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E-Cadherin Antagonizes Transforming Growth Factor B1 Gene Induction in Hepatic Stellate Cells by Inhibiting Rhoa–Dependent Smad3 Phosphorylation

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E-Cadherin Antagonizes Transforming Growth Factor B1 Gene Induction in Hepatic Stellate Cells by Inhibiting Rhoa–Dependent Smad3 Phosphorylation E-Cadherin Antagonizes Transforming Growth Factor b1 Gene Induction in Hepatic Stellate Cells by Inhibiting RhoA–Dependent Smad3 Phosphorylation Il Je Cho,1,2* Young Woo Kim,1* Chang Yeob Han,1 Eun Hyun Kim,3 Richard A. Anderson,4 Young Sok Lee,5 Chang Ho Lee,3 Se Jin Hwang,3 and Sang Geon Kim1 Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fi- brosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibi- tory role of ECAD in hepatic stellate cell activation. Because of ECAD’s potential for in- hibiting the induction of transforming growth factor b1(TGFb1), we investigated whether ECAD overexpression prevents TGFb1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased a-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFb1 gene and its downstream genes. ECAD overexpression decreased Smad3 phos- phorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFb1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the abil- ity of TGFb1 to elicit its own gene induction. Consistently, transfection with a constitu- tively active mutant of RhoA reversed the inhibition of TGFb1-inducible or Smad3- inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFb1 repres- sion. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFb1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Conclusion: Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFb1 and its target genes, and facilitates liver fibrosis. (HEPATOLOGY 2010;52:2053-2064) -cadherin (ECAD), a transmembrane glycopro- responsible for binding cells to neighboring ones and tein that mediates adherens junctions, is devel- maintaining the structural integrity and polarization of opmentally restricted to polarized epithelial epithelia. ECAD also regulates signaling pathways E1,2 cells. Repeated extracellular domains of ECAD are through the intracellular catenin (ctn) binding Abbreviations: aSMA, a-smooth muscle actin; BMP, bone morphogenetic protein; CA-RhoA, constitutively active mutant of ras homolog gene family A; COL1A, collagen type IA; ctn, catenin; DMN, dimethylnitrosamine; DN-RhoA, dominant negative mutant of ras homolog gene family A; ECAD, E-cadherin; ECDT, C-terminal intracellular domain of E-cadherin; ECM, extracellular matrix; EGF, endothelial growth factor; EMT, epithelial-mesenchymal transition; GFAP, glial fibrillar acidic protein; GFP, green fluorescence protein; GTPase, guanosine triphosphatase; H&E, hematoxylin and eosin; HSC, hepatic stellate cell; IB, immunoblotting; Id, inhibitor of DNA binding; IgG, immunoglobulin G; IP,immunoprecipitation; K18, cytokeratin 18; MEF, murine embryonic fibroblast; MMP,matrix metalloproteinase; mRNA, messenger RNA; NCAD, N-cadherin; NS, not significant; PAI-1, plasminogen activator inhibitor 1; PCR, polymerase chain reaction; RAC, ras-related C3 botulinum toxin substrate; RhoA, ras homolog gene family; SBE, Smad binding element; siRNA, small interfering RNA; TGFb, transforming growth factor b. From the 1Innovative Drug Research Center for Metabolic and Inflammatory Disease, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Korea; 2College of Oriental Medicine, Daegu Haany University, Daegu, Korea; 3College of Medicine, Hanyang University, Seoul, Korea; 4Department of Pharmacology, University of Wisconsin Medical School, Madison, WI; and 5Department of Internal Medicine, Catholic University of Korea, Seoul, Korea. Received February 10, 2010; accepted August 10, 2010. This work was supported by the World Class University project (which is funded by the Korean Ministry of Education, Science, and Technology) through grant R32-2008-000-10098-0 and by the National Research Foundation of Korea (which is also funded by the Korean Ministry of Education, Science, and Technology) through grant R11-2007-107-00000-0. Il Je Cho was supported by the Korea Research Foundation (which is funded by the Korean Ministry of Education and Human Resources Development) through grant KRF-2007-355-E00007. *These authors contributed equally to this work. 2053 2054 CHO ET AL. HEPATOLOGY, December 2010 domains.1,2 Cadherin switching is a characteristic moves to the nucleus, in which it promotes the tran- behavior of the process of epithelial-mesenchymal tran- scription of target genes through interactions with spe- sition (EMT). An important phenotypic change in cific Smad binding elements (SBEs; also called the cadherin switching is the loss of ECAD expression. CAGA box).10 It has been reported that single or mul- The loss of ECAD causes cells to dissociate from their tiple copies of SBEs are located in the upstream neighbors and results in a loss of cell polarity. This, in regions of TGFb1’s target genes, such as plasminogen turn, leads to the activation of cell signaling pathways activator inhibitor 1 (PAI-1), matrix metalloproteinases that regulate the mesenchymal transition. On the con- (MMPs), and collagen type I.11,12 trary, an increase in ECAD expression inhibits cell Despite the finding that TGFb1 leads to HSC acti- transformation and tumor cell invasion in an adhe- vation with a phenotypic change of ECAD loss and sion-independent manner.3,4 causes hepatic ECM accumulation, it has not yet been Myofibroblasts play a key role in wound healing and determined whether ECAD overexpression inhibits the pathological organ remodeling.5 The most accepted expression of TGFb1 and its downstream target genes. myofibroblast progenitors in the liver are hepatic stel- We investigated whether ECAD negatively regulates late cells (HSCs),5,6 although various other resident TGFb1 expression; we also examined what the molec- cells are recognized as sources of liver myofibroblasts.5 ular basis could be. Our findings demonstrate tran- As HSCs activate, the level of ECAD expression scriptional repression of the TGFb1 gene by ECAD. decreases.7 Activated HSCs then promote the synthesis Thus, the loss of ECAD initiates TGFb1 induction and deposition of the extracellular matrix (ECM) com- and consequently promotes the expression of genes for ponent and the induction of a-smooth muscle actin ECM accumulation. Moreover, the results of this study (aSMA). In addition, multiple signaling cascades accel- led to the identification of the p120-ctn binding do- erate the growth of activated HSCs6 and contribute to main of ECAD as the site required for complex forma- the development of liver fibrosis. Although the link tion with p120-ctn, which recruits ras homolog gene between cadherin switching and the EMT process in family A (RhoA); this results in the inhibition of HSCs has been studied,7,8 it is yet unclear whether RhoA activity. The data presented here support the ECAD affects the activation of HSCs. Moreover, the ability of ECAD to hinder RhoA activity, which is potential signaling and molecular regulatory mecha- critical for the Smad signaling pathway. nism by which ECAD antagonizes profibrogenic gene expression in quiescent HSCs has not been explored. Materials and Methods Several lines of evidence indicate that transforming growth factor b1(TGFb1) from autocrine or paracrine Materials. Information on the materials used in sources plays a role in activating HSCs and increasing this study is given in the Materials and Methods sec- the synthesis of ECM proteins and cellular receptors tion of the supporting information. for various matrix proteins.6 TGFb1 is regulated tran- Animal Treatment. Animal experiments were con- scriptionally by transcription factors and posttransla- ducted under the guidelines of the institutional animal use tionally by the maturation of the precursors.6 In and care committee at Seoul National University. Male response to TGFb1, type I and II TGFb1 receptors Sprague-Dawley rats at 6 weeks of age (140-160 g) were 13 form a complex and induce receptor autophosphoryla- used for liver fibrosis induction as described previously. tion. TGFb1 is also known as a cytokine that induces Patient Samples. Human liver tissues with fibrosis EMT, which inhibits ECAD expression by up-regulat- were obtained from 81 patients who had been diag- ing transcriptional repressors such as Snail, Zeb, and nosed with liver fibrosis or liver cirrhosis by histologi- Twist.9 Activated TGFb1 receptors transmit the signal cal examination and ultrasonography in seven different by which regulatory Smad molecules (Smad3/2) are hospitals in South Korea.14,15 This human investiga- phosphorylated and form an active complex with co- tion was performed after approval by the institutional Smad (Smad4). The transcription factor complex then review board. Address reprint requests to: Sang Geon Kim, Ph.D., College of Pharmacy, Seoul National University, Sillim-Dong, Kwanak-Gu, Seoul, Korea 151-742. E-mail: [email protected]; fax: þ822-872-1795. Copyright
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