GPNMB And Its Homolog PMEL Regulate The Formation Of Melanocytic Pigment In Mice

The melanosome is a specialized membrane-surrounded organelle,which is involved in the synthesis,storage and transport of melanin.Melanosomes experience four sequential morphological stages(stage ?,?,III and IV)during their mature process.So far more than 150 pigmentation genes have been identified.The vital roles of some of these proteins with regards to enzymatic components in melanosome biogenesis are well known,such as TYR,TYRP1 and TYRP2.However,the functions of some structural proteins in the biosynthesis of melanosome are not clear,including the melanosome-specific structural protein GPNMB and PMEL.Up to now,The studie on the effects of GPNMB and PMEL on the regulation of MITF upstream and downstream related coat color genes affecting pigment generation in mouse melanocytes was not reported.The mechanism of GPNMB and PMEL in the formation of different coat color and follicular period in mice and the regulation of pigment formation was explored by a variety of biological methods.Pur:pose:1.To explore the expression and localization of GPNMB and PMEL in mouse skin in different coat color and hair follicle period;2.1t is clear how the expression of GPNMB and PMEL regulates the production of pigment on MITF upstream and downstream genes;3.Preliminary study on the changed rules of GPNMB and PMEL expression by UVB.Method:1.The skin of mice with different coat color and hair follicle period was taken as the research object.The expression levels of GPNMB and PMEL were detected by immunohistochemical method,Western Blot and real-time fluorescence quantitative PCR;2.In order to deep study the effect of GPNMB and PMEL on pigment production,GPNMB and PMEL were respectively transfected into mouse melanocytes.The MITF upstream and downstream gene expression levels and the changes in melanin content were detected.In addition,the relationship between GPNMB,PMEL and MITF was examined by transfection of the MITF expression vector;3.To preliminary investigate the expression of GPNMB and PMEL,the mouse melanocytes were irradiated with different intensity and irradiation time of UVB to detect the relationship between proliferation curves and the expression of GPNMB and PMEL.Result:1(1)The expression of GPNMB in black and brown mice was significantly higher than that in gray mice and the skin expression of black mice was higher than that of brown;The expression level of PMEL in black and brown mouse skin was also significantly higher than that in gray skin and the skin expression of brown mice was higher than that of black;The results of immunohistochemistry showed that GPNMB was expressed in the hair matrix,inner and outer root sheath,hairy nipples and other areas of skin follicle in different coat mice.(2)The expression of GPNMB in the early and medium period of follicle growth was significantly higher than that in the late period of follicle growth and the early expression of follicle was higher than that of medium;The expression level of PMEL in the early and medium period of follicle growth was significantly higher than that in the late period of follicle growth and the early expression of follicle was higher than that of medium;The results of immunohistochemistry showed that the expression of GPNMB in the hair matrix,inside and outside hair root sheath of different hair follicle growth cycle were obviously different,and the degree of staining was also different.2(1)There was no significant change in the volume and dendrites of mouse melanocytes before and after transfection of GPNMB and PMEL.And the transfection results showed the highest transfection efficiency of both is 5 ?g DNA/well,which was seen as the concentration of highest transfection.(2)Besides the decrease in MITF expression after the transfection of GPNMB,the expression levels of TYR,TYRP1,TYRP2,PMEL,OA1,SLC45A2,MC1R and ?-catenin were increased and the content of melanin was significantly increased;The expression levels of MITF,TYR,TYRP1,TYRP2,GPNMB,OA1,SLC45A2,MC1R and ?-catenin were increased and the melanin content significantly increased after PMEL transfection.(3)In addition,the expression of GPNMB was decreased after MITF transfection,but the expression of PMEL was increased.3.(1)The proliferation curve was determined by MTT:the cell proliferation activity was the highest when the intensity was 100 mJ/cm2 for 1 day by UVB irradiation.(2)The intensity of 100 mJ/cm2 was irradiated 0 d,1 d,3 d,5 d with UVB;The expression level of GPNMB was:UVB irradiation 1 d>3 d>5 d>0 d;The PMEL expression was:UVB irradiation 0 d>3 d>5 d>1 d.(3)The melanocytes were irradiated with UVB at different intensities,and the cell morphology at 200 mJ/cm2 and 300 mJ/cm2 was significantly changed on the 5th day.At this time,the expression of GPNMB was:intensity 300 mJ/cm2>200 mJ/cm2>0 mJ/cm2>50 mJ/cm2>100 mJ/cm2;The expression of PMEL after 5 days of UVB irradiation was as follows:intensity was 100 mJ/cm2>50 mJ/cm2>0 mJ/cm2>200 mJ/cm2>300 mJ/cm2.Conclusion:1.GPNMB and PMEL have a certain function to the formation of coat color and the growth cycle of hair follicles;2.After overexpression of GPNMB,the upstream and downstream genes were regulated by independent MITF pathway to increase the melanin production;After PMEL overexpression,the upstream and downstream genes were regulated by dependent MITF pathway to increase the melanin production;3.The expression of GPNMB and PMEL were affected by UVB irradiation.