Ha-Ras/ERK1-2 pathway is responsible for oxygen species generation in scleroderma fibroblasts

Background

Reactive oxygen species (ROS) are involved in scleroderma (SSc) fibroblast activation, proliferation and type I collagen gene expression [1]. In order to understand the signal trasduction pathway involved in ROS generation by SSc fibroblasts, we have assessed the role of Ha-Ras and Ki-Ras proteins and that of their downstream effectors.

Materials and methods

Intracellular ROS were evaluated with 2',7'-dichlorofluorescein diacetate (DCHF-DA 10 μM). For Ha-Ras and Ki-Ras protein expression whole cell lysates were immunoprecipitated with specific antibodies and subjected to SDS-PAGE or cells were fixed in paraformaldehyde (4%) and observed by confocal microscopy. SSc skin fibroblasts were transiently transfected with the dominant negative form of Ha-Ras (H17Ras) or with the empty vector using the Effectene Trasfection reagents (BioRad).

Results

Quiescent SSc fibroblasts contain an altered ratio between the two Ras isoforms showing a selective increase of the mass and the activity of Ha-Ras protein when compared with normal controls. Moreover ERK1-2 are constitutively phosphorilated in quiescent SSc fibroblasts. Inhibition of either Ras, ERK1-2, ROS with specific inhibitor or trasfecting with the negative Ras variant reverse this phenotype back to normal and reduce collagen overproduction by SSc cells.

Conclusions

These results suggest that ROS production in SSc fibroblasts is linked and ultimately dependent on increased Ha-Ras signalling via ERK1-2. These events are responsible for the characteristic cellular phenotype of scleroderma fibroblasts and explain some of the clinical features of the disease. Moreover, these data provide evidence for the use of Ras inhibitors in the treatment of the disease.