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Arenarol isolated from a marine sponge abrogates endothelin-1-stimulated melanogenesis by interrupting MEK phosphorylation in normal human melanocytes

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Abstract

Using B16 melanoma cells for screening, we found that a marine sponge extract has a potent anti-pigmenting effect and identified arenarol as its major active compound. In normal human melanocytes (NHMs), arenarol significantly abrogated the endothelin 1 (EDN1) stimulated expression of tyrosinase, tyrosinase-related protein 1 and dopachrome tautomerase at the transcriptional, translational and enzymatic activity (only for tyrosinase) levels. That effect was accompanied by the attenuation of the increased expression level of microphthalmia-associated transcription factor (MITF) protein at the transcriptional and translational levels. Analysis of EDN1 signaling demonstrated that arenarol significantly suppressed the EDN1-induced phosphorylation of MEK, ERK, MITF and CREB but not of Raf-1s. In contrast, the forskolin-induced phosphorylation of CREB was not down-regulated by arenarol. As for the mode of action of the suppressed phosphorylation of MEK, Raf-1 activity was not directly inhibited by arenarol in vitro and pretreatment with the protein phosphatase inhibitor okadaic acid did not affect the down-regulated phosphorylation of MEK that was induced by arenarol in NHMs. The sum of these findings suggests that arenarol abrogates the EDN1-stimulated expression of melanocyte-specific proteins by interrupting MEK phosphorylation in an as yet unknown Raf-1 inactivation mechanism.

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Abbreviations

NHMs:

Normal human melanocytes

MITF:

Microphthalmia-associated transcription factor

TYR:

Tyrosinase

TYRP-1:

Tyrosinase-related protein-1

DCT:

Dopachrome tautomerase

EDN1:

Endothelin 1

SCF:

Stem cell factor

PKC:

Protein kinase C

CREB:

Cyclic AMP responsive element binding protein

References

  • Abdel-Malek Z, Swope VB, Suzuki I, Akcali C, Harriger MD, Boyce ST, Urabe K, Hearing VJ (1995) Mitogenic melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proc Natl Acad Sci USA 92:1789–1793

    Article  CAS  Google Scholar 

  • Abraham D, Podar K, Pacher M, Kubicek M, Welzel N, Hemmings BA, Dilworth SM, Mischak H, Kolch W, Baccarini M (2000) Raf-1-associated protein phosphatase 2A as a positive regulator of kinase activation. J Biol Chem 275:22300–22304

    Article  CAS  Google Scholar 

  • Ando H, Funasaka Y, Oka M, Ohashi A, Furumura M, Matsunaga J, Matsunaga N, Hearing VJ, Ichihashi M (1999) Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis. J Lipid Res 40:1312–1316

    CAS  Google Scholar 

  • Barnard D, Diaz B, Clawson D, Marshall M (1998) Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms. Oncogene 17:1539–1547

    Article  CAS  Google Scholar 

  • Bentley NJ, Eisen T, Goding CR (1994) Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator. Mol Cell Biol 14:7996–8006

    CAS  Google Scholar 

  • Bertolotto C, Buscà R, Abbe P, Bille K, Aberdam E, Ortonne JP, Ballotti R (1998) Different cis-acting elements are involved in the regulation of TRP1 and TRP2 promoter activities by cyclic AMP: pivotal role of M boxes (GTCATGTGCT) and of microphthalmia. Mol Cell Biol 18:694–702

    CAS  Google Scholar 

  • Bialojan C, Takai A (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J 256:283–290

    CAS  Google Scholar 

  • Carroll MP, May WS (1994) Protein kinase C-mediated serine phosphorylation directly activates Raf-1 in murine hematopoietic cells. J Biol Chem 269:1249–1256

    CAS  Google Scholar 

  • Chong H, Vikis HG, Guan KL (2003) Mechanisms of regulating the Raf kinase family. Cell Signal 15:463–469

    Article  CAS  Google Scholar 

  • Chung KW, Park YJ, Choi YJ, Park MH, Ha YM, Uehara Y, Yoon JH, Chun P, Moon HR, Chung HY (2012) Evaluation of in vitro and in vivo anti-melanogenic activity of a newly synthesized strong tyrosinase inhibitor (E)-3-(2,4 dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP). Biochim Biophys Acta 1820:962–969

    Article  CAS  Google Scholar 

  • Hachiya A, Kobayashi A, Ohuchi A, Takema Y, Imokawa G (2001) The paracrine role of stem cell factor/c-kit signaling in the activation of human melanocyte in ultraviolet B-induced pigmentation. J Invest Dermatol 116:578–586

    Article  CAS  Google Scholar 

  • Hachiya A, Kobayashi A, Yoshida Y, Kitahara T, Takema Y, Imokawa G (2004) Biphasic expression of two paracrine melanogenic cytokines, stem cell factor and endothelin-1, in ultraviolet B-induced human melanogenesis. Am J Pathol 65:2099–2109

    Article  Google Scholar 

  • Hardie DG (1990) Roles of protein kinases and phosphatases in signal transduction. Symp Soc Exp Biol 44:241–255

    CAS  Google Scholar 

  • Hattori H, Kawashima M, Ichikawa Y, Imokawa G (2004) The epidermal stem cell factor is over-expressed in lentigo senilis: implication for the mechanism of hyperpigmentation. J Invest Dermatol 122:1256–1265

    Article  CAS  Google Scholar 

  • Imokawa G, Mishima Y (1982) Loss of melanogenic properties in tyrosinases induced by glycosylation inhibitors within malignant melanoma cells. Cancer Res 42:1994–2002

    CAS  Google Scholar 

  • Imokawa G, Mishima Y (1984) Functional analysis of tyrosinase isozymes of cultured melanoma cells during the recovery period following interrupted melanogenesis induced by glycosylation inhibitors. J Invest Dermatol 83:196–201

    Article  CAS  Google Scholar 

  • Imokawa G, Yada Y, Miyagishi M (1992) Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem 267:24675–24680

    CAS  Google Scholar 

  • Imokawa G, Miyagishi G, Yada Y (1995) Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol 105:32–37

    Article  CAS  Google Scholar 

  • Imokawa G, Kobayashi T, Miyagishi M (2000) Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes: cross-talk via trans-activation of the tyrosine kinase c-kit receptor. J Biol Chem 275:33321–33328

    Article  CAS  Google Scholar 

  • Kadono S, Manaka I, Kawashima M, Kobayashi T, Imokawa G (2001) The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J Invest Dermatol 116:571–577

    Article  CAS  Google Scholar 

  • Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, Finkenzeller G, Marmé D, Rapp UR (1993) Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 364:249–252

    Article  CAS  Google Scholar 

  • Komiyama K, Takamatsu S, Takahashi Y, Shinose M, Hayashi M, Tanaka H, Iwai Y, Omura S, Imokawa G (1993) New inhibitors of melanogenesis, OH-3984 K1 and K2, I. taxonomy, fermentation, isolation and biological characteristics. J Antibiot 46:1520–1525

    Article  CAS  Google Scholar 

  • Leevers SJ, Paterson HF, Marshall CJ (1994) Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature 369:411–414

    Article  CAS  Google Scholar 

  • Marais R, Light Y, Mason C, Paterson H, Olson MF, Marshall CJ (1998) Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280:109–112

    Article  CAS  Google Scholar 

  • Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ, Marais R (1999) Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation. EMBO J 18:2137–2148

    Article  CAS  Google Scholar 

  • Moodie SA, Willumsen BM, Weber MJ, Wolfman A (1993) Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. Science 260:1658–1661

    Article  CAS  Google Scholar 

  • Paine C, Sharlow E, Liebel F, Eisinger M, Shapiro S, Seiberg M (2001) An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway. J Invest Dermatol 116:587–595

    Article  CAS  Google Scholar 

  • Sato-Jin K, Nishimura EK, Akasaka E, Huber W, Nakano H, Miller A, Du J, Wu M, Hanada K, Sawamura D, Fisher DE, Imokawa G (2008) Epistatic connections between MITF and endothelin signaling in Waardenburg syndrome and other pigmentary disorders. FASEB J 22:1155–1168

    Article  CAS  Google Scholar 

  • Schmiz FJ, Lakshmi V, Powell DR, Helm DVD (1984) Arenarol and arenarone: sesquiterpenoids with rearranged drimane skeletons from the marine sponge Dysidea arenaria. J Org Chem 49:241–244

    Article  Google Scholar 

  • Schönwasser DC, Marais RM, Marshall CJ, Parker PJ (1998) Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol Cell Biol 18:790–798

    Google Scholar 

  • Stokoe D, Macdonald SG, Cadwallader K, Symons M, Hancock JF (1994) Activation of Raf as a result of recruitment to the plasma membrane. Science 264:1463–1467

    Article  CAS  Google Scholar 

  • Suzuki I, Cone RD, Im S, Nordlund J, Abdel-Malek ZA (1996) Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 137:1627–1633

    Article  CAS  Google Scholar 

  • Winder AJ, Harris H (1991) New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. Eur J Biochem 198:317–326

    Article  CAS  Google Scholar 

  • Yada Y, Higuchi K, Imokawa G (1991) Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem 266:18352–18357

    CAS  Google Scholar 

  • Yoo HD, Leung D, Sanghara J, Daley D, Soest R, Andersen RJ (2003) Isoarenarol, a new protein kinase inhibitor from the marine sponge Dysidea arenaria. Pharm Biol 41:223–225

    Article  CAS  Google Scholar 

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Acknowledgments

We thank GCOE and Leading Program in Nagoya University and a scholarship of Mitsubishi UFJ Trust and Banking for their financial supports.

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Authors and Affiliations

  1. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan

    Bong-Keun Choi & Makoto Ojika

  2. Research Institute for Biological Functions, Chubu University, Kasugai, Japan

    Byung-Yoon Cha, Je-Tae Woo & Genji Imokawa

  3. OP Bio Factory Co. Ltd., Okinawa, Japan

    Takeshi Fujiwara & Akihiko Kanamoto

Authors
  1. Bong-Keun Choi
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  2. Byung-Yoon Cha
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  3. Takeshi Fujiwara
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  4. Akihiko Kanamoto
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  5. Je-Tae Woo
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  6. Makoto Ojika
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  7. Genji Imokawa
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Correspondence to Makoto Ojika or Genji Imokawa.

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Genji Imokawa and Makoto Ojika as corresponding authors contributed equally to this work.

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Choi, BK., Cha, BY., Fujiwara, T. et al. Arenarol isolated from a marine sponge abrogates endothelin-1-stimulated melanogenesis by interrupting MEK phosphorylation in normal human melanocytes. Cytotechnology 65, 915–926 (2013). https://doi.org/10.1007/s10616-013-9555-5

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  • DOI: https://doi.org/10.1007/s10616-013-9555-5

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