Abstract
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.
Highlights
R-Ras is a small GTPase of the ras family that was originally identified as a close homolog of the oncogene product v-H-Ras [1]
The analyses with the deletion clones RP1, dP, and RP3 in A2780 cells demonstrated the crucial role of the Ϫ727/ Ϫ476 fragment in activating the basal promoter activity as we demonstrated in endothelial cells and arterial smooth muscle cells; the internal deletion of the 350-bp DNA fragment in the clone dP reduced the promoter activity by 64%, and the combined deletion of this fragment and the basal promoter (RP3) resulted in nearly complete silencing of the R-ras promoter in A2780 cells (Fig. 3A)
GA-binding protein (GABP) Is Bound to the R-ras Promoter in Cultured Endothe-Our result indicated that GABP is a major binder to the EBS in lial Cells, Vascular Smooth Muscle Cells, and A2780 Ovarian the human umbilical cord vein endothelial cells (HUVEC) nuclear extract (Fig. 8A, lanes 6 –9)
Summary
Cell Culture—Human umbilical vein endothelial cells (HUVEC) and human coronary artery smooth muscle cells were purchased from Lonza Inc. (Allendale, NJ). Possible transcription factor-binding sites were predicated on genomic DNA sequences using the TFsearch program with a threshold of 85 and Vertebrate Matrix. The second PCR products were gel-purified and subcloned into the pCR-4-TOPO vector (Invitrogen) and sequenced to determine the 5Ј-end of the R-Ras transcript. The fragments of different lengths in the 5Ј-flanking region of the human R-ras gene were amplified by PCR (see Table 1 for PCR primer sequences) using the human genomic DNA or cloned 5Ј-flanking DNA as templates. To construct the dP promoter fragment, which deletes the segment between Ϫ756 and Ϫ404 from the P7 construct, two DNA fragments were amplified separately using the primers F and L-R3, and L-CR and Hs-R, respectively (see Table 1 for primer sequences). Generation of Mutations in the R-ras Promoter—Site-directed mutagenesis was carried out in the putative transcription factor-binding sites within the Ϫ727/Ϫ476 DNA fragment using the QuikChange XL site-directed mutagenesis kit
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