Nonmetastatic Melanoma Protein Research Articles

Amyloidosis cutis dyschromica cases caused by GPNMB mutations with different inheritance patterns

BackgroundAmyloidosis cutis dyschromica (ACD) is a rare form of primary cutaneous amyloidosis featured by reticulate dotted hypo- and hyperpigmentation. Recently, loss-of-function mutations in GPNMB, encoding glycoprotein (transmembrane) nonmetastatic melanoma protein B, were found in autosomal-recessive or semi-dominant ACD. ObjectiveThis study aims to detect the genetic defect underlying ACD in nine separate cases and to investigate the functional consequences of the mutants. MethodsNine ACD cases were collected including eight with autosomal-recessive pattern and one with autosomal-dominant pattern. Whole-exome sequencing or Sanger sequencing of the GPNMB gene was performed to detect the pathogenic mutations. Haplotype analysis was employed to determine the origin of mutation c.565C > T using adjacent highly polymorphic SNPs. Immunoblotting and subcellular localization assessments were performed to evaluate the expression of the mutants using HEK293 cells transfected with the GPNMB constructs. ResultsWe detected four recurrent mutations (c.393 T > G, p.Y131*; c.565C > T, p.R189*; c.1056delT, p.P353Lfs*20; c.1238 G > C, p.C413S) and two novel mutations (c.935delA, p.N312Tfs*4; c.969 T > A, p.C323*) in GPNMB. Mutation c.565C > T found in six separate ACD cases shared a common haplotype. The two novel mutations caused a decreased abundance of truncated proteins. The c.1238 G > C mutation, which was detected in the autosomal-dominant case, caused abnormal reticular subcellular localization of the protein. A major percentage of wildtype changed its expression pattern when co-expressed with this mutant. ConclusionsOur findings proved that the recurrent mutation c.565C > T originated from a founder effect. The autosomal-dominant ACD associated mutation p.C413S played its pathogenic role through a dominant-negative effect on wild-type GPNMB. This study expands the genotype and inherited modes of ACD and improves our understanding of the pathogenesis of this disorder.

Effect of glycoprotein (transmembrane) nonmetastatic melanoma protein B on the proliferation and migration of as well as melanogenesis in melanoma cells

Objective To estimate the effect of glycoprotein (transmembrane) nonmetastatic melanoma protein B (GPNMB) on the proliferation and migration of as well as melanogenesis in melanoma cells.Methods The expression of GPNMB was detected by immunofluorescence assay in two melanoma cell lines M14 and G-361,as well as in primary human melanocytes.Then,the three kinds of cells each were classified into three groups:experimental group treated with small interfering RNA targeting GPNMB (GPNMB-siRNA),negative control group treated with the negative control siRNA,blank control group remaining untreated.Methyl thiazolyl tetrazolium (MTT) assay,transwell invasion assay and spectrophotometry were performed to evaluate cell proliferation activity,invasion potential and melanin levels,respectively.Statistical analysis was done using Student's t test.Results GPNMB was expressed in both melanoma cells and melanocytes.The transfection with GPNMB-siRNA down-regulated the mRNA and protein expressions of GPNMB in,and markedly suppressed the proliferation and migration of,melanoma cells.In detail,the proliferative activity (expressed as the absorbence value at 570 nm) of M14 and G361 cells was reduced by 35％ and 40％ respectively,the migration activity of M14 and G361 cells by 49％ and 51％ respectively,and the melanin levels in melanocytes,M14 cells and G361 cells by 73％,82％ and 69％ respectively,in the experiment group compared with those in the blank control group.Conclusions The siRNA-mediated silencing of GPNMB could effectively inhibit the proliferation of,invasion of and melanogenesis in melanoma cells,which suggests that GPNMB plays critical roles in the initiation and progression of melanoma. Key words: GPNMB; RNA, small interfering; Melanins; Cell proliferation; Cell movement

Endothelin-1 enhances the melanogenesis via MITF-GPNMB pathway

Endothelin-1 (ET-1) plays an indispensable role in epidermal pigmentation in hyperpigmentary disorders due to a central role in melanogenesis. Nevertheless, precise mechanism involved in ET-1-induced hyperpigmentation is still undefined. Glycoprotein (transmembrane) non-metastatic melanoma protein b (GPNMB) is a key element in melanosome formation. Therefore, we speculated that GPNMB was correlated with ET-1-induced pigmentation. After culturing with ET-1, melanin synthesis was significantly up-regulated, accompanying with increased expression of GPNMB and microphthalmia-associated transcription factor (MITF). Total number of melanosomes and melanin synthesis were sharply reduced via GPNMB-siRNA transfection, indicating ET-1-induced pigmentation by GPNMB-dependent manner. Furthermore, MITFsiRNA transfection strikingly inhibited GPNMB expression and the melanogenesis, and this suppression failed to be alleviated by ET-1 stimulation. All of these results demonstrated that ET-1 can trigger melanogenesis via the MITF-regulated GPNMB pathway. Taken together, these findings will provide a new explanation of how ET-1 induces hyperpigmentation, and possibly supply a new strategy for cosmetic studies. [BMB Reports 2013; 46(7): 364-369]

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

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.