Abstract
We previously reported that activation of mitogen-activated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-1 (ET-1). In the present study, we determined signaling mechanisms upstream of MAPK activation that are involved in ET-1 stimulation and their synergism with stem cell factor (SCF). Pretreatment of cultured NHMC with ET(B) receptor antagonists, pertussis toxin, a specific phospholipase C inhibitor (), or a protein kinase C inhibitor (calphostine) blocked a transient tyrosine phosphorylation of MAPK induced by ET-1, whereas the addition of a calcium chelator (BAPTA) failed to inhibit that tyrosine phosphorylation of MAPK. Treatment with ET-1 and SCF together synergistically 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-trisphosphate stimulated by ET-1 + SCF or by ET-1 alone. Evaluations of the serine phosphorylation of MEK and Raf-1 activity showed a synergistic effect in SCF + ET-1-treated NHMC. Stimulation with SCF + ET-1 induced a more rapid and stronger tyrosyl phosphorylation of proteins corresponding to p52 and p66 Shc than did stimulation with SCF only, and this was accompanied by a stronger 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.
Highlights
We previously reported that activation of mitogenactivated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-1 (ET-1)
ET Binding Initiated Signaling Pathway Leading to the Activation of MAPK—In order to clarify whether the activation of MAPK is mediated via the endothelin-binding ETA or ETB receptor, we looked at the effect of endothelin A and B receptor antagonists, BQ610 and BQ788, respectively, on the tyrosine phosphorylation of ERK2, a hallmark of MAPK activation as assessed by Western blotting using a phosphotyrosine antibody following immunoprecipitation with anti-ERK2
The phospholipase C inhibitor, U73122, downregulates tyrosine phosphorylation of ERK2 in a dosedependent manner (Fig. 4), indicating that phospholipase C is involved in the activation of MAPK during the intracellular signaling initiated by ET and ETB receptor binding
Summary
We previously reported that activation of mitogenactivated protein kinase (MAPK) is involved in the mitogenic stimulation of normal human melanocytes (NHMC) by endothelin-1 (ET-1). A more rapid and marked tyrosine phosphorylation of c-kit was 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 complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop. We report that the synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of trans-activation of c-kit, including its enhanced tyrosine phosphorylation, during the SCF-induced activation process This results in an increase in the formation of the Shc-Grb complex, which leads in turn to synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop
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