Deciphering skin re-pigmentation patterns in vitiligo: an update on the cellular and molecular events involved.

Current treatment of vitiligo is still a great challenge, since most cases of vitiligo have variable re-pigmentation outcomes due to their unpredictable responses to existing therapeutic regimens. There is an urgent need to identify this re-pigmentation process and to develop novel therapies. This review illustrates the most current research and latest understanding of vitiligo skin re-pigmentation and related regulatory mechanisms. Literature was collected from PubMed until January 2020, using the search terms including "vitiligo," "re-pigmentation," "phototherapy," "narrow-band ultraviolet B, " "excimer," "fractional carbon dioxide laser," and "melanocyte stem cells." Literature was mainly derived from English articles. Article type was not limited. Emerging evidence suggests that patients with vitiligo present various re-pigmentation patterns following ultraviolet B phototherapy, which relies on different cell reservoirs from the perilesional margins and/or from uninvolved hair follicles to replenish functional melanocytes that are lost in vitiliginous skin. The following events are likely to be involved in this re-pigmentation process, including: 1) changes in the paracrine secretion and distribution of transforming growth factor-ß1 in the bulge area and in the epidermis; 2) the enhanced transfer of dermal pro-melanogenic growth factors to the epidermis; and 3) the induction of a C-X-C motif chemokine ligand (CXCL) 12-enriched micro-environment that efficiently recruits CXCR4- or CXCR7-positive melanocytes. Ongoing studies on the cellular and molecular events underlying vitiligo re-pigmentation will help design new therapeutic strategies to improve treatment outcomes.

Inhibition of Human Tyrosinase Requires Molecular Motifs Distinctively Different from Mushroom Tyrosinase.

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.

The Evaluation of Noninvasive Measurements of Erythema as a Potential Surrogate for DNA Damage in Repetitively UV-exposed Human Skin.

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.

Molecular and histological characterization of age spots.

Age spots, also called solar lentigines and lentigo senilis, are light brown to black pigmented lesions of various sizes that typically develop in chronically sun-exposed skin. It is well known that age spots are strongly related to chronic sun exposure and are associated with photodamage and an increased risk for skin cancer; however, the mechanisms underlying their development remain poorly understood. We used immunohistochemical analysis and microarray analysis to investigate the processes involved in their formation, focusing on specific markers associated with the functions and proliferation of melanocytes and keratinocytes. A total of 193 genes were differentially expressed in age spots, but melanocyte pigment genes were not among them. The increased expression of keratins 5 and 10, markers of basal and suprabasal keratinocytes, respectively, in age spots suggests that the increased proliferation of basal keratinocytes combined with the decreased turnover of suprabasal keratinocytes leads to the exaggerated formation of rete ridges in lesional epidermis which in turn disrupts the normal processing of melanin upwards from the basal layer. Based on our results, we propose a model for the development of age spots that explains the accumulation of melanin and the development of extensive rete ridges in those hyperpigmented lesions.

Identification of Genes Expressed in Hyperpigmented Skin Using Meta-Analysis of Microarray Data Sets.

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.

NUAK2 Amplification Coupled with PTEN Deficiency Promotes Melanoma Development via CDK Activation.

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.

Photobiological implications of melanin photoprotection after UVB-induced tanning of human skin but not UVA-induced tanning.

Repetitive suberythemal UVA and/or UVB exposures were used to generate comparable UV-induced tans in human skin over the course of 2 weeks. 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 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.

Antibody αPEP13h reacts with lymphangioleiomyomatosis cells in lung nodules.

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.

Oculocutaneous albinism: developing novel antibodies targeting the proteins associated with OCA2 and OCA4.

BACKGROUND: Patients with oculocutaneous albinism (OCA) have severely decreased pigmentation of their skin, hair and eyes. OCA2 and OCA4 result from mutations of the OCA2 and SLC45A2 genes, respectively, both of which disrupt the trafficking of the critical melanogenic enzyme tyrosinase to melanosomes. Both proteins encoded by those loci (termed P and MATP, respectively) have 12 putative transmembrane regions and are thought to function as transporters, although their functions and subcellular localizations remain to be characterized. OBJECTIVE: To generate specific antibodies against unique synthetic peptides encoded by P and MATP that could be used to characterize their functions and subcellular localizations. METHODS: Western blotting and immunohistochemistry were used to assess the specificity of antibodies and to colocalize P and MATP proteins with various subcellular markers. RESULTS: Specific antibodies to the P and MATP proteins were generated that work well for Western blotting and immunohistochemistry. The localizations of P and MATP with various subcellular organelles were characterized using confocal microscopy, which revealed that they colocalize to some extent with LAMP2, but do not significantly colocalize with markers of the ER, Golgi or melanosomes. Interestingly, both P and MATP colocalize significantly with BLOC-1, a sorting component involved in the intracellular trafficking of melanosomal/lysosomal constituents. CONCLUSION: These results provide a basis to understand how disrupted functions of P or MATP result in the misrouting of tyrosinase and cause the hypopigmentation seen in OCA2 and OCA4.

UV exposure modulates hemidesmosome plasticity, contributing to long-term pigmentation in human skin.

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 α6ß4 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.

Epidermal gene expression and ethnic pigmentation variations among individuals of Asian, European and African ancestry.

Differences in visible skin pigmentation give rise to the wide variation of skin colours seen in racial/ethnic populations. Skin pigmentation is important not only from cosmetic and psychological points of view, but more importantly because of its implications for the risk of all types of skin cancers, on photoaging, etc. Despite differences in those parameters in Caucasian and Asian skin types, they are remarkably similar in their production and distribution of melanins, and the mechanism(s) underlying their different characteristics have remained obscure. In this study, we used microarray analysis of skin suction blisters to investigate molecular differences underlying the determination of pigmentation in various skin types, and we used immunohistochemistry to validate the expression patterns of several interesting targets that were identified. Intriguingly, Caucasian and Asian skins had highly similar gene expression patterns that differed significantly from the pattern of African skin. The results of this study suggest the dynamic interactions of different types of cells in human skin that regulate its pigmentation, reveal that the known pigmentation genes have a limited contribution and uncover a new array of genes, including NINL and S100A4, that might be involved in that regulation.

Melanocytes and their diseases.

Human melanocytes are distributed not only in the epidermis and in hair follicles but also in mucosa, cochlea (ear), iris (eye), and mesencephalon (brain) among other tissues. Melanocytes, which are derived from the neural crest, are unique in that they produce eu-/pheo-melanin pigments in unique membrane-bound organelles termed melanosomes, which can be divided into four stages depending on their degree of maturation. Pigmentation production is determined by three distinct elements: enzymes involved in melanin synthesis, proteins required for melanosome structure, and proteins required for their trafficking and distribution. Many genes are involved in regulating pigmentation at various levels, and mutations in many of them cause pigmentary disorders, which can be classified into three types: hyperpigmentation (including melasma), hypopigmentation (including oculocutaneous albinism [OCA]), and mixed hyper-/hypopigmentation (including dyschromatosis symmetrica hereditaria). We briefly review vitiligo as a representative of an acquired hypopigmentation disorder.

Essential role of the molecular chaperone gp96 in regulating melanogenesis.

Through a process known as melanogenesis, melanocyte produces melanin in specialized organelles termed melanosomes, which regulates pigmentation of the skin, eyes, and hair. Gp96 is a constitutively expressed heat shock protein in the endoplasmic reticulum whose expression is further upregulated upon ultraviolet irradiation. However, the roles and mechanisms of this chaperone in pigmentation biology are unknown. In this study, we found that knockdown of gp96 by RNA interference significantly perturbed melanin synthesis and blocked late melanosome maturation. Gp96 knockdown did not impair the expression of tyrosinase, an essential enzyme in melanin synthesis, but compromised its catalytic activity and melanosome translocation. Further, mice with melanocyte-specific deletion of gp96 displayed decreased pigmentation. A mechanistic study revealed that the defect in melanogenesis can be rescued by activation of the canonical Wnt pathway, consistent with the critical roles of gp96 in chaperoning Wnt-coreceptor LRP6. Thus, this work uncovered the essential role of gp96 in regulating melanogenesis.

Non-invasive diffuse reflectance measurements of cutaneous melanin content can predict human sensitivity to ultraviolet radiation.

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.

Evidence for a new paradigm for ultraviolet exposure: a universal schedule that is skin phototype independent.

BACKGROUND: The Food and Drug Administration has published guidelines for manufacturer-recommended exposure schedules for ultraviolet (UV) tanning, intended to limit acute and delayed damage from UV exposure. These guidelines recommend that exposure schedules be adjusted for skin phototype. However, it has been shown that the dose necessary to produce tanning is similar for phototypes 2-4. METHODS: We observed tanning in phototypes 2 and 3 from repeated UV exposures over a 5-week period. Pigmentation was evaluated visually, instrumentally, and through Fontana-Masson staining of biopsies. RESULTS: The resultant pigmentation was equal or greater in phototype 3 compared with phototype 2 - both visually and instrumentally - measured on day 31 of the exposure protocol. The amount of melanin measured in biopsies taken 24 h postexposure was also greater in phototype 3 compared with phototype 2. CONCLUSION: Published data on tanning in phototypes 4 and 5 support our findings that higher phototypes can develop pigmentation more efficiently than lower phototypes. Therefore, a universal exposure schedule (based on sensitivity of phototype 2) can be used for all phototypes that are expected to engage in indoor tanning. This approach will result in a reduction of the UV burden for skin phototypes 3 and above.

Diacylglycerol kinase regulates tyrosinase expression and function in human melanocytes.

Diacylglycerol (DAG) increases the melanin content of human melanocytes in vitro and increases the pigmentation of guinea pig skin in vivo, but the mechanism(s) underlying those effects remain unknown. In this study, we characterized the role of diacylglycerol kinase (DGK), which phosphorylates DAG to generate phosphatidic acid, in the regulation of pigmentation. Ten isoforms of DGK have been identified, and we show that DGKζ is the most abundant isoform expressed by human melanocytic cells. Melanin content, tyrosinase activity, and tyrosinase protein levels were significantly reduced by a DGK inhibitor, but tyrosinase and microphthalmia-associated transcription factor messenger RNA (mRNA) levels were not changed by that inhibition, and there were no effects on the expression of other melanogenesis-related proteins. Isoform-specific small interfering RNAs showed that knockdown of DGKζ decreased melanin content and tyrosinase expression in melanocytic cells. Overexpression of DGKζ increased tyrosinase protein levels, but did not increase tyrosinase mRNA levels. Glycosidase digestion revealed that inhibition of DGK reduced only the mature form of tyrosinase, and the decrease of tyrosinase resulting from DGK inhibition could be blocked partially by protease inhibitors. These results suggest that DGK regulates melanogenesis via modulation of the posttranslational processing of tyrosinase, which may be related with the protein degradation machinery.

Effects of cosmetic formulations containing hydroxyacids on sun-exposed skin: current applications and future developments.

This paper describes recent data on the effects of various skin formulations containing hydroxyacids (HAs) and related products on sun-exposed skin. The most frequently used classes of these products, such as α- and ß-hydroxyacids, polyhydroxy acids, and bionic acids, are reviewed, and their application in cosmetic formulations is described. Special emphasis is devoted to the safety evaluation of these formulations, particularly on the effects of their prolonged use on sun-exposed skin. We also discuss the important contribution of cosmetic vehicles in these types of studies. Data on the effects of HAs on melanogenesis and tanning are also included. Up-to-date methods and techniques used in those explorations, as well as selected future developments in the cosmetic area, are presented.

Characterization of the bioactive motif of neuregulin-1, a fibroblast-derived paracrine factor that regulates the constitutive color and the function of melanocytes in human skin.

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.