Intracellular Signaling Mechanisms Leading to Synergistic Effects of Endothelin-1 and Stem Cell Factor on Proliferation of Cultured Human Melanocytes

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 ETB receptor antagonists, pertussis toxin, a specific phospholipase C inhibitor (U73122), 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-Grb2 complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop. 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 ETB receptor antagonists, pertussis toxin, a specific phospholipase C inhibitor (U73122), 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-Grb2 complex which leads in turn to the synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop. endothelin mitogen-activated protein kinase normal human melanocytes stem cell factor inositol 1,4,5-trisphosphate protein kinase C polyacrylamide gel electrophoresis myosin basic protein Src homology domain 2 4-morpholinepropanesulfonic acid 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid In the long course of studying paracrine mechanisms involved in epidermal hyperpigmentary disorders, we have found that endothelin-1 (ET-1)1 plays a central role in UVB-induced pigmentation (1Imokawa G. Yada Y. Miyagishi M. J. Biol. Chem. 1992; 267: 24675-24680Abstract Full Text PDF PubMed Google Scholar, 2Yada Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar) and in the accentuated pigmentation of senile freckles. 2S. Kadono, I. Manaka, M. Kawashima, T. Kobayashi, and G. Imokawa, submitted for publication.2S. Kadono, I. Manaka, M. Kawashima, T. Kobayashi, and G. Imokawa, submitted for publication.Furthermore, stem cell factor (SCF) plays an important role in the increased pigmentation of the epidermis overlying benign fibroblastic tumors in dermatofibroma 3E. Shishido, S. Kadono, I. Manaka, M. Kawashima, and G. Imokawa, submitted for publication.3E. Shishido, S. Kadono, I. Manaka, M. Kawashima, and G. Imokawa, submitted for publication. as well as in UVB-induced pigmentation. 4A. Hachiya, A. Kobayashi, A. Ohuchi, Y. Takema, and G. Imokawa, submitted for publication.4A. Hachiya, A. Kobayashi, A. Ohuchi, Y. Takema, and G. Imokawa, submitted for publication. We have also observed that aged fibroblasts in culture produce a larger amount of SCF than do younger ones (3Imokawa G. Yada Y. Morisaki N. Kimura M. Biochem. J. 1998; 330: 1235-1239Crossref PubMed Scopus (88) Google Scholar), which probably provides a basis for the tendency of aged skins to be more sensitive to environmental stimuli and to be easily induced to epidermal hyperpigmentation. SCF is also known as a stimulator for epidermal hyperpigmentation in mastocytosis where mast cells undergo hyperproliferation in response to the soluble type of SCF derived from keratinocytes (4Longley Jr., B.J. Morganroth G.S. Tyrrell L. Ding T.G. Anderson D.M. Williams D.E. Halaban R. N. Engl. J. Med. 1993; 328: 1302-1307Crossref PubMed Scopus (252) Google Scholar). In inherited pigmentary diseases such as piebaldism and Hirschsprung disease, mutations of c-kit (5Giebel L.B. Spritz R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8696-8699Crossref PubMed Scopus (313) Google Scholar, 6Spritz R.A. 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Genet. 1997; 34: 656-665Crossref PubMed Scopus (428) Google Scholar, 11Tachibana M. Takeda K. Nobujuni Y. Urabe K. Long J.E. Meyers K.A. Aaronson S.A. Miki T. Nature Gen. 1996; 14: 50-54Crossref PubMed Scopus (227) Google Scholar). Recently, the c-kit signaling pathway was found to be upstream of microphthalmia-associated transcription factor transcription through phosphorylation by MAP kinase (MAPK) (12Hemesath T.J. Price E.R. Takemoto C. Badalian T. Fisher D.E. Nature. 1998; 391: 298-301Crossref PubMed Scopus (552) Google Scholar). In mast cells, many reports (13Tsujimura T. Morii E. Nozaki M. Hashimoto K. Moriyama Y. Takebayashi K. Kondo T. Kanakura Y. Kitamura Y. Blood. 1996; 88: 1225-1233Crossref PubMed Google Scholar, 14Ito A. Morii E. Kim D.K. Kataoka T.R. Jippo T. Maeyama K. Nojima H. Kitamura Y. Blood. 1999; 93: 1189-1196Crossref PubMed Google Scholar) have described a strong link between c-kit expression and microphthalmia-associated transcription factor transcriptional function. Furthermore, a disease termed Shah-Waardenburg syndrome, which combines the Waardenburg type 2 and Hirschsprung phenotypes, shows defects in microphthalmia-associated transcription factor and in the endothelin B receptor (15Edery P. Attie T. Amiel J. Pelet A. Eng C. Hofstra R.M. Martelli H. Bidaud C. Munnich A. Lyonnet S. Nat. Genet. 1996; 12: 442-444Crossref PubMed Scopus (378) Google Scholar). Thus, it is likely that signaling pathways stimulated by binding of ET-1 and SCF to their corresponding receptors have at least some common pathways and are working in a coordinated fashion to regulate melanocyte function. In relation to this, we have recently found that the proliferation of cultured human melanocytes (NHMC) induced by ET-1 is synergistically enhanced by the concomitant addition of SCF (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). Endothelins are unique mitogens and melanogens for human melanocytes (1Imokawa G. Yada Y. Miyagishi M. J. Biol. Chem. 1992; 267: 24675-24680Abstract Full Text PDF PubMed Google Scholar, 2Yada Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar, 17Imokawa G. Kobayashi T. Miyagishi M. Higashi K. Yada Y. Pigment Cell Res. 1997; 10: 218-228Crossref PubMed Scopus (158) Google Scholar). These cellular actions are known to be initiated by binding of ET-1 to G-protein-coupled ETB receptor, followed by sequential signaling processes consisting mainly of protein kinase C (PKC) and MAPK (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar, 17Imokawa G. Kobayashi T. Miyagishi M. Higashi K. Yada Y. Pigment Cell Res. 1997; 10: 218-228Crossref PubMed Scopus (158) Google Scholar). SCF stimulation is important for the survival and proliferation of several cell types in the hematopoietic system (18McNiece I.K. Langley K.E. Zsebo K.M. Exp. Hematol. 1991; 19: 226-231PubMed Google Scholar, 19Zsebo K.M. Wypych J. McNiece I.K. Lu H.S. Smith K.A. Karkare S.B. Sachdev R.K. Yuschenkoff V.N. Birkett N.C. Williams L.R. Cell. 1990; 63: 195-201Abstract Full Text PDF PubMed Scopus (655) Google Scholar), where it may function in combination with other growth factors. In human melanocytes, SCF alone is sufficient to sustain proliferation under serum-free conditions (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). SCF binding to the c-kit receptor mediates dimerization, activation of its intrinsic tyrosine kinase activity, and autophosphorylation (20Blume-Jensen P. Claesson-Welsh L. Siegbahn A. Zsebo K.M. Westermark B. Heldin C.H. EMBO J. 1991; 10: 4121-4128Crossref PubMed Scopus (266) Google Scholar). The activated receptor then phosphorylates various substrates and associates with various signaling molecules, including phosphatidylinositol 3′-kinase (PI 3-kinase), the Shc and Grb2 adaptor proteins, and the guanine nucleotide exchange factor, SOS, all of which lead to the activation of the Ras-MAPK pathway (21Cutler R.L. Liu L. Damen J.E. Krystal G. J. Biol. Chem. 1993; 268: 21463-21465Abstract Full Text PDF PubMed Google Scholar, 22Lennartsson J. Blume-Jensen P. Hermanson M. Ponten E. Carlberg M. Ronnstrand L. Oncogene. 1999; 18: 5546-5553Crossref PubMed Scopus (173) Google Scholar, 23Liu L. Damen J.E. R.L. Krystal G. Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). In the the cells which melanocytes are that are known to produce increased of SCF and ET-1 in response to several stimuli Y. J. Imokawa G. and Scholar). it is of to the cross-talk between ET-1 and signaling in human melanocytes of specific signaling pathways been with the stimulation of (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). The of present was to the mechanisms by which signaling MAPK and its synergism with We that the synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of of c-kit, including its enhanced tyrosine the activation results in an in the formation of the Shc-Grb2 which leads in turn to synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop. NHMC from from was from from and from and and from other of NHMC in growth with basic growth factor, and and at under a In to cellular or signaling NHMC in culture at a of and cultured in for then treated with at various NHMC cultured in with ET-1 SCF at of to the cells for with with the cells and a with and with then The the was These as reported previously Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar). for cells in culture at a of and cultured for The and the was and for at The stimulation was at by acid and the for with for the at for to The to the Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar). The of in was determined from a the binding protein specific for and with ET-1 SCF in culture then in of and The and at for at in the determined by the These as reported previously C. M. Kobayashi T. J. D. Imokawa G. G.S. EMBO J. 1997; PubMed Scopus Google Scholar, T. Imokawa G. Bennett J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). or complex by and then to blocked with in for at and then with in with for and enhanced These as reported previously T. Urabe K. A. C. Imokawa G. T. EMBO J. 1994; PubMed Scopus Google Scholar, T. Urabe K. Higashi Imokawa G. B. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). and c-kit from cell by with of for 2 at The then by with protein for at The with with in and at for and proteins detected with as described Raf-1 was to the reported previously P. A. S. 1995; PubMed Scopus Google Scholar) by its to MAPK which was then by the activation of MAPK P. A. S. 1995; PubMed Scopus Google Scholar). of cell was to of protein to of Raf-1 and was for at a The for at the and the with of and with of and and then as described of inhibitor inhibitor and of and of was to by Raf-1 for at a a was to of and for at the of was determined by of with ET-1 and SCF are at and respectively, there is a synergistic stimulation of DNA in NHMC ET-1 and SCF of a marked synergistic stimulation of DNA is with a to the and by SCF or ET-1 In to the activation of MAPK is the or we at the effect of endothelin and B receptor antagonists, and respectively, the tyrosine phosphorylation of a of MAPK activation as by a with The ETB receptor tyrosine phosphorylation of whereas the receptor to inhibit the phosphorylation that the activation of MAPK is through the endothelin B The addition of pertussis tyrosine phosphorylation of that the is with signaling leading to the activation of MAP the phospholipase C tyrosine phosphorylation of in a that phospholipase C is also involved in the activation of MAPK the signaling initiated by and ETB receptor The addition of the calcium no effect the phosphorylation of there is no of calcium in the activation of MAPK. In the the phosphorylation of that activation of is for the activation of the tyrosine phosphorylation of melanocytes treated with ET-1 in the of pertussis and and the activation of MAPK was then by the tyrosine phosphorylation of as under pertussis phospholipase C the tyrosine phosphorylation of for with the phospholipase C or its and human melanocytes treated with ET-1 and and The activation of MAPK was by the tyrosine phosphorylation of as under phospholipase calcium no effect the phosphorylation of with the calcium human melanocytes treated with ET-1 and and The activation of MAPK was by the tyrosine phosphorylation of as under the phosphorylation of for with human melanocytes treated with ET-1 and and The activation of MAPK was by the tyrosine phosphorylation of as under The time course of the formation of SCF and ET-1 revealed that there is no difference in the level of with ET-1 + SCF or ET-1 no in the In the of tyrosine phosphorylation of and an of MAPK as by with and we found that there is a synergistic effect tyrosine phosphorylation in ET-1 + NHMC with ET-1 or SCF alone. in the and of tyrosine which a stimulation In the of serine phosphorylation of an of as by a MEK there is a synergistic effect in SCF + ET-1-treated melanocytes with a stimulation that the in the activation of the observed in MAPK activation in SCF + ET-1-treated NHMC. of Raf-1 activity, an of as by the phosphorylation of showed that ET-1 Raf-1 activity, which that Raf-1 is at least from the pathway to the MAPK Furthermore, there is a synergistic effect Raf-1 activity in SCF + ET-1-treated melanocytes the that the synergistic between SCF and ET-1 signaling is effect serine phosphorylation of melanocytes stimulated with ET-1 The activation of MEK was by the serine phosphorylation of MEK at the by as under effect Raf-1 melanocytes stimulated for 2 with ET-1 The activation of Raf-1 was by the by a with and as under The of tyrosine kinase MAPK signaling the formation of between the guanine nucleotide exchange protein SOS, and the and adaptor protein Grb2 with growth factor receptors or tyrosine-phosphorylated adaptor protein known as Shc in this signaling pathway tyrosyl we the of SCF and SCF + ET-1 tyrosyl phosphorylation of Shc proteins in NHMC. with with Shc that stimulation with SCF + ET-1 a more rapid and a stronger tyrosyl phosphorylation of proteins corresponding to p52 in and p66 Shc than is with SCF and that this a in activation of tyrosine kinase tyrosine-phosphorylated Shc associates with Grb2 and the guanine nucleotide exchange factor SOS, leading to we the association of tyrosine-phosphorylated Shc with Grb2 also synergistically marked with SCF + ET-1 as with SCF only. in more corresponding to p52 and p66 Shc are observed in from SCF + ET-1-treated melanocytes than from a SCF shows that SCF + ET-1 Grb2 associates more in a complex with increased of Shc than it a SCF effect the association of tyrosine-phosphorylated Shc with Grb2. melanocytes stimulated for 2 with ET-1 The association of Shc and Grb2 was by with Shc or Grb2 followed by as under we the tyrosine phosphorylation of c-kit initiated by SCF binding to the c-kit receptor is stimulated by the concomitant addition of course by a rapid and more marked tyrosine phosphorylation of c-kit, which a with SCF + ET-1 with SCF only. In a ET-1 stimulation tyrosine phosphorylation of c-kit, a which that ET-1 and ETB receptor binding initiated signaling tyrosine phosphorylation of c-kit under conditions where c-kit is activated by SCF binding to its In human melanocytes, it is well binding to its receptor, ET-1 of which and and respectively, which then proliferation and Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar, G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). In addition to the recently been to also the MAPK in human melanocytes (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). In other types of cells, such as of a activation of MAPK by ET-1 been reported in relation to T. I. Y. I. Y. T. K. H. R. H. Y. J. Biol. 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