Silencing of GPNMB by siRNA inhibits the formation of melanosomes in melanocytes in a MITF-independent fashion

PLoS One. 2012;7(8):e42955. doi: 10.1371/journal.pone.0042955. Epub 2012 Aug 13.

Abstract

Background: Melanosomes are specialized membrane-surrounded organelles, which are involved in the synthesis, storage and transport of melanin. Glycoprotein (transmembrane) non-metastatic melanoma protein b (GPNMB), a melanosome-specific structural protein, shares significant amino acid sequence homology with Pmel-17. Proteomic analysis demonstrated that GPNMB is present in all stages (I-IV) of melanosomes. However, little is known about the role of GPNMB in melanosomes.

Methodology/principal findings: Using real-time quantitative PCR, Western blotting and immunofluorescence analysis, we demonstrated that the expression of GPNMB in PIG1 melanocytes was up-regulated by ultraviolet B (UVB) radiation. Transmission electron microscopy analysis showed that the total number of melanosomes in PIG1 melanocytes was sharply reduced by GPNMB-siRNA transfection. Simultaneously, the expression levels of tyrosinase (Tyr), tyrosinase related protein 1 (Trp1), Pmel17/gp100 and ocular albinism type 1 protein (OA1) were all significantly attenuated. But the expression of microphthalmia-associated transcription factor (MITF) was up-regulated. Intriguingly, in GPNMB silenced PIG1 melanocytes, UVB radiation sharply reduced MITF expression.

Conclusion: Our present work revealed that the GPNMB was critical for the formation of melanosomes. And GPNMB expression down-regulation attenuated melanosome formation in a MITF-independent fashion.

MeSH terms

  • Blotting, Western
  • Cell Line
  • DNA Primers / genetics
  • Eye Proteins / metabolism
  • Flow Cytometry
  • Gene Expression Regulation / radiation effects*
  • Gene Silencing
  • Humans
  • Melanocytes / metabolism*
  • Melanosomes / metabolism*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism*
  • Microphthalmia-Associated Transcription Factor / metabolism
  • Microscopy, Electron, Transmission
  • Microscopy, Fluorescence
  • Monophenol Monooxygenase / metabolism
  • Proteomics
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Trypsin / metabolism
  • Ultraviolet Rays

Substances

  • DNA Primers
  • Eye Proteins
  • GPNMB protein, human
  • GPR143 protein, human
  • MITF protein, human
  • Membrane Glycoproteins
  • Microphthalmia-Associated Transcription Factor
  • RNA, Small Interfering
  • Monophenol Monooxygenase
  • PRSS1 protein, human
  • Trypsin

Grant support

The authors have no support or funding to report.