RESUMO
Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.
Assuntos
Endorribonucleases , MicroRNAs , RNA Mensageiro , Humanos , Genes jun/genética , Genes myc/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Endorribonucleases/química , Endorribonucleases/metabolismo , TranscriptomaRESUMO
Chronic inflammation increases the risk of developing one of several types of cancer. Inflammatory responses are currently thought to be controlled by mechanisms that rely on transcriptional networks that are distinct from those involved in cell differentiation. The orphan nuclear receptor NR5A2 participates in a wide variety of processes, including cholesterol and glucose metabolism in the liver, resolution of endoplasmic reticulum stress, intestinal glucocorticoid production, pancreatic development and acinar differentiation. In genome-wide association studies, single nucleotide polymorphisms in the vicinity of NR5A2 have previously been associated with the risk of pancreatic adenocarcinoma. In mice, Nr5a2 heterozygosity sensitizes the pancreas to damage, impairs regeneration and cooperates with mutant Kras in tumour progression. Here, using a global transcriptomic analysis, we describe an epithelial-cell-autonomous basal pre-inflammatory state in the pancreas of Nr5a2+/- mice that is reminiscent of the early stages of pancreatitis-induced inflammation and is conserved in histologically normal human pancreases with reduced expression of NR5A2 mRNA. In Nr5a2+/-mice, NR5A2 undergoes a marked transcriptional switch, relocating from differentiation-specific to inflammatory genes and thereby promoting gene transcription that is dependent on the AP-1 transcription factor. Pancreatic deletion of Jun rescues the pre-inflammatory phenotype, as well as binding of NR5A2 to inflammatory gene promoters and the defective regenerative response to damage. These findings support the notion that, in the pancreas, the transcriptional networks involved in differentiation-specific functions also suppress inflammatory programmes. Under conditions of genetic or environmental constraint, these networks can be subverted to foster inflammation.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica , Inflamação/genética , Pâncreas/metabolismo , Pâncreas/patologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcriptoma , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Cromatina/genética , Cromatina/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Redes Reguladoras de Genes/genética , Genes jun/genética , Heterozigoto , Humanos , Camundongos , Especificidade de Órgãos/genética , Pancreatite/genética , Regiões Promotoras Genéticas/genética , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis-induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1ß, and IL-8 in P. gingivalis-stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre-mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis-induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection-induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis-induced inflammatory immune responses and mapped out a novel SGK1-TRAF2/3-c-Jun-NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.
Assuntos
Perda do Osso Alveolar , Proteínas Imediatamente Precoces , Proteínas Serina-Treonina Quinases , Fator 3 Associado a Receptor de TNF , Perda do Osso Alveolar/genética , Animais , Citocinas/metabolismo , Genes jun , Proteínas Imediatamente Precoces/metabolismo , Imunidade , Inflamação , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Porphyromonas gingivalis/patogenicidade , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismoRESUMO
According to previous research, turmeric seeds exhibit anti-inflammatory, anti-malignancy, and anti-aging properties due to an abundance of terpinen-4-ol (T4O). Although it is still unclear how T4O works on glioma cells, limited data exist regarding its specific effects. In order to determine whether or not glioma cell lines U251, U87, and LN229 are viable, CCK8 was used as an assay and a colony formation assay was performed using different concentrations of T4O (0, 1, 2, and 4 µM). The effect of T4O on the proliferation of glioma cell line U251 was detected through the subcutaneous implantation of the tumor model. Through high-throughput sequencing, a bioinformatic analysis, and real-time quantitative polymerase chain reactions, we identified the key signaling pathways and targets of T4O. Finally, for the measurement of the cellular ferroptosis levels, we examined the relationship between T4O, ferroptosis, and JUN and the malignant biological properties of glioma cells. T4O significantly inhibited glioma cell growth and colony formation and induced ferroptosis in the glioma cells. T4O inhibited the subcutaneous tumor proliferation of the glioma cells in vivo. T4O suppressed JUN transcription and significantly reduced its expression in the glioma cells. The T4O treatment inhibited GPX4 transcription through JUN. The overexpression of JUN suppressed ferroptosis in the cells rescued through T4O treatment. Taken together, our data suggest that the natural product T4O exerts its anti-cancer effects by inducing JUN/GPX4-dependent ferroptosis and inhibiting cell proliferation, and T4O will hope-fully serve as a prospective compound for glioma treatment.
Assuntos
Ferroptose , Glioma , Humanos , Genes jun , Estudos Prospectivos , Linhagem Celular Tumoral , Glioma/tratamento farmacológico , Glioma/genética , Glioma/metabolismo , Proliferação de CélulasRESUMO
BACKGROUND: Nucleosome assembly protein 1-like 1 (NAP1L1) is highly expressed in various types of cancer and plays an important role in carcinogenesis, but its specific role in tumor development and progression remains largely unknown. In this study, we suggest the potential of NAP1L1 as a prognostic biomarker and therapeutic target for the treatment of ovarian cancer (OC). METHODS: In our study, a tissue microarray (TMA) slide containing specimens from 149 patients with OC and 11 normal ovarian tissues underwent immunohistochemistry (IHC) to analyze the correlation between NAP1L1 expression and clinicopathological features. Loss-of- function experiments were performed by transfecting siRNA and following lentiviral gene transduction into SKOV3 and OVCAR3 cells. Cell proliferation and the cell cycle were assessed by the Cell Counting Kit-8, EDU assay, flow cytometry, colony formation assay, and Western blot analysis. In addition, co-immunoprecipitation (Co-IP) and immunofluorescence assays were performed to confirm the relationship between NAP1L1 and its potential targets in SKOV3/OVCAR3 cells. RESULTS: High expression of NAP1L1 was closely related to poor clinical outcomes in OC patients. After knocking down NAP1L1 by siRNA or shRNA, both SKOV3 and OVCAR3 cells showed inhibition of cell proliferation, blocking of the G1/S phase, and increased apoptosis in vitro. Mechanism analysis indicated that NAP1L1 interacted with hepatoma-derived growth factor (HDGF) and they were co-localized in the cytoplasm. Furthermore, HDGF can interact with jun proto-oncogene (C-JUN), an oncogenic transformation factor that induces the expression of cyclin D1 (CCND1). Overexpressed HDGF in NAP1L1 knockdown OC cells not only increased the expression of C-JUN and CCND1, but it also reversed the suppressive effects of si-NAP1L1 on cell proliferation. CONCLUSIONS: Our data demonstrated that NAP1L1 could act as a prognostic biomarker in OC and can interact with HDGF to mediate the proliferation of OC, and this process of triggered proliferation may contribute to the activation of HDGF/C-JUN signaling in OC cells.
Assuntos
Apoptose , Proteína 1 de Modelagem do Nucleossomo , Neoplasias Ovarianas , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Genes jun , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína 1 de Modelagem do Nucleossomo/genética , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Neoplasias Ovarianas/patologiaRESUMO
OBJECTIVE: METTL3 (methyltransferase-like protein 3)-mediated N6-methyladenosine modification is the most abundant RNA modification on eukaryote mRNAs and plays a crucial role in diverse physiological and pathological processes. However, whether N6-methyladenosine modification has function in thrombosis is unknown. This study aims to determine the role of METTL3 in the endothelial cells-mediated thrombosis. Approach and Results: RNA-sequencing and real-time quantitative PCR revealed that the expression of PAI-1 (plasminogen activator inhibitor-1) was downregulated in METTL3 knockdown human umbilical vein endothelial cells. In vitro experiments showed that METTL3 suppressed fibrinolysis. Mechanically, RNA methylation sequencing and meRIP-quantitative real-time PCR showed that METTL3 catalyzed N6-methyladenosine modification on 3' UTR of JUN mRNA. Western blotting analysis showed that METTL3 promoted JUN protein expression. Chromatin immunoprecipitation analysis demonstrated that JUN bound to the PAI-1 promoter in human umbilical vein endothelial cells. Furthermore, mice challenged with lipopolysaccharide resulted in higher METTL3 expression in vessels. Endothelial-specific knockdown of Mettl3 decreased expression of active PAI-1 in plasma and attenuated fibrin deposition in livers and lungs during endotoxemia. CONCLUSIONS: Our study reveals that METTL3-mediated N6-methyladenosine modification plays a crucial role in fibrinolysis and is an underlying target for the therapy of thrombotic disorders.
Assuntos
Endotélio Vascular/metabolismo , Fibrinólise , Metiltransferases/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trombose/metabolismo , Regiões 3' não Traduzidas , Animais , Regulação para Baixo , Genes jun , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Regulação para CimaRESUMO
Eukaryotic mRNAs contain a 5' cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs such as the cell proliferation regulator c-Jun (also known as JUN). The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Capuzes de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ligação Competitiva , Cristalografia por Raios X , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4F em Eucariotos/metabolismo , Genes jun/genética , Humanos , Modelos Moleculares , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Capuzes de RNA/química , Capuzes de RNA/genética , Proteínas de Ligação a RNA/químicaRESUMO
Exposure to silica nanoparticles (SiNPs) is related to the dysregulation of pulmonary surfactant that maintains lung stability and function. Nevertheless, there are limited studies concerning the interaction and influence between SiNPs and pulmonary surfactant, and the damage and mechanism are still unclear. Herein, we used A549 cells to develop an in vitro model, with which we investigated the effect of SiNPs exposure on the expression of pulmonary surfactant and the potential regulatory mechanism. The results showed that SiNPs were of cytotoxicity in regarding of reduced cell viability and promoted the production of excessive reactive oxygen species (ROS). Additionally, the JNK/c-Jun signaling pathway was activated, and the expression of surfactant protein A (SP-A) and surfactant protein B (SP-B) was decreased. After the cells being treated with N-acetyl-L-cysteine (NAC), we found that the ROS content was effectively downregulated, and the expression of proteins related to JNK and c-Jun signaling pathways was suppressed. In contrast, the expression of SP-A and SP-B was enhanced. Furthermore, we treated the cells with JNK inhibitor and c-Jun-siRNA and found that the expression of protein related to JNK and c-Jun signaling pathways, as well as SP-A and SP-B, changed in line with that of NAC treatment. These findings suggest that SiNPs exposure can upregulate ROS and activate the JNK/c-Jun signaling pathway in A549 cells, thereby inhibiting the expression of SP-A and SP-B proteins.
Assuntos
Pulmão , Nanopartículas , Proteína A Associada a Surfactante Pulmonar , Proteína B Associada a Surfactante Pulmonar , Dióxido de Silício , Células A549 , Acetilcisteína/farmacologia , Apoptose , Genes jun/genética , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/metabolismo , Nanopartículas/toxicidade , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína B Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Dióxido de Silício/toxicidadeRESUMO
Our understanding of signaling pathways regulating the cell fate of human embryonic stem cells (hESCs) is limited. Calcineurin-NFAT signaling is associated with a wide range of biological processes and diseases. However, its role in controlling hESC fate remains unclear. Here, we report that calcineurin A gamma and the NFATc3/SRPX2 axis control the expression of lineage and epithelial-mesenchymal transition (EMT) markers in hESCs. Knockdown of PPP3CC, the gene encoding calcineurin A gamma, or NFATC3, downregulates certain markers both at the self-renewal state and during differentiation of hESCs. Furthermore, NFATc3 interacts with c-JUN and regulates the expression of SRPX2, the gene encoding a secreted glycoprotein known as a ligand of uPAR. We show that SRPX2 is a downstream target of NFATc3. Both SRPX2 and uPAR participate in controlling expression of lineage and EMT markers. Importantly, SRPX2 knockdown diminishes the upregulation of multiple lineage and EMT markers induced by co-overexpression of NFATc3 and c-JUN in hESCs. Together, this study uncovers a previously unknown role of calcineurin A gamma and the NFATc3/SRPX2 axis in modulating the fate determination of hESCs.
Assuntos
Calcineurina/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Humanas/citologia , Proteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Diferenciação Celular/genética , Transição Epitelial-Mesenquimal/fisiologia , Genes jun/fisiologia , Humanos , Proteínas do Tecido Nervoso/metabolismoRESUMO
The reason why CD4(+) T helper 17 (Th17) cells, despite their well-known pathogenic role in chronic inflammatory disorders, are very rare in the inflammatory sites remains unclear. We demonstrate that human Th17 cells exhibit low ability to proliferate and to produce the T cell growth factor interleukin-2 (IL-2), in response to combined CD3 and CD28 stimulation. This was due to the upregulated expression of IL-4-induced gene 1 (IL4I1) mRNA, a secreted L-phenylalanine oxidase, which associated with a decrease in CD3ζ chain expression and consequent abnormalities in the molecular pathway that allows IL-2 production and cell proliferation. High IL4I1 mRNA expression was detectable in Th17 cell precursors and was strictly dependent on Th17 cell master gene, the retinoid acid related orphan receptor (RORC). Th17 cells also exhibited RORC-dependent CD28 hyperexpression and the ability to produce IL-17A after CD28 stimulation without CD3 triggering. Our findings suggest that the rarity of human Th17 cells in inflamed tissues results from RORC-dependent mechanisms limiting their expansion.
Assuntos
Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Artrite Juvenil/genética , Artrite Juvenil/imunologia , Artrite Juvenil/patologia , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Proliferação de Células , Criança , Expressão Gênica , Genes fos , Genes jun , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-17/biossíntese , Interleucina-2/biossíntese , L-Aminoácido Oxidase/genética , Fatores de Transcrição NFATC/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologiaRESUMO
Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.
Assuntos
Biologia Computacional/métodos , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-jun/antagonistas & inibidores , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proliferação de Células , Simulação por Computador , Dimerização , Genes fos/fisiologia , Genes jun/fisiologia , Humanos , Oncogenes , Biblioteca de Peptídeos , Peptídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismoRESUMO
Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1ß (IL-1ß) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1ß-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1ß specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.
Assuntos
Neoplasias do Córtex Suprarrenal/metabolismo , Carcinoma Adrenocortical/metabolismo , Interleucina-8/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição AP-1/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genes jun/genética , Humanos , Interleucina-1beta/farmacologia , Interleucina-8/genética , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/metabolismo , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Deleção de Sequência , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Regulação para CimaRESUMO
To investigate the TLR-NF-κB/AP-1 pathways in S. aureus infection-induced mammary gland fibrosis, mice were infected with S. aureus isolated from the mammary glands of cows with mastitis. Lactating mice were divided into three groups: control group (CON); PBS control group (PBS) and the S. aureus-treated group (S. aureus). Pathological observations revealed that neutrophil infiltration into mammary gland tissue was obviously induced by S. aureus at the early stage of infection (1-7 d). With persistent S. aureus infection, mammary gland fibrosis developed and was characterized by infiltration and proliferation of macrophage, lymphocyte and fibroblast and ECM hyperplasia (7-21 d). Immunohistochemistry staining showed upregulation of fibrosis associated cytokines viz bFGF and PDGF-BB. Real-time qPCR and Western blot analysis revealed that transcription and translation of TLR2, TLR4, bFGF, PDGF-BB, α-SMA and COL â α1 was significantly upregulated by S. aureus. NF-κB p65 and AP-I c-jun were translocated into the nucleus after S. aureus infection. There was no remarkable difference between the CON and PBS groups. The datas indicate that mammary gland fibrosis in mice is induced by S. aureus, which promotes cytokine release and the expression of ECM though activating the TLR/NF-κB p65 and TLR/AP-1 c-jun signaling pathways.
Assuntos
Glândulas Mamárias Animais/patologia , Transdução de Sinais , Infecções Estafilocócicas , Animais , Bovinos , Feminino , Fibrose , Genes jun , Lactação , Glândulas Mamárias Animais/microbiologia , Mastite Bovina/microbiologia , Camundongos , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/patogenicidade , Receptores Toll-Like/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição RelA/genéticaRESUMO
Vascular aging has a strong relationship with cardiovascular disease. Fos-related antigen 1 (Fra-1), also referred to as Fos-like antigen 1, is a transcription factor and has been reported to be involved in many pathologic processes. Here, we demonstrate that Fra-1 plays a critical role in angiotensin II (Ang II)-induced vascular senescence. Fra-1 expression is increased significantly in Ang II-induced rat aortic endothelial cell (RAEC) senescence and the arteries from Ang II-infused mice. Interestingly, silencing Fra-1 blocks Ang II-induced senescence phenotypes in RAECs, including decreased senescence-associated ß-galactosidase staining, and mitigated proliferation suppression and senescence-associated secretory phenotype. Further, knocking down Fra-1 inhibits vascular aging phenotypes in an Ang II-infused mice model. The up-regulated Fra-1 also exists in human atherosclerotic plaques and Ang II-induced vascular smooth muscle cells as well as in replicated senescence RAECs. Mechanistic studies reveal that Fra-1 preferentially associates with c-Jun and binds to the cyclin-dependent kinase inhibitor 1a (p21) and cyclin-dependent kinase inhibitor 2a (p16) promoter region, leading to elevated gene expression, which causes senescence-related phenotypes. In conclusion, our results identify that Fra-1 plays a novel and key role in promoting vascular aging by directly binding and transcriptionally activating p21 and p16 signaling, suggesting intervention of Fra-1 is a potential strategy for preventing aging-associated cardiovascular disorders.-Yang, D., Xiao, C., Long, F., Wu, W., Huang, M., Qu, L., Liu, X., Zhu, Y. Fra-1 plays a critical role in angiotensin II-induced vascular senescence.
Assuntos
Angiotensina II/fisiologia , Músculo Liso Vascular/fisiologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Animais , Células Cultivadas , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Genes jun , Genes p16 , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Transdução de SinaisRESUMO
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant that causes cardiovascular toxicity. The phenotypic transformation of vascular smooth muscle cells (VSMCs) from the contractile to the synthetic phenotype is a hallmark of vascular response to injury. However, the precise role and molecular mechanism of TCDD in vascular remodeling remains unknown. In the present study, we found that TCDD treatment promoted VSMC phenotypic transition from contractile to synthetic phenotype and exaggerated vascular neointimal hyperplasia after wire injury in mice. TCDD treatment enhanced VSMC entry into cell cycle from G0/G1 phase to S and G2/M phase. The expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), and its phosphorylation were coordinately increased in response to TCDD treatment. Knocking down of aryl hydrocarbon receptor (AHR) inhibited VSMC phenotypic transition induced by TCDD and promoted S/G2 phase cell cycle arrest. TCDD treatment markedly increased oncogenic c-Jun gene expression in VSMCs. ChIP assay revealed the direct binding of AHR on the promoter of c-Jun to up-regulate the mRNA expression of c-Jun. Silencing of c-Jun gene enhanced the expression of p53 and p21, whereas attenuated the expression of CDK4 and cyclin D1 leading to the decrease in the TCDD-stimulated VSMC proliferation and synthetic phenotype transition in vitro. In vivo study showed that genetic ablation of c-Jun in VSMCs restricted injury-induced neointimal hyperplasia in TCDD-treated mice. Thus, TCDD exposure exaggerated injury-induced vascular remodeling by the activation of AHR and up-regulation of the expression of its target gene c-Jun, indicating that inhibition of AHR may be a promising prevention strategy for TCDD-associated cardiovascular diseases.-Guo, S., Zhang, R., Liu, Q., Wan, Q., Wang, Y., Yu, Y., Liu, G., Shen, Y., Yu, Y., Zhang, J. 2,3,7,8-Tetrachlorodibenzo-p-dioxin promotes injury-induced vascular neointima formation in mice.
Assuntos
Endotélio Vascular/lesões , Poluentes Ambientais/toxicidade , Neointima/induzido quimicamente , Dibenzodioxinas Policloradas/toxicidade , Animais , Aorta/citologia , Ciclo Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/lesões , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Genes Reporter , Genes jun , Humanos , Hiperplasia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neointima/etiologia , Fenótipo , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/genética , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/fisiologia , Remodelação Vascular/efeitos dos fármacosRESUMO
BACKGROUND: Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties. RESULTS: Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content. CONCLUSIONS: This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.
Assuntos
Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Zíper de Leucina , Proteínas Recombinantes de Fusão/biossíntese , Biotecnologia , Sobrevivência Celular , Genes fos , Genes jun , Proteínas de Fluorescência Verde/metabolismo , Nanopartículas/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genéticaRESUMO
Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that Lcc-Jun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the c-Jun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.
Assuntos
Citocinas/genética , Proteínas de Peixes/genética , Expressão Gênica/imunologia , Genes jun/imunologia , Perciformes/genética , Perciformes/imunologia , Fatores de Transcrição/genética , Animais , Clonagem Molecular , Citocinas/imunologia , DNA Complementar/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Fatores de Transcrição/imunologiaRESUMO
BACKGROUND AND AIMS: The unique expression pattern makes oncofetal proteins ideal diagnostic biomarkers and therapeutic targets in cancer. However, few oncofetal proteins have been identified and entered clinical practice. METHODS: Fetal liver, adult liver and hepatocellular carcinoma (HCC) tissues were employed to assess the expression of hepatic leukaemia factor (HLF). The impact of HLF on HCC onset and progression was investigated both in vivo and in vitro. The association between HLF and patient prognosis was determined in patient cohorts. The correlation between HLF expression and sorafenib benefits in HCC was further evaluated in patient cohorts and patient-derived xenografts (PDXs). RESULTS: HLF is a novel oncofetal protein which is reactivated in HCC by SOX2 and OCT4. Functional studies revealed that HLF transactivates c-Jun to promote tumour initiating cell (TIC) generation and enhances TIC-like properties of hepatoma cells, thus driving HCC initiation and progression. Consistently, our clinical investigations elucidated the association between HLF and patient prognosis and unravelled the close correlation between HLF levels and c-Jun expression in patient HCCs. Importantly, HLF/c-Jun axis determines the responses of hepatoma cells to sorafenib treatment, and interference of HLF abrogated c-Jun activation and enhanced sorafenib response. Analysis of patient cohorts and PDXs further suggests that HLF/c-Jun axis might serve as a biomarker for sorafenib benefits in HCC patients. CONCLUSIONS: Our findings uncovered HLF as a novel oncofetal protein and revealed the crucial role of the HLF/c-Jun axis in HCC development and sorafenib response, rendering HLF as an optimal target for the prevention and intervention of HCC.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Carcinoma Hepatocelular/genética , Resistencia a Medicamentos Antineoplásicos , Genes jun/genética , Neoplasias Hepáticas/genética , Sorafenibe/farmacologia , Adulto , Antineoplásicos/farmacologia , Apoptose , Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Progressão da Doença , Feminino , Humanos , Imunoprecipitação , Zíper de Leucina , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Masculino , PrognósticoRESUMO
Neuropathic pain (NP) may cause serious brain diseases, but the genes associated with the metabolic pathway and transcript factors of NP remain unclear. This study is aimed to identify the therapy target genes for NP and to investigate the metabolic pathways and transcript factors associated with NP. The differentially expressed genes of three brain tissues (nucleus accumbens, periaqueductal gray, and prefrontal cortex) dealt with NP stimulation were analyzed. Besides, The Database for Annotation, Visualization, and Integrated Discovery and Tfacts datasets were used in the analysis of the genes related to the metabolic pathway and transcript factors of the brain. Eight genes were found to coexpress in all three tissues. A functional enrichment analysis showed that the upregulated genes were mostly enriched in pathways as inflammatory response, calcium-mediated signaling, cytokine-cytokine receptor interaction, and extracellular matrix (ECM)-receptor interaction, whereas the downregulated genes were mostly enriched in pathways as phospholipid metabolic processes, positive regulation of protein kinase B signaling, and metabolism of xenobiotics by cytochrome P450. Finally, 135 and 98 transcript factors genes were upregulated and downregulated, among which SP1, MYC, CTNNB1, CREB1, JUN were identified as the most critical genes because the number of up- and downregulated gene ranked at the top. In conclusion, the pathways of immune response and cytokine-cytokine receptor interaction were determined as the main metabolic pathways of NP affecting the brain, and SP1, MYC, CTNNB1, CREB1, JUN genes were recognized as the most enriched genes in this process, which may provide evidence for the diagnosis and treatment research of neuropathic pain.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Genes jun/genética , Genes myc/genética , Imunoglobulinas/genética , beta Catenina/genética , Animais , Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Masculino , Camundongos , Mapas de Interação de Proteínas/genética , Receptores de Superfície Celular/genéticaRESUMO
BACKGROUND: Evidence shows that the anticancer effects of microtubule targeting agents are not due solely to their antimitotic activities but also their ability to impair microtubule-dependent oncogenic signalling. METHODS: The effects of microtubule targeting agents on regulators of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) were evaluated in breast cancer cell lines using high content imaging, gene and protein expression, siRNA-mediated knockdown and chromatin immunoprecipitation. RESULTS: Microtubule targeting agents rapidly and differentially alter the expression of Snail and Slug, key EMT-promoting transcription factors in breast cancer. Eribulin, vinorelbine and in some cases, ixabepalone, but not paclitaxel, inhibited TGF-ß-mediated Snail expression by impairing the microtubule-dependent nuclear localisation of Smad2/3. In contrast, eribulin and vinorelbine promoted a TGF-ß-independent increase in Slug in cells with low Smad4. Mechanistically, microtubule depolymerisation induces c-Jun, which consequently increases Slug expression in cells with low Smad4. CONCLUSION: These results identify a mechanism by which eribulin-mediated microtubule disruption could reverse EMT in preclinical models and in patients. Furthermore, high Smad4 levels could serve as a biomarker of this response. This study highlights that microtubule targeting drugs can exert distinct effects on the expression of EMT-regulating transcription factors and that identifying differences among these drugs could lead to their more rational use.