Silencing of GPNMB by siRNA Inhibits the Formation of Melanosomes in Melanocytes in a MITF-Independent Fashion

Ping Zhang,Tianwen Gao,Huimin Ma,Wei Liu,Weijie Gu,Dongguang Li,Cansheng Zhu,Xiaoying Yuan,Xin Xie,Reiner Albert Veitia

doi:10.1371/journal.pone.0042955

Abstract

BackgroundMelanosomes 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 FindingsUsing 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.ConclusionOur 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.

Highlights

The melanosome is a specialized membrane-surrounded organelle, which is involved in the synthesis, storage and transport of melanin
12 proteins have been identified as melanosome-specific proteins, including tyrosinase (Tyr), tyrosinase-related protein 1 (Trp1), tyrosinase-related protein 2 (Trp2, being known as dopachrome tautomerase), ocular albinism type 1 protein (OA1), melanoma-associated antigen recognized by T cells (MART-1), Pmel17/gp100, vesicle amine transport protein 1 homolog (VAT-1), oculospanin, syntenin, coiled-coil-helix-coiled-coil-helix domain containing 3 (CHCHD3), flotillin-1/2 and glycoprotein nonmetastatic melanoma protein b (GPNMB) [1,2]
Given that the ultraviolet B (UVB) radiation could up-regulate the expression of Tyr [17], we determined whether the expression of GPNMB could be modulated by UVB

Summary

Introduction

The melanosome is a specialized membrane-surrounded organelle, which is involved in the synthesis, storage and transport of melanin. Multiple enzymatic and structural proteins are involved in the maturation of melanosomes. 12 proteins have been identified as melanosome-specific proteins, including tyrosinase (Tyr), tyrosinase-related protein 1 (Trp1), tyrosinase-related protein 2 (Trp, being known as dopachrome tautomerase), ocular albinism type 1 protein (OA1), melanoma-associated antigen recognized by T cells (MART-1), Pmel17/gp100, vesicle amine transport protein 1 homolog (VAT-1), oculospanin, syntenin, coiled-coil-helix-coiled-coil-helix domain containing 3 (CHCHD3), flotillin-1/2 and glycoprotein (transmembrane) nonmetastatic melanoma protein b (GPNMB) [1,2]. The vital roles of some of these proteins with regards to enzymatic components in melanosome biogenesis are well known, such as Tyr, Trp and. Glycoprotein (transmembrane) non-metastatic melanoma protein b (GPNMB), a melanosomespecific structural protein, shares significant amino acid sequence homology with Pmel-17. Little is known about the role of GPNMB in melanosomes

Methods

Results

Conclusion