Objectives: 

In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. There is a proven relationship between the signalling pathways of transforming growth factor-beta1 (TGF-beta1) and Ras GTPases. Each of the Ras isoforms (H, N and K) exhibits specific modulator activity on different cellular pathways. K-Ras GTPase (V- Ki-ras2 Kirsten rat sarcoma viral oncogene) is the predominantly expressed isoform in renal fibroblasts. Expression of K-Ras is essential during the developmental stage in mice. Our aim was to assess the individual role of the K-Ras isoform in TGF-beta1-mediated ECM synthesis.

Materials: 

ECM synthesis was studied by evaluating the expression of fibronectin and collagen type I by western blot and RT-PCR, as well as analyzing total collagen synthesis by a colorimetric method. We also analyzed the role of Erk and Akt in the modulation of ECM synthesis using specific inhibitors of both pathways (U0126 and LY294002, respectively).

Results: 

Fibronectin and collagen type I protein expression, as well as total collagen synthesis, were notably higher in K-ras-/- than in control K-ras+/+ fibroblasts in basal conditions. TGF-beta1 treatment induced an increase in ECM protein expression only in K-ras+/+ fibroblasts. MEK/Erk1/2 inhibition reduced fibronectin and collagen I expression only in K-ras+/+ fibroblast in resting conditions and after stimulation with TGF-beta1, whereas PI3K/Akt inhibition reduced collagen I and fibronectin in basal conditions and after stimulation with TGF-beta1 in both K-ras+/+ and K-ras-/- fibroblasts.

Conclusions: 

Our data suggest that the K-Ras isoform might down-regulate fibronectin and collagen synthesis in fibroblasts, in part through down-regulation of Akt activation; moreover Erk activation seems to be necessary to modulate fibronectin and collagen I protein expression only in presence of K-Ras.