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J. Biol. Chem., Vol. 283, Issue 20, 13834-13841, May 16, 2008

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Transforming Growth Factor-β-stimulated Endocardial Cell Transformation Is Dependent on Par6c Regulation of RhoA^*

Todd A. Townsend¹, Jeffrey L. Wrana, George E. Davis^¶, and Joey V. Barnett, Supported by NIH Grant HL52922²

From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, the Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada M56 1X5, and the ^¶Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri 65212

Valvular heart disease due to congenital abnormalities or pathology is a major cause of mortality and morbidity. Understanding the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. In the developing heart, epithelial-mesenchymal transformation (EMT) in a subpopulation of endocardial cells in the atrioventricular cushion (AVC) is an important step in valve formation. Transforming growth factor-β (TGFβ) has been shown to be an important regulator of AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Recently Par6c (Par6) has been shown to function downstream of TGFβ to recruit Smurf1, an E3 ubiquitin ligase, which targets RhoA for degradation to control apical-basal polarity and tight junction dissolution. We tested the hypothesis that Par6 functions in a pathway that regulates endocardial cell EMT. Here we show that the Type I TGFβ receptor ALK5 is required for endocardial cell EMT. Overexpression of dominant negative Par6 inhibits EMT in AVC endocardial cells, whereas overexpression of wild-type Par6 in normally non-transforming ventricular endocardial cells results in EMT. Overexpression of Smurf1 in ventricular endocardial cells induces EMT. Decreasing RhoA activity using dominant negative RhoA or small interfering RNA in ventricular endocardial cells also increases EMT, whereas overexpression of constitutively active RhoA in AVC endothelial cells blocks EMT. Manipulation of Rac1 or Cdc42 activity is without effect. These data demonstrate a functional role for Par6/Smurf1/RhoA in regulating EMT in endocardial cells.

Received for publication, December 31, 2007 , and in revised form, March 13, 2008.

^* This work was supported, in whole or in part, by National Institute of Health grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Figs. S1 and S2, and Table S1.

¹ Supported by National Institutes of Health (NIH) Grant GM007628.

² To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Rm. 476 RRB, 2220 Pierce Ave., Nashville, TN 37232-6600. Tel.: 615-936-1722; Fax: 615-343-6532; E-mail: joey.barnett{at}vanderbilt.edu.