title: Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling.
abstract: BACKGROUND
  Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect.
  
  
  OBJECTIVE
  To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action.
  
  
  RESULTS
  RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity of tyrosinase in melanocytes. Western blot analysis of intracellular signaling factors revealed that withaferin A but not astaxanthin significantly abolished the endothelin-1-stimulated phosphorylation of Raf-1, MEK, ERK, MITF and CREB in human melanocytes.
  
  
  CONCLUSIONS
  These results demonstrate that this co-culture system is an appropriate model to characterize melanogenic paracrine interactions and that astaxanthin and withaferin A serve as potent inhibitors of those interactions. Their effects are caused not only by down-regulating the increased secretion of an intrinsic melanogenic cytokine, endothelin-1, by UVB-exposed human keratinocytes, but also by interrupting the endothelin-1-triggered downstream intracellular signaling between protein kinase C and Raf-1 in human melanocytes (only for withaferin A).
authors.affiliation: Tsuno Rice Fine Chemicals, Co., Ltd., Japan; School of Bioscience and Biotechnology, Tokyo University of Technology, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Niwano
authors.affiliation: Research Institute for Biological Functions, Chubu University, Japan.
authors.forename: Shuko
authors.initials: S
authors.lastname: Terazawa
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakajima
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Wakabayashi
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology, Japan; Research Institute for Biological Functions, Chubu University, Japan. Electronic address: imokawag@dream.ocn.ne.jp.
authors.forename: Genji
authors.initials: G
authors.lastname: Imokawa
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D019332:Endothelin-1 / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D014442:Monophenol Monooxygenase / Q000378:metabolism; D019899:Paracrine Communication / Q000187:drug effects* / Q000528:radiation effects; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D015398:Signal Transduction / Q000187:drug effects* / Q000528:radiation effects; D014466:Ultraviolet Rays; D054358:Withanolides / Q000494:pharmacology*; D024341:Xanthophylls / Q000494:pharmacology
chemical_list: D000906:Antibodies; D016207:Cytokines; D019332:Endothelin-1; D054358:Withanolides; D024341:Xanthophylls; C005948:astaxanthine; D014442:Monophenol Monooxygenase; C009684:withaferin A; D002118:Calcium; D004229:Dithiothreitol
keywords: Endothelin-1; Keratinocytes; Melanocytes; Paracrine interaction; Tyrosinase