Increased Tyrosinase Activity Research Articles

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.

Effects of Latanoprost on Tyrosinase Activity and Mitotic Index of Cultured Melanoma Lines

The intraocular pressure-lowering drug latanoprost, a phenyl-substituted analogue of prostaglandin F2α(PGF2α), increases iris pigmentation in a small number of patients. In theory, this could be due to increased melanogenesis or melanocyte proliferation. To distinguish these two possibilities, the present study examined the effects of latanoprost on tyrosinase activity (the rate-limiting step for melanin synthesis) and mitotic index of cultured melanoma lines.Murine cutaneous melanoma lines (S91 and B16), and human uveal (OCM1, OCM3, and OM431) and cutaneous (SK-MEL5 and M21) melanoma lines were cultured with PGE1, PGE2, PGF2α, latanoprost, or the adenylate cyclase stimulating agent forskolin. After treatment, tyrosinase was assayed with respect to its dopa oxidase activity using a colorimetric assay. PGE1, PGE2, PGF2α, and latanoprost greatly increased tyrosinase activity in murine melanoma lines and caused small increases in tyrosinase activity in human uveal and cutaneous melanoma lines. Similar results were obtained with the cAMP-elevating compound forskolin. Cyclic AMP content, as determined by an enzyme-linked immunoassay, was similarly increased by all treatments, with forskolin being the most potent stimulator. Since the species difference in tyrosinase activity was observed without an apparent difference in induction of cAMP, latanoprost would appear to induce tyrosinase activity through a non-cAMP-dependent pathway. Finally, latanoprost and PGF2αdid not enhance the mitotic index of human uveal or cutaneous melanoma lines, measured by [6-3H] thymidine uptake, although they increased the mitotic index of one murine cutaneous line.Given that latanoprost induced tyrosinase activity, but did not increase the mitotic index in any of the human melanoma lines studied, this suggests that the in vivo iris pigmentation side effect of latanoprost may not result from increased cell division, but from elevated tyrosinase activity.

Ectopic pigmentation in Xenopus in response to DCoH/PCD, the cofactor of HNF1 transcription factor/pterin-4α-carbinolamine dehydratase

DCoH, the dimerization cofactor of the HNF-1 homeodomain proteins (hepatocyte nuclear factor-1α and β), is involved in gene expression by associating with these transcription factors. The protein also called PCD for pterin-4α-carbinolamine dehydratase is a bifunctional factor as it catalyzes also the regeneration of tetrahydrobiopterin. This coenzyme is used by the enzyme phenylalanine hydroxylase, which generates tyrosine, the precursor of catecholamines and melanin. DCoH/PCD presumably cooperates with other partners, because it is expressed earlier than HNF1 and phenylalanine hydroxylase (PAH) in early vertebrate development. It is also found in cells lacking HNF1 and PAH like skin, brain and the pigmented epithelium of the eye suggesting a yet unknown function. We show that the overexpression of DCoH/PCD in Xenopus induces the formation of ectopic pigment cells in the epidermis, that are visible earlier than the endogenous pigmentation and broader distributed. This ectopic pigmentation is accompanied by an increase in tyrosinase activity and the amount of melanin. Overexpression of DCoH/PCD induces the appearance of pigment cells also in animal cap explants, that normally differentiate into atypical epidermis. DCoH/PCD mutants with impaired carbinolamine dehydratase activity retain the potential to induce pigmentation and we propose therefore that DCoH/PCD is not simply an essential enzyme for melanin biosynthesis, but also a regulator for the differentiation of pigment producing cells.

Downregulation of Tyrosinase Activity in Human Melanocyte Cell Cultures by Yohimbine

Treatment of human melanocyte cell cultures with the alpha-2 adrenergic receptor antagonist yohimbine results in a marked down-regulation of tyrosinase activity. A 30% decrease occurs within 12 h of exposure of cells to yohimbine (100 microM), and by 48 h tyrosinase activity in treated melanocytes is less than a fifth that of control cultures. The inhibition is dose dependent and occurs in human melanocytes derived from either black or white skin types, and also in mouse melanoma cells. The yohimbine-induced decrease in tyrosinase activity is reversible, with enzyme levels returning to 90% of control values 48 h after removal of drug. Although tyrosinase activity is markedly suppressed by yohimbine, the compound has no effect on cell proliferation, cellular translation, or DNA synthesis. Treatment of melanocyte cultures with yohimbine blocks the increase in tyrosinase activity by either 3-isobutyl-1-methylxanthine, dibutyryl cAMP, or forskolin. Results of cAMP immunoassays, show that intracellular levels of the cyclic nucleotide are unaffected in cells treated with yohimbine. Tyrosinase inhibition by yohimbine does not involve a decrease in substrate availability since tyrosine uptake studies show that yohimbine has no effect on the amount of tyrosine entering the cell. Incubation of a melanosome-enriched fraction with yohimbine does not cause a lowering of tyrosinase activity, suggesting that an intact cell is required for yohimbine action. In addition, tyrosinase extracts show no reduction in activity when incubated directly with yohimbine, indicating that the drug does not act as a direct inhibitor of the enzyme. Finally, results of western immunoblotting show that yohimbine does not significantly lower the amount of tyrosinase protein in human melanocytes. These findings suggest that yohimbine acts through an as yet unidentified signaling pathway to lower the catalytic activity of pre-existing tyrosinase molecules present in melanocytes.

Biocatalysis of tyrosinase in chloroform medium, using selected phenolic substrates

The biocatalysis of mushroom tyrosinase was optimized in chloroform medium using as substrates selected phenolic compounds, including chlorogenic acid (CA), p-aminophenol (pAP) and hydroquinone (HQ). The specific activity of tyrosinase was found to be much higher in the chloroform medium than that obtained in the aqueous one. The optimal pH and temperature as well as other kinetic parameters for tyrosinase biocatalysis was also determined. The presence of catechol in the chloroform medium resulted in an increase in tyrosinase activity when CA was used as substrate; however, no activatory effect was found with pAP or HQ. The presence of ethylenediamine tetraacetic acid in the chloroform medium showed different effects on tyrosinase activity depending on the nature of the substrate. FT-IR analyses confirmed the bioconversion of selected phenolic substrates into o-quinones by tyrosinase activity in the chloroform medium. © 2000 Society of Chemical Industry

Protein Kinase C-β Activates Tyrosinase by Phosphorylating Serine Residues in Its Cytoplasmic Domain

We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.

Modulation of skin cell functions by transforming growth factor-beta1 and ACTH after ultraviolet irradiation.

The adaptive responses in skin to ultraviolet (UV) radiation include increased cornification of keratinocytes and increased synthesis and distribution of melanin by melanocytes. The possible involvement of paracrine factors in the generation of these responses was studied in a novel two-stage culture model. Human melanocytes or keratinocytes were first irradiated or sham-irradiated and then the conditioned media collected from these cells after 24 h were used to treat unirradiated skin cells. Immunofluorescent staining for transforming growth factor (TGF)-beta1 was increased in UV-irradiated keratinocytes compared with sham-irradiated cells. Increased TGF-beta1 was also detected in the culture media of irradiated keratinocytes. Treatment of unirradiated keratinocytes with conditioned media collected from UV-irradiated keratinocytes resulted in increased absolute numbers and percentages of cornified envelopes per well compared with treatment with conditioned media from sham-irradiated keratinocytes. The magnitude of this effect increased with increased dose of initial irradiation. The effects of conditioned media from UVR-treated cells were mimicked by authentic TGF-beta1. Treatment of conditioned media from irradiated cells with an antibody shown to neutralise the effects of TGF-beta1 but not with a non-immune antibody of similar isotype, abolished this bioactivity of the conditioned media from UV-irradiated cells. Immunofluorescent staining for ACTH was also increased in UV-irradiated keratinocytes. Conditioned media from UV-irradiated keratinocytes increased tyrosinase activity of unirradiated melanocytes, an effect which was mimicked by authentic ACTH. This bioactivity of conditioned media from irradiated keratinocytes was abolished in the presence of an antibody which neutralised the activity of ACTH but not MSH. These results provide evidence to support the involvement of TGF-beta1 and ACTH in the cornification and pigmentary responses respectively of skin cells after UV exposure.

Functional regulation of tyrosinase and LAMP gene family of melanogenesis and cell death in immortal murine melanocytes after repeated exposure to ultraviolet B.

This study characterizes the induction of melanogenesis and the expression of tyrosinase, tyrosinase-related protein (TRP) and lysosome-associated membrane protein (LAMP) gene families in the cultured melanocyte lines of non-agouti mice with four major genetic loci, i.e. melan-a2 (black, wild type), melan-b (brown, TRP-1 mutation), melan-s (black, piebaldism mutation) and melan-c (white, tyrosinase mutation) in response to repeated exposure to ultraviolet (UV) B (5 mJ/cm2, 7 consecutive days). Electron microscopy showed that new melanogenesis was induced in melan-a2, melan-s and melan-b melanocytes. Melan-a2, melan-s and melan-b showed an almost twofold increase in tyrosinase activity and gene expression with increased synthesis of melanosomes, although melan-b showed a minimum increase in tyrosinase activity. There was a twofold upregulation of LAMP-1 mRNA but no alteration in LAMP-2 and LAMP-3 mRNA expression in melan-a2, while there was no alteration in LAMP-1 mRNA expression but increased expression of LAMP-2 and LAMP-3 mRNA in melan-s, LAMP-3 showing a higher increase. Melan-b cells showed the same gene expression of LAMP-1, LAMP-2 and LAMP-3 as that of non-UV exposed cells. All three lines, however, exhibited simultaneously cell death, melan-b reaching the highest rate of cell death (96.5%). In contrast, melan-c, which did not have any tyrosinase activity with failure of melanogenesis induction, expressed all the mRNAs of the tyrosinase and LAMP gene families, but was not associated with any significant melanocyte death. Our study indicated: (i) that melanogenesis induction and melanocyte death are two photobiological processes occurring simultaneously after repeated UVB exposure, (ii) that in response to an upregulation of tyrosinase mRNA and enzymic activity, there was a co-ordinated upregulation of the LAMP-1 gene in wild type melan-a2, while no upregulation was found in melan-s and melan-b mutants, and (iii) that UV-induced melanocyte death is related to the upregulation of the tyrosinase gene, induction of new melanogenesis and mutation of the TRP-1 gene in immortal murine melanocytes.

Lincomycin abrogates dexamethasone-enhanced melanogenesis in B16 melanoma cells.

The effects of lincosamide, and interference between the effects of glucocorticoid and lincosamide, on melanogenesis were determined in B16 melanoma cells. Cells were treated for 4 days with lincomycin (LM) and/or dexamethasone (DX) at equimolar concentrations ranging from 10(-9) M to 10(-5) M, or at various concentrations of DX with 10(-6) M LM. Effects on proliferation, tyrosinase activity, melanin biosynthesis, and levels of mRNA for tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) were examined. Treatment with LM or LM + DX stimulated proliferation of melanoma cells with minimal cytotoxicity, while DX did not influence cell proliferation either alone or in combination with LM. Treatment with LM alone increased tyrosinase activity slightly and reduced melanin content in a dose-dependent manner. However, LM counteracted the pronounced increase in tyrosinase elicited by DX and also abrogated the dose-dependent increase in melanin content elicited by DX. Treatment with LM alone did not affect mRNA levels for tyrosinase, TRP1, or TRP2. Furthermore, LM abrogated the DX-induced up-regulation of mRNAs for tyrosinase and the down-regulation of TRP1 mRNA. These results suggest that LM inhibits melanogenesis post-transcriptionally and abrogates glucocorticoid-induced melanogenesis at the transcriptional level in B16 melanoma cells.

Melanogenesis in Cultured Melanocytes can be Substantially Influenced by L-Tyrosine and L-Cysteine

We investigated the effect of varying concentration of 1-tyrosine and 1-cysteine in culture medium on melanin production by human skin melanocytes (skin phototype II/III). In addition to the analyses of dopa oxidase activity and total melanin, pheomelanin production in the cells was assessed by high-performance liquid chromatography determinations of pheomelanin degradation products, 3-aminotyrosine and 4-amino-3-hydroxyphenylalanine. As another marker for pheomelanin, melanosomal sulfur was determined by the use of X-ray microanalysis. With varying concentration of both amino acids, profound changes in the pigmentation patterns of the melanocytes were observed. A high concentration of 1-tyrosine (0.2 mM) was always connected with increased pigmentation. In combination with a low 1-cysteine content we saw an increase in tyrosinase activity and the highest melanin content. At high concentrations of both 1-tyrosine and 1-cysteine, the melanocytes showed reduced tyrosinase activity and they produced notably more pheomelanin. In case of the pheomelanin measurements by high-performance liquid chromatography and the sulfur detection with X-ray microanalysis, strongly increased concentrations were found when cells were maintained in high 1-tyrosine medium as compared with those grown with low 1-tyrosine. This was especially true for the combination with low 1-cysteine showing that the 1-tyrosine content of the medium strongly influences not only the eumelanin but also the pheomelanin production in the cultured melanocyte. It can be concluded that variations in the concentrations of 1-tyrosine and 1-cysteine in culture medium can be used to regulate the melanogenetic phenotype under in vitro conditions.

Comparative Biological Activities of α-MSH Antagonists in Vertebrate Pigment Cells

We have previously reported that melatonin was an effective lightening agonist in the teleostSynbranchus marmoratus,the amphibiansRana pipiensandBufo ictericus,and in the lizardAnolis carolinensis.The hormone, previously applied to the preparations, effectively inhibited α-MSH darkening activity in a dose-independent manner, and was also able to reverse MSH-induced darkening. We presently describe the inhibitory effect of the indoleamine on the murine melanoma cell proliferation. Interestingly, the hormone also stimulated tyrosinase activity, with a correlated increase in melanin content. We also demonstrate that in a diverse lizard species,Urosaurus ornatus,the indoleamine was totally ineffective. The competitive MSH antagonistic activity of H-His-d-Arg-Ala-Trp-d-Phe-Lys-NH2has been demonstrated previously inR. pipiensandU. ornatus.Herein, its inhibitory activity is also reported in another lizard species,A. carolinensis.However, this MSH analogue was inactive inS. marmoratus,and in murine melanoma cells. On the other hand, the 7 thru 10 α-MSH fragment, Ac-Phe-Arg-Trp-Gly-NH2, although ineffective inS. marmoratusandR. pipiens,was an α-MSH antagonist inA. carolinensis.Surprisingly, in the melanoma cell line, the MSH fragment exhibited no agonist or antagonist activity, but dramatically potentiated the MSH-induced increase in tyrosinase activity. These data might suggest that the fragment is participating either in the process of facilitation or in positive cooperativity. The present results, taken together with our previously reported data, demonstrate a major interspecies diversity of the MC1 subtype of melanocortin receptor, and point out the relevance of the membrane microenvironment for the final receptor configuration.

The influence of extracellular matrix proteins on cutaneous and uveal melanocytes.

Cutaneous and ocular melanocytes are routinely cultured in complex mitogen-rich media. The physiological regulation of melanocyte proliferation and differentiation is not yet fully defined and this study summarises several separate lines of evidence which suggest that, in vivo, some of the signals required for melanocyte proliferation and differentiation may derive from extracellular matrix (ECM) proteins adjacent to these cells. Culture of cutaneous and uveal melanocytes on cell-derived and individual ECM proteins was found to influence cell morphology with such effects being most noticeable in mitogen-deficient media. Similarly, cell-derived and individual ECM proteins increased tyrosinase activity in normal cutaneous melanocytes and effects of these ECM proteins were seen most consistently in mitogen-deficient media. Uveal melanocytes (as has been reported for cutaneous melanocytes) showed preferential attachment to fibronectin over other ECM substrates. This attachment was particularly sensitive to drugs which affected intracellular calcium or calmodulin activity. Acute addition of fibronectin to coverslips of uveal melanocytes loaded with Fura-2 produced an acute and transient increase in intracellular calcium which was more prevalent in low density than higher density cells. We conclude that ECM proteins in vitro are capable of influencing melanocyte morphology, tyrosinase activity, and proliferation and that an ECM-induced elevation in intracellular calcium may be part of the signalling system that transmits ECM information into the cell.

Investigation of the influence of extracellular matrix proteins on normal human melanocyte morphology and melanogenic activity.

Several studies have indicated that extracellular matrix (ECM) proteins can influence melanocyte behaviour in vitro. However, the choice of medium is known to have a profound effect on melanocyte behaviour and it is currently difficult to ascribe which reported effects are due to ECM proteins and those which are attributable to the medium used in these different studies. The purpose of this study was to learn more about the influence of ECM proteins on melanocyte function by examining a range of cell-derived and individual ECM proteins for their impact on melanocyte tyrosinase activity in cells cultured under conditions of varying mitogenic drive. We found that ECM derived from human dermal fibroblasts, bovine endothelial cells and a human endothelial cell line as well as collagen I, collagen IV, fibronectin, and to a lesser extent laminin, were all capable of increasing tyrosinase activity in cultures of normal melanocytes. Effects of these ECM were seen most consistently in media with relatively few mitogens, for example ECM proteins influenced melanocyte morphology and this was seen most readily in cells cultured in medium without any mitogens (which ordinarily fails to support melanocyte survival). This study illustrates that ECM proteins can influence melanocyte morphology, proliferation, and tyrosinase activity in vitro and supports a possible role of ECM proteins in the regulation of melanocyte function in vivo.

Regulation of Tyrosinase mRNA in Mouse Melanoma Cells by α-Melanocyte–Stimulating Hormone

Cloudman S-91 mouse melanoma cells respond to alpha-melanocyte-stimulating hormone) by demonstrating a marked increase in tyrosinase activity (O-diphenol-O2 oxidoreductase, EC 1.14.18.1). This increase is the result of increased levels of tyrosinase mRNA with a subsequent increase in tyrosinase abundance. Our studies were carried out to determine the effect of melanocyte-stimulating hormone on tyrosinase gene transcription and to measure the kinetics of the hormone-induced increase in tyrosinase mRNA. When melanoma cells were exposed continuously to melanocyte-stimulating hormone for 6 d, a large but transient increase in both tyrosinase mRNA abundance and enzyme activity were observed. The maximum increase in tyrosinase mRNA occurred 60 h after melanocyte-stimulating hormone stimulation and was followed by a decline in message levels even though cells were continuously exposed to hormone. Results of nuclear run-off transcription assays showed that melanocyte-stimulating hormone caused a slow increase in the rate of transcription of the tyrosinase gene with a maximal 6-fold stimulation occurring at 48 h. In cells treated with the ribonucleic acid synthesis inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, tyrosinase mRNA levels decayed with a half-life of 4-5 h. This decay rate was unaffected by treatment of cells with melanocyte-stimulating hormone, indicating that the hormone does not act to stabilize tyrosinase ribonucleic acid. Inhibition of protein synthesis by treatment with cycloheximide had no effect on the melanocyte-stimulating hormone-induced increase in tyrosinase messenger ribonucleic acid levels suggesting that ongoing protein synthesis is not required for, at least, the initial stimulation of tyrosinase gene transcription by melanocyte-stimulating hormone.

Tyrosinase may protect human melanocytes from the cytotoxic effects of the superoxide anion.

Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a rôle by utilising the superoxide anion (O2-) in the production of melanin. In the present study, we have examined the cytotoxic effects of O2- and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5-150 mU.ml-1) and glucose oxidase (GO, 0.1-20 mU.ml-1) were used to generate the O2- and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay. 3 h later, dose-related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and fibroblasts showed that these cells were more resistant to the cytotoxic effects of O2- than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melanocyte-stimulating hormone (MSH) Nle4DPhe7 alpha-MSH (10(-8) M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2- and the resistance of the melanocytes to the cytotoxic effects of this ROS, but failed to alter their susceptibility to the damaging effects of H2O2.Nle4DPhe7 alpha-MSH had no effect on the resistance of keratinocytes and fibroblasts to either O2- or H2O2. After 3 h, XO, as opposed to GO, also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2- to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.

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 PKC alpha had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKC alpha 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.

Activation of tyrosinase reduces the cytotoxic effects of the superoxide anion in B16 mouse melanoma cells.

Tyrosinase may protect against oxidative stress by using the superoxide anion (O2-1.) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10-differential deficient (-DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O2.1 and cytotoxicity assessed by measuring cell survival. XO increased O2.- concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10-DD cells. [Nle4, DPhe7]MSH increased tyrosinase activity and melanin content, reduced O2.- concentration and increased the resistance of F10 cells to the cytotoxic effects of O2.-. No such effects were seen in F10-DD cells. The effect of [Nle4, DPhe7]MSH on the resistance of the F10 cells was time-dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O2.-. In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle4, DPhe7]MSH. Moreover, although melanin was capable of scavenging O2.- it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10-DD cells. We conclude that tyrosinase is able to utilise O2.- to produce melanin and this provides pigment cells with a unique anti-oxidant mechanism.

The melanotropic peptide, [Nle4,D-Phe7] alpha-MSH, stimulates human melanoma tyrosinase activity and inhibits cell proliferation.

Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle4,D-Phe7] alpha-MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called "amelanotic" (tyrosinase-negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle4,D-Phe7] alpha-MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and "amelanotic" cell lines incubated with [Nle4,D-Phe7] alpha-MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle4,D-Phe7] alpha-MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.

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.

Stimulation of tyrosinase in human melanocytes by pro-opiomelanocortin-derived peptides.

Human melanocytes, maintained on bovine corneal endothelium-derived extracellular matrix for at least 4 days in the absence of phorbol 12-myristate 13-acetate (PMA) and cholera toxin (CT), displayed increased tyrosinase activity when exposed to several pro-opiomelanocortin-derived (POMC) peptides. Melanocytes from 9 of 14 donors showed significantly increased tyrosinase activity after treatment with adrenocorticotropic hormone (ACTH; mean increase 320 +/- 107 (S.E.M.)% of control, P < 0.005), while melanocytes from 8 of 13 donors increased tyrosinase in the presence of diacetyl-melanocyte stimulating hormone (di-MSH; mean increase 223 +/- 31 (S.E.M.)% of control, P < 0.005). Maximal increases in tyrosinase were seen after treatment with 10(-10) M ACTH and with 10(-6) M di-MSH. In two cell cultures which showed tyrosinase stimulation, melanin synthesis was similarly increased in the presence of added POMC peptides. PMA but not CT increased tyrosinase activity in melanocytes cultured under these conditions. In the presence of staurosporine, an inhibitor of protein kinase C (PKC), the magnitude of the increase in tyrosinase due to PMA, ACTH and di-MSH was significantly reduced. These results indicate that tyrosinase activity in melanocytes from most human donors, under appropriate conditions, is susceptible to the stimulatory effects of POMC peptides, that ACTH is considerably more potent than di-MSH in this test system and that in human cells the PKC pathway may be important in modulating melanogenesis.