Ha-Ras stabilization mediates pro-fibrotic signals in dermal fibroblasts

Forced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous collagen I proteins. Moreover, normal levels of Tgfβ transcripts and active transforming growth factor-beta (TGFβ) implied Ha-Ras stimulation of the canonical Smad2/3 signalling pathway independently of TGFβ production or activation. Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and collagen I production in cells that constitutively express Ha-Ras.Our study shows for the first time that constitutively elevated Ha-Ras protein levels can directly stimulate Smad2/3 signalling and collagen I accumulation independently of TGFβ neo-synthesis and activation. This finding therefore implicates the Ha-Ras pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic targets in SSc.Wound healing is a complex and tightly regulated physiological process that involves several different cell types and a plethora of signalling molecules [1-3]. In the early phase of this process, platelets brought by the blood stream form a fibrin cloth at the site of injury that blocks bleeding (haemostasis). Increased levels of soluble signals, induced by the cell-mediated inflammatory response, subsequently promote migration and proliferation of angiogenic cells and activated fibroblasts (myofibroblasts) that synthesize extracellular matrix (ECM) proteins, chiefly collagen I [1]. By contracting the newly synthesized ECM, myofibroblasts allow the closure of the wound where the provisional matrix is ultimately remodelled to form a scar [1]. Failure of myofibroblasts to