Promoter cloning and characterization of the anti-vascular proliferation gene, R-ras: role of Ets- and Sp-binding motifs.

The R-ras gene encodes a small GTPase of the ras family that is closely related to H-ras and K-ras. Unlike the prototypic ras genes, the disruption of the R-ras gene in mice results in enhanced angiogenesis in tumor implants and sustained neointimal hyperplasia in response to arterial injury, indicating the in vivo role of R-ras as a negative regulator of vascular proliferation. R-ras is abundantly expressed in normal mature blood vessels but significantly down-regulated in pathologically regenerating vasculature. In this study, we investigated the roles of cis-acting elements in the transcription of the human R-ras gene, as well as the transcription factors that interact with these sequences in cultured endothelial cells and arterial smooth muscle cells. The findings from vascular cells were then compared with findings from epithelial tumor cells that aberrantly express R-ras. Deletion analyses on 5 kb of 5'-flanking DNA of the human R-ras gene revealed the functional importance of the region between -727/-476, which contains two Ets and one Sp1 consensus binding motifs. Mutation analyses of various consensus binding motifs within this region suggest both cell type-dependent and -independent regulatory mechanisms for the R-ras gene transcription. Electrophoretic mobility shift and antibody disruption assays demonstrated that an Ets transcription factor family protein, GA-binding protein (GABP), binds to the R-ras-derived sequence. Chromatin immunoprecipitation analyses determined the association of endogenous GABP as well as Sp3 proteins with the -727/-476 region of the R-ras promoter in intact cells grown in culture. Forced expression of GABP significantly enhanced R-ras mRNA expression level in endothelial cells. These results map the functional elements in the R-ras promoter sequence and suggest that the GABP may be critical for transcription of R-ras and for maintenance of normal blood vessel functions through the regulation of this gene.