RESUMO
Psoriasis is a common skin disease, of which pathogenesis involves the increase of inflammatory reaction in epidermal cells. In an attempt to find therapeutics for psoriasis, we found that cucurbitacin B has an inhibitory potential on imiquimod-induced inflammation of keratinocytes. Cucurbitacin B significantly inhibited imiquimod-induced expression of crucial psoriatic cytokines, such as IL-8 and CCL20, via down-regulation of NF-κB and STAT3 signaling pathway in human keratinocytes. In addition, keratinocyte proliferation was markedly inhibited by cucurbitacin B. The potential beneficial effect of cucurbitacin B on psoriasis was further validated in imiquimod-induced psoriasiform dermatitis of experimental animal. Topical application of cucurbitacin B resulted in significant reduction of epidermal hyperplasia and inflammatory cytokines production, and ameliorated the psoriatic symptom. Taken together, these results suggest that cucurbitacin B may be a potential candidate for the treatment of psoriasis.
Assuntos
Aminoquinolinas/farmacologia , Dermatite/tratamento farmacológico , Triterpenos/farmacologia , Sequência de Bases , Primers do DNA , Humanos , Imiquimode , Técnicas In Vitro , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Inhibitor of DNA binding 1 (Id1) is a basic helix-loop-helix (bHLH) protein that has a variety of functional roles in cellular events including differentiation, cell cycle and cancer development. In addition, it has been demonstrated that Id1 is related with TGF-ß and Smad signaling in various biological conditions. In this study, we investigated the effect of Id1 on TGF-ß-induced collagen expression in human dermal fibroblasts. When Id1-b isoform was overexpressed, TGF-ß-induced collagen expression was markedly inhibited. Consistent with this result, Id1-b significantly inhibited TGF-ß-induced collagen gel contraction. In addition, Id1-b inhibited TGF-ß-induced phosphorylation of Smad2 and Smad3. Finally, immunohistochemistry showed that Id1 expression was decreased in fibrotic skin diseases while TGF-ß signaling was increased. Together, these results suggest that Id1 is an inhibitory regulator on TGF-ß-induced collagen expression in dermal fibroblasts.
Assuntos
Colágeno Tipo I/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Células Cultivadas , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Regulação para Baixo , Fibroblastos/metabolismo , Fibrose , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Transdução de Sinais , Pele/citologia , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Genkwadaphnin is a daphnane diterpene ester molecule isolated from the flower buds of Daphne genkwa. In the present study, we investigated the apoptosis-inducing effect of genkwadaphnin in squamous cell carcinoma (SCC) cells. Apoptosis was triggered in SCC12 cells following genkwadaphnin treatment in a time- and concentration-dependent manner. Genkwadaphnin treatment increased phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Knockdown of JNK and p38 MAPK by recombinant adenovirus expressing microRNA (miR) resulted in significant inhibition of genkwadaphnin-induced apoptosis in SCC12 cells. Finally, pretreatment with the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) markedly reduced SCC12 cell apoptosis, concomitant with significant inhibition of MAPK activation. These results indicate that genkwadaphnin has the potential to induce apoptosis in SCC cells, providing information on which to base further research with the aim of developing a cure for SCC.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Diterpenos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação , Neoplasias Cutâneas/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Androgens are important hormones that influence sebum production from the sebaceous glands. Human facial skin can be categorized as T- and U-zones, which are areas with high and low levels of sebum secretion, respectively. This study was performed to investigate whether there are topographical differences in androgen receptor (AR) expression related to regional variations in facial sebum secretion. The results of in vivo analysis indicated a statistically significant increase in AR expression in the sebaceous gland T-zones compared with the U-zones. In vitro experiments using human primary sebocytes also yielded similar results, with higher levels of AR protein and mRNA expression in T-zones. The results of this study suggested that differences in androgen susceptibility may be an important factor influencing regional differences in sebum production in human facial skin.
Assuntos
Androgênios/metabolismo , Sebo/metabolismo , Pele/metabolismo , Idoso , Face , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Glândulas Sebáceas/citologia , Glândulas Sebáceas/metabolismo , Pele/anatomia & histologia , Distribuição TecidualRESUMO
In this study, we investigated the role of glucocorticoid receptor (GR) in epidermal keratinocytes. In adult normal human skin, GR was highly expressed in the upper layers of the epidermis. Consistent with normal skin, GR expression was increased after calcium treatment of HaCaT keratinocytes cultured in vitro, suggesting that GR is involved in keratinocyte differentiation process. Overexpression of GR using an adenovirus showed that expression of involucrin, an early differentiation marker of keratinocytes, was markedly increased due to GR overexpression. However, treatment with dexamethasone, a GR agonist, did not increase involucrin expression. Overexpression of GR led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK) in the absence of glucocorticoid, suggesting that the GR effect on involucrin expression is related to activation of intracellular signaling cascades. This idea was supported by the fact that GR-mediated involucrin induction was abolished after treatment with JNK and ERK inhibitors. In addition, GR mutants lacking the ligand-binding domain increased involucrin expression concomitantly with increase of ERK phosphorylation. Together, these results suggest that GR modulates involucrin expression of keratinocytes by regulating the intracellular signaling network in a ligand-independent manner.
Assuntos
Diferenciação Celular/genética , Precursores de Proteínas/biossíntese , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Regulação da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Queratinócitos/metabolismo , Fosforilação , Transdução de Sinais/genéticaRESUMO
The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Psoríase/metabolismo , Actinas/metabolismo , Estudos de Casos e Controles , Hipóxia Celular , Movimento Celular , Proliferação de Células , Regulação para Baixo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP40/genética , Células HeLa , Proteínas de Choque Térmico , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Chaperonas Moleculares , Fosforilação , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Psoríase/patologia , Transdução de Sinais , Pele/metabolismo , Pele/patologiaRESUMO
The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Autofagia/imunologia , Regulação para Baixo/imunologia , Mediadores da Inflamação/fisiologia , Queratinócitos/imunologia , Queratinócitos/patologia , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Peptídeos Catiônicos Antimicrobianos/biossíntese , Linhagem Celular , Citocinas/biossíntese , Dermatite Atópica/imunologia , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Células HEK293 , Humanos , Queratinócitos/metabolismo , NF-kappa B/fisiologia , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1 , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/fisiologia , Receptor 6 Toll-Like/metabolismo , Receptor 6 Toll-Like/fisiologia , CatelicidinasRESUMO
BACKGROUND: Recently, autologous platelet-rich plasma (PRP) has attracted attention in various medical fields, including plastic and orthopedic surgery and dermatology, for its ability to promote wound healing. PRP has been tested during facelift and hair transplantation to reduce swelling and pain and to increase hair density. OBJECTIVE: To investigate the effects of PRP on hair growth using in vivo and in vitro models. METHODS: PRP was prepared using the double-spin method and applied to dermal papilla (DP) cells. The proliferative effect of activated PRP on DP cells was measured. To understand the mechanisms of activated PRP on hair growth, we evaluated signaling pathways. In an in vivo study, mice received subcutaneous injections of activated PRP, and their results were compared with control mice. RESULTS: Activated PRP increased the proliferation of DP cells and stimulated extracellular signal-regulated kinase (ERK) and Akt signaling. Fibroblast growth factor 7 (FGF-7) and beta-catenin, which are potent stimuli for hair growth, were upregulated in DP cells. The injection of mice with activated PRP induced faster telogen-to-anagen transition than was seen on control mice. CONCLUSIONS: Although few studies tested the effects of activated PRP on hair growth, this research provides support for possible clinical application of autologous PRP and its secretory factors for promotion of hair growth.
Assuntos
Folículo Piloso/crescimento & desenvolvimento , Cabelo/crescimento & desenvolvimento , Plasma Rico em Plaquetas , Pele/citologia , Animais , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator 7 de Crescimento de Fibroblastos/metabolismo , Cabelo/metabolismo , Folículo Piloso/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Pele/metabolismo , Regulação para Cima , beta Catenina/metabolismoRESUMO
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/imunologia , Epiderme/enzimologia , Queratinócitos/enzimologia , Psoríase/enzimologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular , Ciclina D1/imunologia , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/imunologia , Quinase 4 Dependente de Ciclina/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Epiderme/patologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fator 1 Induzível por Hipóxia/imunologia , Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/enzimologia , Indutores de Interferon/farmacologia , Interleucina-8/imunologia , Interleucina-8/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Poli I-C/farmacologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Psoríase/patologia , RNA Interferente Pequeno/imunologia , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologia , Transfecção , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Zimosan/farmacologia , CatelicidinasRESUMO
Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.
Assuntos
Células Epidérmicas , Proteínas de Homeodomínio/metabolismo , Queratinócitos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Humanos , Queratinócitos/citologia , Peixe-Zebra/embriologia , Proteína Homeobox PITX2RESUMO
Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.
Assuntos
Animais Geneticamente Modificados/genética , Técnicas de Transferência de Genes , Genes de Troca , Transgenes , Peixe-Zebra/genética , AnimaisRESUMO
Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.
Assuntos
Interações Hospedeiro-Patógeno , Imunidade Inata , Queratinócitos , Proteínas de Membrana/metabolismo , Mycobacterium ulcerans/patogenicidade , Proteínas do Tecido Nervoso/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Células Epidérmicas , Epiderme/imunologia , Epiderme/microbiologia , Regulação da Expressão Gênica/imunologia , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinócitos/microbiologia , Lectinas Tipo C , Proteínas de Membrana/genética , Mycobacterium ulcerans/imunologia , Proteínas do Tecido Nervoso/genética , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , CatelicidinasRESUMO
BACKGROUND: Atherosclerosis is the main cause of cardiovascular diseases such as ischemic stroke and coronary heart disease. Gene-specific promoter methylation changes have been suggested as one of the causes underlying the development of atherosclerosis. We aimed to identify and validate specific genes that are differentially expressed through promoter methylation in atherosclerotic plaques. We performed the present study in four steps: (1) profiling and identification of gene-specific promoter methylation changes in atherosclerotic tissues; (2) validation of the promoter methylation changes of genes in plaques by comparison with non-plaque intima; (3) evaluation of promoter methylation status of the genes in vascular cellular components composing atherosclerotic plaques; and (4) evaluation of promoter methylation differences in genes among monocytes, T cells, and B cells isolated from the blood of ischemic stroke patients. RESULTS: Upon profiling, AIRE1, ALOX12, FANK1, NETO1, and SERHL2 were found to have displayed changes in promoter methylation. Of these, AIRE1 and ALOX12 displayed higher methylation levels in plaques than in non-plaque intima, but lower than those in the buffy coat of blood. Between inflammatory cells, the three genes were significantly less methylated in monocytes than in T and B cells. In the vascular cells, AIRE1 methylation was lower in endothelial and smooth muscle cells. ALOX12 methylation was higher in endothelial, but lower in smooth muscle cells. Immunofluorescence staining showed that co-localization of ALOX12 and AIRE1 was more frequent in CD14(+)-monocytes than in CD4(+)-T cell in plaque than in non-plaque intima. CONCLUSIONS: Promoter methylation changes in AIRE1 and ALOX12 occur in atherosclerosis and can be considered as novel epigenetic markers.
Assuntos
Araquidonato 12-Lipoxigenase/genética , Aterosclerose/genética , Epigênese Genética , Fatores de Transcrição/genética , Aterosclerose/metabolismo , Biomarcadores/metabolismo , Metilação de DNA , Endotélio Vascular/metabolismo , Linfócitos/metabolismo , Monócitos/metabolismo , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/genética , Regiões Promotoras Genéticas , Proteína AIRERESUMO
Alopecia areata (AA) is a chronic, relapsing hair-loss disorder that is considered to be a T-cell-mediated autoimmune disease. Several animal models for AA have been created to investigate the pathophysiology and screen for effective therapeutic targets. As C3H/HeJ mice develop AA spontaneously in a low frequency, a novel animal model is needed to establish an AA-like condition faster and more conveniently. In this study, we present a novel non-invasive AA rodent model that avoids skin or lymph-node cell transfer. We simply injected C3H/HeJ mice subcutaneously with interferon-gamma (IFNγ) along with polyinosinic:polycytidylic acid (poly[I:C]), a synthetic dsRNA, to initiate innate immunity via inflammasome activation. Approximately 80% of the IFNγ and poly(I:C) co-injected mice showed patchy AA lesions after 8 weeks. None of the mice displayed hair loss in the IFNγ or poly(I:C) solely injection group. Immunohistochemical staining of the AA lesions revealed increased infiltration of CD4+ and CD8+ cells infiltration around the hair follicles. IFNγ and poly(I:C) increased the expression of NLRP3, IL-1ß, CXCL9, CXCL10, and CXCL11 in mouse skin. Taken together, these findings indicate a shorter and more convenient means of AA animal model induction and demonstrate that inflammasome-activated innate immunity is important in AA pathogenesis.
Assuntos
Alopecia em Áreas/imunologia , Modelos Animais de Doenças , Interferon gama/efeitos adversos , Poli I-C/efeitos adversos , Alopecia em Áreas/induzido quimicamente , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Quimiocinas CXC/metabolismo , Sinergismo Farmacológico , Feminino , Humanos , Injeções Subcutâneas , Interferon gama/administração & dosagem , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Poli I-C/administração & dosagemRESUMO
The epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1 is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affects skin homeostasis and hair morphogenesis, we generated epidermis-specific Crif1 cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1 cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis of Crif1 cKO mice was disrupted by down-regulation of Wnt/ß-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.
Assuntos
Proteínas de Ciclo Celular/deficiência , Epiderme/metabolismo , Deleção de Genes , Cabelo/anormalidades , Morfogênese/genética , Anormalidades da Pele/genética , Animais , Apoptose/genética , Proliferação de Células , Epiderme/anormalidades , Marcação de Genes , Homeostase , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinócitos/ultraestrutura , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Via de Sinalização WntRESUMO
Alopecia areata (AA), a chronic, relapsing, hair-loss disorder, is considered to be a T cell-mediated autoimmune disease. It affects approximately 1.7% of the population, but its precise pathogenesis remains to be elucidated. Despite the recent attention focused on the roles of inflammasomes in the pathogenesis of autoinflammatory diseases, little is known about inflammasome activation in AA. Thus, in this study, we investigated the pattern of NLRP3 inflammasome activation in the outer root sheath (ORS) cells of hair follicles. We found that interleukin (IL)-1ß and caspase-1 expression was increased in hair follicle remnants and inflammatory cells of AA tissue specimens. After stimulation of ORS cells with the double-stranded (ds)RNA mimic polyinosinic:polycytidylic acid (poly[I:C]), the activation of caspase-1 and secretion of IL-1ß were enhanced. Moreover, NLRP3 knockdown decreased this poly(I:C)-induced IL-1ß production. Finally, we found that high-mobility group box 1 (HMGB1) translocated from the nucleus to the cytosol and was secreted into the extracellular space by inflammasome activation. Taken together, these findings suggest that ORS cells are important immunocompetent cells that induce NLRP3 inflammasomes. In addition, dsRNA-induced IL-1ß and HMGB1 secretion from ORS cells may contribute to clarifying the pathogenesis and therapeutic targets of AA.
Assuntos
Folículo Piloso/imunologia , NF-kappa B/imunologia , RNA de Cadeia Dupla/imunologia , Transdução de Sinais/imunologia , Linhagem Celular Transformada , Proteína HMGB1/imunologia , Folículo Piloso/patologia , Humanos , Inflamassomos/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Interleucina-1beta/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Poli I-C/farmacologia , RNA de Cadeia Dupla/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Hyaluronic acid (HA) is the major glycosaminoglycan in the extracellular matrix and has been implicated in several functions in skin cells. However, evidence is lacking regarding the HA signaling in sebaceous glands, and its potential role needs to be clarified. We investigated the role of HA in lipid production in sebaceous glands in an experimental study of human sebocytes followed by a clinical study. We first examined the effects of HA on sebaceous glands in hamsters and intradermal injection of HA into hamster auricles decreased both the size of sebaceous glands and the level of lipid production. We demonstrated that human skin sebaceous glands in vivo and sebocytes in vitro express CD44 (HA binding receptor) and that HA downregulates lipid synthesis in a dose-dependent manner. To evaluate the clinical relevance of HA in human skin, 20 oily participants were included in a double-blind, placebo-controlled, split-face study, and the HA-treated side showed a significant decrease in sebum production. The results of this study indicate that HA plays a functional role in human sebaceous gland biology and HA signaling is an effective candidate in the management of disorders in which sebum production is increased.
Assuntos
Ácido Hialurônico/administração & dosagem , Lipídeos/biossíntese , Glândulas Sebáceas/efeitos dos fármacos , Glândulas Sebáceas/metabolismo , Sebo/efeitos dos fármacos , Adulto , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cricetinae , Modelos Animais de Doenças , Método Duplo-Cego , Feminino , Voluntários Saudáveis , Humanos , Ácido Hialurônico/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Pessoa de Meia-Idade , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real , Sebo/metabolismo , Sensibilidade e EspecificidadeRESUMO
Accidental radiation exposures or radiation therapy can cause internal and external damage including radiodermatitis. Even though radiodermatitis is one of the dose limiting factors in radiotherapy, the immunological nature of it is not yet been clearly understood. In this study, we have examined the alteration in immune cell population during the radiodermatitis process. A radiodermatitis model was established in HR-1 mice by locally exposing a posterior dorsal region to 10 Gy X-ray/day for 4 consecutive days. Collagen accumulation, redness, erythema, and dry desquamation of the skin were detected after X-irradiation. The size and total cell number of the spleen decreased immediately after X-irradiation, compared to those of the sham-irradiated mice, and recovered to the normal levels two weeks later. Reduction and recovery of the bone marrow cell population preceded a similar change of the spleen cell population. The proportion of CD4+ T cell increased, while the proportion of CD8+ T cell decreased ahead of the obvious skin damage, in both lymph node and spleen of the irradiated mice. Interestingly, the proportion of splenic monocytes/macrophages was expanded gradually at a similar kinetics with the aggravation of the radiodermatitis. The infiltration of the CD11b+ monocyte/macrophage to the X-irradiated skin also coincided with the development of radiodermatitis. These altered proportions of immune cells may play important roles in radiodermatitis.
Assuntos
Células da Medula Óssea/imunologia , Linfócitos/imunologia , Radiodermite/imunologia , Radiodermite/patologia , Baço/imunologia , Irradiação Corporal Total/efeitos adversos , Adaptação Fisiológica/imunologia , Adaptação Fisiológica/efeitos da radiação , Animais , Células da Medula Óssea/efeitos da radiação , Linfócitos/efeitos da radiação , Camundongos , Camundongos Pelados , Radiodermite/etiologia , Baço/efeitos da radiaçãoRESUMO
BACKGROUND: Interleukin-17A (IL-17A) is mainly secreted from Th17 cells that are activated by various stimuli including CpG oligodeoxynucleotide, a Toll-like receptor 9 (TLR9) ligand. Recently, it has been demonstrated that keratinocytes play an important role in the pathogenesis of psoriasis. OBJECTIVE: To investigate the potential role of keratinocytes, we examined whether TLR9 ligand CpG induces IL-17A expression in keratinocytes. METHODS: We used HaCaT keratinocytes as a model system, and determined CpG-induced IL-17A using enzyme-linked immunosorbent assay and Western blot. RESULTS: When HaCaT keratinocytes were treated with CpG, the expression of several cytokines including IL-17A, tumor necrosis factor-α and CCL20 was markedly increased. Treatment with nuclear factor (NF)-κB inhibitor significantly blocked the CpG-induced IL-17A production, indicating that CpG induced IL-17A expression through the NF-κB signaling pathway. In addition, IL-17A secreted from keratinocytes stimulated the CD4+ T cells, resulting in strong induction of IL-22 production. CONCLUSION: Since IL-22 is an important mediator for psoriatic inflammation, our data suggest that keratinocytes can participate in the pathogenesis of psoriasis via the TLR9-dependent IL-17A production.
RESUMO
BACKGROUND: One of the most serious complications of breast surgery using implants is capsular contracture. Several preventive treatments have been introduced; however, the mechanism of capsule formation has not been resolved completely. The authors previously identified negative effects of botulinum toxin type A on capsule formation, expression of transforming growth factor (TGF)-ß1, and differentiation of fibroblasts into myofibroblasts. Thus, the authors investigated how to prevent capsule formation by using botulinum toxin type A, particularly by means of TGF-ß1 signaling, in human fibroblasts. METHODS: In vitro, cultured human fibroblasts were treated with TGF-ß1 and/or botulinum toxin type A. Expression of collagen, matrix metalloproteinase, and Smad was examined by Western blotting. The activation of matrix metalloproteinase was observed by gelatin zymography. In vivo, the effect of botulinum toxin type A on the phosphorylation of Smad2 in silicone-induced capsule formation was evaluated by immunocytochemistry. RESULTS: In vitro, the phosphorylation of Smad2 was inhibited by botulinum toxin type A treatment. The expression levels of collagen types 1 and 3 were inhibited by botulinum toxin type A treatment, whereas those of matrix metalloproteinase-2 and matrix metalloproteinase-9 were enhanced. Gelatin zymography experiments confirmed enhanced matrix metalloproteinase-2 activity in collagen degradation. In vivo, botulinum toxin type A treatment reduced capsule thickness and Smad2 phosphorylation in silicone-induced capsules. CONCLUSION: This study suggests that botulinum toxin type A plays an important role in the inhibition of capsule formation through the TGF-ß/Smad signaling pathway. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.