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Glucosamine abrogates the stem cell factor + endothelin-1-induced stimulation of melanogenesis via a deficiency in MITF expression due to the proteolytic degradation of CREB in human melanocytes

  • Takao Niwano
  • Shuko Terazawa
  • Yoriko Sato
  • Takahiro Kato
  • Hiroaki Nakajima
  • Genji Imokawa
Original Paper

Abstract

We have already reported that glucosamine (GlcN) distinctly abrogates the pigmentation of human epidermal equivalents stimulated by stem cell factor + endothelin-1 (SE). In this study, we characterized the molecular mechanism involved in the anti-melanogenic effects of GlcN using normal human melanocytes (NHMs) in culture. The SE-stimulated gene (12 h) and protein (24 h) expression levels of melanocyte-specific proteins (at the indicated times post-stimulation) were significantly abrogated by pretreatment with GlcN for 72 h. Western blotting analysis of the phosphorylation of intracellular signaling molecules in the MAPK pathway revealed that despite the significantly decreased level of total CREB protein at all times post-stimulation, the SE-stimulated phosphorylation of ERK, CREB and MITF is not attenuated at 15 min post-stimulation in GlcN-treated NHMs. However, the SE-stimulated protein expression level of total MITF at 2 and 6 h post-stimulation was significantly abrogated by 72 h pretreatment with GlcN. Consistently, pretreatment with GlcN for 72 h abrogated the stimulated gene and protein expression levels of MITF at 1 h and 2 h post-stimulation, respectively. Analysis of gene and protein expression levels also demonstrated that pretreatment with GlcN for 72 h significantly reduced the protein levels of CREB and MITF without affecting their gene expression levels prior to the SE stimulation. Silencing with a CREB siRNA distinctly abrogated the SE-stimulated expression of MITF (at 2 h post-stimulation) and melanocyte-specific proteins (at 24 h post-stimulation). Similarly, transfection of MITF siRNA markedly abrogated the SE-stimulated expression of MITF protein and melanocyte-specific proteins at 2 and 24 h post-stimulation, respectively. Finally, the decreased levels of CREB and MITF proteins induced by 72 h pretreatment with GlcN were abrogated by the co-addition of the proteosomal degradation inhibitor MG132. These findings suggest that the anti-melanogenic effect elicited by GlcN is mediated via the decreased expression of MITF which results from the attenuated transcriptional activity of CREB due to proteolytic degradation.

Keywords

Glucosamine Tyrosinase Stem cell factor Endothelin 1 Melanogenesis Intracellular signaling pathway 

Abbreviations

GlcN

Glucosamine

EDN

Endothelin

SCF

Stem cell factor

MITF

Microphthalmia-associated transcription factor

CREB

Cyclic AMP responsive element binding protein

NHMs

Normal human melanocytes

TYR

Tyrosinase

TRP1

Tyrosinase-related protein-1

TRP2

Tyrosinase-related protein-2 (or dopachrome tautomerase)

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

HEEs

Human epidermal equivalents

Notes

Funding

There is no funding source.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

403_2018_1850_MOESM1_ESM.pdf (218 kb)
Supplementary material 1 (PDF 217 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Center for Bioscience Research and EducationUtsunomiya UniversityTochigiJapan
  2. 2.School of Bioscience and TechnologyTokyo University of TechnologyTokyoJapan
  3. 3.Research Institute for Biological FunctionsChubu UniversityAichiJapan

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