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Microphthalmia Associated Transcription Factor Is a Target of the Phosphatidylinositol-3-Kinase Pathway

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Microphthalmia Associated Transcription Factor Is a Target of the Phosphatidylinositol-3-Kinase Pathway View metadata, citation and similar papers at core.ac.uk brought to you by CORE providedORIGINAL by Elsevier - ARTICLE Publisher Connector Microphthalmia Associated Transcription Factor Is a Target of the Phosphatidylinositol-3-Kinase Pathway Mehdi Khaled, Lionel Larribere, Karine Bille, Jean-Paul Ortonne, Robert Ballotti, and Corine Bertolotto INSERM U385, Biologie et Physiopathologie de la Peau, Nice, France In B16 melanoma cells, cyclic adenosine monopho- scription factor at the transcriptional level through dis- sphate inhibits the phosphatidylinositol-3-kinase and tal regulatory element that remain to be identi¢ed. the phosphatidylinositol-3-kinase inhibitor, LY294002, Interestingly, we have recently reported that cAMP-ele- stimulates melanogenesis. However, the molecular vating agents, through a phosphatidylinositol-3-kinase/ mechanisms, by which phosphatidylinositol-3-kinase AKT inhibition and a glycogen synthase kinase 3b inhibition increases melanogenesis remained to be activation, may stimulate microphthalmia associated identi¢ed. In this study, we show that LY294002 up-reg- transcription factor binding to its target sequence, ulates the expression of the melanogenic enzymes, tyr- suggesting that inhibition of the phosphatidylinositol- osinase and Tyrp1, through a transcriptional mechanism 3-kinase is implicated in the stimulation of melanogen- that involves microphthalmia associated transcription esis at di¡erent levels. Thus, the results presented in this factor, a basic helix-loop-helix transcription factor, report strengthen the importance of the phosphatidyli- which plays a key role in melanocyte survival and dif- nositol-3-kinase pathway in the regulation of mela- ferentiation. Further, we observe that LY294002 in- nogenesis and emphasize the complexity of the cyclic creases the intracellular content of microphthalmia adenosine monophosphate signaling that controls mela- associated transcription factor, thereby demonstrating nocyte di¡erentiation and melanogenesis. Key words: that microphthalmia associated transcription factor is cyclic adenosine monophosphate/microphthalmia associated also a convergence point of the phosphatidylinositol-3- transcription factor /phosphatidylinositol-3-kinase/tyrosinase/ kinase signaling pathway. Finally, our results indicate TYRP1. J Invest Dermatol 121:831 ^836, 2003 that LY294002 controls microphthalmia associated tran- n mammals, pigmentation results from the synthesis and protein kinase A (PKA). Patients in which the cAMP/PKA path- distribution of melanin in the skin, hair bulbs, and eyes. way has been altered, present skin pigmentation defects, thereby Melanin synthesis or melanogenesis occurs in melanocytes demonstrating the importance of the cAMP/PKA pathway in the through an enzymatic process, catalyzed by tyrosinase and regulation of melanogenesis (Schwindinger et al, 1992; Kirschner tyrosinase-related protein 1 (Tyrp1), which converts tyro- et al,2000).In vitro, the melanogenic e¡ects of a-melanocyte-sti- Isine to melanin pigments (Hearing, 1987; Prota, 1988; Jimenez- mulating hormone can be mimicked by pharmacologic agents Cervantes et al,1994;Kobayashiet al,1994).In vivo, these pigments such as forskolin, a direct activator of adenylate cyclase (Englaro play a crucial photoprotective role against the carcinogenous ef- et al, 1995).We have previously shown that cAMP-elevating agents fects of ultraviolet radiation of the solar light. increase the expression of the melanogenic enzymes, tyrosinase Compelling evidence has demonstrated the key role of the and Tyrp1, by stimulating the transcription of their cognate melanotropic hormone a-melanocyte-stimulating hormone and genes. We also reported that the M-box (AGTCATGTGCT), a adrenocorticotropic hormone in the control of pigmentation. highly conserved DNA sequence, identi¢ed in the promoter of a-melanocyte-stimulating hormone and adrenocorticotropic these enzymes, is essential for their cAMP responsiveness. The hormone bind to the Gas-coupled MC1 receptor (MC1R) that leads M-box binds MITF (microphthalmia-associated transcription to adenylate cyclase activation, elevation of the intracellular cyclic factor), a transcription factor of the basic-helix-loop-helix- adenosine monophosphate (cAMP) content, and activation of leucine-zipper family (b-HLH-LZ) that plays a crucial part in melanocyte development (Hodgkinson et al, 1993; Steingrimsson et al, 1994). In the mouse, mutations at the mi locus lead to coat Manuscript received December 9, 2002; revised February 18, 2003; color dilution, white spotting, or complete loss of pigmentation accepted for publication March 25, 2003 due to absence of melanocyte (Hodgkinson et al, 1993; Hughes Address correspondence and reprint requests to: Corine Bertolotto, et al, 1993). Similarly, in humans, mutations in MITF have been INSERM U385, Biologie et Physiopathologie de la peau, IFR 50, 28, ave- linked to abnormal pigmentation observed in Waardenburg syn- nue de Valombrose, 06107 Nice Cedex 2, France. Email: [email protected] drome type IIa (Hughes et al, 1994; Tassabehji et al, 1994). Interest- Abbreviations: CREB, cyclic-AMP response element binding pro- tein; fsk, forskolin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ingly, the cAMP/PKA pathway, through phosphorylation and GSK3b, glycogen synthase kinase 3 b; MITF, microphthalmia-associated activation of the cAMP response element binding protein transcription factor; PKA, protein kinase A, PI3K, phosphatidyl-inositol- (CREB) transcription factor, upregulates the Mitf promoter ac- 3-kinase. tivity, thereby leading to stimulation of MITF expression. 0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc. 831 832 KHALED ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Further, we have previously reported that Mitf is absolutely re- 100 U per mL aprotinin, 10 mM NaF, and 1 mM Na3VO4. Samples (30 mg) quired to mediate the melanogenic e¡ects of cAMP (Bertolotto were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electro- et al, 1998a), indicating that Mitf is also crucial for melanocyte phoresis, transferred to a nitrocellulose membrane and then exposed to the di¡erentiation and melanogenesis. appropriate antibodies. Proteins were visualized with the enhanced chemi- luminescence system from Amersham using horseradish peroxidase-conju- Focusing our interest on the molecular mechanisms involved gated anti-rabbit or anti-mouse secondary antibody. in the regulation of melanin synthesis by cAMP, we have already reported that cAMP promotes an inhibition of the phosphatidy- Expression vectors, transfection, and luciferase assays The luciferase reporter linositol-3 kinase (PI3K) activity (Busca et al, 1996). PI3K is a li- plasmids pTyro, pTyrp1, and pMITF and the expression vector encoding pid kinase that has been implicated in several physiologic the wild-type or the dominant negative form of MITF were previously processes, such as proliferation, survival, intracellular tra⁄c, and described (Bertolotto et al, 1998a, c). TOPFlash luciferase reporter plasmid, cell di¡erentiation (Katso et al, 2001). Additionally, a speci¢c inhi- containing 3 Lef/TCF binding site cloned upstream of the c-fos promoter, bitor of PI3K, LY294002, mimics the e¡ects of cAMP leading to was a kind gift of Dr L. Larue (Orsay, France). the induction of melanogenesis and melanocyte di¡erentiation Brie£y, B16 melanoma cells were seeded in 24-well dishes and transient (Busca et al, 1996). These observations suggest that inhibition of transfections were performed the following day using 2 mL of lipofecta- mine and 0.5 mg of total DNA plasmid. pCMVbGal was transfected with PI3K is an important step of cAMP-induced pigment production. the test plasmids to control the variability in transfection e⁄ciency. After In this study, we have investigated how the inhibition of PI3K 48 h, cells were harvested in 50 mL of lysis bu¡er and assayed for luciferase promotes an induction of melanogenesis. First, we show that the and b-galactosidase activities. All transfections were repeated at least three inhibitor of PI3K, LY294002, increases the expression of tyrosi- times and performed in triplicate. nase and Tyrp1 through a transcriptional mechanism. Further, a dominant negative form of Mitf strongly reduces the stimulation Northern blot analysis Poly(A) þ RNA were isolated from control and for- by LY294002 of the tyrosinase and Ty r p1 promoter activities, de- skolin or LY294002-treated cells using the mRNA puri¢cation kit from monstrating the involvement of Mitf in the regulation of mela- Qiagen Inc.,Valencia, CA. (OligotexTM, mRNA) and total RNA was iso- lated by a modi¢cation of the method of Chomczynski and Sacchi (1987). nogenic enzyme transcription by LY294002.We next observed an 1 increased Mitf binding to the M-box sequence of the tyrosinase RNA was denatured for 5 min at 65 C in a formamide/formaldehyde mix- ture, separated by electrophoresis in a 1% agarose/7% formaldehyde gel, and Ty r p1 promoters following LY294002 treatment, which re- transferred to a nylon membrane (Hybond N, Amersham Bio Sciences, sults from a stimulation of Mitf expression. Finally, our results Orsay, France) in 20 Â sodium citrate/chloride bu¡er (3 M NaCl, 0.3 M so- indicate that elevation of Mitf levels by LY294002 is regulated at dium citrate pH 7) and hybridized to MITF and GAPDH probe labeling the transcriptional level through distal regulatory elements that by random priming with a-[32P] deoxycytidine triphosphate (Amersham). remain to be identi¢ed.
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