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Evidence of a Role for Ki-RAS in the Stimulated Proliferation of Renal Fibroblasts

CLAIRE C. SHARPE^*, MARK E. C. DOCKRELL^*, RIZALDY SCOTT^*, MAZHAR I. NOOR^*, LEX M. COWSERT, BRETT P. MONIA and BRUCE M. HENDRY^*

^* Cell Signalling Group, Department of Renal Medicine, GKT School of Medicine, King's College London, London, United Kingdom
ISIS Pharmaceuticals, Department of Molecular Pharmacology, Carlsbad, California.

Correspondence to Bruce M. Hendry, Department of Renal Medicine, King's College London, Bessemer Road, London SE5 9PJ, United Kingdom. Phone: +44 171 346 3741; Fax: +44 171 346 3742; E-mail: bruce.hendry{at}kcl.ac.uk

Abstract. Progressive renal fibrosis is driven by a range of cytokines that act via membrane receptors and intracellular signaling cascades to evoke gene transcription events and related responses. The Ras family of GTPases has been implicated in many of these signaling cascades in model systems such as 3T3 fibroblasts. However, the role of the specific Ras isoforms Ki, Ha, and N in the stimulation of renal fibroblasts has not been defined. In this study, Ras has been inhibited in primate renal fibroblasts (vero cells) using specific phosphorothioate oligodeoxynucleotides (oligos) targeting the three isoforms. Lipofectin transfection with 200 to 400 nM Ki-Ras oligo inhibited the epidermal growth factor- and fibroblast growth factor-stimulated proliferation of vero cells by 25 to 35% with a lesser effect on serum-stimulated growth. Oligos against Ha-Ras and N-Ras were inactive with respect to control oligo. Total cellular Ras protein (estimated by Western blotting) was reduced by 60 to 90% 24 h after transfection with Ki-Ras oligo. N-Ras, Ha-Ras, and control oligos were inactive. Total Ras synthesis over 4 h measured using [³⁵S]-cys/met pulse chase was reduced by approximately 70% by Ki-Ras oligo and not altered by other oligos. The fractional prenylation of Ras was quantified from the discrete bands on polyacrylamide gel electrophoresis and was increased by the Ki-Ras oligo alone. These data demonstrate that these renal fibroblasts predominantly express the Ki isoform of Ras and that this GTPase plays a role in the stimulated proliferation of these cells. Ras GTPases may be a target for the inhibition of processes leading to renal fibrosis.

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