RESUMO
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.
Assuntos
Melanossomas/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Flavoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melaninas/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Knockout , Organelas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Transporte ProteicoRESUMO
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.
Assuntos
Biomarcadores Tumorais/metabolismo , Gangliosídeos/metabolismo , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Animais , Antígenos CD1d/metabolismo , Biomarcadores Tumorais/imunologia , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Gangliosídeos/imunologia , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/imunologia , Linfangioleiomiomatose/patologia , Camundongos , Células T Matadoras Naturais/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/fisiologia , Células Cultivadas , Eletroquímica , Escherichia coli/efeitos dos fármacos , Fluorescência , Humanos , Queratinócitos/efeitos dos fármacos , Espectrometria de Massas , Compostos Orgânicos/isolamento & purificação , Oxirredução , Pigmentos Biológicos/isolamento & purificação , Percepção de Quorum , Staphylococcus aureus/efeitos dos fármacosRESUMO
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.
Assuntos
Arbutina/análogos & derivados , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Melanócitos/enzimologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Albinismo Oculocutâneo/enzimologia , Animais , Antioxidantes/farmacologia , Apoptose , Arbutina/farmacologia , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Hidroquinonas/farmacologia , Melanócitos/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/metabolismo , Superóxido Dismutase/farmacologia , Tirosina 3-Mono-Oxigenase/antagonistas & inibidores , Vitamina E/farmacologiaRESUMO
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.
Assuntos
Melanócitos/efeitos da radiação , Receptor Tipo 1 de Melanocortina/genética , Raios Ultravioleta , Células Cultivadas , Genótipo , HumanosRESUMO
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.
Assuntos
Antígenos de Neoplasias/imunologia , Citotoxicidade Imunológica , Linfangioleiomiomatose/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/metabolismo , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Oxirredutases Intramoleculares/imunologia , Oxirredutases Intramoleculares/metabolismo , Linfangioleiomiomatose/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Antígeno MART-1 , Melanoma/imunologia , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas de Neoplasias/metabolismo , Oxirredutases/imunologia , Oxirredutases/metabolismo , Neoplasias Cutâneas/imunologia , Antígeno gp100 de MelanomaRESUMO
UVB irradiation has been reported to induce photoaging and suppress systemic immune function that could lead to photocarcinogenesis. However, because of the paucity of an UVB-induced photodamaged skin model, precise and temporal mechanism(s) underlying the deleterious effects of long-term UVB exposure on human skin have yet to be delineated. In this study, we established a model using human skin xenografted onto severe combined immunodeficient mice, which were subsequently challenged by repeated UVB irradiation for 6 weeks. Three-dimensional optical image analysis of skin replicas and noninvasive biophysical measurements illustrated a significant increase in skin surface roughness, similar to premature photoaging, and a significant loss of skin elasticity after long-term UVB exposure. Resembling authentically aged skin, UVB-exposed samples exhibited significant increases in epithelial keratins (K6, K16, K17), elastins, and matrix metalloproteinases (MMP-1, MMP-9, MMP-12) as well as degradation of collagens (I, IV, VII). The UVB-induced deterioration of fibrous keratin intermediate filaments was also observed in the stratum corneum. Additionally, similarities in gene expression patterns between our model and chronologically aged skin substantiated the plausible relationship between photodamage and chronological age. Furthermore, severe skin photodamage was observed when neutralizing antibodies against TIMP-1, an endogenous inhibitor of MMPs, were administered during the UVB exposure regimen. Taken together, these findings suggest that our skin xenograft model recapitulates premature photoaged skin and provides a comprehensive tool with which to assess the deleterious effects of UVB irradiation.
Assuntos
Derme/efeitos da radiação , Epiderme/efeitos da radiação , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Adulto , Animais , Western Blotting , Colágeno/metabolismo , Colágeno/efeitos da radiação , Derme/patologia , Modelos Animais de Doenças , Elasticidade/efeitos da radiação , Elastina/metabolismo , Elastina/efeitos da radiação , Epiderme/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Queratinas/metabolismo , Queratinas/efeitos da radiação , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz/efeitos da radiação , Camundongos , Camundongos SCID , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/antagonistas & inibidores , Transplante HeterólogoRESUMO
Hair greying is one of the most distinct but least comprehended features of senescence. The signalling of stem cell factor (SCF) and its receptor KIT has been documented to regulate essential roles in the maintenance of embryonic melanocyte lineages and postnatal cutaneous melanogenesis, although little is known about its detailed mechanisms in postnatal hair pigmentation. To address this, anagen human hair follicles and C57BL/6 murine pelage were analysed in this study. Molecular biological analyses of murine follicular skin indicated a significant increase of membrane-bound SCF expression, reaching its peak 8-16 days after anagen induction in concert with the escalation of cutaneous tyrosinase activity and corresponding pigmentation. Administration of KIT-neutralizing antibody abolished MITF and tyrosinase expressions, resulting in a reversible hair depigmentation in murine regenerated hair and human hair organ culture. Quantitative RT-PCR of human hair follicles indicated that KIT expression as well as the expression of several melanogenic factors, including MITF, was significantly lower in unpigmented than in pigmented follicles. Taken together, these data revealed a pivotal role of SCF-KIT signalling in the maintenance of human hair follicle melanogenesis during the anagen cycle and its involvement in physiological ageing of the hair follicle pigmentary unit.
Assuntos
Cor de Cabelo/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/fisiologia , Fator de Células-Tronco/metabolismo , Adulto , Idoso , Animais , Feminino , Expressão Gênica , Folículo Piloso/metabolismo , Humanos , Imuno-Histoquímica , Melanócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas Proto-Oncogênicas c-kit/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Células-Tronco/análise , Fator de Células-Tronco/genética , Técnicas de Cultura de Tecidos , Adulto JovemRESUMO
The pathogenesis of vitiligo is complex and not well understood. Genes play a role in all aspects of vitiligo pathogenesis, and studies are ongoing to identify these genes and understand their biology. There is a body of interlocking, compelling evidence supporting an autoimmune basis for most or all cases of generalized vitiligo. The development of an autoimmune disease generally involves three components; the immune system, environmental triggers and other exogenous precipitating factors, and the target tissue. In vitiligo, precipitating factors could induce melanocyte damage in genetically susceptible individuals and consequent cell death, loss of tolerance, and induction of melanocyte-directed autoimmunity. Future research will more precisely define the multiple biological events that regulate development of vitiligo.
Assuntos
Doenças Autoimunes/etiologia , Vitiligo/etiologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Cálcio/metabolismo , Humanos , Melanócitos/imunologia , Melanócitos/metabolismo , Melanócitos/patologia , Espécies Reativas de Oxigênio/metabolismo , Pigmentação da Pele/genética , Pigmentação da Pele/imunologia , Vitiligo/genética , Vitiligo/imunologiaRESUMO
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.
Assuntos
Pigmentação da Pele , Animais , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/fisiologia , Melanossomas/efeitos dos fármacos , Melanossomas/fisiologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de SinaisRESUMO
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.
Assuntos
Colágeno Tipo VII/biossíntese , Fibroblastos , Regulação da Expressão Gênica , Queratinócitos , Pele Artificial , Engenharia Tecidual , Adulto , Animais , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/transplante , Xenoenxertos , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/transplante , Masculino , Camundongos , Mutação , Cicatrização , Ferimentos e Lesões/genética , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologiaRESUMO
Skin color is one of the most distinct features in the human race. To assess the mechanisms of skin color variation, human skin substitutes (HSS) were constructed by grafting mixtures of cultured keratinocytes and melanocytes from a combination of donor skin types, together with light skin derived fibroblasts, into chambers inserted onto the back skin of severe combined immunodeficient (SCID) mice. The resulting complexion coloration of the HSS was relatively darker and lighter when dark and light skin derived keratinocytes, respectively, were combined with melanocytes derived from either light or dark skin. The melanin content in the epidermis and the maturation stage of melanosomes in basal keratinocytes were significantly increased in the HSS composed of dark compared to light skin derived keratinocytes. In addition, the ratio of individual/clustered melanosomes in recipient keratinocytes was increased in the former as opposed to the latter HSS. The genetic expression of endothelin-1, proopiomelanocortin, microphthalmia-associated transcription factor, tyrosinase, GP100, and MART1 were increased in HSS composed of dark vs. light skin derived keratinocytes. These data suggest that our HSS is a promising melanogenic model that demonstrates the role of the keratinocyte in regulating in part both melanogenesis and distribution of transferred melanosomes.
Assuntos
Queratinócitos/metabolismo , Modelos Animais , Pigmentação da Pele/fisiologia , Pele Artificial , Pele/patologia , Animais , Biomarcadores/metabolismo , Western Blotting , Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Queratinócitos/citologia , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Camundongos SCID , Análise de Sequência com Séries de OligonucleotídeosRESUMO
PURPOSE: Whereas cutaneous pigmentation increases after exposure to ultraviolet (UV) irradiation, ocular pigmentation does not. This study was designed to examine the evidence that alpha-melanocyte-stimulating hormone (alpha-MSH), which is thought to be the mediator of UV response in the skin, has any role to play in uveal melanocytes. METHODS: Human uveal melanocytes derived from the choroid and the iris were cultivated by using eyes harvested from adult cadaveric donors and were assessed by Northern blot analysis for growth and melanogenic response to alpha-MSH and expression of the receptor for alpha-MSH (MC1-R). In addition, expression of alpha-MSH was evaluated in ocular tissue by immunocytochemistry. RESULTS: Uveal melanocytes, unlike cutaneous melanocytes in vitro, exhibited no stimulation of proliferation in response to alpha-MSH at dosages ranging from 0.1 to 100 muM. In addition, tyrosine hydroxylase, DOPA oxidase, and protein levels for tyrosinase, TRP-1, and TRP-2 were not influenced by alpha-MSH. Associated with the lack of alpha-MSH response in cultured uveal melanocytes was the absence of expression of the receptor for alpha-MSH (MC1-R), as assessed by Northern blot analysis. Also in contrast to the skin, pigmented ocular tissue lacked expression of the alpha-MSH ligand, as assessed by immunocytochemistry. CONCLUSIONS: In conclusion, ocular pigmentation does not appear to be regulated by melanocyte stimulating hormone.
Assuntos
Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Receptor Tipo 1 de Melanocortina/metabolismo , Úvea/citologia , alfa-MSH/farmacologia , Northern Blotting , Células Cultivadas , Cor de Olho , Humanos , Oxirredutases Intramoleculares/metabolismo , Melaninas/metabolismo , Glicoproteínas de Membrana/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Oxirredutases/metabolismo , Pele/citologia , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-MSH/metabolismoRESUMO
Several tyrosinase inhibitors have been developed and utilized to ameliorate various cutaneous hyperpigmentary disorders and complexion discolorations. Deoxyarbutin (dA) (i.e., 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), designed using quantitative structure-activity relationships (QSAR), demonstrates effective inhibition of mushroom tyrosinase and skin-lightening capability (1). However, its comparative safety, effectiveness, and reversibility to other known tyrosinase inhibitors in human melanocytes had not been determined. The effect of dA was assessed in cultured human skin cells, on xenographs, and with a clinical trial. Using cultured human melanocytes, the maximum concentration of dA that allowed 95% viability was fourfold greater than for hydroquinone (HQ), indicating that dA is less cytotoxic/cytostatic than HQ. The viability of cultured human keratinocytes and fibroblasts was also less compromised by increasing concentrations of dA as opposed to HQ. At the maximum concentration allowing normal cellular viability, dA effectively inhibited tyrosinase activity and melanin content in human melanocytes, whereas HQ was marginally inhibitory. Upon removal of dA, tyrosinase activity and melanin content was normalized within five days. Topical application of dA on human xenografts resulted in a gradual and visually apparent skin lightening effect during an eight-week period. In a clinical trial, dA facilitated fading of pre-tanned skin to a statistically significant greater extent than either HQ or no treatment. These results demonstrate that dA is a potentially safe, effective, and reversible tyrosinase inhibitor.
Assuntos
Arbutina/análogos & derivados , Hiperpigmentação/tratamento farmacológico , Monofenol Mono-Oxigenase/antagonistas & inibidores , Pigmentação da Pele/efeitos dos fármacos , Administração Tópica , Adolescente , Adulto , Animais , Arbutina/farmacologia , Biópsia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Hidroquinonas/farmacologia , Hiperpigmentação/enzimologia , Masculino , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Monofenol Mono-Oxigenase/metabolismo , Pele/efeitos dos fármacos , Pele/enzimologia , Organismos Livres de Patógenos Específicos , Transplante HeterólogoRESUMO
The neural crest gives rise to numerous cell types, including Schwann cells, neurons, and melanocytes. The extent to which adult neural crest-derived cells retain plasticity has not been tested previously. We report that cutting adult mouse sciatic nerve induces pigmentation around nerve fascicles, among muscle bundles, and in the hypodermis. Pigmented cells are derived from adult nerve, because pigmentation occurs even when nerve fragments are grafted into tyrosinase null albino mice. Pigmentation defects are pervasive in patients with neurofibromatosis type 1 (NF1). Mice hemizygous for Nf1 mutations show enhanced pigmentation after nerve lesion and occasionally form pigmented and unpigmented tumors. The Nf1 nerve and the Nf1 host environment both contribute to enhanced pigmentation. Grafted purified Nf1 mutant glial cells [S100(+)-p75NGFR(+)-GFAP(+)-EGFR(+) or S100(+)-p75NGFR(+)-GFAP(+)-EGFR(-)] mimic nerve-derived pigmentation. The NF1 protein, neurofibromin, is a Ras-GAP that acts downstream of a few defined receptor tyrosine kinases, including [beta-common (beta(c))] the shared common receptor for granulocyte and monocyte colony-stimulating factor, interleukin-3 (IL3), and IL5. Cytokines in the environment have the potential to suppress pigmentation as shown by nerve injury experiments in null mice; when is beta(c) absent or Nf1 is mutant, melanogenesis is increased. Thus, the adult nerve glial cell phenotype is maintained after nerve injury by response to cytokines, through neurofibromin.
Assuntos
Citocinas/metabolismo , Melaninas/biossíntese , Neuroglia/metabolismo , Neoplasias do Sistema Nervoso Periférico/patologia , Neuropatia Ciática/fisiopatologia , Transdução de Sinais/fisiologia , Animais , Axotomia , Diferenciação Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/transplante , Heterozigoto , Masculino , Melanócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Compressão Nervosa , Crista Neural/citologia , Crista Neural/embriologia , Neurofibromina 1/deficiência , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Neuroglia/citologia , Neoplasias do Sistema Nervoso Periférico/complicações , Pigmentação , Células de Schwann/citologia , Células de Schwann/metabolismo , Células de Schwann/transplante , Nervo Isquiático/lesões , Nervo Isquiático/transplante , Neuropatia Ciática/complicações , Neuropatia Ciática/patologiaRESUMO
Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder in which mutations in one of several genes interrupts biogenesis of melanosomes, platelet dense bodies, and lysosomes. Affected patients have oculocutaneous albinism, a bleeding diathesis, and sometimes develop granulomatous colitis or pulmonary fibrosis. In order to assess the role of HPS genes in melanosome biogenesis, melanocytes cultured from patients with HPS subtypes 1, 2, or 3 were assessed for the localization of various melanocyte proteins. Tyrosinase, Tyrp1, and Dct/Tyrp2 were atypically and distinctly expressed in HPS-1 and HPS-3 melanocytes, whereas only tyrosinase showed an atypical distribution in HPS-2 melanocytes. The HPS1 and AP3B1 (i.e., HPS-2) gene products showed no expression in HPS-1 and HPS-2 melanocytes, respectively, whereas HPS-3 melanocytes exhibited normal expression for both proteins. In normal human melanocytes, the HPS1 protein was expressed as an approximately 80 kDa molecule with both granular and reticular intracellular profiles. In HPS-1, lysosome associated membrane protein 1 (LAMP1), and LAMP3 were localized to abnormal large granules; in HPS-2, all LAMPs exhibited a normal granular expression; and in HPS-3, LAMP1, and LAMP3 exhibited a distinct less granular and more floccular pattern. In contrast, the expressions of Rab 27, transferrin, and cKit were unaffected in all three HPS genotypes. These data demonstrate that the three initially identified subtypes of human HPS exhibit distinct defects in the trafficking of various melanocyte-specific proteins.
Assuntos
Síndrome de Hermanski-Pudlak/metabolismo , Síndrome de Hermanski-Pudlak/patologia , Melanócitos/metabolismo , Melanócitos/patologia , Transporte Proteico/fisiologia , Complexo 3 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Antígenos CD/metabolismo , Proteínas de Transporte/metabolismo , Células Cultivadas , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Oxirredutases Intramoleculares/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Proteínas de Neoplasias/metabolismo , Oxirredutases/metabolismoRESUMO
The trigger initiating an autoimmune response against melanocytes in vitiligo remains unclear. Patients frequently experience stress to the skin prior to depigmentation. 4-tertiary butyl phenol (4-TBP) was used as a model compound to study the effects of stress on melanocytes. Heat shock protein (HSP)70 generated and secreted in response to 4-TBP was quantified. The protective potential of stress proteins generated following 4-TBP exposure was examined. It was studied whether HSP70 favors dendritic cell (DC) effector functions as well. Melanocytes were more sensitive to 4-TBP than fibroblasts, and HSP70 generated in response to 4-TBP exposure was partially released into the medium by immortalized vitiligo melanocyte cell line PIG3V. Stress protein HSP70 in turn induced membrane tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and activation of DC effector functions towards stressed melanocytes. Melanocytes exposed to 4-TBP demonstrated elevated TRAIL death receptor expression. DC effector functions were partially inhibited by blocking antibodies to TRAIL. TRAIL expression and infiltration by CD11c+ cells was abundant in perilesional vitiligo skin. Stressed melanocytes may mediate DC activation through release of HSP70, and DC effector functions appear to play a previously unappreciated role in progressive vitiligo.
Assuntos
Células Dendríticas/imunologia , Irritantes/farmacologia , Melanócitos/efeitos dos fármacos , Fenóis/farmacologia , Vitiligo/patologia , Adenoviridae/genética , Anticorpos/farmacologia , Proteínas Reguladoras de Apoptose , Morte Celular/imunologia , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Células Dendríticas/citologia , Expressão Gênica , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Melanócitos/citologia , Melanócitos/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Pele/citologia , Pele/imunologia , Pigmentação da Pele/imunologia , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vitiligo/imunologiaRESUMO
BACKGROUND: Hermansky-Pudlak syndrome (HPS) is a disorder of lysosome-related organelle biogenesis characterized by oculocutaneous albinism and prolonged bleeding. These clinical findings reflect defects in the formation of melanosomes in melanocytes and dense bodies in platelets. HPS type-3 (HPS-3) results from mutations in the HPS3 gene, which encodes a 1004 amino acid protein of unknown function that contains a predicted clathrin-binding motif (LLDFE) at residues 172-176. RESULTS: Clathrin was co-immunoprecipitated by HPS3 antibodies from normal but not HPS3 null melanocytes. Normal melanocytes expressing a GFP-HPS3 fusion protein demonstrated partial co-localization of GFP-HPS3 with clathrin following a 20 degrees C temperature block. GFP-HPS3 in which the predicted clathrin-binding domain of HPS3 was mutated (GFP-HPS3-delCBD) did not co-localize with clathrin under the same conditions. Immunoelectron microscopy of normal melanocytes expressing GFP-HPS3 showed co-localization of GFP-HPS3 with clathrin, predominantly on small vesicles in the perinuclear region. In contrast, GFP-HPS3-delCBD did not co-localize with clathrin and exhibited a largely cytoplasmic distribution. CONCLUSION: HPS3 associates with clathrin, predominantly on small clathrin-containing vesicles in the perinuclear region. This association most likely occurs directly via a functional clathrin-binding domain in HPS3. These results suggest a role for HPS3 and its protein complex, BLOC-2, in vesicle formation and trafficking.
Assuntos
Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Sítios de Ligação/genética , Proteínas de Transporte/fisiologia , Núcleo Celular/química , Células Cultivadas , Clatrina/fisiologia , Citoplasma/química , Vesículas Citoplasmáticas/química , Proteínas de Fluorescência Verde , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular , Melanócitos/química , Melanócitos/citologia , Mutação , Ligação ProteicaRESUMO
Parathyroid hormone-related protein (PTHrP) regulates a wide variety of developmental processes. Keratin 14 (K14) promoter-mediated overexpression of PTHrP in the epidermis during development converts the entire murine ventral skin to hairless nipple-like skin. In this report, we characterize the morphology and processes that influence the development of nipple connective tissue. The connective tissue of the nipple displayed increased levels of proteoglycans, and collagen bundles with atypical morphology, as well as increased numbers of mast cells, capillaries, nerve fibers and dermal melanocytes. The unique characteristics of nipple connective tissue were not present until mice reach 3-4 weeks of age. The adult male K14-PTHrP mouse has a less dramatic ventral skin phenotype, and does not manifest a nipple-like dermis. Ovariectomy or orchiedectomy prior to sexual maturity had no impact on the ventral skin of the male or female K14-PTHrP mice, but exposure to androgens in utero repressed many of the nipple-like characteristics in the ventral skin of the female K14-PTHrP mice.
Assuntos
Tecido Conjuntivo/patologia , Queratinas/fisiologia , Hormônios Peptídicos/fisiologia , Pele/citologia , Animais , Diferenciação Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Hormônios Esteroides Gonadais/metabolismo , Queratina-14 , Queratinas/genética , Masculino , Camundongos , Proteína Relacionada ao Hormônio Paratireóideo , Hormônios Peptídicos/genética , Esteroides/metabolismoRESUMO
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.