Faculty Bibliography

BACKGROUND: Oculocutaneous albinism type 2 (OCA2) is caused by mutations of the OCA2 gene. Individuals affected by OCA2 as well as other types of albinism are at a significantly increased risk for sun-induced skin-cancers, including malignant melanoma (MM). OBJECTIVE: To identify the molecular etiology of oculocutaneous albinism in a previously uncharacterized melanoma pedigree and to investigate the relationship between two OCA2 variants and melanoma predisposition in this pedigree. METHODS: DNA and RNA were isolated from the peripheral blood of seven patients in a familial melanoma pedigree. Electron microscopy was performed on the individual with clinical oculocutaneous albinism. OCA2, TYRP1, MC1R, CDKN2A/p16, CDKN2A/p19ARF, and CDK4 genes were sequenced in affected individuals. The relationship between OCA2 variants and melanoma was assessed using a pedigree likelihood-based method. RESULTS: The proband was determined to be an OCA2 compound heterozygous mutation carrier with a previously reported conservative missense mutation (V443I) and a novel non-conservative missense mutation (L734R). The pedigree contained individuals diagnosed with both cutaneous and iris melanoma. Based on co-segregation analysis, the odds of these OCA2 variants being high penetrance loci for melanoma was: 1.3-to-1 if we include the iris melanoma as affected and 6.5-to-1 if we only consider cutaneous melanoma as affected. CONCLUSION: The discovery of this novel OCA2 variant adds to the body of evidence on the detrimental effects of OCA2 gene mutations on pigmentation, supports existing GWAS data on the relevance of the OCA2 gene in melanoma predisposition, and may ultimately assist in the development of targeted molecular therapies in the treatment of OCA and melanoma.

Vitiligo is characterized by depigmented skin patches caused by loss of epidermal melanocytes. Oxidative stress may have a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study, we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol and monobenzyl ether of hydroquinone, known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box-binding protein 1 (XBP1), is increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators IL6 and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while overexpression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity.Journal of Investigative Dermatology advance online publication, 14 June 2012; doi:10.1038/jid.2012.181.

Safety is a major concern in developing commercial skin-lightening agents. Here, we report the modulating effects of deoxyArbutin (dA) and its second-generation derivatives - deoxyFuran (dF), 2-fluorodeoxyArbutin (fdA), and thiodeoxyArbutin (tdA) - on tyrosinase, and consequently, on melanization. Results demonstrate that dA and its derivatives inhibit tyrosine hydroxylase and dopa oxidase activity of tyrosinase. The inhibition is dose-dependent, thereby inhibiting melanin synthesis in intact melanocytes, when used at concentrations that retain 95% viability of the treated cells in culture. Herein we demonstrate that dA, and its second-generation derivatives dF, fdA, and tdA, exhibit dose-dependent reductions in melanocyte cell number, primarily due to inhibition of proliferation rather than initiation of apoptosis as exemplified by hydroquinone (HQ), ie, cytostatic as opposed to cytotoxic. Human and murine melanocytes with functional mutations in either tyrosinase or tyrosinase-related protein 1 (Tyrp1) are less sensitive to the cytostatic effects of dA and its derivatives. Minimal amounts of reactive oxygen species (ROS) were generated upon treatment with dA and its derivatives, in contrast to a dramatic amount of ROS induced by HQ. This increase in ROS subsequently induced the expression of the endogenous antioxidant catalase in treated melanocytes. Treatment with exogenous antioxidants provided protection for melanocytes treated with HQ, but not dA and its derivatives, suggesting that HQ exerts more oxidative stress. These studies demonstrate that dA and its derivatives are relatively safe tyrosinase inhibitors for skin lightening or for ameliorating hyperpigmented lesions.

The pathobiology of vitiligo, characterized by the spread of depigmented skin patches due to localized melanocyte loss, is not fully understood. Oxidative stress is thought to play a role in disease onset with a subsequent autoimmune response underlying progression. We therefore sought to identify mechanisms that linked oxidative stress and autoimmune responses. Melanocytes at the periphery of vitiligo lesions have distended endoplasmic reticuli (ER). We hypothesized that oxidative stress disrupted homeostasis of the ER where oxidation/reduction reactions facilitate disulfide bond formation. As a result, misfolded peptides would accumulate, dilating the ER and activating the unfolded protein response (UPR). The UPR is a stress response pathway, initiated by three regulators (IRE1, PERK and ATF6). It first promotes cell survival, however sustained activation induces apoptosis. In order to identify a potential role for the UPR in vitiligo we dosed melanocytes with 4-tertiary butyl phenol (4- TBP) and monobenzyl ether of hydroquinone (MBEH), phenols known to trigger vitiligo. The phenols caused an increase in expression of IRE1 and PERK. PERK activation leads to enhancement of the antioxidant response by recruitment of the transcription factor NRF2 to the nucleus and increased expression of the antioxidant HMOX1. The IRE1 effector, X-box binding protein-1 (XBP1) was also activated by phenol treatment which led to increased production of interleukin-6 (IL6) and IL8, cytokines expressed at increased levels in perilesional skin in vitiligo. Treatment with XBP1 inhibitors reduced phenol-induced IL6 and IL8 production, while over-expression of active XBP1 increased their expression. Thus, chemicals known to cause vitiligo trigger a UPR-mediated increase in cytokine production. There are a number of potential roles for the UPR in vitiligo. The UPR may (i) induce apoptosis following sustained oxidative stress causing release of melanocyte specific antigens, (ii) be dysregulated resulting in a muted PERK-!

Although the precise mechanisms that trigger vitiligo remain elusive, autoimmune responses mediate its progression. The development of therapies has been impeded by a paucity of animal models, since mice lack interfollicular melanocytes, the primary targets in vitiligo. In this issue, Harris et al. describe a mouse model in which interfollicular melanocytes are retained by Kit ligand overexpression and an immune response is initiated by transplanting melanocyte-targeting CD8+ T cells.

Oculocutaneous albinism (OCA) is a group of genetic disorders characterized by hypopigmentation of the skin, hair, and eyes. Affected individuals experience reduced visual acuity and substantially increased skin cancer risk. There are four major types of OCA (OCA1-OCA4) that result from disruption in production of melanin from tyrosine. Current treatment options for individuals with OCA are limited to attempts to correct visual problems and counseling to promote use of sun protective measures. However, Onojafe et al., reporting in this issue of the JCI, provide hope for a new treatment approach for OCA, as they demonstrate that treating mice that model OCA-1b with nitisinone, which is FDA approved for treating hereditary tyrosinemia type 1, elevates plasma tyrosine levels, and increases eye and hair pigmentation

Stress induced by buildup of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR). Failure by the UPR to restore homeostasis can trigger apoptosis. We have shown chronic UPR activation in tyrosinase and Tyrp1 mutant melanocytes, where tyrosinase is retained in the ER, without concomitant loss of viability, suggesting that melanocytes adapt to ER stress. Identifying adaptive mechanisms has implications for treatment of melanocyte disorders. For example, UPR activation may permit adaptation to hypoxia and play a role in melanomagenesis. Thus melanoma therapies targeting the UPR are being tested. The UPR may also play a role in vitiligo. Xbp1 (a key UPR transcription factor) polymorphisms are associated with increased risk of vitiligo, while ER dilation in perilesional melanocytes suggests ER stress. The UPR consists of three pathways regulated by IRE1, ATF6 and PERK. Prolonged IRE1 signaling in stressed cells promotes survival, while sustained PERK activity promotes apoptosis. OCA2 mutations result in hypopigmentation due, in part, to ER retention of tyrosinase, but do not result in loss of viability. We therefore investigated the UPR in Oca2-(null) melanocytes. Wildtype and Oca2-melanocytes were treated with ER stressors thapsigargin or tunicamycin and UPR activation was monitored by RT-PCR and Western blot analysis. Ire1 expression was increased in Oca2-melanocytes compared to wildtype cells. However, downstream signaling was not activated, with no splicing of Ire1 targeted Xbp1. Expression of Perk and its downstream effector Atf4 were decreased in Oca2-melanocytes. Upon ER stress, Ire1 expression and Xbp1 splicing increased in both cell lines indicating UPR activation. Typically, UPR activation leads to Perk-mediated phosphorylation of eIF2alpha. Remarkably, levels of p-eIF2alpha were markedly diminished in stressed Oca2-cells. Expression of proapoptotic CHOP was still induced in Oca2-melanocytes, but did not result in significant cell death. Our da!