Two-pore channel 2 is required for soluble adenylyl cyclase-dependent regulation of melanosomal pH and melanin synthesis.

Melanosomal pH is important for the synthesis of melanin as the rate-limiting enzyme, tyrosinase, is very pH-sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC-dependent regulation of melanosomal pH were undefined. We now systematically examine four well-characterized melanosomal membrane proteins to determine whether any of them are required for sAC-dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC-dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC-dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP-AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC-dependent regulation of melanosomal pH and pigmentation.

Race, pigmentation, and the human skin barrier-considerations for dermal absorption studies.

A functional human skin barrier is critical in limiting harmful exposure to environmental agents and regulating the absorption of intentionally applied topical drug and cosmetic products. Inherent differences in the skin barrier between consumers due to extrinsic and intrinsic factors are an important consideration in the safety assessment of dermatological products. Race is a concept often used to describe a group of people who share distinct physical characteristics. The observed predisposition of specific racial groups to certain skin pathologies highlights the potential differences in skin physiology between these groups. In the context of the human skin barrier, however, the current data correlating function to race often conflict, likely as a consequence of the range of experimental approaches and controls used in the existing works. To date, a variety of methods have been developed for evaluating compound permeation through the human skin, both in vivo and in vitro. Additionally, great strides have been made in the development of reconstructed human pigmented skin models, with the flexibility to incorporate melanocytes from donors of different race and pigmentation levels. Together, the advances in the production of reconstructed human skin models and the increased adoption of in vitro methodologies show potential to aid in the standardization of dermal absorption studies for discerning racial- and skin pigmentation-dependent differences in the human skin barrier. This review analyzes the existing data on skin permeation, focusing on its interaction with race and skin pigmentation, and highlights the tools and research opportunities to better represent the diversity of the human populations in dermal absorption assessments.

Parallel evaluation of alternative skin barrier models and excised human skin for dermal absorption studies in vitro.

Skin permeation is a primary consideration in the safety assessment of cosmetic ingredients, topical drugs, and human users handling veterinary medicinal products. While excised human skin (EHS) remains the 'gold standard' for in vitro permeation testing (IVPT) studies, unreliable supply and high cost motivate the search for alternative skin barrier models. In this study, a standardized dermal absorption testing protocol was developed to evaluate the suitability of alternative skin barrier models to predict skin absorption in humans. Under this protocol, side-by-side assessments of a commercially available reconstructed human epidermis (RhE) model (EpiDerm-200-X, MatTek), a synthetic barrier membrane (Strat-M, Sigma-Aldrich), and EHS were performed. The skin barrier models were mounted on Franz diffusion cells and the permeation of caffeine, salicylic acid, and testosterone was quantified. Transepidermal water loss (TEWL) and histology of the biological models were also compared. EpiDerm-200-X exhibited native human epidermis-like morphology, including a characteristic stratum corneum, but had an elevated TEWL as compared to EHS. The mean 6 h cumulative permeation of a finite dose (6 nmol/cm2) of caffeine and testosterone was highest in EpiDerm-200-X, followed by EHS and Strat-M. Salicylic acid permeated most in EHS, followed by EpiDerm-200-X and Strat-M. Overall, evaluating novel alternative skin barrier models in the manner outlined herein has the potential to reduce the time from basic science discovery to regulatory impact.

The Many Faces of G Protein-Coupled Receptor 143, an Atypical Intracellular Receptor.

GPCRs transform extracellular stimuli into a physiological response by activating an intracellular signaling cascade initiated via binding to G proteins. Orphan G protein-coupled receptors (GPCRs) hold the potential to pave the way for development of new, innovative therapeutic strategies. In this review we will introduce G protein-coupled receptor 143 (GPR143), an enigmatic receptor in terms of classification within the GPCR superfamily and localization. GPR143 has not been assigned to any of the GPCR families due to the lack of common structural motifs. Hence we will describe the most important motifs of classes A and B and compare them to the protein sequence of GPR143. While a precise function for the receptor has yet to be determined, the protein is expressed abundantly in pigment producing cells. Many GPR143 mutations cause X-linked Ocular Albinism Type 1 (OA1, Nettleship-Falls OA), which results in hypopigmentation of the eyes and loss of visual acuity due to disrupted visual system development and function. In pigment cells of the skin, loss of functional GPR143 results in abnormally large melanosomes (organelles in which pigment is produced). Studies have shown that the receptor is localized internally, including at the melanosomal membrane, where it may function to regulate melanosome size and/or facilitate protein trafficking to the melanosome through the endolysosomal system. Numerous additional roles have been proposed for GPR143 in determining cancer predisposition, regulation of blood pressure, development of macular degeneration and signaling in the brain, which we will briefly describe as well as potential ligands that have been identified. Furthermore, GPR143 is a promiscuous receptor that has been shown to interact with multiple other melanosomal proteins and GPCRs, which strongly suggests that this orphan receptor is likely involved in many different physiological actions.

Evidence for Protein-Protein Interaction between Dopamine Receptors and the G Protein-Coupled Receptor 143.

Protein-protein interactions between G protein-coupled receptors (GPCRs) can augment their functionality and increase the repertoire of signaling pathways they regulate. New therapeutics designed to modulate such interactions may allow for targeting of a specific GPCR activity, thus reducing potential for side effects. Dopamine receptor (DR) heteromers are promising candidates for targeted therapy of neurological conditions such as Parkinson's disease since current treatments can have severe side effects. To facilitate development of such therapies, it is necessary to identify the various DR binding partners. We report here a new interaction partner for DRD2 and DRD3, the orphan receptor G protein-coupled receptor 143 (GPR143), an atypical GPCR that plays multiple roles in pigment cells and is expressed in several regions of the brain. We previously demonstrated that the DRD2/ DRD3 antagonist pimozide also modulates GPR143 activity. Using confocal microscopy and two FRET methods, we observed that the DRs and GPR143 colocalize and interact at intracellular membranes. Furthermore, co-expression of wildtype GPR143 resulted in a 57% and 67% decrease in DRD2 and DRD3 activity, respectively, as determined by ß-Arrestin recruitment assay. GPR143-DR dimerization may negatively modulate DR activity by changing affinity for dopamine or delaying delivery of the DRs to the plasma membrane.

Biophysical Compatibility of a Heterotrimeric Tyrosinase-TYRP1-TYRP2 Metalloenzyme Complex.

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.

The unfolded protein and integrated stress response in melanoma and vitiligo.

Epidermal melanocytes are constantly exposed to environmental stressors such as ultraviolet light (UV) and chemotoxins. Several evolutionarily conversed survival mechanisms are deployed to ensure melanocyte recovery after damage including the unfolded protein response (UPR) and integrated stress response (ISR). The UPR/ISR promote restoration of homeostasis, by modulating transcription and translation as well as activating nuclear factor erythroid 2-related factor 2 (NRF2)-mediated antioxidant activity. If repair fails, the UPR/ISR either stimulate cell death, or adaptation that can lead to survival of damaged cells and promote disease. For example, the UPR/ISR may support melanomagenesis by allowing UV-damaged, mutated cells to survive and adapt to a hostile tumor microenvironment that subjects cells to hypoxia, nutrient deprivation, and sub-optimal pH. The UPR and ISR can also promote transcriptional changes that support tumor growth and/or metastasis. Furthermore, these pathways may also underlie acquisition of chemoresistance and modulation of protein expression that alters the efficacy of immunotherapies. UPR activation has also been implicated in the pathogenesis of vitiligo and may promote increased expression of chemokines such as interleukin 6 and interleukin 8 that trigger an autoimmune response against melanocytes. We herein review the potential roles of the UPR/ISR in the etiology of melanoma and vitiligo.

Vitiligo and Melanoma-Associated Vitiligo: Understanding Their Similarities and Differences.

BACKGROUND: There has been a significant increase in the number and efficacy of therapies for advanced melanoma. Immunotherapies, such as anti-cytotoxic T-lymphocyte antigen-4 and programmed cell death-1 inhibitors, have improved the prognosis for patients with advanced melanoma. While spontaneous melanoma-associated vitiligo is a known phenomenon, the occurrence of melanoma-associated vitiligo following melanoma therapy is now recognized to associate with favorable outcomes. OBJECTIVE: The objective of this article is to provide a comprehensive literature review of melanoma-associated vitiligo and explore the insights these findings provide about the pathobiology of vitiligo and mechanisms underlying melanoma therapies. METHODS: PubMed and Science Direct databases were searched for studies pertaining to melanoma-associated vitiligo. The 36 studies reviewed included meta-analyses (n = 2), prospective cohort studies (n = 4), prospective observational studies (n = 3), retrospective studies (n = 12), case series (n = 2), and case reports (n = 13). RESULTS: The basic mechanisms underlying melanoma-associated vitiligo and vitiligo may be shared. Characterization of these mechanisms will identify new biomarkers and therapeutic targets for both melanoma and vitiligo. CONCLUSIONS: Co-opting the immune system to target tumor antigens highlights the potential overlap between anti-tumor immunity and autoimmunity. The development of vitiligo-like depigmentation in association with immunotherapy for melanoma may provide insights into both the immune response against melanoma as well as the pathogenesis of vitiligo.

Frontiers in pigment cell and melanoma research.

In this perspective, we identify emerging frontiers in clinical and basic research of melanocyte biology and its associated biomedical disciplines. We describe challenges and opportunities in clinical and basic research of normal and diseased melanocytes that impact current approaches to research in melanoma and the dermatological sciences. We focus on four themes: (1) clinical melanoma research, (2) basic melanoma research, (3) clinical dermatology, and (4) basic pigment cell research, with the goal of outlining current highlights, challenges, and frontiers associated with pigmentation and melanocyte biology. Significantly, this document encapsulates important advances in melanocyte and melanoma research including emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, epidemiology, pigment biophysics and chemistry, and evolution.

The triennial International Pigment Cell Conference (IPCC).

The International Federation of Pigment Cell Societies (IFPCS) held its XXIII triennial International Pigment Cell Conference (IPCC) in Denver, Colorado in August 2017. The goal of the summit was to provide a venue promoting a vibrant interchange among leading basic and clinical researchers working on leading-edge aspects of melanocyte biology and disease. The philosophy of the meeting, entitled Breakthroughs in Pigment Cell and Melanoma Research, was to deliver a comprehensive program in an inclusive environment fostering scientific exchange and building new academic bridges. This document provides an outlook on the history, accomplishments, and sustainability of the pigment cell and melanoma research community. Shared progress in the understanding of cellular homeostasis of pigment cells but also clinical successes and hurdles in the treatment of melanoma and dermatological disorders continue to drive future research activities. A sustainable direction of the societies creates an international forum identifying key areas of imminent needs in laboratory research and clinical care and ensures the future of this vibrant, diverse and unique research community at the same time. Important advances showcase wealth and breadth of the field in melanocyte and melanoma research and include emerging frontiers in melanoma immunotherapy, medical and surgical oncology, dermatology, vitiligo, albinism, genomics and systems biology, precision bench-to-bedside approaches, epidemiology, pigment biophysics and chemistry, and evolution. This report recapitulates highlights of the federate meeting agenda designed to advance clinical and basic research frontiers from melanoma and dermatological sciences followed by a historical perspective of the associated societies and conferences.

Identification of Novel G Protein-Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism.

Purpose: GPR143 regulates melanosome biogenesis and organelle size in pigment cells. The mechanisms underlying receptor function remain unclear. G protein-coupled receptors (GPCRs) are excellent pharmacologic targets; thus, we developed and applied a screening approach to identify potential GPR143 ligands and chemical modulators. Methods: GPR143 interacts with ß-arrestin; we therefore established a ß-arrestin recruitment assay to screen for compounds that modulate activity. Because GPR143 is localized intracellularly, screening with the wild-type receptor would be restricted to agents absorbed by the cell. For the screen we used a mutant receptor, which shows similar basal activity as the wild type but traffics to the plasma membrane. We tested two compound libraries and investigated validated hits for their effects on melanocyte pigmentation. Results: GPR143, which showed high constitutive activity in the ß-arrestin assay, was inhibited by several compounds. The three validated inhibitors (pimozide, niclosamide, and ethacridine lactate) were assessed for impact on melanocytes. Pigmentation and expression of tyrosinase, a key melanogenic enzyme, were reduced by all compounds. Because GPR143 appears to be constitutively active, these compounds may turn off its activity. Conclusions: X-linked ocular albinism type I, characterized by developmental eye defects, results from GPR143 mutations. Identifying pharmacologic agents that modulate GPR143 activity will contribute significantly to our understanding of its function and provide novel tools with which to study GPCRs in melanocytes and retinal pigment epithelium. Pimozide, one of three GPR143 inhibitors identified in this study, maybe be a good lead structure for development of more potent compounds and provide a platform for design of novel therapeutic agents.

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) antioxidant response promotes melanocyte viability and reduces toxicity of the vitiligo-inducing phenol monobenzone.

Vitiligo, characterised by progressive melanocyte death, can be initiated by exposure to vitiligo-inducing phenols (VIPs). VIPs generate oxidative stress in melanocytes and activate the master antioxidant regulator NRF2. While NRF2-regulated antioxidants are reported to protect melanocytes from oxidative stress, the role of NRF2 in the melanocyte response to monobenzone, a clinically relevant VIP, has not been characterised. We hypothesised that activation of NRF2 may protect melanocytes from monobenzone-induced toxicity. We observed that knockdown of NRF2 or NRF2-regulated antioxidants NQO1 and PRDX6 reduced melanocyte viability, but not viability of keratinocytes and fibroblasts, suggesting that melanocytes were preferentially dependent upon NRF2 activity for growth compared to other cutaneous cells. Furthermore, melanocytes activated the NRF2 response following monobenzone exposure and constitutive NRF2 activation reduced monobenzone toxicity, supporting NRF2's role in the melanocyte stress response. In contrast, melanocytes from individuals with vitiligo (vitiligo melanocytes) did not activate the NRF2 response as efficiently. Dimethyl fumarate-mediated NRF2 activation protected normal and vitiligo melanocytes against monobenzone-induced toxicity. Given the contribution of oxidant-antioxidant imbalance in vitiligo, modulation of this pathway may be of therapeutic interest.

Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis.

Recent advances in understanding vitiligo.

Vitiligo, an acquired depigmentation disorder, manifests as white macules on the skin and can cause significant psychological stress and stigmatization. Recent advances have shed light on key components that drive disease onset and progression as well as therapeutic approaches. Vitiligo can be triggered by stress to the melanin pigment-producing cells of the skin, the melanocytes. The triggers, which range from sunburn to mechanical trauma and chemical exposures, ultimately cause an autoimmune response that targets melanocytes, driving progressive skin depigmentation. The most significant progress in our understanding of disease etiology has been made on three fronts: (1) identifying cellular responses to stress, including antioxidant pathways and the unfolded protein response (UPR), as key players in disease onset, (2) characterizing immune responses that target melanocytes and drive disease progression, and (3) identifying major susceptibility genes. The current model for vitiligo pathogenesis postulates that oxidative stress causes cellular disruptions, including interruption of protein maturation in the endoplasmic reticulum (ER), leading to the activation of the UPR and expression of UPR-regulated chemokines such as interleukin 6 (IL-6) and IL-8. These chemokines recruit immune components to the skin, causing melanocytes to be targeted for destruction. Oxidative stress can further increase melanocyte targeting by promoting antigen presentation. Two key components of the autoimmune response that promote disease progression are the interferon (IFN)-γ/CXCL10 axis and IL-17-mediated responses. Several genome-wide association studies support a role for these pathways, with the antioxidant gene NRF2, UPR gene XBP1, and numerous immune-related genes including class I and class II major histocompatibility genes associated with a risk for developing vitiligo. Novel approaches to promote repigmentation in vitiligo are being investigated and may yield effective, long-lasting therapies.

EdnrB Governs Regenerative Response of Melanocyte Stem Cells by Crosstalk with Wnt Signaling.

Delineating the crosstalk between distinct signaling pathways is key to understanding the diverse and dynamic responses of adult stem cells during tissue regeneration. Here, we demonstrate that the Edn/EdnrB signaling pathway can interact with other signaling pathways to elicit distinct stem cell functions during tissue regeneration. EdnrB signaling promotes proliferation and differentiation of melanocyte stem cells (McSCs), dramatically enhancing the regeneration of hair and epidermal melanocytes. This effect is dependent upon active Wnt signaling that is initiated by Wnt ligand secretion from the hair follicle epithelial niche. Further, this Wnt-dependent EdnrB signaling can rescue the defects in melanocyte regeneration caused by Mc1R loss. This suggests that targeting Edn/EdnrB signaling in McSCs can be a therapeutic approach to promote photoprotective-melanocyte regeneration, which may be useful for those with increased risk of skin cancers due to Mc1R variants.

Variation in Hsp70-1A Expression Contributes to Skin Color Diversity.

The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes, and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either white or African American skin. Two-dimensional gel electrophoresis and mass spectrometry demonstrated that heat shock protein 70-1A (Hsp70-1A) protein levels were significantly higher in African American melanocytes compared with white melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70 family members in tyrosinase post-translational modification. In addition, pharmacologic inhibition and small interfering RNA-mediated downregulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes, and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes.