SOX5影响小鼠皮肤黑色素细胞MITF-M表达和黑色素生成
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摘要
旨在探讨SOX5在小鼠皮肤毛色中的作用。本研究随机选取出生后12d的C57BL/6品系黑色、棕色、灰色小鼠各3只,采用实时荧光定量PCR、Western blotting和免疫组织化学方法对SOX5在黑色、棕色和灰色小鼠皮肤中的表达差异进行分析;并构建SOX5真核表达载体;运用体外转染的方法,转染小鼠黑色素细胞并检测与色素形成相关基因的表达水平,同时测定黑色素含量。结果表明:1)SOX5在不同毛色小鼠皮肤中均有表达,SOX5在黑色和灰色小鼠皮肤中mRNA和蛋白相对表达量高于棕色小鼠皮肤,均差异极显著(P<0.01);且其主要表达部位为毛囊外根鞘。2)转染组与空载组相比,MITF-M、TYR、TYRP1、TYRP2、PMEL、OA1表达量均显著(P<0.05)或极显著(P<0.01)升高,黑色素含量也极显著增加(P<0.01)。3)过表达MITF-M对SOX5存在负反馈作用。综上表明,SOX5通过调控MITF-M影响色素的生成进而参与毛色的形成。
To investigate the function of SOX5 in mouse skins and the regulation of hair color formation,the differential expression of SOX5 in skins of different coat color mouse skin was examined.In this study,C57 BL/6 strains of black,brown and gray mice were selected each of the 3 for 12 days after birth,randomly.Quantitative real-time PCR(qRT-PCR),Western blotting and immunohistochemistry methods were used in this study to analyze the expression of SOX5;we also constructed the eukaryotic expression vector of SOX5 and transfected the mouse melanocytes to detect the expression level of the pigmentation-related genes,and measured the melanin content.The results showed that:1)SOX5 could be expressed in mouse skin with different hair color.The mRNA and protein expression levels of SOX5 in black and gray mouse skin were higher than that in brown mouse skin with very significantly difference(P<0.01).And the main expression site of SOX5 was hair follicle outer root sheath.2)Compared with empty vector group(VectorGFP),the mRNA and protein expression levels of MITF-M,TYR,TYRP1,TYRP2,PMEL and OA1 were increased with significant difference(P<0.05)and very significantly difference(P<0.01)in SOX5 transfected group;the content of melanin was significantly increased with verysignificantly difference(P<0.01)in SOX5 transfected group.3)MITF-M had a negative feedback on SOX5 by overexpression of MITF-M.These suggest that SOX5 affect the formation of pigments and then involve in the formation of coat color by regulating MITF-M.
To investigate the function of SOX5 in mouse skins and the regulation of hair color formation,the differential expression of SOX5 in skins of different coat color mouse skin was examined.In this study,C57 BL/6 strains of black,brown and gray mice were selected each of the 3 for 12 days after birth,randomly.Quantitative real-time PCR(qRT-PCR),Western blotting and immunohistochemistry methods were used in this study to analyze the expression of SOX5;we also constructed the eukaryotic expression vector of SOX5 and transfected the mouse melanocytes to detect the expression level of the pigmentation-related genes,and measured the melanin content.The results showed that:1)SOX5 could be expressed in mouse skin with different hair color.The mRNA and protein expression levels of SOX5 in black and gray mouse skin were higher than that in brown mouse skin with very significantly difference(P<0.01).And the main expression site of SOX5 was hair follicle outer root sheath.2)Compared with empty vector group(VectorGFP),the mRNA and protein expression levels of MITF-M,TYR,TYRP1,TYRP2,PMEL and OA1 were increased with significant difference(P<0.05)and very significantly difference(P<0.01)in SOX5 transfected group;the content of melanin was significantly increased with verysignificantly difference(P<0.01)in SOX5 transfected group.3)MITF-M had a negative feedback on SOX5 by overexpression of MITF-M.These suggest that SOX5 affect the formation of pigments and then involve in the formation of coat color by regulating MITF-M.
引文
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[20]BAXTER L L,PAVAN W J.Pmel17expression is Mitf-dependent and reveals cranial melanoblast migration during murine development[J].Gene Expr Patterns,2003,3(6):703-707.
[21]WATT B,VAN NIEL G,RAPOSO G,et al.PMEL:apigment cell-specific model for functional amyloid formation[J].Pigment Cell Melanoma Res,2013,26(3):300-315.
[22]CHEN T Z,WANG H D,LIU Y,et al.Ocular Albinism Type 1regulates melanogenesis in mouse melanocytes[J].Int J Mol Sci,2016,17(10):1596.
[23]FALLETTA P,BAGNATO P,BONO M,et al.Melanosome-autonomous regulation of size and number:the OA1receptor sustains PMEL expression[J].Pigment Cell Melanoma Res,2014,27(4):565-579.
[24]STEINGRMSSON E,COPELAND N G,JENKINS N A.Melanocytes and the microphthalmiatranscription factor network[J].Annu Rev Genet,2004,38(1):365-411.
[25]THODY A J,HIGGINS E M,WAKAMATSU K,et al.Pheomelanin as well as eumelanin is present in human epidermis[J].J Invest Dermatol,1991,97(2):340-344.
[26]WANG P C,ZHAO Y Y,FAN R W,et al.microRNA-21a-5p functions on the regulation of melanogenesis by targeting Sox5in mouse skin melanocytes[J].Int J Mol Sci,2016,17(7):959.
[27]GERSTENBLITH M R,SHI J X,LANDI M T.Genome-wide association studies of pigmentation and skin cancer:a review and meta-analysis[J].Pigment Cell Melanoma Res,2010,23(5):587-606.
[28]BURGOYNE T,O′CONNOR M N,SEABRA M C,et al.Regulation of melanosome number,shape and movement in the zebrafish retinal pigment epithelium by OA1and PMEL[J].J Cell Sci,2015,128(7):1400-1407.
[29]杨玉静,张丹瑾,聂瑞强,等.绵羊MITF-M在黑素细胞中过表达后的功能分析[J].中国农业科学,2016,49(21):4214-4221.YANG Y J,ZHANG D J,NIE R Q,et al.The function analysis of over-expression of oar MITF-M in melanocytes[J].Scientia Agricultura Sinica,2016,49(21):4214-4221.(in Chinese)
[2]LEFEBVRE V.The SOXD transcription factorsSOX5,SOX6,and SOX13-are key cell fate modulators[J].Int J Biochem Cell Biol,2010,42(3):429-432.
[3]SMITS P,DY P,MITRA S,et al.SOX5and SOX6are needed to develop and maintain source,columnar,and hypertrophic chondrocytes in the cartilage growth plate[J].J Cell Biol,2004,164(5):747-758.
[4]MARTINEZ-MORALES P L,QUIROGA A C,BARBAS J A,et al.SOX5controls cell cycle progression in neural progenitors by interfering with the WNT-β-catenin pathway[J].EMBO Rep,2010,11(6):466-472.
[5]STOLT C C,LOMMES P,HILLGRTNER S,et al.The transcription factor SOX5 modulates SOX10function during melanocyte development[J].Nucleic Acids Res,2008,36(17):5427-5440.
[6]KORDAΒT,WEBER C E M,OSWALD M,et al.SOX5is involved in balanced MITFregulation in human melanoma cells[J].BMC Med Genomics,2016,9:10.
[7]HEMESATH T J,STEINGRMSSON E,MCGILL G,et al.microphthalmia,a critical factor in melanocyte development,defines a discrete transcription factor family[J].Genes Dev,1994,8(22):2770-2780.
[8]LEVY C,KHALED M,FISHER D E.MITF:master regulator of melanocyte development and melanoma oncogene[J].Trends Mol Med,2006,12(9):406-414.
[9]HERSHEY C L,FISHER D E.Genomic analysis of the Microphthalmia locus and identification of the MITF-J/Mitf-J isoform[J].Gene,2005,347(1):73-82.
[10]FUSE N,YASUMOTO K,SUZUKI H,et al.Identification of a melanocyte-type promoter of the microphthalmia-associated transcription factor gene[J].Biochem Biophys Res Commun,1996,219(3):702-707.
[11]ROH E,YUN C Y,YUN J Y,et al.cAMP-binding site of PKA as a molecular target of bisabolangelone against melanocyte-specific hyperpigmented disorder[J].J Invest Dermatol,2013,133(4):1072-1079.
[12]HARTMAN M L,CZYZ M.Pro-survival role of MITF in melanoma[J].J Invest Dermatol,2015,135(2):352-358.
[13]WELLBROCK C,AROZARENA I.Microphthalmiaassociated transcription factor in melanoma development and MAP-kinase pathway targeted therapy[J].Pigment Cell Melanoma Res,2015,28(4):390-406.
[14]YASUMOTO K,YOKOYAMA K,TAKAHASHI K,et al.Functional analysis of microphthalmia-associated transcription factor in pigment cell-specific transcription of the human tyrosinase family genes[J].J Biol Chem,1997,272(1):503-509.
[15]FANG D,TSUJI Y,SETALURI V.Selective downregulation of tyrosinase family gene TYRP1by inhibition of the activity of melanocyte transcription factor,MITF[J].Nucleic Acids Res,2002,30(14):3096-3106.
[16]DU J Y,MILLER A J,WIDLUND H R,et al.MLANA/MART1 and SILV/PMEL17/GP100 are transcriptionally regulated by MITF in melanocytes and melanoma[J].Am J Pathol,2003,163(1):333-343.
[17]MCKAY B S,SCHWARTZ S G.Pigmentation and macular degeneration:is there a role for GPR143?[J].J Ocul Pharmacol Ther,2016,32(1):3-4.
[18]GIORDANO F,SIMOES S,RAPOSO G.The ocular albinism type 1(OA1)GPCR is ubiquitinated and its traffic requires endosomal sorting complex responsible for transport(ESCRT)function[J].Proc Natl Acad Sci U S A,2011,108(29):11906-11911.
[19]BENTLEY N J,EISEN T,GODING C R.Melanocyte-specific expression of the human tyrosinase promoter:activation by the microphthalmia gene product and role of the initiator[J].Mol Cell Biol,1994,14(12):7996-8006.
[20]BAXTER L L,PAVAN W J.Pmel17expression is Mitf-dependent and reveals cranial melanoblast migration during murine development[J].Gene Expr Patterns,2003,3(6):703-707.
[21]WATT B,VAN NIEL G,RAPOSO G,et al.PMEL:apigment cell-specific model for functional amyloid formation[J].Pigment Cell Melanoma Res,2013,26(3):300-315.
[22]CHEN T Z,WANG H D,LIU Y,et al.Ocular Albinism Type 1regulates melanogenesis in mouse melanocytes[J].Int J Mol Sci,2016,17(10):1596.
[23]FALLETTA P,BAGNATO P,BONO M,et al.Melanosome-autonomous regulation of size and number:the OA1receptor sustains PMEL expression[J].Pigment Cell Melanoma Res,2014,27(4):565-579.
[24]STEINGRMSSON E,COPELAND N G,JENKINS N A.Melanocytes and the microphthalmiatranscription factor network[J].Annu Rev Genet,2004,38(1):365-411.
[25]THODY A J,HIGGINS E M,WAKAMATSU K,et al.Pheomelanin as well as eumelanin is present in human epidermis[J].J Invest Dermatol,1991,97(2):340-344.
[26]WANG P C,ZHAO Y Y,FAN R W,et al.microRNA-21a-5p functions on the regulation of melanogenesis by targeting Sox5in mouse skin melanocytes[J].Int J Mol Sci,2016,17(7):959.
[27]GERSTENBLITH M R,SHI J X,LANDI M T.Genome-wide association studies of pigmentation and skin cancer:a review and meta-analysis[J].Pigment Cell Melanoma Res,2010,23(5):587-606.
[28]BURGOYNE T,O′CONNOR M N,SEABRA M C,et al.Regulation of melanosome number,shape and movement in the zebrafish retinal pigment epithelium by OA1and PMEL[J].J Cell Sci,2015,128(7):1400-1407.
[29]杨玉静,张丹瑾,聂瑞强,等.绵羊MITF-M在黑素细胞中过表达后的功能分析[J].中国农业科学,2016,49(21):4214-4221.YANG Y J,ZHANG D J,NIE R Q,et al.The function analysis of over-expression of oar MITF-M in melanocytes[J].Scientia Agricultura Sinica,2016,49(21):4214-4221.(in Chinese)