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Inhibition of the Phosphatidylinositol 3-Kinase/p70S6-Kinase Pathway Induces B16 Melanoma Cell Differentiation*

  • Roser Buscà
    Correspondence
    To whom correspondence should be addressed:
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    INSERM U385, Faculté de Médecine, 06107 Nice, Cedex 02, France
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  • Corine Bertolotto
    Affiliations
    INSERM U385, Faculté de Médecine, 06107 Nice, Cedex 02, France
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  • Jean-Paul Ortonne
    Affiliations
    INSERM U385, Faculté de Médecine, 06107 Nice, Cedex 02, France
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  • Robert Ballotti
    Affiliations
    INSERM U385, Faculté de Médecine, 06107 Nice, Cedex 02, France
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  • Author Footnotes
    * This work was supported in part by the Center de Recherche et d'Investigations Epidermiques et Sensorielles (C.E.R.I.E.S.) and by the Association pour la Recherche sur le Cancer (grant 6760), Ligue Contre le Cancer, Fondation de France, Fondation pour la Recherche Médicale, Institut National de la Santé et de la Recherche Médicale, and Université de Nice Sophia-Antipolis. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 13, 1996DOI:https://doi.org/10.1074/jbc.271.50.31824
      α-Melanocyte-stimulating hormone and cAMP-elevating agents are known to induce B16 cell differentiation, characterized by increased melanin synthesis and dendrite outgrowth. In order to elucidate intracellular signaling pathways involved in this differentiation process, we focused our interest on the phosphatidylinositol 3-kinase/p70S6-kinase pathway. The specific inhibition of phosphatidylinositol 3-kinase by LY294002 markedly stimulated dendrite outgrowth, thus mimicking the action of cAMP-elevating agents on B16 cell morphology. In addition, LY294002 and rapamycin, a specific p70S6-kinase inhibitor, were found to independently stimulate tyrosinase expression, thus increasing melanin synthesis. In an attempt to better dissect the molecular mechanisms triggered by cAMP to induce melanoma cell differentiation, we examined the effects of a cAMP-elevating agent forskolin, on both phosphatidylinositol 3-kinase and p70S6-kinase activities. Specific kinase assays revealed that forskolin partially inhibited phosphatidylinositol 3-kinase activity and completely blocked p70S6-kinase activity and phosphorylation. In conclusion, our results clearly demonstrate that the inhibition of phosphatidylinositol 3-kinase and p70S6-kinase is involved in the regulation of B16 cell differentiation. Furthermore, we provide evidence which suggests that cAMP-induced melanogenesis and dendricity are, at least partially, mediated by the cAMP inhibition of the phosphatidylinositol 3-kinase/p70S6-kinase signaling pathway.

      INTRODUCTION

      In the epidermis, melanocytes synthesize melanin, which is responsible for skin pigmentation. Melanin synthesis is carried out by a cell-specific enzymatic pathway controlled by tyrosinase (EC 1.14.18.1), the enzyme that catalyzes the initial two rate-limiting reactions of this process, the hydroxylation of tyrosine to dopa and its subsequent oxidation to dopaquinone (
      • Hearing V.J.
      • Jimenez M.
      ,
      • Hearing V.J.
      • Jimenez M.
      ,
      • Prota G.
      ,
      • Hearing V.J.
      • Tsukamoto K.
      ). In vivo, melanogenesis is induced mainly by ultraviolet A and B radiation of sunlight and α-melanocyte-stimulating hormone (α-MSH)
      The abbreviations used are: α-MSH
      α-melanocyte-stimulating hormone
      AEBSF
      4-(2-aminoethyl)benzenesulfonyl fluoride
      ERK1
      extracellular signal regulated kinase 1
      PNPP
      p-nitrophenolphosphate
      PI
      phosphatidylinositol
      PI3-K
      phosphatidylinositol 3-kinase
      p70S6K
      p70S6-kinase
      MOPS
      4-morpholinepropanesulfonic acid
      PBS
      phosphate-buffered saline
      bp
      base pair(s).
      (
      • Levine N.
      • Sheftel N.
      • Eytan T.
      • Dorr R.T.
      • Hadley M.E.
      • Weinrach G.A.
      • Ertl G.A.
      • Toth K.
      • McGee D.L.
      • Hruby V.J.
      ) which binds to a specific G protein-coupled receptor. In cultured melanocytes or in melanoma cells, melanogenesis can be induced by ultraviolet A and B radiation and by a large array of effectors including α-MSH (
      • Hearing V.J.
      • Tsukamoto K.
      ) and pharmacological agents such as forskolin, cholera toxin, or isobutylmethylxanthine (
      • Wong G.
      • Pawelek J.
      ,
      • Halaban R.
      • Pomerantz S.H.
      • Marshall S.
      • Lambert D.T.
      • Lerner A.B.
      ,
      • Hunt G.
      • Todd C.
      • Cresswell J.E.
      • Thody A.J.
      ,
      • Englaro W.
      • Rezzonico R.
      • Durand-Clément M.
      • Lallemand D.
      • Ortonne J.-P.
      • Ballotti R.
      ). These agents increase the intracellular cAMP content, thereby indicating the importance of the cAMP pathway in melanogenesis. The stimulation of melanogenesis by cAMP-elevating agents seems to occur through the induction of tyrosinase expression and stimulation of its intrinsic enzymatic activity ensuing post-translational modifications (
      • Fuller B.B.
      • Lundsford J.B.
      • Iman D.S.
      ). However, few data are available concerning molecular mechanisms that connect the cAMP signaling pathway and tyrosinase regulation. Recently, we have shown in B16 melanoma cells that cAMP-elevating agents stimulate ERK1 activity and induce its translocation to the nucleus (
      • Englaro W.
      • Rezzonico R.
      • Durand-Clément M.
      • Lallemand D.
      • Ortonne J.-P.
      • Ballotti R.
      ), whereas in the majority of cell systems, cAMP has been described to inhibit this kinase (
      • Burgering B.M.
      • Pronk G.J.
      • van Weeren P.C.
      • Chardin P.
      • Bos J.L.
      ). Furthermore, concomitantly to the stimulation of ERK1 and melanogenesis, cAMP induces a morphological differentiation characterized by dendrite outgrowth (
      • Englaro W.
      • Rezzonico R.
      • Durand-Clément M.
      • Lallemand D.
      • Ortonne J.-P.
      • Ballotti R.
      ) and an inhibition of B16 melanoma cell proliferation. Similar effects including ERK1 activation, neurite outgrowth, and cell growth inhibition have been observed during cAMP-induced differentiation of rat pheochromocytoma PC12 cells (
      • Cowley S.
      • Paterson H.
      • Kemp P.
      • Marshall J.C.
      ,
      • Sale E.M.
      • Atkinson P.G.P.
      • Sale G.J.
      ), which, like melanocytes, are derived from the neural crest. The mechanisms of differentiation in PC12 cells have been thoroughly investigated. Recently, two reports have shown that wortmannin (
      • Arcaro A.
      • Whymann M.
      ), a potent phosphatidylinositol-3-kinase (PI3-K) inhibitor, as well as dominant negative mutants of PI3-K, clearly inhibit nerve growth factor-induced neurite outgrowth in PC12 cells, thus demonstrating a positive involvement of PI3-K in PC12 cell differentiation (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ,
      • Jackson T.R.
      • Blader I.J.
      • Latanya P.H.-O.
      • Burga C.R.
      • Cooke F.
      • Hawkins P.T.
      • Wolf A.G.
      • Heldman K.A.
      • Theibert A.B.
      ).
      PI3-K belongs to a family of signal transducer heterodimeric enzymes composed of a 85-kDa regulatory subunit (p85 α or β) containing SH2 and SH3 domains and a 110-kDa catalytic subunit (p110 α or β) (
      • Hu P.
      • Schlessinger J.
      ,
      • Volinia S.
      • Hiles I.
      • Ortmondroyd E.
      • Nizetic D.
      • Antonacci R.
      • Rocchi M.
      • Waterfield M.D.
      ) that phosphorylates the D3 hydroxyl in the inositol ring of phosphatidylinositol. PI3-K is activated after association of the p85 regulatory subunit with tyrosine-phosphorylated proteins, including activated tyrosine kinase receptors, non-receptor tyrosine kinases (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Bout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz-Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtneidge S.A.
      • Waterfield M.D.
      ,
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayotou G.
      • Thompson A.
      • Dhand R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Stephens L.R.
      • Hughes K.T.
      • Irvine R.F.
      ), and docking proteins such IRS-1 (
      • Giorgetti S.
      • Ballotti R.
      • Kowalski-Chauvel A.
      • Tartare S.
      • Van Obberghen E.
      ). The role of PI3-K in transducing mitogenic signals is currently clearly confirmed, and recently the kinase has been found to be implicated in differentiation (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ,
      • Jackson T.R.
      • Blader I.J.
      • Latanya P.H.-O.
      • Burga C.R.
      • Cooke F.
      • Hawkins P.T.
      • Wolf A.G.
      • Heldman K.A.
      • Theibert A.B.
      ). Other studies have involved PI3-K in membrane transport and intracellular traffic through the regulation of membrane and cytoskeleton rearrangements occurring in response to growth factor stimulation (
      • Arcaro A.
      • Whymann M.
      ,
      • Wymann M.
      • Arcaro A.
      ,
      • Wennström S.
      • Hawkins P.
      • Cooke F.
      • Hara K.
      • Yonezawa K.
      • Kasuga M.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • et al.
      ).
      Recent studies have demonstrated that the serine/threonine kinase, p70S6-kinase (p70S6K) acts downstream of PI3-K (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ,
      • Chou M.
      • Blenis J.
      ). P70S6K phosphorylates its main target, the 40 S ribosomal protein S6, which is involved in translational up-regulation of an essential family of mRNAs, including transcripts for ribosomal proteins and elongation factors. Inhibition of the p70S6K activity by the immunosupressant rapamycin or microinjection of neutralizing antibodies severely block cell cycle progression at the middle G1 phase, indicating that p70S6K is necessary for cells to enter the S phase (
      • Chung J.
      • Kuo C.J.
      • Crabtree G.R.
      • Blenis J.
      ).
      Taking into account the involvement of the PI3-K pathway in PC12 cell differentiation and in an attempt to search for intracellular signaling pathways involved in melanogenesis, we focused our interest on the implication of PI3-K in the B16 melanoma cell differentiation process. In this report, using the specific PI3-K inhibitor LY294002 (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ) and the p70S6K-specific inhibitor rapamycin (
      • Price D.J.
      • Grove J.R.
      • Calvo V.
      • Avruch J.
      • Bierer B.E.
      ), we show that the inhibition of the PI3-K/p70S6K pathway mimics the effect of cAMP-elevating agents and leads to a strong stimulation of melanogenesis in B16 melanoma cells. Furthermore, cAMP triggers a significant inhibition of PI3-K activity and a strong blockage of p70S6K activity, thus revealing the implication of the PI3-K/p70S6K pathway in the transmission of the melanogenic effect of cAMP.

      DISCUSSION

      In the present work we provide evidence for the role of the PI3-K/p70S6K pathway in the control of B16 melanoma cell differentiation which is characterized by a stimulation of melanin synthesis and dendrite outgrowth. In PC12 cells, the inhibition of PI3-K activity has been shown to prevent nerve growth factor-induced neurite outgrowth (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ,
      • Jackson T.R.
      • Blader I.J.
      • Latanya P.H.-O.
      • Burga C.R.
      • Cooke F.
      • Hawkins P.T.
      • Wolf A.G.
      • Heldman K.A.
      • Theibert A.B.
      ). Since PC12 cells share numerous features with B16 cells, we therefore expected that the inhibition of PI3-K would block the cAMP-induced dendrite outgrowth in this melanoma cell line. Strikingly, our studies revealed that, in B16 cells, the inhibition of PI3-K by LY294002 did not block forskolin effects but was sufficient by itself to induce a strong cell dendricity. However, the inhibition of p70S6K that functions downstream PI3-K (
      • Chou M.
      • Blenis J.
      ) did not lead to any morpholological changes. This observation suggests the existence of PI3-K targets acting upstream or independently of p70S6K that might play a regulatory role in the induction of these morphological modifications. The protein kinase B/Akt which is a serine/threonine protein kinase encoded by the proto-oncogene akt (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ) has been found to function downstream PI3-K and to participate to the p70S6K activation (
      • Boudewijn M.
      • Burgering T.
      • Coffer P.J.
      ); it is tempting to propose that other unknown targets of protein kinase B/AKT could be involved in the induction of the dendritic phenotype. Similarly, some members of the protein kinase C family such as protein kinase C δ, ϵ, θ, and ζ, which are directly activated by phosphatidylinositol phosphate (
      • Nakanishi H.
      • Brewer K.A.
      • Exton J.H.
      ,
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Moriya S.
      • Kazlauskas A.
      • Akimoto K.
      • Hirai S.
      • Mizuno K.
      • Takenawa T.
      • Fukui Y.
      • Watanabe Y.
      • Ozaki S.
      • Ohno S.
      ) are also putative candidates that could participate in this phenomenon. Moreover, it has recently been reported that proteins Rho and Rac, which belong to the superfamily of small G proteins, are indirectly regulated by PI3-K and mediate cytoskeleton rearrangements triggering events such as membrane ruffling and vesicle reorganization (
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Olson M.F.
      • Ashworth A.
      • Hall A.
      ,
      • Kotani K.
      • Hara K.
      • Kotani K.
      • Yonezawa K.
      • Kasuga M.
      ,
      • Hawkins P.T.
      • Eguinoa A.
      • Qui R.G.
      • Stokoe D.
      • Cooke F.T.
      • Walters R.
      • Wennström S.
      • Claesson-Welsh L.
      • Evans T.
      • Symons M.
      • Stephens L.
      ). This points to the putative involvement of these molecules in eliciting dendrite outgrowth processes after cAMP or LY294002 stimulation. Future research is certainly required to identify the pathway leading to this morphological modification (dendricity) in B16 melanoma cells.
      On the other hand, our work demonstrates that the inhibition of PI3-K led to a stimulation of melanin synthesis that appeared to result from an increased tyrosinase activity and expression. In addition, similar effects were observed with rapamycin, indicating that the inhibition of p70S6K is sufficient to induce melanogenesis. However, rapamycin, which is as potent as forskolin or LY294002 in stimulating tyrosinase activity, is markedly less efficient in inducing cell tanning. A possible explanation of this finding might reside in the fact that the synthesis of black melanin (eumelanin) involves other specific regulated enzymes such as tyrosinase-related proteins 1 and 2 (
      • Jackson J.I.
      • Cambers D.M.
      • Tsukamoto K.
      • Copeland N.
      • Gilbert D.J.
      • Jenkins N.A.
      • Hearing V.
      ,
      • Kameyama K.
      • Takemura T.
      • Hamada Y.
      • Sakai C.
      • Kondoh S.
      • Nishiyama S.
      • Urabe K.
      • Hearing V.J.
      ,
      • Yokoyama K.
      • Yasumoto K.
      • Suzuki H.
      • Shibahara S.
      ). Thus, we can hypothesize that rapamycin may somehow inhibit the expression or the activity of these tyrosinase-related enzymes or down-regulate other steps downstream of tyrosinase in the melanin production pathway.
      It is worth emphasizing that forskolin, LY294002, and rapamycin produced similar effects on melanogenesis. This supports the hypothesis that the induction of melanogenesis by cAMP could be due to a down-regulation of the PI3-K/p70S6K pathway. Indeed an inhibition of PI3-K activity by cAMP has been already described in T lymphocytes (
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.
      • Vlahos C.
      • Blenis J.
      ) and in neutrophils (
      • Ahmed M.U.
      • Hazeki K.
      • Hazeki O.
      • Katada T.
      • Ui M.
      ). Furthermore, cAMP-elevating agents have been shown to inhibit p70S6K activity in Swiss 3T3 cells (
      • Petritsch C.
      • Woscholski R.
      • Edelmann H.M.L.
      • Ballou L.M.
      ) and in T lymphocytes (
      • Monfar M.
      • Lemon K.P.
      • Grammer T.C.
      • Cheatham L.
      • Chung J.
      • Vlahos C.
      • Blenis J.
      ). In B16 melanoma cells, forskolin partially inhibits PI3-K activity and completely blocks p70S6K activity. In addition, this cAMP-elevating agent as well as α-MSH (not shown), a physiological melanogenic agent that increases the intracellular cAMP levels, induced a dramatic inhibition of p70S6K phosphorylation which constitutes an essential event for the activation of the kinase. Since the inhibition of PI3-K activity by cAMP appears rather weak, this could not entirely explain the strong inhibition of p70S6K activity found after the treatment with cAMP elevating agents. Therefore it can be proposed that cAMP could act through the regulation of the activity of protein kinase B or other kinases involved in phosphorylation and activation of p70S6K.
      Interestingly, we observed a significant diminution in the cell number and in DNA synthesis, without cytotoxic effects, after forskolin, LY294002, or rapamycin treatments (not shown), revealing an arrest of cell growth. This could be due to the inhibitory effects of forskolin, LY294002, or rapamycin on p70S6K activity, which has been found essential for progression through the G1 phase of the cell cycle (
      • Lane H.A.
      • Fernandez A.
      • Lamb N.J.C.
      • Thomas G.
      ). In contrast, serum or growth factors, such as 12-O-tetradecanoylphorbol-13-acetate or epidermal growth factor that activate p70S6K, display an inhibitory effect on melanogenesis in B16 cells (not shown). These findings suggest that the induction of melanogenesis and morphological differentiation in B16 cells might require the arrest of cell growth at the G1 phase. A similar conclusion has been reached concerning the induction of neurite outgrowth in the neuroblastoma cell line NE1-115 (
      • Kranenburg O.
      • Scharnhorst V.
      • Van der Eb A.J.
      • Zantema A.
      ).
      The precise molecular mechanisms that connect the inhibition of the PI3-K/p70S6K pathway to the induction of melanogenesis and dendrite outgrowth remain to be elucidated. Nevertheless, the existence of a nuclear isoform of the S6 kinase (p85S6K) (
      • Reinhard C.
      • Fernandez A.
      • Lamb N.J.C.
      • Thomas G.
      ) is consistent with the idea that this kinase could modulate the activity of transcription factors. Indeed, cAMP-responsive element modulator τ has been reported to be phosphorylated and activated by p70S6K (
      • Groot R.P.
      • Ballou L.M.
      • Sassone-Corsi P.
      ). On the other hand, microphthalmia, a tissue-specific transcription factor that has been recently shown to play a key role in the stimulation of tyrosinase gene expression by cAMP (
      • Bertolotto C.
      • Bille K.
      • Ortonne J.-P.
      • Ballotti R.
      ), could also be a target of p70S6K. Thus the inhibition of this kinase by cAMP would lead to a decreased activation of microphthalmia, or other transcription factors involved in B16 cell differentiation, and thereby regulate their transcriptional activities.
      Taken together our results demonstrate the involvement of the PI3-K/p70S6K pathway in B16 melanoma cells differentiation and indicate for the first time that the inhibition of this signaling cascade by cAMP is likely to be a key event for the cAMP-induced melanogenesis and dendrite outgrowth. Furthermore, the novel finding that the inhibition of the PI3-K/p70S6K pathway displays a positive effect on B16 melanoma cell differentiation whereas the PI3-K inhibition blocks the differentiation process in PC12 cells, reveals that the study of several cell systems is required to fully understand the mechanisms of cell differentiation.

      Acknowledgments

      We thank Dr. G. Thomas for providing the antibody M6 against p70S6K and Dr. V. Hearing for the antibody anti-tyrosinase PEP7. We are grateful to Naïma Biagioli for the preparation of culture reagents and to C. Minghelli for his help in the illustration work, and we thank Dr. Kim Boulukos for critical reading of this manuscript.

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