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.     

Regulation of tyrosinase processing and trafficking by organellar pH and by proteasome activity

Pigmentation of the hair, skin, and eyes of mammals results from a number of melanocyte-specific proteins that are required for the biosynthesis of melanin. Those proteins comprise the structural and enzymatic components of melanosomes, the membrane-bound organelles in which melanin is synthesized and deposited. Tyrosinase (TYR) is absolutely required for melanogenesis, but other melanosomal proteins, such as TYRP1, DCT, and gp100, also play important roles in regulating mammalian pigmentation. However, pigmentation does not always correlate with the expression of TYR mRNA/protein, and thus its function is also regulated at the post-translational level. Thus, TYR does not necessarily exist in a catalytically active state, and its post-translational activation could be an important control point for regulating melanin synthesis. In this study, we used a multidisciplinary approach to examine the processing and sorting of TYR through the endoplasmic reticulum (ER), Golgi apparatus, coated vesicles, endosomes and early melanosomes because those organelles hold the key to understanding the trafficking of TYR to melanosomes and thus the regulation of melanogenesis. In pigmented cells, TYR is trafficked through those organelles rapidly, but in amelanotic cells, TYR is retained within the ER and is eventually degraded by proteasomes. We now show that TYR can be released from the ER in the presence of protonophore or proton pump inhibitors which increase the pH of intracellular organelles, after which TYR is transported correctly to the Golgi, and then to melanosomes via the endosomal sorting system. The expression of TYRP1, which facilitates TYR processing in the ER, is down-regulated in the amelanotic cells; this is analogous to a hypopigmentary disease known as oculocutaneous albinism type 3 and further impairs melanin production. The sum of these results shows that organellar pH, proteasome activity, and down-regulation of TYRP1 expression all contribute to the lack of pigmentation in TYR-positive amelanotic melanoma cells.     

A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37 degrees C but exits the ER and accumulates in endosomal structures in cells grown at 31 degrees C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37 degrees C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37 degrees C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.     

25-hydroxycholesterol acts in the Golgi compartment to induce degradation of tyrosinase

Oxysterols play a significant role in cholesterol homeostasis. 25-Hydroxycholesterol (25HC) in particular has been demonstrated to regulate cholesterol homeostasis via oxysterol-binding protein and oxysterol-related proteins, the sterol regulatory element binding protein, and the rate-limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl coenzyme A reductase. We have examined the effect of 25HC on pigmentation of cultured murine melanocytes and demonstrated a decrease in pigmentation with an IC(50) of 0.34 microM and a significant diminution in levels of melanogenic protein tyrosinase. Pulse-chase studies of 25HC-treated cells demonstrated enhanced degradation of tyrosinase, the rate-limiting enzyme of melanin synthesis, following endoplasmic reticulum (ER) and Golgi maturation. Protein levels of GS28, a member of an ER/cis-Golgi SNARE protein complex, were also diminished in 25HC-treated melanocytes, however levels of the ER chaperone calnexin and the cis-Golgi matrix protein GM130 were unaffected. Effects of 25HC on tyrosinase were completely reversed by 4 alpha-allylcholestan-3 alpha-ol, a sterol identified by its ability to reverse effects of 25HC on cholesterol homeostasis. Finally, the addition of 25HC to lipid deficient serum inhibited correct processing of tyrosinase. We conclude that 25HC acts in the Golgi compartment to regulate pigmentation by a mechanism shared with cholesterol homeostasis.     

Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator.

The tyrosinase gene is expressed specifically in melanocytes and the cells of the retinal pigment epithelium, which together are responsible for skin, hair, and eye color. By using a combination of DNase I footprinting and band shift assays coupled with mutagenesis of specific DNA elements, we examined the requirements for melanocyte-specific expression of the human tyrosinase promoter. We found that as little as 115 bp of the upstream sequence was sufficient to direct tissue-specific expression. This 115-bp stretch contains three positive elements: the M box, a conserved element found in other melanocyte-specific promoters; an Sp1 site; and a highly evolutionarily conserved element located between -14 and +1 comprising an E-box motif and an overlapping octamer element. In addition, two further elements, one positive and one negative, are located between positions -185 and -150 and positions -150 and -115, respectively. We also found that the basic helix-loop-helix factor encoded by the microphthalmia gene, which is essential for melanocyte differentiation, can transactivate the tyrosinase promoter via the M box and the conserved E box located close to the initiator. Since in vitro assays failed to identify any melanocyte-specific DNA-binding activity, the possibility that the specific arrangement of elements within the basal tyrosinase promoter determines melanocyte-specific expression is discussed.     

Functional activation of plasma membrane anion exchangers occurs in a pre-Golgi compartment

Folding and oligomerization of most plasma membrane glycoproteins, including those involved in ion transport, occur in the ER and are frequently required for their exit from this organelle. It is currently unknown, however, where or when in the biosynthetic pathway these proteins become functionally active. AE1 and AE2 are tissue-specific, plasma membrane anion transport proteins. Transient expression of AE2 in a eukaryotic cell line leads to an increase in stilbene inhibitable whole cell 35SO4(2-)-efflux consistent with its function as a plasma membrane anion exchanger. No such increased transport activity was observed in AE1 transfectants, despite the fact that the two proteins were synthesized in roughly equal portions. In contrast, both AE1 and AE2 expression resulted in significant increase in Cl-/SO4(2-)-exchange in crude microsomes demonstrating that both AE1 and AE2 cDNAs encode functional proteins. Immunofluorescence staining and pulse-chase labeling experiments revealed that while 60% of AE2 is processed to the cell surface of transfectants, AE1 is restricted to an intracellular compartment and never acquires mature oligosaccharides. Crude microsomes from transfected cells were fractionated into plasma membrane and ER-derived vesicles by con A affinity chromatography. All of the AE1 and approximately half of the cellular AE2 was eluted with the ER vesicles, confirming their intracellular localization. Anion transport measurements on these fractions confirmed that the ER- restricted anion exchangers were functional. We conclude that AE1 and AE2 acquire the ability to mediate anion exchange at an early stage of their biosynthesis, before their exit from the ER.     

Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.     

The inhibition of early N-glycan processing targets TRP-2 to degradation in B16 melanoma cells

Tyrosinase-related protein-2 (TRP-2) is a DOPAchrome tautomerase catalyzing a distal step in the melanin synthesis pathway. Similar to the other two melanogenic enzymes belonging to the TRP gene family, tyrosinase and TRP-1, TRP-2 is expressed in melanocytes and melanoma cells. Despite the increasing evidence of its efficiency as a melanoma antigen, little is known about the maturation and intracellular trafficking of TRP-2. Here we show that TRP-2 is mainly distributed in the TGN of melanoma cells instead of being confined solely to melanosomes. This, together with the plasma membrane occasional localization observed by immunofluorescence, suggest the TRP-2 participation in a recycling pathway, which could include or not the melanosomes. Using pulse-chase experiments we show that the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi. If N-glycosylation inhibitors prevent the association with calnexin, the TRP-2 nascent chain undergoes an accelerated degradation process. This process is delayed in the presence of proteasomal inhibitors, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. This is a rare example in which calnexin although indispensable for the nascent chain folding is not required for its targeting to degradation. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins. Clearly, TRP-2 has a distinct maturation pathway from tyrosinase and TRP-1 and possibly a second regulatory function within the cell.     

Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

25‐Hydroxycholesterol Acts in the Golgi Compartment to Induce Degradation of Tyrosinase

Oxysterols play a significant role in cholesterol homeostasis. 25‐Hydroxycholesterol (25HC) in particular has been demonstrated to regulate cholesterol homeostasis via oxysterol‐binding protein and oxysterol‐related proteins, the sterol regulatory element binding protein, and the rate‐limiting enzyme of cholesterol biosynthesis, hydroxymethylglutaryl coenzyme A reductase. We have examined the effect of 25HC on pigmentation of cultured murine melanocytes and demonstrated a decrease in pigmentation with an IC₅₀ of 0.34 μM and a significant diminution in levels of melanogenic protein tyrosinase. Pulse‐chase studies of 25HC‐treated cells demonstrated enhanced degradation of tyrosinase, the rate‐limiting enzyme of melanin synthesis, following endoplasmic reticulum (ER) and Golgi maturation. Protein levels of GS28, a member of an ER/cis‐Golgi SNARE protein complex, were also diminished in 25HC‐treated melanocytes, however levels of the ER chaperone calnexin and the cis‐Golgi matrix protein GM130 were unaffected. Effects of 25HC on tyrosinase were completely reversed by 4α‐allylcholestan‐3α‐ol, a sterol identified by its ability to reverse effects of 25HC on cholesterol homeostasis. Finally, the addition of 25HC to lipid deficient serum inhibited correct processing of tyrosinase. We conclude that 25HC acts in the Golgi compartment to regulate pigmentation by a mechanism shared with cholesterol homeostasis.     

Tyrosinase maturation through the mammalian secretory pathway: bringing color to life

Tyrosinase has been extensively utilized as a model substrate to study the maturation of glycoproteins in the mammalian secretory pathway. The visual nature of its enzymatic activity (melanin production) has facilitated the identification and characterization of the proteins that assist it becoming a functional enzyme, localized to its proper cellular location. Here, we review the steps involved in the maturation of tyrosinase from when it is first synthesized by cytosolic ribosomes until the mature protein reaches its post-Golgi residence in the melanosomes. These steps include protein processing, covalent modifications, chaperone binding, oligomerization, and trafficking. The disruption of any of these steps can lead to a wide range of pigmentation disorders.     

Molecular diagnostics of malignant melanoma: molecular staging,minimal residual disease

Nucleic acid based molecular techniques have been introduced into the diagnosis of malignant melanoma similarly to other cancers. They were applied for refinement of staging and to detect minimal residual disease. There are several good melanocyte-specific genetic markers such as tyrosinase, gp100, Melan-A/MART-1 and MIA. Unlike in the case of the lymph nodes, peripheral blood or bone marrow do not contain melanocytes excluding the possibility of fals positive reactions. Considering the pronounced heterogeneity of melanoma cells the most reliable molecular marker is the expression of tyrosinase. Several studies indicate that the quantity of circulating melanoma cells correlates with tumor burden and disease progression and reflects the effect of therapy. On the other hand, molecular techniques detect circulating melanoma cells much more frequently than the clinical manifestation of the disease progression (molecular recurrence), questioning the clinical significance of the detection of a small number of melanoma cells in the circulation. Based on these data molecular diagnostics is not part of the melanoma protocols yet and further studies are necessary to define its diagnostic role.     

Excess tyrosine rescues the reduced activity of proliferation and differentiation of cultured recessive yellow melanocytes derived from neonatal mouse epidermis

The murine recessive yellow (Mc1r(e)) is a loss-of-function mutation in the receptor for alpha-melanocyte-stimulating hormone, melanocortin receptor 1 (Mc1r) and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes. In this study, the proliferation and differentiation of recessive yellow epidermal melanocytes cultured in dibutyryl cyclic AMP-supplemented serum-free medium were investigated in detail. The melanocytes produced mainly eumelanin in this culture system. The proliferation of recessive yellow melanocytes was decreased compared with that of wild-type at the e-locus, black melanocytes. The differentiation of melanocytes was also delayed and inhibited in recessive yellow mice. Tyrosinase (TYR) activity and TYR-related protein 1 (TRP1) and TRP2 (dopachrome tautomerase, DCT) expressions were decreased and, in addition, the maturation of stage IV melanosomes was inhibited. Excess l-tyrosine (l-Tyr) added to the culture media rescued the reduced activity of proliferation of melanocytes. l-Tyr also stimulated TYR activity and TRP1 and TRP2 expressions as well as the maturation of stage IV melanosomes and pigmentation. These results suggest that the Mc1r(e) mutation affects the proliferation and differentiation of melanocytes and l-Tyr rescues the reduced proliferative and differentiative activities by stimulating TYR activity and TRP1 and TRP2 expressions as well as melanosome maturation.     

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。