Stimulation Of Melanogenesis Research Articles

The degree of pigmentation modulates the radiosensitivity of human melanoma cells.

The relationship between cell pigmentation and radiosensitivity was investigated in two selected human melanoma cell lines with different melanin content (mixed type: eumelanin and pheomelanin, and pheomelanotic phenotypes). The same study was also done after stimulation of melanogenesis (1) by addition of the melanin precursor l-tyrosine to each of the cell lines separately and (2) by irradiation alone with doses ranging from 0 to 10 Gy. We found that a decrease in cell radiosensitivity was correlated with the type of melanin, with a clear involvement of eumelanin rather than pheomelanin. Increasing the intracellular content of both melanins promoted the growth of irradiated cells. Moreover, at a dose of 10 Gy, both tyrosinase activity and melanin cell content were significantly increased in the absence of any other melanogenesis promoter. Our data suggest that the amount of intracellular melanin is inversely related to the radiosensitivity of melanoma cells and may explain at least in part the controversial responses to ionizing radiations reported for melanoma.

Endothelin 3 induces the reversion of melanocytes to glia through a neural crest-derived glial-melanocytic progenitor.

Functional signaling of endothelin 3 (ET3) and its receptor B (ETRB) has been shown to be required for the development of neural crest (NC)-derived pigment cells in mouse, but the precise role of ET3 is not completely understood. Using the avian embryo as a model, we previously reported that ET3 promotes the survival and proliferation of unipotent melanocyte and bipotent glia-melanocyte precursors in trunk NC cultures. Here we investigated whether, at later stages, embryonic pigment cells respond to ET3. Such a possibility is supported by the previous finding that, in vivo, avian melanocytes express endothelin receptor B2 (ETRB2) during migration and after their differentiation in the skin. We found that in vitro ET3 exerts a dose-dependent stimulation of proliferation and melanogenesis in NC cells that had homed to the epidermis of embryonic quail dorsal skin. Moreover, in clonal cultures of skin-derived pigment cells, ET3 induces rapid cell divisions of clonogenic melanocytes that generate a mixed progeny of melanocytes and cells devoid of pigment granules and expressing glial markers in more than 40% of the colonies. It can therefore be concluded that ET3 is strongly mitogenic to embryonic pigment cells and able to alter their differentiation program, leading them to recapitulate the glial-melanocyte bipotentiality of their NC ancestors.

Effect of Prostaglandins A2, E1, F2 αand Latanoprost on Cultured Human Iridal Melanocytes

The effect of various prostaglandins on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium, cAMP-deleted medium with or without various prostaglandins at different concentrations. After 5–7 days, the numbers of cells and dendrites were counted and melanin content was measured and compared to controls. Prostaglandin A2and E1stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. Prostaglandin F2 α, PhXA85 and latanoprost showed no effect under similar experimental conditions. These results indicate that prostaglandin A2and E1stimulate growth and differentiation of iridal melanocytes through the activation of cAMP system. Failure of stimulation of melanogenesis of iridal melanocytes by prostaglandin F2 αand PhXA85 in vitro may be related to a different second messenger system activated by these prostaglandins and the selective stimulating effect of these prostaglandins on iridal melanocytes from mixed-colored irides.

The Human Melanocyte as a Particular Target for UVA Radiation and an Endpoint for Photoprotection Assessment

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.

Does alpha-MSH have a role in regulating skin pigmentation in humans?

Over the years there has been much debate as to whether alpha-MSH has a role as a pigmentary hormone in humans. There are two main reasons for this. First, despite the observations in the 1960s that alpha-MSH increased skin darkening in humans, there are reports that the peptide has no effect on melanogenesis in cultured human melanocytes. Second, the human pituitary, unlike that of most mammals, secretes very little alpha-MSH and circulatory levels of the peptide in humans are extremely low. However, there is now evidence from several groups that alpha-MSH is capable of stimulating melanogenesis in cultured human melanocytes. Rather than producing an overall increase in melanin production, it appears that the peptide acts specifically to increase the synthesis of eumelanin. Such an action could well explain the previously observed skin darkening effects of alpha-MSH. It is also now known that alpha-MSH is not produced exclusively in the pituitary but has been found at numerous sites, including the skin where it is produced by several cell types. Related Proopiomelanocortin (POMC) peptides such as ACTH are also produced in human skin. The ACTH peptides act at the same receptor (MC-1) as alpha-MSH and certain of these would appear to be more potent than alpha-MSH in stimulating melanogenesis. The ACTH peptides are also present in greater amounts than alpha-MSH in human epidermis and it is likely that they play an important role in regulating pigmentary responses. These POMC peptides are released from keratinocytes in response to ultraviolet radiation (UVR) and it has been proposed that they serve as paracrine factors in mediating UV induced pigmentation. Their production by keratinocytes could therefore be critical in determining pigmentary responses and any changes in the availability of these POMC peptides might explain the variations in tanning ability seen in different individuals. However, the possibility that tanning ability is also dependent upon differences at the level of the MC-1 receptor cannot be ruled out and it has been suggested that an inability to tan may depend upon the presence of non-functional changes at the MC-1 receptor. alpha-MSH does, of course, affect human melanocytes in several ways and its stimulation of melanogenesis could be the consequence of some other fundamental action in the melanocyte. The peptide also has many other target sites in the skin and while it may have a role in regulating skin pigmentation in humans, it should not be viewed solely as a pigmentary peptide. alpha-MSH clearly has many different actions and its primary role in the skin may be to maintain homeostasis.

Tyrosine Transport into Melanosomes Is Increased Following Stimulation of Melanocyte Differentiation

A variety of physiological factors can stimulate differentiation of melanocytes to increase pigmentation, and critical to this process is the transport of the melanogenic substrate (tyrosine) into melanosomes. In this study, we examined whether stimulation of melanogenesis affects melanosomal tyrosine transport. Tyrosine uptake increased almost 2-fold in melanosomes derived from melanocytes treated with melanocyte-stimulating hormone (MSH), which acts to increase intracellular cAMP levels, resulting in the up-regulation of many genes involved in melanogenesis. Stimulation of melanoma cells with dibutyryl cAMP increased melanosomal tyrosine transport 2- to 3-fold after 24 to 48 hrs, with peak levels occurring after 3 to 5 days of treatment, suggesting thatde novogene expression may be required. The cAMP-induced increase in melanosomal tyrosine transport could be effectively competed with phenylalanine or tryptophan, but not with dopamine or proline, suggesting either that a pool of transporters with greater tyrosine transporting ability pre-exists, or that a greater number of tyrosine transporters reside within the melanosomal membrane. These results illustrate a rare example of hormonal plasma membrane stimulation which transduces a signal for increased vesicular transport of an amino acid.

Stimulation of Melanogenesis in a Human Melanoma Cell Line by Bistratene A

The polyether toxin, bistratene A, induced morphological and functional differentiation of a human melanoma cell line (MM96E). The cells became blocked at the G2/M transition and elaborated a number of processes. Tyrosinase activity and melanin content were substantially increased. Northern blot analysis showed up-regulation of mRNA for several genes known to be involved in melanin biosynthesis (pmel17, pmel34, and tyrosinase related proteins, TRP-1 and TRP-2). Bistratene A induced the phosphorylation of several proteins as assessed by 2D gel electrophoresis and one of these was identified as stathmin (oncoprotein 18), a cell-cycle regulated phosphoprotein. Bistratene A specifically induced the translocation of protein kinase C δ (PKCδ) from a soluble to a particulate fraction without affecting other isoforms. These results implicate a role for protein kinase C δ in the induction of differentiation of this human melanoma cell line.

Binding of alpha-melanocyte-stimulating hormone to its G-protein-coupled receptor on B-lymphocytes activates the Jak/STAT pathway.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a 13-amino-acid peptide with a variety of physiological effects, including the stimulation of melanocyte proliferation and melanogenesis, temperature control, control of prolactin release and the modulation of cytokine action in the immune system. There are five known subtypes of G-protein-coupled receptors, which bind with different affinities to alpha-MSH. This paper provides evidence that Ba/F3 pro-B-lymphocyte cells express the gene for the melanocortin 5 (MC5) receptor and specifically bind alpha-MSH. Western-blot analysis reveals that alpha-MSH binding stimulates Janus kinase 2 (JAK2) and signal transducers and activators of transcription (STAT1) tyrosine phosphorylation in both Ba/F3 cells and human cultured IM-9 lymphocytes. alpha-MSH is further revealed to activate JAK2 in mouse L-cells stably expressing the human MC5 receptor. Finally, alpha-MSH binding is shown to result in an enhancement of cellular proliferation. These findings identify a new protein tyrosine kinase pathway in the action of alpha-MSH, and suggest that alpha-MSH plays an important role in B-lymphocyte function via the activation of the same intracellular phosphorylation pathway used by cytokines and growth factors.

Characterization of corticotropin-releasing hormone (CRH) in human skin.

We have confirmed the expression of CRH and CRH receptor type 1 genes in human skin, cultured HaCaT keratinocytes, squamous cell carcinoma, and melanoma cells. The size of CRH messenger ribonucleic acid (mRNA), estimated by Northern blot hybridization, was 1.5 kilobases. CRH peptide was identified by reverse phase high pressure liquid chromatography separation in both whole skin and cultured cells. Forskolin and dexamethasone at concentrations of 10 micromol/L stimulated and inhibited, respectively, CRH peptide production in squamous cell carcinoma and melanoma cells, but had no significant effect on the CRH mRNA level. In melanoma cells, stimulation of melanogenesis down-regulated CRH receptor type 1 mRNA expression, but was without effect on CRH mRNA production. We suggest that in human skin the CRH signaling system is both operative and under regulatory control.

Different cis-acting elements are involved in the regulation of TRP1 and TRP2 promoter activities by cyclic AMP: pivotal role of M boxes (GTCATGTGCT) and of microphthalmia.

In melanocytes and in melanoma cells, cyclic AMP (cAMP)-elevating agents stimulate melanogenesis and increase the transcription of tyrosinase, the rate-limiting enzyme in melanin synthesis. However, two other enzymes, tyrosinase-related protein 1 (TRP1) and TRP2, are required for a normal melanization process leading to eumelanin synthesis. In B16 melanoma cells, we demonstrated that stimulation of melanogenesis by cAMP-elevating agents results in an increase in tyrosinase, TRP1, and TRP2 expression. cAMP, through a cAMP-dependent protein kinase pathway, stimulates TRP1 and TRP2 promoter activities in both B16 mouse melanoma cells and normal human melanocytes. Regulation of the TRP1 and TRP2 promoters by cAMP involves a M box and an E box. Further, a classical cAMP response element-like motif participates in the cAMP responsiveness of the TRP2 promoter, demonstrating that the TRP2 gene is subjected to different regulatory processes, which could account for its different expression patterns during embryonic development or under specific physiological and pathological conditions. We also found that microphthalmia, a basic helix-loop-helix transcription factor, strongly stimulates the transcriptional activities of the TRP1 and TRP2 promoters, mainly through binding to the M boxes. Additionally, we demonstrated that cAMP increases microphthalmia expression and thereby its binding to TRP1 and TRP2 M boxes. These convergent and compelling results disclose at least a part of the molecular mechanism involved in the regulation of melanogenic gene expression by cAMP and emphasize the pivotal role of microphthalmia in this process.

Immunoreactive alpha-melanotropin as an autocrine effector in human melanoma cells.

Melanotropin is a peptide having several functions, including the stimulation of melanogenesis and the modulation of proliferation of melanocytes and melanoma cells. It acts through binding to high-affinity receptors of the melanocortin-1 subtype, exclusively expressed in cells of the melanocytic lineage. Elevated levels of immunoreactive alpha-melanotropin were previously reported in melanoma cell lines, tumours and plasma from patients with melanoma. Here, we show that this high ectopic production of melanotropin is restricted to melanoma and non-pituitary tumours with the same neuroectodermic origin. The occurrence of a melanotropin-specific autocrine loop was further investigated in human melanoma cells. Immunoreactive alpha-melanotropin was spontaneously released from a melanoma cell line (HBL) expressing melanotropin receptors on the cell surface. This release was significantly increased in the presence of melanotropin-related peptides such as corticotropin-(4-10)-peptide and beta-melanotropin, competing for binding to the melanotropin receptor and was directly correlated to the displacement potential of these peptides. Both spontaneous and induced releases of immunoreactive alpha-melanotropin could be blocked at low temperatures, suggesting the involvement of intracellular protein movement in the release mechanism. The release of immunoreactive alpha-melanotropin was not significant in melanoma cells expressing very low levels of melanotropin receptors (IGR3) or in non-melanoma cells (SCC1). However, upon expression of the melanocortin-1 receptor cDNA into IGR3 cells, spontaneous and competition-induced releases of immunoreactive alpha-melanotropin were both increased and also blocked at low temperatures. This observation further underlines a role for the melanotropin receptor in the release of immunoreactive alpha-melanotropin. These experiments indicate that an autocrine loop between the melanocortin-1 receptor and immunoreactive alpha-melanotropin may be functional in human melanoma cells.

The role of alpha-MSH, its agonists, and C-AMP in in vitro avian melanocytes.

Little is known about the effect of alpha-MSH and other melanogenic stimulators on avian melanocytes. Tissue cultures of Barred Plymouth Rock regenerating feather melanocytes were established and the culture medium contained selected concentrations of alpha-MSH and other melanogenic stimulators in Ham's F-10 medium supplemented with antibiotics and 10% new born calf serum. Cultures were maintained at 37 degrees C in 95% air/5% CO2. No increase in melanogenesis over control levels due to the addition of 10(-5) M Forskolin, 10(-4) M IBMX, 10(-3) M c-GMP, and 10(-3) M db-c-AMP was observed in the cultures on days 5 and 7. However, 2.5 (optimum), 5, and 10 micrograms/ml alpha-MSH and 10(-3) M 8-bromo-c-AMP significantly increased melanogenesis over control levels on days 5 and 7. The stimulation of melanogenesis was detectable by a significantly increased number of melanocytes containing numerous stage IV melanosomes. No increase in melanocyte cell number was observed in any of the experimental cultures. The addition of 1, 2 (optimum), or 3 mM calcium did enhance the increased pigmentation effect of 2.5 micrograms/ml alpha-MSH. Two very convincing experiments showed that c-AMP was the second messenger for alpha-MSH in these birds. First, the c-AMP inhibitor, 10(-3) M Rp-c-AMPS, completely inhibited the stimulatory effect of alpha-MSH in these in vitro melanocytes. Second, direct measurements of c-AMP levels in feather tissue showed a significant increase in c-AMP levels 10.min after alpha-MSH treatment. Controls received no alpha-MSH. The results showed that these avian melanocytes have alpha-MSH receptors and were able to respond to the hormone. C-AMP was the second messenger in this system. Apparently db-c-AMP was not able to enter these mature, highly-differentiated cells and c-AMP agonists, Forskolin and IBMX, were also either unable to enter these older cells or, if they did enter the cells, were unable to stimulate c-AMP production. Evidently the more lipophilic 8-bromo-c-AMP was able to enter these cells and stimulate melanogenesis.

Ultraviolet B Radiation Acts through the Nitric Oxide and cGMP Signal Transduction Pathway to Stimulate Melanogenesis in Human Melanocytes

Ultraviolet B (UVB) radiation is the main physiological stimulus for human skin pigmentation; however, the molecular mechanisms underlying this process are still unclear. Recently, nitric oxide (NO) and cGMP have been involved in mediation of skin erythema induced by UVB. Therefore, we investigated the role of NO and cGMP in UVB-induced melanogenesis. In this study, we demonstrated that UVB stimulation of melanogenesis was mimicked by exogenous NO donors. Additionally, we showed that NO stimulated cGMP synthesis and that cGMP was also a potent stimulator of melanogenesis. Furthermore, the inhibition of the melanogenic effect of NO by guanylate cyclase inhibitor demonstrated that NO mediated its effect through the activation of guanylyl cyclase. Interestingly, 1 min after UVB irradiation, we observed a significant increase in cGMP content in melanocytes. The effects of UVB on cGMP production and on melanogenesis were blocked by both guanylate cyclase and NO synthase inhibitors. Additionally, inhibition of cGMP-dependent kinase also prevented the stimulation of melanogenesis by UVB and NO. Therefore, we concluded that NO and cGMP production is required for UVB-induced melanogenesis and that cGMP mediated its melanogenic effects mainly through the activation of cGMP-dependent kinase.

PUVA (5-Methoxypsoralen Plus UVA) Enhances Melanogenesis and Modulates Expression of Melanogenic Proteins in Cultured Melanocytes

PUVA (combination of psoralens and ultraviolet A radiation) is a potent inducer of melanogenesis in normal human skin. The molecular mechanisms underlying this effect are poorly characterized. This study was undertaken to investigate the action of PUVA on melanogenesis in S91 murine melanoma cells and in cultured normal human melanocytes. Tyrosinase and DOPAchrome tautomerase (DCT) activities as well as melanin neosynthesis were measured in PUVA-treated pigment cells. To determine whether a correlation exists between PUVA-induced melanogenesis and expression of melanogenic enzymes, we analyzed the levels of tyrosinase, DCT, and tyrosinase-related protein-1 (TRP-1 or gp75) by western blotting in PUVA-treated cells. We demonstrate that UVA upregulates tyrosinase activity and melanin content with 5-methoxypsoralen at 1 microM. This phenomenon depends on the energy delivered during phototreatment. In both human and mouse cells, stimulation of melanogenesis correlated with an increase of the amount of tyrosinase. In PUVA-treated S91 cells, tyrosinase mRNA was increased, but no stimulation of DCT activity occurred in these cells, in agreement with the unchanged amount of DCT protein in cell extracts. On the contrary, in melanocytes treated with PUVA, a decrease in DCT protein was observed. Finally, the amount of TRP-1 protein was not affected by PUVA in either S91 cells or melanocytes. These results show that melanogenesis induced by PUVA is related to an increase in expression of tyrosinase. In melanocytes, melanogenesis and DCT are negatively correlated, which suggests that PUVA favors the metabolic pathway of dark-eumelanins with high UV-protective properties. This study also suggests that PUVA regulates tyrosinase, DCT, and TRP-1 expression in a noncoordinate manner.

Effects on Intraocular Pressure and Side Effects of 0.005% Latanoprost Applied Once Daily, Evening or Morning: A Comparison with Timolol

Purpose: To compare the effect on intraocular pressure (IOP) and side effects of 0.005% latanoprost applied once daily, morning or evening, with 0.5% timolol applied twice daily.Methods: A 6-month randomized, double-masked, multicenter study with three parallel groups was undertaken. Two hundred sixty-seven patients were randomized, 84 to timolol, 89 to latanoprost in the morning for 3 months and then in the evening for another 3 months, and 94 to latanoprost with the treatment schedule reversed.Results: After 6 months, timolol reduced diurnal IOP from 24.6 to 17.9 mmHg (27%); latanoprost applied in the morning, from 25.5 to 17.7 mmHg (31%); and Latanoprost applied in the evening, from 24.8 to 16.2 mmHg (35%). The efficacy of latanoprost applied in the evening was statistically superior to latanoprost applied in the morning and to timolol (P< 0.001). Latanoprost induced a slight increase in conjunctival hyperemia in 31.4% of treated patients, compared with 15.9% for timolol. Sporadic episodes of mild punctate corneal epithelial erosions were three times as frequent in latanoprost-treated eyes as in timolol-treated eyes. The most significant ocular side effect was increased pigmentation of the iris observed in five and suspected in seven more latanoprost-treated eyes. All these eyes had a mixed green-brown or blue/gray-brown iris color. Timolol reduced heart rate by 3 beats/minute (P< 0.005).Conclusions: The effect on diurnal IOP of latanoprost applied once daily in the evening is superior to that of timolol. The main difference in side effects is increased pigmentation of the iris induced by latanoprost, most likely due to stimulation of melanogenesis in iris stromal melanocytes.

Mitogen-activated Protein Kinase Pathway and AP-1 Are Activated during cAMP-induced Melanogenesis in B-16 Melanoma Cells

In mammalian melanocytes, melanin synthesis is controlled by tyrosinase, the critical enzyme in the melanogenic pathway. We and others showed that the stimulation of melanogenesis by cAMP is due to an increased tyrosinase expression at protein and mRNA levels. However, the molecular events connecting the rise of intracellular cAMP and the increase in tyrosinase activity remain to be elucidated. In this study, using B16 melanoma cells, we showed that cAMP-elevating agents stimulated mitogen-activated protein (MAP) kinase, p44mapk. This effect was mediated by the activation of MAP kinase kinase. cAMP-elevating agents induced a translocation of p44mapk to the nucleus and an activation of the transcription factor AP-1. cAMP-induced AP-1 contained FOS-related antigen-2 in association with JunD, while after phorbol ester stimulation AP-1 complexes consist mainly of JunD/c-Fos heterodimers. In an attempt to connect these molecular events to the control of tyrosinase expression that appears to be the pivotal point of melanogenesis regulation, we hypothesized that following its activation by cAMP, p44mapk activates AP-1. Then AP-1 could stimulate tyrosinase expression through the interaction with specific DNA sequences present in the mouse tyrosinase promoter.

Melatonin antagonizes alpha-melanocyte-stimulating hormone enhancement of melanogenesis in mouse melanoma cells by blocking the hormone-induced accumulation of the c locus tyrosinase.

Melatonin was found to have a small inhibitory effect on tyrosinase activity and a slight stimulatory action on dopachrome tautomerase activity in B16 mouse melanoma cells. These effects were time and dose dependent, with the maximal response being observed after 24-48 h treatment and at concentrations of melatonin higher than the physiologic levels of the circulating hormone. Although these effects on the melanogenic activities were modest, incubation of melanocytes with melatonin prior to the addition of the melanotropin mediated a dramatic inhibition of alpha-melanocyte-stimulating-hormone-(alpha-MSH)-induced melanogenesis. This inhibitory effect was evident at melatonin concentrations as low as 10 nM. Inhibition was nearly total at 0.1 mM melatonin, even at high concentrations of alpha-MSH (1 microM). The inhibitory effect of melatonin on alpha-MSH stimulation of melanogenesis was investigated. Melatonin appeared to act at least at two stages. Pharmacological concentrations of melatonin diminished the number of alpha-MSH receptors to about 75% of the control values without an apparent effect on receptor affinity, as determined by receptor-binding studies using 125I-[N-Leu4-D-Phe7]alpha-MSH as a probe. Physiological concentrations of melatonin also appeared to interfere with the intracellular events coupling increased cAMP levels and induction of the c locus tyrosinase, since it strongly inhibited the theophylline-mediated stimulation of melanogenesis. The inhibition of tyrosinase stimulation was higher in the microsomal than in the melanosomal fractions of cells which were treated with melatonin, then exposed to either alpha-MSH (1 microM) or theophylline (1 mM), suggesting that one of the main effects of melatonin might be inhibition of the induction of tyrosinase de novo synthesis.

Analysis of the UV-induced melanogenesis and growth arrest of human melanocytes.

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm2 UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G2-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 microM alpha-melanocyte stimulating hormone (alpha-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of alpha-MSH on the UV-induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.

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 x 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.

Repeated UVB irradiations do not have the same potential to promote stimulation of melanogenesis in cultured normal human melanocytes

The major stimulus for human melanin production is ultraviolet (UV) radiation. Little is known about the mechanisms underlying this response and the eventual enzyme regulation resulting from this activation. We treated normal human melanocytes in culture with daily UVB radiations. Cumulative increases in UVB doses resulted in proportional increases in tyrosinase activity over the first few days whereas an intermittent pattern of tyrosinase activation was observed after the fifth day of irradiation. This intermittent pattern consisted of latency periods where no melanogenic response was elicited despite exposure to UVB. Tyrosinase activity in cellular extracts increased shortly after an effective irradiation, peaked at 3 hours and thereafter decreased to below basal levels. Increased tyrosinase activity was associated with increased amounts of both the newly synthesized and mature forms of the enzyme. Decreased tyrosinase activity following an activation period was correlated with decreases in both the expression of tyrosinase mRNA and the amount of the newly synthesized form of the enzyme present in the melanocytes 24 hours after six irradiations. This particular pattern of stimulation of tyrosinase was not observed in S-91 murine melanoma cells after repeated UVB irradiations. Taken together these results may suggest a photo-protective mechanism developed by irradiated normal human melanocytes.