Identification of H-Ras, RhoA, Rac1 and Cdc42 responsive genes.

The superfamily of small GTP-binding proteins has expanded dramatically in recent years. The Ras family has long been associated with signaling pathways contributing to normal and aberrant cell growth, while Rho-related protein function is to integrate extracellular signals with specific targets regulating cell morphology, cell aggregation, tissue polarity, cell motility and cytokinesis. Recent findings suggest that certain Rho proteins, including RhoA, Rac1 and Cdc42, can also play a role in signal transduction to the nucleus and cell growth control. However, the nature of the genes regulated by Ras and Rho GTPases, as well as their contribution to their numerous biological effects is still largely unknown. To approach these questions, we investigated the global gene expression pattern induced by activated forms of H-Ras, RhoA, Rac1 and Cdc42 using cDNA microarrays comprising 19 117 unique elements. Using this approach, we identified 1184 genes that were up- or downregulated by at least twofold. Hierarchical cluster analysis revealed the existence of patterns of gene regulation both unique and common to H-Ras V12, RhoA QL, Rac1 QL and Cdc42 QL activation. For example, H-Ras V12 upregulated osteopontin and Akt 1, and H-Ras and RhoA stimulated cyclin G1, cyclin-dependent kinase 8, cyclin A2 and HMGI-C, while Rac1 QL and Cdc42 QL upregulated extracellular matrix and cell adhesion proteins such as alpha-actinin 4, procollagen type I and V and neuropilin. Furthermore, H-Ras V12 downregulated by >eightfold 52 genes compared to only three genes by RhoA QL, Rac1 QL and Cdc42 QL. These results provide key information to begin unraveling the complexity of the molecular mechanisms underlying the transforming potential of Ras and Rho proteins, as well as the numerous morphological and cell cycle effects induced by these small GTPases.64