PP60C-SRC IS REQUIRED FOR THE INDUCTION OF A QUIESCENT MESANGIAL CELLPHENOTYPE

The tyrosine kinase c-src associates with growth factor receptors, foc al contacts and cytoskeletal proteins and is involved in signaling eve nts. The aim of this study was to investigate the role of src in the r egulation of mesangial cell (MC) proliferation and differentiation in three-dimensional (3D) culture in collagen gels. Using retroviral gene transfer we have overexpressed wild-type c-src, a kinase-negative c-s rc mutant (c-src295) and transforming v-src in MC. The MC differentiat ion in 3D culture was characterized by the formation oi a nonprolifera ting multicellular network in control cells and in cells expressing wi ld-type c-src. Immunoblotting demonstrated a rapid down-regulation of the alpha-smooth muscle actin expression. The kinase-negative MC (c-sr c295) failed to differentiate, maintained a significant proliferative rate, and the alpha-smooth muscle actin expression remained stable dur ing 3D culture. MC transformed with v-src showed a high level of tyros ine phosphorylation and proliferation in 3D culture. Analysis of prote ins involved in cell cycle regulation demonstrated dephosphorylation o f the retinoblastoma protein (Rb) during 3D culture in control and c-s rc transfected cells. Expression of v-src resulted in sustained Rb pho sphorylation. Zymographic analysis of plasminogen activator (u-PA) rev ealed an inhibition of u-PA secretion in MC transfected with c-src295. These results indicate that c-src exerts regulatory effects on MC pro liferation, cytoskeletal organization, matrix proteases and differenti ation. Targeted manipulation of the c-src kinase may be useful in modu lating MIC behavior in vivo.