Up-regulation of tyrosinase gene by nitric oxide in human melanocytes

Ultraviolet light (UV) radiation causes skin-tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO-induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S-nitroso-N-acetyl-L-arginine. An increase of tyrosinase activity was also detected time-dependently within the 24-hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO-induced melanogenesis.     

Possible Involvement of ERK 1/2 in UVA‐Induced Melanogenesis in Cultured Normal Human Epidermal Melanocytes

UV‐induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m² and examined the potential involvement of mitogen‐activated protein kinases (MAPK) as UVA‐responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal‐related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c‐Jun N‐terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre‐treated with N‐acetyl‐l ‐cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6‐4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation‐induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.     

A Transgenic Mouse Model with Inducible Tyrosinase Gene Expression Using the Tetracycline (Tet‐on) System Allows Regulated Rescue of Abnormal Chiasmatic Projections Found in Albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET‐ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

Possible involvement of ERK 1/2 in UVA-induced melanogenesis in cultured normal human epidermal melanocytes

UV-induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m2 and examined the potential involvement of mitogen-activated protein kinases (MAPK) as UVA-responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal-related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c-Jun N-terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre-treated with N-acetyl-L-cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6-4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation-induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.     

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.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Regulation of Melanogenesis Induced by 5-Methoxypsoralen Without Ultraviolet Light in Murine Melanoma Cells

Melanogenesis in melanoma cells can be enhanced by psoralens in the absence of UV light. Melanin biosynthesis is regulated by a number of melanocyte-specific proteins, including tyrosinase, DOPAchrome tautomerase (DCT), and tyrosinase-related protein-1 (TRP-1, gp75). To get more insight on the molecular mechanisms involved in psoralens-induced melanogenesis, we determined tyrosinase and DCT activities as well as mRNA and protein levels of tyrosinase, DCT, and TRP-1 in S91 mouse melanoma cells treated by 5-MOP. High concentration of 5-MOP (5 × 10^-5 M) induced a time-dependent increase of tyrosinase activity and melanin content, which was correlated to an increase of both mRNA and protein levels of tyrosinase. These results demonstrate that the 5-MOP stimulation of melanogenesis is related to increased tyrosinase synthesis. In addition, 5-MOP stimulated TRP-1 synthesis and induced a dose-dependent decrease of DCT activity without any modification in the expression of the protein. We explored then the signalling pathways involved in 5-MOP-induced melanogenesis and, particularly, the role of cyclic AMP and protein kinase C (PKC). A small stimulation of cyclic AMP production was observed in presence of 5-MOP. Furthermore, 1-oleoyl-2-acetylglycerol (OAG), a PKC activator, potentiated the 5-MOP stimulation of tyrosinase activity, while calphostin, a specific PKC inhibitor, inhibited the 5-MOP induction of tyrosinase activity. Phorbol-myristate acetate (PMA), described as a strong activator of PKC, inhibited also the effect of 5-MOP when used at long term. Taken together, these results demonstrate that in murine melanoma cells 5-MOP stimulates melanogenesis by increasing activity and synthesis of tyrosinase. Tyrosinase and TRP-1 expression are coordinately regulated by 5-MOP Furthermore, a negative correlation between melanogenesis and DCT activity was observed under 5-MOP stimulation. At least, PKA and PKC systems appear to play an important role in the melanogenic effect of 5-MOP.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKCα had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKCα in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1. © 1996 Wiley-Liss, Inc.     

Mutational Analysis of the Modulation of Tyrosinase by Tyrosinase‐Related Proteins 1 and 2 In Vitro

The albino (tyrosinase, Tyr^c), brown (tyrosinase‐related protein 1, Tyrp1^b) and slaty (tyrosinase‐related protein 2, tyrp2^slt) loci are all involved in the regulation of melanogenesis. Phenotypes of inbred mice mutant at two or more of these loci are not always explicable by simple summation of the established or suspected catalytic functions of the gene products. These phenotypes suggest that relationships among the proteins extend beyond the obvious fact that they catalyze different steps in the same melanogenic pathway, and that they may also interact intimately in such a way that a mutation in one impacts the function of the other(s). Previous studies have attributed catalytic activities to each member of this trio; however, it has been difficult to study the proteins individually, either in vivo or in tissues or cells. Therefore, we undertook to transfect the genes, in revealing combinations, into COS‐7 cells (which have no melanogenic apparatus of their own) to clarify the interacting functions of their encoded proteins. Specifically, we attempted to evaluate the effects of Tyrp1 and Tyrp2 proteins on tyrosinase protein. We report evidence that Tyrp1 stabilizes tyrosinase, confirming previous observations, and, in addition, demonstrate that Tyrp1 decreases tyrosinase activity. By contrast, Tyrp2 increases tyrosinase activity by stabilizing the protein. We conclude that both Tyrp1 and Tyrp2, in addition to other catalytic functions they may possess, act together to modulate tyrosinase activity.     

Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO)

Background

Excessive melanin production and accumulation are characteristics of a large number of skin diseases, including melasma, and post-inflammatory hyperpigmentation. During our on-going search for new agents with an inhibitory effect on tyrosinase, we synthesized a new type of tyrosinase inhibitor, 4-(thiazolidin-2-yl)benzene-1,2-diol (MHY-794), which directly inhibits mushroom tyrosinase.

Methods

The inhibitory effect of MHY-794 on tyrosinase activity and nitric oxide (NO) scavenging activity was evaluated in cell free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of MHY-794 in vitro. HRM2 hairless mice were used to evaluate anti-melanogenic effects of MHY-794 in vivo.

Results

MHY-794 effectively inhibited mushroom tyrosinase activity in cell free system. In silico docking simulation also supported the inhibitory effects of MHY-794 on mushroom tyrosinase. MHY-794 also proved to be effective at scavenging nitric oxide (NO), which serves as an important modulator in the melanogenesis signaling pathway. In addition, MHY-794 effectively inhibited SNP (NO donor)-induced melanogenesis by directly inhibiting tyrosinase and diminishing NO-mediated melanogenesis signaling in B16 melanoma cells. The anti-melanogenic effects of MHY-794 were further confirmed in HRM2 hairless mice. Ultraviolet light (UV) significantly up-regulated NO-mediated melanogenesis signaling in HRM2 hairless mice, but MHY-794 effectively inhibited both melanogenesis and diminished UV-induced NO-signaling.

Conclusions

Our results indicate that MHY-794 is highly effective at inhibiting NO-mediated melanogenesis in vitro and in vivo by direct NO scavenging and directly inhibiting tyrosinase activity, and suggest that MHY-794 be considered a new developmental candidate for the treatment of hyper-pigmentation disorders.

General significance

MHY-794, which showed great efficacy on NO-mediated melanogenesis by direct NO scavenging as well as direct inhibition of tyrosinase catalytic activity, might be utilized for the development of a new candidate for treatment of the hyper-pigmentation disorders.