Collagen VII Expression Is Required in Both Keratinocytes and Fibroblasts for Anchoring Fibril Formation in Bilayer Engineered Skin Substitutes.

The blistering disease recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding collagen VII (COL7), which forms anchoring fibrils that attach the epidermis to the dermis. Cutaneous gene therapy to restore COL7 expression in RDEB patient cells has been proposed, and cultured epithelial autograft containing COL7-modified keratinocytes was previously tested in clinical trials. Because COL7 in normal skin is expressed in both fibroblasts and keratinocytes, cutaneous gene therapy using a bilayer skin substitute may enable faster restoration of anchoring fibrils. Hypothetically, COL7 expression in either dermal fibroblasts or epidermal keratinocytes might be sufficient for functional anchoring fibril formation in a bilayer skin substitute. To test this, engineered skin substitutes (ESS) were prepared using four combinations of normal + RDEB cells: (1) RDEB fibroblasts + RDEB keratinocytes; (2) RDEB fibroblasts + normal keratinocytes; (3) normal fibroblasts + RDEB keratinocytes; and (4) normal fibroblasts + normal keratinocytes. ESS were incubated in vitro for 2 weeks prior to grafting to full-thickness wounds in immunodeficient mice. Biopsies were analyzed in vitro and at 1, 2, or 3 weeks after grafting. COL7 was undetectable in ESS prepared using all RDEB cells (group 1), and macroscopic blistering was observed by 2 weeks after grafting in ESS containing RDEB cells. COL7 was expressed, in vitro and in vivo, in ESS prepared using combinations of normal + RDEB cells (groups 2 and 3) or all normal cells (group 4). However, transmission electron microscopy revealed structurally normal anchoring fibrils, in vitro and by week 2 in vivo, only in ESS prepared using all normal cells (group 4). The results suggest that although COL7 protein is produced in engineered skin when cells in only one layer express the COL7 gene, formation of structurally normal anchoring fibrils appears to require expression of COL7 in both dermal fibroblasts and epidermal keratinocytes.

PIKfyve regulates melanosome biogenesis.

PIKfyve, VAC14, and FIG4 form a complex that catalyzes the production of PI(3,5)P2, a signaling lipid implicated in process ranging from lysosome maturation to neurodegeneration. While previous studies have identified VAC14 and FIG4 mutations that lead to both neurodegeneration and coat color defects, how PIKfyve regulates melanogenesis is unknown. In this study, we sought to better understand the role of PIKfyve in melanosome biogenesis. Melanocyte-specific PIKfyve knockout mice exhibit greying of the mouse coat and the accumulation of single membrane vesicle structures in melanocytes resembling multivesicular endosomes. PIKfyve inhibition blocks melanosome maturation, the processing of the melanosome protein PMEL, and the trafficking of the melanosome protein TYRP1. Taken together, these studies identify a novel role for PIKfyve in controlling the delivery of proteins from the endosomal compartment to the melanosome, a role that is distinct from the role of PIKfyve in the reformation of lysosomes from endolysosomes.

Tyrosinase Depletion Prevents the Maturation of Melanosomes in the Mouse Hair Follicle.

The mechanisms that lead to variation in human skin and hair color are not fully understood. To better understand the molecular control of skin and hair color variation, we modulated the expression of Tyrosinase (Tyr), which controls the rate-limiting step of melanogenesis, by expressing a single-copy, tetracycline-inducible shRNA against Tyr in mice. Moderate depletion of TYR was sufficient to alter the appearance of the mouse coat in black, agouti, and yellow coat color backgrounds, even though TYR depletion did not significantly inhibit accumulation of melanin within the mouse hair. Ultra-structural studies revealed that the reduction of Tyr inhibited the accumulation of terminal melanosomes, and inhibited the expression of genes that regulate melanogenesis. These results indicate that color in skin and hair is determined not only by the total amount of melanin within the hair, but also by the relative accumulation of mature melanosomes.

Melanocytic galectin-3 is associated with tyrosinase-related protein-1 and pigment biosynthesis.

Galectin-3 is a family member of the carbohydrate-binding proteins widely expressed by many cell types and exhibits multiple cellular functions. We demonstrate that melanocytes express galectin-3, which is predominantly localized to the cell body peripherally along the Golgi zone. Downregulation of galectin-3 in human melanocytes using short hairpin RNA technology resulted in the reduction of both melanin synthesis and expression/activity of tyrosinase-related protein-1 (Tyrp-1). In the cell body, galectin-3 colocalizes with melanosome-destined cargo, specifically tyrosinase and Tyrp-1. We studied melanocytes cultured from patients with forms of Hermansky-Pudlak syndrome (HPS) containing defects in trafficking steps governed by biogenesis of lysosome-related organelle complex-2 (BLOC-2) (HPS-5), BLOC-3 (HPS-1), and adaptin-3 (HPS-2). We found that galectin-3 expression mimicked the defective expression of the tyrosinase cargo in dendrites of HPS-5 melanocytes, but it was not altered in HPS-1 or HPS-2 melanocytes. In addition, galectin-3 colocalized predominantly with the HPS-5 component of BLOC-2 in normal human melanocytes. These data indicate that galectin-3 is a regulatory component in melanin synthesis affecting the expression of Tyrp-1.

Epistatic and combinatorial effects of pigmentary gene mutations in the domestic pigeon.

Understanding the molecular basis of phenotypic diversity is a critical challenge in biology, yet we know little about the mechanistic effects of different mutations and epistatic relationships among loci that contribute to complex traits. Pigmentation genetics offers a powerful model for identifying mutations underlying diversity and for determining how additional complexity emerges from interactions among loci. Centuries of artificial selection in domestic rock pigeons (Columba livia) have cultivated tremendous variation in plumage pigmentation through the combined effects of dozens of loci. The dominance and epistatic hierarchies of key loci governing this diversity are known through classical genetic studies, but their molecular identities and the mechanisms of their genetic interactions remain unknown. Here we identify protein-coding and cis-regulatory mutations in Tyrp1, Sox10, and Slc45a2 that underlie classical color phenotypes of pigeons and present a mechanistic explanation of their dominance and epistatic relationships. We also find unanticipated allelic heterogeneity at Tyrp1 and Sox10, indicating that color variants evolved repeatedly though mutations in the same genes. These results demonstrate how a spectrum of coding and regulatory mutations in a small number of genes can interact to generate substantial phenotypic diversity in a classic Darwinian model of evolution.

Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy, and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus, whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes.

Cyclic voltammetric, fluorescence and biological analysis of purified aeruginosin A, a secreted red pigment of Pseudomonas aeruginosa PAO1.

The opportunistic pathogen Pseudomomas aeruginosa produces multiple pigments during in vitro culture and in vivo during colonization of burn wounds and in the airways of cystic fibrosis (CF) patients. One pigment is a deep 'merlot'-coloured compound known as aeruginosin A (AA). However, the red pigment(s) of P. aeruginosa are often collectively called pyorubrin, of which there is no known chemical composition. Here, we purified and confirmed by MS and assessed the physicochemical properties of AA (2-amino-6-carboxy-10-methylphenazinium betaine) by first focusing on its ability to redox-cycle using cyclic voltammetry and its spectroscopic (as well as fluorescent) properties, experiments that were conducted at physiological pH. AA exhibited reversible electrochemistry at a glassy carbon electrode within a potential range of -500 to -200 mV. Electrochemical anodic and cathodic peak currents were observed at -327 and -360 mV, respectively, with a low formal reduction potential of -343.5 mV versus Ag/AgCl. AA absorbed at 516 nm and fluoresced at 606 nm. Results from the spectro-electrochemistry of pyorubrin revealed that its strongest fluorescence was in its parent or oxidized form. Production of AA by P. aeruginosa was found to be controlled by the rhl component of the intercellular signalling system known as quorum sensing and was produced maximally during the stationary growth phase. However, unlike its downstream blue redox-active toxin, pyocyanin, AA had no adverse effects on methicillin-resistant Staphylococcus aureus USA300, Escherichia coli DH5-α or human keratinocytes. We close with some thoughts on the potential commercial use(s) of AA.

Positioning ganglioside D3 as an immunotherapeutic target in lymphangioleiomyomatosis.

Tumors that develop in lymphangioleiomyomatosis (LAM) as a consequence of biallelic loss of TSC1 or TSC2 gene function express melanoma differentiation antigens. However, the percentage of LAM cells expressing these melanosomal antigens is limited. Here, we report the overexpression of ganglioside D3 (GD3) in LAM. GD3 is a tumor-associated antigen otherwise found in melanoma and neuroendocrine tumors; normal expression is largely restricted to neuronal cells in the brain. We also observed markedly reduced serum antibody titers to GD3, which may allow for a population of GD3-expressing LAM cells to expand within patients. This is supported by the demonstrated sensitivity of cultured LAM cells to complement mediated cytotoxicity via GD3 antibodies. GD3 can serve as a natural killer T (NKT) cell antigen when presented on CD1d molecules expressed on professional antigen-presenting cells. Although CD1d-expressing monocyte derivatives were present in situ, enhanced NKT-cell recruitment to LAM lung was not observed. Cultured LAM cells retained surface expression of GD3 over several passages and also expressed CD1d, implying that infiltrating NKT cells can be directly cytotoxic toward LAM lung lesions. Immunization with antibodies to GD3 may thus be therapeutic in LAM, and enhancement of existing NKT-cell infiltration may be effective to further improve antitumor responses. Overall, we hereby establish GD3 as a suitable target for immunotherapy of LAM.

Impaired PIASy-Tip60 signaling weakens activation of p53 in melanoma.

The tumor suppressor p53 plays a central role in preventing tumor development by promoting transcription of genes that stall cell cycle and induce cell death. Although the majority of melanomas express wild-type p53, the molecular mechanisms that impede its activation remain unclear. We previously reported that the SUMO E3 ligase PIASy and the histone acetyltransferase Tip60 signaling cascade promote p53-dependent autophagy and apoptosis. We hypothesized that impairment in this signaling attenuates p53, thus disabling its apoptotic function in melanoma. Here, we show that human melanoma patient samples and cell lines maintain p53 expression but PIASy and/or Tip60 are frequently lost. We observed dysregulation of Tip60-mediated p53 transcription program in melanoma cell lines. Reconstitution of PIASy and Tip60 in melanoma cells increased genotoxic stress-induced apoptosis. Our study provides a clinical link of how sumoylation signaling may activate p53-mediated cell death in melanoma.

Report of a novel OCA2 gene mutation and an investigation of OCA2 variants on melanoma risk in a familial melanoma pedigree.

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.

Hydrolytic enzymes of the interfollicular epidermis differ in expression and correlate with the phenotypic difference observed between light and dark skin.

Degradation of melanosomes in light skin (LS, i.e. phototype I/II) appears to occur more rapidly than dark skin (DS, i.e. phototype IV/V). Hydrolytic enzymes known to reside and be expressed in a differential pattern within the interfollicular epidermis are implicated in playing a role in epidermal differentiation and potentially melanosome degradation. The aim of this present study was to evaluate the differential expression of hydrolytic enzymes that may correlate with physiological and phenotypic differences seen between DS and LS. Expression of six hydrolytic enzymes was confirmed by microarray analysis of the suprabasal epidermis from LS and DS. Specific lysosomal hydrolases identified by microarray analysis were analyzed by indirect immunofluorescence (IIF) and immunoblot analysis. Immunogold electron microscopy (IEM) was completed to visualize cellular expression of the hydrolytic enzyme cathepsin L2 (Cath L2) and biochemical assay was performed to ascertain Cath L2 activity. Immunoblotting of light and dark epidermal lysates demonstrated that of the six enzymes initially analyzed, both prostatic acid phosphatase (ACPP) and Cath L2 were reproducibly upregulated in DS and LS, respectively. IIF and IEM analyses of Cath L2 in tissue confirmed this differential expression. Biochemical analysis of Cath L2 in light and dark epidermal lysates displays increased activity of Cath L2 in LS. The results of this study confirm differential expression of ACPP and Cath L2 in DS and LS at gene and protein level. Additionally, Cath L2 displays increased activity in LS-derived epidermal lysates. This study indentified two acid hydrolases that may play a role in melanosome degradation and pigment processing.

DeoxyArbutin and its derivatives inhibit tyrosinase activity and melanin synthesis without inducing reactive oxygen species or apoptosis.

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.

On the pathophysiology of vitiligo: possible treatment options.

Vitiligo is an acquired depigmenting disorder usually classified as non-segmental and segmental types with a higher incidence of the non-segmental ones. The cause of non-segmental vitiligo is still unknown. Currently, it is a dogma that there are several genes affecting the immune system and the pigment system that predisposes someone to develop vitiligo. A precipitating factor must then ellicit an interaction between the immune system and the melanocyte, resulting in destruction of the melanocyte population in discrete areas of the skin. Starting from the overlapping but distinct pathomechanisms, treatment should be finalized to the cellular targets and possibly related to the disease phase.

Epidermal keratinocytes from light vs. dark skin exhibit differential degradation of melanosomes.

Modification of skin complexion coloration has traditionally been accomplished by interruption or attenuation of melanogenesis and/or melanosome transfer. Post-transfer modification of pigmented melanosomes provides an attractive and distinct avenue of modulating skin pigmentation. The processing of melanosomes during keratinocyte (KC) terminal differentiation and the degradative variability observed between light and dark skin (LS and DS) remains enigmatic. To evaluate this, we developed a model system to investigate the loss of fluorescently labeled and isolated melanosomes by cultured human KCs. The extent of melanosome loss has been qualitatively assessed using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Results show that melanosomes are incorporated into the cytoplasm of both light and dark keratinocytes (LKCs and DKCs) and trafficked to a perinuclear region. Within 48 hours, confocal microscopy images suggest that LKCs display accelerated melanosome loss. This time-dependent decrease in carboxyfluorescein diacetate (CFDA) fluorescence was then quantitatively analyzed using flow cytometry. Consistent with the results of the confocal analysis, over a 48-hour time frame, LKCs appear to lose melanosomes more efficiently than DKCs. These experiments show that melanosomes are more rapidly lost in KCs derived from LS as opposed to DS.

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation.

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.

Monobenzyl ether of hydroquinone and 4-tertiary butyl phenol activate markedly different physiological responses in melanocytes: relevance to skin depigmentation.

Monobenzyl ether of hydroquinone (MBEH) is a Food and Drug Administration approved drug used for depigmentation therapy of advanced vitiligo. Here, the working mechanism of MBEH is explored in comparison to 4-tertiary butyl phenol (4-TBP), a known causative agent for occupational vitiligo mediating apoptotic melanocytic death. Cytotoxic experiments reveal that similar to 4-TBP, MBEH induces specific melanocyte death. To compare death pathways initiated by 4-TBP and MBEH, classical apoptotic hallmarks were evaluated in treated melanocytes. MBEH induced cell death without activating the caspase cascade or DNA fragmentation, showing that the death pathway is non-apoptotic. Release of High Mobility Group Box-1 protein by MBEH-treated melanocytes and ultrastructural features further confirmed a necrotic death pathway mediated by MBEH. A negative correlation between MBEH-induced cell death and cellular melanin content supports a cytoprotective role for melanin. Moreover, MBEH exposure upregulated the levels of melanogenic enzymes in cultured melanocytes and skin explants, whereas 4-TBP reduced the expression of the same. In summary, exposure to MBEH or 4-TBP has profoundly different consequences for melanocyte physiology and activates different death pathways. As the mode of cell death defines the nature of the immune response that follows, these findings help to explain the relative efficacy of these agents in mediating depigmentation.

Melanoma-associated antigen expression in lymphangioleiomyomatosis renders tumor cells susceptible to cytotoxic T cells.

The antibody HMB45 is used to diagnose lymphangioleiomyomatosis, a hyperproliferative disorder of lung smooth muscle cells with mutations in both alleles of either TSC1 or TSC2. A subset of these tumor cells expresses the melanoma-associated antigens gp100 and melanoma antigen recognized by T cells (MART-1). To explore the feasibility of targeting tumors in lymphangioleiomyomatosis by melanoma immunotherapy, we therefore assessed melanoma target antigen expression and existing immune infiltration of affected tissue compared with normal lung and melanoma as well as the susceptibility of cultured lymphangioleiomyomatosis cells to melanoma reactive cytotoxic T lymphocytes in vitro. Tumors expressed tyrosinase-related proteins 1 and 2 but not tyrosinase, in addition to gp100 and MART-1, and were densely infiltrated by macrophages, but not dendritic cells or T cell subsets. Although CD8(+) lymphocytes were sparse compared with melanoma, cells cultured from lymphangioleiomyomatosis tissue were susceptible to cytotoxic, gp100 reactive, and major histocompatibility complex class I restricted CD8(+) T cells in functional assays. Responder T cells selectively clustered and secreted interferon-gamma in response to HLA-matched melanocytes and cultured lymphangioleiomyomatosis cells. This reactivity exceeded that based on detectable gp100 expression; thus, tumor cells in lymphangioleiomyomatosis may process melanosomal antigens different from melanocytic cells. Therefore, boosting immune responses to gp100 in lymphangioleiomyomatosis may offer a highly desirable treatment option for this condition.

Mechanisms regulating skin pigmentation: the rise and fall of complexion coloration.

Skin pigmentary abnormalities are seen as aesthetically unfavorable and have led to the development of cosmetic and therapeutic treatment modalities of varying efficacy. Hence, several putative depigmenting agents aimed at modulating skin pigmentation are currently being researched or sold in commercially available products. In this review we will discuss the regulation of processes that control skin complexion coloration. This includes direct inhibition of tyrosinase and related melanogenic enzymes, regulation of melanocyte homeostasis, alteration of constitutive and facultative pigmentation and down-regulation of melanosome transfer to the keratinocytes. These various processes, in the complex mechanism of skin pigmentation, can be regulated individually or concomitantly to alter complexion coloration and thus ameliorate skin complexion diseases.