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 LHX2 LIM-homeobox gene 2 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).