ABSTRACT
Inhibition of quorum sensing is considered to be an effective strategy of control and treatment of a wide range of acute and persistent infections. Pseudomonas aeruginosa is an opportunistic bacterium with a high adaptation potential that contributes to healthcare-associated infections. In the present study, the effects of the synthesized hybrid structures bearing sterically hindered phenolic and heterocyclic moieties in a single scaffold on the production of virulence factors by P. aeruginosa were determined. It has been shown that the obtained compounds significantly reduce both pyocyanin and alginate production and stimulate the biosynthesis of siderophores in vitro, which may be attributed to their iron-chelating properties. The results of docking-based inverse high-throughput virtual screening indicate that transcription regulator LasR and Cu-transporter OPRC could be potential molecular targets for these compounds. Investigation of the impact small molecules exert on the molecular mechanisms of the production of bacterial virulence factors may pave the way for the design and development of novel antibacterial agents.
Subject(s)
Pseudomonas aeruginosa , Virulence Factors , Trans-Activators/pharmacology , Quorum Sensing , Pyocyanine , Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , BiofilmsABSTRACT
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease characterised by aberrant fibroblast/myofibroblast accumulation and excessive collagen matrix deposition in the alveolar areas of lungs. As the first approved IPF medication, pirfenidone (PFD) significantly decelerates lung function decline while its underlying anti-fibrotic mechanism remains elusive. METHODS: We performed transcriptomic and immunofluorescence analyses of primary human IPF tissues. RESULTS: We showed that myocardin-related transcription factor (MRTF) signalling is activated in myofibroblasts accumulated in IPF lungs. Furthermore, we showed that PFD inhibits MRTF activation in primary human lung fibroblasts at clinically achievable concentrations (half-maximal inhibitory concentration 50-150â µM, maximal inhibition >90%, maximal concentration of PFD in patients <100â µM). Mechanistically, PFD appears to exert its inhibitory effects by promoting the interaction between MRTF and actin indirectly. Finally, PFD-treated IPF lungs exhibit significantly less MRTF activation in fibroblast foci areas than naïve IPF lungs. CONCLUSIONS: Our results suggest MRTF signalling as a direct target for PFD and implicate that some of the anti-fibrotic effects of PFD may be due to MRTF inhibition in lung fibroblasts.
Subject(s)
Idiopathic Pulmonary Fibrosis , Transcription Factors , Humans , Fibrosis , Trans-Activators/pharmacology , Lung/pathology , Idiopathic Pulmonary Fibrosis/drug therapy , Idiopathic Pulmonary Fibrosis/pathology , Fibroblasts , MyofibroblastsABSTRACT
Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), a syndromic autism spectrum disorder associated with craniofacial abnormalities, intellectual disability, and behavioral problems. There is currently no cure for SMS. Here, we generated a genetic mouse model to determine the reversibility of SMS-like neurobehavioral phenotypes in Rai1 heterozygous mice. We show that normalizing the Rai1 level 3-4 wk after birth corrected the expression of genes related to neural developmental pathways and fully reversed a social interaction deficit caused by Rai1 haploinsufficiency. In contrast, Rai1 reactivation 7-8 wk after birth was not beneficial. We also demonstrated that the correct Rai1 dose is required in both excitatory and inhibitory neurons for proper social interactions. Finally, we found that Rai1 heterozygous mice exhibited a reduction of dendritic spines in the medial prefrontal cortex (mPFC) and that optogenetic activation of mPFC neurons in adults improved the social interaction deficit of Rai1 heterozygous mice. Together, these results suggest the existence of a postnatal temporal window during which restoring Rai1 can improve the transcriptional and social behavioral deficits in a mouse model of SMS. It is possible that circuit-level interventions would be beneficial beyond this critical window.
Subject(s)
Disease Models, Animal , Haploinsufficiency , Interpersonal Relations , Smith-Magenis Syndrome/genetics , Social Behavior Disorders/prevention & control , Trans-Activators/pharmacology , Adolescent , Animals , Dendritic Spines/metabolism , Dendritic Spines/pathology , Heterozygote , Humans , Male , Mice , Mutation , Phenotype , Smith-Magenis Syndrome/pathology , Social Behavior Disorders/geneticsABSTRACT
OBJECTIVE: Hepatitis B virus X protein (HBx) is a pivotal factor for HBV-induced hepatitis. Herein, we sought to investigate HBx-mediated NLR pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis under oxidative stress. METHODS: The effect of HBx on the NLRP3 inflammasome was analyzed by enzyme-linked immunosorbent assays, quantitative reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence in hepatic HL7702 cells. Pyroptosis was evaluated by western blotting, lactate dehydrogenase release, propidium iodide staining, and transmission electron microscopy. NLRP3 expression in the inflammasome from liver tissues was assessed by immunohistochemistry. RESULTS: In hydrogen peroxide (H2O2)-stimulated HL7702 cells, HBx triggered the release of pro-inflammatory mediators apoptosis-associated speck-like protein containing a CARD (ASC), interleukin (IL)-1ß, IL-18, and high-mobility group box 1 (HMGB1); activated NLRP3; and initiated pro-inflammatory cell death (pyroptosis). HBx localized to the mitochondria, where it induced mitochondrial damage and production of mitochondrial reactive oxygen species (mitoROS). Treatment of HL7702 cells with a mitoROS scavenger attenuated HBx-induced NLRP3 activation and pyroptosis. Expression levels of NLRP3, ASC, and IL-1ß in liver tissues from patients were positively correlated with HBV DNA concentration. CONCLUSIONS: The NLRP3 inflammasome was activated by elevated mitoROS levels and mediated HBx-induced liver inflammation and hepatocellular pyroptosis under H2O2-stress conditions.
Subject(s)
Hepatocytes/pathology , Inflammasomes/physiology , NLR Family, Pyrin Domain-Containing 3 Protein/physiology , Oxidative Stress , Pyroptosis/drug effects , Trans-Activators/pharmacology , Viral Regulatory and Accessory Proteins/pharmacology , CARD Signaling Adaptor Proteins/blood , Carcinoma, Hepatocellular/virology , Cell Line , DNA, Viral/analysis , Gene Expression , Hepatitis B virus/genetics , Hepatocytes/metabolism , Humans , Hydrogen Peroxide/pharmacology , Liver Neoplasms/virology , Mitochondria, Liver/metabolism , Reactive Oxygen Species/metabolism , Trans-Activators/genetics , Transfection , Viral Regulatory and Accessory Proteins/geneticsABSTRACT
Retinitis pigmentosa (RP) is a generic term for a group of genetic diseases characterized by loss of rod and cone photoreceptor cells. Although the genetic causes of RP frequently only affect the rod photoreceptor cells, cone photoreceptors become stressed in the absence of rods and undergo a secondary degeneration. Changes in the gene expression profile of cone photoreceptor cells are likely to occur prior to observable physiological changes. To this end, we sought to achieve greater understanding of the changes in cone photoreceptor cells early in the degeneration process of the Rho-/- mouse model. To account for gene expression changes attributed to loss of cone photoreceptor cells, we normalized PCR in the remaining number of cones to a cone cell reporter (OPN1-GFP). Gene expression profiles of key components involved in the cone phototransduction cascade were correlated with tests of retinal cone function prior to cell loss. A significant downregulation of the photoreceptor transcription factor Crx was observed, which preceded a significant downregulation in cone opsin transcripts that coincided with declining cone function. Our data add to the growing understanding of molecular changes that occur prior to cone dysfunction in a model of rod-cone dystrophy. It is of interest that gene supplementation of CRX by adeno-associated viral vector delivery prior to cone cell loss did not prevent cone photoreceptor degeneration in this mouse model.
Subject(s)
Cone-Rod Dystrophies/genetics , Cone-Rod Dystrophies/physiopathology , Animals , Cone-Rod Dystrophies/therapy , Disease Models, Animal , Electroretinography , Gene Expression Regulation , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Genetic Vectors/pharmacology , Green Fluorescent Proteins/genetics , HEK293 Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/pharmacology , Humans , Mice, Transgenic , Ophthalmoscopy , Retinal Cone Photoreceptor Cells/pathology , Retinal Cone Photoreceptor Cells/physiology , Rhodopsin/genetics , Rod Opsins/genetics , Tomography, Optical Coherence , Trans-Activators/genetics , Trans-Activators/pharmacology , Vision, Ocular/geneticsABSTRACT
The transcription factor PU.1 plays multiple context and concentration dependent roles in lymphoid and myeloid cell development. Here we showed that PU.1 (encoded by Sfpi1) was essential for dendritic cell (DC) development in vivo and that conditional ablation of PU.1 in defined precursors, including the common DC progenitor, blocked Flt3 ligand-induced DC generation in vitro. PU.1 was also required for the parallel granulocyte-macrophage colony stimulating factor-induced DC pathway from early hematopoietic progenitors. Molecular studies demonstrated that PU.1 directly regulated Flt3 in a concentration-dependent manner, as Sfpi1(+/-) cells displayed reduced expression of Flt3 and impaired DC formation. These studies identify PU.1 as a critical regulator of both conventional and plasmacytoid DC development and provide one mechanism how altered PU.1 concentration can have profound functional consequences for hematopoietic cell development.
Subject(s)
Dendritic Cells/immunology , Membrane Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Animals , Base Sequence , Cell Differentiation , Cells, Cultured , Dendritic Cells/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Membrane Proteins/drug effects , Membrane Proteins/genetics , Mice , Mice, Transgenic , Models, Immunological , Molecular Sequence Data , Polymerase Chain Reaction , Promoter Regions, Genetic , Proto-Oncogene Proteins/pharmacology , Trans-Activators/pharmacologyABSTRACT
Side effects of methotrexate (MTX) especially hepatotoxicity limits clinical applications of this anticancer agent. Carboxypeptidase G2 (CPG2) is administrated for the treatment of elevated plasma concentrations of MTX. In this study, we have investigated the intracellular delivery of CPG2 fused to the transactivator transduction domain (TAT) and its protective effects against MTX-induced cell death of HepG2 cells. We have observed that both native and denatured forms of the enzyme transduced into the HepG2 cells efficiently in a concentration and time-dependent manner. The denatured protein transduced with higher efficiency than the native form and was functional inside the cells. MTX exposure significantly decreased HepG2 cell viability in a dose- and time-dependent manner. The cell viability after 24 and 48â¯h of incubation with 100⯵M MTX was reduced to 44.37% and 17.69%, respectively. In cells pretreated with native and denatured TAT-CPG2 protein the cell viability was 98.63% and 86.31% after 24 and 48â¯h, respectively. Treatment with MTX increased the number of apoptotic HepG2 cells to 90.23% after 48â¯h. However, the apoptosis percentage in cells pretreated with native and denatured TAT-CPG2 was 21.49% and 22.28%, respectively. Our results showed that TAT-CPG2 significantly prevents MTX-induced oxidative stress by decreasing the formation of ROS and increasing the content of glutathione (GSH) and catalase activity. Our finding indicates that both native and denatured TAT-CPG2 strongly protect HepG2 cells against MTX-induced oxidative stress and apoptosis. Hence, intracellular delivery of CPG2 might provide a new therapeutic strategy for protecting against MTX mediated cytotoxicity.
Subject(s)
Cell Death/drug effects , Methotrexate/adverse effects , Protective Agents/pharmacology , Trans-Activators/pharmacology , gamma-Glutamyl Hydrolase/pharmacology , Apoptosis/drug effects , Catalase/metabolism , Cell Line, Tumor , Glutathione/metabolism , Hep G2 Cells , Humans , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolismABSTRACT
Hepatitis B virus (HBV) infection is the primary cause of cirrhosis and liver cancer. Protein-protein interactions (PPIs) between HBV x protein (HBx) and its host targets, including Bcl-2, are important for cell death and viral replication. No modulators targeting these PPIs have been reported yet. Here, we developed HBx-derived constrained peptides generated by a facile macrocyclization method by joining two methionine side chains of unprotected peptides with chemoselective alkylating linkers. The resulting constrained peptides with improved cell permeability and binding affinity were effective anti-HBV modulators by blocking the secretion of viral antigens. This study clearly demonstrated HBx as a potentially important PPI target and the potential application of this efficient peptide macrocyclization strategy for targeting key PPIs.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B Surface Antigens/metabolism , Hepatitis B virus/metabolism , Peptides/pharmacology , Trans-Activators/pharmacology , Antiviral Agents/chemistry , Cell Membrane Permeability , Cyclization , Hep G2 Cells , Hepatitis B/drug therapy , Hepatitis B/immunology , Hepatitis B/virology , Hepatitis B Surface Antigens/immunology , Hepatitis B virus/immunology , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/immunology , Humans , Peptides/chemistry , Protein Binding/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Trans-Activators/chemistry , Viral Regulatory and Accessory ProteinsABSTRACT
Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands, that is, MHC class I-related chain (MIC) A/B and UL16-binding proteins (ULBP)1-6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3, and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter via its binding to an IE2-responsive element that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation via a mechanism independent of IE DNA binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3-related protein, and DNA-dependent protein kinase did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host antiviral response.
Subject(s)
Gene Expression Regulation , Histocompatibility Antigens Class I/genetics , Immediate-Early Proteins/metabolism , Receptors, Virus/genetics , Trans-Activators/metabolism , Antigens, Differentiation, T-Lymphocyte/genetics , Cell Line , Cytotoxicity, Immunologic , DNA Replication/drug effects , Fibroblasts/drug effects , Fibroblasts/virology , Foscarnet/pharmacology , GPI-Linked Proteins/genetics , HEK293 Cells , Host-Pathogen Interactions , Humans , Immediate-Early Proteins/pharmacology , Intercellular Signaling Peptides and Proteins/genetics , Killer Cells, Natural/immunology , NK Cell Lectin-Like Receptor Subfamily K/genetics , Trans-Activators/pharmacology , Transcriptional Activation , Up-Regulation , Viral Proteins/genetics , Virus Replication/drug effectsABSTRACT
Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.
Subject(s)
Cell Differentiation/drug effects , Estrogen Receptor alpha/metabolism , Leiomyoma/metabolism , Nuclear Proteins/pharmacology , Trans-Activators/pharmacology , Animals , Cell Differentiation/physiology , Gene Expression Regulation/genetics , Humans , Leiomyoma/chemically induced , Leiomyoma/drug therapy , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Nuclear Proteins/metabolism , Promoter Regions, Genetic/genetics , Rats , Serum Response Factor/metabolism , Trans-Activators/metabolismABSTRACT
Hepatitis B virus (HBV) infection plays a crucial role and is a major cause of hepatocellular carcinoma (HCC) in China. microRNAs (miRNAs) have emerged as key players in hepatic steatosis and carcinogenesis. We found that down-regulation of miR-384 expression was a common event in HCC, especially HBV-related HCC. However, the possible function of miR-384 in HBV-related HCC remains unclear. The oncogene pleiotrophin (PTN) was a target of miR-384. HBx inhibited miR-384, increasing PTN expression. The PTN receptor N-syndecan was highly expressed in HCC. PTN induced by HBx acted as a growth factor via N-syndecan on hepatocytes and further promoted cell proliferation, metastasis and lipogenesis. PTN up-regulated sterol regulatory element-binding protein 1c (SREBP-1c) through the N-syndecan/PI3K/Akt/mTORC1 pathway and the expression of lipogenic genes, including fatty acid synthesis (FAS). PTN-mediated de novo lipid synthesis played an important role in HCC proliferation and metastasis. PI3K/AKT and an mTORC1 inhibitor diminished PTN-induced proliferation, metastasis and lipogenesis. Taken together, these data strongly suggest that the dysregulation of miR-384 could play a crucial role in HBV related to HCC, and the target gene of miR-384, PTN, represents a new potential therapeutic target for the prevention of hepatic steatosis and further progression to HCC after chronic HBV infection.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carrier Proteins/genetics , Cytokines/genetics , Gene Expression Regulation, Neoplastic , Hepatitis B/genetics , Host-Pathogen Interactions , Liver Neoplasms/genetics , MicroRNAs/genetics , Adult , Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carrier Proteins/metabolism , Cell Proliferation , Chromones/pharmacology , Cytokines/metabolism , Female , Hep G2 Cells , Hepatitis B/complications , Hepatitis B/metabolism , Hepatitis B/pathology , Hepatitis B virus/pathogenicity , Hepatitis B virus/physiology , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Lipogenesis/drug effects , Lipogenesis/genetics , Liver Neoplasms/etiology , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Lymphatic Metastasis , Male , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , MicroRNAs/metabolism , Middle Aged , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Sirolimus/pharmacology , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolism , Syndecan-3/genetics , Syndecan-3/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Trans-Activators/pharmacology , Viral Regulatory and Accessory Proteins , fas Receptor/genetics , fas Receptor/metabolismABSTRACT
Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis. In the 2-year carcinogenicity study with tofacitinib, increased incidence of hibernoma (a neoplasm of brown adipose tissue [BAT]) was noted in female rats at ≥30 mg/kg/day (≥41x human exposure multiples). Thus, signaling pathways within BAT were investigated by measuring BAT: weight, cell proliferation biomarkers, content of basal and prolactin-induced phosphorylated Signal Transducer and Activator of Transcription (STAT), and uncoupling protein 1 (UCP-1). The relationship between cardiovascular hemodynamics and plasma norepinephrine (NE) levels was also investigated. Tofacitinib administered to female rats at doses of 10, 30, or 75 mg/kg/day for 14 days increased BAT weight at 75 mg/kg/day and cell proliferation at ≥30 mg/kg/day. JAK inhibition, observed as lower pSTAT3 and pSTAT5 in BAT, was noted at ≥10 mg/kg/day, while lower activity of BAT was observed as lower UCP-1 protein at ≥30 mg/kg/day. In cultured brown adipocytes, prolactin-induced increase in pSTAT5 and pSTAT3 were inhibited by tofacitinib in a concentration-dependent manner. Tofacitinib lowered blood pressure, increased heart rate, and resulted in dose-dependent increases in circulating NE. Thus, JAK/STAT inhibition in BAT and sympathetic stimulation are two factors which might contribute to the genesis of hibernomas by tofacitinib in rats.
Subject(s)
Lipoma/chemically induced , Piperidines/adverse effects , Protein Kinase Inhibitors/adverse effects , Pyrimidines/adverse effects , Pyrroles/adverse effects , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Animals , Blood Pressure/drug effects , Cell Proliferation/drug effects , Female , Heart Rate/drug effects , Janus Kinase Inhibitors/adverse effects , Janus Kinase Inhibitors/pharmacology , Lipoma/metabolism , Male , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Rats , Rats, Sprague-Dawley , STAT Transcription Factors/antagonists & inhibitors , Signal Transduction/drug effects , Trans-Activators/adverse effects , Trans-Activators/pharmacologyABSTRACT
BACKGROUND/AIMS: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. METHODS: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. RESULTS: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. CONCLUSIONS: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.
Subject(s)
Epithelial Cells/drug effects , Fas Ligand Protein/agonists , Hepatitis B virus/chemistry , Signal Transduction/genetics , Trans-Activators/pharmacology , fas Receptor/agonists , Animals , Apoptosis/drug effects , Carbazoles/pharmacology , Caspase 8/genetics , Caspase 8/metabolism , Cell Line , Cell Proliferation/drug effects , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fas Ligand Protein/genetics , Fas Ligand Protein/metabolism , Gene Expression Regulation , Indole Alkaloids/pharmacology , Kidney Tubules/cytology , Kidney Tubules/drug effects , Kidney Tubules/metabolism , MAP Kinase Kinase 7/genetics , MAP Kinase Kinase 7/metabolism , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase 10/genetics , Mitogen-Activated Protein Kinase 10/metabolism , Plasmids/chemistry , Plasmids/metabolism , Rats , Trans-Activators/isolation & purification , Transfection , Viral Regulatory and Accessory Proteins , fas Receptor/genetics , fas Receptor/metabolism , Mitogen-Activated Protein Kinase Kinase Kinase 11ABSTRACT
Epstein-Barr virus (EBV) infection is closely associated with several human malignancies including nasopharyngeal carcinoma (NPC). The EBV immediate-early protein BZLF1 is the key mediator that switches EBV infection from latent to lytic forms. The lytic form of EBV infection has been implicated in human carcinogenesis but its molecular mechanisms remain unclear. BZLF1 has been shown to be a binding partner of several DNA damage response (DDR) proteins. Its functions in host DDR remain unknown. Thus, we explore the effects of BZLF1 on cellular response to DNA damage in NPC cells. We found that expression of BZLF1 impaired the binding between RNF8 and MDC1 (mediator of DNA damage checkpoint 1), which in turn interfered with the localization of RNF8 and 53BP1 to the DNA damage sites. The RNF8-53BP1 pathway is important for repair of DNA double-strand breaks and DNA damage-induced G2/M checkpoint activation. Our results showed that, by impairing DNA damage repair as well as abrogating G2/M checkpoint, BZLF1 induced genomic instability and rendered cells more sensitive to ionizing radiation. Moreover, the blockage of 53BP1 and RNF8 foci formation was recapitulated in EBV-infected cells. Taken together, our study raises the possibility that, by causing mis-localization of important DDR proteins, BZLF1 may function as a link between lytic EBV infection and impaired DNA damage repair, thus contributing to the carcinogenesis of EBV-associated human epithelial malignancies.
Subject(s)
DNA Damage/drug effects , DNA Repair/drug effects , DNA-Binding Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Trans-Activators/metabolism , Trans-Activators/pharmacology , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins , Cell Line, Tumor , DNA Breaks, Double-Stranded , DNA Damage/physiology , DNA Repair/physiology , HeLa Cells , Herpesvirus 4, Human , Host-Pathogen Interactions , Humans , Nasopharyngeal Neoplasms , Nuclear Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1 , Ubiquitin-Protein LigasesABSTRACT
Nuclear receptors and transcription factors regulate the mRNA expression of many drug metabolizing enzymes, including the carboxylesterases (Ces). However, there are few data regarding whether these changes in mRNA expression result in alteration of protein levels or activity. In the present study, we sought to determine the isoform-specific regulation of hepatic Ces mRNA expression and activity following the administration of pharmacological activators of the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and nuclear factor E2-related protein (Nrf2) to mice. The CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and PXR ligand pregnenolone-16a-carbonitrile (PCN) increased Ces mRNA expression of various Ces2 isoforms, whereas the Nrf2 activator butylated hydroxyanisole primarily reduced Ces3a mRNA expression and induced Ces1g mRNA. TCPOBOP and PCN increased Ces2 hydrolytic activity in an isoform-specific manner. Taken together, these data demonstrate that activation of CAR, PXR, and Nrf2 regulates not only Ces mRNA expression, but also isoform-specific activity.
Subject(s)
Carboxylesterase/genetics , Gene Expression Regulation, Enzymologic/drug effects , Isoenzymes/genetics , Trans-Activators/pharmacology , Animals , Male , Mice , Mice, Inbred C57BL , Pregnenolone Carbonitrile/pharmacology , Pyridines/pharmacology , RNA, Messenger/geneticsABSTRACT
Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/pharmacology , Cell-Penetrating Peptides/pharmacology , Homeodomain Proteins/pharmacology , Induced Pluripotent Stem Cells/drug effects , Insulin-Secreting Cells/drug effects , Maf Transcription Factors, Large/pharmacology , Nerve Tissue Proteins/pharmacology , Trans-Activators/pharmacology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation/drug effects , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/metabolism , Cellular Reprogramming/drug effects , Cinnamates/pharmacology , Gene Expression , Glucagon-Like Peptide 1/pharmacology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Maf Transcription Factors, Large/genetics , Maf Transcription Factors, Large/metabolism , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Niacinamide/pharmacology , Trans-Activators/genetics , Trans-Activators/metabolism , Tretinoin/pharmacology , Veratrum Alkaloids/pharmacologyABSTRACT
OBJECTIVE: To explore the involvement of hepatitis B X protein (HBx) in promoter 3 (P3)-driven mRNA overexpression of the insulin-like growth factor II gene (IGF-II) and investigate the underlying epigenetic mechanism. METHODS: Levels of P3 and HBx mRNA and status of P3 methylation were analyzed in human hepatocellular carcinoma (HCC) samples, with and without hepatitis B virus (HBV) infection, using quantitative reverse transcription-PCR and bisulfite sequencing. In addition, the levels of P3 mRNA and P3 methylation were examined in HepG2 cells stably overexpressing HBx (HepG2-HBx). Finally, P3 promoter-luciferase constructs were cotransfected into HepG2 cells along with an HBx-expressing plasmid, and the effects of HBx on transcriptional activity and methylation of P3 were analyzed. Statistical analyses of the data were conducted by chi square test, Fisher's exact test, Student's t-test, Marn-Whitney U test, and Pearson's correlation coefficient test. RESULTS: The HBV-positive HCC specimens had significantly higher levels of P3 mRNA than the HBV-negative HCC specimens (-9.59 ± 3.22 vs. -12.97 ± 3.08 delta CT; P=0.006) but significantly lower levels of P3 methylation (mean values for the 17 CpG sites (36.9% ± 15.5% vs. 52.1% ± 19.1%; P=0.025). The P3 transcript abundance was positively correlated with the level of HBx expression and negatively correlated with the level of P3 methylation. The epigenetic results from experiments with the HepG2-HBx cells were similar. Transfection of HBx significantly decreased P3 methylation level and increased its activity. CONCLUSION: HBx expression may promote IGF-II expression by inducing hypomethylation of its P3 promoter in hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular/genetics , DNA Methylation , Insulin-Like Growth Factor II/metabolism , Liver Neoplasms/genetics , Promoter Regions, Genetic , Trans-Activators/pharmacology , Carcinoma, Hepatocellular/metabolism , Epigenesis, Genetic , Female , Gene Expression , Hep G2 Cells , Humans , Insulin-Like Growth Factor II/genetics , Liver Neoplasms/metabolism , Male , RNA, Messenger/genetics , Viral Regulatory and Accessory ProteinsABSTRACT
Alkyl glycosides (AGs), commonly used nonionic surfactants, may have toxic effects on the environmental organisms. However, the complex concentration-response patterns of AGs with varying alkyl side chains and their mixtures have not been thoroughly studied. Therefore, the luminescence inhibition toxicities of six AGs with different alkyl side chains, namely, ethyl (AG02), butyl (AG04), hexyl (AG06), octyl (AG08), decyl (AG10), and dodecyl (AG12) glucosides, were determined in Vibrio qinghaiensis sp. -Q67 (Q67) at 0.25, 3, 6, 9, and 12 h. The six AGs exhibited time- and side-chain-dependent nonmonotonic concentration- responses toward Q67. AG02, with a short side chain, presented a concentration-response curve (CRC) with two peaks after 6 h and stimulated the luminescence of Q67 at both 6 and 9 h. AG04, AG06, and AG08 showed S-shaped CRCs at five exposure time points, and their toxicities increased with the side-chain length. AG10 and AG12, with long side chains, exhibited hormesis at 9 and 12 h. Molecular docking was performed to explore the mechanism governing the possible influence of AGs on the luminescence response. The effects of AGs on Q67 could be attributed to multiple luminescence-regulatory proteins, including LuxA, LuxC, LuxD, LuxG, LuxI, and LuxR. Notably, LuxR was identified as the primary binding protein among the six AGs. Given that they may co-exist, binary mixtures of AG10 and AG12 were designed to explore their concentration-response patterns and interactions. The results revealed that all AG10-AG12 binary mixture rays showed time-dependent hormesis on Q67, similar to that shown by their individual components. The interactions of these binary mixtures were mainly characterized by low-concentration additive action and high-concentration synergism at different times.
Subject(s)
Glycosides , Vibrio , Glycosides/toxicity , Molecular Docking Simulation , Drug Interactions , Trans-Activators/pharmacologyABSTRACT
Nasopharyngeal carcinoma (NPC) is a specific human malignancy with unique geographic distribution and genetic backgrounds. Although early treatment with radio-chemotherapy has been proven effective for NPC therapy, its therapeutic efficacy substantially diminishes in the late stages of this malignancy. In the tumor microenvironment of NPC, PD-L1 has been demonstrated as a critical factor in impairing T cell activation. As an etiological role for NPC development, it is found that Epstein-Barr virus (EBV) latent proteins upregulated PD-L1 expression. However, whether EBV lytic protein affects PD-L1 expression remains unclear. In this study, through monitoring the mRNA expression pattern of lytic genes and PD-L1 in EBV-positive NPC cell line NA, EBV immediately-early gene BRLF1(Rta) was found to have the potential for PD-L1 activation. Furthermore, we identified that Rta expression enhanced PD-L1 expression in mRNA and protein levels through quantitative real-time polymerase chain reaction and western blotting analysis. The luciferase reporter assay revealed that Rta expression enhanced PD-L1 promoter activity. We also demonstrated that Rta-induced PD-L1 expressions could impair interleukin 2 secretion of T cells, and this mechanism may be through ERK activation. These results displayed the importance of EBV Rta in PD-L1 expression in NPC and may give an alternative target for NPC therapy.
Subject(s)
Epstein-Barr Virus Infections , Immediate-Early Proteins , Nasopharyngeal Neoplasms , Humans , Nasopharyngeal Carcinoma/genetics , Herpesvirus 4, Human/genetics , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/genetics , B7-H1 Antigen/genetics , Nasopharyngeal Neoplasms/genetics , Nasopharyngeal Neoplasms/drug therapy , Nasopharyngeal Neoplasms/pathology , RNA, Messenger/genetics , Tumor Microenvironment , Trans-Activators/genetics , Trans-Activators/metabolism , Trans-Activators/pharmacology , Immediate-Early Proteins/geneticsABSTRACT
The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.