Function Of A Novel Melanosome-specific Protein GPNMB On Melanin Production

Melanin metabolic process is the research focus in pigmentary disorders and skinwhitening fields. Melanin is mainly produced by melanocytes. Thus, the key of whiteningis to inhibit the ability of melanocytes to produce melanin. It is considered traditionallythat, for the inhibition of the melanin synthesis, it is vital to suppress the activity oftyrosinase (Tyr), the key enzyme for melanin synthesis. With the progress of the melaninsynthesis mechanism, people have been able to inhibit melanin formation through multipletarget molecules. In recent years, some new melanosome-specific proteins with unknownfunctions in melanin metabolism were discovered, including non-metastatic melanomaglycoprotein B (GPNMB). However, their function on melanin metabolism was unclear.This study was aimed to explore the role of GPNMB in melanin synthesis.Aim: To discover modulation of GPNMB by ultraviolet B (UVB). To explore the roleof GPNMB in melanin synthesis by siRNA interference, and its possible mechanisms.Methods: GPNMB was selected as the object in the following studies. Real-time quantitative RT-PCR, Western blot and immunofluorescence were used to determine themodulation of UVB on GPNMB expression in melanin epithelial cell line PIG1. Tyr, aprotein known to be modulated by UVB was used as a positive control. A mixture ofsiRNA target GPNMB was used to transfect PIG1melanocytes to inhibit the GPNMBexpression. Then, Western blot was used to determine the transfection efficiency, and thetransmission electron microscope was used to observe the melanosome formation. ThemRNA and protein expression of Tyr were determined by real-time quantitative RT-PCRand Western blot. We also analyzed the expression of microphthalmia-associatedtranscription factor (MITF) as well as several melanosome-specific proteins regulated byMITF, such as Trp1, and OA1by real-time quantitative RT-PCR.Results:1.GPNMB was up-regulated by UVB with a variation tendency similar to that of Tyr.2.Total number of melanosome in PIG1melanocytes was sharply reduced afterGPNMB expression was knocked down by GPNMB-siRNA transfection. Simultaneously,the mRNA and protein expression of Tyr were both significantly reduced.3.We also demonstrated that UVB irradiation or the down-regulation of GPNMB could raise the expression of MITF. But, when melanocytes PIG1were treated with UVB irradiation and GPNMB-siRNA, the expression of MITF was sharply declined.Conclusions:1. GPNMB expression could be up-regulated by UVB.2. The inhibition of GPNMB expression led to the reduction of melanosome formation and tyrosinaseexpression.3. And the reduction induced by GPNMB inhibition was disconnected withMITF pathway.