Role of Ras Isoforms in the Stimulated Proliferation of Human Renal Fibroblasts in Primary Culture

Abstract. The proliferation of renal fibroblasts is implicated in the pathophysiologic processes of renal fibrosis. Many of the growth factors involved in proliferation are known to activate intracellular signaling pathways that converge on Ras monomeric GTPases. Although three ras family genes exist, their functional specificity is not yet known. Using antisense oligonucleotides, a role for Kirsten (Ki)-Ras in the stimulated proliferation of a primate renal fibroblast cell line was previously demonstrated. This study examines Ras in primary cultures of adult human renal fibroblasts. Using reverse transcription-PCR, mRNA for Harvey (Ha)- ras , Ki(4B)- ras , and neural (N)- ras , but not Ki(4A)- ras , were detected. Antisense oligonucleotides targeting Ha-, Ki-, and N- ras mRNA, which were used for liposomal transfection at 100 to 200 nM, were demonstrated to be active and isoform-specific in quantitative reverse transcription-PCR assays. Cellular Ras protein levels, as estimated using isoform-specific monoclonal antibodies, indicated that Ki-Ras was the predominantly expressed isoform (>95% of total Ras protein) under both serum-containing and serum-free conditions, with N- and Ha-Ras being detected in small amounts. Consistent with this finding, the antisense oligonucleotide directed against Ki-Ras reduced total cellular Ras levels by >70%, whereas Ha-Ras, N-Ras, and control oligonucleotides had no significant effect. Proliferation of oligonucleotide-transfected cells was measured using epidermal growth factor (EGF) and serum stimulation. The Ki-Ras oligonucleotide at 100 nM reduced serum-stimulated proliferation by >50% and EGF-stimulated proliferation by 25%, compared with data obtained with the control oligonucleotide ( P < 0.01). The N-Ras oligonucleotide was not active, compared with the control oligonucleotide. The Ha-Ras oligonucleotide was not significantly active at 100 nM but reduced serum-stimulated proliferation by 13% and EGF-stimulated growth by 40% at 200 nM ( P < 0.01). These results demonstrate that Ki-Ras(4B) is the predominantly expressed Ras isoform in human renal fibroblasts in primary culture and is important for both serum- and EGF-stimulated proliferation. Ha-Ras appears to be expressed at low levels but may also play a distinct role in stimulated proliferation.