Microphthalmia Associated Transcription Factor Is a Target of the Phosphatidylinositol-3-Kinase Pathway
2003; Elsevier BV; Volume: 121; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2003.12420.x
ISSN1523-1747
AutoresMehdi Khaled, Lionel Larribère, Karine Bille, Jean‐Paul Ortonne, Robert Ballotti, Corine Bertolotto,
Tópico(s)Mast cells and histamine
ResumoIn B16 melanoma cells, cyclic adenosine monophosphate inhibits the phosphatidylinositol-3-kinase and the phosphatidylinositol-3-kinase inhibitor, LY294002, stimulates melanogenesis. However, the molecular mechanisms, by which phosphatidylinositol-3-kinase inhibition increases melanogenesis remained to be identified. In this study, we show that LY294002 up-regulates the expression of the melanogenic enzymes, tyrosinase and Tyrp1, through a transcriptional mechanism that involves microphthalmia associated transcription factor, a basic helix-loop-helix transcription factor, which plays a key role in melanocyte survival and differentiation. Further, we observe that LY294002 increases the intracellular content of microphthalmia associated transcription factor, thereby demonstrating that microphthalmia associated transcription factor is also a convergence point of the phosphatidylinositol-3-kinase signaling pathway. Finally, our results indicate that LY294002 controls microphthalmia associated transcription factor at the transcriptional level through distal regulatory element that remain to be identified. Interestingly, we have recently reported that cAMP-elevating agents, through a phosphatidylinositol-3-kinase/AKT inhibition and a glycogen synthase kinase 3β activation, may stimulate microphthalmia associated transcription factor binding to its target sequence, suggesting that inhibition of the phosphatidylinositol-3-kinase is implicated in the stimulation of melanogenesis at different levels. Thus, the results presented in this report strengthen the importance of the phosphatidylinositol-3-kinase pathway in the regulation of melanogenesis and emphasize the complexity of the cyclic adenosine monophosphate signaling that controls melanocyte differentiation and melanogenesis. In B16 melanoma cells, cyclic adenosine monophosphate inhibits the phosphatidylinositol-3-kinase and the phosphatidylinositol-3-kinase inhibitor, LY294002, stimulates melanogenesis. However, the molecular mechanisms, by which phosphatidylinositol-3-kinase inhibition increases melanogenesis remained to be identified. In this study, we show that LY294002 up-regulates the expression of the melanogenic enzymes, tyrosinase and Tyrp1, through a transcriptional mechanism that involves microphthalmia associated transcription factor, a basic helix-loop-helix transcription factor, which plays a key role in melanocyte survival and differentiation. Further, we observe that LY294002 increases the intracellular content of microphthalmia associated transcription factor, thereby demonstrating that microphthalmia associated transcription factor is also a convergence point of the phosphatidylinositol-3-kinase signaling pathway. Finally, our results indicate that LY294002 controls microphthalmia associated transcription factor at the transcriptional level through distal regulatory element that remain to be identified. Interestingly, we have recently reported that cAMP-elevating agents, through a phosphatidylinositol-3-kinase/AKT inhibition and a glycogen synthase kinase 3β activation, may stimulate microphthalmia associated transcription factor binding to its target sequence, suggesting that inhibition of the phosphatidylinositol-3-kinase is implicated in the stimulation of melanogenesis at different levels. Thus, the results presented in this report strengthen the importance of the phosphatidylinositol-3-kinase pathway in the regulation of melanogenesis and emphasize the complexity of the cyclic adenosine monophosphate signaling that controls melanocyte differentiation and melanogenesis. cyclic-AMP response element binding protein forskolin glyceraldehyde-3-phosphate dehydrogenase glycogen synthase kinase 3 β microphthalmia-associated transcription factor protein kinase A, PI3K, phosphatidyl-inositol-3-kinase In mammals, pigmentation results from the synthesis and distribution of melanin in the skin, hair bulbs, and eyes. Melanin synthesis or melanogenesis occurs in melanocytes through an enzymatic process, catalyzed by tyrosinase and tyrosinase-related protein 1 (Tyrp1), which converts tyrosine to melanin pigments (Hearing, 1987Hearing Jr, V.J. Mammalian monophenol monooxygenase (tyrosinase): Purification, properties, and reactions catalyzed.Methods Enzymol. 1987; 142: 154-165Crossref PubMed Scopus (207) Google Scholar; Prota, 1988Prota G. Some new aspects of eumelanin chemistry.Prog Clin Biol Res. 1988; 256: 101-124PubMed Google Scholar; Jimenez-Cervantes et al., 1994Jimenez-Cervantes C. Solano F. Kobayashi T. Urabe K. Hearing V.J. Lozano J.A. Garcia-Borron J.C. A new enzymatic function in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1 (TRP1).J Biol Chem. 1994; 269: 17993-18000Abstract Full Text PDF PubMed Google Scholar; Kobayashi et al., 1994Kobayashi T. Urabe K. Winder A. et al.DHICA oxidase activity of TRP1 and interactions with other melanogenic enzymes.Pigment Cell Res. 1994; 7: 227-234Crossref PubMed Scopus (48) Google Scholar). In vivo, these pigments play a crucial photoprotective role against the carcinogenous effects of ultraviolet radiation of the solar light. Compelling evidence has demonstrated the key role of the melanotropic hormone α-melanocyte-stimulating hormone and adrenocorticotropic hormone in the control of pigmentation. α-melanocyte-stimulating hormone and adrenocorticotropic hormone bind to the Gαs-coupled MC1 receptor (MC1R) that leads to adenylate cyclase activation, elevation of the intracellular cyclic adenosine monophosphate (cAMP) content, and activation of protein kinase A (PKA). Patients in which the cAMP/PKA pathway has been altered, present skin pigmentation defects, thereby demonstrating the importance of the cAMP/PKA pathway in the regulation of melanogenesis (Schwindinger et al., 1992Schwindinger W.F. Francomano C.A. Levine M.A. Identification of a mutation in the gene encoding the alpha subunit of the stimulatory G protein of adenylyl cyclase in McCune-Albright syndrome.Proc Natl Acad Sci USA. 1992; 89: 5152-5156Crossref PubMed Scopus (416) Google Scholar; Kirschner et al., 2000Kirschner L.S. Carney J.A. Pack S.D. et al.Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit in patients with the Carney complex.Nat Genet. 2000; 26: 89-92Crossref PubMed Scopus (835) Google Scholar). In vitro, the melanogenic effects of α-melanocyte-stimulating hormone can be mimicked by pharmacologic agents such as forskolin, a direct activator of adenylate cyclase (Englaro et al., 1995Englaro W. Rezzonico R. Durand-Clement 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). We have previously shown that cAMP-elevating agents increase the expression of the melanogenic enzymes, tyrosinase and Tyrp1, by stimulating the transcription of their cognate genes. We also reported that the M-box (AGTCATGTGCT), a highly conserved DNA sequence, identified in the promoter of these enzymes, is essential for their cAMP responsiveness. The M-box binds MITF (microphthalmia-associated transcription factor), a transcription factor of the basic-helix-loop-helix-leucine-zipper family (b-HLH-LZ) that plays a crucial part in melanocyte development (Hodgkinson et al., 1993Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (918) Google Scholar; Steingrimsson et al., 1994Steingrimsson E. Moore K.J. Lamoreux M.L. et al.Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences.Nat Genet. 1994; 8: 256-263Crossref PubMed Scopus (432) Google Scholar). In the mouse, mutations at the mi locus lead to coat color dilution, white spotting, or complete loss of pigmentation due to absence of melanocyte (Hodgkinson et al., 1993Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (918) Google Scholar; Hughes et al., 1993Hughes M.J. Lingrel J.B. Krakowsky J.M. Anderson K.P. A helix-loop-helix transcription factor-like gene is located at the mi locus.J Biol Chem. 1993; 268: 20687-20690Abstract Full Text PDF PubMed Google Scholar). Similarly, in humans, mutations in MITF have been linked to abnormal pigmentation observed in Waardenburg syndrome type IIa (Hughes et al., 1994Hughes A.E. Newton V.E. Liu X.Z. Read A.P. A gene for Waardenburg syndrome type 2 maps close to the human homologue of the microphthalmia gene at chromosome 3p12-p14.1.Nat Genet. 1994; 7: 509-512Crossref PubMed Scopus (158) Google Scholar; Tassabehji et al., 1994Tassabehji M. Newton V.E. Read A.P. Waardenburg syndrome type 2 caused by mutations in the human microphthalmia (MITF) gene.Nat Genet. 1994; 8: 251-255Crossref PubMed Scopus (546) Google Scholar). Interestingly, the cAMP/PKA pathway, through phosphorylation and activation of the cAMP response element binding protein (CREB) transcription factor, upregulates the Mitf promoter activity, thereby leading to stimulation of MITF expression. Further, we have previously reported that Mitf is absolutely required to mediate the melanogenic effects of cAMP (Bertolotto et al., 1998aBertolotto C. Abbe P. Hemesath T.J. Bille K. Fisher D.E. Ortonne J.P. Ballotti R. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.J Cell Biol. 1998; 142: 827-835Crossref PubMed Scopus (395) Google Scholar), indicating that Mitf is also crucial for melanocyte differentiation and melanogenesis. Focusing our interest on the molecular mechanisms involved in the regulation of melanin synthesis by cAMP, we have already reported that cAMP promotes an inhibition of the phosphatidylinositol-3 kinase (PI3K) activity (Busca et al., 1996Busca R. Bertolotto C. Ortonne J.P. Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70 (S6)-kinase pathway induces B16 melanoma cell differentiation.J Biol Chem. 1996; 271: 31824-31830Crossref PubMed Scopus (220) Google Scholar). PI3K is a lipid kinase that has been implicated in several physiologic processes, such as proliferation, survival, intracellular traffic, and cell differentiation (Katso et al., 2001Katso R. Okkenhaug K. Ahmadi K. White S. Timms J. Waterfield M.D. Cellular function of phosphoinositide 3-kinases: Implications for development, homeostasis, and cancer.Annu Rev Cell Dev Biol. 2001; 17: 615-675Crossref PubMed Scopus (933) Google Scholar). Additionally, a specific inhibitor of PI3K, LY294002, mimics the effects of cAMP leading to the induction of melanogenesis and melanocyte differentiation (Busca et al., 1996Busca R. Bertolotto C. Ortonne J.P. Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70 (S6)-kinase pathway induces B16 melanoma cell differentiation.J Biol Chem. 1996; 271: 31824-31830Crossref PubMed Scopus (220) Google Scholar). These observations suggest that inhibition of PI3K is an important step of cAMP-induced pigment production. In this study, we have investigated how the inhibition of PI3K promotes an induction of melanogenesis. First, we show that the inhibitor of PI3K, LY294002, increases the expression of tyrosinase and Tyrp1 through a transcriptional mechanism. Further, a dominant negative form of Mitf strongly reduces the stimulation by LY294002 of the tyrosinase and Tyrp1 promoter activities, demonstrating the involvement of Mitf in the regulation of melanogenic enzyme transcription by LY294002. We next observed an increased Mitf binding to the M-box sequence of the tyrosinase and Tyrp1 promoters following LY294002 treatment, which results from a stimulation of Mitf expression. Finally, our results indicate that elevation of Mitf levels by LY294002 is regulated at the transcriptional level through distal regulatory elements that remain to be identified. Forskolin, sodium fluoride, sodium orthovanadate, 4-(2-aminoethyl)-benzene-sulfonyl fluoride (AEBSF), aprotinin, and leupeptin were purchased from Sigma (Sigma Chem Co. St. Louis, MO). LY294002 was from MERCK Eurolab (Darmstadt, Germany). Dulbecco's modified Eagle's medium, trypsin, and lipofectamine reagent were from Invitrogen (San Diego, CA) and fetal bovine serum was from Hyclone (Lagan, UT). All clinical studies were approved by the IRB for animal use and patient consent forms were signed. The monoclonal anti-MITF antibody was from Dr D. Fisher (Boston, Massachusetts). The rabbit polyclonal anti-tyrosinase (pep 7) and anti-Tyrp1 (pep1) antibodies were from Dr V. Hearing (Bethesda, Maryland). The polyclonal phospho-specific AKT (S473), CREB (S133), p42/44 mitogen-activated protein kinase (Thr202/Tyr204), and β-catenin (S33/S37/T41) antibodies were from Cell Signaling (Cell Signaling Technology, Beverly, MA) and the monoclonal ERK2 (D-2) antibody was from Santa Cruz (Santa Cruz Biotech, Santa Cruz, CA). The phospho-glycogen synthase antibody (Ab-1) and the phospho-specific Tau antibody (S396) were purchased from Oncogene Research Products (Darmstadt, Germany). Horseradish peroxidase or fluorescein isothiocyanate-conjugated anti-rabbit or anti-mouse antibodies were from Dakopatts (Glostrup, Denmark). B16/F10 murine melanoma cells were grown at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 7% fetal bovine serum and penicillin (100 U per mL)/streptomycin (50 μg per mL). B16 were seeded in six-well dishes and exposed to 20 μM forskolin, or LY294002 for the concentration and time indicated in the legends. Then, melanins were solubilized in 0.5 M NaOH for 1 h at 65°C. Absorbance at 405 nm was compared with a standard curve of known concentrations of fungal melanin prepared in a final NaOH concentration of 0.5 M. The melanin content was corrected by the total cell number of the dish and expressed as fold stimulation of melanin of control cells. B16 melanoma cells were grown in six-well dishes with forskolin or LY294002. Cells were lyzed in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 10 μM leupeptin, 1 mM AEBSF, 100 U per mL aprotinin, 10 mM NaF, and 1 mM Na3VO4. Samples (30 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and then exposed to the appropriate antibodies. Proteins were visualized with the enhanced chemiluminescence system from Amersham using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody. The luciferase reporter plasmids pTyro, pTyrp1, and pMITF and the expression vector encoding the wild-type or the dominant negative form of MITF were previously described (Bertolotto et al., 1998aBertolotto C. Abbe P. Hemesath T.J. Bille K. Fisher D.E. Ortonne J.P. Ballotti R. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.J Cell Biol. 1998; 142: 827-835Crossref PubMed Scopus (395) Google Scholar). TOPFlash luciferase reporter plasmid, containing 3 Lef/TCF binding site cloned upstream of the c-fos promoter, was a kind gift of Dr L. Larue (Orsay, France). Briefly, B16 melanoma cells were seeded in 24-well dishes and transient transfections were performed the following day using 2 μL of lipofectamine and 0.5 μg of total DNA plasmid. pCMVβGal was transfected with the test plasmids to control the variability in transfection efficiency. After 48 h, cells were harvested in 50 μL of lysis buffer and assayed for luciferase and β-galactosidase activities. All transfections were repeated at least three times and performed in triplicate. Poly(A)+ RNA were isolated from control and forskolin or LY294002-treated cells using the mRNA purification kit from Qiagen Inc., Valencia, CA. (OligotexTM, mRNA) and total RNA was isolated by a modification of the method ofChomczynski and Sacchi, 1987Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62289) Google Scholar. RNA was denatured for 5 min at 65°C in a formamide/formaldehyde mixture, separated by electrophoresis in a 1% agarose/7% formaldehyde gel, transferred to a nylon membrane (Hybond N, Amersham Bio Sciences, Orsay, France) in 20×sodium citrate/chloride buffer (3 M NaCl, 0.3 M sodium citrate pH 7) and hybridized to MITF and GAPDH probe labeling by random priming with α-[32P] deoxycytidine triphosphate (Amersham). B16 melanoma cells were grown on glass coverslips (2×104 cells per point) in 12-well dishes and treated for 6 h with 20 μM forskolin or 10 μM LY294002. Cells were then washed, fixed at room temperature for 20 min with 3% paraformaldehyde, and permeabilized by a 2 min treatment with phosphate-buffered saline 1% Triton before being exposed to an anti-MITF antibody for 1 h at room temperature. Cells were next incubated with FITC coupled anti-mouse antibody for 1 h at room temperature and the cells were washed with phosphate-buffered saline in which bisbenzidine (0.5 μg per mL) was added. Finally, coverslips were mounted in moviol immunofluorescence mounting medium and examined with the 40×objective using Zeiss Axiophot microscope equipped with epifluorescence illumination. Nuclear extracts from control cells or cells incubated with 20 μM forskolin or 10 μM LY294002 were prepared as previously described. Double-stranded synthetic M-box, 5′-GAAAAAGTCATGTGCTTTGCAGAAGA-3′ was γ32P end-labeled using T4 polynucleotide kinase. Five micrograms of nuclear proteins were preincubated in a binding buffer containing 10 mM Tris pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM ethylenediamine tetraacetic acid, 4% glycerol, 80 μg per mL of salmon sperm DNA, 0.1 μg poly(dIdC), 10% fetal bovine serum, 2 mM MgCl2, and 2 mM spermidine for 15 min on ice. Then, 30,000 to 50,000 cpm of 32P-labeled probe were added to the binding reaction for 10 min at room temperature. DNA-protein complexes were resolved by electrophoresis on a 4% polyacrylamide gel (37.5:1 acrylamide/bisacrylamide) in TBE buffer (22.5 mM Tris-borate, 0.5 mM ethylenediamine tetraacetic acid, pH 8) for 90 min at 100 V. For supershift assays, 0.3 μL of preimmune serum or anti-MITF antibody were preincubated with nuclear extracts in the binding reaction buffer before adding the labeled probe. In this study, we investigated the molecular mechanisms by which the inhibition of the PI3K pathway stimulates melanogenesis. With this aim we used LY294002, a pharmacologic inhibitor of PI3K that binds to the adenosine triphosphate binding site of PI3K and specifically blocks the activity of this kinase. In Figure 1a, we showed that LY294002 inhibited the phosphorylation of AKT that is a direct target of PI3K; however, LY294002 did not affect the phos-phorylation of p42/44 mitogen-activated protein kinase or CREB transcription factor. These results indicate that LY294002 specifically inhibits the PI3K signaling but has no effect on the p42/44 mitogen-activated protein kinase or PKA pathways. Dose-response and time course experiments revealed a maximal induction of melanin synthesis after 48 h of cell exposure to 10 μM of LY294002 (Figure 1b). Melanin synthesis assays were corrected by the total cell number as forskolin and LY294002 decreased cell growth by about 22%±1% and 30%±3%, respectively. Next, dose-response and time course assays of the LY294002 effects on tyrosinase and Tyrp1 were performed. Consistent with previous observations, western blot experiments revealed a maximal stimulation of tyrosinase and Tyrp1 expression after cell treatment with 10 μM of LY294002 for 48 h (Figure 1c). These observations were not due to differences in loading as shown by ERK2 detection. Then, plasmids containing a fragment of the tyrosinase or Tyrp1 promoter, cloned upstream of the luciferase reporter gene, were transiently transfected in B16 cells. Cells were incubated with LY294002, and luciferase activity, reflecting the activity of the promoters, was measured. We observed that LY294002 induced a 5- and 4-fold stimulation of the transcriptional activity of the tyrosinase and Tyrp1 promoters, respectively (Figure 1d). As a control, we used forskolin, which has been described to increase the expression of tyrosinase and Tyrp1 through a transcriptional mechanism (Figure 1b,c). Taken together, our results show that inhibition of PI3K upregulates the transcriptional activity of the tyrosinase and Tyrp1 promoters, thereby leading to stimulation of tyrosinase and Tyrp1 expression and induction of melanogenesis. Mitf, that plays a central role in melanocyte fate and differentiation, has been demonstrated to be an essential signal transducer in cAMP-induced melanogenesis. We thus studied the involvement of this transcription factor in the regulation of melanogenesis by LY294002. The effect of a dominant negative form of Mitf, lacking the N-terminal transactivation domain (Mi-ΔNT), was assayed on the response of the tyrosinase and Tyrp1 promoters to LY294002. We showed that the Mi-ΔNT transfection strongly reduced the sensitivity of the tyrosinase and Tyrp1 promoters to LY294002, which indicates the important role of Mitf in LY294002-induced melanogenic gene expression (Figure 2a). We next performed gel shift experiments using as labeled probe the M-box, which is the target sequence of Mitf in the tyrosinase and Tyrp1 promoters, respectively (Figure 2b). Nuclear extracts from control cells formed complexes with the labeled M-box (lane 1), that were greatly increased when reactions were performed with nuclear extracts from forskolin (lane 3) or LY294002 (lane 5) treated cells. These complexes were strongly displaced by a monoclonal anti-MITF antibody (lanes 2, 4, and 6), demonstrating the specific interaction of Mitf with the labeled M-box. Together, our results indicate that the LY294002-increased Mitf binding to its target sequence leads to a stimulation of tyrosinase and Tyrp1 gene expression. Immuno-fluorescence studies with the monoclonal anti-MITF antibody showed that forskolin or LY294002 increased the level of Mitf compared with nontreated cells (Figure 3a). On the other hand, no change in Mitf cellular localization could be detected. Phase contrast microscopy analysis showed, as previously described, that forskolin or LY294002 dramatically increased B16 melanoma cell dendricity. Next, western blot experiments using the monoclonal anti-MITF antibody were carried out to determine the effects of PI3K inhibition on Mitf expression. Mitf, appeared as a doublet in control cells, which was increased after LY294002 exposure (Figure 3b). Stimulation of Mitf expression by LY294002 or forskolin reached its maximum levels after 4 h to 8 h of treatment. Noteworthy, stimulation of Mitf expression by forskolin appeared more sustained than that evoked by LY294002. Finally, we investigated how LY294002 upregulated Mitf expression. Northern blot experiments revealed that LY294002, like forskolin, increased Mitf mRNA levels (Figure 4a, upper panel). The equal loading of each lane was shown by GAPDH detection (Figure 4a, lower panel). Further, the effect of actinomycin D, an inhibitor of the transcriptional processes, was assayed on the regulation of Mitf mRNA by forskolin and LY294002. We observed that incubation of control B16 melanoma cells with actinomycin D decreased Mitf mRNA below the basal level. As expected, actinomycin D completely abolished the induction of Mitf mRNA by forskolin (Figure 4b, upper panel). Additionally, actinomycin D totally blocked the stimulation of the Mitf mRNA evoked by LY294002, indicating that LY294002 as well as forskolin stimulate Mitf gene transcription rather than Mitf messenger stability. These observations were not due to a difference in loading as shown by the bromide ethidium staining of the 28S and 18S RNA (Figure 4b, lower panel). In conclusion, these results demonstrate that LY294002 increases Mitf mRNA through a transcriptional process.Figure 4LY294002 controls MITF transcription. (A) mRNA from control B16 cells or cells exposed for 2 h to 20 μM forskolin or 10 μM LY294002 were analyzed by Northern blot using MITF and GAPDH probes. (B) B16 cells were left untreated or were incubated with actinomycin D 2.5 μg per mL before adding 20 μM forskolin or 10 μM LY294002 for 2 h. Then, total mRNA was extracted and analyzed by Northern blot using MITF and GAPDH probes.View Large Image Figure ViewerDownload (PPT) cAMP elevating agents upregulate the activity of the Mitf promoter through phosphorylation and activation of the CREB transcription factor. As shown in Figure 1a) LY294002 did not promote CREB phosphorylation. Further, we have recently demonstrated that LY294002 and forskolin lead to the activation of the downstream AKT target, glycogen synthase kinase 3 β (GSK3β). Then, activated GSK3β phosphorylates its substrates such as β-catenin, glycogen synthase, and tau (Figure 5a,b). β-catenin, is one of the factors that controls Mitf transcription. LY294002, however, did not affect the intracellular levels of β-catenin or the activity of a TOPFlash reporter plasmid. These observations preclude a possible involvement of β-catenin/Lef complex in Mitf activation by LY294002 (Figure 5a). Next, B16 cells were transiently transfected with a reporter plasmid containing a 2.1 kb fragment of the Mitf promoter upstream of the luciferase gene and then were incubated with LY294002 (Figure 5c). A time course experiment indicated that LY294002 had no effect on the activity of the Mitf promoter, compared with forskolin, which caused, as previously described, a 7-fold stimulation of this promoter activity. These observations indicate that LY294002 does not act on the proximal region of the Mitf promoter and, suggest that LY294002 regulates Mitf transcription through distal regulatory elements. In a previous study, it was shown that cAMP, a potent inducer of melanogenesis, promotes an inhibition of the PI3K, and that LY294002, a specific inhibitor of PI3K, increases melanin synthesis (Busca et al., 1996Busca R. Bertolotto C. Ortonne J.P. Ballotti R. Inhibition of the phosphatidylinositol 3-kinase/p70 (S6)-kinase pathway induces B16 melanoma cell differentiation.J Biol Chem. 1996; 271: 31824-31830Crossref PubMed Scopus (220) Google Scholar). To investigate how inhibition of PI3K stimulates pigment production, we studied the effects of LY294002 on different parameters of melanin synthesis. In this study, we first show that LY294002, by regulating the activity of the tyrosinase and Tyrp1 promoter activities leads to an increase in tyrosinase and Tyrp1 expression and stimulation of melanogenesis. Further, we demonstrate that Mitf, a transcription factor that plays a key role in melanocyte survival and differentiation (Hodgkinson et al., 1993Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (918) Google Scholar; Steingrimsson et al., 1994Steingrimsson E. Moore K.J. Lamoreux M.L. et al.Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences.Nat Genet. 1994; 8: 256-263Crossref PubMed Scopus (432) Google Scholar; McGill et al., 2002McGill G.G. Horstmann M. Widlund H.R. et al.Bcl2 regulation by the melanocyte master regulator mitf modulates lineage survival and melanoma cell viability.Cell. 2002; 109: 707-718Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar) is implicated in the responsiveness of the melanogenic gene promoters to LY294002. Finally, our results indicate that LY294002 stimulates Mitf expression through a transcriptional mechanism that involves distal regulatory elements. Indeed, Northern blot experiments revealed that LY294002 increases Mitf mRNA levels in B16 melanoma cells. Further, actinomycin D, an inhibitor of transcription, prevents LY294002-induced upregulation of Mitf mRNA, suggesting that LY294002 regulates Mitf expression at the transcriptional level. To understand how LY294002 controls Mitf transcription, we focused our attention on the CREB transcription factor. First, we have previously reported that phosphorylation and activation of CREB transcription factor by PKA stimulates Mitf expression (Bertolotto et al., 1998aBertolotto C. Abbe P. Hemesath T.J. Bille K. Fisher D.E. Ortonne J.P. Ballotti R. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.J Cell Biol. 1998; 142: 827-835Crossref PubMed Scopus (395) Google Scholar). Secondly, AKT, a downstream target of PI3K, has been shown to promote the phosphorylation and activation of the phosphodiesterase 3B (PDE3B), which down-modulates the intracellular cAMP content (Kitamura et al., 1999Kitamura T. Kitamura Y. Kuroda S. et al.Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt.Mol Cell Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (294) Google Scholar). Thus, it was tempting to hypothesize that LY294002, through the inhibition of the PI3K/AKT/PDE3B cascade, maintained high intracellular cAMP levels, thereby leading to PKA activation, CREB phosphorylation, and stimulation of the Mitf promoter activity; however, we show that LY294002 does not affect CREB phosphorylation, demonstrating that CREB is not implicated in the regulation of Mitf transcription by LY294002. Next, we focused our attention on β-catenin, which has been involved in Mitf activation through its interaction with the transcription factors of the Lef/TCF family. We observed that LY294002 induces the phosphorylation of β-catenin; however, LY294002 does not significantly affect the activity of a TOPFlash reporter plasmid that is controlled by the β-catenin/Lef/TCF complex, meaning that phosphorylation of β-catenin by LY294002 is not sufficient to induce its degradation. We thus hypothesized that, LY294002 could trigger another regulatory element in the Mitf promoter. Sox10 and Pax3 transcription factors have been shown to play an important part in Mitf promoter activity, indicating that these factors could be potential targets of LY294002 action (Watanabe et al., 1998Watanabe A. Takeda K. Ploplis B. Tachibana M. Epistatic relationship between Waardenburg syndrome genes MITF and PAX3.Nat Genet. 1998; 18: 283-286Crossref PubMed Scopus (226) Google Scholar; Takeda et al., 2000bTakeda K. Yasumoto K. Takada R. et al.Induction of melanocyte-specific microphthalmia-associated transcription factor by Wnt-3a.J Biol Chem. 2000; 275: 14013-14016Crossref PubMed Scopus (251) Google Scholar; Verastegui et al., 2000Verastegui C. Bille K. Ortonne J.P. Ballotti R. Regulation of microphthalmia-associated transcription factor gene by the Waardenburg syndrome type 4 gene, Sox10.J Biol Chem. 2000; 275: 30757-30760Crossref PubMed Scopus (135) Google Scholar). A 2.1 kb fragment of the Mitf promoter containing such regulatory sequences, however, was not stimulated by LY294002, demonstrating that the proximal regulatory region does not mediate the response of the Mitf promoter to LY294002. Recently, a distal regulatory region located 14.5 kb upstream from exon 1 of the human MITF gene has been shown to enhance the activity of the Mitf promoter (Watanabe et al., 2002Watanabe K. Takeda K. Yasumoto K. et al.Identification of a distal enhancer for the melanocyte-specific promoter of the MITF gene.Pigment Cell Res. 2002; 15: 201-211Crossref PubMed Scopus (56) Google Scholar). These data are in agreement with the presence of crucial regulatory elements upstream of the 2.1 kb fragment of the Mitf promoter and suggest that these sequences could mediate the sensitivity of the Mitf promoter to LY294002. Mitf has been shown to be the target of different signaling pathways that regulate pigment production (Bertolotto et al., 1998aBertolotto C. Abbe P. Hemesath T.J. Bille K. Fisher D.E. Ortonne J.P. Ballotti R. Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes.J Cell Biol. 1998; 142: 827-835Crossref PubMed Scopus (395) Google Scholar; Price et al., 1998Price E.R. Ding H.F. Badalian T. et al.Lineage-specific signaling in melanocytes. C-kit stimulation recruits p300/CBP to microphthalmia.J Biol Chem. 1998; 273: 17983-17986Crossref PubMed Scopus (162) Google Scholar; Kim et al., 2002Kim D.S. Kim S.Y. Chung J.H. Kim K.H. Eun H.C. Park K.C. Delayed ERK activation by ceramide reduces melanin synthesis in human melanocytes.Cell Signal. 2002; 14: 779-785Crossref PubMed Scopus (133) Google Scholar). Interestingly, our results demonstrate that Mitf is also a convergence point of the PI3K pathway, thus reinforcing the key role of Mitf in melanocyte differentiation and melanogenesis. Furthermore, we have recently shown that cAMP, through AKT inhibition, activates the GSK3β, and inhibition of GSK3β by lithium decreases forskolin-induced tyrosinase promoter activity (Khaled et al., 2002Khaled M. Larribere L. Bille K. Aberdam E. Ortonne J.P. Ballotti R. Bertolotto C. Glycogen synthase kinase 3beta is activated by cAMP and plays an active role in the regulation of melanogenesis.J Biol Chem. 2002; 277: 33690-33697Crossref PubMed Scopus (144) Google Scholar). In agreement with the report fromTakeda et al., 2000aTakeda K. Takemoto C. Kobayashi I. Watanabe A. Nobukuni Y. Fisher D.E. Tachibana M. Ser298 of MITF, a mutation site in Waardenburg syndrome type 2, is a phosphorylation site with functional significance.Hum Mol Genet. 2000; 9: 125-132Crossref PubMed Scopus (143) Google Scholar, we proposed that activation of GSK3β by cAMP would lead to phosphorylation of Mitf, thus increasing its binding to the M-box sequence in the tyrosinase and Tyrp1 promoters, respectively. Therefore, the PI3K pathway stimulates melanogenesis by at least two mechanisms. First, cAMP, through inhibition of PI3K and AKT, and activation of GSK3β, stimulates Mitf binding to the M-box of the tyrosinase promoter, increasing the melanogenic enzyme expression (Khaled et al., 2002Khaled M. Larribere L. Bille K. Aberdam E. Ortonne J.P. Ballotti R. Bertolotto C. Glycogen synthase kinase 3beta is activated by cAMP and plays an active role in the regulation of melanogenesis.J Biol Chem. 2002; 277: 33690-33697Crossref PubMed Scopus (144) Google Scholar). Second, LY294002 would control the activity of the Mitf promoter through distal regulatory elements that remain to be identified. Interestingly, forskolin gives rise to a more important and sustained expression of Mitf compared with the LY294002 treatment. Taken together, our results suggest that forskolin mediates its effects through the combined activation of the PKA/CREB pathway and inhibition of PI3K, whereas LY294002 action is only the result of PI3K inhibition. The report fromKhaled et al., 2002Khaled M. Larribere L. Bille K. Aberdam E. Ortonne J.P. Ballotti R. Bertolotto C. Glycogen synthase kinase 3beta is activated by cAMP and plays an active role in the regulation of melanogenesis.J Biol Chem. 2002; 277: 33690-33697Crossref PubMed Scopus (144) Google Scholar, mentioned that cAMP inhibits AKT in a PKA-independent manner reinforcing the notion that PKA and PI3K regulate melanogenesis through separate pathways. These differences could explain why forskolin is a stronger inducer of tyrosinase and Tyrp1 promoter activities. It should be noted, however, that the effects of forskolin and LY294002 on tyrosinase and Tyrp1 expression and melanin synthesis were roughly comparable, suggesting that LY294002 could also control melanogenesis through post-transcriptional modifications of tyrosinase or Tyrp1. Post-transcriptional modifications of tyrosinase and Tyrp1 have already been proposed to upregulate their enzyme activity (Imokawa and Mishima, 1982Imokawa G. Mishima Y. Loss of melanogenic properties in tyrosinase induced by glucosylation inhibitors within malignant melanoma cells.Cancer Res. 1982; 42: 1994-2002PubMed Google Scholar; Martinez-Esparza et al., 1998Martinez-Esparza M. Jimenez-Cervantes C. Solano F. Lozano J.A. Garcia-Borron J.C. Mechanisms of melanogenesis inhibition by tumor necrosis factor-alpha in B16/F10 mouse melanoma cells.Eur J Biochem. 1998; 255: 139-146Crossref PubMed Scopus (94) Google Scholar; Park et al., 1999Park H.Y. Perez J.M. Laursen R. Hara M. Gilchrest B.A. Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain.J Biol Chem. 1999; 274: 16470-16478Crossref PubMed Scopus (120) Google Scholar). Whether LY294002 could promote such modifications remains to be elucidated. In conclusion, we demonstrate in this study that the PI3K inhibitor, LY294002, increases Mitf expression, leading to a stimulation of tyrosinase and Tyrp1 expression and finally induction of melanogenesis. Our data suggest that activation of the PKA/CREB cascade and inhibition of PI3K during cAMP-induced melanogenesis cooperate to trigger a stronger induction of melanin synthesis. Taken together, our results bring new information on the molecular mechanisms involved in the cAMP-induced melanogenesis and help in the understanding of the signaling events that govern pigmentation. This work was supported by INSERM, The Ligue Nationale contre le Cancer and the Association pour la Recherche sur le Cancer grant 5808.
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