Rab7 is a critical mediator in vesicular transport of tyrosinase-related protein 1 in melanocytes

How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.     

Increased sensitivity of melanocytes to oxidative stress and abnormal expression of tyrosinase-related protein in vitiligo

BACKGROUND: Vitiligo is a depigmenting disease of the skin, which may derive from programmed melanocyte death or destruction due to inherent sensitivity to oxidative stress arising from either toxic intermediates of melanin, a melanocyte-specific protein, or other sources. Tyrosinase-related protein (TRP) -1 has been shown to be involved not only in melanin biosynthesis but also in the prevention of premature melanocyte death in animals. OBJECTIVES: To clarify the biological role of human TRP-1 in melanocyte survival. METHODS: Cultured melanocyte strains from an active advancing border of vitiligo were established and studied. RESULTS: The established 'vitiligo melanocytes' showed large perikaryon and stubby dendrites. They showed early cell death when exposed to oxidative stress (ultraviolet B) and increased and abnormal immunostaining and immunoprecipitation by antibodies against human and mouse TRP-1, indicating an altered synthesis and processing of TRP-1. In pulse-chase and sequential immunoprecipitation experiments, vitiligo melanocytes revealed abnormal protein-protein interaction with calnexin, a melanogenesis-associated chaperone, suggesting altered folding and maturation of nascent TRP-1 polypeptides. Northern blot analysis indicated a decreased expression of TRP-1 mRNA, but heteroduplex analysis and verification of the mutation at the carboxy terminus of TRP-1 by restriction enzyme analysis did not show any abnormality. CONCLUSIONS: Our study suggests that the early cell death of vitiligo melanocytes is related to their increased sensitivity to oxidative stress, which may arise from complex processes of abnormal synthesis and processing of TRP-1 and its interaction with calnexin.     

PGE2 is a UVR‐inducible autocrine factor for human melanocytes that stimulates tyrosinase activation

Please cite this paper as: PGE₂ is a UVR‐inducible autocrine factor for human melanocytes that stimulates tyrosinase activation. Experimental Dermatology 2010; 19: 682–684. Abstract: Prostaglandins activate signalling pathways involved in growth, differentiation and apoptosis. Prostaglandin E₂ (PGE₂) is released by keratinocytes following ultraviolet irradiation (UVR) and stimulates the formation of dendrites in melanocytes. We show that multiple irradiations of human melanocytes with UVR‐activated cPLA₂, the rate‐limiting enzyme in eicosanoid synthesis and stimulated PGE₂ secretion. PGE₂ increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE₂ binds to four distinct G‐protein coupled receptors (EP_1–4). We show that PGE₂ stimulates EP₄ receptor signalling in melanocytes, resulting in cAMP production. Conversely, PGE₂ also stimulated the EP₃ receptor in melanocytes, resulting in lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE₂ on cAMP in melanocytes. The data are the first to identify PGE₂ as an UVR‐inducible autocrine factor for melanocytes. These data also show that PGE₂ activates EP₃ and EP₄ receptor signalling, resulting in opposing effects on cAMP production, a critical signalling pathway that regulates proliferation and melanogenesis in melanocytes.     

Placental extracts regulate melanin synthesis in normal human melanocytes with alterations of mitochondrial respiration

Placental extracts have been widely used as skin lightening agents in the Japanese cosmetic market. Here, we show that placental extracts contain factors that can decrease or increase melanin synthesis by normal human melanocytes in vitro in possible association with mitochondrial respiration. When normal human melanocytes were treated with a whole porcine placental extract, melanin synthesis was decreased. In contrast, a porcine placental extract in which exudates and insoluble materials including lipids had been removed increased melanin synthesis. In addition, the amount of tyrosinase, the enzyme critical for melanin synthesis, changed in accordance with the alteration of melanin synthesis. Interestingly, the amount of manganese‐dependent superoxide dismutase (MnSOD), a mitochondrial‐resident antioxidant enzyme, was increased when melanin synthesis was decreased by the whole placental extract. Mitochondrial respiration and glycolysis also changed following treatment with the placental extracts. These results suggest that placental extracts contain factors that can increase or decrease melanin synthesis by normal human melanocytes and that mitochondrial function may be associated with the placental extract‐induced regulation of melanogenesis.     

Heat shock protein 27 is expressed in normal and malignant human melanocytes in vivo

BACKGROUND: Heat shock proteins (HSPs) are a family of highly conserved proteins found ubiquitously in mammalian cells, believed to be regulators of normal cell physiology and the cellular stress response. In addition, the small 27-kDa heat shock protein (HSP27) has previously been found to be a differentiation marker for keratinocytes and a prognostic marker associated with increased survival in certain cancerous tumors. METHODS: Using immunohistochemistry on routinely processed paraffin sections, we examined skin biopsies from 15 invasive melanomas, 13 intradermal nevi, and two compound nevi immunostained with a mouse monoclonal antibody to HSP27. In addition, cultured melanocytes were heat stressed at 45 degrees C for 1 h and then fixed and immunostained in order to localize HSP27 expression intracellularly. RESULTS: We found cytoplasmic and strong perinuclear staining of HSP27 in melanocytes in normal skin, in melanomas, and in nevi. Nuclear reactivity was absent. In addition, in cultured non-malignant melanocytes, HSP27 expression relocated from the cytoplasm to the nucleus with heat stress. CONCLUSIONS: To our knowledge, this investigation is the first to demonstrate that HSP27 is expressed in melanocytes in normal skin, in nevi, and in non-malignant cultured melanocytes.     

芪白合剂对培养的人黑素细胞生长及酪氨酸酶、MITF基因表达的影响

目的 研究芪白合剂对黑素细胞生长及黑素细胞酪氨酸酶、小眼相关转录因子（MITF）基因表达的影响。方法 芪白合剂组方黄芪、党参、白芷、白蒺藜、鸡血藤，水煎煮浓缩成3 g/mL，取青紫蓝兔，按12 mL&;#8226;kg-1&;#8226;d-1连续灌胃3天后于末次给药1 h，2 h和5 h无菌采血，收集血清；采用体外培养正常人黑素细胞，观察不同采血时间的芪白合剂含药血清对黑素细胞生长、酪氨酸酶的活性、黑素合成以及用RT-PCR的方法测定其对酪氨酸酶、MITF基因表达的影响。结果 含药血清与对照血清比较，能明显促进黑素细胞生长，上调酪氨酸酶的活性和黑素合成（P < 0.05），以1 h和2 h所采血清作用明显。并可以促进酪氨酸酶和 MITF的基因表达。结论 芪白合剂可以通过直接作用于黑素细胞和酪氨酸酶通道来促进黑素合成     

Abnormal translocation of tyrosinase and tyrosinase-related protein 1 in cutaneous melanocytes of Hermansky-Pudlak Syndrome and in melanoma cells transfected with anti-sense HPS1 cDNA

Hermansky-Pudlak syndrome is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and, in some patients, ceroid storage and progressive lung disease. Although Hermansky-Pudlak syndrome exhibits locus heterogeneity, most patients have mutations in the HPS1 gene. Melanocytes in the basal epithelial layer of skin from patients with different mutations in the HPS1 gene exhibited occasional large complexes containing dihydroxyphenylalanine-positive cisterna and 50 nm vesicles. To characterize the role of the HPS1 protein in cells, human HPS1 cDNA was transfected into pigmented SK-MEL-188 melanoma cells (M-188) in either the sense (S-188) or the antisense (A-188) orientation. Expression of the 79 kDa HPS1 protein (in M-188 and S-188 cells) or lack of expression (in A-188 cells) was confirmed by Western blotting using two HPS1-protein-specific polyclonal antibodies. Significant reduction in expression of HPS1 protein in A-188 cells resulted in a significant decrease in tyrosinase activity and melanin content compared with M-188 and S-188 cells using an intact cell assay for tyrosinase. In contrast, tyrosinase activities in cell lysates of M-188, S-188, and A-188 cells were not significantly different. Knockout of HPS1 protein expression in A-188 cells caused both tyrosinase and tyrosinase-related protein 1 to be localized to large granular complexes in the cell cytosol and dendrites. Electron microscope analysis of the A-188 cells revealed that absence of HPS1 protein resulted in the deposition of dihydroxyphenylalanine reaction products (i.e., tyrosinase) confined to large membrane-bound structures with limiting membranes. We conclude that lack of HPS1 protein expression results in mistranslocation of tyrosinase and tyrosinase-related protein 1 to large granular complexes rather than melanosomes, compromising melanin synthesis.     

Placental glutathione-S-transferase-pi mRNA is abundantly expressed in human skin

Four overlapping cDNA clones were isolated from a lambda gt11 human placenta cDNA library using purified human IgG antibody, from a patient with bullous pemphigoid. The sequence was homologous to human placenta glutathione-S-transferase-pi (GST-pi). Using the placenta clone, epidermal cDNA clones were isolated from a human keratinocyte library. Expression of GST-pi mRNA in human skin, cultured keratinocytes and fibroblasts, and disorders of squamous hyperplasia was demonstrated by Northern blotting and in situ hybridization. Human epidermal and placental cDNA clones hybridized to the same genomic DNA fragments. Hybridization of placental cDNA to interspecific somatic cell hybrids showed retention of chromosome 11, confirming the assignment of GST 3 to the long arm of chromosome 11 by molecular means. Anti-GST-pi antibody did not give a basement membrane zone pattern, although some normal and BP sera contained antibodies to GST-pi. Human skin expresses glutathione-S-transferase-pi, which belongs to an enzyme family important for detoxification and carcinogenesis.     

他卡西醇对人表皮黑素细胞增殖、黏附、迁移及c-kit表达的影响

【摘要】 目的 探讨他卡西醇对体外培养的人表皮黑素细胞增殖、黏附、迁移及c-kit mRNA相对表达的影响。 方法 用不同浓度的他卡西醇干预体外培养的人表皮黑素细胞,采用四唑氮氢氧化物（XTT）法分别检测培养24、48、72 h后黑素细胞的增殖活性;用纤维连接蛋白（FN）包被细胞培养板检测培养72 h后黑素细胞的黏附率;用Transwell微孔膜法检测培养24 h后黑素细胞在FN上的迁移情况;逆转录-聚合酶链反应（RT-PCR）检测培养72 h后黑素细胞c-kit mRNA相对表达量。 采用重复测量方差分析及完全随机设计方差分析进行统计学检验。 结果 重复测量方差分析结果显示,10-10、10-9、10-8、10-7、10-6 mol/L他卡西醇促进黑素细胞增殖作用的差异有统计学意义（F = 9.47,P < 0.01）,上述浓度他卡西醇在培养24、48、72 h后促进黑素细胞增殖作用的差异有统计学意义（F = 14.44,P < 0.01）,药物浓度和培养时间之间存在交互作用（F = 2.47,P < 0.01）,其中10-8 mol/L他卡西醇与黑素细胞共同培养72 h黑素细胞增殖活性最高。10-8 ～ 10-7 mol/L他卡西醇可显著促进黑素细胞在FN上的黏附（均P < 0.01）;10-9 ～ 10-8 mol/L他卡西醇在培养24 h能显著促进黑素细胞迁移（均P < 0.01）;RT-PCR显示10-9 ～ 10-7 mol/L他卡西醇在培养72 h均能显著增加黑素细胞c-kit mRNA的相对表达量（均P < 0.01）。 结论 他卡西醇可促进培养的人表皮黑素细胞增殖及在FN上的黏附、迁移作用,并可上调黑素细胞c-kit mRNA的表达。 【关键词】 他卡西醇; 黑素细胞; 细胞增殖; 细胞运动; 原癌基因蛋白质c-kit     

Different expression patterns of p27 and p57 proteins in benign and malignant melanocytic neoplasms and in cultured human melanocytes

Background: The p27^KIP1 and p57^KIP2 proteins belong to the CIP/KIP family of cyclin‐dependent kinase inhibitors involved in the growth arrest and cellular senescence. High levels of p27^KIP1 unexpectedly have been detected in invasive malignant melanomas (MM), whereas the role of p57^KIP2 in melanocytic lesions is unknown. We therefore chose to study the expression of p27^KIP1 and p57^KIP2 in melanocytic neoplasms. Design: The expression of p27^KIP1 and p57^KIP2 were examined by immunohistochemistry in 40 melanocytic neoplasms and by Western blot analysis in cultured human melanocytes. Results: Expression of both nuclear p27^KIP1 and p57^KIP2 (> 10% of cells with nuclear labeling) was observed in most cases with non‐proliferating melanocytes (8/10, benign nevi and 9/10 DN, dysplastic nevi), but in only a few cases containing proliferating melanocytes (3/11 RN, recurrent nevi and 2/9 MM, melanoma) (p < 0.002). In proliferating melanocytes, there was an inverse correlation of nuclear expression of p27^KIP1 and p57^KIP2 in both RN (p27^KIP1 = 3/11 RN and p57^KIP2 = 8/11 RN) and MM (p27^KIP1 = 7/9 MM and p57^KIP2 = 2/9 MM) (p < 0.05). Western blot analysis detected p57^KIP2 only in proliferating melanocytes. p27^KIP1 was detected in both proliferating and senescent melanocytes. Conclusion: The difference in expression patterns of p27^KIP1 and p57^KIP2 in proliferating and senescent melanocytes suggests the interplay between these proteins may play a functional role in melanocytic tumorigenesis.     

Semaphorin 7a promotes spreading and dendricity in human melanocytes through beta1-integrins

Described as secreted and membrane-bound proteins important for neural pathfinding, the class of proteins called Semaphorins are expressed in multiple tissue types and are involved in diverse biologic processes. In this study, we describe the function of Semaphorin 7a, a membrane-bound Semaphorin known to stimulate neurite outgrowth, on human melanocytes. We show that Semaphorin 7a is expressed by human keratinocytes and fibroblasts in vitro and in vivo and that melanocytes express Plexin C1, a receptor for Semaphorin 7a. Upregulation of Semaphorin 7a was observed in fibroblasts treated with UV irradiation, a potent stimulus for melanocyte dendricity. Because of the importance of melanocyte dendrites in cutaneous photoprotection, we performed functional studies examining the effect of Semaphorin 7a in melanocyte dendrite formation. We also examined the contribution of beta1-integrin and Plexin C1 receptor signaling in mediating effects of Semaphorin 7a in melanocytes. We show that Semaphorin 7a induces significant melanocyte spreading and dendricity in human melanocytes. Furthermore, we show that beta1-integrins and Plexin C1 receptors are ligands for Semaphorin 7a, and that signaling by these receptors has opposing effects on Semaphorin 7a-induced dendrite formation.     

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:  Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.     

Acid phosphatase in melanosome formation: a cytochemical study in normal human melanocytes

A cytochemical study of acid phosphatase (AcPase) activity was conducted in normal epidermal melanocytes from both sun-exposed and sun-protected human skin to define the relationship between enzyme activity and melanosome formation. In the perikarya of melanocytes of both sun-exposed and sun-protected skin, it was determined that only a small proportion of stage 1 and 2 melanosomes had AcPase activity (20-33% and 9-26%, respectively). The proportion of AcPase-positive melanosomes in perikarya increased in stage 3 (39-56%), reaching a maximum in stage 4 (67-84%). In the dendrite of melanocytes, where melanosomes were mostly in stages 3 and 4, the vast majority of melanosomes demonstrated AcPase activity (79-87% and 88-93%, respectively). The preferential incorporation of AcPase in the later stages of melanogenesis is more consistent with a possible role for this enzyme in the degradation or transfer of melanosomes, rather than as an essential component in the early process of melanization.