RESUMO
Vitiligo is a non-contagious skin disorder with loss of pigmentation, often impairing patients' well-being. This study used Dermatology Life Quality Index (DLQI), Adjustment to Chronic Skin Disorders Questionnaire (ACS), Beck Depression Inventory (BDI) and additional questions to explore quality of life (QoL), coping, depression and stigmatisation and included 96 patients with vitiligo and 23 controls. Stigmatisation was common: 87/96 patients (90%) reported questions/approaches, 23/96 (24%) experienced nasty comments. Sixty-four out of 96 (66.7%) had avoided situations because of vitiligo or concealed their white spots. Sixty patients (62.5%) implied psychological stress as influential on disease's course. Patients scored higher in all questionnaires than controls (DLQI = 4.9/1.6, ACS-social anxiety/avoidance = 36.9/22.1, ACS-helplessness = 27.3/16.0, ACS-anxious-depressive mood = 19.4/15.6), except BDI (6.8/7.3). QoL of 65 patients (67.7%) was hardly impaired, 70 (72.9%) were not depressed. Treatment with pro-pseudocatalase PC-KUS reduced social anxiety/avoidance, anxious-depressive mood and depression. Patients without low-key stigmatisation scored highest in DLQI and social anxiety/avoidance. Avoidance and concealing behavior correlated with all questionnaires' scores.
Assuntos
Adaptação Psicológica , Qualidade de Vida , Comportamento Social , Estereotipagem , Vitiligo/psicologia , Adulto , Fatores Etários , Análise de Variância , Estudos de Casos e Controles , Depressão/epidemiologia , Depressão/psicologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição de Risco , Fatores Sexuais , Perfil de Impacto da Doença , Estresse Psicológico , Inquéritos e Questionários , Vitiligo/diagnóstico , Vitiligo/terapiaRESUMO
Prohormone convertases (PCs) are endoproteases that process many substrates in addition to hormone precursors. Although overexpression of PCs is linked to carcinogenesis in some solid tumors, the role of subtilisin-kexin isoenzyme-1 (SKI-1) in this context is unknown. We show that SKI-1 is constitutively expressed in human pigment cells with higher SKI activity in seven out of eight melanoma cell lines compared with normal melanocytes. SKI-1 immunoreactivity is also detectable in tumor cells of melanoma metastases. Moreover, tissue samples of the latter display higher SKI-1 mRNA levels and activity than normal skin. From various stimuli tested, 12-O-tetradecanoylphorbol-13-acetate and tunicamycin affected SKI-1 expression. Importantly, SKI-1 inhibition by the cell-permeable enzyme inhibitor decanoyl-RRLL-chloromethylketone (dec-RRLL-CMK) not only suppressed proliferation and metabolic activity of melanoma cells in vitro but also reduced tumor growth of melanoma cells injected intracutaneously into immunodeficient mice. Mechanistic studies revealed that dec-RRLL-CMK induces classical apoptosis of melanoma cells in vitro and affects expression of several SKI-1 target genes including activating transcription factor 6 (ATF6). However, ATF6 gene silencing does not result in apoptosis of melanoma cells, suggesting that dec-RRLL-CMK induces cell death in an ATF6-independent manner. Our findings encourage further studies on SKI-1 as a potential target for melanoma therapy.
Assuntos
Melanoma/metabolismo , Melanoma/secundário , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fator 6 Ativador da Transcrição/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico/genética , Humanos , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos Mutantes , Camundongos SCID , Pró-Proteína Convertases/genética , Serina Endopeptidases/genética , Neoplasias Cutâneas/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nonsegmental vitiligo (NSV) is characterized by loss of inherited skin color. The cause of the disease is still unknown despite accumulating in vivo and in vitro evidence of massive epidermal oxidative stress via H2O2 and peroxynitrite (ONOO(-)) in affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Strictly segmental vitiligo (SSV) with dermatomal distribution is a rare entity, often associated with stable outcome. Recently, it was documented that this form can be associated with NSV (mixed vitiligo). We here asked the question whether ROS and possibly ONOO(-) could be players in the pathogenesis of SSV. Our in situ results demonstrate for the first time epidermal biopterin accumulation together with significantly decreased epidermal catalase, thioredoxin/thioreoxin reductase, and MSRA/MSRB expression. Moreover, we show epidermal ONOO(-) accumulation. In vivo FT-Raman spectroscopy reveals the presence of H2O2, methionine sulfoxide, and tryptophan metabolites; i.e., N-formylkynurenine and kynurenine, implying Fenton chemistry in the cascade (n=10). Validation of the basic data stems from successful repigmentation of skin and eyelashes in affected individuals, regardless of SSV or segmental vitiligo in association with NSV after reduction of epidermal H2O2 (n=5). Taken together, our contribution strongly supports H2O2/ONOO-mediated stress in the pathogenesis of SSV. Our findings offer new treatment intervention for lost skin and hair color.
Assuntos
Catalase/metabolismo , Pestanas/metabolismo , Peróxido de Hidrogênio/metabolismo , Ácido Peroxinitroso/metabolismo , Pele/metabolismo , Vitiligo/metabolismo , Adolescente , Adulto , Biopterinas/metabolismo , Catalase/uso terapêutico , Criança , Pré-Escolar , Ativação Enzimática/efeitos da radiação , Epiderme/metabolismo , Epiderme/patologia , Pestanas/efeitos dos fármacos , Pestanas/patologia , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nitratos/metabolismo , Oxirredução/efeitos dos fármacos , Pele/efeitos dos fármacos , Pele/patologia , Pigmentação da Pele/efeitos dos fármacos , Análise Espectral Raman , Raios Ultravioleta , Vitiligo/tratamento farmacológico , Vitiligo/patologia , Adulto JovemRESUMO
Vitiligo is characterized by a progressive loss of inherited skin color. The cause of the disease is still unknown. To date, there is accumulating in vivo and in vitro evidence for massive oxidative stress via hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)) in the skin of affected individuals. Autoimmune etiology is the favored theory. Since depletion of the essential amino acid L-tryptophan (Trp) affects immune response mechanisms, we here looked at epidermal Trp metabolism via tryptophan hydroxylase (TPH) with its downstream cascade, including serotonin and melatonin. Our in situ immunofluorescence and Western blot data reveal significantly lower TPH1 expression in patients with vitiligo. Expression is also low in melanocytes and keratinocytes under in vitro conditions. Although in vivo Fourier transform-Raman spectroscopy proves the presence of 5-hydroxytryptophan, epidermal TPH activity is completely absent. Regulation of TPH via microphthalmia-associated transcription factor and L-type calcium channels is severely affected. Moreover, dopa decarboxylase (DDC) expression is significantly lower, in association with decreased serotonin and melatonin levels. Computer simulation supports H(2)O(2)/ONOO(-)-mediated oxidation/nitration of TPH1 and DDC, affecting, in turn, enzyme functionality. Taken together, our data point to depletion of epidermal Trp by Fenton chemistry and exclude melatonin as a relevant contributor to epidermal redox balance and immune response in vitiligo.
Assuntos
Dopa Descarboxilase/metabolismo , Epiderme/metabolismo , Peróxido de Hidrogênio/metabolismo , Melatonina/metabolismo , Ácido Peroxinitroso/metabolismo , Serotonina/metabolismo , Triptofano Hidroxilase/metabolismo , Triptofano/metabolismo , Vitiligo/metabolismo , Adulto , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Humanos , Pessoa de Meia-Idade , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Triptofano/química , Vitiligo/imunologiaRESUMO
Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H(2)O(2)) and peroxynitrite (ONOO(-)) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid L-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H(2)O(2)/ONOO(-) deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H(2)O(2) supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.
Assuntos
Epiderme/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/metabolismo , Vitiligo/metabolismo , Simulação por Computador , Humanos , Peróxido de Hidrogênio/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Estresse Oxidativo , Ácido Peroxinitroso/metabolismo , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Triptofano/metabolismo , Vitiligo/imunologiaRESUMO
Quality of life in patients with vitiligo is impaired. This study explored the immediate effect of 20 days of climatotherapy at the Dead Sea on quality of life, coping with the disease, general well-being and individual stress levels in a group of 71 patients with vitiligo and 42 matched controls. The long-term effect was assessed after 12 months in 33/71 patients and 12/42 controls. Study instruments were Dermatology Life Quality Index, Beck Depression Inventory and the Adjustment to Chronic Skin Disorders Questionnaire. Stress measurements were based on cortisol and ß-endorphin concentrations in saliva samples. Quality of life was significantly improved at day 20 at the Dead Sea compared with day 1, and this was still significant after 12 months. Moreover, social anxiety/avoidance, anxious-depressive mood and helplessness as measured by the Adjustment to Chronic Skin Disorders Questionnaire were significantly reduced. There was no difference in levels of cortisol and ß-endorphin between patients and controls, indicating that stress per se is not a significant contributor in vitiligo. In conclusion, therapy in patient groups offers an effective tool for long-lasting improvement in quality of life and patients' well-being.
Assuntos
Antioxidantes/uso terapêutico , Catalase/uso terapêutico , Climatoterapia/psicologia , Psicoterapia de Grupo , Qualidade de Vida/psicologia , Vitiligo/psicologia , Vitiligo/terapia , Adaptação Psicológica , Adulto , Ansiedade/prevenção & controle , Terapia Combinada , Depressão/prevenção & controle , Feminino , Humanos , Hidrocortisona/metabolismo , Masculino , Pessoa de Meia-Idade , Saliva/metabolismo , Estresse Psicológico/metabolismo , Inquéritos e Questionários , Fatores de Tempo , beta-Endorfina/metabolismoRESUMO
The biochemistry of hair pigmentation is a complex field involving a plethora of protein and peptide mechanisms. The in loco factory for melanin formation is the hair follicle melanocyte, but it is common knowledge that melanogenesis results from a fine tuned concerted interaction between the cells of the entire dermal papilla in the anagen hair follicle. The key enzyme is tyrosinase to initiate the active pigmentation machinery. Hence, an intricate understanding from transcription of mRNA to enzyme activity, including enzyme kinetics, substrate supply, optimal pH, cAMP signaling, is a must. Moreover, the role of reactive oxygen species on enzyme regulation and functionality needs to be taken into account. So far our knowledge on the entire hair cycle relies on the murine model of the C57BL/6 mouse. Whether this data can be translated into humans still needs to be shown. This article aims to focus on the effect of H(2)O(2)-redox homeostasis on hair follicle pigmentation via tyrosinase, its substrate supply and signal transduction as well as the role of methionine sulfoxide repair via methionine sulfoxide reductases A and B (MSRA and B).
Assuntos
Cabelo/fisiologia , Pigmentação/fisiologia , Animais , Antioxidantes/metabolismo , Catecolaminas/biossíntese , AMP Cíclico/metabolismo , Cabelo/efeitos dos fármacos , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/fisiologia , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Modelos Biológicos , Monofenol Mono-Oxigenase/metabolismo , Oxirredução/efeitos dos fármacos , Fenilalanina/metabolismo , Pigmentação/efeitos dos fármacos , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismoRESUMO
Acne is the most common inflammatory skin disease in which IL-1 plays a central role. Although alpha-melanocyte-stimulating hormone has immunomodulatory effects, its usefulness as an anti-inflammatory agent in acne is hampered owing to its lipid- and pigment-inducing effects via activation of melanocortin receptors (MC-Rs). We used the immortalized human sebocyte line SZ95 as an in vitro model to investigate the anti-inflammatory potential of KdPT, a tripeptide derivative of the C-terminal end of alpha-melanocyte-stimulating hormone. KdPT potently suppressed IL-1beta-induced IL-6 and IL-8 expression. Mechanistically, KdPT decreased IL-1beta-mediated IkappaBalpha degradation, reduced nuclear accumulation of p65, and attenuated DNA binding of NF-kappaB. Moreover, KdPT reduced IL-1beta-mediated generation of intracellular reactive oxygen species, which contributed to IL-1beta-mediated cytokine induction. KdPT also reduced cell surface binding of fluorochrome-labeled IL-1beta in SZ95 sebocytes. Analysis of the crystal structure of the complex between IL-1beta/IL-1R type I (IL-1RI), followed by computer modeling of KdPT and subsequent modeling of the peptide receptor complex with the crystal structure of IL-1RI via manual docking, further predicted that the tripeptide, through several H-bonds and one hydrophobic bond, interacts with the IL-1RI. Importantly, KdPT did not bind to MC-1Rs, as demonstrated by blocking experiments with a peptide analog of Agouti signaling protein and by binding assays using MC-1R-expressing B16 melanoma cells. Accordingly, KdPT failed to induce melanogenesis. Our data demonstrate a promising anti-inflammatory potential of KdPT and point toward novel future directions in the treatment of acne-as well as of various other IL-1-mediated inflammatory diseases-with this small molecule.
Assuntos
Citocinas/antagonistas & inibidores , Imunossupressores/farmacologia , Interleucina-1beta/antagonistas & inibidores , Fragmentos de Peptídeos/fisiologia , Glândulas Sebáceas/citologia , Glândulas Sebáceas/imunologia , Transdução de Sinais/imunologia , alfa-MSH/fisiologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Citocinas/biossíntese , Proteínas de Ligação a DNA/fisiologia , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/metabolismo , Interleucina-1beta/fisiologia , Interleucina-6/antagonistas & inibidores , Interleucina-8/antagonistas & inibidores , Melanoma Experimental , Camundongos , Inibidor de NF-kappaB alfa , Glândulas Sebáceas/metabolismoRESUMO
Piebaldism is characterised by the absence of pigment in patches on the skin, usually present at birth. Mutations in the kit gene are documented. Clinically this disorder can mimic vitiligo. Here, we show for the first time the presence of oxidised pteridine-induced fluorescence in association with H2O2-mediated stress in piebald patches employing Wood's light and in vivo FT-Raman spectroscopy. In situ immunofluorescence data revealed low catalase and methionine sulphoxide reductase A (MSRA) levels whereas thioredoxin reductase and methionine sulphoxide reductase B (MSRB) are not affected. We also show low superoxide dismutase levels in these patients. The presence of thioredoxin reductase provides capacity to reduce H2O2, a mechanism which is absent in vitiligo. Importantly, this enzyme reduces biopterin back to the functioning cofactor 6-tetrahydrobiopterin. The absence of MSRA indicates deficient methionine sulphoxide repair in the cytosol, meanwhile the presence of MSRB is helpful to protect the nucleus. Taken together, we have identified H2O2-mediated stress in piebald skin with distinct differences to vitiligo.
Assuntos
Epiderme/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo/fisiologia , Piebaldismo/metabolismo , Adulto , Biópsia , Catalase/metabolismo , Epiderme/patologia , Imunofluorescência , Humanos , Técnicas In Vitro , Masculino , Metionina/análogos & derivados , Metionina/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Proteínas dos Microfilamentos , Piebaldismo/patologia , Pteridinas/metabolismo , Análise Espectral Raman , Superóxido Dismutase/metabolismo , Fatores de Transcrição/metabolismo , Vitiligo/metabolismo , Vitiligo/patologiaRESUMO
Vitiligo is characterized by a patchy loss of inherited skin color affecting approximately 0.5% of individuals of all races. Despite the absence of the protecting pigment and the overwhelming evidence for hydrogen peroxide (H(2)O(2))-induced oxidative stress in the entire epidermis of these patients, there is neither increased photodamage/skin aging nor a higher incidence for sun-induced nonmelanoma skin cancer. Here we demonstrate for the first time increased DNA damage via 8-oxoguanine in the skin and plasma in association with epidermal up-regulated phosphorylated/acetylated p53 and high levels of the p53 antagonist p76(MDM2). Short-patch base-excision repair via hOgg1, APE1, and polymerasebeta DNA repair is up-regulated. Overexpression of Bcl-2 and low caspase 3 and cytochrome c levels argue against increased apoptosis in this disease. Moreover, we show the presence of high epidermal peroxynitrite (ONOO(-)) levels via nitrotyrosine together with high nitrated p53 levels. We demonstrate by EMSA that nitration of p53 by ONOO(-) (300 x 10(-6) M) abrogates DNA binding, while H(2)O(2)-oxidized p53 (10(-3) M) enhances DNA binding capacity and prevents ONOO(-)-induced abrogation of DNA binding. Taken together, we add a novel reactive oxygen species to the list of oxidative stress inducers in vitiligo. Moreover, we propose up-regulated wild-type p53 together with p76(MDM2) as major players in the control of DNA damage/repair and prevention of photodamage and nonmelanoma skin cancer in vitiligo.
Assuntos
Dano ao DNA/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Vitiligo/tratamento farmacológico , Adulto , Apoptose/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Caspase 3/biossíntese , Proteínas de Ciclo Celular/metabolismo , Citocromos c/biossíntese , DNA/metabolismo , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Epiderme/efeitos dos fármacos , Epiderme/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Pessoa de Meia-Idade , Oxirredução , Ácido Peroxinitroso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Vitiligo/genética , Vitiligo/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor beta, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.
Assuntos
Colesterol/metabolismo , Células Epidérmicas , Melanócitos/citologia , Melanoma/patologia , Apolipoproteína B-100/metabolismo , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Modelos Biológicos , Pigmentação , Receptores de Estrogênio/metabolismo , Receptores de LDL/metabolismo , Fatores de Tempo , Vitamina D/metabolismoRESUMO
BACKGROUND: The epidermal accumulation of hydrogen peroxide (H(2)O(2)) has been documented in vitiligo. AIM: To assess the effect on disease cessation and repigmentation of the reduction/removal of H(2)O(2) using low-dose, narrow-band, ultraviolet-B (UV-B)-activated pseudocatalase PC-KUS in 71 children with vitiligo. METHODS: This uncontrolled and retrospective study included 45 girls and 26 boys (mean age, 10.3 years) who applied topical PC-KUS twice daily to the entire body surface without narrow-band UV-B dose increments. The affected body areas were documented by special photography at the first visit and after 8-12 months. The response was evaluated by two independent physicians as > 75% vs. < 75% total repigmentation of the face/neck, trunk, extremities, and hands/feet. Generalized (n = 61) and segmental (n = 10) vitiligo were evaluated as different entities. The effect of total-body, low-dose, narrow-band UV-B (0.15 mJ/cm(2)) monotherapy once daily without any increments and without application of PC-KUS was tested over 6 months in 10 children with vitiligo vulgaris (mean age, 8.4 years). RESULTS: One hundred per cent cessation was observed in 70 of the 71 children. More than 75% repigmentation was achieved in 66 of 71 patients on the face/neck, 48 of 61 on the trunk, and 40 of 55 on the extremities; however, repigmentation on the hands/feet was disappointing (five of 53). The response was independent of skin color, age of onset, duration of disease, other demographic features, and previous treatments. The follow-up after narrow-band UV-B monotherapy showed no significant repigmentation in all areas. Seven of 10 patients showed progression of their vitiligo. CONCLUSION: A reduction in epidermal H(2)O(2) using low-dose, narrow-band UV-B-activated pseudocatalase PC-KUS is an effective treatment for childhood vitiligo which can be safely performed at home.
Assuntos
Catalase/uso terapêutico , Terapia PUVA/métodos , Vitiligo/diagnóstico , Vitiligo/tratamento farmacológico , Administração Tópica , Adolescente , Fatores Etários , Distribuição de Qui-Quadrado , Criança , Pré-Escolar , Estudos de Coortes , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Seguimentos , Humanos , Masculino , Pesquisa , Estudos Retrospectivos , Medição de Risco , Fatores Sexuais , Resultado do TratamentoRESUMO
This review highlights the importance of enzymology, a field of great neglect in current cutaneous biology research. It was therefore the aim by using selected examples of epidermal enzymes and their action including some open questions to demonstrate the importance of this area. Clearly a thorough understanding of basic knowledge in this field is needed which in turn offers a plethora of innovative research projects for a curious mind. Moreover, in order to gain the closest understanding to the truth instead of generating esoteric results, emphasis is put forward on a holistic view utilizing a combination of modern and sometimes old methods to get the answer. Last but not least the bench work is only useful for the welfare of our patients if we can apply our basic knowledge.
Assuntos
Antioxidantes/metabolismo , Epiderme/enzimologia , Homeostase , Queratinócitos/enzimologia , Melanócitos/enzimologia , Fenômenos Fisiológicos da Pele , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Cálcio/metabolismo , Epiderme/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Sistemas Neurossecretores/enzimologiaRESUMO
Patients with vitiligo have low levels/activities of catalase in their lesional and non-lesional epidermis as well as in their epidermal melanocytes under in vitro conditions while the levels of catalase mRNA are unaltered. This defect leads to a build-up of hydrogen peroxide (H(2)O(2)) in the 10(-3) m range in the epidermis of these patients. In this context, it was realized that 10(-3) m H(2)O(2) deactivates catalase. Along this line, it was also suspected that catalase in patients with vitiligo possesses a special sensitivity to this reactive oxygen species (ROS), and indeed several heterozygous single nucleotide polymorphisms (SNPs) have been documented in the cat gene of these patients. Based on the 3D structure of human catalase monomer, we have modelled the influence of three selected SNPs on the enzyme active site, on the NADPH- as well as the tetramerization-binding domains. Our results show that these SNPs severely alter catalase structurally, which in turn should make the enzyme more susceptible to ROS compared with wild-type enzyme. Taken together, the work presented herein together with the earlier results on SNPs in the cat gene suggests a genetic predisposition for an altered catalase in patients with vitiligo.
Assuntos
Catalase/genética , Simulação por Computador , Modelos Químicos , Polimorfismo de Nucleotídeo Único , Vitiligo/genética , Substituição de Aminoácidos/genética , Sítios de Ligação , Catalase/química , Bases de Dados de Proteínas , Predisposição Genética para Doença , Humanos , Peróxido de Hidrogênio/metabolismo , NADP/química , Conformação Proteica , Vitiligo/enzimologiaRESUMO
Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H(2)O(2) yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H(2)O(2) up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription-polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography/mass spectrometry analysis were carried out. Our results identified the presence of XDH/XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H(2)O(2) in a concentration-dependent manner, where concentrations of 10(-6 )m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H(2)O(2)-mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10(-3 )m H(2)O(2). Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H(2)O(2)-mediated oxidative stress in vitiligo via H(2)O(2)-production and allantoin formation in the epidermal compartment.
Assuntos
Queratinócitos/enzimologia , Melanócitos/enzimologia , Estresse Oxidativo , Vitiligo/metabolismo , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo , Alantoína/biossíntese , Western Blotting , Estudos de Casos e Controles , Domínio Catalítico , Células Cultivadas , Simulação por Computador , Epiderme/metabolismo , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Imuno-Histoquímica , Modelos Químicos , Estrutura Molecular , Oxirredução , RNA Mensageiro/metabolismo , Ácido Úrico/metabolismoRESUMO
Despite many efforts, regulation of skin and hair pigmentation is still not fully understood. This article focuses mainly on controversial aspects in pigment cell biology which have emerged over the last decade. The central role of tyrosinase as the key enzyme in initiation of melanogenesis has been closely associated with the 6BH4 dependent phenylalanine hydroxylase (PAH) and tyrosine hydroxylase isoform I (THI) providing evidence for an old concept of the three enzyme theory in the initiation of the pigmentation process. In this context, it is noteworthy that intracellular L-phenylalanine uptake and turnover to L-tyrosine via PAH is vital for substrate supply of THI and tyrosinase. While PAH acts in the cytosol of melanocytes, THI and tyrosinase are sitting side by side in the melanosomal membrane. THI at low pH provides L-3,4-hydroxyphenylalanine L-DOPA which in turn is required for activation of met-tyrosinase. After an intramelanosomal pH change, possibly by the p-protein, has taken place, tyrosinase is subject to control by 6/7BH4 and the proopiomelanocortin (POMC) peptides alpha-MSH melanocyte stimulating hormone and beta-MSH in a receptor independent manner. cAMP is required for the activation of microphthalmia-associated transcription factor to induce expression of tyrosinase, for transcription of THI and for activation of PAH. The redundancy of the cAMP signal is discussed. Finally, we propose a novel mechanism involving H2O2 in the regulation of tyrosinase via p53 through transcription of hepatocyte nuclear factor 1alpha which in turn can also affect the POMC response.
Assuntos
Melaninas/metabolismo , Melanócitos/fisiologia , Transdução de Sinais/fisiologia , Pigmentação da Pele/fisiologia , Biopterinas/análogos & derivados , Biopterinas/fisiologia , Cálcio/fisiologia , AMP Cíclico/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , Melanócitos/enzimologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Melanossomas/fisiologiaRESUMO
Quinones are potentially dangerous substances generated from quinols via the intermediates semiquinone and hydrogen peroxide. Low semiquinone radical concentrations are acting as radical scavengers while high concentrations produce reactive oxygen species and quinones, leading to oxidative stress, apoptosis, and/or DNA damage. Recently it was recognised that thioredoxin reductase/thioredoxin (TR/T) reduces both p- and o-quinones. In this report we examine additional reduction mechanisms for p- and o-quinones generated from hydroquinone (HQ) and coenzyme Q10 and by 17beta-estradiol by the common cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)). Our results confirmed that TR reduces the p-quinone 1,4 benzoquinone and coenzyme Q10-quinone back to HQ and coenzyme Q10-quinol, respectively, while 6BH(4) has the capacity to reduce coenzyme Q10-quinone and the o-quinone produced from 17beta-estradiol. 6BH(4) is present in the cytosol and in the nucleus of epidermal melanocytes and keratinocytes as well as melanoma cells and colocalises with TR/T. Therefore we conclude that both mechanisms are major players in the prevention of quinone-mediated oxidative stress and DNA damage.
Assuntos
Biopterinas/análogos & derivados , Queratinócitos/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Quinonas/metabolismo , Biopterinas/fisiologia , Células Cultivadas , Dano ao DNA , Humanos , Peróxido de Hidrogênio/metabolismo , Oxirredução , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Patients with the depigmentation disorder vitiligo have low catalase expression/activities and constantly accumulate 10(-3) M hydrogen peroxide (H(2)O(2)) in their skin. Such high concentrations of H(2)O(2) oxidize L-methionine residues in proteins and peptides to (R and S)-methionine sulfoxide diasteriomers. In vivo FT-Raman Spectroscopy revealed the presence of methionine sulfoxide in the depigmented skin of patients with active vitiligo. In normal healthy human skin, methionine sulfoxide reductases A and B specifically reduce methionine sulfoxides (S) and (R), respectively, back to L-methionine consequently repairing oxidatively damaged proteins and peptides. In this report, we show that the expression/activities of MSRA and MSRB are significantly decreased in the epidermis of patients with vitiligo compared to healthy controls. Also, we used recombinant human MSRA and MSRB1 to show that both enzymes are deactivated by 10(-3) M H(2)O(2) by 85 and 40%, respectively. Structural modelling based on the crystal structure of human MSRA revealed that the active site of this enzyme is significantly altered after H(2)O(2)-mediated oxidation of L-methionine, L-tryptophan, and L-cysteine residues in its active site. Taken together, our results confirm that very important anti-oxidant enzymes are seriously affected in acute vitiligo.
Assuntos
Peróxido de Hidrogênio/metabolismo , Metionina/metabolismo , Oxirredutases/metabolismo , Fatores de Transcrição/metabolismo , Vitiligo/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Epiderme/enzimologia , Feminino , Humanos , Peróxido de Hidrogênio/toxicidade , Masculino , Metionina/análogos & derivados , Metionina/análise , Metionina Sulfóxido Redutases , Proteínas dos Microfilamentos , Modelos Moleculares , Estresse Oxidativo , Oxirredutases/análise , Oxirredutases/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Análise Espectral Raman , Estereoisomerismo , Fatores de Transcrição/análise , Fatores de Transcrição/efeitos dos fármacosRESUMO
The density of beta2-adrenoceptors is significantly decreased in both keratinocytes and peripheral blood lymphocytes from patients with atopic eczema. Furthermore both cell types showed a sixfold increase in the K(D) for the specific binding of the non-specific antagonists (-)-[(3)H]CGP 12177 and [(125) I]CYP to keratinocytes and lymphocytes respectively compared with healthy controls. Based on these results polymorphism in the beta2-adrenoceptor gene was suspected. Consequently the entire intronless beta2-adrenoceptor gene was isolated from whole blood and by RT-PCR from keratinocyte extracts of nine patients with atopic eczema and four healthy controls. DNA sequence analysis of nine atopic eczema patients confirmed a substitution in codon (1618) GCC (Ala(119)) to GAC (Asp(119)). This point mutation is expressed on the third transmembrane helix only 13A away from the established agonist/antagonist binding site at Asp(113). Computer modelling of this third transmembrane helix revealed substantial structural changes in the mutant compared with the wild type. Epidermal keratinocytes were established from one patient with atopic eczema (homozygote), the mother (heterozygote) and one age-matched healthy control. Cells were grown in media containing different concentrations of l-phenylalanine and receptor densities were determined. The results showed that cells with atopic eczema showed an increased sensitivity to l-phenylalanine concentrations with a narrow homeostasis compared with healthy controls. The heterozygous mother was only 50% as sensitive as the child. In summary, the results indicate that atopic eczema is associated with a single point mutation in the beta2-adrenoceptor gene leading to an impaired adrenergic response in the epidermis of these patients.
Assuntos
Dermatite Atópica/genética , Mutação Puntual , Receptores Adrenérgicos beta 2/genética , Adolescente , Antagonistas Adrenérgicos beta/farmacologia , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Ligação Competitiva/efeitos dos fármacos , Criança , Análise Mutacional de DNA , Dermatite Atópica/patologia , Feminino , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Fenilalanina/farmacologia , Propanolaminas/metabolismo , Estrutura Secundária de Proteína , Ensaio Radioligante , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismoRESUMO
This article focuses on recent advances in melanocyte biology and physiology. The major function of this neural crest-derived cell is the production of melanins. A "three enzyme theory" in the initiation of pigmentation is put forward and backed up by recent findings. A receptor-independent role for alpha-MSH and the cofactor (6R)-l-erythro-5,6,7,8-terahydrobiopterin (6BH(4)) in the control of tyrosinase is described. The importance of intramelanosomal pH for melanogenesis is covered. Finally, the redundancy of the cAMP and IP3/DAG/calcium signal in melanocytes together with the downstream events are highlighted. The main message of this article is that the intracellular H(2)O(2)- redox-equilibrium controls melanocyte function in a concentration-dependent manner.