Essential role of the molecular chaperone gp96 in regulating melanogenesis

Through a process known as melanogenesis, melanocyte produces melanin in specialized organelles termed melanosomes, which regulates pigmentation of the skin, eyes, and hair. Gp96 is a constitutively expressed heat shock protein in the endoplasmic reticulum whose expression is further upregulated upon ultraviolet irradiation. However, the roles and mechanisms of this chaperone in pigmentation biology are unknown. In this study, we found that knockdown of gp96 by RNA interference significantly perturbed melanin synthesis and blocked late melanosome maturation. Gp96 knockdown did not impair the expression of tyrosinase, an essential enzyme in melanin synthesis, but compromised its catalytic activity and melanosome translocation. Further, mice with melanocyte‐specific deletion of gp96 displayed decreased pigmentation. A mechanistic study revealed that the defect in melanogenesis can be rescued by activation of the canonical Wnt pathway, consistent with the critical roles of gp96 in chaperoning Wnt‐coreceptor LRP6. Thus, this work uncovered the essential role of gp96 in regulating melanogenesis.     

Tyrosinase-related proteins suppress tyrosinase-mediated cell death of melanocytes and melanoma cells

The synthesis of melanin intermediates through tyrosinase (TYR) involves the production of cytotoxic free radicals. By using recombinant adenoviruses that express TYR, tyrosinase-related protein 1 (TYRP1) or DOPAchrome tautomerase (DCT), we analyzed the biological function of these proteins with regard to melanin production and the growth of melanocytes, fibroblasts, melanoma cells and nonmelanoma cancer cells. High-level expression of TYR produced newly synthesized melanin and induced cell death in all of these cells. However, when TYRP1 or DCT was coexpressed with TYR in melanocytes and melanoma cells, TYR-mediated cell death was clearly decreased. This decrease was not observed in nonmelanocytic cells. Western blot analysis and measurement of enzyme activity revealed that the expression of TYRP1 or DCT had little effect on the amount or activity of cointroduced TYR in either the melanocytic or nonmelanocytic cells. In cells expressing both TYR and TYRP1 or TYR and DCT, the total amount of melanin and/or eumelanin increased substantially more than that in cells expressing TYR alone. On the other hand, the level of pheomelanin was similar in these three cell types. These findings suggest that TYRP1 and DCT play an important role in suppressing TYR-mediated cytotoxicity in melanocytic cells without decreasing TYR expression and/or activity. These biological activities of TYRP1 and DCT may work through the interaction with TYR in melanosomal compartment.     

miR-146a抑制酪氨酸酶基因家族在小鼠黑色素细胞中表达

miRNA是在许多生物过程中都起着至关重要作用的一类内源性非编码的小RNA,与癌症、肿瘤的发生有关。现发现很多miRNA在黑色素生成中都有重要的调控作用,但miR-146a是否对黑色素的生成具有影响未见报道。本研究发现miR-146a通过靶向抑制酪氨酸酶相关蛋白1(tyrosinase related protein 1,TYRP1)的表达而使黑色素生成降低。在小鼠黑色素细胞中分别转染miR-146a mimic和miR-146a 抑制剂,通过qRT-PCR与Western印迹分析比较各实验组中TYRP1基因与酪氨酸家族相关基因酪氨酸酶(tyrosinase, TYR)、酪氨酸酶相关蛋白2(tyrosinase related protein 2, TYRP2)的表达差异。双荧光报告实验验证TYRP1与miR-146a的靶向关系,双荧光酶活性结果显示,实验组相比对照组,荧光素酶活性明显降低,说明TYRP1是miR-146a的靶基因之一;qRT-PCR和Western印迹结果显示实验组TYR、TYRP1及TYRP2 在mRNA水平和蛋白质水平表达均显著降低;紫外分光光度法检测黑色素含量,结果显示miR-146a mimic转染组黑色素含量明显下降,而抑制组的黑色素含量呈上升趋势。综上所述,miR-146a通过靶向抑制TYRP1基因的表达,而影响TYR家族成员的表达,调控黑色素的生物合成。     

miRNA-338-3p通过靶向抑制MC1R基因表达抑制羊驼黑色素细胞黑色素的生成

黑色素皮质素1受体MC1R）是在黑色素细胞内表达的G蛋白耦合受体（G protein coupled receptor, GPCR）家族成员,参与黑色素细胞中黑色素的生成。微RNAs（miRNAs）是一类非编码RNA,通过与靶基因3′-UTR结合抑制基因表达。已有研究证明,miR-338-3p 在多种人类肿瘤细胞中（过）表达,可通过下调靶基因表达抑制肿瘤细胞的侵袭迁移能力。然而,有关miR-338-3p对羊驼皮肤黑色素细胞的黑色素合成影响却罕见报道。本研究证明,miRNA-338-3p通过靶向抑制MC1R基因表达,抑制羊驼黑色素细胞黑色素的生成。采用生物信息学预测MC1R基因是miRNA-338-3p的靶基因,其基因表达抑制羊驼黑色素细胞黑色素合成。随后构建miR-338-3p真核表达载体。其基因转染结合qPT-PCR和Western印迹结果揭示,与对照细胞比较,过表达miRNA-338-3p的羊驼黑色素细胞的MC1R基因,及其下游与黑色素生成相关的小眼相关性转录因子（MITF)、酪氨酸酶(TYR)、酪氨酸酶相关蛋白1（TYRP1）、酪氨酸酶相关蛋白2（TYRP2）编码基因mRNA及蛋白质表达水平明显下调。酶联免疫吸附分析显示,过表达miRNA-338-3p的羊驼皮肤黑色素细胞的黑色素产量,较对照细胞显著下降（P＜0.01）。综上结果,miR-338-3p可通过抑制靶基因MC1R表达,下调其下游基因MITF、TYR、TYRP1和TYRP2基因的表达,从而抑制羊驼皮肤黑色素细胞黑色素的合成。miRNA-338-3p在羊驼生长发育过程中,是否参与调控体内皮肤黑色素细胞的黑色素生成尚待进一步研究。     

Melanosomal pH controls rate of melanogenesis, eumelanin/phaeomelanin ratio and melanosome maturation in melanocytes and melanoma cells.

The skin pigment melanin is produced in melanocytes in highly specialized organelles known as melanosomes. Melanosomes are related to the organelles of the endosomal/lysosomal pathway and can have a low internal pH. In the present study we have shown that melanin synthesis in human pigment cell lysates is maximal at pH 6.8. We therefore investigated the role of intramelanosomal pH as a possible control mechanism for melanogenesis. To do this we examined the effect of neutralizing melanosomal pH on tyrosinase activity and melanogenesis in 11 human melanocyte cultures and in 3 melanoma lines. All melanocyte cultures (9 of 9) from Caucasian skin as well as two melanoma cell lines with comparable melanogenic activity showed rapid (within 24 h) increases in melanogenesis in response to neutralization of melanosomal pH. Chemical analysis of total melanin indicated a preferential increase in eumelanin production. Electron microscopy revealed an accumulation of melanin and increased maturation of melanosomes in response to pH neutralization. In summary, our findings show that: (i) near neutral melanosomal pH is optimal for human tyrosinase activity and melanogenesis; (ii) melanin production in Caucasian melanocytes is suppressed by low melanosomal pH; (iii) the ratio of eumelanin/phaeomelanin production and maturation rate of melanosomes can be regulated by melanosomal pH. We conclude that melanosomal pH is an essential factor which regulates multiple stages of melanin production. Furthermore, since we have recently identified that pink locus product (P protein) mediates neutralization of melanosomal pH, we propose that P protein is a key control point for skin pigmentation. We would further propose that the wide variations in both constitutive and facultative skin pigmentation seen in the human population could be associated with the high degree of P-locus polymorphism.     

lpa-miR-nov-66通过调控cAMP路径抑制α-MSH介导的羊驼黑色素生成

α-黑素细胞刺激素(α-MSH)和lpa-miR-nov-66在羊驼黑色素细胞产生黑色素过程中均起重要的调控作用,但二者之间的关系尚未报道.本研究在体外培养的羊驼黑色素细胞中通过转染lpamiR-nov-66和添加α-MSH处理,用实时定量PCR和Western印迹检测黑色素细胞内基因表达水平,ELISA法检测c AMP和cGMP的产量,RTCA实时无标记细胞功能分析黑色素细胞增殖以及紫外分光光度法检测黑色素产量,证实二者在调控羊驼黑色素细胞产生黑色素颗粒过程中的关系.结果显示,与单纯α-MSH处理相比,lpa-miR-nov-66转染结合α-MSH处理组中,小眼转录因子(MITF)和酪氨酸酶(TYR)在转录水平和翻译水平的表达均降低,而酪氨酸酶相关蛋白2(TYRP2)在转录和翻译水平的表达均升高;cGMP的产量升高,cAMP的产量下降;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量下降.与单纯转染lpa-miR-nov-66相比,lpa-miR-nov-66转染结合α-MSH处理组中,MITF、TYR和TYRP2在转录水平和翻译水平的表达均升高;cGMP的产量下降,cAMP的产量升高;黑色素细胞增殖没有显著变化;黑色素细胞内黑色素产量升高.上述结果证明,lpa-miR-nov-66通过调控羊驼黑色素细胞中毛色形成的c AMP路径,抑制α-MSH对黑色素细胞产生黑色素的促进作用.     

DKK3调控羊驼黑色素细胞中黑色素生成

Dickkopf-3(DKK3),Wnt/p-catenin信号通路中一个重要的抑制因子,可能参与调控黑色素生成过程.本文研究了DKK3在羊驼黑色素细胞中黑色素生成的作用.在羊驼黑色素细胞中,过表达DKK3显著下调Wntl,Lefl,Myc和黑色素生成相关基因MITF及其下游基因TYR,TYRP1和TYRP2的表达,在...     

小鼠毛囊不同生长周期中MITF下游色素相关基因的定位表达及相关性分析

小眼畸形转录因子（MITF）不仅是黑色素细胞发育、增殖和存活的必要调节因子,而且对调节相关酶和黑素体蛋白表达来确保黑色素产生具有至关重要的作用。MITF下游色素相关基因在小鼠毛囊生长周期中的表达及相关性仍有待研究。HE染色结果表明不同毛囊时期的小鼠毛囊呈现典型的组织形态学结构;免疫组织化学显示,MITF、GPNMB、OA1、TYR、TYRP2在不同毛囊生长周期中的毛基质及内外毛根鞘均有不同程度的阳性表达。黑色素测定结果表明,在毛囊生长初期和中期,碱性可溶性总黑色素（ASM）、真黑素（EM）以及褐黑素（PM）相对含量高于毛囊生长末期。蛋白免疫印迹结果表明,MITF、GPNMB、OA1、TYR、TYRP2在毛囊生长初期和中期蛋白质相对水平明显高于毛囊生长末期。实时荧光定量PCR结果表明, MITF、GPNMB、OA1、TYR、TYRP2、PMEL在毛囊生长初期和中期,mRNA相对表达量显著高于毛囊生长末期。在不同毛囊生长周期小鼠皮肤的MITF下游色素相关基因表达存在显著差异,表明上述因子在维持黑色素细胞色素生成是不可或缺的因素。     

Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle

The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.     

The repeat domain of the melanosomal matrix protein PMEL17/GP100 is required for the formation of organellar fibers

Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles.     

Differential radiosensitivity in cultured B-16 melanoma cells following interrupted melanogenesis induced by glucosamine

The relationship between cell pigmentation and radiosensitivity was investigated in a cell model in which melanogenesis was suppressed by a glycosylation inhibitor. It was found that X-irradiation of melanotic B-16 melanoma cells and their amelanotic counterparts, obtained by glucosamine treatment, showed an inverse correlation between radiosensitivity and melanin contents. Since melanogenesis interruption by glucosamine does not affect the DNA repair capacity of nonpigmented cells, it is likely that intracellular melanins play a role in the relative resistance of pigmented cells to X-irradiation.     

Activation of melanogenesis by vacuolar type H(+)-ATPase inhibitors in amelanotic, tyrosinase positive human and mouse melanoma cells

In this study, we describe the activation of melanogenesis by selective vacuolar type H(+)-ATPase inhibitors (bafilomycin A1 and concanamycin A) in amelanotic human and mouse melanoma cells which express tyrosinase but show no melanogenesis. Addition of the inhibitors activated tyrosinase within 4 h, and by 24 h the cells contained measurable amounts of melanin. These effects were not inhibited by cycloheximide (2 microgram/ml) which is consistent with a post-translational mechanism of activation. Our findings suggest that melanosomal pH could be an important and dynamic factor in the control of melanogenesis in mammalian cells.     

Assembly, target-signaling and intracellular transport of tyrosinase gene family proteins in the initial stage of melanosome biogenesis

Assembly, target-signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein-3 (AP-3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di-leucine motif in their cytoplasmic tail, to which AP-3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes

A mutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype "chocolate" (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38(G19V) is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.