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Supplementary Figures S1-9 and Tables S1-3. Supplementary Figure S1 - Effects of knockdown of NUAK2 on melanoma cells and specificity of the p-AKT antibody. Supplementary Figure S2 - Immunohistochemical analyses of p-Akt and NUAK2 expression in clinical specimens. Supplementary Figure S3 - Kaplan-Meier curves for overall survival of Acral and Non-CSD melanoma patients. Supplementary Figure S4 - Knockdown of NUAK2 induces apoptosis in SM2-1 melanoma cells. Supplementary Figure S5 - Immunohistochemistry showing the expression of p21 following inhibition of the PI3K pathway by LY294002. Supplementary Figure S6 - Schematic diagram of the regulation of the cell cycle machinery by NUAK2 and PI3K pathways. Supplementary Figure S7 - Knockdown of CDK2 by siCDK2 in C32 and mel18 melanoma cells. Supplementary Figure S8 - Immunohistochemical analyses of the expression of CDK2 in clinical specimens. Supplementary Figure S9 - Expression of NUAK2, PTEN, p-Akt, Akt and CDK2, and cell proliferation in various melanoma cell lines treated with Roscovitine. Supplementary Table S1 - Clinical parameters and expressions of CDK2, p-Akt and NUAK2 of 56 acral melanomas and 35 non-CSD melanomas with survival information. Supplementary Table S2 - Genomic status of NUAK2 and PTEN gene in each cell line. Supplementary Table S3 - Expression of p27 and CDK2 in primary melanomas with or without over-expression of NUAK2 and p-Akt.

Tyrosinase is the rate-limiting enzyme of melanin production and, accordingly, is the most prominent target for inhibiting hyperpigmentation. Numerous tyrosinase inhibitors have been identified, but most of those lack clinical efficacy because they were identified using mushroom tyrosinase as the target. Therefore, we used recombinant human tyrosinase to screen a library of 50,000 compounds and compared the active screening hits with well-known whitening ingredients. Hydroquinone and its derivative arbutin only weakly inhibited human tyrosinase with a half-maximal inhibitory concentration (IC50) in the millimolar range, and kojic acid showed a weak efficacy (IC50 > 500 μmol/L). The most potent inhibitors of human tyrosinase identified in this screen were resorcinyl-thiazole derivatives, especially the newly identified Thiamidol (Beiersdorf AG, Hamburg, Germany) (isobutylamido thiazolyl resorcinol), which had an IC50 of 1.1 μmol/L. In contrast, Thiamidol only weakly inhibited mushroom tyrosinase (IC50 = 108 μmol/L). In melanocyte cultures, Thiamidol strongly but reversibly inhibited melanin production (IC50 = 0.9 μmol/L), whereas hydroquinone irreversibly inhibited melanogenesis (IC50 = 16.3 μmol/L). Clinically, Thiamidol visibly reduced the appearance of age spots within 4 weeks, and after 12 weeks some age spots were indistinguishable from the normal adjacent skin. The full potential of Thiamidol to reduce hyperpigmentation of human skin needs to be explored in future studies.

More than 375 genes have been identified that are involved in regulating skin pigmentation and these act during development, survival, differentiation, and/or responses of melanocytes to the environment. Many of these genes have been cloned, and disruptions of their functions are associated with various pigmentary diseases; however, many remain to be identified. We have performed a series of microarray analyses of hyperpigmented compared with less pigmented skin to identify genes responsible for these differences. The rationale and goal for this study was to perform a meta-analysis on these microarray databases to identify genes that may be significantly involved in regulating skin phenotype either directly or indirectly that might not have been identified due to subtle differences by any of these individual studies alone. The meta-analysis demonstrates that 1,271 probes representing 921 genes are differentially expressed at significant levels in the 5 microarray data sets compared, providing new insights into the variety of genes involved in determining skin phenotype. Immunohistochemistry was used to validate two of these markers at the protein level (TRIM63 and QPCT), and we discuss the possible functions of these genes in regulating skin physiology.

The AMPK-related kinase NUAK2 has been implicated in melanoma growth and survival outcomes, but its therapeutic utility has yet to be confirmed. In this study, we show how its genetic amplification in PTEN-deficient melanomas may rationalize the use of CDK2 inhibitors as a therapeutic strategy. Analysis of array-CGH data revealed that PTEN deficiency is coupled tightly with genomic amplification encompassing the NUAK2 locus, a finding strengthened by immunohistochemical evidence that phospho-Akt overexpression was correlated with NUAK2 expression in clinical specimens of acral melanoma. Functional studies in melanoma cells showed that inactivation of the PI3K pathway upregulated p21 expression and reduced the number of cells in S phase. NUAK2 silencing and inactivation of the PI3K pathway efficiently controlled CDK2 expression, whereas CDK2 inactivation specifically abrogated the growth of NUAK2-amplified and PTEN-deficient melanoma cells. Immunohistochemical analyses confirmed an association of CDK2 expression with NUAK2 amplification and p-Akt expression in melanomas. Finally, pharmacologic inhibition of CDK2 was sufficient to suppress the growth of NUAK2-amplified and PTEN-deficient melanoma cells in vitro and in vivo. Overall, our results show how CDK2 blockade may offer a promising therapy for genetically defined melanomas, where NUAK2 is amplified and PTEN is deleted. Cancer Res; 75(13); 2708–15. ©2015 AACR.

BACKGROUND Lymphangioleiomyomatosis (LAM) is characterized by the proliferation in the lung, axial lymphatics (eg, lymphangioleiomyomas), and kidney (eg, angiomyolipomas) of abnormal smooth muscle-like LAM cells, which express melanoma antigens such as Pmel17/gp100 and have dysfunctional tumor suppressor tuberous sclerosis complex (TSC) genes TSC2 or TSC1 . Histopathologic diagnosis of LAM in lung specimens is based on identification of the Pmel17 protein with the monoclonal antibody HMB-45. METHODS We compared the sensitivity of HMB-45 to that of antipeptide antibody αPEP13h, which reacts with a C-terminal peptide of Pmel17. LAM lung nodules were laser-capture microdissected to identify proteins by Western blotting. RESULTS HMB-45 recognized approximately 25% of LAM cells within the LAM lung nodules, whereas αPEP13h identified > 82% of LAM cells within these structures in approximately 90% of patients. Whereas HMB-45 reacted with epithelioid but not with spindle-shaped LAM cells, αPEP13h identified both spindle-shaped and epithelioid LAM cells, providing greater sensitivity for detection of all types of LAM cells. HMB-45 recognized Pmel17 in premelanosomal organelles; αPEP13h recognized proteins in the cytoplasm as well as in premelanosomal organelles. Both antibodies recognized a Pmel17 variant of approximately 50 kDa. CONCLUSIONS Based on its sensitivity and specificity, αPEP13h may be useful in the diagnosis of LAM and more sensitive than HMB-45.

Human skin colour, ie pigmentation, differs widely among individuals, as do their responses to various types of ultraviolet radiation (UV) and their risks of skin cancer. In some individuals, UV-induced pigmentation persists for months to years in a phenomenon termed long-lasting pigmentation (LLP). It is unclear whether LLP is an indicator of potential risk for skin cancer. LLP seems to have similar features to other forms of hyperpigmentation, eg solar lentigines or age spots, which are clinical markers of photodamage and risk factors for precancerous lesions. To investigate what UV-induced molecular changes may persist in individuals with LLP, clinical specimens from non-sunburn-inducing repeated UV exposures (UVA, UVB or UVA + UVB) at 4 months post-exposure (short-term LLP) were evaluated by microarray analysis and dataset mining. Validated targets were further evaluated in clinical specimens from six healthy individuals (three LLP+ and three LLP-) followed for more than 9 months (long-term LLP) who initially received a single sunburn-inducing UVA + UVB exposure. The results support a UV-induced hyperpigmentation model in which basal keratinocytes have an impaired ability to remove melanin that leads to a compensatory mechanism by neighbouring keratinocytes with increased proliferative capacity to maintain skin homeostasis. The attenuated expression of SOX7 and other hemidesmosomal components (integrin α6β4 and plectin) leads to increased melanosome uptake by keratinocytes and points to a spatial regulation within the epidermis. The reduced density of hemidesmosomes provides supporting evidence for plasticity at the epidermal-dermal junction. Altered hemidesmosome plasticity, and the sustained nature of LLP, may be mediated by the role of SOX7 in basal keratinocytes. The long-term sustained subtle changes detected are modest, but sufficient to create dramatic visual differences in skin colour. These results suggest that the hyperpigmentation phenomenon leading to increased interdigitation develops in order to maintain normal skin homeostasis in individuals with LLP.

Repetitive suberythemal UVA and/or UVB exposures were used to generate comparable UV-induced tans in human skin over the course of 2 weeks. In order to evaluate the potential photoprotective values of those UVA- and/or UVB- induced tans and to avoid the confounding issue of residual UV-induced DNA damage, we waited 1 week before challenging those areas with a 1.5 MED dose of UVA+UVB after which we measure DNA damage. The results show that the type of UV used to induce skin pigmentation affects the redistribution of melanin in the skin and/or de novo melanin synthesis. The UVA-induced tans failed to even provide a minimal SPF of 1.5, which suggests that producing a tan with UVA-rich sunlamps prior to a holiday or vacation is completely counterproductive.

The diversity of human skin phenotypes and the ubiquitous exposure to ultraviolet radiation (UVR) underscore the need for a non-invasive tool to predict an individual's UVR sensitivity. We analysed correlations between UVR sensitivity, melanin content, diffuse reflectance spectroscopy (DR) and UVR-induced DNA damage in the skin of subjects from three racial/ethnic groups: Asian, black or African American and White. UVR sensitivity was determined by evaluating each subject's response to one minimal erythemal dose (MED) of UVR one day after the exposure. Melanin content was measured using DR and by densitometric analysis of Fontana-Masson staining (FM) in skin biopsies taken from unexposed areas. An individual's UVR sensitivity based on MED was highly correlated with melanin content measured by DR and by FM. Therefore, a predictive model for the non-invasive determination of UVR sensitivity using DR was developed. The MED precision was further improved when we took race/ethnicity into consideration. The use of DR serves as a tool for predicting UVR sensitivity in humans that should be invaluable for determining appropriate UVR doses for therapeutic, diagnostic and/or cosmetic devices.

Erythema (i.e. visible redness) and DNA damage caused by ultraviolet radiation (UVR) in human skin have similar action spectra and show good correlation after a single exposure to UVR. We explored the potential to use instrumental assessments of erythema as a surrogate for DNA damage after repeated exposures to UVR. We exposed 40 human subjects to three different exposure schedules using two different UVR sources. Cyclobutane-pyrimidine dimers (CPDs) in skin biopsies were measured by immunofluorescence, and erythema was assessed by both the Erythemal Index (EI) and the Oxy-hemoglobin (Oxy-Hb) content. Surprisingly, the skin with the highest cumulative dose ended up with the lowest level of DNA damage, and with the least erythema, as assessed by Oxy-Hb (but not EI) 24 h after the last UV exposure. Although the level of CPDs, on average, paralleled Oxy-Hb (R2 = 0.80-0.94, P = 0.03-0.11), the correlation did not hold for the pooled individual measurements (R2 = 0.009, P = 0.37) due to potential individual differences in UV-induced photoadaptation. We suggest that the methodology may be optimized to improve the correlation between DNA damage level and erythema to enable noninvasive risk assessment based on erythema/Oxy-Hb content for individual human subjects.

The Fitzpatrick system of skin phototyping (1) has been used for decades, mainly to determine appropriate phototherapy treatment regimens. It is also used in the indoor tanning industry for adjusting the exposure time and, therefore, the UV dose. The Fitzpatrick system assigns human skin to one of 6 distinct phototypes and is based on responses to 2 questions about an individual’s sensitivity to sunburn and ability to tan. The indoor tanning industry has developed its own questionnaire [http://www.tanningtraining.com/btf/stQuestionnarie.pdf] that uses responses to 8 questions, including questions on phenotype and racial background. The US Food and Drug Administration (FDA) has regulated indoor tanning equipment since 1979 (2). The deleterious effects of UV exposure are well known and the purpose of the FDA regulations is to limit both acute and delayed damage from such exposure. Current regulations (3) stipulate that the manufacturer of the tanning equipment provide a recommended exposure schedule based on the UV output of the device. FDA provided additional guidance in a policy letter (4) which recommends that exposure schedules be tailored to different skin phototypes. This was largely based on the photobiological knowledge at the time, which indicated that UV sensitivity was significantly associated with skin phototype. According to Pathak and Fanselow (5) phototypes 2, 3 and 4 had mean Minimal Erythema Doses (MED) of 210 J/m2, 360 J/m2 and 780 J/m2, respectively. As a result, tanning exposure schedules were designed to expose skin phototype 2 to doses lower than the MED for the most sensitive individuals in this group, and allowed higher doses for higher skin phototypes. Skin phototype 1 individuals were not included, as they are not able to tan. This exposure scheme operates under the paradigm of giving the highest dose possible, while avoiding erythema. There was a limited amount of data on melanogenesis available at the time the FDA guidelines were written. The minimal melanogenic dose (MMD) is defined as the dose of UV radiation that produces detectable pigmentation of the skin 5 to 7 days after exposure. Pathak and Fanselow (5) found that the MMD was essentially the same for skin phototypes 2, 3 and 4. When exposures are repeated, a much lower UV dose (per exposure) can induce melanogenesis as compared to what is needed for a single exposure (6). The efficiency of the tanning process strongly depends on the UV spectrum used. UVA (320 – 400 nm) radiation (especially UVA1 (340 – 400 nm)) is more melanogenic than UVB (290 – 320 nm) or solar simulated radiation (7, 8). We found that a 2% UVB/98% UVA tanning source produced darker pigmentation more rapidly than did a 5% UVB/95% UVA source for equally erythemogenic doses (9). In a survey of over 29,000 adults, it was reported that the majority of indoor tanners in the US are UV-sensitive non-Hispanic whites (10) which includes phototypes 1 thru 4. In this study, we compared the tanning ability of skin phototypes 2 and 3. We employed equally erythemogenic repeated doses, as determined by spectral weighting with the CIE reference erythema action spectrum (11). Our exposure protocols followed both the FDA guidance for sunlamp exposure schedules (5) and the International Electrotechnical Commission (IEC) standard (12) in defining the first and maximum allowable doses using an erythema action spectrum. However, as shown in our previous publication (9), the exposure protocols employed in this study result in cumulative doses that are a factor of 2 to 3 lower than that recommended by current FDA guidelines. We used two different UV sources, one with a relatively high proportion (5%; Source 1) and the other with a relatively low proportion (2%; Source 2) of UVB. Both lamps are commonly used in tanning devices. We believe that a new paradigm should be used when designing exposure schedules for indoor tanning, i.e. the exposure schedule should be based on the minimum dose necessary for tanning, not burning. This paradigm leads to a universal exposure schedule for all skin phototypes that are expected to engage in indoor tanning. In addition, the use of such a schedule would reduce the UV burden for all phototypes, especially 3 and above, which should result in a reduction of the long-term deleterious effects of UV exposure for this population.

Interactions between melanocytes and neighboring cells in the skin (keratinocytes and fibroblasts) play important roles in regulating human skin color. We recently reported that neuregulin-1 (NRG1) is highly expressed in fibroblasts from Fitzpatrick type VI skin (the darkest) and at least in part determines the constitutive color of human skin. We have now characterized the bioactive motif of NRG1 that is involved in modulating melanin production in human melanocytes. We found that 8-mer motifs (PSRYLCKC and LCKCPNEF) increased melanin production but did not increase the proliferation of melanocytes; the minimum fragment that could elicit that effect was the tetrapeptide LCKC. This smaller bioactive peptide might have an advantage in clinical applications in which it modulates only pigmentation and does not stimulate melanocyte proliferation.

Visible pigmentation of the skin, hair, and eyes depends primarily on the presence of melanin(s) in those tissues. Melanins are produced by specific cells called melanocytes. Not only is the type of melanin produced important, but also its eventual distribution in the tissue dramatically affects visible color, which ultimately determines the functions of the pigment, such as photoprotection (Gilchrest, 2011). Clearly, the specification, migration, and differentiation during development of melanocyte precursors (‘‘melanoblasts’’) in specific patterns are essential for eventual pigmentation in adults (Kawakami and Fisher, 2011). Following is a synopsis of critical findings that have led to our current understanding of the biochemical pathways and melanogenic factors involved in melanin synthesis. The key enzyme involved in the synthesis of all types of melanins from the initial precursor tyrosine is tyrosinase (EC 1.14.18.1). Tyrosinases have been described in many species, including mammals and lower animals, plants, and even fungi; in fact, the earliest observations of the catalytic function of tyrosinase were made in extracts of mushrooms (Bourquelot and Bertrand, 1895), which are still widely used today as a highly enriched source of that enzyme. All tyrosinases depend on the binding of copper for their catalytic function (Lerner et al., 1950; Lerch et al., 1986), although their substrate specificities and physical properties can differ dramatically depending on the species (Lerner et al., 1951; Hearing et al., 1980). The ratelimiting initial step in the biosynthesis of melanin was initially thought to be the hydroxylation of tyrosine to L-3,4dihydroxyphenylalanine (DOPA) and its immediate subsequent oxidation to DOPAquinone (DQ). In melanocytic cells, the DQ formed will be spontaneously converted to an orangecolored intermediate known as DOPAchrome. In vitro, the DOPAchrome will spontaneously lose its carboxylic acid group to form 5,6-dihydroxyindole (DHI), which can then further oxidize and polymerize to form a dense, highmolecular-weight complex now known as DHI-melanin. This was initially reported by Raper (1926), and the pathway was later refined by Mason (1948); hence, the biosynthetic pathway is frequently referred to as the Raper–Mason pathway. Throughout the 1950s, 1960s, and 1970s, the collaborative research groups at Yale (headed by AB Lerner) and Harvard (headed by TB Fitzpatrick) played key roles in defining the involvement of tyrosinase in the human skin pigmentation (Fitzpatrick et al., 1950), how its activities were confined to melanosomes and how those organelles developed (Seiji et al., 1961; Szabo et al., 1969), and the disruptions that occurred in those processes in many skin pigmentary diseases (Breathnach et al., 1965; Kawamura et al., 1971; Lerner and Nordlund, 1978; Rees, 2011; Spritz, 2011). Those findings, plus the training of many post-doctoral fellows and clinicians in their groups, played a major role in establishing research centers in Asia, Europe, and the Americas, which still have a strong influence on studies of skin pigmentation and related pigmentary diseases. As summarized in Figure 1, recent revisions of this melanogenic pathway have shown that DOPA is not a distinct intermediate produced initially from tyrosine, but is in fact produced later in the pathway owing to the paired reduction of DQ (Riley, 1999), and that downstream tyrosinase-related enzymes can rearrange the DOPAchrome to form a carboxylated intermediate (DHI-2-carboxylic acid) known as DHICA, as discussed below. Analysis of the structures of the high-molecular-weight polymers of melanins depended on the development of new techniques to analyze these intractable pigments, and gradual progress was made in defining those structures, initially by Nicolaus’s group in Naples and Swan’s group in the United Kingdom (Swan, 1963; Nicolaus et al., 1964). In working with melanins found in nature, it quickly became apparent that there were two major types, the brown–black melanins now collectively known as eumelanins, and the yellow–red melanins now collectively known as pheomelanins. Prota’s group in Naples took the lead in defining the structure of pheomelanin and the involvement of sulfur as responsible for its unique color and properties (Prota, 1980). Prota and colleagues, as well as Ito and Wakamatsu in Japan (Ito et al., 1984), developed a series of more sensitive and specific assays for eumelanin and pheomelanin intermediates that gradually formed the basis for our understanding of how they are formed in melanocytes and how they are copolymerized in situ. The critical role of sulfhydryl groups in reacting immediately with DQ upon its formation to form various combinations of cysteinylDOPAs and downstream reactions of those intermediates, via cysteinylDOPA-quinones and benzothiazine intermediates, to produce

Takeshi Namiki 1,2 , Sergio G. Coelho 1 , Vincent J. Hearing 1 1 Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20814, USA 2 Department of Dermatology, Yokohama Minato Red Cross Hospital, Yokohama, Kanagawa 231-0801, Japan Received: September 9, 2011; Accepted: September 10, 2011; Published: September 10, 2011; Keywords: NUAK2, acral melanoma, migration, metastasis, oncogene Correspondence: Dr. Takeshi Namiki, email: // // Abstract Recent technological advances in cancer genomics make it possible to dissect complicated genomic aberrations of melanomas. In particular, several specific genomic aberrations including 11q13 amplification and KIT aberrations have been identified in acral melanomas. We recently identified NUAK2 at 1q32 as a promising oncogene in acral melanomas and reported its significant roles in tumorigenesis in melanoma cells using both in vitro and in vivo analyses. NUAK2 as a member of the AMPK family has several intriguing aspects both as an oncogene and as a tumor suppressor gene. Here we review genomic aberrations of melanomas focusing on acral melanomas to emphasize the possible roles of NUAK2 in tumorigenesis in general and suggest that NUAK2 has pivotal roles in acral melanomagenesis.

Melanogenesis is the unique process of producing pigmented biopolymers that are sequestered within melanosomes, which provides color to the skin, hair and eyes of animals and, in the case of human skin, also protects the underlying tissues from UV damage. We review the current understanding of melanogenesis, focusing on factors important to the biochemistry of pigment synthesis, the biogenesis of melanosomes, signaling pathways and factors that regulate melanogenesis, intramelanosomal pH, transport and transfer of melanosomes, and pigmentary disorders related to the dysfunction of melanosome-related proteins. Although it has been known for some time that many of the factors that affect melanogenesis are derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells and nerves, a number of new factors that are involved in that regulation have recently been reported, such as factors that regulate melanosome pH and ion transport.

The relationship between human skin pigmentation and protection from ultraviolet (UV) radiation is an important element underlying differences in skin carcinogenesis rates. The association between UV damage and the risk of skin cancer is clear, yet a strategic balance in exposure to UV needs to be met. Dark skin is protected from UV-induced DNA damage significantly more than light skin owing to the constitutively higher pigmentation, but an as yet unresolved and important question is what photoprotective benefit, if any, is afforded by facultative pigmentation (i.e. a tan induced by UV exposure). To address that and to compare the effects of various wavelengths of UV, we repetitively exposed human skin to suberythemal doses of UVA and/or UVB over 2 weeks after which a challenge dose of UVA and UVB was given. Although visual skin pigmentation (tanning) elicited by different UV exposure protocols was similar, the melanin content and UV-protective effects against DNA damage in UVB-tanned skin (but not in UVA-tanned skin) were significantly higher. UVA-induced tans seem to result from the photooxidation of existing melanin and its precursors with some redistribution of pigment granules, while UVB stimulates melanocytes to up-regulate melanin synthesis and increases pigmentation coverage, effects that are synergistically stimulated in UVA and UVB-exposed skin. Thus, UVA tanning contributes essentially no photoprotection, although all types of UV-induced tanning result in DNA and cellular damage, which can eventually lead to photocarcinogenesis.

Interactions between melanocytes and neighboring cells in the skin are important in regulating skin color in humans. We recently demonstrated that the less pigmented and thicker skin on the palms and soles is regulated by underlying fibroblasts in those areas, specifically via a secreted factor (DKK1) that modulates Wnt signaling. In this study, we tested the hypothesis that dermal fibroblasts regulate the constitutive skin color of individuals ranging from very light to very dark. We used microarray analysis to compare gene expression patterns in fibroblasts derived from lighter skin types compared to darker skin types, with a focus on secreted proteins. We identified a number of genes that differ dramatically in expression and, among the expressed proteins, neuregulin-1, which is secreted by fibroblasts derived from dark skin, effectively increases the pigmentation of melanocytes in tissue culture and in an artificial skin model and regulates their growth, suggesting that it is one of the major factors determining human skin color.

Melanosomes are organelles specialized for the production of melanin pigment and are specifically produced by melanocytic cells. More than 150 pigmentation-related genes have been identified, including glycoprotein nonmetastatic melanoma protein b (GPNMB). A recent proteomics analysis revealed that GPNMB is localized in melanosomes, and GPNMB is a membrane-bound glycoprotein that shows high homology with a well-known melanosomal structural protein, Pmel17/gp100. In this study, we show that GPNMB is expressed in melanocytes of normal human skin, as well as in human melanoma cells. GPNMB is heavily glycosylated and is enriched in mature (stage III and IV) melanosomes in contrast to MART-1 and Pmel17, which are abundant in early (stage I and II) melanosomes. MART-1 and Pmel17 play critical roles in the maturation of early melanosomes; thus, we speculate that GPNMB might be important in the functions of late melanosomes, possibly their transport and/or transfer to keratinocytes. We also demonstrate that a secreted form of GPNMB is released by ectodomain shedding from the largely Golgi-modified form of GPNMB and that the PKC and Ca2+ intracellular signaling pathways regulate that shedding. We conclude that GPNMB is a melanosomal protein that is released by proteolytic ectodomain shedding and might be a useful and specific histological marker of melanocytic cells.—Hoashi, T., Sato, S., Yamaguchi, Y., Passeron, T., Tamaki, K., Hearing, V. J. Glycoprotein nonmetastatic melanoma protein b, a melanocytic cell marker, is a melanosome-specific and proteolytically released protein.

Melanomas are the most prevalent cancers in 25-29 yr old females and compose roughly 12% of cancers in 20-40 yr old women; under the age of 40, women have a higher incidence of melanomas than do men. Within the past few decades, the alarming trend to use commercial sunlamps for cosmetic pigmentation is of particular concern, especially since 71% of those patrons are women with 50% of patrons under the age of 29. A major problem may be the use of UVA-rich sunlamps which produce a visible tan but afford little to no protection from subsequent UV exposure. We hypothesize that the additional exposure of adolescents to unnaturally large amounts of UVA from artificial UV sources is implicated in the increasing incidence of malignant melanomas disproportionately in young women.

Because melanomas are intrinsically resistant to conventional radiotherapy and chemotherapy, many alternative treatment approaches have been developed such as biochemotherapy and immunotherapy. The most common cause of multidrug resistance (MDR) in human cancers is the expression and function of one or more ATP-binding cassette (ABC) transporters that efflux anticancer drugs from cells. Melanoma cells express a group of ABC transporters (such as ABCA9, ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, and ABCD1) that may be associated with the resistance of melanoma cells to a broad range of anticancer drugs and/or of melanocytes to toxic melanin intermediates and metabolites. In this review, we propose a model (termed the ABC-M model) in which the intrinsic MDR of melanoma cells is at least in part because of the transporter systems that may also play a critical role in reducing the cytotoxicity of the melanogenic pathway in melanocytes. The ABC-M model suggests molecular strategies to reverse MDR function in the context of the melanogenic pathway, which could open therapeutic avenues towards the ultimate goal of circumventing clinical MDR in patients with melanoma.

Ectodomain shedding is a proteolytic mechanism by which a transmembrane protein is converted into a secreted form. Pmel17/gp100 is a melanocyte-specific membrane-bound glycoprotein that has amyloid characteristics and forms fibrillar structures in melanosomes after a complex sequence of post-translational processing and trafficking events, including cleavage by a furin-like proprotein convertase (PC). A secreted form of Pmel17 (termed sPmel17) was also thought to be released due to cleavage by a PC. We used multidisciplinary approaches to demonstrate that sPmel17 is released by ectodomain shedding at the juxtamembrane and/or intramembrane motif and to show that this is independent of cleavage by a PC. We further show that sPmel17 consists of 2 fragments linked by disulfide bonds and that the shedding is inhibited at low temperature but not by metalloproteinase inhibitors. Moreover, treatment with a phorbol ester or a calmodulin inhibitor induces Pmel17 shedding. We also refine the reactivity of HMB50 and NKI/beteb, 2 monoclonal antibodies commonly used as melanoma-specific markers. The fact that those antibodies require physically separated domains of Pmel17 sheds interesting light on its 3-dimensional conformation. We conclude that sPmel17 is released by regulated proteolytic ectodomain shedding.—Hoashi, T., Tamaki, K., Hearing, V. J. The secreted form of a melanocyte membrane-bound glycoprotein (Pmel17/gp100) is released by ectodomain shedding.

Pigmentation of the skin, hair and eyes is regulated by tyrosinase, the critical rate-limiting enzyme in melanin synthesis by melanocytes. Tyrosinase is degraded endogenously, at least in part, by the ubiquitin proteasome system (UPS). Several types of inherited hypopigmentary diseases, such as oculocutaneous albinism and Hermansky-Pudlak syndrome, involve the aberrant processing and/or trafficking of tyrosinase and its subsequent degradation which can occur due to the quality-control machinery. Studies on carbohydrate modifications have revealed that tyrosinase in the endoplasmic reticulum (ER) is proteolyzed via ER-associated protein degradation and that tyrosinase degradation can also occur following its complete maturation in the Golgi. Among intrinsic factors that regulate the UPS, fatty acids have been shown to modulate tyrosinase degradation in contrasting manners through increased or decreased amounts of ubiquitinated tyrosinase that leads to its accelerated or decelerated degradation by proteasomes.

Background Malignant melanomas are intrinsically resistant to many conventional treatments, such as radiation and chemotherapy, for reasons that are poorly understood. Here we propose and test a model that explains drug resistance or sensitivity in terms of melanosome dynamics. Methods The growth and sensitivity to cisplatin of MNT-1 cells, which are melanotic and enriched with mature stage III and IV melanosomes, and SK-MEL-28 cells, which have only immature stage I and II melanosomes, were compared using clonogenic assays. Differences in pigmentation, melanosome stages, melanosome number, and cellular structures in different cell lines in response to various treatments were examined by electron microscopy. The relative numbers of melanosomes of different stages were compared after treatment with 1-phenyl-2-thiourea. The relationship between drug transporter function and endogenous melanogenic toxicity was assessed by treating cells with the cyclosporin analog PSC-833 and by assessing vacuole formation and cell growth inhibition. All statistical tests were two-sided. Results Endogenous melanogenic cytotoxicity, produced by damaged melanosomes, resulted in pronounced cell growth inhibition in MNT-1 cells compared with amelanotic SK-MEL-28 cells. The sensitivity to CDDP of MNT-1 cells was 3.8-fold higher than that of SK-MEL-28 cells (mean IC(50) for SK-MEL-28 and MNT-1 = 2.13 microM and 0.56 microM, respectively; difference = 1.57 microM, 95% confidence interval = 1.45 to 1.69; P = .0017). After treatment with 6.7 microM CDDP for 72 hours, the number of stage II-III melanosomes in surviving MNT-1 cells was 6.8-fold that of untreated cells. Modulation of MNT-1 cells to earlier-stage (II, II-III, III) melanosomes by treatment with the tyrosinase inhibitor 1-phenyl-2-thiourea dramatically increased CDDP resistance. Furthermore, PSC-833 principally suppressed MNT-1 melanotic cell growth via an elevation of autophagosome-like vacuolar structures, possibly by inhibiting melanosome membrane transporters. Conclusions Melanosome dynamics (including their biogenesis, density, status, and structural integrity) regulate the drug resistance of melanoma cells. Manipulation of melanosome functions may be an effective way to enhance the therapeutic activity of anticancer drugs against melanoma.

The melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor (GPCR) expressed in melanocytes, is a major determinant of skin pigmentation and phototype. MC1R activation stimulates melanogenesis and increases the ratio of black, strongly photoprotective eumelanins to reddish, poorly photoprotective pheomelanins. Several MC1R alleles are associated with red hair, fair skin, increased sensitivity to ultraviolet radiation (the RHC phenotype) and increased skin cancer risk. Three highly penetrant RHC variants, R151C, R160W, and D294H are loss-of-function MC1R mutants with altered cell surface expression. In this study, we show that forward trafficking was normal for D294H. Conversely, export traffic was impaired for R151C, which accumulated in the endoplasmic reticulum (ER), and for R160W, which was enriched in the cis-Golgi. This is the first report of steady-state retention in a post-ER secretory compartment of a GPCR mutant found in the human population. Residues R151 and R160 are located in the MC1R second intracellular loop (il2). Two other mutations in il2, T157A preventing T157 phosphorylation and R162P disrupting a 160RARR163 motif, also caused intracellular retention. Moreover, T157 was phosphorylated in wild-type MC1R and a T157D mutation mimicking constitutive phosphorylation allowed normal traffic, and rescued the retention phenotype of R160W and R162P. Therefore, MC1R export is likely regulated by T157 phosphorylation and the 160RARR163 arginine-based motif functions as an ER retrieval signal. These elements are conserved in mammalian MC1Rs and in all five types of human melanocortin receptors. Thus, members of this GPCR subfamily might share common mechanisms for regulation of plasma membrane expression. J. Cell. Physiol. 220: 640–654, 2009. © 2009 Wiley-Liss, Inc.

The incidence of skin cancer, including cutaneous melanoma, has risen substantially in recent years, and epidemiological and laboratory studies demonstrate that ultraviolet (UV) radiation is a major causative factor for this increase. UV damage also underlies photoaging of the skin, and those deleterious effects of UV can be, in part, prevented in skin with higher levels of constitutive pigmentation. We review the findings of a series of clinical studies we have made in recent years regarding the rapid and the long-term responses of the pigmentary system in human skin to UV exposure. Key words: ultraviolet, skin, photoprotection, pigmentation, photoaging

The increased popularity of commercially available three-dimensional human skin equivalents in recent years has allowed for assessment of melanogenesis modulated by compounds topically applied to the skin or directly incorporated from the medium. These skin equivalents provide a suitable model for elucidating the mechanisms of action of various factors that modulate skin pigmentation or other properties of the skin. As such, researchers need to objectively quantify cutaneous responses at the macroscopic level. A simple method to standardize macrophotography images is reported that can quantify cutaneous responses in human skin equivalents of Asian, Black or African American, and Caucasian or White racial/ethnic origin. Macrophotographs are analyzed using the Commission Internationale de l'Eclairage L * a * b * color space system in combination with a personal computer and image editing software. Pigmentation changes monitored over a 9 day period showed a high correlation with melanin content evaluated in Fontana―Masson-stained sections. These results indicate the feasibility of using a macrophotography setup in a sterile tissue culture environment to objectively assess in vitro cutaneous responses in human skin equivalents. This serves as an adjunct tool to biochemical and morphological methods to effectively quantify changes in pigmentation over time.

Background α-Hydroxy acids (αHAs) are reported to reduce signs of aging in the skin and are widely used cosmetic ingredients. Several studies suggest that αHA can increase the sensitivity of skin to ultraviolet radiation. More recently, β-hydroxy acids (βHAs), or combinations of αHA and βHA have also been incorporated into antiaging skin care products. Concerns have also arisen about increased sensitivity to ultraviolet radiation following use of skin care products containing β-HA. Objective To determine whether topical treatment with glycolic acid, a representative αHA, or with salicylic acid, a βHA, modifies the short-term effects of solar simulated radiation (SSR) in human skin. Methods Fourteen subjects participated in this study. Three of the four test sites on the mid-back of each subject were treated daily Monday–Friday, for a total of 3.5 weeks, with glycolic acid (10%), salicylic acid (2%), or vehicle (control). The fourth site received no treatment. After the last treatment, each site was exposed to SSR, and shave biopsies from all four sites were obtained. The endpoints evaluated in this study were erythema (assessed visually and instrumentally), DNA damage and sunburn cell formation. Results Treatment with glycolic acid resulted in increased sensitivity of human skin to SSR, measured as an increase in erythema, DNA damage and sunburn cell formation. Salicylic acid did not produce significant changes in any of these biomarkers. Conclusions Short-term topical application of glycolic acid in a cosmetic formulation increased the sensitivity of human skin to SSR, while a comparable treatment with salicylic acid did not.

It is known that UV modulates the expression of paracrine factors that regulate melanocyte function in the skin. We investigated the consequences of repetitive UV exposure of human skin in biopsies of 10 subjects with phototypes 2-3.5 taken 1-4 years later. The expression of melanogenic factors (TYR, MART1, MITF), growth factors/receptors (SCF/KIT, bFGF/FGFR1, ET1/EDNRB, HGF, GM-CSF), adhesion molecules (beta-catenin, E-cadherin, N-cadherin), cell cycle proteins (PCNA, cyclins D1, E2) as well as Bcl-2, DKK1, and DKK3, were analyzed by immunohistochemistry. Most of those markers showed no detectable changes ator = 1 year after the repetitive UV irradiation. Although increased expression of EDNRB protein was detected in 3 of 10 UV-irradiated subjects, there was no detectable change in the expression of ET1 protein or in EDNRB mRNA levels. In summary, only the expression of TYR, MART1, and/or EDNRB, and only in some subjects, was elevated ator = 1 year after UV irradiation. Thus the long-term effects of repetitive UV irradiation on human skin did not lead to significant changes in skin morphology and there is considerable subject-to-subject variation in responses. The possibility that changes in the expression and function of EDNRB triggers downstream activation of abnormal melanocyte proliferation and differentiation deserves further investigation.

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes, and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells, and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A, and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2, and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va, and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including alpha-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins, and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Melanin is a common pigment in animals. In humans, melanin is produced in melanocytes, in retinal pigment epithelium (RPE) cells, in the inner ear, and in the central nervous system. Previously, we noted that human adipose tissue expresses several melanogenesis-related genes. In the current study, we confirmed the expression of melanogenesis-related mRNAs and proteins in human adipose tissue using real-time polymerase chain reaction and immunohistochemical staining. TYR mRNA signals were also detected by in situ hybridization in visceral adipocytes. The presence of melanin in human adipose tissue was revealed both by Fontana-Masson staining and by permanganate degradation of melanin coupled with liquid chromatography/ultraviolet/mass spectrometry determination of the pyrrole-2,3,5-tricarboxylic acid (PTCA) derivative of melanin. We also compared melanogenic activities in adipose tissues and in other human tissues using the L-[U-14C] tyrosine assay. A marked heterogeneity in the melanogenic activities of individual adipose tissue extracts was noted. We hypothesize that the ectopic synthesis of melanin in obese adipose may serve as a compensatory mechanism that uses its anti-inflammatory and its oxidative damage-absorbing properties. In conclusion, our study demonstrates for the first time that the melanin biosynthesis pathway is functional in adipose tissue.—Randhawa, M., Huff, T., Valencia, J. C., Younossi, Z., Chandhoke, V., Hearing, V. J., Baranova, A. Evidence for the ectopic synthesis of melanin in human adipose tissue.

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, alpha-melanocyte stimulating hormone (alphaMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and alphaMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). alphaMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu- and pheo-melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized, but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, tyrosinase (TYR) and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical, and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system, which is involved with the sorting of cargo from the plasma membrane. These results provide important clues toward understanding the processes involved with early events in melanosome formation and transport.

Ultraviolet (UV)-induced skin cancers, including melanomas and basal/squamous cell carcinomas, occur more frequently in individuals with fair skin than in those with dark skin. Melanin plays an important role in protecting the skin against UV radiation and levels of melanin correlate inversely with amounts of DNA damage induced by UV in human skin of different racial/ethnic groups. The objectives of this study are to review recent progress in our understanding of mechanisms underlying differences in cancer incidence in skins of different colors, particularly between Black and White skin. More specifically, we review DNA damage and apoptosis in various types of skin before and after exposure to UV in our human study protocols using a single UV dose, either one minimal erythema dose (MED) or a similar low dose of 180-200 J/m2. Our data and other published reports indicate that several major mechanisms underlie the increased rates of photocarcinogenesis in fair/light skin. First, UV-induced DNA damage in the lower epidermis (including keratinocyte stem cells and melanocytes) is more effectively prevented in darker skin. Second, rates of repair of DNA damage can differ significantly in individuals. Third, UV-induced apoptosis to remove potentially precancerous cells is significantly greater in darker skin. These results suggest that pigmented epidermis is an efficient UV filter and that UV damaged cells are removed more efficiently in darker skin. The combination of decreased DNA damage and more efficient removal of UV-damaged cells may play a critical role in the decreased photocarcinogenesis seen in individuals with darker skin.

The epidermis (containing primarily keratinocytes and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, microphthalmia-associated transcription factor (MITF) and beta-catenin. We now report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase-polymerase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and alpha-Kelch-like ECT2 interacting protein (alphaKLEIP) but down-regulated the expression of beta-catenin, glycogen synthase kinase 3beta, protein kinase C, and proteinase-activated receptor-2 (PAR-2), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 reproduced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site-specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis.

SOX ( SRY type HMG box) proteins are transcription factors that are predominantly known for their roles during development. During melanocyte development from the neural crest, SOX10 regulates microphthalmia-associated transcription factor, which controls a set of genes critical for pigment cell development and pigmentation, including dopachrome tautomerase and tyrosinase. We report here that another SOX factor, SOX9, is expressed by melanocytes in neonatal and adult human skin and is up-regulated by UVB exposure. We demonstrate that this regulation is mediated by cAMP and protein kinase. We also show that agouti signal protein, a secreted factor known to decrease pigmentation, down-regulates SOX9 expression. In adult and neonatal melanocytes, SOX9 regulates microphthalmia-associated transcription factor, dopachrome tautomerase, and tyrosinase promoters, leading to an increase in the expression of these key melanogenic proteins and finally to a stimulation of pigmentation. SOX9 completes the complex and tightly regulated process leading to the production of melanin by acting at a very upstream level. This role of SOX9 in pigmentation emphasizes the poorly understood impact of SOX proteins in adult tissues.

Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players as they produce and distribute melanin that protects the skin from ultraviolet (UV) radiation. In vitro experiments on melanocytic cell lines are useful to study melanogenesis and their progression towards melanoma. However, interactions of melanocytes with keratinocytes and with other types of cells in the skin, such as fibroblasts and Langerhans cells, are also crucial. We describe two techniques, immunohistochemistry (IHC) and tissue in situ hybridization (TISH), that can be used to identify and study melanocytes in the skin and their responses to UV or other stimuli in situ. We describe a practical method to localize melanocytic antigens on formalin-fixed, paraffin-embedded tissue sections and in frozen sections using indirect immunofluorescence with conjugated secondary antibodies. In addition, we detail the use of TISH and its combination with IHC to study mRNA levels of genes expressed in the skin at cellular resolution. This methodology, along with relevant tips and troubleshooting items, are important tools to identify and study melanocytes in the skin.

Pigmentation of human skin is closely involved in protection against environmental stresses, in particular exposure to ultraviolet (UV) radiation. It is well known that darker skin is significantly more resistant to the damaging effects of UV, such as photocarcinogenesis and photoaging, than is lighter skin. Constitutive skin pigmentation depends on the amount of melanin and its distribution in that tissue. Melanin is significantly photoprotective and epidermal cells in darker skin incur less DNA damage than do those in lighter skin. This review summarizes current understanding of the regulation of constitutive human skin pigmentation and responses to UV radiation, with emphasis on physiological factors that influence those processes. Further research is needed to characterize the role of skin pigmentation to reduce photocarcinogenesis and to develop effective strategies to minimize such risks.

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis -diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an ≈8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells.

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms μ1A and μ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of μ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (μ1A or μ1B) showed two distinct distribution patterns that involved Pmel17, and only μ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking μ1B expression. Finally, we established that expression of μ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.

Dopachrome tautomerase (Dct) is a type I membrane protein and an important regulatory enzyme that plays a pivotal role in the biosynthesis of melanin and in the rapid metabolism of its toxic intermediates. Dct-mutant melanocytes carrying the slaty or slaty light mutations were derived from the skin of newborn congenic C57BL/6J non-agouti black mice and were used to study the effect(s) of these mutations on the intracellular trafficking of Dct and on the pigmentation of the cells. Dct activity is 3-fold lower in slaty cells compared with non-agouti black melanocytes, whereas slaty light melanocytes have a surprisingly 28-fold lower Dct activity. Homology modelling of the active site of Dct suggests that the slaty mutation [R194Q (Arg194→Gln)] is located in the active site and may alter the ability of the enzyme to transform the substrate. Transmembrane prediction methods indicate that the slaty light mutation [G486R (Gly486→Arg)] may result in the sliding of the transmembrane domain towards the N-terminus, thus interfering with Dct function. Chemical analysis showed that both Dct mutations increase pheomelanin and reduce eumelanin produced by melanocytes in culture. Thus the enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis.

Among more than 120 genes that are now known to regulate mammalian pigmentation, one of the key genes is MC1R, which encodes the melanocortin 1 receptor, a seven transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Since the monoexonic sequence of the gene was cloned and characterized more than a decade ago, tremendous efforts have been dedicated to the extensive genotyping of mostly red-haired populations all around the world, thus providing allelic variants that may or may not account for melanoma susceptibility in the presence or absence of ultraviolet (UV) exposure. Soluble factors, such as proopiomelanocortin (POMC) derivatives, agouti signal protein (ASP) and others, regulate MC1R expression, leading to improved photoprotection via increased eumelanin synthesis or in contrast, inducing the switch to pheomelanin. However, there is an obvious lack of knowledge regarding the numerous and complex regulatory mechanisms that govern the expression of MC1R at the intra-cellular level, from gene transcription in response to an external stimulus to the expression of the mature receptor on the melanocyte surface.

Many approaches have been tried to establish assays for melanosome transfer to keratinocytes. In this report, we describe and summarize various novel attempts to label melanosomes in search of a reliable, specific, reproducible and quantitative assay system. We tried to fluorescently label melanosomes by transfection of GFP-labeled melanosomal proteins and by incubation of melanocytes with fluorescent melanin intermediates or homologues. In most cases a weak cytoplasmic fluorescence was perceived, which was probably because of incorrect sorting or deficient incorporation of the fluorescent protein and different localization. We were able to label melanosomes via incorporation of 14C-thiouracil into melanin. Consequently, we tried to develop an assay to separate keratinocytes with transferred radioactivity from melanocytes after co-culture. Differential trypsinization and different magnetic bead separation techniques were tested with unsatisfactory results. An attempt was also made to incorporate fluorescent thiouracil, since this would allow cells to be separated by FACS. In conclusion, different methods to measure pigment transfer between donor melanocytes and acceptor keratinocytes were thoroughly examined. This information could give other researchers a head start in the search for a melanosome transfer assay with said qualities to better understand pigment transfer.

ATP-binding cassette (ABC) transporters play a pivotal role in physiology and pathology. We identified and cloned two novel mRNA isoforms (ABCB 5alpha and ABCB 5beta) of the ABC transporter ABCB 5 in human melanoma cells. The deduced ABCB 5alpha protein appears to be an altered splice variant containing only a putative ABC, whereas the ABCB 5beta isoform shares approximately 70% similarity with ABCB1 (MDR1) and has a deduced topological arrangement similar to that of the whole carboxyl terminal half of the ABCB1 gene product, P-glycoprotein, including an intact ABC. Northern blot, real-time PCR, and conventional RT-PCR were used to verify the expression profiles of ABCB 5alpha/beta. We found that the melanomas included among the NCI-60 panel of cell lines preferentially expressed both ABCB 5alpha and ABCB 5beta. However, ABCB 5alpha/beta expression was undetectable in two amelanotic melanomas (M14 and LOX-IMVI). The expression profile of ABCB 5alpha/beta in all of the other melanomas of the panel was confirmed both by RT-PCR and by sequencing. Neither ABCB 5alpha nor ABCB 5beta expression was found in normal tissues such as liver, spleen, thymus, kidney, lung, colon, small intestines or placenta. ABCB 5alpha/beta mRNAs were also expressed in normal melanocytes and in retinal pigment epithelial cells, suggesting that ABCB 5alpha/beta expression is pigment cell-specific and might be involved in melanogenesis. Our findings indicate that expression of ABCB 5alpha/beta might possibly provide two novel molecular markers for differential diagnosis of melanomas and constitute potential molecular targets for therapy of melanomas.

The constitutive color of our skin plays a dramatic role in our photoprotection from solar ultraviolet radiation (UVR) that reaches the Earth and in minimizing DNA damage that gives rise to skin cancer. More than 120 genes have been identified and shown to regulate pigmentation, one of the key genes being melanocortin 1 receptor (MC1R) that encodes the melanocortin 1 receptor (MC1R), a seven-transmembrane G protein-coupled receptor expressed on the surface of melanocytes. Modulation of MC1R function regulates melanin synthesis by melanocytes qualitatively and quantitatively. The MC1R is regulated by the physiological agonists alpha-melanocyte-stimulating hormone (alphaMSH) and adrenocorticotropic hormone (ACTH), and antagonist agouti signaling protein (ASP). Activation of the MC1R by binding of an agonist stimulates the synthesis of eumelanin primarily via activation of adenylate cyclase. The significance of cutaneous pigmentation lies in the photoprotective effect of melanin, particularly eumelanin, against sun-induced carcinogenesis. Epidermal melanocytes and keratinocytes respond to UVR by increasing their expression of alphaMSH and ACTH, which up-regulate the expression of MC1R, and consequently enhance the response of melanocytes to melanocortins. Constitutive skin pigmentation dramatically affects the incidence of skin cancer. The pigmentary phenotype characterized by red hair, fair complexion, inability to tan and tendency to freckle is an independent risk factor for all skin cancers, including melanoma. The MC1R gene is highly polymorphic in human populations, and allelic variation at this locus accounts, to a large extent, for the variation in pigmentary phenotypes and skin phototypes (SPT) in humans. Several allelic variants of the MC1R gene are associated with the red hair and fair skin (RHC) phenotype, and carrying one of these variants is thought to diminish the ability of the epidermis to respond to DNA damage elicited by UVR. The MC1R gene is considered a melanoma susceptibility gene, and its significance in determining the risk for skin cancer is of tremendous interest.

Summary Background Diabetic foot ulcers with exposed bones commonly result in amputation. Objectives To determine whether exposure of bone marrow cells and subsequent grafting of epidermal sheets accelerates healing and reduces the need for amputation. Methods Thirty-eight patients with chronic wounds caused by diabetes mellitus were enrolled in this study. Epidermal sheets obtained from suction blisters of each patient were grafted on to diabetic foot ulcers without exposed bones (n = 10) and were compared with the standard treatment of local wound care, debridement with a scalpel when indicated, bed rest and parenteral antibiotics (n = 8). In another group of patients, diabetic wounds with exposed bones were treated either with the standard procedure (n = 9) or with a newly developed experimental procedure (n = 11). In that new procedure, the affected bone was initially exposed by debridement with a scalpel, followed by partial excision with a bone scraper until fresh bleeding was observed from the exposed bone. The lesions were then immediately covered with an occlusive dressing, and finally the wound was covered with an epidermal graft of skin harvested from suction blisters. Patients in each group were matched with their counterparts by age, sex, wound size, wound infection and wound duration, to compare the time needed for total skin repair and rates of amputation. Results Epidermal grafting significantly accelerated the healing of diabetic foot ulcers (P = 0·042) without exposed bones, with site-specific differentiation. The newly developed combination therapy resulted in the healing of all diabetic ulcers with exposed bones without the occurrence of osteomyelitis or the necessity for amputation (P

Melanin protects the skin against DNA damage induced by direct absorption of sunlight's UV radiation. Yet, irradiating melanin in vitro or in cultured cells also generates active oxygen species such as superoxide, which can indirectly induce oxidative base lesions and DNA strand breaks. This photosensitization is greater for pheomelanin (yellow and red melanin) than for eumelanin (brown and black). The in vivo photosensitizing ability of melanin is unknown. We used congenic mice of black, yellow, and albino coat colors to investigate the induction of DNA lesions and apoptosis after exposure to predominantly UVB (280–320 nm) or UVA (320–400 nm) radiation. Cyclobutane pyrimidine dimers induced by direct UVB absorption were equal in all three strains, as was apoptosis measured as sunburn cells or as keratinocytes containing active caspase-3. However, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL)-positive cells were ≈3-fold more frequent in black and yellow mice after UVB or UVA irradiation than in albino. In epidermal sheets, TUNEL-positive cells lined the upper portion of the hair follicle, consistent with UV-induced photosensitization by melanin in the hair shaft. Because the concentration of eumelanin in black mice was three times that of pheomelanin in yellow mice, pheomelanin had 3-fold greater specific activity. We conclude that UV-irradiated melanin, particularly pheomelanin, photosensitizes adjacent cells to caspase-3 independent apoptosis, and this occurs at a frequency greater than the apoptosis induced by direct DNA absorption of UV. Melanin-induced apoptosis may contribute to the increased sensitivity of individuals with blonde and red hair to sunburn and skin cancer.