Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoske leton. The changes in the actin cytoskeleton comprise a loss of actin stres s fibers and enhanced cortical actin, Using MDCK cells expressing a conditi onally active form of Raf, we have explored the molecular mechanisms that u nderlie these observations. Raf activation elicited a robust increase in Ra d activity consistent with the observed increase in cortical actin, Loss of actin stress fibers is indicative of attenuated Rho function, but no chang e in Rho-GTP levels was detected following Raf activation, However, the los s of actin stress fibers in Raf-transformed cells was preceded by the induc ed expression of Rnd3, an endogenous inhibitor of Rho protein function. Exp ression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, tells expressing activated RhoA failed to multilayer in response to Raf, Ph armacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consist ent with a role for induced Rnd3 expression downstream of the Raf-MEK-extra cellular signal-regulated kinase pathway in epithelial oncogenesis.