ABSTRACT
Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/ß-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724. Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly. PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/ß-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.
Subject(s)
Antineoplastic Agents , Liver Neoplasms , Non-alcoholic Fatty Liver Disease , Animals , Mice , Non-alcoholic Fatty Liver Disease/drug therapy , Matrix Metalloproteinase 9/genetics , beta Catenin , Liver Cirrhosis/drug therapyABSTRACT
Intranasal vaccines that elicit mucosal immunity are deemed effective against respiratory tract infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but their ability to induce humoral immunity characterized by immunoglobulin A (IgA) and IgG production is low. It has been reported that vaccination with a mixture of a viscous base carboxyvinyl polymer (CVP) and viral antigens induced robust systemic and mucosal immune responses. In this study, we analyzed the behavior of immunocompetent cells in the nasal cavity over time by spatial transcriptome profiling induced immediately after antigen vaccination using CVP. We established a method for performing spatial transcriptomics using the Visium system in the mouse nasal cavity and analyzed gene expression profiles within the nasal cavity after intranasal vaccination. Glycoprotein 2 (Gp2)-, SRY-box transcription factor 8 (Sox8)-, or Spi-B transcription factor (Spib)-expressing cells were increased in the nasal passage (NP) region at 3-6 hr after SARS-CoV-2 spike protein and CVP (S-CVP) vaccination. The results suggested that microfold (M) cells are activated within a short period of time (3-6 hr). Subsequent cluster analysis of cells in the nasal cavity showed an increase in Cluster 9 at 3-6 hr after intranasal vaccination with the S-CVP. We found that Il6 in Cluster 9 had the highest log2 fold values within the NP at 3-6 hr. A search for gene expression patterns similar to that of Il6 revealed that the log2 fold values of Edn2, Ccl20, and Hk2 also increased in the nasal cavity after 3-6 hr. The results showed that the early response of immune cells occurred immediately after intranasal vaccination. In this study, we identified changes in gene expression that contribute to the activation of M cells and immunocompetent cells after intranasal vaccination of mice with antigen-CVP using a time-series analysis of spatial transcriptomics data. The results facilitated the identification of the cell types that are activated during the initial induction of nasal mucosal immunity.
Subject(s)
COVID-19 , Transcriptome , Humans , Animals , Mice , Nasal Cavity/chemistry , Interleukin-6 , Antibodies, Viral , SARS-CoV-2 , Vaccination/methods , Gene Expression ProfilingABSTRACT
BACKGROUND: Tokyo, the capital of Japan, is a densely populated city of >13 million people, so the population is at high risk of epidemic severe acute respiratory coronavirus 2 (SARS-CoV-2) infection. A serologic survey of anti-SARS-CoV-2 IgG would provide valuable data for assessing the city's SARS-CoV-2 infection status. Therefore, this cross-sectional study estimated the anti-SARS-CoV-2 IgG seroprevalence in Tokyo. METHODS: Leftover serum of 23,234 hospital visitors was tested for antibodies against SARS-CoV-2 using an iFlash 3000 chemiluminescence immunoassay analyzer (Shenzhen YHLO Biotech, Shenzhen, China) with an iFlash-SARS-CoV-2 IgG kit (YHLO) and iFlash-SARS-CoV-2 IgG-S1 kit (YHLO). Serum samples with a positive result (≥10 AU/mL) in either of these assays were considered seropositive for anti-SARS-CoV-2 IgG. Participants were randomly selected from patients visiting 14 Tokyo hospitals between September 1, 2020 and March 31, 2021. No participants were diagnosed with coronavirus disease 2019 (COVID-19), and none exhibited COVID-19-related symptoms at the time of blood collection. RESULTS: The overall anti-SARS-CoV-2 IgG seroprevalence among all participants was 1.83% (95% confidence interval [CI], 1.66-2.01%). The seroprevalence in March 2021, the most recent month of this study, was 2.70% (95% CI, 2.16-3.34%). After adjusting for population age, sex, and region, the estimated seroprevalence in Tokyo was 3.40%, indicating that 470,778 individuals had a history of SARS-CoV-2 infection. CONCLUSIONS: The estimated number of individuals in Tokyo with a history of SARS-CoV-2 infection was 3.9-fold higher than the number of confirmed cases. Our study enhances understanding of the SARS-CoV-2 epidemic in Tokyo.
Subject(s)
COVID-19 , Antibodies, Viral , Cross-Sectional Studies , Hospitals , Humans , Immunoglobulin G , SARS-CoV-2 , Seroepidemiologic Studies , Tokyo/epidemiologyABSTRACT
The 3' untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5' and 3' RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5'UTR and 177 nucleotides of the 3'UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3'UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3'-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45-4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.
ABSTRACT
Chronic hepatitis C virus (HCV) infection is one of the major causes of serious liver diseases, including liver cirrhosis. There are no anti-fibrotic drugs with efficacy against liver cirrhosis. Wnt/ß-catenin signaling has been implicated in the pathogenesis of a variety of tissue fibrosis. In the present study, we investigated the effects of a ß-catenin/CBP (cyclic AMP response element binding protein) inhibitor on liver fibrosis. The anti-fibrotic activity of PRI-724, a selective inhibitor of ß-catenin/CBP, was assessed in HCV GT1b transgenic mice at 18 months after HCV genome expression. PRI-724 was injected intraperitoneally or subcutaneously in these mice for 6 weeks. PRI-724 reduced liver fibrosis, which was indicated by silver stain, Sirius Red staining, and hepatic hydroxyproline levels, in HCV mice while attenuating αSMA induction. PRI-724 led to increased levels of matrix metalloproteinase (MMP)-8 mRNA in the liver, along with elevated levels of intrahepatic neutrophils and macrophages/monocytes. The induced intrahepatic neutrophils and macrophages/monocytes were identified as the source of MMP-8. In conclusion, PRI-724 ameliorated HCV-induced liver fibrosis in mice. We hypothesize that inhibition of hepatic stellate cells activation and induction of fibrolytic cells expressing MMP-8 contribute to the anti-fibrotic effects of PRI-724. PRI-724 is a drug candidate which possesses anti-fibrotic effect.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Cyclic AMP Response Element-Binding Protein/antagonists & inhibitors , Enzyme Inhibitors/administration & dosage , Hepatitis C, Chronic/complications , Liver Cirrhosis/pathology , Pyrimidinones/administration & dosage , Wnt Signaling Pathway , beta Catenin/antagonists & inhibitors , Animals , Disease Models, Animal , Histocytochemistry , Injections, Intraperitoneal , Mice, Transgenic , Treatment OutcomeABSTRACT
UNLABELLED: Macrophages in liver tissue are widely defined as important inflammatory cells in chronic viral hepatitis due to their proinflammatory activity. We reported previously that interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) play significant roles in causing chronic hepatitis in hepatitis C virus (HCV) transgenic mice (S. Sekiguchi et al., PLoS One 7:e51656, 2012, http://dx.doi.org/10.1371/journal.pone.0051656). In addition, we showed that recombinant vaccinia viruses expressing an HCV nonstructural protein (rVV-N25) could protect against the progression of chronic hepatitis by suppression of macrophage activation. Here, we focus on the role of macrophages in liver disease progression in HCV transgenic mice and examine characteristic features of macrophages following rVV-N25 treatment. The number of CD11b(+) F4/80(+) CD11c(-) CD206(+) (M2) macrophages in the liver of HCV transgenic mice was notably increased compared to that of age-matched control mice. These M2 macrophages in the liver produced elevated levels of IL-6 and TNF-α. rVV-N25 infection suppressed the number and activation of M2 macrophages in liver tissue. These results suggested that inflammatory cytokines produced by M2-like macrophages contribute to the induction of chronic liver inflammation in HCV transgenic mice. Moreover, the therapeutic effect of rVV-N25 might be induced by the suppression of the number and activation of hepatic macrophages. IMPORTANCE: HCV causes persistent infections that can lead to chronic liver diseases, liver fibrosis, and hepatocellular carcinoma; the search for an HCV curative is the focus of ongoing research. Recently, effective anti-HCV drugs have been developed; however, vaccine development still is required for the prevention and therapy of infection by this virus. We demonstrate here that M2 macrophages are important for the pathogenesis of HCV-caused liver diseases and additionally show that M2 macrophages contribute to the therapeutic mechanism observed following rVV-N25 treatment.
Subject(s)
Hepacivirus/immunology , Hepatitis C, Chronic/pathology , Macrophages/immunology , Macrophages/virology , Animals , Disease Models, Animal , Disease Progression , Hepacivirus/genetics , Hepatitis C, Chronic/immunology , Hepatitis C, Chronic/virology , Interleukin-6/metabolism , Mice, Transgenic , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Hepatitis C virus (HCV) is a causative agent of chronic hepatitis. Although the standard therapy for HCV-infected patients consists of pegylated interferon plus ribavirin, this treatment is associated with serious side effects and high costs, and fails in some patients infected with specific HCV genotypes. To address this problem, we are developing small-molecule inhibitors of cyclin-dependent kinases (CDKs) as novel anti-HCV drug candidates. Previous data showed that HCV replication is inhibited by retinoblastoma protein, which is itself inactivated by CDK-mediated phosphorylation. Here, we report that CDK inhibitors suppress HCV replication in vitro and in vivo, and that CDK4 is required for efficient HCV replication. These findings shed light on the development of novel anti-HCV drugs that target host factors.
Subject(s)
Antiviral Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Hepacivirus/drug effects , Hepacivirus/physiology , Virus Replication/drug effects , Cell Line , Drug Discovery/methods , Humans , Inhibitory Concentration 50ABSTRACT
Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.
Subject(s)
Genes, Viral , Genotype , Hepacivirus/isolation & purification , Virus Replication/genetics , Animals , Hepacivirus/genetics , Humans , Mice , Mutation , RNA, Viral/geneticsABSTRACT
BACKGROUND & AIMS: Host cell lipid rafts form a scaffold required for replication of hepatitis C virus (HCV). Serine palmitoyltransferases (SPTs) produce sphingolipids, which are essential components of the lipid rafts that associate with HCV nonstructural proteins. Prevention of the de novo synthesis of sphingolipids by an SPT inhibitor disrupts the HCV replication complex and thereby inhibits HCV replication. We investigated the ability of the SPT inhibitor NA808 to prevent HCV replication in cells and mice. METHODS: We tested the ability of NA808 to inhibit SPT's enzymatic activity in FLR3-1 replicon cells. We used a replicon system to select for HCV variants that became resistant to NA808 at concentrations 4- to 6-fold the 50% inhibitory concentration, after 14 rounds of cell passage. We assessed the ability of NA808 or telaprevir to inhibit replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in mice with humanized livers (transplanted with human hepatocytes). NA808 was injected intravenously, with or without pegylated interferon alfa-2a and HCV polymerase and/or protease inhibitors. RESULTS: NA808 prevented HCV replication via noncompetitive inhibition of SPT; no resistance mutations developed. NA808 prevented replication of all HCV genotypes tested in mice with humanized livers. Intravenous NA808 significantly reduced viral load in the mice and had synergistic effects with pegylated interferon alfa-2a and HCV polymerase and protease inhibitors. CONCLUSIONS: The SPT inhibitor NA808 prevents replication of HCV genotypes 1a, 1b, 2a, 3a, and 4a in cultured hepatocytes and in mice with humanized livers. It might be developed for treatment of HCV infection or used in combination with pegylated interferon alfa-2a or HCV polymerase or protease inhibitors.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepatocytes/virology , Serine C-Palmitoyltransferase/antagonists & inhibitors , Virus Replication/drug effects , Animals , Hepacivirus/classification , Hepacivirus/genetics , Humans , Mice , RNA, Viral/analysisABSTRACT
BACKGROUND & AIMS: The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). METHODS: This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. RESULTS: Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-ß in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS: These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.
Subject(s)
Chimera/immunology , Hepacivirus/physiology , Hepatitis B virus/physiology , Interferons/genetics , Liver/immunology , Liver/virology , Transcriptional Activation , Animals , Apoptosis/immunology , Cell Line , Humans , Immunity, Innate/genetics , Interleukins/genetics , Liver/cytology , Liver/metabolism , Mice , Polymorphism, Single Nucleotide , RNA, Double-Stranded/genetics , Species SpecificityABSTRACT
Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.
Subject(s)
Hepacivirus/physiology , Hepatitis C/metabolism , Sphingolipids/biosynthesis , Virus Replication/physiology , Animals , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Hepatitis C/genetics , Humans , Membrane Proteins/biosynthesis , Mice , Nerve Tissue Proteins/biosynthesis , Serine C-Palmitoyltransferase/antagonists & inhibitors , Serine C-Palmitoyltransferase/genetics , Serine C-Palmitoyltransferase/metabolism , Sphingolipids/genetics , Transferases (Other Substituted Phosphate Groups)/biosynthesis , Virus Replication/drug effectsABSTRACT
Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.
Subject(s)
End Stage Liver Disease/immunology , Hepacivirus/immunology , Hepatitis C, Chronic/immunology , Recombinant Proteins/therapeutic use , Vaccinia virus/immunology , Viral Core Proteins/metabolism , Viral Hepatitis Vaccines/therapeutic use , Viral Nonstructural Proteins/immunology , Animals , Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/metabolism , Cytokines/metabolism , End Stage Liver Disease/complications , End Stage Liver Disease/metabolism , Female , Gene Expression Regulation, Viral , Humans , Immunization , Immunoenzyme Techniques , Liver Neoplasms, Experimental/etiology , Liver Neoplasms, Experimental/metabolism , Mice , Mice, Transgenic , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
BACKGROUND & AIMS: We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. METHODS: We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. RESULTS: Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. CONCLUSIONS: DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target.
Subject(s)
Hepacivirus/physiology , Hepatitis C/virology , Nerve Tissue Proteins/metabolism , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Virus Replication/physiology , Androstenes/pharmacology , Androstenes/therapeutic use , Animals , Cholesterol/metabolism , Hep G2 Cells , Hepatitis C/drug therapy , Hepatitis C/metabolism , Humans , Interferon-alpha/therapeutic use , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/physiology , Oxidoreductases Acting on CH-CH Group Donors/drug effects , Oxidoreductases Acting on CH-CH Group Donors/physiology , Polyethylene Glycols/therapeutic use , RNA, Small Interfering/pharmacology , Recombinant Proteins/therapeutic use , Up-RegulationABSTRACT
Persistent infection with hepatitis C virus (HCV) induces tumorigenicity in hepatocytes. To gain insight into the mechanisms underlying this process, we generated monoclonal antibodies on a genome-wide scale against an HCV-expressing human hepatoblastoma-derived cell line, RzM6-LC, showing augmented tumorigenicity. We identified 3beta-hydroxysterol Delta24-reductase (DHCR24) from this screen and showed that its expression reflected tumorigenicity. HCV induced the DHCR24 overexpression in human hepatocytes. Ectopic or HCV-induced DHCR24 overexpression resulted in resistance to oxidative stress-induced apoptosis and suppressed p53 activity. DHCR24 overexpression in these cells paralleled the increased interaction between p53 and MDM2 (also known as HDM2), a p53-specific E3 ubiquitin ligase, in the cytoplasm. Persistent DHCR24 overexpression did not alter the phosphorylation status of p53 but resulted in decreased acetylation of p53 at lysine residues 373 and 382 in the nucleus after treatment with hydrogen peroxide. Taken together, these results suggest that DHCR24 is elevated in response to HCV infection and inhibits the p53 stress response by stimulating the accumulation of the MDM2-p53 complex in the cytoplasm and by inhibiting the acetylation of p53 in the nucleus.
Subject(s)
Cell Transformation, Viral , Hepacivirus , Hepatitis C/metabolism , Hepatocytes/metabolism , Nerve Tissue Proteins/biosynthesis , Oxidoreductases Acting on CH-CH Group Donors/biosynthesis , Tumor Suppressor Protein p53/metabolism , Acetylation/drug effects , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Enzyme Induction/genetics , Genome-Wide Association Study , Hep G2 Cells , Hepatitis C/genetics , Humans , Hydrogen Peroxide/pharmacology , Mice , Mice, Inbred BALB C , Mice, Nude , Nerve Tissue Proteins/genetics , Oxidants/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/genetics , Oxidoreductases Acting on CH-CH Group Donors/genetics , Phosphorylation/drug effects , Phosphorylation/genetics , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/geneticsABSTRACT
Vascular endothelial growth factor (VEGF-A) and its family proteins are crucial regulators of blood vessel formation and vascular permeability. Snake venom has recently been shown to be an exogenous source of unique VEGF (known as VEGF-F), and now, two types of VEGF-F with distinct biochemical properties have been reported. Here, we show that VEGF-Fs (venom-type VEGFs) are highly variable in structure and function among species, in contrast to endogenous tissue-type VEGFs (VEGF-As) of snakes. Although the structures of tissue-type VEGFs are highly conserved among venomous snake species and even among all vertebrates, including humans, those of venom-type VEGFs are extensively variegated, especially in the regions around receptor-binding loops and C-terminal putative coreceptor-binding regions, indicating that highly frequent variations are located around functionally key regions of the proteins. Genetic analyses suggest that venom-type VEGF gene may have developed from a tissue-type gene and that the unique sequence of its C-terminal region was generated by an alteration in the translation frame in the corresponding exons. We further verified that a novel venom-type VEGF from Bitis arietans displays unique properties distinct from already known VEGFs. Our results may provide evidence of a novel mechanism causing the generation of multiple snake toxins and also of a new model of molecular evolution.
Subject(s)
Snake Venoms/metabolism , Vascular Endothelial Growth Factor A/metabolism , Animals , Cloning, Molecular , Exons , Humans , Introns , Kinetics , Models, Genetic , Molecular Sequence Data , Phylogeny , Protein Binding , Protein Structure, Tertiary , Species Specificity , Time FactorsABSTRACT
VEGF-A165 plays a central role in neovascularization. The biological activities of VEGF-A165 are largely mediated through KDR. VEGF-A165 also binds to cellular coreceptors, neuropilin-1 (NP-1), and heparin, via its C-terminal domain, resulting in functional modulation. Parapoxvirus-encoded VEGFs (PV-VEGFs), which recognize KDR, possess basic amino acid clusters in their C-terminal regions. Some PV-VEGFs may interact with NP-1; however, the NP-1- and heparin-binding properties have not been fully characterized. Here, we demonstrate that the heparin- and NP-1-binding region of PV-VEGFs is located in its C-terminal tail. Furthermore, the two arginine residues adjacent to the C-terminus greatly contribute to both interactions.
Subject(s)
Heparin/metabolism , Neuropilin-1/metabolism , Parapoxvirus/metabolism , Vascular Endothelial Growth Factors/metabolism , Binding Sites , Parapoxvirus/chemistry , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Vascular Endothelial Growth Factors/chemistryABSTRACT
Vascular endothelial growth factor exerts multiple functions through binding to two distinct receptor tyrosine kinases, Flt-1 and KDR. We previously demonstrated that snake venom VEGFs, designated VEGF-Fs, exhibit potent biological activities when compared with VEGF165 and that they selectively recognize KDR. We herein report the screening of several snake venoms for VEGF-Fs using antibodies against vammin and HF, VEGF-Fs derived from the venom of Vipera ammodytes ammodytes and Vipera aspis aspis, respectively. Specific immunoreactivity was observed in the venoms of Vipera and Daboia species. The present results suggest that VEGF-Fs are specific components of venoms from the Viperinae snakes that inhabit Europe, India, and Asia. We also report the isolation and characterization of an additional VEGF-F from Daboia russelli siamensis venom.
Subject(s)
Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factors/metabolism , Viper Venoms/chemistry , Amino Acid Sequence , Animals , Cell Proliferation/drug effects , Cells, Cultured , Cross Reactions , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Molecular Sequence Data , Sequence Analysis, Protein , Vascular Endothelial Growth Factor A/immunology , Vascular Endothelial Growth Factor A/isolation & purification , Vascular Endothelial Growth Factors/chemistry , Vascular Endothelial Growth Factors/pharmacology , Viper Venoms/immunology , Viper Venoms/metabolism , Viper Venoms/pharmacology , ViperidaeABSTRACT
VEGF-A165 displays multiple effects through binding to KDR (VEGFR-2). Heparin/heparan sulfate-like molecules are known to greatly modulate their interaction. In fact, VEGF-A lacking a C-terminal heparin-binding region exhibits significantly reduced mitogenic activity. We recently found novel heparin-binding VEGFs in snake venom, designated VEGF-Fs, which specifically recognize KDR, rather than other VEGF receptors. VEGF-Fs virtually lack the C-terminal heparin-binding region when compared with other heparin-binding VEGF subtypes, despite their heparin-binding potential. The C-terminal region does not exhibit any significant homology with other known proteins or domains. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. The C-terminal peptide of vammin (one of the VEGF-Fs, 19 residues) bound to heparin with similar affinity as native vammin. We then evaluated the effects of this peptide on the biological activity of VEGF-A165. The C-terminal peptide of VEGF-F exhibited specific blockage of VEGF-A165 activity both in vitro and in vivo. These observations demonstrate that the short C-terminal region of VEGF-F functions fully as the active heparin-binding domain and the corresponding peptide specifically blocks VEGF-A165, thus suggesting that the C-terminal heparin-binding region of VEGF-F recognizes similar heparin/heparan sulfate molecules as VEGF-A165. The present results will provide novel insight into VEGF-heparin interaction and may facilitate the design of new anti-VEGF drugs based on novel strategies.
Subject(s)
Heparin/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/chemistry , Vascular Endothelial Growth Factors/chemistry , Viper Venoms/chemistry , Viper Venoms/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Affinity , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Humans , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Umbilical Veins , Vascular Endothelial Growth Factors/pharmacology , Viper Venoms/isolation & purification , ViperidaeABSTRACT
Vascular endothelial growth factor (VEGF-A165) displays diverse effects through binding to its receptor, KDR (kinase domain-containing receptor). Heparan sulfate/heparin-like molecules greatly contribute to their interaction. Indeed, the mitogenic potency of VEGF-A165 lacking the C-terminal heparin-binding region is less than 1% compared with intact VEGF-A165. We previously found novel heparin-binding VEGFs, designated VEGF-F that specifically recognizes KDR in snake venoms. VEGF-Fs almost completely lack the C-terminal heparin-binding region compared with VEGF-A165, despite their heparin-binding potential. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. We have demonstrated that the heparin-binding site of VEGF-F is located in its C-terminal region, particularly localized on the N-terminal portion of this region. Furthermore, a synthetic peptide of this region blocks the biological activity of VEGF-A165 in vitro and in vivo.
Subject(s)
Heparin/metabolism , Peptide Fragments/metabolism , Snake Venoms/chemistry , Vascular Endothelial Growth Factor A/metabolism , Animals , Binding Sites , Heparin/pharmacology , Peptide Fragments/antagonists & inhibitors , Snakes , Vascular Endothelial Growth Factor A/antagonists & inhibitorsABSTRACT
Vascular endothelial growth factor-A165 (VEGF-A165) exhibits diverse biological effects through binding to its receptor KDR (VEGFR-2). Heparin-like molecules are known to modulate their interaction. There have been reports that VEGF-A lacking the C-terminal heparin binding region significantly reduced mitogenic activity. Recently, we found novel heparin-binding VEGFs from snake venoms, designated VEGF-Fs, which specifically recognize kinase domain containing receptor (KDR). The C-terminal heparin-binding region is almost completely absent in VEGF-Fs when compared with other heparin-binding VEGFs, despite their heparin-binding potential. In this congress, we report that the C-terminal heparin-binding region of VEGF-F specifically/preferentially interacts with the VEGF-bondable heparin/heparan sulfate, but not with those associated with bFGF or TFPI. We also present the identification of a VEGF receptor-binding protein from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49PLA2, an inactive PLA2 homologue with strong myoxicity. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. The interaction between KDR-bp and KDR was blocked by VEGF-A165, and KDR-bp specifically inhibited VEGF-A165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. This is the first observation demonstrating that an exogenous factor antagonizes the VEGF receptor, furthermore, it is the first identification of the target molecule of the myotoxic PLA2 from viper venom.