ABSTRACT
Abnormal neovascularization is an important cause of blindness in many ocular diseases, for which the etiology and pathogenic mechanisms remain incompletely understood. Recent studies have revealed the diverse roles of noncoding RNAs in various biological processes and facilitated the research and development of the clinical application of numerous RNA drugs, including microRNAs. Here, we report the antiangiogenic activity of microRNA-29a (miR-29a) in three animal models of ocular neovascularization. The miR-29a knockout (KO) mice displayed enhanced vessel pruning, resulting in a decreased vascularized area during retinal development. In contrast, miR-29a deletion in adult mice accelerated angiogenesis in preclinical disease models, including corneal neovascularization, oxygen-induced retinopathy, and choroidal neovascularization, while the administration of agomir-29a ameliorated pathological neovascularization. Furthermore, miR-29a exerted inhibitory effects on endothelial cell proliferation, migration, and tube formation capacities. RNA sequencing analysis of retinas from miR-29a KO mice and RNA interference experiments identified platelet-derived growth factor C and several extracellular matrix genes as downstream targets of miR-29a involved in regulating ocular angiogenesis. Our data suggest that miR-29a may be a promising clinical candidate for the treatment of neovascular diseases.
Subject(s)
Choroidal Neovascularization , MicroRNAs , Mice , Animals , MicroRNAs/metabolism , Cell Proliferation , RNA Interference , Eye/metabolism , Choroidal Neovascularization/metabolism , Mice, KnockoutABSTRACT
BK polyomavirus (PyV) infects the genitourinary tract of >90% of the adult population. Immunosuppression increases the risk of viral reactivation, making BKPyV a leading cause of graft failure in kidney transplant recipients. Polyomaviruses have a small double-stranded DNA (dsDNA) genome that requires host replication machinery to amplify the viral genome. Specifically, polyomaviruses promote S phase entry and delay S phase exit by activating the DNA damage response (DDR) pathway via an uncharacterized mechanism requiring viral replication. BKPyV infection elevates expression of MutSα, a mismatch repair (MMR) pathway protein complex that senses and repairs DNA mismatches and can activate the DDR. Thus, we investigated the role of the MMR pathway by silencing the MutSα component, Msh6, in BKPyV-infected primary cells. This resulted in severe DNA damage that correlated with weak DNA damage response activation and a failure to arrest the cell cycle to prevent mitotic entry during infection. Furthermore, silencing Msh6 expression resulted in significantly fewer infectious viral particles due to significantly lower levels of VP2, a minor capsid protein important for trafficking during subsequent infections. Since viral assembly occurs in the nucleus, our findings are consistent with a model in which entry into mitosis disrupts viral assembly due to nuclear envelope breakdown, which disperses VP2 throughout the cell, reducing its availability for encapsidation into viral particles. Thus, the MMR pathway may be required to activate the ATR (ATM-Rad3-related) pathway during infection to maintain a favorable environment for both viral replication and assembly. IMPORTANCE Since there are no therapeutics that target BKPyV reactivation in organ transplant patients, it is currently treated by decreasing immunosuppression to allow the natural immune system to fight the viral infection. Antivirals would significantly improve patient outcomes since reducing immunosuppression carries the risk of graft failure. PyVs activate the DDR, for which there are several promising inhibitors. However, a better understanding of how PyVs activate the DDR and what role the DDR plays during infection is needed. Here, we show that a component of the mismatch repair pathway is required for DDR activation during PyV infection. These findings show that the mismatch repair pathway is important for DDR activation during PyV infection and that inhibiting the DDR reduces viral titers by generating less infectious virions that lack the minor capsid protein VP2, which is important for viral trafficking.
Subject(s)
BK Virus , DNA Mismatch Repair , BK Virus/genetics , Capsid Proteins/genetics , DNA Damage , DNA Mismatch Repair/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Polyomavirus Infections/virology , Virus Replication/geneticsABSTRACT
Phosphocholine phosphatase-1 (PHOSPHO1) is a phosphocholine phosphatase that catalyzes the hydrolysis of phosphocholine (PC) to choline. Here we demonstrate that the PHOSPHO1 transcript is highly enriched in mature brown adipose tissue (BAT) and is further induced by cold and isoproterenol treatments of BAT and primary brown adipocytes. In defining the functional relevance of PHOPSPHO1 in BAT thermogenesis and energy metabolism, we show that PHOSPHO1 knockout mice are cold-tolerant, with higher expression of thermogenic genes in BAT, and are protected from high-fat diet-induced obesity and development of insulin resistance. Treatment of mice with the PHOSPHO1 substrate phosphocholine is sufficient to induce cold tolerance, thermogenic gene expression, and allied metabolic benefits. Our results reveal a role of PHOSPHO1 as a negative regulator of BAT thermogenesis, and inhibition of PHOSPHO1 or enhancement of phosphocholine represent innovative approaches to manage the metabolic syndrome.
Subject(s)
Adipose Tissue, Brown/physiology , Phosphoric Monoester Hydrolases/genetics , Phosphorylcholine/metabolism , Thermogenesis , Adipocytes, Brown/enzymology , Adipocytes, Brown/metabolism , Adipose Tissue, Brown/enzymology , Animals , Cold Temperature , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphoric Monoester Hydrolases/deficiencyABSTRACT
The soft rot disease caused by Rhizopus stolonifer is an important disease in cherry tomato fruit. In this study, the effect of iturin A on soft rot of cherry tomato and its influence on the storage quality of cherry tomato fruit were investigated. The results showed that 512 µg/mL of iturin A could effectively inhibit the incidence of soft rot of cherry tomato fruit. It was found that iturin A could induce the activity of resistance-related enzymes including phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), glucanase (GLU), and chitinase (CHI), and active oxygen-related enzymes including ascorbate peroxidases (APX), superoxide dismutases (SOD), catalases (CAT), and glutathione reductase (GR) of cherry tomato fruit. In addition, iturin A treatment could slow down the weight loss of cherry tomato and soften the fruit. These results indicated that iturin A could retard the decay and improve the quality of cherry tomato fruit by both the inhibition growth of R. stolonifera and the inducing the resistance.
Subject(s)
Drug Resistance/drug effects , Fruit/metabolism , Peptides, Cyclic/pharmacology , Plant Diseases/microbiology , Plant Roots/metabolism , Solanum lycopersicum/metabolism , Fruit/microbiology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Solanum lycopersicum/microbiology , Plant Proteins/biosynthesis , Plant Roots/microbiology , Rhizopus/growth & developmentABSTRACT
The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), ß-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.
Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Fruit/microbiology , Rhizopus/drug effects , Rhizopus/growth & development , Solanum lycopersicum/microbiology , Chitinases/metabolism , Fruit/enzymology , Glucan 1,3-beta-Glucosidase/metabolism , Solanum lycopersicum/enzymology , Peroxidase/metabolism , Phenylalanine Ammonia-Lyase/metabolism , Plant Diseases/microbiology , Spores, Fungal/drug effects , Spores, Fungal/growth & developmentABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver disease. The incidence of NAFLD has increased steadily due to its close association with the global epidemic of obesity and type 2 diabetes. However, there is no effective pharmacological therapy approved for NAFLD. Farnesoid X receptor (FXR), a member of the nuclear receptor subfamily, plays important roles in maintaining the homeostasis of bile acids, glucose, and lipids. FXR agonists have shown promise for the treatment of NAFLD. In this study, we report altenusin (2076A), a natural nonsteroidal fungal metabolite, as a novel selective agonist of FXR with an EC50 value of 3.2 ± 0.2 µM. Administration of 2076A protected mice from high-fat diet (HFD)-induced obesity by reducing the body weight and fat mass by 22.9% and 50.0%, respectively. Administration of 2076A also decreased the blood glucose level from 178.3 ± 12.4 mg/dl to 116.2 ± 4.1 mg/dl and the serum insulin level from 1.4 ± 0.6 ng/dl to 0.4 ± 0.1 ng/dl. Moreover, 2076A treatment nearly reversed HFD-induced hepatic lipid droplet accumulation and macrovesicular steatosis. These metabolic effects were abolished in FXR knockout mice. Mechanistically, the metabolic benefits of 2076A might have been accounted for by the increased insulin sensitivity and suppression of genes that are involved in hepatic gluconeogenesis and lipogenesis. In summary, we have uncovered a new class of nonsteroidal FXR agonist that shows promise in treating NAFLD and the associated metabolic syndrome.
Subject(s)
Biphenyl Compounds/pharmacology , Biphenyl Compounds/therapeutic use , Non-alcoholic Fatty Liver Disease/drug therapy , Non-alcoholic Fatty Liver Disease/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Biphenyl Compounds/chemistry , Dose-Response Relationship, Drug , HEK293 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Docking Simulation/methods , Protein Structure, Secondary , Receptors, Cytoplasmic and Nuclear/chemistryABSTRACT
UNLABELLED: The APOBEC3 family of DNA cytosine deaminases has important roles in innate immunity and cancer. It is unclear how DNA tumor viruses regulate these enzymes and how these interactions, in turn, impact the integrity of both the viral and cellular genomes. Polyomavirus (PyVs) are small DNA pathogens that contain oncogenic potentials. In this study, we examined the effects of PyV infection on APOBEC3 expression and activity. We demonstrate that APOBEC3B is specifically upregulated by BK polyomavirus (BKPyV) infection in primary kidney cells and that the upregulated enzyme is active. We further show that the BKPyV large T antigen, as well as large T antigens from related polyomaviruses, is alone capable of upregulating APOBEC3B expression and activity. Furthermore, we assessed the impact of A3B on productive BKPyV infection and viral genome evolution. Although the specific knockdown of APOBEC3B has little short-term effect on productive BKPyV infection, our informatics analyses indicate that the preferred target sequences of APOBEC3B are depleted in BKPyV genomes and that this motif underrepresentation is enriched on the nontranscribed stand of the viral genome, which is also the lagging strand during viral DNA replication. Our results suggest that PyV infection upregulates APOBEC3B activity to influence virus sequence composition over longer evolutionary periods. These findings also imply that the increased activity of APOBEC3B may contribute to PyV-mediated tumorigenesis. IMPORTANCE: Polyomaviruses (PyVs) are a group of emerging pathogens that can cause severe diseases, including cancers in immunosuppressed individuals. Here we describe the finding that PyV infection specifically induces the innate immune DNA cytosine deaminase APOBEC3B. The induced APOBEC3B enzyme is fully functional and therefore may exert mutational effects on both viral and host cell DNA. We provide bioinformatic evidence that, consistent with this idea, BK polyomavirus genomes are depleted of APOBEC3B-preferred target motifs and enriched for the corresponding predicted reaction products. These data imply that the interplay between PyV infection and APOBEC proteins may have significant impact on both viral evolution and virus-induced tumorigenesis.
Subject(s)
Cytidine Deaminase/metabolism , Gene Expression Regulation , Genome, Viral , Kidney Tubules/enzymology , Minor Histocompatibility Antigens/metabolism , Polyomavirus Infections/virology , Polyomavirus/pathogenicity , Virus Replication , Cells, Cultured , Cytidine Deaminase/antagonists & inhibitors , Cytidine Deaminase/genetics , Humans , Kidney Tubules/virology , Minor Histocompatibility Antigens/genetics , Polyomavirus/genetics , Polyomavirus Infections/pathology , RNA, Small Interfering/genetics , Transcriptional Activation , Up-RegulationABSTRACT
Sulfonation and desulfation are two opposing processes that represent an important layer of regulation of estrogenic activity via ligand supplies. Enzymatic activities of families of enzymes, known as sulfotransferases and sulfatases, lead to structural and functional changes of the steroids, thyroids, xenobiotics, and neurotransmitters. Estrogen sulfotransferase (EST) and steroid sulfatase (STS) represent negative and positive regulation of the estrogen activity, respectively. This is because EST-mediated sulfation deactivates estrogens, whereas STS-mediated desulfation converts the inactive estrogen sulfates to active estrogens. In addition to the known functions of estrogens, EST and STS in reproductive processes, regulation of estrogens and other signal molecules especially at the local tissue levels has gained increased attention in the context of metabolic disease in recent years. EST expression is detectable in the subcutaneous adipose tissue in both obese women and men, and the expression of EST is markedly induced in the livers of rodent models of obesity and type 2 diabetes. STS was found to be upregulated in patients with chronic inflammatory liver diseases. Interestingly, the tissue distribution and the transcriptional regulation of EST and STS exhibit obvious sex and species specificity. EST ablation produces completely opposite metabolic phenotype in female and male obese mice. Adipogenesis is also differentially regulated by EST in murine and human adipocytes. This chapter focuses on the recent progress in our understanding of the expression and regulation EST and STS in the context of metabolic homeostasis.
Subject(s)
Energy Metabolism , Estrogens/metabolism , Steryl-Sulfatase/metabolism , Sulfotransferases/metabolism , Animals , Female , Homeostasis , Humans , Male , Sex Characteristics , Sex Factors , Signal Transduction , Species SpecificityABSTRACT
OBJECTIVE: The diagnosis of coronary artery disease (CAD) in atrial fibrillation (AF) patients using coronary computed tomography angiography (CCTA) requires a large exposure dosage or repeated examinations. This study evaluates the feasibility of using low-dose CCTA in the double prospectively ECG-triggered high-pitch spiral acquisition mode (Double Flash Spiral mode). METHODS: Twenty-eight AF patients with suspected CAD were recruited. Double Flash Spiral mode (tube voltage 100 kVp) and iterative reconstruction was used for CCTA examination. Two radiologists cross-evaluated the CCTA image quality. The effective radiation dose was measured for each patient. RESULTS: Twenty-eight AF patients (10 female, 18 male, mean age 68.8 ± 13.9 y, body mass index 24.3 ± 2.3â¯kg/cm2) were recruited and 337 artery segments were evaluated. In total, 98.5% (332/337) of the coronary artery segments and 96.4% (27/28) of the AF patients were rated as diagnostically evaluable. Of these 27 diagnosable patients, 17â¯patients (63%) were diagnosed with multi-vessel stenosis. Besides, 5 of 28â¯patients (17.9%) have left atrial appendage thrombus. The quality of the integrated image was significantly better than either of the individual first or the second scans, based on segments (P < 0.001) and patients (P < 0.05). The mean effective radiation dose was 1.5 mSv ±0.4 mSv. CONCLUSIONS: Using the Double Flash Spiral mode at low radiation dose (mean 1.5 mSv), 98.5% of the coronary segments and 96.4% of the scans were of sufficient diagnostic quality.
Subject(s)
Atrial Fibrillation/diagnosis , Coronary Angiography/methods , Coronary Artery Disease/diagnosis , Coronary Vessels/diagnostic imaging , Heart Rate/physiology , Adult , Aged , Aged, 80 and over , Atrial Fibrillation/etiology , Atrial Fibrillation/physiopathology , Computed Tomography Angiography/methods , Coronary Artery Disease/complications , Coronary Artery Disease/physiopathology , Dose-Response Relationship, Radiation , Feasibility Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Reproducibility of Results , Retrospective StudiesABSTRACT
Estrogen sulfotransferase (EST) regulates estrogen homeostasis by sulfonating and deactivating estrogens. Liver ischemia and reperfusion (I/R) involves both hypoxia during the ischemic phase and oxidative damage during the reperfusion phase. In this report, we showed that the expression of EST was markedly induced by I/R. Mechanistically, oxidative stress-induced activation of Nrf2 was responsible for the EST induction, which was abolished in Nrf2(-/-) mice. EST is a direct transcriptional target of Nrf2. In female mice, the I/R-responsive induction of EST compromised estrogen activity. EST ablation attenuated I/R injury as a result of decreased estrogen deprivation, whereas this benefit was abolished upon ovariectomy. The effect of EST ablation was sex-specific because the EST(-/-) males showed heightened I/R injury. Reciprocally, both estrogens and EST regulate the expression and activity of Nrf2. Estrogen deprivation by ovariectomy abolished the I/R-responsive Nrf2 accumulation, whereas the compromised estrogen deprivation in EST(-/-) mice was associated with increased Nrf2 accumulation. Our results suggested a novel I/R-responsive feedback mechanism to limit the activity of Nrf2 in which Nrf2 induces the expression of EST, which subsequently increases estrogen deactivation and limits the estrogen-responsive activation of Nrf2. Inhibition of EST, at least in females, may represent an effective approach to manage hepatic I/R injury.
Subject(s)
Liver/pathology , Oxidative Stress , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Sulfotransferases/genetics , Animals , Cells, Cultured , Estrogens/metabolism , Female , Gene Deletion , Hep G2 Cells , Humans , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Reperfusion Injury/metabolism , Sex Factors , Sulfotransferases/metabolism , Up-RegulationABSTRACT
BACKGROUND & AIMS: Chronic inflammatory liver diseases are associated with estrogen excess and feminization in men, which is thought to be due to compromised liver function to break down estrogens. The goal of this study is to determine whether the inflammatory induction of steroid sulfatase (STS), which converts inactive estrogen sulfates to active estrogens, may have contributed to the estrogen excess in chronic liver disease. METHODS: We performed bioinformatic analysis, real-time PCR, immunohistochemistry, and UPLC/MS-MS to analyze hepatic STS expression and serum estrogen levels in patients with chronic liver diseases. The crosstalk between NF-κB pathway and STS-regulated estrogen signaling was investigated by electrophoretic mobility shift assay, chromatin immunoprecipitation, luciferase assay and gene knockdown experiments in human hepatocytes. RESULTS: Hepatic STS was induced in patients with chronic inflammatory liver diseases, which was accompanied by increased circulating estrogen levels. The human STS gene, but not the mouse Sts gene, was induced by inflammatory stimuli in hepatic cells. Mechanistically, STS was established as a novel NF-κB target gene, whose induction facilitated the conversion of inactive estrogen sulfates to active estrogens, and consequently attenuated the inflammatory response. In contrast, genetic or pharmacological inhibition of STS or a direct blockade of estrogen signaling sensitized liver cells to the transcriptional activation of NF-κB and inflammatory response, possibly through the inhibition of IκB kinase activation. CONCLUSIONS: Our results suggest a negative feedback loop in chronic inflammatory liver diseases, in which the inflammatory activation of NF-κB induces STS gene expression. The induced STS facilitates the conversion of inactive estrogen sulfates to active estrogens, which in return attenuates the NF-κB-mediated inflammation.
Subject(s)
Estrogens/metabolism , Homeostasis , Inflammation/etiology , Liver Diseases/metabolism , Steryl-Sulfatase/physiology , Cells, Cultured , Chronic Disease , Computational Biology , Humans , Liver Cirrhosis, Alcoholic/metabolism , NF-kappa B/physiology , Signal TransductionABSTRACT
Understanding the life cycle and pathogenesis of animal viruses requires that we have systems in which the viruses can replicate and cause disease. For the latter, we rely upon animal models or information that we can obtain from studying natural infections of humans and other animals. For the former, however, we are largely dependent on the availability of cell culture systems in which viruses can be propagated to investigate the molecular mechanisms of viral replication. For many years, it was assumed that replication in culture provided an accurate description of the life cycle of the organism. In this Gem, we will discuss two viruses, polyomavirus and cytomegalovirus, in which cell culture systems have accidentally provided unique potential insights into viral replication and persistence in their hosts.
Subject(s)
Cytomegalovirus/physiology , Gene Rearrangement , Genome, Viral , Polyomavirus/physiology , Virus Replication , Animals , Cytomegalovirus/genetics , DNA, Viral/genetics , Humans , Polyomavirus/geneticsABSTRACT
UNLABELLED: BK polyomavirus (BKPyV) reactivation is associated with severe human disease in kidney and bone marrow transplant patients. The interplay between viral and host factors that regulates the productive infection process remains poorly understood. We have previously reported that the cellular DNA damage response (DDR) is activated upon lytic BKPyV infection and that its activation is required for optimal viral replication in primary kidney epithelial cells. In this report, we set out to determine what viral components are responsible for activating the two major phosphatidylinositol 3-kinase-like kinases (PI3KKs) involved in the DDR: ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3-related (ATR) kinase. Using a combination of UV treatment, lentivirus transduction, and mutant virus infection experiments, our results demonstrate that neither the input virus nor the expression of large T antigen (TAg) alone is sufficient to trigger the activation of ATM or ATR in our primary culture model. Instead, our data suggest that the activation of both the ATM- and ATR-mediated DDR pathways is linked to viral DNA replication. Intriguingly, a TAg mutant virus that is unable to activate the DDR causes substantial host DNA damage. Our study provides insight into how DDRs are activated by polyomaviruses in primary cells with intact cell cycle checkpoints and how the activation might be linked to the maintenance of host genome stability. IMPORTANCE: Polyomaviruses are opportunistic pathogens that are associated with several human diseases under immunosuppressed conditions. BK polyomavirus (BKPyV) affects mostly kidney and bone marrow transplant patients. The detailed replication mechanism of these viruses remains to be determined. We have previously reported that BKPyV activates the host DNA damage response (DDR), a response normally used by the host cell to combat genotoxic stress, to aid its own replication. In this study, we identified that the trigger for DDR activation is viral replication. Furthermore, we show that the virus is able to cause host DNA damage in the absence of viral replication and DDR activation. These results suggest an intricate relationship between viral replication, DDR activation, and host genome instability.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , BK Virus/physiology , DNA Damage , DNA Repair , Virus Replication , Cells, Cultured , Epithelial Cells/virology , Humans , Mutation , Transduction, Genetic , Ultraviolet RaysABSTRACT
Polyomaviruses are a family of small DNA viruses that are associated with a number of severe human diseases, particularly in immunocompromised individuals. The detailed virus-host interactions during lytic polyomavirus infection are not fully understood. Here, we report the first nuclear proteomic study with BK polyomavirus (BKPyV) in a primary renal proximal tubule epithelial cell culture system using stable isotope labeling by amino acids in cell culture (SILAC) proteomic profiling coupled with liquid chromatography-tandem mass spectrometry. We demonstrated the feasibility of SILAC labeling in these primary cells and subsequently performed reciprocal labeling-infection experiments to identify proteins that are altered by BKPyV infection. Our analyses revealed specific proteins that are significantly up- or down-regulated in the infected nuclear proteome. The genes encoding many of these proteins were not identified in a previous microarray study, suggesting that differential regulation of these proteins may be independent of transcriptional control. Western blotting experiments verified the SILAC proteomic findings. Finally, pathway and network analyses indicated that the host cell DNA damage response signaling and DNA repair pathways are among the cellular processes most affected at the protein level during polyomavirus infection. Our study provides a comprehensive view of the host nuclear proteomic changes during polyomavirus lytic infection and suggests potential novel host factors required for a productive polyomavirus infection.
Subject(s)
BK Virus/physiology , Cell Nucleus/metabolism , DNA Repair , Epithelial Cells/metabolism , Proteome/metabolism , Cell Nucleus/chemistry , Cell Nucleus/pathology , Cell Nucleus/virology , Chromatography, Liquid , DNA Damage , Epithelial Cells/pathology , Epithelial Cells/virology , Gene Expression Regulation , Gene Regulatory Networks , Host-Pathogen Interactions , Humans , Isotope Labeling , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Kidney Tubules, Proximal/virology , Molecular Sequence Annotation , Primary Cell Culture , Proteome/genetics , Proteome/isolation & purification , Signal Transduction , Tandem Mass Spectrometry , Transcription, GeneticABSTRACT
The steroid sulfatase (STS)-mediated desulfation is a critical metabolic mechanism that regulates the chemical and functional homeostasis of endogenous and exogenous molecules. In this report, we first showed that the liver expression of Sts was induced in both the high fat diet (HFD) and ob/ob models of obesity and type 2 diabetes and during the fed to fasting transition. In defining the functional relevance of STS induction in metabolic disease, we showed that overexpression of STS in the liver of transgenic mice alleviated HFD and ob/ob models of obesity and type 2 diabetes, including reduced body weight, improved insulin sensitivity, and decreased hepatic steatosis and inflammation. Interestingly, STS exerted its metabolic benefit through sex-specific mechanisms. In female mice, STS may have increased hepatic estrogen activity by converting biologically inactive estrogen sulfates to active estrogens and consequently improved the metabolic functions, whereas ovariectomy abolished this protective effect. In contrast, the metabolic benefit of STS in males may have been accounted for by the male-specific decrease of inflammation in white adipose tissue and skeletal muscle as well as a pattern of skeletal muscle gene expression that favors energy expenditure. The metabolic benefit in male STS transgenic mice was retained after castration. Treatment with the STS substrate estrone sulfate also improved metabolic functions in both the HFD and ob/ob models. Our results have uncovered a novel function of STS in energy metabolism and type 2 diabetes. Liver-specific STS induction or estrogen/estrogen sulfate delivery may represent a novel approach to manage metabolic syndrome.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Liver/enzymology , Obesity/genetics , Steryl-Sulfatase/genetics , Up-Regulation , Animals , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/pathology , Energy Metabolism , Estrogens/metabolism , Fatty Liver/enzymology , Fatty Liver/genetics , Fatty Liver/pathology , Female , Insulin Resistance , Liver/metabolism , Liver/pathology , Male , Mice , Mice, Transgenic , Obesity/enzymology , Obesity/pathology , Steryl-Sulfatase/metabolismABSTRACT
BACKGROUND & AIMS: Fatty acid binding protein 4 (FABP4) has been known as a mediator of inflammatory response in the macrophages and adipose tissue, but its hepatic function is poorly understood. The goal of this study is to investigate the role of FABP4 in liver ischemia/reperfusion (I/R), a clinical condition that involves both hypoxia and inflammation. METHODS: To examine the I/R regulation of FABP4, mice were subjected to I/R surgery before being measured for FABP4 gene expression. Both loss-of-function (by using a pharmacological FABP4 inhibitor) and gain-of-function (by adenoviral overexpression of FABP4) were used to determine the functional relevance of FABP4 expression and its regulation during I/R. To determine the hypoxia responsive regulation of FABP4, primary mouse hepatocytes were exposed to hypoxia. The FABP4 gene promoter was cloned and its regulation by hypoxia inducible factor 1α (HIF-1α) was characterized by luciferase reporter gene, electrophoretic mobility shift, and chromatin immunoprecipitation assays. RESULTS: We found that the hepatic expression of FABP4 was markedly induced by I/R. At the functional level, pharmacological inhibition of FABP4 alleviated the I/R injury, whereas adenoviral overexpression of FABP4 sensitized mice to I/R injury. We also showed that exposure of primary hepatocytes to hypoxia or transgenic overexpression of HIF-1α in the mouse liver was sufficient to induce the expression of FABP4. Our promoter analysis established FABP4 as a novel transcriptional target of HIF-1α. CONCLUSIONS: FABP4 is a hypoxia inducible gene that sensitizes mice to liver I/R injury. FABP4 may represent a novel therapeutic target, and FABP4 inhibitors may be used as therapeutic agents to manage hepatic I/R injury.
Subject(s)
DNA/genetics , Fatty Acid-Binding Proteins/genetics , Gene Expression Regulation , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Ischemia/genetics , Liver/blood supply , Reperfusion Injury/genetics , Animals , Blotting, Western , Cell Hypoxia , Cells, Cultured , Chromatin Immunoprecipitation , Disease Models, Animal , Fatty Acid-Binding Proteins/biosynthesis , Female , Hepatocytes/metabolism , Hepatocytes/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Ischemia/etiology , Ischemia/metabolism , Liver/pathology , Mice , Mice, Knockout , Real-Time Polymerase Chain Reaction , Reperfusion Injury/complications , Reperfusion Injury/metabolismABSTRACT
BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells.
Subject(s)
BK Virus/physiology , Endoplasmic Reticulum-Associated Degradation , Simian virus 40/physiology , Viral Proteins/metabolism , Virus Replication , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/virology , Humans , Protein TransportABSTRACT
BK polyomavirus (BKPyV) is an emerging pathogen whose reactivation causes severe disease in transplant patients. Unfortunately, there is no specific anti-BKPyV treatment available, and host cell components that affect the infection outcome are not well characterized. In this report, we examined the relationship between BKPyV productive infection and the activation of the cellular DNA damage response (DDR) in natural host cells. Our results showed that both the ataxia-telangiectasia mutated (ATM)- and ATM and Rad-3-related (ATR)-mediated DDR were activated during BKPyV infection, accompanied by the accumulation of polyploid cells. We assessed the involvement of ATM and ATR during infection using small interfering RNA (siRNA) knockdowns. ATM knockdown did not significantly affect viral gene expression, but reduced BKPyV DNA replication and infectious progeny production. ATR knockdown had a slightly more dramatic effect on viral T antigen (TAg) and its modified forms, DNA replication, and progeny production. ATM and ATR double knockdown had an additive effect on DNA replication and resulted in a severe reduction in viral titer. While ATM mainly led to the activation of pChk2 and ATR was primarily responsible for the activation of pChk1, knockdown of all three major phosphatidylinositol 3-kinase-like kinases (ATM, ATR, and DNA-PKcs) did not abolish the activation of γH2AX during BKPyV infection. Finally, in the absence of ATM or ATR, BKPyV infection caused severe DNA damage and aberrant TAg staining patterns. These results indicate that induction of the DDR by BKPyV is critical for productive infection, and that one of the functions of the DDR is to minimize the DNA damage which is generated during BKPyV infection.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , BK Virus/physiology , DNA Damage , Polyomavirus Infections/virology , Tumor Virus Infections/virology , Ataxia Telangiectasia Mutated Proteins/genetics , BK Virus/genetics , Cell Cycle , Cells, Cultured , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , DNA Replication , Gene Knockdown Techniques , Histones/genetics , Histones/metabolism , Humans , Models, Molecular , Polyploidy , RNA, Small Interfering , Signal Transduction , Virus ReplicationABSTRACT
The nuclear receptor liver X receptor (LXR) plays an important role in the metabolism and homeostasis of cholesterol, lipids, bile acids, and steroid hormones. In this study, we uncovered a function of LXRα (NR1H3) in regulating the human hydroxysteroid sulfotransferase SULT2A1, a phase II conjugating enzyme known to sulfonate bile acids, hydroxysteroid dehydroepiandrosterone, and related androgens. We showed that activation of LXR induced the expression of SULT2A1 at mRNA, protein, and enzymatic levels. A combination of promoter reporter gene and chromatin immunoprecipitation assays showed that LXRα transactivated the SULT2A1 gene promoter through its specific binding to the -500- to -258-base pair region of the SULT2A1 gene promoter. LXR small interfering RNA knockdown experiments suggested that LXRα, but not LXRß, played a dominant role in regulating SULT2A1. In primary human hepatocytes, we found a positive correlation between the expression of SULT2A1 and LXRα, which further supported the regulation of SULT2A1 by LXRα. In summary, our results established human SULT2A1 as a novel LXRα target gene. The expression of LXRα is a potential predictor for the expression of SULT2A1 in human liver.
Subject(s)
Gene Expression Regulation , Orphan Nuclear Receptors/metabolism , Sulfotransferases/genetics , Transcription, Genetic , Cells, Cultured , Gene Expression Regulation/drug effects , Hepatocytes/metabolism , Humans , Liver X Receptors , Promoter Regions, Genetic/genetics , Protein Isoforms/metabolism , RNA, Small Interfering/pharmacology , Transcription, Genetic/drug effectsABSTRACT
Food turns out to be not only the nutrient supplier for our body but also a carrier of regulatory information. Interestingly, a recent study made the discovery that some plant/food-derived microRNAs (miRNAs) accumulate in the serum of humans or plant-feeding animals, and regulate mammalian gene expression in a sequence-specific manner. The authors provided striking evidence that miRNAs could function as active signaling molecules to transport information across distinct species or even kingdoms. Although the mechanism of how miRNAs are shuttled between different organisms is still not well characterized, initial results point to the involvement of microvesicles and specific RNA-transporter-like proteins. These findings raise both speculation about the potential impact that plants may have on animal physiology at the molecular level, and an appealing possibility that food-derived miRNAs may offer us another means to deliver necessary nutrients or therapeutics to our bodies.