title: Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes.
abstract: To understand the signalling mechanisms involved in the dual stimulatory effects of endothelin-1 (ET-1) on DNA synthesis and melanization in cultured human melanocytes, we analysed the biological profile of ET-1 receptor and determined the effects of ET-1 on the protein kinase C, cyclic AMP system and mitogen-activated protein kinase (MAP kinase) in comparison with their relevant stimulants. The photoaffinity labelling of ET-1 receptors with Denny-Jaff reagents revealed an ET-1 receptor with a molecular mass of 51 kDa in human melanocytes. The ET(A) receptor subtype-sensitive antagonist BQ123(50 nM) or pertussis toxin (100 ng/ml) significantly suppressed the ET-1-induced intracellular calcium mobilization, indicating the presence of pertussis toxin-sensitive G-protein-coupled ET(A) receptors. An assay of protein kinase C activity revealed that 10nM ET-1 translocated cytosolic protein kinase C to membrane-bound protein kinase C within 5 min of the start of incubation. In contrast, receptor-mediated melanocyte activation by ET-1 was accompanied by an elevated level of cyclic AMP (4-fold over control) after 10-60 min of incubation, whereas 60 min of incubation of human melanocytes with c-Kit or c-Met ligands such as stem cell factor (10 nM) or basic fibroblast growth factor (10 nM) did not elevate the cyclic AMP level. We have also demonstrated that a specific tyrosine kinase inhibitor, tyrphostin B-42 (10 microM), inhibited the ET-1-induced growth stimulation, suggesting the involvement of the tyrosine kinase pathway in growth stimulation. Consistently, an assay of MAP kinase revealed that ET-1 caused a 10-fold activation of MAP kinase after 5 min of incubation with human melanocytes in a similar way to tyrosine kinase ligands such as stem cell factor and hepatocyte growth factor. Further, the DNA synthesis stimulated by the c-Kit ligand stem cell factor at a concentration of 1 nM was synergistically enhanced by 5 nM ET-1. These results suggest that ET-induced dual cellular events in human melanocytes are closely associated with cross-talk between the protein kinase C and A and tyrosine kinase pathways.
authors.affiliation: Institute for Fundamental Research, Kao Corporation, Tochigi, Japan.
authors.forename: G
authors.initials: G
authors.lastname: Imokawa
authors.forename: Y
authors.initials: Y
authors.lastname: Yada
authors.forename: M
authors.initials: M
authors.lastname: Kimura
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000494:pharmacology*; D006801:Humans; D008543:Melanins / Q000096:biosynthesis*; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000494:pharmacology; D037342:Pertussis Toxin; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D019089:Stem Cell Factor / Q000494:pharmacology; D013889:Thiouracil / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D008543:Melanins; D010456:Peptides, Cyclic; D010726:Phosphodiesterase Inhibitors; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D019089:Stem Cell Factor; D010566:Virulence Factors, Bordetella; D013889:Thiouracil; D004247:DNA; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C072247:cyclo(Trp-Asp-Pro-Val-Leu); D015056:1-Methyl-3-isobutylxanthine