RESUMO
Clouston syndrome (OMIM #129500), also known as hidrotic ectodermal dysplasia type 2, is a rare autosomal dominant skin disorder. To date, four mutations in the GJB6 gene, G11R, V37E, A88V, and D50N, have been confirmed to cause this condition. In previous studies, the focus has been mainly on gene sequencing, and there has been a lack of research on clinical manifestations and pathogenesis. To confirm the diagnosis of this pedigree at the molecular level and summarize and analyse the clinical phenotype of patients and to provide a basis for further study of the pathogenesis of the disease, we performed whole-exome and Sanger sequencing on a large Chinese Clouston syndrome pedigree. Detailed clinical examination included histopathology, hair microscopy, and scanning electron microscopy. We found a novel heterozygous missense variant (c.134G>C:p.G45A) for Clouston syndrome. We identified a new clinical phenotype involving all nail needling pain in all patients and found a special honeycomb hole structure in the patients' hair under scanning electron microscopy. Our data reveal that a novel variant (c.134G>C:p.G45A) plays a likely pathogenic role in this pedigree and highlight that genetic testing is necessary for the diagnosis of Clouston syndrome.
Assuntos
Conexinas , Displasia Ectodérmica , Humanos , Conexina 30/genética , Conexinas/genética , População do Leste Asiático , Displasia Ectodérmica/genética , Displasia Ectodérmica/diagnóstico , Displasia Ectodérmica/patologia , FenótipoRESUMO
Septic acute kidney injury (AKI) is characterized by inflammation. Pyroptosis often occurs during AKI and is associated with the development of septic AKI. This study found that induction of insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to a higher level can induce pyroptosis in renal tubular cells. Meanwhile, macrophage migration inhibitory factor (MIF), a subunit of NLRP3 inflammasomes, was essential for IGF2BP1-induced pyroptosis. A putative m6A recognition site was identified at the 3'-UTR region of E2F transcription factor 1 (E2F1) mRNA via bioinformatics analyses and validated using mutation and luciferase experiments. Further actinomycin D (Act D) chase experiments showed that IGF2BP1 stabilized E2F1 mRNA dependent on m6A. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) indicated that E2F1 acted as a transcription factor to promote MIF expression. Thus, IGF2BP1 upregulated MIF through directly upregulating E2F1 expression via m6A modification. Experiments on mice with cecum ligation puncture (CLP) surgery verified the relationships between IGF2BP1, E2F1, and MIF and demonstrated the significance of IGF2BP1 in MIF-associated pyroptosis in vivo. In conclusion, IGF2BP1 was a potent pyroptosis inducer in septic AKI through targeting the MIF component of NLRP3 inflammasomes. Inhibiting IGF2BP1 could be an alternate pyroptosis-based treatment for septic AKI.
Assuntos
Injúria Renal Aguda , Fatores Inibidores da Migração de Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Injúria Renal Aguda/metabolismo , Inflamassomos , Inflamação , Rim/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , RNA MensageiroRESUMO
Raddeanin A (RA) has indicated suppressive effects on various human tumor cells, and insufficient vitamin D was associated with human papillomavirus (HPV) persistence and gynecological tumors. However, combined effects of RA and vitamin D on HPV-positive cells remain elusive. Herein, we aimed to investigate the combined effects of RA and 1É,25(OH)2D3 (VD3) on cellular viability and modulation of HPV18E6/E7, programmed cell death 1 ligand (PD-L1) and vitamin D receptor (VDR) expression in HeLa cells in vitro. HeLa cells were treated with RA alone or VD3 combined with RA. Cell viability was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and apoptosis was detected by flow cytometry. Real-time PCR (qRT-PCR) and Western blot were used to determine the gene/protein expression levels. The autophagosomes were observed by Transmission electron microscopy (TEM). The result showed that cell viability was inhibited by RA, and apoptosis in HeLa cells treated with RA was elevated accordingly. The expression of Bax, Cleaved-caspase-3, Cleaved-caspase-9 and Cleaved-PARP increased, and Bcl-2 decreased. The autophagy was induced by RA, as evidenced by elevated autophagosomes and the increased LC3-II/I ratio and Beclin-1. The expression of HPV18E6/E7, PD-L1 and VDR was reduced by RA. Moreover, RA combined with VD3 had a stronger effect on HeLa cells than RA alone. In conclusion, RA inhibits HeLa proliferation and induces apoptosis and autophagy via suppressing HPV18E6/E7, PD-L1 and VDR, and VD3 showed reinforced effects of RA on HeLa cells. Therefore, combined usage of VD3 with RA might be a potential novel immunotherapy strategy for HPV-related diseases.
Assuntos
Antígeno B7-H1/metabolismo , Calcitriol/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Receptores de Calcitriol/metabolismo , Saponinas/farmacologia , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Microscopia Eletrônica de TransmissãoRESUMO
Androgenetic alopecia (AGA) is the most common type of hair loss dysfunction. Secreted frizzled related protein 1 (SFRP1) is found to be associated with hair loss, but its role in AGA and the regulation mechanism of its transcription level is unclear. The aim of our study is to explore the expression of SFRP1 in AGA samples and its transcriptional mechanism. Male frontal and occipital scalp hair follicles from AGA patients were collected, and human dermal papilla cells (DPCs) were isolated and cultured. SFRP1 gene was cloned and constructed into recombinant plasmids to perform dual-luciferase reporter assay. Transcription factor binding sites were predicted through the Jaspar website and further confirmed by the chromatin immunoprecipitation (ChIP) assay. Expression of genes in DPCs was determined by immunofluorescence (IF) staining, quantitative real-time PCR (qRT-PCR) and western blotting. Our findings showed that SFRP1 was highly expressed in DPCs of AGA patients. The core promoter region of SFRP1 was from -100 to +50 bp and was found to be positively regulated by forkhead box C1 (FOXC1), a transcription factor related to hair growth, both at mRNA and protein level in DPCs. Our study suggests that FOXC1 plays an important role in regulating SFRP1 transcription, which may provide new insights into the development of therapeutic strategies for the treatment of AGA.
Assuntos
Alopecia/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Folículo Piloso/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Alopecia/tratamento farmacológico , Alopecia/genética , Derme/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fatores de Transcrição/metabolismoRESUMO
Sepsis is a systemic inflammatory response syndrome caused by infection, resulting in organ dysfunction. Sepsis-induced acute kidney injury (AKI) is one of the most common potential complications. Increasing reports have shown that M1 and M2 macrophages both take part in the progress of AKI by influencing the level of inflammatory factors and the cell death, including pyroptosis. However, whether M1 and M2 macrophages regulate AKI by secreting exosome remains unknown. In the present study, we isolated the exosomes from M1 and M2 macrophages and used Western blot and enzyme-linked immunosorbent assay (ELISA) to investigate the effect of M1 and M2 exosomes on cell pyroptosis. miRNA sequencing was used to identify the different miRNA in M1 and M2 exosomes. Luciferase reporter assay was used to verify the target gene of miRNA. We confirmed that exosomes excreted by macrophages regulated cell pyroptosis in vitro by using Western blot and ELISA. miRNA sequencing revealed the differentially expressed level of miRNAs in M1 and M2 exosomes, among which miR-93-5p was involved in the regulation of pyroptosis. By using bioinformatics predictions and luciferase reporter assay, we found that thioredoxin-interacting protein (TXNIP) was a direct target of miR-93-5p. Further in vitro and in vivo experiments indicated that exosomal miR-93-5p regulated the TXNIP directly to influence the pyroptosis in renal epithelial cells, which explained the functional difference between different phenotypes of macrophages. This study might provide new targets for the treatment of sepsis-induced AKI.
Assuntos
Injúria Renal Aguda/patologia , Exossomos/patologia , Macrófagos/patologia , MicroRNAs/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Sepse/complicações , Tiorredoxinas/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Exossomos/genética , Exossomos/metabolismo , Feminino , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Tiorredoxinas/genéticaRESUMO
Oxygen therapy is a common treatment in neonatal intensive care units, but long-term continuous hyperoxia ventilation may induce acute lung injury (ALI). Gasdermin D (GSDMD)-mediated pyroptosis participates in various diseases including ALI, but the role of GSDMD in hyperoxia-induced ALI is yet understood. Here, we showed a significant increase in GSDMD after exposure to high oxygen. To elucidate the molecular mechanisms involved in GSDMD regulation, we identified the core promoter of GSDMD, -98 ~ -12 bp relative to the transcriptional start site (TSS). The results of mutational analysis, overexpression or siRNA interference, EMSA and ChIP demonstrated that E2F4 and TFAP2A positively regulate the transcriptional activity of the GSDMD by binding to its promoter. However, only TFAP2A showed a regulatory effect on the expression of GSDMD. Moreover, TFAP2A was increased in the lung tissues of rats exposed to hyperoxia and showed a strong linear correlation with GSDMD. Our results indicated that TFAP2A positively regulates the GSDMD expression via binding to the promoter region of GSDMD.
Assuntos
Lesão Pulmonar Aguda/genética , Fator de Transcrição E2F4/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oxigênio/efeitos adversos , Proteínas de Ligação a Fosfato/genética , Fator de Transcrição AP-2/genética , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Animais Recém-Nascidos , Hipóxia Celular/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Unidades de Terapia Intensiva Neonatal , Oxigênio/uso terapêutico , Regiões Promotoras Genéticas/genética , Piroptose/genética , Ratos , Sítio de Iniciação de TranscriçãoAssuntos
Alopecia/epidemiologia , Assistência Ambulatorial/estatística & dados numéricos , Adulto , Idade de Início , Alopecia/diagnóstico , Alopecia/etiologia , China/epidemiologia , Feminino , Humanos , Masculino , Anamnese , Pacientes Ambulatoriais , Vigilância da População , Fatores de Risco , Adulto JovemRESUMO
Interferon regulatory factor 3 (IRF-3) is an important transcription factor for interferon genes. Although its functional activation by viral infection has been widely explicated, the regulatory mechanism of IRF-3 gene expression in cancer cells is poorly understood. In this study, we demonstrated treatment of lung adenocarcinoma A549 cells with trichostatin A (TSA) and valproic acid (VPA), two different classes of histone deacetylase inhibitors, strongly stimulated IRF-3 gene expression. Truncated and mutated IRF-3 promoter indicated that a specific GATA-1 element was responsible for TSA-induced activation of IRF-3 promoter. Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TSA treatment increased the binding affinity of GATA-1 to IRF-3 promoter. Using immunoprecipitation assay and immunoblotting, we demonstrated that TSA increased the level of acetylated GATA-1 in A549 cells. In summary, our study implied that TSA enhanced IRF-3 gene expression through increased GATA-1 recruitment to IRF-3 promoter and the acetylation level of GATA-1 in lung adenocarcinoma A549 cells.
RESUMO
Retinopathy of prematurity, a leading cause of visual impairment in low birth-weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all-trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose- and time-dependent manner, and PKA signaling pathway is necessary for ATRA-induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen-induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.
Assuntos
Fator Neurotrófico Ciliar/biossíntese , Células Epiteliais/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Animais , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/patologia , Humanos , Camundongos , Regiões Promotoras Genéticas , Epitélio Pigmentado da Retina/patologia , Retinopatia da Prematuridade/metabolismo , Retinopatia da Prematuridade/patologiaRESUMO
Interferon regulatory factor 5 (IRF5) plays a critical role in the induction of type I interferon, proinflammatory cytokines and chemokines, and participates in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE). However, the relationship between IRF5 and childhood-onset SLE remains elusive. In the present study, we demonstrated that levels of mRNA expression of IRF5, IFN-α, and Sp1 were significantly increased in childhood-onset SLE, as seen on quantitative real-time PCR, and the expression of Sp1 and IFN-α was positively correlated with IRF5. In addition to being used as antitumor drugs, a number of histone deacetylase inhibitors (HDACi) display potent anti-inflammatory properties; however, their effects on IRF5 expression remain unclear. In this study, we identified that HDACi trichostatin A (TSA) and histone acetyltransferase (HAT)-p300 downregulated IRF5 promoter activity, mRNA expression, and protein level, whereas the HAT-p300/CBP-associated factor had no effect. Moreover, TSA inhibited the production of TNF-α and IL-6 in differentiated THP-1cells. Furthermore, chromatin immunoprecipitation assays revealed that TSA inhibited DNA binding of Sp1, RNA polymerase II, HDAC3, and p300 to the core promoter region of IRF5. Our results suggest that HDACi may have therapeutic potential in patients with autoimmune diseases such as SLE through repression of IRF5 expression.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Fatores Reguladores de Interferon/genética , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Idade de Início , Autoanticorpos/imunologia , Autoimunidade , Estudos de Casos e Controles , Linhagem Celular , Criança , Pré-Escolar , Feminino , Expressão Gênica , Genes Reporter , Humanos , Fatores Reguladores de Interferon/metabolismo , Interleucina-6/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Masculino , Regiões Promotoras Genéticas , Ligação Proteica , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The transcription factor, interferon regulatory factor 5 (IRF5), is important in the induction of type I interferon, proinflammatory cytokines and chemokines, and is involved in autoimmune diseases and tumourigenesis. However, the mechanisms underlying the transcriptional regulation of wildtype IRF5 remain to be fully elucidated. The present study was primarily designed to clarify whether specificity protein 1 (Sp1) was involved in the regulation of IRF5. Initially, the IRF5 promoter region was cloned and its promoter activity was examined using Hela and HEK 293 cells. Deletion analyses revealed that the region spanning 179 to +62 was the minimal promoter of IRF5. Bioinformatics analyses showed that this region contained three putative Sp1 binding sites, and mutational analyses revealed that all the Sp1 sites contributed to transcriptional activity. Secondly, the overexpression of Sp1 was found to increase the activity of the IRF5 promoter and the mRNA level of IRF5, determined using reporter gene assays and polymerase chain reaction analysis, respectively. By contrast, treatment with mithramycin and Sp1 small interfering RNA significantly reduced the activity of the IRF5 promoter and the mRNA level of IRF5. Finally, the results of an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated that Sp1 bound to the promoter region of IRF5 in vitro and in vivo. These results suggested that the Sp1 transcription factor is the primary determinant for activating the basal transcription of the IRF5.