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

2000; Elsevier BV; Volume: 275; Issue: 43 Linguagem: Inglês

10.1074/jbc.m004346200

1083-351X

Genji Imokawa, Takeshi Kobayasi, Makoto Miyagishi,

Mast cells and histamine

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. Giebel L.B. Holmes S.A. Am. J. Hum. Genet. 1992; 50: 261-269PubMed Google Scholar, 7Ezoe K. Holmes S.A. Ho I. Bennett C.P. Bolognia J.L. Brueton L. Burn J. Falabella R. Gatto E.M. Ishii N. Moss C. Pittelkow M.R. Thompson E. Ward K.A. Spritz R.A. Am. J. Hum. Genet. 1995; 56: 58-66PubMed Google Scholar) or endothelin B receptor (8Puffenberger E.G. Hosoda K. Washington S.S. Nakao K. deWit D. Yanagisawa M. Chakravarti A. Cell. 1994; 79: 1257-1266Abstract Full Text PDF PubMed Scopus (790) Google Scholar, 9Amiel J. Attie T. Jan D. Pelet A. Edery P. Bidaud C. Lacombe D. Tam P. Simeoni J. Flori E. Nihoul-Fekete C. Munnich A. Lyonnet S. Hum. Mol. Genet. 1996; 5: 355-357Crossref PubMed Scopus (169) Google Scholar), respectively, have been documented. In Waardenburg syndrome type 2, mutation of microphthalmia-associated transcription factor is considered to be a central event leading to the dysfunction or loss of melanocytes (10Read A.P. Newton P.E. J. Med. 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. Cutler R.L. Krystal G. Mol. Cell. Biol. 1994; 14: 6926-6935Crossref PubMed Scopus (160) Google Scholar). In the epidermis, the most abundant cells which surround melanocytes are keratinocytes, that are known to produce increased amounts of SCF and ET-1 in response to several stimuli (24Hori Y. Hearing V.J. Nakayama J. Imokawa G. Melanogenesis and Malignant Melanoma. Elsevier Science B.V., Amsterdam1996: 35-48Google Scholar). Therefore, it is of particular value to clarify the cross-talk mechanism between ET-1 and SCF-mediated signaling in human melanocytes because each of those specific signaling pathways has been associated with the physiological stimulation of melanogenesis (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). The aim of our present study was to evaluate the mechanisms by which ET signaling activates MAPK and its underlying synergism with SCF. We now 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-Grb2 complex, which leads in turn to synergistic activation of the Ras/Raf-1/MEK/MAP kinase loop. NHMC were obtained from Morinaga Co. Endothelin derivatives were purchased from Sigma. Anti-phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology Inc. (New York), αERK1/2, α-phospho-specific ERK1/2, αMEK, α-phospho-specific antibodies from New England Labs Inc., α−c-kit and αGRB2 antibodies were from Santa Cruz Biotech. Inc., and α-Shc antibodies and anti-phosphotyrosine (clone RC20) from Transduction Lab. All other chemicals were of reagent grade. NHMC were maintained in modified MCDB 153 growth medium supplemented with 1 ng/ml recombinant basic fibroblast growth factor, 5 μg/ml insulin, 0.5 μg/ml hydrocortisone, 10 ng/ml phorbol 12-myristate 13-acetate, antibiotics (50 μg/ml gentamycin and 0.25 μg/ml amphotericin B), 0.5% fetal calf serum, and 0.2% bovine pituitary extract at 37 °C under a 5% CO2 atmosphere. In experiments to evaluate cellular effects or signaling changes, NHMC were seeded in culture trays at a density of 5–8 × 104 cells/ml, and were cultured in keratinocyte-SFM (Life Technologies, Inc.) containing bovine pituitary extract for 48 h, then treated with reagents at various concentrations. NHMC cultured in 96-well trays were incubated with ET-1 and/or SCF at concentrations of 0 to 100 nm. Twenty hours later, the cells were labeled for 4 h with 1.0 μCi/ml [3H]thymidine. After three washes with phosphate-buffered saline, the cells were trypsinized and harvested on a glass fiber filter, washed three times with distilled water, and twice with ice-cold ethanol, then dried. The radioactivity on the filter was directly measured using MATRIX 96 (Packard Bioscience Co.). These techniques were performed as reported previously (2Yada Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar). Briefly, for IP3 assay, cells were seeded in 24-well culture trays at a density of 3 × 104-105 cells/ml and cultured for 24–48 h. The media were aspirated and the MCDB 153 medium containing 10 mm LiCl was added and incubated for 10 min at 37 °C before stimulation. The ligand stimulation was terminated at designed times by adding 10% perchloric acid and the samples were kept on ice for 15 min. After neutralization with ice-cold 1.5 m KOH for 60 min on ice, the samples were centrifuged at 2,000 ×g for 10 min to remove KClO4 precipitate. The supernatants (100 μl each) were subjected to IP3 assay using the IP3 assay kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) (2Yada Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar). The content of IP3 in each sample was quantitatively determined from a calibration curve established using the binding protein specific for IP3 and [3H]IP3. Cells were incubated with ET-1 and/or SCF in 60-mm diameter culture dishes, then solubilized in 500 μl of ice-cold RIPA (radioimmunoprecipitation) buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.25% Na deoxycholate, 1 mm EDTA, 50 mm NaF, 1 mm sodium orthovanadate, 50 mm phenylmethylsulfonyl fluoride, and 1 mg/ml aprotinin. The mixtures were sonicated briefly and centrifuged at 14,000 ×g for 30 min at 4 °C. Protein concentrations in the supernatants were determined by the BCAassay kit(Pierce Chemical Co.). These techniques were performed as reported previously (25Sakai C. Ollmann M. Kobayashi T. Muller J. Vieira D. Imokawa G. Barsh G.S. Hearing V.J. EMBO J. 1997; 16: 3544-3552Crossref PubMed Scopus (86) Google Scholar, 26Kobayashi T. Imokawa G. Bennett D.C. Hearing V.J. J. Biol. Chem. 1998; 273: 31801-31805Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Lysates (20 μg/lane) or immunoprecipitated complex were separated by 10% SDS-PAGE and then transferred to Immobilon-P PVDF membranes (Millipore, Eschborn, Germany). Membranes were blocked with 3% bovine serum albumin in Tris-buffered saline containing Tween 20 (TBS-T) buffer (20 mm Tris-HCl, pH 7.2, 0.14 m NaCl, 0.1% Tween 20) for 3 h at room temperature, and then probed with primary antibodies in TBS-T buffer. After washing, blots were incubated with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech) for 1 h, and signals were visualized using enhanced chemiluminescence (ECL) detection reagents (Amersham Pharmacia Biotech). These techniques were performed as reported previously (27Kobayashi T. Urabe K. Winder A. Jimenez-Cervantes C. Imokawa G. Brewington T. Solano F. Garcia-Borron J.C. Hearing V.C. EMBO J. 1994; 13: 5818-5825Crossref PubMed Scopus (431) Google Scholar, 28Kobayashi T. Urabe K. Orlow S.J. Higashi K Imokawa G. Kwon B.S. Potterf B. Hearing V.J. J. Biol. Chem. 1994; 269: 29198-29205Abstract Full Text PDF PubMed Google Scholar). Grb2, Shc, and c-kit were immunoprecipitated from whole cell lysates by incubation with 4 μg of antibodies for 2 h at 4 °C. The resultant immune complexes were then precipitated by incubation with protein G-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C. The pellets were washed three times with RIPA buffer, once with phosphate-buffered saline, resuspended in SDS sample buffer (125 mm Tris-HCl, pH 6.8, 20%(v/v) glycerol, 4% (w/v) SDS, 100 mm dithiothreitol, 0.1% (w/v) bromphenol blue), and heated at 95 °C for 5 min. Immunoprecipitated and associated proteins were detected with immunoblotting as described above. Raf-1 was assayed according to the method reported previously (29Alessi D.R. Cohen P. Ashworth A. Cowley S. Leevers S.J. Marshall C.J. Methods Enzymol. 1995; 255: 279-290Crossref PubMed Scopus (155) Google Scholar) by its ability to activate MAPK kinase, which was then assayed by the activation of MAPK (29Alessi D.R. Cohen P. Ashworth A. Cowley S. Leevers S.J. Marshall C.J. Methods Enzymol. 1995; 255: 279-290Crossref PubMed Scopus (155) Google Scholar). An aliquot of each cell lysate was added to 5 μl of protein G-Sepharose conjugated to 1.0 μg of Raf-1 antibody (Transduction Laboratories) and was incubated for 60 min at 4 °C on a shaking platform. The suspensions were centrifuged for 1 min at 14,000 × g, the supernatants discarded, and the immunoprecipitates washed twice with 1.0 ml of buffer (20 mm Tris acetate, pH 7.5, 0.27m sucrose, 1 mm EDTA, 1 mm EGTA, 1 mm sodium orthovanadate, 10 mm sodium β-glycerophosphate, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 1% Triton X-100, 0.1% 2-mercaptoethanol, 1 mm benzamidine, 0.2 mmphenylmethylsulfonyl fluoride, and leupeptin 5 μg/ml), twice with 1.0 ml of buffer (50 mm Tris-HCl, pH 7.5, 0.03% Brij 35, 0.1 mm EGTA, and 0.1% 2-mercaptoethanol) and then assayed as described below. Eighteen μl of buffer (20 mm MOPS, pH 7.2, 5 mm EGTA, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 25 mm sodium β-glycerophosphate, 5 mm PKC inhibitor peptide (Upstate Biotechnology Inc.), 0.5 mm PKA inhibitor peptide (Upstate Biotechnology Inc.), 5 mm compound R24571 (Upstate Biotechnology Inc.), 20 mm magnesium chloride, and 0.13 mm ATP) containing 0.2 μg of glutathioneS-transferase-MAPKK1 and 0.7 μg of glutathioneS-transferase-MAPK was added to immunoprecipitates by Raf-1 antibody and, after incubation for 30 min at 30 °C on a shaking platform, a 2-μl aliquot was added to 15 μl of 20 mmMOPS (pH 7.2), 5 mm EGTA, 1 mm dithiothreitol, 1 mm sodium orthovanadate, 25 mm sodium β-glycerophosphate, MBP (0.67 mg/ml), 25 mm magnesium chloride, and 0.1 mm [γ-32P]ATP (∼1000 Ci/mmol). After incubation for 10 min at 30 °C, the incorporation of phosphate into MBP was determined by autoradiography of MBP separated with SDS-PAGE. When ET-1 and SCF are concomitantly added at constant and varied concentrations, respectively, there is a synergistic stimulation of DNA synthesis in NHMC (Fig.1). At ET-1 and SCF concentrations of 10 nm each, a marked synergistic stimulation of DNA synthesis is elicited with a 62-fold increase relative to the 1.2- and 7.2-fold increases elicited by SCF or ET-1 treatments alone, respectively. In order to clarify whether the activation of MAPK is mediated via the endothelin-binding ETA or ETBreceptor, 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 ETB receptor antagonist BQ788 completely abolishes endothelin-induced tyrosine phosphorylation of ERK2 whereas the ETA receptor antagonist BQ610 fails to inhibit the phosphorylation (Fig.2), indicating that the activation of MAPK is mediated through the endothelin B receptor. The addition of 1 μg/ml pertussis toxin abolishes tyrosine phosphorylation of ERK2 (Fig. 3), showing that the Giprotein is associated with endothelin-induced signaling leading to the activation of MAP kinase. Similarly, the phospholipase C inhibitor,U73122, down-regulates tyrosine phosphorylation of ERK2 in a dose-dependent manner (Fig.4), indicating that phospholipase C is also involved in the activation of MAPK during the intracellular signaling initiated by ET and ETB receptor binding. The addition of the calcium chelator, BAPTA, has no effect on the phosphorylation of ERK2 (Fig. 5), showing there is no involvement of calcium mobilization in the activation of MAPK. In contrast, the PKC inhibitor, calphostine, abolishes the phosphorylation of ERK2 (Fig. 6), indicating that activation of PKC is required for the activation of MAPK.Figure 3Pertussis toxin abolishes the tyrosine phosphorylation of ERK2. Human melanocytes were treated with 10 nm ET-1 in the presence of pertussis toxin (1 μg/ml) and 5 min later were harvested and solubilized; the activation of MAPK was then evaluated by measuring the tyrosine phosphorylation of ERK2 as detailed under "Experimental Procedures." PXT, pertussis toxin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4The phospholipase C inhibitor, U73122, down-regulates the tyrosine phosphorylation of ERK2. Following treatment for 15 min with the phospholipase C inhibitor, U73122, or its inactive analogue, U73343 (1 and 5 μg/ml each), human melanocytes were treated with 10 nm ET-1 and 5 min later were harvested and solubilized. The activation of MAPK was evaluated by measuring the tyrosine phosphorylation of ERK2 as detailed under "Experimental Procedures." PLC, phospholipase C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5The calcium chelator, BAPTA , has no effect on the phosphorylation of ERK2. Following incubation with the intracellular calcium chelator, BAPTA-AM (at 30 μm), human melanocytes were treated with 10 nm ET-1 and 5 min later were harvested and solubilized. The activation of MAPK was evaluated by measuring the tyrosine phosphorylation of ERK2 using Western immunoblotting as detailed under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Calphostine abolishes the phosphorylation of ERK2. Following incubation for 1 h with calphostine (at 3 μm), human melanocytes were treated with 10 nm ET-1 and 5 min later were harvested and solubilized. The activation of MAPK was evaluated by measuring the tyrosine phosphorylation of ERK2 as detailed under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) The time course of the formation of IP3 following SCF and ET-1 treatment (Fig.7) revealed that there is no difference in the level of IP3 following treatment with ET-1 + SCF or ET-1 alone, indicating no synergy occurring in the PKC pathway. In the evaluation of tyrosine phosphorylation of ERK-1 and -2, an indicator of MAPK activation, as assessed by Western blotting using phosphotyrosine antibodies following immunoprecipitation with anti-ERK-1 and -2, we found that there is a synergistic effect on tyrosine phosphorylation in ET-1 + SCF-treated NHMC compared with ET-1 or SCF alone. There were increases both in the duration and intensity of tyrosine phosphorylation, which reached a peak within 10 min after stimulation (Fig. 8). In the evaluation of serine phosphorylation of MEK, an indicator of MAPKK activation, as assessed by Western blotting using a phospho-specific MEK antibody, there is a synergistic effect in SCF + ET-1-treated melanocytes with a peak within 4 min after stimulation (Fig.9). This indicates that the synergy in the activation of MAPKK precedes the observed synergy in MAPK activation in SCF + ET-1-treated NHMC. Analysis of Raf-1 activity, an indicator of MAPKKK, as assessed by the final phosphorylation of MBP, showed that 10 nm ET-1 significantly stimulates Raf-1 activity, which indicates that Raf-1 is at least one convergence point from the PKC pathway to the MAPK pathway. Furthermore, there is a synergistic effect on Raf-1 activity in SCF + ET-1-treated melanocytes (Fig. 10), suggesting the possibility that the synergistic convergence between SCF and ET-1 signaling is located nearby Raf-1.Figure 9Synergistic effect on serine phosphorylation of MEK1/2. Human melanocytes were stimulated with 10 nm ET-1 and/or 10 nm SCF. The activation of MEK was evaluated by measuring the serine phosphorylation of MEK at the indicated times by Western immunoblotting using phospho-specific MEK1/2 antibody as detailed under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 10Synergistic effect on Raf-1 activity.Human melanocytes were stimulated for 2 min with 10 nm ET-1 and/or 10 nm SCF. The activation of Raf-1 was evaluated by measuring the phosphorylated MBP by a coupled assay with inactive MAPK-glutathione S-transferase and inactive MEK1-glutathioneS-transferase as detailed under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) The mechanism of tyrosine kinase receptor-stimulated MAPK signaling involves the formation of complexes between the guanine nucleotide exchange protein SOS, and the SH2 and SH3 domain-containing adaptor protein Grb2 either with autophosphorylated growth factor receptors or another tyrosine-phosphorylated adaptor protein known as Shc. Since Shc involvement in this signaling pathway requires tyrosyl phosphorylation, we compared the effects of SCF only and SCF + ET-1 on tyrosyl phosphorylation of Shc proteins in NHMC. Western blotting with phosphotyrosine antibodies following immunoprecipitation with Shc antibodies (Fig. 11) demonstrates that stimulation with SCF + ET-1 induces a more rapid and a stronger tyrosyl phosphorylation of proteins corresponding to p52 (1.2-fold in densitometric intensity) and p66 Shc than is elicited with SCF alone, and that this reaches a maximum (p52: 2.0-fold in densitometric intensity) within 5 min after stimulation. Upon activation of tyrosine kinase receptors, tyrosine-phosphorylated Shc associates with Grb2 and the guanine nucleotide exchange factor SOS, thereby leading to Ras activation. Therefore, we next examined whether the association of tyrosine-phosphorylated Shc with Grb2 also becomes synergistically marked following treatment with SCF + ET-1 as compared with SCF only. As shown in Fig. 12, more distinct bands corresponding to p52 and p66 Shc are observed in immunoprecipitates from SCF + ET-1-treated melanocytes than from a single SCF stimulation. This shows that upon SCF + ET-1 stimulation, Grb2 associates more strongly in a complex with increased amounts of tyrosyl-phosphorylated Shc than it does upon a single SCF stimulation.Figure 12Synergistic effect on the association of tyrosine-phosphorylated Shc with Grb2. Human melanocytes were stimulated for 2 min with 10 nm ET-1 and/or 10 nm SCF. The association of phosphorylated Shc and Grb2 was evaluated by immunoprecipitation with Shc or Grb2 antibodies, followed by Western immunoblotting as detailed under "Experimental Procedures." IP, immunoprecipitation; WB,Western immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Finally, we examined whether the tyrosine phosphorylation of c-kit initiated by SCF binding to the c-kit receptor is stimulated by the concomitant addition of ET-1. Time course experiments assessed by Western blotting (Fig. 13) reveal a rapid and more marked tyrosine phosphorylation of c-kit, which reaches a maximum within 5 min after stimulation, with SCF + ET-1 compared with SCF only. In contrast, a single ET-1 stimulation does not elicit any tyrosine phosphorylation of c-kit, a finding which strongly suggests that ET-1 and ETB receptor binding initiated signaling stimulates tyrosine phosphorylation of c-kit only under conditions where c-kit is activated by SCF binding to its receptor. In human melanocytes, it is well established that, after binding to its receptor, ET-1 triggers hydrolysis of polyphosphoinositide, which generates IP3 and diacylglycerol, mobilizing intracellular Ca2+ and activating PKC, respectively, which then stimulate proliferation and melanization (2Yada Y. Higuchi K. Imokawa G. J. Biol. Chem. 1991; 266: 18352-18357Abstract Full Text PDF PubMed Google Scholar, 16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar). In addition to the PKC pathway, ET has recently been shown to also activate the MAPK cascade 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 cardiomyocytes of neonatal rats, a similar activation of MAPK by ET-1 has been reported in relation to mechanical stress-induced cardiac hypertrophy (30Yamazaki T. Issei-Komuro I. Kudoh Sm Zou Y. Shiojima I. Hiroi Y. Mizuno T. Maemura K. Kurihara H. Aikawa R. Takano H. Yazaki Y. J. Biol. Chem. 1996; 271: 3221-3228Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). However, the mechanism by which the Giprotein-coupled ET receptor activates the MAPK cascade is still poorly characterized. The site at which the PKC pathway stimulated by ET signaling enters the tyrosine kinase pathway (including the MAPK cascade) remains to be elucidated in human melanocytes. Such an understanding would provide a basis for cross-talk mechanisms between ET-1 and SCF initiated signaling. Therefore, we have attempted to determine whether the ET signaling cascade, which consists of its receptor binding, G protein, phospholipase C, calcium mobilization, and PKC activation is really associated with the activation of MAPK. The present study demonstrates clearly that ET activates Erk1/2 in a time-dependent manner in NHMC that express the ETB receptor and that the ET-induced activation of Erk1/2 is independent of calcium mobilization, but is largely associated with the PKC pathway via binding to ETB receptor, Giprotein, and the activation of phospholipase C. This strongly suggests that activation of MAPK is involved in the molecular mechanism associated with protein-coupled receptors. In contrast to another study which showed that the formation of the Shc-Grb2 complex can be mediated by G protein activation by ET-1 in astrocytes (31Cazaubon S.M. Ramos-Morales F. Fischer S. Schweighoffer F. Strosberg A.D. Couraud P.O. J. Biol. Chem. 1994; 269: 24805-24809Abstract Full Text PDF PubMed Google Scholar), we found that Erk1/2 activation by ET alone was not accompanied by tyrosine phosphorylation of Shc and the formation of Shc-Grb2 complexes, whereas the prior sequential activation of MEK and Raf-1 were detectable. Since in other cells, Raf-1 is reported to be a target for PKC (32Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1156) Google Scholar, 33Zou Y. Komuro I. Yamazaki T. Aikawa R. Kudoh S. Shiojima I. Hiroi Y. Mizuno T. Yazaki Y. J. Biol. Chem. 1996; 271: 33592-33597Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 34Marais R. Light Y. Mason C. Paterson H. Olson M.F. Marshall C.J. Science. 1998; 280: 109-112Crossref PubMed Scopus (398) Google Scholar, 35Van Dijk M.C. Hilkmann H. van Blitterswijk W.J. Biochem. J. 1997; 325: 303-307Crossref PubMed Scopus (63) Google Scholar, 36Cacace A.M. Ueffing M. Philipp A. Han E.K. Kolch W. Weinstein I.B. Oncogene. 1996; 13: 2517-2526PubMed Google Scholar, 37Ueffing M. Lovric J. Philipp A. Mischak H. Kolch W. Oncogene. 1997; 15: 2921-2927Crossref PubMed Scopus (71) Google Scholar), Raf-1 activation, probably via serine/threonine phosphorylation which modifies its catalytic activity, appears to be a convergent point between the PKC and MAPK pathways. Regarding the association between those two pathways, the sum of the above findings indicate that activation of MAPK during the ET signaling pathway is mediated through ETB receptor/Gi protein/phospholipase C/PKC and the Raf-1 loop. SCF and ET-1 binding initiated intracellular signaling that leads to melanization and cell growth of NHMC are transmitted by two major classes of cell surface receptors, tyrosine kinase growth factor receptors (38Rottapel R. Reedijk M. Williams D.E. Lyman S.D. Anderson D.M. Pawson T. Bernstein A. Mol. Cell. Biol. 1991; 11: 3043-3051Crossref PubMed Scopus (201) Google Scholar) and G protein-coupled receptors (16Imokawa G. Yada Y. Kimura M. Biochem. J. 1996; 314: 305-312Crossref PubMed Scopus (154) Google Scholar), respectively. Therefore, it is of interest to determine which signaling pathway is responsible for this synergistic effect. At first, we assumed that the convergence between ET-1 and SCF signaling occurs at the activation of MAPK, because we had previously found that ET-1-induced stimulation of melanization and proliferation in NHMC is associated with the activation of MAPK, a pathway very similar to that mediated by SCF (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. Cutler R.L. Krystal G. Mol. Cell. Biol. 1994; 14: 6926-6935Crossref PubMed Scopus (160) Google Scholar). In this connection, Western blotting using tyrosyl MAPK antibodies demonstrates that the activation of MAPK occurs synergistically between SCF and ET-1-initiated signaling. Similar synergisms to the activation of MAPK have been reported between ET-1 and angiotensin II in cultured cardiomyocytes (30Yamazaki T. Issei-Komuro I. Kudoh Sm Zou Y. Shiojima I. Hiroi Y. Mizuno T. Maemura K. Kurihara H. Aikawa R. Takano H. Yazaki Y. J. Biol. Chem. 1996; 271: 3221-3228Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) and between SCF and erythropoietin in human erythroid colony-forming cells (39Sui X. Krantz S.B. You M. Zhao Z. Blood. 1998; 92: 1142-1149Crossref PubMed Google Scholar), in relation to mechanical stress-induced cardiac hypertrophy and expanded erythropoiesis, respectively. Since MAPK activation is generally accompanied by the prior sequential activation of MEK and Raf-1 (40Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2278) Google Scholar), we determined whether the synergism in the activation of MAPK is reflected by the synergistic activation of MEK and Raf-1. Related experiments using Western blotting and kinase assays revealed that there is a sequential synergistic activation in the MAPK cascade consisting of Raf-1, MEKK, MEK, and MAPK. This suggests that the convergence point between ET-1 and SCF-initiated signaling is located upstream of Raf-1. As we confirmed a role for Raf-1 in the cross-talk mechanism between ET-1-associated PKC and SCF-associated tyrosine kinase pathways, we next determined signaling mechanisms leading to the synergistic Raf-1 activation by examining the influence of SCF on the ET-1-dependent PKC pathway. The formation of IP3 following stimulation is a hallmark for evaluation of PKC activation because IP3 and the protein kinase C activator, diacylglycerol, are simultaneously generated at an equimolar ratios. The time course of formation of IP3 following SCF and ET-1 treatment revealed that there is no difference in the raised level of IP3 induced by ET-1 + SCF or ET-1 alone, indicating no synergy occurs in the PKC pathway. The mechanism of tyrosine kinase receptor-stimulated mitogenic signaling involves the formation between complexes of the guanine nucleotide exchange protein SOS, and the SH2 and SH3 domain-containing adaptor protein Grb2 with another tyrosine-phosphorylated adaptor protein Shc (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. Cutler R.L. Krystal G. Mol. Cell. Biol. 1994; 14: 6926-6935Crossref PubMed Scopus (160) Google Scholar). Recent studies have shown that some G protein-coupled receptors utilize the same effectors as the tyrosine kinase receptor pathway (e.g. Shc-Grb-SOS), resulting in Ras and MAPK activation (41Dabrowski A. VanderKuur J.A. Carter-Su C. Williams J.A. J. Biol. 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In this study, the synergism between ET-1 and SCF was found to be accompanied by synergistic tyrosyl phosphorylation of proteins corresponding to p52 and p66 Shc, leading to synergistic association of tyrosine-phosphorylated Shc with Grb2. It has also been shown that ET-1 signaling through heterotrimeric G protein-coupled receptors stimulates MAPK activity in primary cultures of astrocytes (46Cazaubon S. Parker P.J. Strosberg A.D. Couraud P.O. Biochem. J. 1993; 293: 381-386Crossref PubMed Scopus (68) Google Scholar) via an increase in the tyrosine phosphorylation of Shc, which is followed by its stable association with Grb2 (31Cazaubon S.M. Ramos-Morales F. Fischer S. Schweighoffer F. Strosberg A.D. Couraud P.O. J. Biol. Chem. 1994; 269: 24805-24809Abstract Full Text PDF PubMed Google Scholar). Those studies suggested that ET-1-induced MAPK activation is a G protein-coupled pathway that involves Shc, Grb2, and probably Raf-1. Thus, the Shc-Grb2 complex may be involved in activation of the MAPK pathway, not only by several receptor tyrosine kinases but also by heterotrimeric G protein-coupled receptors, such as ET-1 receptors. In contrast to those studies using astrocytes, our study using human melanocytes showed that ET-1 does not stimulate tyrosine phosphorylation of Shc and its association with Grb2 even at its mitogenic and melanogenic concentrations. Interestingly, Western blotting analysis of tyrosine phosphorylation of c-kit upstream of Shc-Grb2 association revealed that this synergistic activation with adaptor molecules is initiated by the synergistic tyrosine phosphorylation of the SCF receptor, c-kit. Again, it should be noted that in human melanocytes the combination of ET-1 and SCF (but not ET-1 alone) enhances tyrosine phosphorylation of c-kit. A similar activation of receptor tyrosine kinases through intracellular signal cross-talk with ET-1-associated G-protein-coupled receptors has been documented for the epidermal growth factor receptor of Rat-1 cells (47Daub H. Weiss F.U. Wallasch C. Ullrich A. Nature. 1996; 379: 557-560Crossref PubMed Scopus (1318) Google Scholar) and of smooth muscle cells (48Iwasaki H. Eguchi S. Ueno H. Marumo F. Hirata Y. Endocrinology. 1999; 140: 4659-4668Crossref PubMed Google Scholar) in which only ET-1 can stimulate tyrosine phosphorylation of the receptor. In conclusion, the sum of the above findings indicates that synergistic cross-talk between SCF and ET-1 signaling is initiated through the pathway of tyrosine phosphorylation of c-kit. This results in the synergistically enhanced formation of Shc-Grb2 complex, which leads to the synergistic activation of the Ras/Raf-1/MEK/MAPK loop. ET-1 associated activation of PKC probably plays a role in the enhanced tyrosine phosphorylation of c-kit although the detailed mechanism is not clear. Thus, our results demonstrate a role for the c-kit tyrosine kinase receptor as a downstream mediator in synergistic mitogenic signaling induced by ET-1 + SCF and suggest a ligand-independent mechanism for c-kit activation through a synergistic intracellular signal cross-talk.