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


RESULTS
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.


CONCLUSIONS
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.
Department of Dermatology, the General Hospital of the Air Force, Beijing, China.
Ping
P
Zhang
Wei
W
Liu
Cansheng
C
Zhu
Xiaoying
X
Yuan
Dongguang
D
Li
Weijie
W
Gu
Huimin
H
Ma
Xin
X
Xie
Tianwen
T
Gao
D015153:Blotting, Western; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000528:radiation effects*; D020868:Gene Silencing; D006801:Humans; D008544:Melanocytes / Q000378:metabolism*; D020460:Melanosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051739:Microphthalmia-Associated Transcription Factor / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D014442:Monophenol Monooxygenase / Q000378:metabolism; D040901:Proteomics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D017931:DNA Primers; D005136:Eye Proteins; C503042:GPNMB protein, human; C095276:GPR143 protein, human; C497234:MITF protein, human; D008562:Membrane Glycoproteins; D051739:Microphthalmia-Associated Transcription Factor; D034741:RNA, Small Interfering; D014442:Monophenol Monooxygenase; C489303:PRSS1 protein, human; D014357:Trypsin