RESUMO
Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor ß1 (TGF-ß1) signaling, since both KLF6 and Sp1 are key players in TGF-ß1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-ß1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-ß1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-ß1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.
Assuntos
Dipeptidases , Fator 6 Semelhante a Kruppel , Transdução de Sinais , Fator de Transcrição Sp1 , Animais , Humanos , Camundongos , Colágeno/metabolismo , Fator 6 Semelhante a Kruppel/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.
Assuntos
Vírus da Febre Suína Clássica , Peste Suína Clássica , Endopeptidases , Histona Desacetilase 1 , Imunidade Inata , Complexo de Endopeptidases do Proteassoma , Fator de Transcrição Sp1 , Proteínas Virais , Animais , Peste Suína Clássica/imunologia , Peste Suína Clássica/metabolismo , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/enzimologia , Vírus da Febre Suína Clássica/imunologia , Vírus da Febre Suína Clássica/metabolismo , Vírus da Febre Suína Clássica/patogenicidade , Endopeptidases/química , Endopeptidases/metabolismo , Histona Desacetilase 1/biossíntese , Histona Desacetilase 1/metabolismo , Fator Regulador 3 de Interferon , Proteínas do Nucleocapsídeo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Transcrição Sp1/metabolismo , Suínos/virologia , Proteínas do Core Viral/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Ubiquitinas/metabolismo , Citocinas/metabolismo , Vírus da Diarreia Epidêmica Suína/imunologia , Vírus da Diarreia Epidêmica Suína/metabolismo , Domínios ProteicosRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic heterogeneous disease. This study explored the mechanism of specificity protein 1/3 (Sp1/3) in T-ALL cells through ß-catenin by acting as targets of miR-495-3p. Expression levels of miR-495-3p, Sp1, Sp3, and ß-catenin in the serum from T-ALL children patients, healthy controls, and the T-ALL cell lines were measured. The cell proliferation ability and apoptosis rate were detected. Levels of proliferation-related proteins proliferating cell nuclear antigen (PCNA)/cyclinD1 and apoptosis-related proteins B-cell lymphoma-2 associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) were determined. The binding of Sp1/3 and ß-catenin promoter and the targeted relationship between miR-495-3p with Sp1/3 were analyzed. Sp1/3 were upregulated in CD4+ T-cells in T-ALL and were linked with leukocyte count and risk classification. Sp1/3 interference prevented proliferation and promoted apoptosis in T-ALL cells. Sp1/3 transcription factors activated ß-catenin expression. Sp1/3 enhanced T-ALL cell proliferation by facilitating ß-catenin expression. miR-495-3p targeted and repressed Sp1/3 expressions. miR-495-3p overexpression inhibited T-ALL cell proliferation and promoted apoptosis. Conjointly, Sp1/3, as targets of miR-495-3p limit apoptosis and promote proliferation in T-ALL cells by promoting ß-catenin expression.
Assuntos
Apoptose , Proliferação de Células , MicroRNAs , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Fator de Transcrição Sp1 , Fator de Transcrição Sp3 , beta Catenina , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , beta Catenina/genética , beta Catenina/metabolismo , Linhagem Celular Tumoral , Regulação Leucêmica da Expressão Gênica , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp3/metabolismoRESUMO
Nickel (Ni) is a human carcinogen with genotoxic and epigenotoxic effects. Environmental and occupational exposure to Ni increases the risk of cancer and chronic inflammatory diseases. Our previous findings indicate that Ni alters gene expression through epigenetic regulation, specifically impacting E-cadherin and angiopoietin-like 4 (ANGPTL4), involved in epithelial-mesenchymal transition and migration. GST-M2, a member of the glutathione S-transferase (GST) enzyme family, plays a crucial role in cellular defense against oxidative damage and has been increasingly associated with cancer. GST-M2 overexpression inhibits lung cancer invasion and metastasis in vitro and in vivo. Hypermethylation of its promoter in cancer cells reduces gene expression, correlating with poor prognosis in non-small-cell lung cancer patients. The impact of Ni on GST-M2 remains unclear. We will investigate whether nickel exerts regulatory effects on GST-M2 through epigenetic modifications. Additionally, metformin, an antidiabetic drug, is being studied as a chemopreventive agent against nickel-induced damage. Our findings indicate that nickel chloride (NiCl2 ) exposure, both short-term and long-term, represses GST-M2 expression. However, the expression can be restored by demethylation agent 5-aza-2'-deoxycytidine and metformin. NiCl2 promotes hypermethylation of the GST-M2 promoter, as confirmed by methylation-specific PCR and bisulfite sequencing. Additionally, NiCl2 also influences histone acetylation, and metformin counteracts the suppressive effect of NiCl2 on histone H3 expression. Metformin reestablishes the binding of specificity protein 1 to the GST-M2 promoter, which is otherwise disrupted by NiCl2 . These findings elucidate the mechanism by which Ni reduces GST-M2 expression and transcriptional activity, potentially contributing to Ni-induced lung carcinogenesis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Metformina , Humanos , Níquel , Carcinoma Pulmonar de Células não Pequenas/genética , Epigênese Genética , Neoplasias Pulmonares/patologia , Glutationa Transferase/metabolismoRESUMO
BACKGROUND: Cervical cancer remains one of the most prevalent cancers worldwide. Accumulating evidence suggests that specificity protein 1 (Sp1) plays a pivotal role in tumour progression. The underlying role and mechanism of Sp1 in tumour progression remain unclear. METHODS: The protein level of Sp1 in tumour tissues was determined by immunohistochemistry. The effect of Sp1 expression on the biological characteristics of cervical cancer cells was assessed by colony, wound healing, transwell formation, EdU, and TUNEL assays. Finally, the underlying mechanisms and effects of Sp1 on the mitochondrial network and metabolism of cervical cancer were analysed both in vitro and in vivo. RESULTS: Sp1 expression was upregulated in cervical cancer. Sp1 knockdown suppressed cell proliferation both in vitro and in vivo, while overexpression of Sp1 had the opposite effects. Mechanistically, Sp1 facilitated mitochondrial remodelling by regulating mitofusin 1/2 (Mfn1/2), OPA1 mitochondrial dynamin-like GTPase (Opa1), and dynamin 1-like (Drp1). Additionally, the Sp1-mediated reprogramming of glucose metabolism played a critical role in the progression of cervical cancer cells. CONCLUSIONS: Our study demonstrates that Sp1 plays a vital role in cervical tumorigenesis by regulating the mitochondrial network and reprogramming glucose metabolism. Targeting Sp1 could be an effective strategy for the treatment of cervical cancer.
Assuntos
MicroRNAs , Neoplasias do Colo do Útero , Feminino , Humanos , Neoplasias do Colo do Útero/patologia , MicroRNAs/metabolismo , Transformação Celular Neoplásica , Glucose/metabolismo , Proliferação de Células , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular TumoralRESUMO
Cisplatin resistance is a major therapeutic challenge in advanced head and neck squamous cell carcinoma (HNSCC). Here, we aimed to investigate the key signaling pathway for cisplatin resistance in HNSCC cells. Vomeronasal type-1 receptor 5 (VN1R5) was identified as a cisplatin resistance-related protein and was highly expressed in cisplatin-resistant HNSCC cells and tissues. The long noncoding RNA (lncRNA) lnc-POP1-1 was confirmed to be a downstream target induced by VN1R5. VN1R5 transcriptionally regulated lnc-POP1-1 expression by activating the specificity protein 1 (Sp1) transcription factor via the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. VN1R5 promoted cisplatin resistance in HNSCC cells in a lnc-POP1-1-dependent manner. Mechanistically, lnc-POP1-1 bound to the minichromosome maintenance deficient 5 (MCM5) protein directly and decelerated MCM5 degradation by inhibiting ubiquitination of the MCM5 protein, which facilitated the repair of DNA damage caused by cisplatin. In summary, we identified the cisplatin resistance-related protein VN1R5 and its downstream target lnc-POP1-1. Upon upregulation by VN1R5, lnc-POP1-1 promotes DNA repair in HNSCC cells through interaction with MCM5 and deceleration of its degradation.
Assuntos
Neoplasias de Cabeça e Pescoço , RNA Longo não Codificante , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , RNA Longo não Codificante/genética , Ribonucleoproteínas , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
Aging with dysregulated metabolic and immune homeostasis stimulates pyroptosis, neuroinflammation, and cellular senescence, thus contributing to etiopathogenesis of Alzheimer's disease. GATA-binding protein 4 (GATA4) functions as a transcriptional factor in response to DNA damage, and is associated with neuroinflammation and cellular senescence. The role of GATA4 in Alzheimer's disease was investigated. GATA4 was elevated in hippocampus of Aß1-42 fibril-infused rats. Injection with shRNA targeting GATA4 reduced escape latency with increase of time in target quadrant and number of platform crossings in Aß1-42 fibril-infused rats. Moreover, knockdown of GATA4 ameliorated morphological changes of hippocampus and reduced amyloid plaque deposition in Aß1-42 fibril-infused rats. Silence of GATA4 repressed neuroinflammation and apoptosis in Aß1-42 fibril-infused rats. Loss of GATA4 in Aß1-42 fibril-infused rats reduced the expression of specificity protein 1 (Sp1) to downregulate long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) and upregulated miR-361-3p. Loss of SNHG1 ameliorated learning and memory impairments in Aß1-42 fibril-infused rats. Overexpression of Sp1 attenuated GATA4 silence-induced decrease of escape latency, increase of time in target quadrant, and number of platform crossings in Aß1-42 fibril-infused rats. In conclusion, silence of GATA4 ameliorated cognitive dysfunction and inhibited hippocampal inflammation and cell apoptosis through regulation of Sp1/SNHG1/miR-361-3p.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Fator de Transcrição GATA4 , MicroRNAs , Animais , Ratos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , MicroRNAs/genética , Doenças Neuroinflamatórias , RNA Nucleolar Pequeno , Fator de Transcrição GATA4/metabolismoRESUMO
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor that orchestrates cellular responses to oxidative stress. Because the dysregulation of Nrf2 has been implicated in many diseases, precise regulation of its protein level is crucial for maintaining homeostasis. Kelch-like-ECH-associated protein 1 (Keap1) and WD40 repeat protein 23 (WDR23) directly regulate Nrf2 levels via similar but distinct proteasome-dependent pathways. WDR23 forms a part of the WDR23-Cullin 4A-RING ubiquitin ligase complex (CRL4AWDR23), whereas Keap1 serves as a substrate adaptor for the Cullin 3-containing ubiquitin ligase complex. However, the mechanisms underlying crosstalk between these Keap1 and WDR23 pathways for the regulation of Nrf2 levels have not been investigated. Here, we showed that knockdown (KD) of Keap1 upregulated the expression of Cullin4A (CUL4A) in a specificity protein 1 (Sp1)-dependent manner. We also revealed that Sp1 interacted with Keap1, leading to ubiquitination of Sp1. Increases in Sp1 by Keap1 KD triggered Sp1 binding to the fourth Sp1 binding site (Sp1_M4) within the -230/+50 region of the CUL4A gene. We also demonstrated that the overexpression and KD of Sp1 reduced and increased Nrf2 protein levels, respectively. These effects were abrogated by the WDR23 KD, suggesting that Sp1 also regulates Nrf2 levels via the ubiquitin ligase complex CRL4AWDR23. In conclusion, we discovered Sp1 as a novel substrate of Keap1 and provided evidence that Sp1 regulates the expression of CUL4A. We revealed a novel role for Sp1 in mediating crosstalk between two independent regulators of Nrf2 protein levels.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Transcrição Sp1/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , CinéticaRESUMO
Specificity protein (Sp) is a famous family of transcription factors including Sp1, Sp2 and Sp3. Sp1 is the first one of Sp family proteins to be characterized and cloned in mammalian. It has been proposed that Sp1 acts as a modulator of the expression of target gene through interacting with a series of proteins, especially with transcriptional factors, and thereby contributes to the regulation of diverse biological processes. Notably, growing evidence indicates that Sp1 is involved in the main events in the development of atherosclerosis (AS), such as inflammation, lipid metabolism, plaque stability, vascular smooth muscle cells (VSMCs) proliferation and endothelial dysfunction. This review is designed to provide useful clues to further understanding roles of Sp1 in the pathogenesis of AS, and may be helpful for the design of novel efficacious therapeutics agents targeting Sp1.
Assuntos
Aterosclerose , Fator de Transcrição Sp1 , Animais , Aterosclerose/genética , Humanos , Mamíferos/metabolismo , Regiões Promotoras Genéticas , Proteínas/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismoRESUMO
Cadmium (Cd) is an environmental pollutant and pregnant women are especially susceptible to the effects of exposure to Cd. Our previous study found Cd can be accumulated in the placenta and causes fetal growth restriction (FGR) through damage the placental glucocorticoid barrier. Selenium (Se), as an essential micronutrient, can allivate Cd-induced toxicity. In this study, we aim to explore the protective mechanism of Se against Cd-induced the placental glucocorticoid barrier damage and FGR. Pregnant Sprague Dawley (SD) rats were exposed to CdCl2 (1 mg/kg/day) and Na2 SeO3 (0.1-0.2-0.3 mg/kg/day) by gavage from gestational day (GD) 0 to GD 19. The results showed that reduced fetal weight, increased corticosterone concentrations in the maternal and fetal serum, and impaired placental labyrinth layer blood vessel development, appeared in pregnant rats after Cd exposure and improved after treated with Se. In cell experiments, we confirmed that Se reduces Cd-induced apoptosis. Moreover, Se can abolish Cd-induced 11ß-HSD2 and specificity protein 1 (Sp1) decreasing in vivo and vitro. In human JEG-3 cells, the knockdown of Sp1 expression by small interfering RNA can suppressed the protective effect of Se on Cd-induced 11ß-HSD2 decreasing. In general, our results demonstrated that Se is resistant to Cd-induced FGR through upregulating the placenta barrier via activation of the transcription factor Sp1.
Assuntos
Intoxicação por Cádmio , Selênio , Fator de Transcrição Sp1 , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/farmacologia , Animais , Cádmio/toxicidade , Intoxicação por Cádmio/metabolismo , Linhagem Celular Tumoral , Feminino , Retardo do Crescimento Fetal/induzido quimicamente , Retardo do Crescimento Fetal/metabolismo , Glucocorticoides/farmacologia , Humanos , Placenta/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Selênio/efeitos adversos , Fator de Transcrição Sp1/biossínteseRESUMO
Neuroinflammation is a leading cause for neurological disorders. Carbazole alkaloids, isolated from the medicinal plants of Murraya species (Rutaceae), have exhibited wide pharmacological activities particularly for neuroinflammation. However, its underlying cellular targets and molecular mechanisms still remain unclear. In current study, we found that murrayafoline A (MA), a carbazole alkaloid obtained from Murraya tetramera, potently inhibited the production of neuroinflammation mediators, such as nitric oxide (NO), TNF-α, IL-6 and IL-1ß in LPS-induced BV-2 microglial cells. Then, we performed thermal proteome profiling (TPP) strategy to identify Specificity protein 1 (Sp1) as a potential cellular target of MA. Moreover, we performed surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DRATS) assays to confirm the direct interaction between MA and Sp1. Furthermore, we downregulated Sp1 expression in BV2 cells using siRNA transfection, and observed that Sp1 knockdown significantly antagonized MA-mediated inhibition of neuroinflammation mediator production. Meanwhile, Sp1 knockdown also markedly reversed MA-mediated inactivation of IKKß/NF-κB and p38/JNK MAPKs pathways. Finally, in vivo studies revealed that MA significantly suppressed the expression of Iba-1, TNF-α, and IL-6, while increased the number of Nissl bodies in the brains of LPS-induced mice. Taken together, our study demonstrated that MA exerted obvious anti-neuroinflammation effect by directly targeting Sp1, thereby inhibiting NF-κB and MAPK signaling pathways. Our findings also provided a promising direction of pharmacological targeting Sp1 for anti-neuroinflammation therapeutics as well as novel agent development.
Assuntos
Alcaloides , Anti-Inflamatórios , Carbazóis , Murraya , Doenças Neuroinflamatórias , Fator de Transcrição Sp1 , Animais , Camundongos , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Carbazóis/metabolismo , Carbazóis/uso terapêutico , Interleucina-6/metabolismo , Lipopolissacarídeos , Microglia/efeitos dos fármacos , Murraya/química , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Transcrição Sp1/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológicoRESUMO
Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)-directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation.
Assuntos
RNA Polimerase III/metabolismo , Fator de Transcrição Sp1/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fatores de Transcrição TFIII/metabolismo , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Proteína p300 Associada a E1A/metabolismo , Filaminas/antagonistas & inibidores , Filaminas/genética , Filaminas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Interferência de RNA , RNA Polimerase III/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Fatores Associados à Proteína de Ligação a TATA/antagonistas & inibidores , Fatores Associados à Proteína de Ligação a TATA/genética , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição TFIII/antagonistas & inibidores , Fatores de Transcrição TFIII/genética , Transcrição Gênica , Regulação para CimaRESUMO
GM2-synthase produces sialic acid-containing glycosphingolipids called gangliosides, and its mRNA overexpression and the gangliosides it generates are linked to tumor progression, migration, and suppression of tumor-specific host immune responses. However, the mechanism underlying GM2-synthase de-repression in renal cell carcinoma (RCC) is poorly understood. Here, we demonstrate that higher GM2-synthase mRNA expression levels in various cancer cells and in human RCC tumors correlate with higher histone acetylation levels (H3K9, H3K14, or both) at region +38/+187 relative to the transcription start site (TSS) of the GM2-synthase gene than in normal kidney epithelial (NKE) cells or healthy adjacent tissues. An increase in GM2-synthase mRNA expression in cells treated with a histone deacetylase (HDAC) inhibitor was accompanied by increased histone acetylation levels at this promoter region. DNA methylation around the TSS was absent in both RCC cell lines and NKE cells. Of note, both the transcription factor Sp1 and corepressor HDAC1 associated with the +38/+187 region when the GM2-synthase gene was repressed in NKE and tumor-adjacent tissues, indicating plausible site-specific repressive roles of HDAC1 and Sp1 in GM2-synthase mRNA expression. Site-directed mutagenesis of the Sp1-binding site within the +38/+187 region relieved repressed luciferase activity of this region by limiting HDAC1 recruitment. Moreover, Sp1 or HDAC1 knock down increased GM2-synthase transcription, and butyrate-mediated activation of GM2-synthase mRNA expression in SK-RC-45 cells was accompanied by Sp1 and HDAC1 loss from the +38/+187 region. Taken together, we have identified an epigenetic mechanism for the de-repression of the GM2-synthase gene in RCC.
Assuntos
Carcinoma de Células Renais/metabolismo , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 1/metabolismo , Histonas/metabolismo , Neoplasias Renais/metabolismo , N-Acetilgalactosaminiltransferases/biossíntese , Proteínas de Neoplasias/metabolismo , Fator de Transcrição Sp1/metabolismo , Acetilação , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Células HEK293 , Histona Desacetilase 1/genética , Histonas/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Células MCF-7 , N-Acetilgalactosaminiltransferases/genética , Proteínas de Neoplasias/genética , Fator de Transcrição Sp1/genéticaRESUMO
BACKGROUND: Cell division cycle associated protein-3 (CDCA3) has been reported frequently upregulated in various cancers. It has been progressively realized that changed DNA methylations occur in diverse carcinomas. However, the concrete involvement of CDCA3 and DNA methylation in gastric cancer (GC) still needs to be further elucidated. METHODS: In this study, quantitative reverse-transcription polymerase chain reaction (PCR) was utilized to determine the relative expressions of CDCA3 in GC and normal tissue samples. The methylation condition of CDCA3 was determined by bisulfite-sequencing PCR (BSP) and methylation-specific PCR (MSP). A chromatin immunoprecipitation (ChIP) assay and luciferase activity assay was used for the interaction between transcription factors and promoters and binding site determination, respectively. The effects of knockdown or overexpression of specificity protein 1 (SP1) or CDCA3 on GC cells in vitro were further assessed via wound healing assay, colony formation assay, and matrigel invasion assay. RESULTS: In comparison to paired normal tissues, CDCA3 expressions were significantly increased in the GC tissues. The CDCA3 expression was regulated by DNA methylation, with the CpG island hypomethylation responsible for CDCA3 upregulation of GC. ChIP assays verified that the activity of SP1 binding to the CDCA3 promoter was dramatically increased. When the CDCA3 expression was downregulated in MKN45 cells by knockdown SP1, the proliferation ability, healing ability, and invasive ability were significantly suppressed. CONCLUSION: The process by which SP1 bound to the nearest promoter region was expedited in GC cells, by which DNA was hypomethylated and CDCA3 expression was promoted. The effect on cell proliferation and invasion by CDCA3 was under the regulation of SP1 and also affected by hypomethylation of DNA.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição Sp1/metabolismo , Neoplasias Gástricas/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ilhas de CpG , Regulação para Baixo , Mucosa Gástrica/metabolismo , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estômago , Neoplasias Gástricas/patologia , Ativação Transcricional , CicatrizaçãoRESUMO
Hepatitis B virus (HBV) infection remains a global public health problem. Nearly 257 million people worldwide have been infected with HBV, resulting in 887,000 people dying of cirrhosis or liver cancer caused by chronic hepatitis B (CHB) annually. Therefore, identification of new targets against HBV is urgently needed. Long noncoding RNAs (LncRNAs) have gained widespread attention in recent years due to their function in cancer, inflammation and other diseases. Notably, a growing number of lncRNAs have been found to play a role in HBV development. In the present study, we first identified a famous lncRNA, HOTAIR, which was significantly up-regulated in HBV-infected cells and PBMCs from CHB patients. Furthermore, we evaluated the clinical relevance of HOTAIR in 20 CHB patients and found that higher levels of HOTAIR expression were associated with higher ALT/AST levels and were positively correlated with HBsAg and HBV DNA levels. In addition, functional analysis showed that HOTAIR promoted HBV transcription and replication by elevating the activities of HBV promoters via modulation of the levels of cccDNA-bound SP1. In conclusion, our study reveals that HOTAIR expression is correlated with the clinicopathological and physiological characteristics of HBV. Thus, HOTAIR may serve as a novel HBV diagnostic and therapeutic biomarker based on its ability to facilitate HBV transcription and replication.
Assuntos
Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , RNA Longo não Codificante/metabolismo , Fator de Transcrição Sp1/metabolismo , Transcrição Viral/genética , Replicação Viral/genética , Adulto , Feminino , Redes Reguladoras de Genes , Inativação Gênica , Células Hep G2 , Hepatite B Crônica/genética , Hepatite B Crônica/virologia , Humanos , Masculino , Regiões Promotoras Genéticas/genéticaRESUMO
Postoperative cognitive dysfunction (POCD) is a common complication induced by anesthesia or surgery, which affects the concentration, cognition and memory of patients. Sevoflurane, a clinical anesthetic, could stimulate neuro-inflammation and lead to POCD. Recent studies found that specificity protein 1 (SP1) participates in the development of neurological diseases. Our study aims to elucidate the role of SP1 in sevoflurane-induced POCD pathogenesis. We anesthetized Sprague-Dawley rats and treated the primary hippocampal neurons with sevoflurane to construct the in vivo and in vitro POCD models. Besides, the expression and regulatory mechanism of SP1 in the pathogenesis of POCD were explored. According to the results, sevoflurane anesthesia impaired the cognitive functions of rat, significantly elevated SP1 expression and inactivated the cholinergic anti-inflammatory pathway (CAP) both in vivo and in vitro. Moreover, the sevoflurane-treated rats and neurons also exhibited obvious inflammatory responses and enhanced apoptosis. Loss-of-function assay indicated that SP1 knockdown rescued the deactivation of CAP and alleviated the sevoflurane-induced neuro-inflammation and apoptosis in rat hippocampus. Generally, our study documented that the sevoflurane-induced SP1 up-regulation affected the activation of CAP, leading to the aggravated neuro-inflammation and apoptosis. This may provide a novel sight for POCD therapy.
Assuntos
Anestésicos Inalatórios/efeitos adversos , Inflamação/etiologia , Complicações Cognitivas Pós-Operatórias/etiologia , Sevoflurano/efeitos adversos , Fator de Transcrição Sp1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Inflamação/induzido quimicamente , Teste do Labirinto Aquático de Morris/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Complicações Cognitivas Pós-Operatórias/induzido quimicamente , Ratos Sprague-Dawley , Fator de Transcrição Sp1/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismoRESUMO
Retinal cell damage caused by diabetes leads to retinal microvascular injury. Roundabout 4 (ROBO4) is involved in angiogenesis, which varies with the development of diabetic retinopathy (DR). Here, we explored the transcriptional regulation and microRNA-mediated modulation of ROBO4 expression and related retinal cell function in DR. A streptozotocin-induced type I diabetic animal model was established to detect the expression of hypoxia inducible factor-1α (HIF-1α), specificity protein 1 (SP1) and ROBO4. Retinal pigment epithelium (RPE) cells were cultured under hyperglycaemia or hypoxia and used for mechanistic analysis. Furthermore, roles of miR-125b-5p and miR-146a-5p were evaluated, and their targets were identified using luciferase assays. The cell functions were evaluated by MTS assays, permeability analysis and migration assays. The development of DR increased the levels of HIF-1α, SP1 and ROBO4 both in the DR model and in hyperglycaemic/hypoxic RPE cells. They were co-expressed and up-regulated in diabetic retinas and in RPE cells under hyperglycaemia/hypoxia. Knockdown of HIF-1α significantly inhibited SP1 and ROBO4, whereas SP1 down-regulation abolished ROBO4 expression in RPE cells under hyperglycaemia/hypoxia. miR-125b-5p and miR-146a-5p were down-regulated by hyperglycaemia and/or hypoxia. Up-regulation of miRNAs reversed these changes and resulted in recovery of target gene expression. Moreover, luciferase assays confirmed miR-125b-5p targeted SP1 and ROBO4, and miR-146a-5p targeted HIF-1α and ROBO4 directly. The decreased cell viability, enhanced permeability, and increased cell migration under DR conditions were mitigated by knockdown of HIF-1α/SP1/ROBO4 or up-regulation of miR-125b-5p/miR-146a-5p. In general, our results identified a novel mechanism that miR-125b-5p/miR-146a-5p targeting HIF-1α/SP1-dependent ROBO4 expression could retard DR progression.
Assuntos
Retinopatia Diabética/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MicroRNAs/metabolismo , Receptores de Superfície Celular/metabolismo , Fator de Transcrição Sp1/metabolismo , Regulação para Cima/genética , Animais , Sequência de Bases , Hipóxia Celular/genética , Células Cultivadas , Retinopatia Diabética/complicações , Retinopatia Diabética/patologia , Regulação para Baixo , Humanos , Hiperglicemia/complicações , Hiperglicemia/patologia , Masculino , MicroRNAs/genética , Ratos Sprague-Dawley , Retina/patologia , Transcrição GênicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle. KSHV-related diseases are associated with deregulated expression of inflammatory cytokines, including IL-6 and IL-10, but the mechanisms underlying this dysregulation are unclear. Herein, we report a molecular mechanism for KSHV-induced IL-10 gene expression. KSHV replication and transcription activator (K-RTA) is a molecular switch for the initiation of expression of viral lytic genes, and we describe, for the first time, that K-RTA significantly activates the promoter of the human IL-10 gene. Of note, mutations involving a basic region of K-RTA reduced the association of K-RTA with the IL-10 promoter. Moreover, the host-cell transcription factors, specificity proteins (SP) 1 and 3, play a pivotal cooperative role in K-RTA-mediated transactivation of the IL-10 promoter. K-RTA can interact with SP1 and SP3 directly in vitro, and electrophoresis mobility shift assays (EMSAs) revealed co-operative interaction involving K-RTA, SP1, and SP3 in binding to the IL-10 promoter. As DNase I footprinting assays indicated that K-RTA did not affect SP3 binding to the IL-10 promoter, SP3 can function to recruit K-RTA to the IL-10 promoter. These findings indicate that K-RTA can directly contribute to IL-10 up-regulation via a functional interplay with the cellular transcription factors SP1 and SP3.
Assuntos
Interleucina-10/genética , Interleucina-10/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Regiões Promotoras Genéticas/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/fisiologia , Proteínas Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.
Assuntos
Fator de Ligação a CCAAT/metabolismo , Genômica , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Fator de Transcrição Sp2/metabolismo , Animais , Fator de Ligação a CCAAT/química , Linhagem Celular , Histonas/metabolismo , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Transporte Proteico , Fator de Transcrição Sp2/química , Dedos de ZincoRESUMO
The non-steroidal anti-inflammatory drug, Tolfenamic acid (TA) acts as an anti-cancer agent in several adult and pediatric cancer models. Copper (Cu) is an important element with multiple biological functions and has gained interest in medical applications. Recently, [Cu(TA)2(bpy)] (Cu-TA) has been synthesized in order to enhance therapeutic activity. In this study, we synthesized Cu-TA using an established method, characterized it by UV visible spectroscopy and Fourier-transform infrared spectroscopy (FTIR), and tested its anti-cancer activity using twelve cell lines representing various cancers, such as Ewing sarcoma, glioblastoma, medulloblastoma, neuroblastoma, pancreatic and prostate. The anti-proliferative activity of Cu-TA was determined at 48 h post-treatment and compared with the parental compound, TA. The IC50 values were calculated using GraphPad Prism software. The biological stability of Cu-TA was evaluated using twelve-month-old powder and six-month-old stock solution. Cardiomyocytes (H9C2) were used to test the cytotoxicity in non-malignant cells. Cu-TA showed higher anti-proliferative activity, and the IC50 values were 30 to 80% lower when compared with TA. H9C2 cells were non-responsive to Cu-TA, suggesting that it is selective towards malignant cells. Comparison of the twelve-month-old powder and six-month-old stock solution using the Panc1 cell line showed similar IC50 values (<5% variation), confirming the stability of Cu-TA either in powder or solution form. These findings demonstrate the potential of Cu-TA as an effective anti-cancer agent. Further studies to delineate the detailed mechanism of action of Cu-TA for specific cancer model are underway.