Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes. Cross-talk via 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-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.
Kao Biological Science Laboratories, Haga, Tochigi 321-3497, Japan. 073733@kastanet.kao.co.jp
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Imokawa
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Kobayasi
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Miyagishi
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004357:Drug Synergism; D019332:Endothelin-1 / Q000494:pharmacology*; D051380:GRB2 Adaptor Protein; D006801:Humans; D008544:Melanocytes / Q000187:drug effects* / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019089:Stem Cell Factor / Q000494:pharmacology*; D015533:Transcriptional Activation
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D019332:Endothelin-1; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019089:Stem Cell Factor; D004247:DNA; D019009:Proto-Oncogene Proteins c-kit; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases