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Originally published In Press as doi:10.1074/jbc.M009534200 on February 2, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15362-15368, May 4, 2001
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The Activity of Guanine Exchange Factor NET1 Is Essential for Transforming Growth Factor-beta -mediated Stress Fiber Formation*

Xing Shen, Jianming Li, Patrick Pei-chih Hu, David Waddell, Ji Zhang, and Xiao-Fan WangDagger

From the Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

To examine signaling pathways underlying transforming growth factor-beta (TGF-beta )-mediated changes in cell morphology, we used a microarray system to identify downstream target genes that may play a role in this process. Through this approach, we found that the NET1 gene was induced upon TGF-beta treatment in several cell types. NET1 is a guanine nucleotide exchange factor for RhoA whose activity has been implicated in stress fiber formation. In the Swiss 3T3 cell line, TGF-beta induces NET1 expression, and this correlated with an increase in stress fiber formation. Overexpression of the wild type NET1 gene increases stress fiber formation, and overexpression of a dominant negative NET1 mutant (L392E) prevented TGF-beta dependent increase in stress fiber formation. Furthermore, treatment of the cells with a RhoA kinase inhibitor Y-27632 blocks TGF-beta -induced stress fiber formation. By using a stable cell line expressing dominant negative Smad3, we found that the Smad signaling pathway is essential for the induction of NET1, which in turn leads to the increase of Rho activity. Taken together, those data suggest that induction of NET1 is important for the increase of Rho activity upon TGF-beta treatment, which may represent the critical trigger for a variety of downstream events in different cells. Our results support the presence of a novel signaling pathway by which TGF-beta may regulate the formation of stress fibers and reorganization of cytoskeletal structures.

* This work was supported by National Institutes of Health Grants DK45746 and CA75368 (to X. F. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710. Tel.: 919-681-4861; Fax: 919-681-7152; E-mail: wang@galactose.mc.duke.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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