Tyrosinase, could it be a missing link in ochronosis in alkaptonuria?

The hypothesis that is proposed is that tyrosinase, an enzyme widely found within the human body is implicated in the ochronosis that occurs in alkaptonuria; an autosomal recessive condition first used by Archibald Garrod to describe the theory of "Inborn Errors of Metabolism." The disease results from the absence of a single enzyme in the liver that breaks down homogentisic acid; this molecule becomes systemically elevated in sufferers. The condition is characterised by a clinical triad of symptoms; homogentisic aciduria from birth, ochronosis (darkening) of collagenous tissues (from ∼30years of age) and ochronotic osteoarthropathy in weight bearing joints due to long term ochronosis in them (from ∼40years of age). Tyrosinase, a polyphenol oxidase has been shown in many species to contribute to the darkening of tissues in many organisms; including humans in the production of melanin. Tyrosinase under the right conditions shows alterations in its substrate specificity and may contribute to the darkening seen in AKU where it moves away from polymerising tyrosine but also homogentisic acid, the causative molecule in alkaptonuria, that is present in excess.

Identification of a novel microRNA important for melanogenesis in alpaca (Vicugna pacos).

The molecular mechanisms underlying the formation of coat colors in animals are poorly understood. Recent studies have demonstrated that microRNA play important roles in the control of melanogenesis and coat color in mammals. In a previous study, we characterized the miRNA expression profiles in alpaca skin with brown and white coat color and identified a novel miRNA (named lpa-miR-nov-66) that is expressed significantly higher in white skin compared to brown skin. The present study was conducted to determine the functional roles of this novel miRNA in the regulation of melanogenesis in alpaca melanocytes. lpa-miR-nov-66 is predicted to target the soluble guanylate cyclase (sGC) gene based on presence of a binding site in the sGC coding sequence (CDS). Overexpression of lpa-miR-nov-66 in alpaca melanocyes upregulated the expression of sGC both at the mRNA and protein level. Overexpression of lpa-miR-nov-66 in melanocyes also resulted in decreased expression of key melanogenic genes including tyrosinase (TYR), tyrosinase related protein 1 (TYRP1), and microphthalmia transcription factor (MITF). Our ELISA assays showed increased cyclic guanosine monophosphate (cGMP) but decreased cyclic adenosine monophosphate (cAMP) production in melanocytes overexpressing lpa-miR-nov-66. In addition, overexpression of lpa-miR-nov-66 also reduced melanin production in cultured melanocytes. Results support a role of lpa-miR-nov-66 in melanocytes by directly or indirectly targeting , which regulates melanogenesis via the cAMP pathway.

[Leukoderma caused by chemicals: mechanisms underlying 4-alkyl/aryl-substituted phenols- and rhododendrol-induced melanocyte loss].

Chemical leukoderma is a skin depigmentation disorder known to occur in manufactural workplace through contact with chemicals, such as monobenzyl ether of hydroquinone (MBEH) and 4-tert- butylphenol (4-TBP). In the skin depigmented -legions induced by these chemicals, the number of melanocyte was severely decreased. Anti-melanoma agent 4-cysteaminylphenol (4-SCAP) and its derivatives are also known to cause leukoderma. Evidence has accumulated supporting that typical class of chemicals causing leukoderma is "4-alkyl/aryl-substituted phenols/catechols", which are structurally similar to melanin precursor tyrosine. Tyrosinase-mediated oxidation of these chemicals yields toxic ortho-quinones which bind to cellular proteins and produce reactive oxygen species. Accordingly, this tyrosinase-dependent metabolic activation is thought to cause melanocyte-specific damage and subsequent immune reactions toward melanocytes. Recently, rhododendrol, an inhibitor of tyrosinase developed for so-called lightening/whitening cosmetics, was shown to cause leukoderma in the users. In this review, I document the causes of known chemical leukoderma and rhododendrol- induced leukoderma, focusing on their common mechanisms underlying melanocyte loss.

L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions.

There is evidence that L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor-mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as regulate the melanocyte functions through the activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate-induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate an older theory proposing that receptors for amino acid-derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates.

AAV-mediated tyrosinase gene transfer restores melanogenesis and retinal function in a model of oculo-cutaneous albinism type I (OCA1).

Oculo-cutaneous albinism type 1 (OCA1) is characterized by congenital hypopigmentation and is due to mutations in the TYROSINASE gene (TYR). In this study, we have characterized the morpho-functional consequences of the lack of tyrosinase activity in the spontaneous null mouse model of OCA1 (Tyr(c-2j)). Here, we show that adult Tyr(c-2j) mice have several retinal functional anomalies associated with photoreceptor loss. To test whether these anomalies are reversible upon TYR complementation, we performed intraocular administration of an adeno-associated virus (AAV)-based vector, encoding the human TYR gene, in adult Tyr(c-2j) mice. This resulted in melanosome biogenesis and ex novo synthesis of melanin in both neuroectodermally derived retinal pigment epithelium (RPE) and in neural crest-derived choroid and iris melanocytes. Ocular melanin accumulation prevented progressive photoreceptor degeneration and resulted in restoration of retinal function. Our results reveal novel properties of pigment cells and show that the developmental anomalies of albino mice are associated with defects occurring in postnatal life, adding novel insights on OCA1 disease pathogenesis. In addition, we provide proof-of-principle of an effective gene-based strategy relevant for future application in albino patients.

Probing into the role of conserved N-glycosylation sites in the Tyrosinase glycoprotein family.

N-linked glycosylation has a profound effect on the proper folding, oligomerization and stability of glycoproteins. These glycans impart many properties to proteins that may be important for their proper functioning, besides having a tendency to exert a chaperone-like effect on them. Certain glycosylation sites in a protein however, are more important than other sites for their function and stability. It has been observed that some N-glycosylation sites are conserved over families of glycoproteins over evolution, one such being the tyrosinase related protein family. The role of these conserved N-glycosylation sites in their trafficking, sorting, stability and activity has been examined here. By scrutinizing the different glycosylation sites on this family of glycoproteins it was inferred that different sites in the same family of polypeptides can perform distinct functions and conserved sites across the paralogues may perform diverse functions.

Inhibition of MITF and tyrosinase by paeonol-stimulated JNK/SAPK to reduction of phosphorylated CREB.

Tyrosinase and its transcriptional regulator microphthalmia-associated transcription factor (MITF) play critical roles in regulation of melanogenesis, and are required for environmental cues or agents in modulation of melanin synthesis. Identifying the signals regulating tyrosinase and MITF is crucial to understanding how pigmentation responds to extracellular stimuli. In this report, we discovered that paeonol down-regulated melanin production via decreasing MITF expression and consequent mRNA and protein levels of tyrosinase. We also found that paeonol reduced phosphorylation of a cAMP responsive element binding protein (phospho-CREB), which binds and activates MITF. A selective inhibitor of c-jun N-terminal or stress-activated protein kinases (JNK/SAPK)-SP600125 significantly reversed paeonol-induced down-regulation of melanogenesis. Inhibition of cAMP/PKA pathway intensified the hypopigmentation response to paeonol. These results identify a mechanism in which paeonol induces the down-regulation of melanogenesis through inhibition of CREB phosphorylation, leading to the expression reduction of MITF and subsequently tyrosinase. The key kinase mediating the effects of paeonol on melanogenesis in B16F10 cells is JNK/SAPK. Additionally, the cAMP/PKA pathway may take part in this process.

Parkin protects against tyrosinase-mediated dopamine neurotoxicity by suppressing stress-activated protein kinase pathways.

Parkinson's disease (PD) motor symptoms are caused by degeneration of nigrostriatal dopaminergic (DAergic) neurons. The most common causes of hereditary PD are mutations in the PARKIN gene. The ubiquitin ligase parkin has been shown to mediate neuroprotection in cell culture and in vivo, but the molecular mechanisms are not well understood. We investigated the effects of parkin in a human SH-SY5Y neuroblastoma cell culture model of PD, in which transcriptional induction of the enzyme tyrosinase causes a neurotoxic overproduction of cellular DA and its oxidative metabolites. Tyrosinase induction caused formation of reactive oxygen species in the cytosol and mitochondria, and neurotoxicity via activation of apoptotic stress-activated protein kinases and caspase 3. Stable transfection of wild-type parkin suppressed tyrosinase-induced apoptosis, and PD-associated mutations abolished the neuroprotective effect of parkin. Expression of wild-type parkin did not affect reactive oxygen species production, but attenuated the tyrosinase-induced activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase as well as their cognate mitogen-activated protein kinase kinases. PD-associated mutations differentially affected the anti-apoptotic signaling of parkin. Thus, parkin contributes to DAergic neuroprotection by suppression of apoptotic stress-activated protein kinase pathways.

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells.

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.

Advances in melanocyte basic science research.

This article focuses on recent advances in melanocyte biology and physiology. The major function of this neural crest-derived cell is the production of melanins. A "three enzyme theory" in the initiation of pigmentation is put forward and backed up by recent findings. A receptor-independent role for alpha-MSH and the cofactor (6R)-l-erythro-5,6,7,8-terahydrobiopterin (6BH(4)) in the control of tyrosinase is described. The importance of intramelanosomal pH for melanogenesis is covered. Finally, the redundancy of the cAMP and IP3/DAG/calcium signal in melanocytes together with the downstream events are highlighted. The main message of this article is that the intracellular H(2)O(2)- redox-equilibrium controls melanocyte function in a concentration-dependent manner.

Involvement of extracellular signal-regulated kinase in nobiletin-induced melanogenesis in murine B16/F10 melanoma cells.

Nobiletin contributes to pharmacological activities such as anti-cancer and anti-inflammatory effects, but little is known about its effect on melanogenesis. In this study, we found that nobiletin increased melanin content and tyrosinase activity in murine B16/F10 melanoma cells. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK) pathway with U0216 resulted in inhibition of nobiletin-induced melanin synthesis and tyrosinase expression.

Survey and mechanism of skin depigmenting and lightening agents.

The type and amount of melanin synthesized by the melanocyte, and its distribution pattern in the surrounding keratinocytes, determines the actual color of the skin. Melanin forms through a series of oxidative reactions involving the amino acid tyrosine in the presence of the enzyme tyrosinase. Tyrosinase catalyses three different reactions in the biosynthetic pathway of melanin in melanocytes: the hydroxylation of tyrosine to l-DOPA and the oxidation of l-DOPA to dopaquinone; furthermore, in humans, dopaquinone is converted by a series of complex reactions to melanin. Among the skin-lightening and depigmenting agents, magnesium-l-ascorbyl-2-phosphate (MAP), hydroxyanisole, N-acetyl-4-S-cysteaminylphenol, arbutin (hydroquinone-beta-d-glucopyranoside) and hydroquinone (HQ) are the most widely prescribed worldwide. However, with reports of potential mutagenicity and epidemics of ochronosis, there has been an increasing impetus to find alternative herbal and pharmaceutical depigmenting agents. A review of the literature reveals that numerous other depigmenting or skin-lightening agents are either in use or in investigational stages. Some of these, such as kojic, glycolic and azelaic acids, are well known to most dermatologists. Others have been discovered and reported in the literature more recently. Several depigmentation and lightening agents are discussed, including their historical background, biochemical characteristics, type of inhibition and activators from various sources. In addition, the clinical importance of mushroom tyrosinase as a recent prospect is discussed in this paper.

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata).

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.

Tyrosinase exacerbates dopamine toxicity but is not genetically associated with Parkinson's disease.

Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.

Transcriptional control: an essential component of cancer gene therapy strategies?

The therapeutic index of cancer gene therapy approaches will, at least in part, be dictated by the spatial and temporal control of expression of the therapeutic transgenes. Strategies which allow precise control of gene transcription are likely to play a crucial role in the future pre-clinical and clinical development of gene therapy. In this review, we discuss these issues as they relate to tissue and tumor specific promoters. In addition, the exciting opportunities offered by the development of regulated gene expression systems using small molecules, radiation and heat are reviewed. It is realistic to expect that the future offers the prospect of amalgamating elements of a number of these different systems in a co-ordinated gene delivery approach with the potential to increase the efficacy and reduce the toxicity of treatment.

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.

Selective incorporation and specific cytocidal effect as the cellular basis for the antimelanoma action of sulphur containing tyrosine analogs.

Tyrosine analogs are good candidates for developing melanoma chemotherapy because melanogenesis is inherently toxic and uniquely expressed in melanocytic cells. Sulphur containing substrate (tyrosine) analogs, N-acetyl-4-S-cysteaminylphenol (NAcCAP) and N-propionyl-4-S-cysteaminylphenol (NPrCAP), have been shown to have potent antimelanoma activity in mice bearing melanoma. Both NAcCAP and NPrCAP show selective cytotoxicity towards melanoma cell lines. But the mechanism leading to selectivity is not clear as these drugs are also toxic to other cell lines to a lesser extent. Here we show that these drugs have both cytostatic and cytocidal effects, which could account for this. Cytostatic effect is suggested by DNA flow cytometry. The drug causes cell cycle changes in four human cell lines (normal skin fibroblasts, HeLa cells, and melanoma cell lines, C32 and SK-MEL-23) in a dose-dependent manner blocking cells in S phase with concomitant decrease in the number of cells in G1 phase. There is also a gradual decrease in cells in G2 + M phases. The dose-concentration curves give IC50 values in the range of 50-400 microM and the melanotic melanoma cell line SK-MEL-23 has the lowest IC50 value consistent with our hypothesis that these drugs are selective towards melanoma cells. The concentration-dependent accumulation of cells in S phase suggest a cytostatic effect as a consequence of inhibition of DNA synthesis in agreement with [3H] thymidine incorporation assay. There is a highly specific uptake of [14C]NAcCAP and irreversible damage to DNA synthesis machinery in SK-MEL-23 cells, indicating a melanotic-specific cytocidal effect as well. Trypan blue exclusion study and competitive inhibition assay indicated that visible cytocidal effect occurs slowly and oxidative stress resulting from tyrosinase mediated oxidation of the drug appears to be the underlying mechanism. The primary antimelanoma effect of cysteaminylphenols derives from a selective cytostatic effect, but is followed by a specific cytocidal action rendering the drugs useful for targeted melanoma chemotherapy.

Hepatocyte growth factor/scatter factor and hepatocytes are potent downregulators of tyrosinase expression in B16 melanoma cells.

Reiterated selection in vivo of B16 murine melanoma cells for enhanced liver metastatic ability yielded a cel line (B16-LS9) dramatically overexpressing a constitutively active hepatocyte growth factor/scatter factor (HGF/SF) receptor, the product of the c-met proto-oncogene. Most likely because of their overexpressing c-met, B16-LS9 cells appear to be more responsive than parental B16-F1 cells to HGF stimulation, in terms of motility, invasion, and growth. They are also more pigmented, and express higher levels of tyrosinase as compared to parental B1 6-F1 cells. Therefore, we set out to explore whether HGF/SF and the liver might influence the differentiation state of B1 6 cells. We found that HGF/SF and MSH, two factors which reportedly have a strong influence on the phenotype and the malignant behavior of melanoma cells, may act at different levels, and with opposite results, on the regulation of gene expression. In fact, while MSH induces, at the transcriptional level, an increase in the production of both c-met and tyrosinase, HGF/SF, in contrast, promotes a decrease in the expression of both c-met and tyrosinase, however at a posttranscriptional level. These two opposite effects can counter-balance each other, when the cells are treated with both factors at the same time, apparently through a mechanism involving MAP kinase activation. The effects were, however, additive when morphological changes were considered. Most intriguingly, we also describe a very strong downregulatory activity, limited to tyrosinase expression, by hepatocytes in coculture with B16 cells. This activity, also at the posttranscriptional level, is much stronger than that exerted by HGF/SF, and appears to be due to a labile soluble factor produced by the hepatocytes.

Induction of melanogenesis during the various melanoma growth phases and the role of tyrosinase, lysosome-associated membrane proteins, and p90 calnexin in the melanogenesis cascade.

Melanin biosynthesis (melanogenesis) is a metabolic pathway exclusively expressed by melanocytes and melanoma cells, and is often altered and/or markedly elevated in the latter cells. The changes in melanogenesis may be responsible for some of the clinical and histopathological features unique to melanoma. Melanogenesis may also contribute to the malignant transformation of melanoma precursors (i.e., atypical moles [or dysplastic nevi]) to melanoma as seen in patients with the familial atypical multiple-mole-melanoma (FAMMM) syndrome. However, it does not appear to affect the multi-step growth phases of melanoma cells from radial to vertical and lastly metastatic growth phases. Within the melanosomal compartment, eu- and pheomelanin pigments are synthesized. Both tyrosinase and lysosome-associated membrane protein (LAMP) gene products play important roles in this process. A coordinated interaction between these two gene family products is required for melanogenesis to occur properly. p90 calnexin is a new melanosome-associated molecule that is presumed to function as a melanogenesis chaperone by controlling the assembly and folding of glycoprotein intermediates of tyrosinase and LAMP gene families.

Melanocyte differentiation marker gp75, the brown locus protein, can be regulated independently of tyrosinase and pigmentation.

Human melanoma arises from epidermal melanocytes and displays remarkable phenotypic heterogeneity. This heterogeneity in part reflects the ability of melanoma cells to undergo differentiation along a pathway parallel to differentiation of normal melanocytes. Tyrosinase, encoded by the albino (c), and the tyrosinase-related protein-1 or gp75, encoded by the brown (b) locus, are two of the best-characterized markers for melanocyte differentiation. Both molecules are glycoproteins expressed in melanosomes, the site of pigment synthesis. We studied the regulation of these proteins in human melanoma cells induced by the polar-planar compound hexamethylene bisacetamide (HMBA). In well-differentiated melanoma cell lines, HMBA induced dendritic morphology and specifically regulated the expression of melanosomal glycoproteins (but not a panel of other molecules expressed by melanoma cells). HMBA specifically down-regulated gp75 expression by rapidly decreasing the steady-state level of gp75 mRNA and gp75 synthesis. HMBA was able to down-regulate gp75 expression even in the presence of cholera toxin, which when added alone induced a two- to threefold increase in gp75 expression. In contrast to uniform down-regulation of gp75 expression, HMBA could either up-regulate or down-regulate tyrosinase expression and pigmentation. Based on the differential regulation of gp75 and tyrosinase, melanoma cells could be classified into two groups. In one group, gp75 expression was coordinately regulated with tyrosinase activity and pigmentation. In the other group, gp75 expression and tyrosinase activity and pigmentation were dissociated (with pigmentation coupling to tyrosinase activity, not to gp75 expression). These results show that in mature melanocytic cells, regulation of gp75 expression follows a pattern that can be independent of regulation of tyrosinase and pigmentation.