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Signaling and Regulation

Raf and RhoA Cooperate to Transform Intestinal Epithelial Cells and Induce Growth Resistance to Transforming Growth Factor ß¹

¹ Department of Pediatrics, Center for Cell and Vascular Biology, Columbus Children's Research Institute and Ohio State College of Medicine and Public Health, Columbus, Ohio and ² Division of Gastroenterology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee

Requests for reprints: John Barnard, Columbus Children's Research Institute, W502, 700 Children's Drive, Columbus, OH 43205. Phone: (614) 722-2753; Fax: (614) 722-3273. E-mail: barnardj{at}pediatrics.ohio-state.edu

Although unregulated activation of the Ras/Raf/mitogen-activated protein kinase kinase/Erk signaling pathway is believed to be a central mechanism by which many cell types undergo oncogenic transformation, recent studies indicate that activation of Raf kinase by oncogenic Ras is not sufficient to cause tumorigenic transformation in intestinal epithelial cells. Thus, identification of signaling proteins and pathways that interact with Raf to transform intestinal epithelial cells may be critical for understanding aberrant growth control in the intestinal epithelium. Functional interactions between Raf and the small GTPase RhoA were studied in RIE-1 cells overexpressing both activated Raf(22W) and activated RhoA(63L). Double transfectants were morphologically transformed, formed colonies in soft agar, grew in nude mice, overexpressed cyclin D1 and cyclooxygenase-2 (COX-2), and were resistant to growth inhibition by transforming growth factor (TGF) ß. RIE-Raf and RIE-RhoA single transfectants showed none of these characteristics. Expression of a dominant-negative RhoA(N19) construct in RIE-Ras(12V) cells was associated with markedly reduced COX-2 mRNA, COX-2 protein, and prostaglandin E₂ levels when compared with RIE-Ras(12V) cells transfected with vector alone. However, no change in transformed morphology, growth in soft agar, cyclin D1 expression, TGF expression, or TGFß sensitivity was observed. In summary, coexpression of activated Raf and RhoA induces transformation and TGFß resistance in intestinal epithelial cells. Although blockade of RhoA signaling reverses certain well-described characteristics of RIE-Ras cells, it is insufficient to reverse the transformed phenotype and restore TGFß sensitivity. Blockade of additional Rho family members or alternate Ras effector pathways may be necessary to fully reverse the Ras phenotype.

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