMP9 promoter to effectively enhance MMP9
expression (Fig.S-2). As expression levels of junB and phosphorylated Smads decline at
later time point of TGF-β stimulation, MMP9 expression also decreases. TGF-β signaling
through the Smad pathway delicately balances the levels of Cited2, Smad and junB,
leading to well controlled expression of MMP9. More intriguingly, MMP9 has been
shown to convert latent to active form of TGF-β and amplify TGF-β signaling (Yu and
Stamenkovic, 2000). As TGF-β modulates Cited2 expression, downregulation of Cited2
may represent a negative feedback control to attenuate signaling from the positive
feedback loop of MMP9-TGF-β. In addition, as Cited2 expression resumes at later time
points of TGF-β stimulation to regulate MMP9 expression, TGF-β specifically and
temporally controls the downstream signaling network. 173
Results from this thesis research provide new knowledge about the role of Cited2 in TGF-β signaling and the mechanism how it is regulated by TGF-β. The data from this thesis research also suggest that Cited2 could participate in tumor progression. As Cited2 modulates MMP9 and MMP13 expression, which are highly involved in tumorigenesis, our research proposes that Cited2 could also be a potential anticancer target.
174
Figure S-1
175
Figure S-1 Proposed model of TGF-β mediated downregulation of Cited2
Cited2 mRNA is protected by CRD binding protein (CRD-BP), which interacts with the
C-terminus of Cited2 mRNA to prevent degradation from polysome-associated endonucleases during translation process. Upon TGF-β stimulation, through both Smad and p38MAPK pathways, CDR-BP dissociates from Cited2 mRNA. Cited2 mRNA is degraded by polysome associated endonucleases while Cited2 coupled translation is processing.
176
Figure S-2
177
Figure S-2 Proposed self-balancing model of MMP9 upregulation by TGF-β
In MDA-MB-231 cells, TGF-β autocrine signaling maintains basal MMP9 expression through the interaction between Cited2 and the Smad complex on MMP9 promoter.
Under exogenous treatment of TGF-β, junB expression is induced and accumulated junB becomes recruited through the Smad complex to the MMP9 promoter. At the meantime,
Cited2 is downregulated, which leads the attenuation of transactiavtion of the Smad complex on MMP9 promoter. 24 hours after TGF-β stimulation, Cited2 level resumes and is recruited to the Smad-junB complex to effectively enhance MMP9 expression.
178
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