Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes- Cross-talk vla trans-activation of the tyrosine kinase c-kit receptor

We previously reported that activation of mitogen-activated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-l (ET-1). In the present study, we determined signali ng mechanisms upstream of MAPK activation that are involved in ET-1 stimula tion and their synergism with stem cell factor (SCF). Pretreatment of cultu red NHMC with ETB receptor antagonists, pertussis toxin, a specific phospho lipase C inhibitor (U73122), or a protein kinase C inhibitor (calphostine) blocked a transient tyrosine phosphorylation of MAPK induced by ET-1, where as the addition of a calcium chelator (BAPTA) failed to inhibit that tyrosi ne phosphorylation of MAPK. Treatment with ET-1 and SCF together synergisti cally increased DNA synthesis, which was accompanied by synergism for MAPK phosphorylation. The time course of inositol 1,4,5-trisphosphate formation revealed that there is no difference in the level of inositol 1,4,5-trispho sphate stimulated by ET-1 + SCF or by ET-1 alone. Evaluations of the serine phosphorylation of MEK and Raf-l activity showed a synergistic effect in S CF + ET-l-treated NHMC. Stimulation with SCF + ET-1 induced a more rapid an d stronger tyrosyl phosphorylation of proteins corresponding to p52 and p66 Shc than did stimulation with SCF only, and this was accompanied by a stro nger association of tyrosine-phosphorylated Shc with Grb2. Interestingly, a more rapid and marked tyrosine phosphorylation of c-kit was also detected in NHMC-treated with SCF + ET-1 than NHMC treated with SCF only. These data indicate that the synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of tyrosine phosphorylation of c-kit, which results in the enhanced formation of the Shc-Grb(2) complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop.