RESUMO
BACKGROUND: Wrinkles represent a characteristic symptom of skin aging. In recent years, various studies have focused on their prevention and/or cure. However, clinical tests are still the only method available to directly detect and evaluate the anti-wrinkle efficacy of various substances. Moreover, no in vitro strategy for such anti-aging skin analysis has been reported. Therefore, in this study, we aimed to develop a novel technology to overcome these limitations. MATERIALS AND METHODS: Full-thickness (FT) skin wrinkle mimics with various widths and depths were fabricated using a collagen stamping method. These were analyzed and compared using 2D and 3D Swept Source-Optical Coherence Tomography (SS-OCT) imaging technologies. RESULTS: SS-OCT demonstrated superficial and cross-sectional images of the wrinkle mimics, and the size of the wrinkles was validated using image analysis. Retinoic acid treatment significantly decreased both the depth and width of wrinkles formed in the FT skin wrinkle mimics. CONCLUSIONS: Using 3D tissue engineering and SS-OCT imaging technologies, we developed a novel in vitro technique that can directly detect skin wrinkles. This significantly efficient method could lead to an alternative strategy for animal experiments and preclinical anti-aging research on the skin.
Assuntos
Envelhecimento da Pele , Tomografia de Coerência Óptica , Tomografia de Coerência Óptica/métodos , Pele/diagnóstico por imagem , Imageamento Tridimensional/métodosRESUMO
Particulate matter 2.5 (PM2.5), an atmospheric pollutant with an aerodynamic diameter of <2.5 µm, can cause serious human health problems, including skin damage. Since sebocytes are involved in the regulation of skin homeostasis, it is necessary to study the effects of PM2.5 on sebocytes. We examined the role of PM2.5 via the identification of differentially expressed genes, functional enrichment and canonical pathway analysis, upstream regulator analysis, and disease and biological function analysis through mRNA sequencing. Xenobiotic and lipid metabolism, inflammation, oxidative stress, and cell barrier damage-related pathways were enriched; additionally, PM2.5 altered steroid hormone biosynthesis and retinol metabolism-related pathways. Consequently, PM2.5 increased lipid synthesis, lipid peroxidation, inflammatory cytokine expression, and oxidative stress and altered the lipid composition and expression of factors that affect cell barriers. Furthermore, PM2.5 altered the activity of sterol regulatory element binding proteins, mitogen-activated protein kinases, transforming growth factor beta-SMAD, and forkhead box O3-mediated pathways. We also suggest that the alterations in retinol and estrogen metabolism by PM2.5 are related to the damage. These results were validated using the HairSkin® model. Thus, our results provide evidence of the harmful effects of PM2.5 on sebocytes as well as new targets for alleviating the skin damage it causes.
Assuntos
Poluentes Ambientais , Material Particulado , Citocinas/genética , Estrogênios , Perfilação da Expressão Gênica , Humanos , Lipídeos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Material Particulado/química , Material Particulado/toxicidade , RNA Mensageiro , Esteroides , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Fator de Crescimento Transformador beta/genética , Vitamina A , XenobióticosRESUMO
To overcome dermatological concerns causing abnormally excessive melanin synthesis, highly effective and safe skin depigmentation compounds have been identified in the cosmetic and pharmaceutical industries. Among several methods used to achieve skin depigmentation, inhibition of tyrosinase is one of the most effective, since tyrosinase is a crucial enzyme in melanogenesis. Herein, isolindleyin, a novel inhibitor of human tyrosinase, was introduced and evaluated for its anti-melanogenic effects in human epidermal melanocytes. The results revealed that isolindleyin was directly bound to tyrosinase and it suppressed melanin synthesis. The binding mode between isolindleyin and the active sites of human tyrosinase was investigated using computational molecular docking at the atomic level. Isolindleyin binding was found to be stabilized by hydrophobic interactions between His 367 and Val 377 and by hydrogen bonds between Ser 380 and Asn 364. The results of this study revealed the anti-melanogenic effects of isolindleyin that could contribute toward overcoming dermatological concerns that cause abnormally excessive melanin synthesis.
Assuntos
Glucosídeos/farmacologia , Melanócitos/efeitos dos fármacos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epidérmicas/efeitos dos fármacos , Glucosídeos/química , Glucosídeos/metabolismo , Humanos , Melaninas/metabolismo , Melanócitos/metabolismo , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/química , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: The in vivo evaluation of antipollution products has attracted attention due to increasing global pollution levels; however, it is expensive, time-consuming, and dangerous because of the harmful nature of fine dust. Therefore, this paper proposes an alternative in vitro assessment method and compares the fine dust blocking effectiveness of both methods for different antipollution products. MATERIALS AND METHODS: Initially, tests were conducted by spraying fine dust on human forearms and artificial leather without pretreatment for in vivo and in vitro samples, respectively. However, the same results were not obtained for both the methods. Therefore, we evaluated different leather conditions (color, drying time, and temperature) to determine the optimal artificial material for testing antipollution products before adopting beige artificial leather dried at 32°C for 30 minutes for further tests. RESULTS: The initial tests exhibited a significant difference (P < .05) between the two methods; however, the revised tests exhibited no significant difference (P > .05) between the two methods for either beige leather dried at room temperature (20°C-25°C) for 60 minutes or at 32°C for 30-60 minutes or white leather dried at 32°C for 60 min. Therefore, the in vitro method was deemed equivalent to the in vivo method. The effectiveness of fine dust blocking (P < .05) and the equivalence between the evaluation methods (P > .05) were confirmed for each antipollution product. CONCLUSION: The proposed method is economical, efficient, and safe, making it a novel and valid alternative for the evaluation of antipollution products.
Assuntos
Poeira , Material Particulado , HumanosRESUMO
Precise measurement of particulate matter (PM) on skin is important for managing and preventing PM-related skin diseases. This study aims to directly visualize the deposition and penetration of PM into human skin using a multimodal nonlinear optical (MNLO) imaging system. We successfully obtained PM particle signals by merging two different sources, C-C vibrational frequency and autofluorescence, while simultaneously visualizing the anatomical features of the skin via keratin, collagen, and elastin. As a result, we found morphologically dependent PM deposition, as well as increased deposition following disruption of the skin barrier via tape-stripping. Furthermore, PM penetrated more and deeper into the skin with an increase in the number of tape-strippings, causing a significant increase in the secretion of pro-inflammatory cytokines. Our results suggest that MNLO imaging could be a useful technique for visualizing and quantifying the spatial distribution of PM in ex vivo human skin tissues.
Assuntos
Imagem Multimodal/métodos , Imagem Óptica/métodos , Material Particulado/análise , Dermatopatias/diagnóstico , Pele/metabolismo , Humanos , Dermatopatias/metabolismoRESUMO
Particulate matters (PMs) increase oxidative stress and inflammatory response in different tissues. PMs disrupt the formation of primary cilia in various skin cells, including keratinocytes and melanocytes. In this study, we found that 2-isopropylmalic acid (2-IPMA) promoted primary ciliogenesis and restored the PM2.5-induced dysgenesis of primary cilia in dermal fibroblasts. Moreover, 2-IPMA inhibited the generation of excessive reactive oxygen species and the activation of stress kinase in PM2.5-treated dermal fibroblasts. Further, 2-IPMA inhibited the production of pro-inflammatory cytokines, including IL-6 and TNF-α, which were upregulated by PM2.5. However, the inhibition of primary ciliogenesis by IFT88 depletion reversed the downregulated cytokines by 2-IPMA. Moreover, we found that PM2.5 treatment increased the MMP-1 expression in dermal fibroblasts and a human 3-D-skin model. The reduced MMP-1 expression by 2-IPMA was further reversed by IFT88 depletion in PM2.5-treated dermal fibroblasts. These findings suggest that 2-IPMA ameliorates PM2.5-induced inflammation by promoting primary ciliogenesis in dermal fibroblasts.
Assuntos
Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Malatos/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Cílios/metabolismo , Cílios/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaloproteinase 1 da Matriz/genética , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Demands for safe depigmentation compounds are constantly increasing in the pharmaceutical and cosmetic industry, since the numerous relevant compounds reported to date have shown undesirable side effects or low anti-melanogenic effects. In this study, we reported three novel inhibitors of tyrosinase, which is the key enzyme in melanogenesis, identified using docking-based high throughput virtual screening of an in-house natural compound library followed by mushroom tyrosinase inhibition assay. Of the three compounds, gallacetophenone showed high anti-melanogenic effect in both human epidermal melanocytes and a 3D human skin model, MelanoDerm. The inhibitory effect of gallacetophenone on tyrosinase was elucidated by computational molecular modeling at the atomic level. Binding of gallacetophenone to the active site of tyrosinase was found to be stabilized by hydrophobic interactions with His367, Ile368, and Val377; hydrogen bonding with Ser380 and a water molecule bridging the copper ions. Thus, our results strongly suggested gallacetophenone as an anti-melanogenic ingredient that inhibits tyrosinase.
Assuntos
Acetofenonas/farmacologia , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Monofenol Mono-Oxigenase/antagonistas & inibidores , Pirogalol/análogos & derivados , Acetofenonas/química , Agaricales/enzimologia , Sequência de Aminoácidos , Biomarcadores , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Monofenol Mono-Oxigenase/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Pirogalol/química , Pirogalol/farmacologia , Pele/citologia , Pele/efeitos dos fármacos , Pele/metabolismo , Relação Estrutura-AtividadeRESUMO
Sugars are ubiquitous in organisms and well-known cosmetic ingredients for moisturizing skin with minimal side-effects. Glucose, a simple sugar used as an energy source by living cells, is often used in skin care products. Several reports have demonstrated that sugar and sugar-related compounds have anti-melanogenic effects on melanocytes. However, the underlying molecular mechanism by which glucose inhibits melanin synthesis is unknown, even though glucose is used as a whitening as well as moisturizing ingredient in cosmetics. Herein, we found that glucose significantly reduced the melanin content of α-melanocyte-stimulating hormone (MSH)-stimulated B16 cells and darkly pigmented normal human melanocytes with no signs of cytotoxicity. Furthermore, topical treatment of glucose clearly demonstrated its whitening efficacy through photography, Fontana-Masson (F&M) staining, and multi-photon microscopy in a pigmented 3D human skin model, MelanoDerm. However, glucose did not alter the gene expression or protein levels of major melanogenic proteins in melanocytes. While glucose potently decreased intracellular tyrosinase activity in melanocytes, it did not reduce mushroom tyrosinase activity in a cell-free experimental system. However, glucose was metabolized into lactic acid, which can powerfully suppress tyrosinase activity. Thus, we concluded that glucose indirectly inhibits tyrosinase activity through conversion into lactic acid, explaining its anti-melanogenic effects in melanocytes.
Assuntos
Glucose/farmacologia , Melanócitos/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Humanos , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Camundongos , Pele/citologia , Pele/metabolismo , alfa-MSH/farmacologiaRESUMO
Ageing is characterized by the accumulation of chronic and irreversible oxidative damage, chronic inflammation and organ dysfunction. To attenuate these ageing-related changes, various natural phytochemicals are often applied. Trans-communic acid (TCA), an active component of brown pine leaf extract, has antimicrobial and cancer chemopreventive activity and inhibits ultraviolet B (UVB)-induced MMP-1 expression. To determine whether the phytochemical TCA could affect the lifespan of an ageing model, Caenorhabditis elegans prevent ageing-related phenotypes of the skin. Caenorhabditis elegans (C. elegans) wild-type N2 and mutant strains were used in this study to explore the lifespan extension effect of TCA and its mechanism. We estimated lipofuscin accumulation and melanin levels, which are closely associated with skin senescence. Moreover, we explored the mechanism of action associated with ageing attenuation. We performed oxidative stress resistance and thermotolerance assays in C. elegans and surface plasmon resonance analysis of TCA binding with the forkhead box-O3a (FoxO3a) protein. TCA, which is the active component in Korean red pine (Pinus densiflora), attenuated ageing-related changes in skin cells. TCA lowered lipofuscin accumulation in fibroblasts and decreased melanin levels in melanocytes. These protective effects were mediated by activation of the representative longevity gene FoxO3a, which was induced by direct binding with TCA. Interestingly, TCA extended the lifespan of C. elegans, although it did not affect stress resistance, oxidative stress or thermotolerance. These results strongly suggest that TCA prevents the senescent phenotype of model organisms and exhibits beneficial effects on ageing-related skin phenotypes through direct FoxO3a activation.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Diterpenos/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Longevidade/efeitos dos fármacos , Animais , Caenorhabditis elegans , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Estudos de Viabilidade , Fibroblastos/efeitos dos fármacos , Humanos , Melanócitos/efeitos dos fármacos , Fitoterapia , PinusRESUMO
Chemical leukoderma is an acquired type of vitiligo that can be initiated by various exogenous chemicals such as hydroquinone (HQ), rhododendrol (RD), or 4-tertiary butyl phenol (4-TBP). Despite the importance of epidermal keratinocytes in diverse dermatological conditions, their toxicological role in chemical leukoderma is poorly understood. To elucidate their role in the pathogenesis of chemical leukoderma, genome-scale transcriptional analysis was performed in human epidermal keratinocytes (HEKs) treated with a sub-cytotoxic HQ concentration (10 µM). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-based functional enrichment analysis of HQ-induced differentially expressed genes (DEGs) revealed that HQ significantly upregulated DEGs related to the IL-17 signaling pathway and significantly downregulated DEGs associated with melanogenesis in HEKs. The meta-analysis between the HQ-induced and cytokine-induced transcriptional data (GSE53751) showed that 58 DEGs were commonly upregulated between HQ- and IL-17A-treated HEKs. Notably, the expression of IL36G was significantly increased in HEKs in response to both HQ and IL-17A. IL-36γ (2 µg/ml) directly inhibits melanin biosynthesis in cultured human epidermal melanocytes (HEMs) and downregulates the gene transcription of key enzymes in the melanogenesis pathway including TYR, DCT, and TYRP1. Moreover, IL-36γ autocrinally regulated keratinocyte function to produce the proinflammatory cytokines IL-36γ, IL-6, and CXCL8/IL-8 in a concentration-dependent manner, suggesting that IL-36γ may stimulate the amplification cycle of cutaneous inflammation. In this regard, hydroquinone-induced IL-36γ from human keratinocytes plays a pivotal role in the development of chemical leukoderma by autocrinally or paracrinally modulating the crosstalk between keratinocytes and melanocytes.
Assuntos
Hidroquinonas/toxicidade , Hipopigmentação/induzido quimicamente , Interleucina-1/fisiologia , Queratinócitos/fisiologia , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Citocinas/biossíntese , Humanos , Interleucina-17/farmacologia , Melanócitos/metabolismo , Transdução de Sinais , Vitiligo/etiologiaRESUMO
Melanin synthesis in melanocytes is affected by various cytokines. Here, we reported for the first time that tumor necrosis factor superfamily member 14 (TNFSF14) inhibits melanogenesis in the primary culture of human epidermal melanocytes. TNFSF14 is known to bind to its receptors herpes virus entry mediator (HVEM) and lymphotoxin ß receptor (LTßR) for signal transduction, but TNFSF14-induced hypopigmentation was independent of HVEM and LTßR in melanocytes. To explore signaling in melanocytes treated with TNFSF14, we performed RNA-seq and found that nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling is activated by TNFSF14. Further, we observed that inhibition of NF-kB effectively blocks the hypopigmentation induced by TNFSF14. We conclude that TNFSF14 inhibits melanogenesis in melanocytes via NF-κB signaling and could be applied in the treatment of cutaneous pigment disorders.
Assuntos
Melanócitos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Linhagem Celular , Humanos , Ativação Linfocitária/fisiologia , Receptor beta de Linfotoxina/metabolismo , Melaninas/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismoRESUMO
Benzophenone-3 (BP-3), which is extensively used in organic sunscreen, has phototoxic potential in human skin. Phosphodiesterase 4B (PDE4B) has a well-established role in inflammatory responses in immune cells. Currently, it is unknown if PDE4B is associated with BP-3-induced phototoxicity in normal human keratinocytes (NHKs). We found that BP-3 significantly increased PDE4B expression in ultraviolet B (UVB)-irradiated NHKs. Notably, BP-8, a sunscreen agent that shares the 2-hydroxy-4-methoxyphenyl methanone moiety with BP-3, also upregulated PDE4B expression in NHKs. Upon UVB irradiation, BP-3 upregulated the expression of pro-inflammatory factors, such as prostaglandin endoperoxide synthase 2, tumor necrosis factor α, interleukin 8, and S100A7, and downregulated the level of cornified envelope associated proteins, which are important in the development of the epidermal permeability barrier. The additive effects of UVB-activated BP-3 on the expression of both pro-inflammatory mediators and cornified envelope associated proteins were antagonized by treatment with the PDE4 inhibitor rolipram. The BP-3 and UVB co-stimulation-induced PDE4B upregulation and its association with the upregulation of pro-inflammatory mediators and the downregulation of epidermal differentiation markers were confirmed in a reconstituted three dimensional human epidermis model. Therefore, PDE4B has a role in the mechanism of BP-3-induced phototoxicity.
Assuntos
Benzofenonas/toxicidade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Dermatite Fototóxica/etiologia , Queratinócitos/efeitos dos fármacos , AMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Dinoprostona/biossíntese , Humanos , Interleucina-8/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Raios UltravioletaRESUMO
Adiponectin is an adipocytokine with insulin-sensitizing, anti-inflammatory, anti-atherosclerotic, and anti-aging properties. Compounds with the ability to promote adiponectin secretion are of interest for the development of anti-aging drugs to improve skin-aging phenotypes. In the phenotypic assay to measure adiponectin secretion during adipogenesis in human adipose tissue-derived mesenchymal stem cells (hAT-MSCs), kojyl cinnamate ester derivatives increased adiponectin secretion. A target identification study showed that the kojyl cinnamate ester derivatives competitively bound to peroxisome proliferator-activated receptor α/γ (PPARα/γ). The upregulation of adiponectin production induced by kojyl cinnamate ester derivatives was significantly correlated with PPARα and PPARγ binding activities. Kojyl cinnamate ester derivatives significantly increased the transcription of genes encoding cholesterol and fatty acid synthesizing enzymes in hAT-MSCs. Notably, the kojyl cinnamate esters upregulated the gene transcription of lipid metabolic enzymes in human epidermal keratinocytes, which are important in the integrity of skin permeability barrier. In addition, the kojyl cinnamate esters that function as PPARα/γ dual modulators inhibited ultraviolet B irradiation-induced inflammation in human epidermal keratinocytes. Therefore, kojyl cinnamate ester derivatives are a novel class of PPARα/γ dual agonists with the potential to improve skin-aging phenotypes.
Assuntos
Cinamatos/farmacologia , PPAR alfa/agonistas , PPAR gama/agonistas , Pironas/farmacologia , Adipogenia/efeitos dos fármacos , Adiponectina/genética , Cinamatos/síntese química , Cinamatos/química , Dinoprostona/metabolismo , Humanos , Inflamação/prevenção & controle , Queratinócitos/efeitos dos fármacos , Metaloproteinase 1 da Matriz/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , PPAR alfa/química , PPAR gama/química , Pironas/síntese química , Pironas/química , RNA Mensageiro/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Lipin-1 is an Mg(2+)-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1.
Assuntos
Diferenciação Celular , Regulação Enzimológica da Expressão Gênica , Queratinócitos/citologia , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Diglicerídeos/metabolismo , Regulação para Baixo , Ativação Enzimática , Células Epidérmicas , Técnicas de Silenciamento de Genes , Humanos , Recém-Nascido , Fosfatidato Fosfatase/deficiência , Proteína Quinase C-alfa/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
NO regulates a variety of physiological processes, including cell proliferation, differentiation, and inflammation. S-nitrosylation, a NO-mediated reversible protein modification, leads to changes in the activity and function of proteins. In particular, the role of S-nitrosylation during adipogenesis is largely unknown. We hypothesized that the normal physiological levels of NO, but not the excess levels generated under severe conditions, such as inflammation, may be critically involved in the proper regulation of adipogenesis. We found that endogenous S-nitrosylation of proteins was required for adipocyte differentiation. By performing a biotin-switch assay, we identified FAS, a key lipogenic enzyme in adipocytes, as a target of S-nitrosylation during adipogenesis. Interestingly, we also observed that the dimerization of FAS increased in parallel with the amount of S-nitrosylated FAS during adipogenesis. In addition, we found that exogenous NO enhanced the dimerization and the enzymatic activity of FAS. Moreover, site-directed mutagenesis of three predicted S-nitrosylation sites indicated that S-nitrosylation of FAS at Cys(1471)and Cys(2091), but not at Cys(1127), increased its enzymatic activity. Taken together, these results suggest that the S-nitrosylation of FAS at normal physiological levels of NO increases its activity through dimerization and may contribute to the proper regulation of adipogenesis.
Assuntos
Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Óxido Nítrico/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Adipócitos/citologia , Adipogenia , Cisteína/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Estrutura Quaternária de Proteína , Enxofre/metabolismoRESUMO
Atmospheric oxygen is important for the epidermis, as the skin epidermis is not greatly affected by blood circulation. Therefore, it is necessary to understand the effect of hypoxic signals on the epidermis as some environmental stimuli can induce skin hypoxia. Here, we investigated how hypoxia (1% O2) affected skin equivalents (SEs) and normal human epidermal keratinocytes. We found that hypoxia specifically decreased the protein levels of keratin 1 (K1)/keratin 10 (K10), a representative marker of the epidermal spinous layer in the epidermis. However, hypoxia-inducible factors, the major regulators of hypoxia, did not affect hypoxia-induced down-regulation of K1/K10. We also found that N-acetyl-l-cysteine (NAC), a reactive oxygen species scavenger, antagonized the hypoxia-induced reduction of K1/K10 in keratinocytes and SEs. In contrast to the findings for NAC, inhibitors that blocked reactive oxygen species generation did not cause recovery of K1/K10 protein levels under hypoxic conditions. Taken together, these results indicate that hypoxia leads to abnormal keratinocyte differentiation by down-regulating K1/K10 and that this phenomenon can be ameliorated by NAC.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Queratinas Tipo II/metabolismo , Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Diferenciação Celular/fisiologia , Hipóxia Celular/fisiologia , Células Cultivadas , HumanosRESUMO
Low-level formaldehyde exposure is inevitable in industrialized countries. Although daily-life formaldehyde exposure level is practically impossible to induce cell death, most of mechanistic studies related to formaldehyde toxicity have been performed in cytotoxic concentrations enough to trigger cell death mechanism. Currently, toxicological mechanisms underlying the sub-cytotoxic exposure to formaldehyde are not clearly elucidated in skin cells. In this study, the genome-scale transcriptional analysis in normal human keratinocytes (NHKs) was performed to investigate cutaneous biological pathways associated with daily life formaldehyde exposure. We selected the 175 upregulated differentially expressed genes (DEGs) and 116 downregulated DEGs in NHKs treated with 200µM formaldehyde. In the Gene Ontology (GO) enrichment analysis of the 175 upregulated DEGs, the endoplasmic reticulum (ER) unfolded protein response (UPR) was identified as the most significant GO biological process in the formaldeyde-treated NHKs. Interestingly, the sub-cytotoxic formaldehyde affected NHKs to upregulate two enzymes important in the cellular transsulfuration pathway, cystathionine γ-lyase (CTH) and cystathionine-ß-synthase (CBS). In the temporal expression analysis, the upregulation of the pro-inflammatory DEGs such as MMP1 and PTGS2 was detected earlier than that of CTH, CBS and other ER UPR genes. The metabolites of CTH and CBS, l-cystathionine and l-cysteine, attenuated the formaldehyde-induced upregulation of pro-inflammatory DEGs, MMP1, PTGS2, and CXCL8, suggesting that CTH and CBS play a role in the negative feedback regulation of formaldehyde-induced pro-inflammatory responses in NHKs. In this regard, the sub-cytotoxic formaldehyde-induced CBS and CTH may regulate inflammation fate decision to resolution by suppressing the early pro-inflammatory response.
Assuntos
Cistationina/metabolismo , Formaldeído/toxicidade , Inflamação/patologia , Queratinócitos/patologia , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , HumanosRESUMO
Psoriasin (S100A7), a member of the S100 protein family, is a well-known antimicrobial peptide and a signalling molecule which regulates cellular function and is highly expressed in hyperproliferative skin conditions such as atopic dermatitis (AD) and psoriasis with disrupted skin barrier function. However, its role in epidermal differentiation remains unknown. We examined the effect of S100A7 on epidermal differentiation in normal human keratinocytes (NHKs) and on a reconstituted human epidermis model. When NHKs were exposed to disruptive stimuli such as Staphylococcus aureus, ultraviolet irradiation and retinoic acid, the secretion of S100A7 into the culture medium increased and the expression of epidermal differentiation markers decreased. Treatment of NHKs with S100A7 significantly inhibited epidermal differentiation by reducing the expression of keratin 1, keratin 10, involucrin and loricrin and by increasing the expression of abnormal differentiation markers (keratin 6 and keratin 16). We verified that the MyD88-IκB/NF-κB signal cascade was activated via RAGE after S100A7 treatment, resulting in the upregulation of interleukin-6. Finally, we confirmed that S100A7 is a negative regulator of epidermal differentiation using a reconstituted human epidermis model. This study suggests that S100A7-related signalling molecules could be potent targets for recovering skin barrier function in AD and psoriasis where S100A7 is accumulated excessively.
Assuntos
Diferenciação Celular , Epiderme/metabolismo , Interleucina-6/metabolismo , Queratinócitos/metabolismo , Proteína A7 Ligante de Cálcio S100/metabolismo , Células Cultivadas , Células Epidérmicas , Humanos , Queratinócitos/citologia , Transdução de Sinais , Estresse FisiológicoRESUMO
(-)-Epigallocatechin-3-O-gallate (EGCG) has long been known as a potent inducer of keratinocyte differentiation. Although its molecular mechanisms have been extensively studied, its actions on human skin remain to be elucidated. In this study, we demonstrated that methylated EGCG and EGCG increase the expression of klotho, and that klotho functions as a downstream target of EGCG and methylated EGCG in keratinocyte differentiation. We demonstrated that methylated EGCG3 and EGCG induce morphological changes in normal human epidermal keratinocytes (NHEKs) that are related to up-regulation of klotho expression. We also demonstrated that a klotho-induced keratinocyte differentiation marker in NHEKs is inhibited by H-89, a protein kinase (PKA) inhibitor. These results suggest that methylated EGCG and EGCG may function as inducers of keratinocyte differentiation via transcriptional regulation of the klotho protein.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ácido Gálico/análogos & derivados , Glucuronidase/biossíntese , Queratinócitos/citologia , Biomarcadores , Linhagem Celular , Sobrevivência Celular , Ácido Gálico/farmacologia , Regulação da Expressão Gênica , Glucuronidase/genética , Células HEK293 , Liberação de Histamina/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Proteínas Klotho , Preparações de Plantas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Pele/efeitos dos fármacos , Sulfonamidas/farmacologia , Chá/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para CimaRESUMO
Subcutaneous adipose tissue (SAT), a vital energy reservoir and endocrine organ for maintaining systemic glucose, lipid, and energy homeostasis, undergoes significant changes with age. However, among the existing aging-related markers, only few genes are associated with SAT aging. In this study, weighted gene co-expression network analysis was used on a transcriptome of SAT obtained from the Genotype-Tissue Expression portal to identify biologically relevant, SAT-specific, and age-related marker genes. We found modules that exhibited significant changes with age and identified GYG2 as a novel key aging associated gene. The link between GYG2 and mitochondrial function as well as brown/beige adipocytes was supported using additional bioinformatics and experimental analyses. Additionally, we identified PPARG as the transcription factor of GYG2 expression. The newly discovered GYG2 marker can be used to not only determine the age of SAT but also uncover new mechanisms underlying SAT aging.