Induced Expression of Rnd3 Is Associated with Transformation of Polarized Epithelial Cells by the Raf–MEK–Extracellular Signal-Regulated Kinase Pathway

Abstract

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf–MEK–extracellular signal-regulated kinase pathway in epithelial oncogenesis.

ACKNOWLEDGMENTS

We are grateful to Patrice Bouquet (INSERM, Nice, France) for purified CNF1, Channing Der (University of North Carolina at Chapel Hill) for RhoA expression vectors, and Karl Matlin for MAbs against the 58- and 114-kDa marker proteins as well as MDCK cells expressing K-Ras. We also acknowledge Shubha Bagrodia and Richard A. Cerione (Cornell University, Ithaca, N.Y.), and Xiang-Dong Ren and Martin A. Schwartz (University of California, San Diego) for generously providing reagents and advice for the Pak3 CRIB and rhotekin RBD assays, respectively. In addition, we thank Yen Hoang Nguyen and Tyler Rootlieb for excellent technical assistance and David Dankort (UCSF Cancer Research Institute) for critical reading of the manuscript.

S.H.H. is the recipient of a senior research fellowship from the Weimann Foundation, Denmark, a grant from the Novo Nordic Foundation, and an award from the Danish Medical Research Council. This investigation was supported by a fellowship from the California Division of the American Cancer Society (2-7-99 to M.M.P.Z.) and by a DAMD grant (17-97-1-7326 to K.E.M.). M.M. acknowledges the generous support of the UCSF Cancer Research Institute for funding to support these studies.