Objectives: 

Tubulointerstitial fibrosis is characterized by the presence and proliferation of renal myofibroblasts. Ras-GTPases are crucial for the TGF-beta induced renal fibrosis. Several authors have elucidated the importance of K-Ras in renal fibroblast proliferation. K-Ras regulates two pathways involved in renal fibrosis such as MEK-ERK and PI3K-Akt pathways. In this study we assessed the role of K-Ras in fibroblasts proliferation and motility.

Materials: 

We have generated and characterized knockout (KO) mouse embryonic fibroblasts for the K-Ras isoform (K-ras-/-). Cell proliferation was evaluated by MTT assay. PCNA expression was evaluated by Western-blot. Cell motility was evaluated by a wound healing assay in fibroblast monolayers.

Results: 

The lack of the K-Ras isoform leads to a reduced Ras-GTP activation and reduced ERK phosphorylation, as well as to a higher Akt phosphorylation. The absence of K-Ras isoform induces a reduction in fibroblasts proliferation in basal conditions and a lower expression of PCNA. Treatment with TGF-beta1 increases fibroblasts proliferation in control fibroblasts but not in K-ras-/- fibroblasts. Treatment with the MEK inhibitor U0126 and the PI3K-Akt inhibitor LY294002 decreases cell proliferation in both wild type and KO fibroblasts. LY294002 treatment reduced PCNA expression only in K-ras-/- fibroblasts. The absence of the K-Ras isoform partially inhibited fibroblasts migration. Treatment with U0126 decreases fibroblasts migration in control fibroblasts but not in K-ras-/- fibroblasts, while LY294002 treatment decreased fibroblasts migration in both wild type and K-ras-/- fibroblasts.

Conclusions: 

These data show the involvement of K-Ras isoform in fibroblasts proliferation and migration. We hypothesize that lower proliferation and migration observed in the absence of K-Ras may be due to a lower activation of Ras-GTP and MEK-ERK pathways. Moreover, higher PI3K-Akt activation observed in the absence of K-Ras may be involved in improving survival, proliferation and migration.