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Regulation of Melanogenesis through Phosphatidylinositol 3-Kinase-Akt Pathway in Human G361 Melanoma Cells

2000; Elsevier BV; Volume: 115; Issue: 4 Linguagem: Inglês

10.1046/j.1523-1747.2000.00095.x

1523-1747

Masahiro Oka, Hiroshi Nagai, Hideya Ando, Mizuho Fukunaga, Miyoko Matsumura, Keishi Araki, Wataru Ogawa, Takeshi Miki, Motoyoshi Sakaue, Katsuhiko Tsukamoto, Hiroaki Konishi, Ushio Kikkawa, Masamitsu Ichihashi,

Bioactive natural compounds

The involvement of the phosphatidylinositol 3-kinase pathway in the regulation of melanogenesis was examined using human G361 melanoma cells. In the cells treated with wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, the melanin content increased concomitant with the elevated protein level of tyrosinase, a key enzyme in melanogenesis. Northern blot analysis revealed that the mRNA level of tyrosinase increased transiently on treatment of the cells with the phosphatidylinositol 3-kinase inhibitor. When the cells were infected with the adenovirus vector encoding the mutant adapter subunit of phosphatidylinositol 3-kinase, which acts as a dominant negative of phosphatidylinositol 3-kinase, both the melanin content and the expression of tyrosinase increased. In cells infected with the adenovirus vector encoding the constitutively active mutant of the lipid kinase, a decrease in melanin content as well as reduced expression of tyrosinase was observed. In cells expressing the constitutively active mutant of the serine-threonine protein kinase Akt, one of the downstream targets of phosphatidylinositol 3-kinase, the melanin content decreased as in the cells overproducing the constitutively active mutant of phosphatidylinositol 3-kinase. These results indicate that phosphatidylinositol 3-kinase regulates melanogenesis by modulating the expression of tyrosinase, and that activation of Akt is sufficient for suppression of melanin production in G361 melanoma cells. The involvement of the phosphatidylinositol 3-kinase pathway in the regulation of melanogenesis was examined using human G361 melanoma cells. In the cells treated with wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, the melanin content increased concomitant with the elevated protein level of tyrosinase, a key enzyme in melanogenesis. Northern blot analysis revealed that the mRNA level of tyrosinase increased transiently on treatment of the cells with the phosphatidylinositol 3-kinase inhibitor. When the cells were infected with the adenovirus vector encoding the mutant adapter subunit of phosphatidylinositol 3-kinase, which acts as a dominant negative of phosphatidylinositol 3-kinase, both the melanin content and the expression of tyrosinase increased. In cells infected with the adenovirus vector encoding the constitutively active mutant of the lipid kinase, a decrease in melanin content as well as reduced expression of tyrosinase was observed. In cells expressing the constitutively active mutant of the serine-threonine protein kinase Akt, one of the downstream targets of phosphatidylinositol 3-kinase, the melanin content decreased as in the cells overproducing the constitutively active mutant of phosphatidylinositol 3-kinase. These results indicate that phosphatidylinositol 3-kinase regulates melanogenesis by modulating the expression of tyrosinase, and that activation of Akt is sufficient for suppression of melanin production in G361 melanoma cells. phosphatidylinositol Src homology Melanin is a mixture of pigmented bipolymers synthesized by specialized cells such as melanocytes and melanoma cells, and has a number of important roles including protection from ultraviolet light, the determination of phenotypic appearance, and absorption of toxic drugs and chemicals (Quevedo et al., 1987Quevedo Jr, W.C. Fitzpatrick T.B. Szabó G. Jimbow K. Biology of melanocytes.in: Fitzpatrick T.B. Eisen A.Z. Wolff K. Freedberg I.M. Austen K.F. Dermatology in General Medicine. 3rd edn. McGraw-Hill, New York1987: 224-251Google Scholar;Hearing, 1999Hearing V.J. Biochemical control of melanogenesis and melanosomal organization.J Invest Dermatol Symp Proc The. 1999; 4: 24-28Abstract Full Text PDF PubMed Scopus (179) Google Scholar). The initial step of melanin synthesis begins with oxidation of tyrosine that is catalyzed by tyrosinase, a rate-limiting enzyme of melanogenesis. It has been shown that melanogenesis is modulated by various intracellular signaling mechanisms including protein kinases such as cAMP-dependent protein kinase (Körner and Pawelek, 1977Körner A. Pawelek J. Activation of melanoma tyrosinase by a cyclic AMP-dependent protein kinase in a cell-free system.Nature. 1977; 267: 444-447Crossref PubMed Scopus (64) Google Scholar), protein kinase C-α (Gruber et al., 1992Gruber J.R. Ohno S. Niles R.M. Increased expression of protein kinase Cα plays a key role in retinoic acid-induced melanoma differentiation.J Biol Chem. 1992; 267: 13356-13360Abstract Full Text PDF PubMed Google Scholar;Oka et al., 1993Oka M. Ogita K. Saito N. Mishima Y. Selective increase of the α subspecies of protein kinase C and inhibition of melanogenesis induced by retinoic acid in melanoma cells.J Invest Dermatol. 1993; 100: 204s-208sCrossref PubMed Scopus (19) Google Scholar), protein kinase C-β (Park et al., 1993Park H.-Y. Russakovsky V. Ohno S. Gilchrest B.A. The β isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells.J Biol Chem. 1993; 268: 11742-11749Abstract Full Text PDF PubMed Google Scholar), and mitogen-activated protein kinase (Englaro et al., 1995Englaro W. Rezzonico R. Durand-Clément M. Lallemand D. Ortonne J.-P. Ballotti R. Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells.J Biol Chem. 1995; 270: 24315-24320Crossref PubMed Scopus (195) Google Scholar). Recently, phosphatidylinositol (PI) 3-kinase has also been suggested to be involved in the regulation of melanogenesis, as a potent inhibitor of PI 3-kinase, LY294002, stimulates melanin production in mouse B16 melanoma cells (Buscà et al., 1996Buscà R. Bertolotto C. Ortonne J.-P. Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70S6-kinase pathway induces B16 melanoma cell differentiation.J Biol Chem. 1996; 271: 31824-31830Crossref PubMed Scopus (220) Google Scholar). Direct evidence for the involvement of PI 3-kinase in the regulation of melanogenesis has not been available, however. PI 3-kinase phosphorylates the D3 position of the inositol ring of phosphoinositides, and intracellular concentrations of D3-phosphorylated phosphoinositides rise sharply in response to a wide variety of extracellular stimuli, suggesting a possible function of these lipids as second messengers (Toker and Cantley, 1997Toker A. Cantley L.C. Signalling through the lipid products of phosphoinositide-3-OH kinase.Nature. 1997; 387: 673-676Crossref PubMed Scopus (1192) Google Scholar;Vanhaesebroeck et al., 1997Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Phosphoinositide 3-kinases: a conserved family of signal transducers.Trends Biochem Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (814) Google Scholar;Wymann and Pirola, 1998Wymann M.P. Pirola L. Structure and function of phosphoinositide 3-kinases.Biochim Biophys Acta. 1998; 1436: 127-150Crossref PubMed Scopus (566) Google Scholar). Molecular cloning has revealed that PI 3-kinase is a family of multiple members, which are grouped into three classes on the basis of their lipid substrate specificity, the subunit structure, and the regulation mode of the enzymatic activity. Class I PI 3-kinase phosphorylates PI, PI 4-phosphate [PI(4)P], and PI 4,5-bisphosphate [PI(4,5)P2], whereas Class II PI 3-kinase recognizes PI and PI(4)P, but not PI(4,5)P2. Class III PI 3-kinase has a substrate specificity restricted to PI. Class I PI 3-kinase is further classified into two subclasses A and B according to the subunit structure. Class IA PI 3-kinase is composed of a 110 kDa catalytic subunit (p110α, β, and δ) and an 85 kDa adapter subunit (p85) containing Src homology (SH) 2 domain. This class of PI 3-kinase is established to be a downstream effector of a variety of receptor type and nonreceptor type tyrosine kinases, and has been studied extensively in a variety of cellular functions including mitogenic response, apoptosis, and vesicular trafficking/secretion. Class IB PI 3-kinase, activated by the βγ subunits of the heterotrimeric G-protein, also comprises a 110 kDa catalytic subunit (p110γ) but does not have an SH2-domain-containing adapter subunit. Both Class IA and Class IB enzymes are sensitive to the cell-permeable pharmacologic inhibitors of PI 3-kinase, wortmannin and LY294002, at relatively low concentrations (Arcaro and Wymann, 1993Arcaro A. Wymann M.P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-triphosphate in neutrophil responses.Biochem J. 1993; 296: 297-301Crossref PubMed Scopus (1027) Google Scholar;Vlahos et al., 1994Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one (LY294002).J Biol Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar;Wymann and Pirola, 1998Wymann M.P. Pirola L. Structure and function of phosphoinositide 3-kinases.Biochim Biophys Acta. 1998; 1436: 127-150Crossref PubMed Scopus (566) Google Scholar). Several downstream targets of Class I PI 3-kinase have been identified (Toker and Cantley, 1997Toker A. Cantley L.C. Signalling through the lipid products of phosphoinositide-3-OH kinase.Nature. 1997; 387: 673-676Crossref PubMed Scopus (1192) Google Scholar;Vanhaesebroeck et al., 1997Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Phosphoinositide 3-kinases: a conserved family of signal transducers.Trends Biochem Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (814) Google Scholar;Wymann and Pirola, 1998Wymann M.P. Pirola L. Structure and function of phosphoinositide 3-kinases.Biochim Biophys Acta. 1998; 1436: 127-150Crossref PubMed Scopus (566) Google Scholar). A prime target of the lipid kinase is a serine-threonine kinase Akt (also known as PKB), the cellular homolog of a rodent transforming oncogene product v-Akt (Hemmings, 1997Hemmings B.A. Akt signaling: linking membrane events to life and death decisions.Science. 1997; 275: 628-630Crossref PubMed Scopus (423) Google Scholar;Coffer et al., 1998Coffer P.J. Jin J. Woodgett J.R. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.Biochem J. 1998; 335: 1-13Crossref PubMed Scopus (953) Google Scholar;Downward, 1998Downward J. Mechanisms and consequences of activation of protein kinase B/Akt.Curr Opin Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1165) Google Scholar;Chan et al., 1999Chan T.O. Rittenhouse S.E. Tsichlis P.E. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylayion.Annu Rev Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (824) Google Scholar). Akt has a catalytic domain and a pleckstrin homology domain in the carboxyl- and amino-terminal regions, respectively, and three subtypes of Akt have been isolated thus far. Class I PI 3-kinase is proposed to activate Akt by the association of PI 3-kinase products to the pleckstrin homology domain and/or by promoting phosphorylation of Akt in the catalytic domain. It has been generally accepted that Akt plays a central role in the promotion of survival of a wide range of cell types, in modulation of the metabolic actions regulated by insulin (Hemmings, 1997Hemmings B.A. Akt signaling: linking membrane events to life and death decisions.Science. 1997; 275: 628-630Crossref PubMed Scopus (423) Google Scholar;Coffer et al., 1998Coffer P.J. Jin J. Woodgett J.R. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.Biochem J. 1998; 335: 1-13Crossref PubMed Scopus (953) Google Scholar;Downward, 1998Downward J. Mechanisms and consequences of activation of protein kinase B/Akt.Curr Opin Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1165) Google Scholar;Chan et al., 1999Chan T.O. Rittenhouse S.E. Tsichlis P.E. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylayion.Annu Rev Biochem. 1999; 68: 965-1014Crossref PubMed Scopus (824) Google Scholar), as well as in nitric oxide synthesis in endothelial cells (Dimmeler et al., 1999Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation.Nature. 1999; 399: 601-605Crossref PubMed Scopus (2910) Google Scholar;Fulton et al., 1999Fulton D. Gratton J.-P. McCabe T.J. et al.Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.Nature. 1999; 399: 597-601Crossref PubMed Scopus (2086) Google Scholar). In this study, we investigated whether Class IA PI 3-kinase, the first identified and the best characterized member of the family, is involved in melanogenesis by using a melanotic human melanoma cell line, G361. To this end, adenovirus vectors encoding the dominant negative and constitutively active mutants of Class IA PI 3-kinase were employed. Moreover, the role of Akt in melanin production was studied by overproducing a constitutively active mutant of the enzyme in the melanoma cells. We report here direct evidence that melanogenesis is regulated through the PI 3-kinase-Akt pathway. Human G361 melanoma cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan), and cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum. Where indicated, the cells were treated with wortmannin (Sigma, St. Louis, MO), which had been stored at -20°C in the dark as a dimethyl sulfoxide solution until use. Adenovirus vectors encoding the dominant negative mutant of PI 3-kinase (Δp85), the constitutively active mutant of PI 3-kinase (Myr-p110), and the constitutively active mutant of Akt (Myr-Akt) were developed as described previously (Sakaue et al., 1997Sakaue H. Ogawa W. Takata M. et al.Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3-L1 adipocytes.Mol Endocrinol. 1997; 11: 1552-1562Crossref PubMed Scopus (115) Google Scholar;Kotani et al., 1999Kotani K. Ogawa W. Hino Y. et al.Dominant negative forms of Akt (protein kinase B) and atypical protein kinase Cλ do not prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene transcription.J Biol Chem. 1999; 274: 21305-21312Crossref PubMed Scopus (94) Google Scholar). These virus vectors were termed AxCAΔp85, AxCA Myr-p110, and AxCAMyrAkt, respectively. The adenovirus vector encoding the lacZ gene designated as AxCALacZ (Kanegae et al., 1995Kanegae Y. Lee G. Sato Y. et al.Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase.Nucl Acids Res. 1995; 23: 3816-3821Crossref PubMed Scopus (595) Google Scholar) was a gift from Dr. Izumu Saito (University of Tokyo). The cells infected with each adenovirus vector at 10 plaque-forming units (PFU) per cell were cultured for 72 h and were then employed for the experiments. The melanin content was determined by the method ofGordon and Gilchrest, 1989Gordon P.R. Gilchrest B.A. Human melanogenesis is stimulated by diacylglycerol.J Invest Dermatol. 1989; 93: 700-702Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Briefly, the cells (approximately 1 × 105 cells) were pelleted, solubilized in 1 ml of 1 N NaOH/10% dimethyl sulfoxide for 2 h at 80°C, and centrifuged at 12,000 × g for 10 min. The absorbance of the supernatant was measured at 470 nm and compared with the standard curve for synthetic melanin (Sigma). The melanin content was expressed as pg per cell. The PI 3-kinase activity was measured as described byEndemann et al., 1990Endemann G. Yonezawa K. Roth R.A. Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase.J Biol Chem. 1990; 265: 396-400Abstract Full Text PDF PubMed Google Scholar. The cells (approximately 1 × 106 cells) were lyzed in 20 mM Tris-HCl, pH 7.6, containing 1% (wt/vol) Nonidet P-40, 10% (vol/vol) glycerol, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. The lysate was centrifuged, and the supernatant was subjected to immunoprecipitation with a monoclonal antibody against phosphotyrosine (PY20, Transduction Laboratories, Lexington, KY). The immunoprecipitate was incubated with 0.2 mg per ml PI for 5 min at room temperature, and the PI 3-kinase reaction was carried out in the reaction mixture (50 μl) containing 20 mM HEPES, pH 7.1, 50 mM MgCl2, and 250 μM[γ-32P]adenosine-5′-triphosphate (ATP). The reaction was stopped by the addition of 15 μl of 4 N HCl and 130 μl of chloroform:methanol (1:1). Tubes were mixed for 30 s, and 20 μl of the lower layer was subjected to thin-layer chromatography in chloroform/methanol/4 M ammonium hydroxide (9:7:2); the generation of radioactive PI 3-phosphate [PI(3)P] was detected with a bioimaging analyzer (BAS 2000, Fuji Film, Tokyo, Japan). For the detection of tyrosinase, the cells (approximately 1 × 106 cells) were lyzed at 0–4°C in 100 mM Tris-HCl, pH 7.2, containing 1% (wt/vol) Nonidet P-40, 0.01% sodium dodecyl sulfate (SDS), 0.1 mM phenylmethylsulfonyl fluoride, 10 μg per ml aprotinin, and 10 μg per ml leupeptin. The lysate was centrifuged, and the supernatant (20 μg protein) was subjected to SDS-polyacrylamide gel electrophoresis using a 7.5% polyacrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-p, Millipore, Bedford, MA). After blocking with 5% bovine serum albumin, the membrane was incubated with a polyclonal antibody αPEP7 (Jiménez et al., 1991Jiménez M. Tsukamoto K. Hearing V.J. Tyrosinases from two different loci are expressed by normal and by transformed melanocytes.J Biol Chem. 1991; 266: 1147-1156Abstract Full Text PDF PubMed Google Scholar) at 1:1000 dilution as a primary antibody. The αPEP7 antibody, which is specific to tyrosinase, was donated by Dr. Vincent J. Hearing (National Institutes of Health). The membrane was then incubated with a peroxidase-conjugated antirabbit antibody at 1:4000 dilution and visualization was carried out with an enhanced chemiluminescence kit according to the manufacturer's protocol (Amersham, Buckinghamshire, U.K.). Total RNA (25 μg) isolated by the guanidium isothiocynate method (Chirgwin et al., 1979Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16547) Google Scholar) was electrophoresed on a 1% agarose-formaldehyde gel and blotted onto a nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany). After baking for 2 h at 80°C, the membrane was prehybridized for 1 h at 42°C in 40% (vol/vol) formamide, 1 × Denhardt's solution, 4 × sodium citrate/chloride buffer (SSC) (1 × SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 7 mM Tris-HCl, pH 7.5, and 20 μg per ml sheared salmon sperm DNA. The blot was then hybridized overnight at 42°C with randomly primed 32P-labeled murine tyrosinase cDNA clone Tyrs-J (Yamamoto et al., 1989Yamamoto H. Takeuchi S. Kudo T. Sato C. Takeuchi T. Melanin production in cultured albino melanocytes transfected with mouse tyrosinase cDNA.Jpn J Genet. 1989; 64: 121-135Crossref PubMed Scopus (90) Google Scholar) in a solution containing 4 × SSC, 40% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, 20 mM Tris-HCl, pH 7.5, 1 × Denhardt's solution, 250 μg per ml yeast tRNA, and 20 μg per ml salmon sperm DNA. The membrane was washed at 65°C in 0.1 × SSC and 0.5% SDS, dried, and autoradiographed. After boiling for 5 min in 0.1% SDS, the membrane was rehybridized with 32P-labeled human β-actin cDNA (Clontech Laboratories, CA) under the conditions described above as an internal loading control. The protein kinase activity of Akt was measured essentially as described previously (Kitamura et al., 1998Kitamura T. Ogawa W. Sakaue H. et al.Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport.Mol Cell Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (294) Google Scholar). Briefly, the cells (approximately 1 × 106 cells) were lyzed in 50 mM HEPES-NaOH, pH 7.6, containing 150 mM NaCl, 1% (wt/vol) Triton X-100, 1 mg per ml bacitracin, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, and 30 mM sodium pyrophosphate. The lysate was centrifuged and the resultant supernatant was subjected to immunoprecipitation with a polyclonal antibody raised against a glutathione S-transferase fusion protein containing the amino acid sequence 428–480 of rat Akt1 (PKBα) (Konishi et al., 1994Konishi H. Shinomura T. Kuroda S. Ono Y. Kikkawa U. Molecular cloning of rat RAC protein kinase α and β and their association with protein kinase C ζ.Biochem Biophys Res Commun. 1994; 205: 817-825Crossref PubMed Scopus (70) Google Scholar), which recognizes three subtypes of Akt thus far isolated (Kitamura et al., 1998Kitamura T. Ogawa W. Sakaue H. et al.Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport.Mol Cell Biol. 1998; 18: 3708-3717Crossref PubMed Scopus (294) Google Scholar). After washing three times with HEPES-buffered saline, pH 7.5, containing 0.1% (wt/vol) Triton X-100, the immunoprecipitate was incubated for 30 min at 30°C in the reaction mixture (30 μl) containing 20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 25 μM ATP, 3.0 μCi of [γ-32P]ATP, 1 μM protein kinase inhibitor (Promega, Madison, WI), and 0.2 mg per ml histone H2B (Boehringer Mannheim, Germany). The reaction was terminated by the addition of SDS-sample buffer, and the sample was subjected to SDS-polyacrylamide gel electrophoresis on a 15% gel. The radioactivity incorporated into histone H2B was determined using the BAS 2000 bioimaging analyzer. We first investigated the effect of wortmannin on the melanin content in G361 cells, which inhibits the activities of both Class IA and Class IB PI 3-kinases. G361 cells were employed, because the cells contain moderate amounts of melanin and are suitable for observation of the pigment (Kobayashi et al., 1993Kobayashi N. Muramatsu T. Yamashina Y. Shirai T. Ohnishi T. Mori T. Melanin reduces ultraviolet-induced DNA damage formation and killing rate in cultured human melanoma cells.J Invest Dermatol. 1993; 101: 685-689Abstract Full Text PDF PubMed Google Scholar;Oka et al., 1996Oka M. Ogita K. Ando H. et al.Deletion of specific protein kinase C subspecies in human melanoma cells.J Cell Physiol. 1996; 167: 406-412Crossref PubMed Scopus (33) Google Scholar). The cells contained approximately 8 pg per cell of the pigment when cultured in the presence of fetal bovine serum (Figure 1a), and this value is roughly one-fifth of the melanin content of human melanocytes (Gordon and Gilchrest, 1989Gordon P.R. Gilchrest B.A. Human melanogenesis is stimulated by diacylglycerol.J Invest Dermatol. 1989; 93: 700-702Abstract Full Text PDF PubMed Google Scholar). In G361 cells treated with 0.1 μM wortmannin, the melanin content increased approximately 2.5-fold within 72 h (Figure 1a). The PI 3-kinase activity was markedly inhibited after the treatment with wortmannin (Figure 1b). Furthermore, the treatment of the cells with the PI 3-kinase inhibitor increased the protein level of tyrosinase, the rate-limiting enzyme of melanogenesis, as assessed by Western blot analysis (Figure 1c). Essentially the same results were obtained by using 1 μM LY294002, another potent inhibitor of Class I PI 3-kinase (data not shown). These results suggest that Class I PI 3-kinase is involved in melanogenesis through regulation of the protein level of tyrosinase, in accordance with the previous report using B16 melanoma cells (Buscà et al., 1996Buscà R. Bertolotto C. Ortonne J.-P. Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70S6-kinase pathway induces B16 melanoma cell differentiation.J Biol Chem. 1996; 271: 31824-31830Crossref PubMed Scopus (220) Google Scholar). Northern blot analysis indicated that the mRNA level of tyrosinase transiently increased 8–24 h after the addition of wortmannin (Figure 2). As the PI 3-kinase inhibitor enhances the transcription of tyrosinase, it is most likely that the PI 3-kinase is involved in the negative control of tyrosinase through the transcriptional regulation of the enzyme.Figure 2Effect of wortmannin on transcription of tyrosinase mRNA. After treatment with 0.1 μM wortmannin for the indicated time, total RNA was isolated and Northern blot analysis was carried out using the 32P-labeled probes of tyrosinase and β-actin as described in Materials and Methods. The time (0) indicates the untreated control cells.View Large Image Figure ViewerDownload (PPT) To further study the role of Class I PI 3-kinase in melanogenesis, we examined the effects of a mutant adapter subunit of Class IA PI 3-kinase (Δp85), which lacks the binding site for the catalytic subunit and specifically inhibits the activity of this class of PI 3-kinase by preventing the association of the kinase to tyrosine phosphorylated proteins (Ogawa et al., 1998Ogawa W. Matozaki T. Kasuga M. Role of binding proteins to IRS-1 in insulin signalling.Mol Cell Biochem. 1998; 182: 13-22Crossref PubMed Scopus (98) Google Scholar). The melanin content of the cells infected with the adenovirus vector encoding Δp85 (AxCAΔp85) was markedly higher than that in the cells without infection, whereas the control virus that encodes β-galactosidase (AxCALacZ) did not affect the melanin content (Figure 3a). Indeed, infection of the cells with AxCAΔp85 inhibited the PI 3-kinase activity precipitated with the antibody against phosphotyrosine (Figure 3b). Furthermore, the amount of tyrosinase protein was also increased by the infection with AxCAΔp85 (Figure 3c). These results suggest that Class IA PI 3-kinase participates in the regulation of melanin content probably through regulating the expression of tyrosinase in G361 cells. We next examined the effects of the constitutively active mutant of Class IA PI 3-kinase (Myr-p110), which is a chimeric protein consisting of the catalytic subunit of Class IA PI 3-kinase (p110α) ligated to a myristoylation signal sequence at its amino terminal. Myr-p110 was expressed in the cells with the use of the adenovirus vector encoding the mutant protein (AxCAMyr-p110). Infection of the cells with AxCAMyr-p110 resulted in a significant decrease in melanin content, indicating that signal from Class IA PI 3-kinase decreases the melanin content. The protein level of tyrosinase in the cells infected with AxCAMyr-p110 was also decreased (Figure 3c). It was also shown that protein kinase activity of the endogenous Akt increased in cells infected with AxCA Myr-p110 (Figure 3d) compared with that in cells infected with AxCALacZ and in cells without infection. Because Akt has been shown to be a downstream target of Class IA PI 3-kinase, we examined whether the protein kinase is involved in melanogenesis. A mutant Akt containing a myristoylation signal sequence at its amino terminal showed much higher kinase activity than the wild type enzyme (Kohn et al., 1996Kohn A.D. Takeuchi F. Roth R.A. Akt, a pleckstrin homology domain containing kinase, is activated primarily by phosphorylation.J Biol Chem. 1996; 271: 21920-21926Crossref PubMed Scopus (398) Google Scholar). Infection of the cells with AxCAMyr-Akt, the adenovirus vector encoding the myristoylated Akt mutant, resulted in a decrease of melanin content in G361 cells, indicating that activation of Akt is sufficient to decrease the melanin content in the cells (Figure 4). Cell-permeable inhibitors of PI 3-kinase, such as wortmannin and LY294002, are the potent tools for dissecting intracellular signaling networks. It is a disadvantage of these pharmacologic inhibitors that they fail to discriminate between the Class IA and Class IB enzymes. The members of this class of PI 3-kinase are inhibited by the reagents at similar concentrations (Arcaro and Wymann, 1993Arcaro A. Wymann M.P. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-triphosphate in neutrophil responses.Biochem J. 1993; 296: 297-301Crossref PubMed Scopus (1027) Google Scholar;Vlahos et al., 1994Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one (LY294002).J Biol Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar;Wymann and Pirola, 1998Wymann M.P. Pirola L. Structure and function of phosphoinositide 3-kinases.Biochim Biophys Acta. 1998; 1436: 127-150Crossref PubMed Scopus (566) Google Scholar). Moreover, wortmannin inhibits some protein kinases structurally related to PI 3-kinase such as DNA-dependent protein kinase (Hartley et al., 1995Hartley K.O. Gell D. Smith G.C.M. et al.DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product.Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (656) Google Scholar) and mammalian target of rapamycin (Brunn et al., 1996Brunn G.J. Williams J. Sabers C. Wiederrecht G. Lawrence Jr, J.C. Abraham R.T. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002.EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (604) Google Scholar), whereas other protein kinases such as cAMP-dependent protein kinase, protein kinase C, and cGMP-dependent protein kinase are insensitive to wortmannin at concentrations up to 1 μM (Ui et al., 1995Ui M. Okada T. Hazeki K. Hazeki O. Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase.Trends Biochem Sci. 1995; 20: 303-307Abstract Full Text PDF PubMed Scopus (508) Google Scholar). Thus, findings based solely on the use of the pharmacologic inhibitors must be interpreted cautiously. In this study, we have shown that the wortmannin treatment and the expression of the dominant negative mutant of PI 3-kinase increase the production of melanin as well as the protein level of tyrosinase in human G361 melanoma cells. Although the regulation of Class IA PI 3-kinase is not fully understood, the association of the SH2-domain-containing adapter subunit with tyrosine phosphorylated proteins is regarded as an essential process for its activation. Δp85, the mutant p85 adapter subunit that lacks the binding site for p110, inhibits the growth-factor-induced increase in PI 3-kinase activity that coimmunoprecipitates with tyrosine phosphorylated proteins as well as the cellular D3-phosphorylated phosphoinositides (Hara et al., 1994Hara K. Yonezawa K. Sakaue H. et al.1-phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells.Proc Natl Acad Sci USA. 1994; 91: 7415-7419Crossref PubMed Scopus (414) Google Scholar). Because Class IB PI 3-kinase does not interact with the SH2-domain-containing adapter subunit, Δp85 is a specific inhibitor for Class IA PI 3-kinase. Furthermore, we have shown that the constitutively active form of Class IA PI 3-kinase (Myr-p110) decreased both the melanin content and the protein level of tyrosinase. These results clearly demonstrate that Class IA PI 3-kinase is involved in the regulation of the melanin content and the expression of tyrosinase in G361 cells. Akt, the serine-threonine kinase containing a pleckstrin homology domain, is a prime downstream target of Class IA PI 3-kinase. Akt has been shown to participate in protection of the cells from apoptosis (Hemmings, 1997Hemmings B.A. Akt signaling: linking membrane events to life and death decisions.Science. 1997; 275: 628-630Crossref PubMed Scopus (423) Google Scholar;Coffer et al., 1998Coffer P.J. Jin J. Woodgett J.R. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.Biochem J. 1998; 335: 1-13Crossref PubMed Scopus (953) Google Scholar;Downward, 1998Downward J. Mechanisms and consequences of activation of protein kinase B/Akt.Curr Opin Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1165) Google Scholar;Chan et al., 1999Chan T.O. Rittenhouse S.E. Tsichlis P.E. 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McCabe T.J. et al.Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.Nature. 1999; 399: 597-601Crossref PubMed Scopus (2086) Google Scholar). We showed here that the constitutively active form of Akt (Myr-Akt) markedly decreased the melanin content in G361 cells, indicating that signal transduction through Akt is sufficient for the suppression of melanogenesis in these cells. The amount of tyrosinase protein and the melanin content were correlated in the cells treated with wortmannin and in the cells infected with AxCAΔp85 and AxCAMyr-p110, suggesting the possibility that Class IA PI 3-kinase is involved in the regulation of melanogenesis through modulation of the protein level of tyrosinase. It has been shown, however, that melanogenesis can be regulated not only by transcriptional control but also by its glycosylation (Imokawa and Mishima, 1982Imokawa G. Mishima Y. Loss of melanogenic properties in tyrosinases induced by glycosylation inhibitors within malignant melanoma cells.Cancer Res. 1982; 42: 1994-2002PubMed Google Scholar) and degradation (Halaban et al., 1997Halaban R. Cheng E. Zhang Y. et al.Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells.Proc Natl Acad Sci USA. 1997; 94: 6210-6215Crossref PubMed Scopus (224) Google Scholar;Ando et al., 1999Ando H. Funasaka Y. Oka M. et al.Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis.J Lipid Res. 1999; 40: 1312-1316Abstract Full Text Full Text PDF PubMed Google Scholar) of tyrosinase. Thus, it is still possible that the Class IA PI 3-kinase affects the melanin content not only by modulating the protein level of tyrosinase but also by covalent modifications of the enzyme. Furthermore, it has been reported that other enzymes in the pathway of melanin synthesis, including tyrosinase related proteins 1 and 2, also regulate melanogenesis (Zdarsky et al., 1990Zdarsky E. Favor J. Jackson I.J. The molecular basis of brown, an old mouse mutation, and of an induced revertant to wild type.Genetics. 1990; 126: 443-449Crossref PubMed Google Scholar;Tsukamoto et al., 1992Tsukamoto K. Jackson I.J. Urabe K. Montague P.M. Hearing V.J. A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase.EMBO J. 1992; 11: 519-526Crossref PubMed Scopus (443) Google Scholar). It is necessary to study the regulation of tyrosinase related proteins 1 and 2 by the PI 3-kinase pathway. Several other protein kinases such as cAMP-dependent protein kinase (Körner and Pawelek, 1977Körner A. Pawelek J. Activation of melanoma tyrosinase by a cyclic AMP-dependent protein kinase in a cell-free system.Nature. 1977; 267: 444-447Crossref PubMed Scopus (64) Google Scholar), protein kinase C-α (Gruber et al., 1992Gruber J.R. Ohno S. Niles R.M. Increased expression of protein kinase Cα plays a key role in retinoic acid-induced melanoma differentiation.J Biol Chem. 1992; 267: 13356-13360Abstract Full Text PDF PubMed Google Scholar;Oka et al., 1993Oka M. Ogita K. Saito N. Mishima Y. Selective increase of the α subspecies of protein kinase C and inhibition of melanogenesis induced by retinoic acid in melanoma cells.J Invest Dermatol. 1993; 100: 204s-208sCrossref PubMed Scopus (19) Google Scholar), and protein kinase C-β (Park et al., 1993Park H.-Y. Russakovsky V. Ohno S. Gilchrest B.A. The β isoform of protein kinase C stimulates human melanogenesis by activating tyrosinase in pigment cells.J Biol Chem. 1993; 268: 11742-11749Abstract Full Text PDF PubMed Google Scholar) have been shown to participate in the regulation of melanin synthesis in pigment cells. The involvement of protein kinase C in melanogenesis, however, still needs to be clarified (Carsberg et al., 1994Carsberg C.J. Warenius H.M. Friedmann P.S. Ultraviolet radiation-induced melanogenesis in human melanocytes: effects of modulating protein kinase C.J Cell Sci. 1994; 107: 2591-2597PubMed Google Scholar). Furthermore, mitogen-activated protein kinase has been shown to be involved in cAMP-induced melanogenesis (Englaro et al., 1995Englaro W. Rezzonico R. Durand-Clément M. Lallemand D. Ortonne J.-P. Ballotti R. Mitogen-activated protein kinase pathway and AP-1 are activated during cAMP-induced melanogenesis in B-16 melanoma cells.J Biol Chem. 1995; 270: 24315-24320Crossref PubMed Scopus (195) Google Scholar) and in c-Kit-mediated activation of the microphthalmia-associated transcription factor, a trans-acting factor that regulates the gene transcription of tyrosinase (Hemesath et al., 1998Hemesath T.J. Price E.R. Takemoto C. Badalian T. Fisher D.E. MAP kinase links the transcription factor microphthalmia to c-Kit signalling in melanocytes.Nature. 1998; 391: 298-301Crossref PubMed Scopus (531) Google Scholar). Taken together, it is obvious that melanogenesis is regulated by a network of several intracellular signaling mechanisms including the PI 3-kinase-Akt pathway. It has been shown that the transcription of several genes is regulated by PI 3-kinase. For instance, the insulin-elicited increase in the glucose transport protein GLUT1 has been shown to be mediated at the level of transcription via the PI 3-kinase-Akt cascade (Taha et al., 1999Taha C. Liu Z. Jin J. Al-Hasani H. Sonenberg N. Klip A. Opposite translational control of GLUT1 and GLUT4 glucose transporter mRNAs in response to insulin.J Biol Chem. 1999; 274: 33085-33091Crossref PubMed Scopus (126) Google Scholar). Platelet-derived growth factor-stimulated monocyte chemoattractant protein-1 gene expression has been shown to be mediated by the PI 3-kinase-Akt pathway (Alberta et al., 1999Alberta J.A. Auger K.R. Batt D. et al.Platelet-derived growth factor stimulation of monocyte chemoattractant protein-1 gene expression is mediated by transient activation of the phosphoinositide 3-kinase signal transduction pathway.J Biol Chem. 1999; 274: 31062-31067Crossref PubMed Scopus (21) Google Scholar). It has also been shown that insulin suppresses the transcription of glucose-6-phosphatase through the PI 3-kinase pathway (Dickens et al., 1998Dickens M. Svitek C.A. Culbert A.A. O'brien R.M. Tavaré J.M. Central role for phosphatidylinositide 3-kinase in the repression of glucose-6-phosphatase gene transcription by insulin.J Biol Chem. 1998; 273: 20144-20149Crossref PubMed Scopus (84) Google Scholar). Therefore, it is clear that the PI 3-kinase pathway has an important role in the transcriptional regulation of various proteins. The results obtained in this study clearly demonstrate that melanogenesis is regulated through the Class IA PI 3-kinase-Akt pathway in G361 melanoma cells. It has been shown that some signaling mechanisms differ between melanoma cells and normal melanocytes. For example, it is well known that 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, has opposite effects on the growth of these cells (Herlyn et al., 1987Herlyn M. Clark W.H. Rodeck U. Mancianti M.L. Jambrosic J. Koprowski H. Biology of disease. Biology of tumor progression in human melanocytes.Lab Invest. 1987; 56: 461-474PubMed Google Scholar;Becker et al., 1990Becker D. Beebe S.J. Herlyn M. Differential expression of protein kinase C and cAMP-dependent protein kinase in normal melanocytes and malignant melanomas.Oncogene. 1990; 5: 1133-1139PubMed Google Scholar). Thus, it is necessary to confirm that melanogenesis is regulated by the PI 3-kinase-Akt pathway in normal melanocytes as in the melanoma cells. Studies of the downstream substrates of Akt in the pigment cells would be helpful for an understanding of the regulatory mechanisms of melanogenesis. We thank Dr. Izumu Saito for the adenovirus vector AxCALacZ, and Dr. Vincent J. Hearing for the polyclonal antibody αPEP7. This work was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science, Sports, and Culture of Japan.