Opposing Roles of the Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Cascades in Ras-Mediated Downregulation of Tropomyosin

Abstract

We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for α-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.

We thank Richard Janssen for the tropomyosin expression vectors, Robert Coffey and David Beauchamp for the untransformed and H-Ras(12V)-inducible RIE-1 cell lines, and Eric Stanbridge for the DLD-1 and HT1080 cell lines. We thank Kelley Rogers-Graham for technical assistance and Misha Rand for assistance in manuscript preparation.

Our research was supported by grants from the National Institutes of Health to C.J.D. (CA42978, CA55008, and CA63071) and from the Department of Defense to J.M.S (DAMD17-00-1-0552).