RESUMEN

The World Health Organization reports that 99% of the global population are exposed to pollution levels higher than the recommended air quality guidelines. Pollution-induced changes in the skin have begun to surface; however, the effects require further investigation so that effective protective strategies can be developed. This study aimed to investigate some of the aging-associated effects caused by ozone and particulate matter (PM) on human skin equivalents. Full-thickness skin equivalents were exposed to 0.01 µg/µL PM, 0.05 µg/µL PM, 0.3 ppm ozone, or a combination of 0.01 µg/µL PM and 0.3 ppm ozone, before skin equivalents and culture medium were harvested for histological/immunohistochemical staining, gene and protein expression analysis using qPCR, Western blotting, and ELISA. Markers include MMP-1, MMP-3, COL1A1, collagen-I, 4-HNE, HMGCR, and PGE2. PM was observed to induce a decrease in epidermal thickness and an enhanced matrix building phenotype, with increases in COL1A1 and an increase in collagen-I protein expression. By contrast, ozone induced an increase in epidermal thickness and was found to induce a matrix-degrading phenotype, with decreases in collagen-I gene/protein expression and increases in MMP-1 and MMP-3 gene/protein expression. Ozone was also found to induce changes in lipid homeostasis and inflammation induction. Some synergistic damage was also observed when combining ozone and 0.01 µg/µL PM. The results presented in this study identify distinct pollutant-induced effects and show how pollutants may act synergistically to augment damage; given individuals are rarely only exposed to one pollutant type, exposure to multiple pollutant types should be considered to develop effective protective interventions.

RESUMEN

The damaging effects of air pollution on the skin are becoming increasingly researched and the outcomes of this research are now a major influence in the selection and development of protective ingredients for skincare formulations. However, extensive research has not yet been conducted into the specific cellular defense systems that are being affected after exposure to such pollutants. Research investigating the affected systems is integral to the development of suitable interventions that are capable of augmenting the systems most impacted by air pollutant exposure. The following studies involved exposing primary human dermal fibroblasts to different concentrations of particulate matter and analyzing its effects on mitochondrial complex activity, nuclear factor erythroid 2-related factor 2 localization using immunocytochemistry and protein expression of electron transport chain complex proteins, sirtuin-1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) using western blotting. Particulate matter-induced alterations in both mitochondrial complex protein and activity, indicating oxidative stress, which was also complimented by increased expression of antioxidant proteins GSTP1/2 and SOD2. Particulate matter also seemed to modify expression of the proteins SIRT1 and PGC-1α which are heavily involved in the regulation of mitochondrial biogenesis and energy metabolism. Given the reported results indicating that particulate matter induces damage through oxidative stress and has a profound effect on mitochondrial homeostasis, interventions involving targeted mitochondrial antioxidants may help to minimize the damaging downstream effects of pollutant-induced oxidative stress originating from the mitochondria.

RESUMEN

With a large proportion of the world's population living in areas where air quality does not meet current WHO guidelines, combined with the knowledge that pollutants can interact with human skin, it is now of even greater importance that the effects of air pollutant exposure on human skin be investigated. To evaluate the damaging effects of a known component of air pollution (particulate matter) on human primary dermal fibroblasts. These studies were undertaken by exposing primary human dermal fibroblasts to different concentrations of particulate matter and analyzing the effects over time using resazurin reduction assays. Immunofluorescence microscopy was used to determine if particulate matter caused activation of the aryl hydrocarbon receptor, and phosphorylation of histone H2AX, a known marker of double-strand DNA breaks. Dot blotting was also used to analyze expression changes in secreted MMP-1, MMP-3, and TGFß. Particulate matter was found to dose-dependently increase cellular viability, activate the aryl hydrocarbon receptor, increase double-strand DNA breaks, and increase the expression of MMP-1, MMP-3, and TGFß. With the potential of air pollutants such as particulate matter to not only modulate the expression of proteins implicated in skin aging, but also affect cells at a genetic level, brings a pressing need for further investigation so protective strategies can be implemented.

Asunto(s)

Fibroblastos/efectos de los fármacos , Material Particulado/toxicidad , Envejecimiento de la Piel/efectos de los fármacos , Células Cultivadas , Roturas del ADN de Doble Cadena , Dermis/citología , Fibroblastos/metabolismo , Histonas/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Material Particulado/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

The ability of solar ultraviolet (UV) to induce skin cancer and photoaging is well recognized. The effect of the infrared (IR) and visible light (Vis) components of solar radiation on skin and their interaction with UV is less well known. This study compared the effects of physiologically relevant doses of complete (UV + Vis + IR) solar-simulated light and its individual components on matched primary dermal fibroblasts and epidermal keratinocytes from human donors on three biomarkers of cellular damage (reactive oxygen species (ROS) generation, mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) damage). There was a greater induction of ROS, mtDNA, and nDNA damage with the inclusion of the visible and IR components of solar-simulated light in primary fibroblast cells compared to primary keratinocytes (P < .001). Experiments using exposure to specific components of solar light alone or in combination showed that the UV, Vis, and IR components of solar light synergistically increased ROS generation in primary fibroblasts but not primary keratinocytes (P < .001). Skin cell lines were used to confirm these findings. These observations have important implications for different skin cell type responses to the individual and interacting components of solar light and therefore photodamage mechanisms and photoprotection interventions.

Asunto(s)

Biomarcadores/metabolismo , Rayos Infrarrojos , Queratinocitos/efectos de la radiación , Luz , Piel/citología , Rayos Ultravioleta , Células Cultivadas , Ensayo Cometa , ADN/metabolismo , ADN Mitocondrial/efectos de la radiación , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Queratinocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Air pollution is increasing beyond previous estimates and is viewed as the world's largest environmental health risk factor. Numerous clinical and epidemiological studies have highlighted the adverse effects of environmental pollutants on health. Although there is comparatively less research investigating the cutaneous effects of ambient pollution, there is growing recognition of the adverse effects on skin. In this article, we provide an overview of the nature of environmental pollution and highlight the current evidence detailing the effects on cutaneous health. There is convincing evidence demonstrating that air pollution has a detrimental impact on skin and can exacerbate skin disease. Further epidemiological and experimental studies are required to assess the short- and long-term deleterious effects of ambient pollutant exposure on skin. The future challenge would be to use this evidence to develop specific strategies to protect against pollution-induced damage and prevent the effects of "bad air getting under our skin."

Asunto(s)

Contaminantes Atmosféricos/toxicidad , Contaminación del Aire/efectos adversos , Piel/efectos de los fármacos , Humanos , Dióxido de Nitrógeno/toxicidad , Ozono/toxicidad , Material Particulado/toxicidad , Ácidos Ftálicos/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Piel/metabolismo

RESUMEN

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.

Asunto(s)

Daño del ADN/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Vitíligo/tratamiento farmacológico , Adulto , Apoptosis/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Caspasa 3/biosíntesis , Proteínas de Ciclo Celular/metabolismo , Citocromos c/biosíntesis , ADN/metabolismo , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Persona de Mediana Edad , Oxidación-Reducción , Ácido Peroxinitroso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regulación hacia Arriba , Vitíligo/genética , Vitíligo/metabolismo , Factores de Transcripción p300-CBP/metabolismo

RESUMEN

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.

Asunto(s)

Colesterol/metabolismo , Células Epidérmicas , Melanocitos/citología , Melanoma/patología , Apolipoproteína B-100/metabolismo , Línea Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Modelos Biológicos , Pigmentación , Receptores de Estrógenos/metabolismo , Receptores de LDL/metabolismo , Factores de Tiempo , Vitamina D/metabolismo

RESUMEN

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.

Asunto(s)

Catalasa/genética , Simulación por Computador , Modelos Químicos , Polimorfismo de Nucleótido Simple , Vitíligo/genética , Sustitución de Aminoácidos/genética , Sitios de Unión , Catalasa/química , Bases de Datos de Proteínas , Predisposición Genética a la Enfermedad , Humanos , Peróxido de Hidrógeno/metabolismo , NADP/química , Conformación Proteica , Vitíligo/enzimología

RESUMEN

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.

Asunto(s)

Melaninas/metabolismo , Melanocitos/fisiología , Transducción de Señal/fisiología , Pigmentación de la Piel/fisiología , Biopterinas/análogos & derivados , Biopterinas/fisiología , Calcio/fisiología , AMP Cíclico/fisiología , Humanos , Peróxido de Hidrógeno/metabolismo , Melanocitos/enzimología , Melanocitos/metabolismo , Melanosomas/metabolismo , Melanosomas/fisiología

RESUMEN

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.

Asunto(s)

Peróxido de Hidrógeno/metabolismo , Metionina/metabolismo , Oxidorreductasas/metabolismo , Factores de Transcripción/metabolismo , Vitíligo/enzimología , Sitios de Unión , Cristalografía por Rayos X , Epidermis/enzimología , Femenino , Humanos , Peróxido de Hidrógeno/toxicidad , Masculino , Metionina/análogos & derivados , Metionina/análisis , Metionina Sulfóxido Reductasas , Proteínas de Microfilamentos , Modelos Moleculares , Estrés Oxidativo , Oxidorreductasas/análisis , Oxidorreductasas/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Espectrometría Raman , Estereoisomerismo , Factores de Transcripción/análisis , Factores de Transcripción/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Biopterinas/análogos & derivados , Queratinocitos/metabolismo , Melanocitos/metabolismo , Melanoma/metabolismo , Quinonas/metabolismo , Biopterinas/fisiología , Células Cultivadas , Daño del ADN , Humanos , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Reductasa de Tiorredoxina-Disulfuro/metabolismo

RESUMEN

The human epidermis holds the full machinery for cholinergic signal transduction. However, the presence of the vesicular transporter (vesicular acetylcholine (ACh) transporter (VAChT)) for both choline and ACh has never been shown in this compartment. The results of this study confirm the presence of VAChT in cutaneous nerves and in both epidermal melanocytes and keratinocytes as well as in their nuclei using immunofluorescence labelling in situ and in vitro, Western blot analysis of cellular and nuclear extracts and reverse transcription-PCR. These results underline that ACh/choline transport in the non-neuronal epidermis is no different from the neuronal pathway. However, the function of VAChT in the nucleus remains to be shown.

Asunto(s)

Epidermis/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Acetilcolina/metabolismo , Especificidad de Anticuerpos , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Fibras Colinérgicas/metabolismo , Células Epidérmicas , Epidermis/inervación , Humanos , Queratinocitos/citología , Melanocitos/citología , ARN Mensajero/análisis , Proteínas de Transporte Vesicular de Acetilcolina/genética , Proteínas de Transporte Vesicular de Acetilcolina/inmunología

RESUMEN

(6R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) is crucial in the hydroxylation of L-phenylalanine-, L-tyrosine-, and L-tryptophan-regulating catecholamine and serotonin synthesis as well as tyrosinase in melanogenesis. The rate-limiting step of 6BH4 de novo synthesis is controlled by guanosine triphosphate (GTP) cyclohydrolase I (GTPCHI) and its feedback regulatory protein (GFRP), where binding of L-phenylalanine to GFRP increases enzyme activities, while 6BH4 exerts the opposite effect. Earlier it was demonstrated that the human epidermis holds the full capacity for autocrine 6BH4 de novo synthesis and recycling. However, besides the expression of epidermal mRNA for GFRP, the presence of a functioning GFRP feedback has never been shown. Therefore, it was tempting to investigate whether this important mechanism is present in epidermal cells. Our results identified indeed a functioning GFRP/GTPCHI axis in epidermal keratinocytes and melanocytes in the cytosol, adding the missing link for 6BH4 de novo synthesis which in turn controls cofactor supply for catecholamine and serotonin biosynthesis as well as melanogenesis in the human epidermis. Moreover, GFRP expression and GTPCHI activities have been found in the nucleus of both cell types. The significance of this result warrants further investigation.

Asunto(s)

Biopterinas/análogos & derivados , Epidermis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Biopterinas/biosíntesis , Western Blotting , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/química , Citosol/metabolismo , Epidermis/ultraestructura , GTP Ciclohidrolasa/análisis , GTP Ciclohidrolasa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Péptidos y Proteínas de Señalización Intracelular/genética , Queratinocitos/ultraestructura , Melanocitos/ultraestructura

RESUMEN

To date there is ample in vivo and in vitro evidence for increased epidermal and systemic hydrogen peroxide (H(2)O(2)) levels in vitiligo, which can be reduced with a topical application of a pseudocatalase-K.U. Schallreuter (PC-KUS) leading to the recovery of epidermal catalase levels as well as other enzymes in peripheral blood cells. Recently, the generation of H(2)O(2) by oxidative metabolism of estrogens and other aromatic steroids was documented. Therefore, it was tempting to follow estrogen-generated H(2)O(2) and its possible effect on DNA damage in peripheral blood lymphocytes from patients with vitiligo before and after the reduction of epidermal H(2)O(2) with pseudocatalase PC-KUS compared to controls. For this purpose, 20 Caucasian patients were grouped in treated responders (group A, n=11) and untreated active/acute disease (group B, n=9) and compared to Caucasian healthy controls (group C, n=7). Consequently, epidermal catalase protein expression in full skin biopsies was assessed using immunofluorescence labelling together with determination of basal H(2)O(2) levels in peripheral blood lymphocytes. To test the influence of estrogen on H(2)O(2) generation and DNA damage, freshly prepared peripheral blood lymphocytes from all three groups were used for the alkaline comet assay in the presence and absence of catalase. The results of this study demonstrated that reduction of epidermal H(2)O(2) leads to both increased epidermal catalase protein expression as well as decreased H(2)O(2) concentrations in lymphocytes. Moreover, a direct estrogen-mediated DNA damage was identified in both patient groups, which was absent in healthy controls. This effect was not abolished by catalase pointing to direct quinone-mediated DNA damage by estrogens in peripheral blood lymphocytes in vitiligo.

Asunto(s)

Daño del ADN , Estrógenos/toxicidad , Peróxido de Hidrógeno/metabolismo , Linfocitos/metabolismo , Quinonas/toxicidad , Vitíligo/metabolismo , Adulto , Catalasa/metabolismo , Catalasa/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo

RESUMEN

Oxidation of methionine residues by reactive oxygen (ROS) in protein structures leads to the formation of methionine sulfoxide which can consequently lead to a plethora of impaired functionality. The generation of methionine sulfoxide yields ultimately a diastereomeric mixture of the S and R sulfoxides. So far two distinct enzyme families have been identified. MSRA reduces methionine S-sulfoxide, while MSRB reduces the R-diastereomer. It has been shown that these enzymes are involved in regulation of protein function and in elimination of ROS via reversible methionine formation besides protein repair. Importantly, both enzymes require coupling to the NADPH/thioredoxin reductase/thioredoxin electron donor system. In this report, we show for the first time the expression and function of both sulfoxide reductases together with thioredoxin reductase in the cytosol as well as in the nucleus of epidermal melanocytes which are especially sensitive to ROS. Since this cell resides in the basal layer of the epidermis and its numbers and functions are reduced upon ageing and for instance also in depigmentation processes, we believe that this discovery adds an intricate repair mechanism to melanocyte homeostasis and survival.

Asunto(s)

Núcleo Celular/enzimología , Citosol/enzimología , Células Epidérmicas , Melanocitos/enzimología , Oxidorreductasas/metabolismo , Fraccionamiento Celular , Células Cultivadas , Transporte de Electrón , Epidermis/enzimología , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Metionina Sulfóxido Reductasas , Proteínas de Microfilamentos , Oxidorreductasas/genética , ARN Mensajero/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Factores de Transcripción

RESUMEN

The human skin holds the full machinery for pro-opiomelanocortin processing. The alpha-melanocyte-stimulating hormone (alpha-MSH)/melanocortin-1-receptor cascade has been implicated as a major player via the cAMP signal in the control of melanogenesis. Only very recently the beta-endorphin/mu-opiate receptor signal has been added to the list of regulators of melanocyte dendricity and melanin formation. In this context it was reported that (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) can act as an allosteric inhibitor of tyrosinase, the key enzyme in melanogenesis, and this inhibition is reversible by both alpha- and beta-MSH. It was also shown earlier that 7BH(4), the isomer of 6BH(4), is twice as active in this inhibition reaction. However, as yet it is not known whether 7BH(4) is indeed present in loco in the melanosome. We here provide evidence that this isomer is present in this organelle in a concentration range up to 50 x 10(-6) M. Determination of beta-MSH in melanosomal extracts yielded 10 pg/mg protein. Moreover, we demonstrate reactivation of the 7BH(4)/tyrosinase inhibitor complex by beta-MSH, whereas alpha-MSH failed to do so. Furthermore, we show intra-melanosomal l-dopa formation from dopachrome by 7BH(4) in a concentration range up to 134 x 10(-6) M. Based on these results, we propose a new receptor-independent mechanism in the control of tyrosinase/melanogenesis by beta-MSH and the pterin 7BH(4).

Asunto(s)

Biopterinas/análogos & derivados , Células Epidérmicas , Melanocitos/metabolismo , Pigmentación de la Piel/efectos de los fármacos , beta-MSH/farmacología , Animales , Biopterinas/farmacología , Western Blotting/métodos , Línea Celular Tumoral , Células Cultivadas , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/farmacología , Humanos , Indolquinonas/metabolismo , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Levodopa/análisis , Levodopa/metabolismo , Melanocitos/efectos de los fármacos , Ratones , Microscopía Inmunoelectrónica/métodos , Monofenol Monooxigenasa/antagonistas & inhibidores

RESUMEN

The human epidermis has the full machinery for autocrine L-phenylalanine turnover to L-tyrosine in keratinocytes and melanocytes. Phenylalanine hydroxylase (PAH) activities increase linearly with inherited skin colour (skin phototype I-VI, Fitzpatrick classification) yielding eightfold more activities in black skin compared to white skin. Moreover, UVB irradiation (1 MED) significantly increases epidermal PAH activities 24 h after exposure. Importantly, L-phenylalanine uptake and turnover in the pigment forming melanocytes is vital for initiation of melanogenesis. In this context it was shown that the uptake of this amino acid is regulated by calcium. The depigmentation disorder vitiligo provides a unique model to follow impaired L-phenylalanine turnover in the skin as well as in serum because affected individuals hold an impaired epidermal 6BH4 de novo synthesis/recycling and regulation including low epidermal PAH activities. After overnight fasting and oral loading with L-phenylalanine (100 mg/kg body weight), 29.6% of 970 patients tested (n=287/970) yielded serum phenylalanine/tyrosine ratios >or=4 and 35.3% (n=342/970) had mild to moderate hyperphenylalaninaemia (HPA), while 9.3% (n=90/970) had both serum L-phenylalanine levels >or=2.0 mg/dl and phe/tyr ratios >or=4.0. Isolated HPA was found in 26% (n=252/970), whereas 20.3% had only increased ratios (n=197/970). None of the patients had phenylketonuria and the family history for this metabolic disease was negative. The IQ followed normal Gaussian distribution. In vitro L-phenylalanine uptake/turnover studies on primary epidermal melanocytes originating from these patients demonstrated a significantly decreased calcium dependent L-phenylalanine uptake and turnover compared to healthy control cells. Based on our observation, we would like to propose that phenylalanine uptake/turnover is under tight control by calcium which in turn could offer an additional novel mechanism in the aetiology of HPA.

Asunto(s)

Epidermis/metabolismo , Fenilalanina/metabolismo , Vitíligo/metabolismo , Adolescente , Adulto , Calcio/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenilalanina/sangre , Fenilalanina/farmacocinética , Tirosina/sangre

Asunto(s)

Monofenol Monooxigenasa/metabolismo , Pteridinas/farmacología , Animales , Humanos

RESUMEN

Recently two alternative mechanisms have been put forward for the inhibition of tyrosinase by 6R-l-erythro 5,6,7,8-tetrahydrobiopterin (6BH(4)). Initially allosteric uncompetitive inhibition was demonstrated due to 1:1 binding of 10(-6)M 6BH(4) to a specific domain 28 amino acids away from the Cu(A) active site of the enzyme. Alternatively it was then shown that 10(-3)M 6BH(4) inhibit the reaction by the reduction of the product dopaquinone back to l-dopa. In the study presented herein we have used two structural analogues of 6BH(4) (i.e., 6,7-(R,S)-dimethyl tetrahydrobiopterin and 6-(R,S)-tetrahydromonapterin) confirming classical uncompetitive inhibition due to specific binding of the pyrimidine ring of the pterin moiety to the regulatory domain on tyrosinase. Under these conditions there was no reduction of l-dopaquinone back to l-dopa by both cofactor analogues. Inhibition of tyrosinase by 6BH(4) occurs in the concentration range of 10(-6)M after preactivation with l-tyrosine and this mechanism uncouples the enzyme reaction producing H(2)O(2) from O(2). Moreover, a direct oxidation of 6BH(4) to 7,8-dihydrobiopterin by tyrosinase in the absence of the substrate l-tyrosine was demonstrated. The enzyme was activated by low concentrations of H(2)O(2) (<0.3 x 10(-3)M), but deactivated at concentrations in the range 0.5-5.0 x 10(-3)M. In summary, our results confirm a major role for 6BH(4) in the regulation of human pigmentation.

Asunto(s)

Biopterinas/análogos & derivados , Biopterinas/química , Peróxido de Hidrógeno/química , Monofenol Monooxigenasa/química , Tirosina/química , Sitios de Unión , Activación Enzimática , Inhibidores Enzimáticos/química , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxidación-Reducción , Unión Proteica , Pteridinas/química