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.     

Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells

Melanogenesis is a principal parameter of differentiation in melanocytes and melanoma cells. Our recent study has demonstrated that phospholipase D1 (PLD1) regulates the melanogenic signaling through modulating the expression of tyrosinase, the rate-limiting step enzyme in the melanin biosynthesis. The current study was designed to gain more insight into the involvement of PLD1 in the regulation of melanogenesis. To investigate the role of PLD1, we examined the effect of knockdown of endogenous PLD1 by small interference RNA (siRNA) on melanogenesis in B16 melanoma cells. It was shown that the melanin synthesis was induced in PLD1-knockdowned cells, and also that the level of melanin synthesis was well correlated with increases in expression level of tyrosinase and its related proteins (Tyrp1 and Dct). Furthermore, the reduction of expression levels of PLD1 by siRNA transfection was accompanied by diminution of ribosomal S6 kinase 1 (S6K1) phosphorylation. The activity of mammalian target of rapamycin (mTOR) is essential for phosphorylation of S6K1 and the treatment malanoma cells with rapamycin, a potent inhibitor of mTOR effectively induced melanogenesis. The results obtained here provide possible evidence that PLD1 exerts a negative regulatory role in the melanogenic process through mTOR/S6K1 signaling.     

Effect of Cl- on tyrosinase: complex inhibition kinetics and biochemical implication

Tyrosinase plays a core role in melanogenesis of the various organisms. Therefore, the regulation of the tyrosinase activity is directly related with melanin synthesis. In this study, we investigated the Cl(-)-induced inhibition of human tyrosinase and the potent role of Cl(-) as a negative regulator in melanogenesis. For the inhibition kinetic studies, human tyrosinase was differently prepared from the TXM13 melanotic cells as well as from cells that had undergone gene transfection. We found that Cl(-) inhibited tyrosinase in a slope-parabolic competitive manner and tyrosinase gene transfection into HEK293 cell significantly down-regulated the expression levels of solute carrier family 12, member 4 (potassium/chloride transporters, SLC12A7) and solute carrier family 12, member 7 (potassium/chloride transporters, SLC12A7), which are known to be Cl(-) transporters. From the results of the inhibition kinetic studies and the Cl(-) transporter expression level, we suggested that Cl(-) might act as a potent regulatory factor in melanogenesis. It is worth notice that a high content of Cl(-) exists physiologically and tyrosinase reacts sensitively to Cl- in a complex interaction manner.     

MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.     

Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors

Melanogenesis is a process to synthesize melanin, which is a primary responsible for the pigmentation of human skin, eye and hair. Although numerous enzymatic catalyzed and chemical reactions are involved in melanogenesis process, the enzymes such as tyrosinase and tyrosinase-related protein-1 (TRP-1) and TRP-2 played a major role in melanin synthesis. Specifically, tyrosinase is a key enzyme, which catalyzes a rate-limiting step of the melanin synthesis, and the downregulation of tyrosinase is the most prominent approach for the development of melanogenesis inhibitors. Therefore, numerous inhibitors that target tyrosinase have been developed in recent years. The review focuses on the recent discovery of tyrosinase inhibitors that are directly involved in the inhibition of tyrosinase catalytic activity and functionality from all sources, including laboratory synthetic methods, natural products, virtual screening and structure-based molecular docking studies.     

Endothelin-1-induced macrophage inflammatory protein-1beta expression in monocytic cells involves hypoxia-inducible factor-1alpha and AP-1 and is negatively regulated by microRNA-195

Patients with sickle cell disease (SCD) exhibit a chronic inflammatory state manifested by leukocytosis and increased circulating levels of proinflammatory cytochemokines. Our studies show that placenta growth factor levels are high in SCD, and placental growth factor induces the release of the vasoconstrictor endothelin-1 (ET-1) from pulmonary microvascular endothelial cells. In this study, we observed that ET-1 increased the expression of the chemokines MIP-1β or CCL4. ET-1-induced MIP-1β mRNA expression in THP-1 cells and human peripheral blood monocytes occurred via the activation of PI3K, NADPH oxidase, p38 MAPK, and JNK-1 but not JNK-2. ET-1-induced MIP-1β expression involved hypoxia-inducible factor-1α (HIF-1α), independent of hypoxia, as demonstrated by silencing with HIF-1α small interfering RNA, EMSA, and chromatin immunoprecipitation analysis. ET-1-induced MIP-1β promoter luciferase activity was attenuated when any of the five hypoxia-response elements, AP-1, or NF-κB binding motifs in the proximal MIP-1β promoter (-1053/+43 bp) were mutated. Furthermore, ET-1 significantly downregulated the expression of a key microRNA, microRNA-195a, which showed a complementary binding site in the 3' untranslated region of MIP-1β mRNA. Moreover, ET-1-induced MIP-1β mRNA expression in either THP-1 cells or peripheral blood monocytes was reduced upon expression of microRNA-195a. Conversely, transfection of monocytes with anti-microRNA-195a oligonucleotide augmented several-fold ET-1-induced MIP-1β expression. Taken together, these studies showed that ET-1-mediated MIP-1β gene expression is regulated via hypoxia-response elements, AP-1, and NF-κB cis-binding elements in its promoter and negatively regulated by microRNA-195, which targets the 3' untranslated region of MIP-1β RNA. These studies provide what we believe are new avenues, based on targets of HIF-1α and microRNAs, for ameliorating inflammation in SCD.     

Coordinated mRNA and Protein Expression of Human LAMP-1 in Induction of Melanogenesis After UV-B Exposure and Co-Transfection of Human Tyrosinase and TRP-1 cDNAs

In order to better understand the cascade of melanogenic events in melanocytes, this report has introduced our two recent approaches for the expression of melanogenesis/or melanosome-associated genes and encoded proteins in melanocytes (melanoma cells) after repeated exposure to UV -B and after cotransfection of two human genes, i.e., tyrosinase and tyrosinase-related protein-1 (TRP-1). Repeated exposure of UV B (2.5–5.0 mJ/cm²) caused not only upregulation of tyrosinase and TRP-1 genes but also coordinated increase in the gene and protein synthesis expression of Lamp-1 (lysosome-associated membrane protein-1). When COS-7 kidney cells and amelanotic melanoma (C32 and SKMEL-24) and melanotic melanoma (G361 and SK-MEL-23) cells were exposed to cotransfection of human tyrosinase and TRP-1 cDNAs, there was also an increased expression of Lamp-1 mRNA and protein along with tyrosinase activation and new melanin synthesis. Importantly, single transfectants of human tyrosinase cDNA revealed marked cellular degeneration, whereas this degeneration was not seen in single transfectants of TRP-1 cDNA or cotransfectants of human tyrosinase and TRP-1 cDNAs, indicating that TRP-1 prevented, along with Lamp-1, programmed death of melanocytes after transfection of tyrosinase gene. The coordinated expression of TRP-1 and Lamp-1 was further confirmed by antisense oligodeoxynucleotide hybridization experiment against Lamp-1 gene, showing the decreased expression of TRP-1 as identified by three different types of anti-TRP-1 monoclonal antibodies. We propose therefore that human tyrosinase and TRP-l, when activated or expressed together, will coordinate to upregulate the mRNA expression and protein synthesis of Lamp-1. The Lamp-1 molecules will, in turn, cover the inner surface of melanosomal membrane, together with TRP-1 molecules, thus protecting the melanosomal membrane from toxic melanin intermediates generated during melanogenesis in the presence of active tyrosinase. In contrast, the expression of other lysosome-related proteins, e.g., β-galactosidase and CD63 is not stimulated in new melanogenesis.     

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.     

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.