Interferon-stimulated Response Element Research Articles

SAHA down-regulates the expression of indoleamine 2,3-dioxygenase via inhibition of the JAK/STAT1 signaling pathway in gallbladder carcinoma cells

The aim of the present study was to investigate the role of the JAK/STAT1 signaling pathway in suberoylanilide hydroxamic acid (SAHA)-mediated down-regulation of indoleamine 2,3-dioxygenase (IDO) in gallbladder carcinoma cells. We treated SGC-996 gallbladder carcinoma cells with IFN-γ and SAHA. Western blotting was used to detect the expression of IDO, signal transducer and activator of transcription1 (STAT1) phosphorylation and interferon regulatory factor genes-1 (IRF-1). Confocal microscopy analysis was used to detect STAT1 translocation. Transient transfection and reporter gene assay was used for detecting the activation of γ-activated sites (GAS) and interferon-stimulated response elements (ISRE). The results revealed that IDO was expressed in SGC-996 cells in a dose- and time-dependent manner when stimulated with IFN-γ and SAHA down-regulated the expression of IDO induced by IFN-γ in a dose-dependent manner. SAHA blocked the expression of IRF-1 induced by IFN-γ and SAHA inhibited IFN-γ-induced STAT1 phosphorylation and nuclear translocation. In addition, SAHA down-regulated IFN-γ-induced activation of GAS and ISRE. In conclusion, SAHA down-regulated IDO expression via inhibition of the activation of members of the JAK/STAT1 signaling pathway. Therefore, regulation of the JAK/STAT1 signaling pathway may provide a new gallbladder carcinoma immunotherapeutic strategy to break tumor immune tolerance.

Single-nucleotide polymorphisms in GALNT8 are associated with the response to interferon therapy for chronic hepatitis C

New anti-hepatitis C virus (HCV) therapeutics developed recently are more effective and lead to improvements in sustained viral response. However, interferon (IFN) monotherapy is still used to a limited extent for fear of adverse effects. This study investigated host genetic factors affecting the IFN response in patients with chronic hepatitis C (CHC). Using a two-step design, a large-scale association screening including 1088 Japanese CHC patients treated with IFN was performed employing ~70 000 gene-based single-nucleotide polymorphisms (SNPs). Replication was tested in an independent Japanese cohort of 328 patients. Fine-mapping and functional analyses were also performed. Through two-step screening, it was found that rs2286580 in intron 6 of the gene encoding N-acetylgalactosaminyltransferase 8 (GALNT8) on chromosome 12 was significantly associated with a sustained viral response (combined P = 3.9×10(-6), odds ratio 1.52, 95 % confidence interval 1.29-1.82). The association was replicated in an additional cohort of 328 Japanese patients. In subgroup analysis, GALNT8 variants were associated with treatment outcome independently of HCV genotype. By contrast, the outcome of pegylated IFN and ribavirin combined therapy was not affected by the SNP. Fine-mapping analysis revealed that the association peak was at rs10849138 in intron 6 of GALNT8. Allele-specific transcription analysis demonstrated that GALNT8 expression was upregulated by an unfavourable allele of the variant. A luciferase reporter assay demonstrated that overexpression of GALNT8 attenuated IFN-α-induced gene transcription via the IFN-stimulated response element. These results suggest that GALNT8 variants contribute to the response to IFN therapy against CHC, providing a new insight into antiviral mechanisms of IFN.

Tetraspanin 6 (TSPAN6) Negatively Regulates Retinoic Acid-inducible Gene I-like Receptor-mediated Immune Signaling in a Ubiquitination-dependent Manner

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.

Silencing of microRNA-122 enhances interferon-α signaling in the liver through regulating SOCS3 promoter methylation

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. Although novel drugs against HCV are under development, the current standard therapy consists principally of interferon (IFN). To improve the response to IFN treatment by enhancing interferon-stimulated response element (ISRE)-mediated gene transcription, we screened 75 microRNAs highly expressed in hepatocytes for their ability to modulate ISRE activity. Overexpression of microRNA-122 (miR122) significantly suppressed ISRE activity. Conversely, silencing of miR122 function enhanced IFN-induced ISRE activity, by decreasing expression of suppressor of cytokine signaling 3 (SOCS3). This decrease in SOCS3 level was not mediated by microRNA target gene suppression, but rather by enhanced methylation at SOCS3 gene promoter. Taken together, our data, along with the fact that antisense oligonucleotides of miR122 also directly inhibit HCV replication, suggest that a combination therapy comprising IFN and silencing of miR122 function may be a promising therapeutic option in the near future.

Functional analysis of the interferon-stimulated response element of porcine circovirus type 2 and its role during viral replication in vitro and in vivo

BackgroundPorcine circovirus type 2 (PCV2) is associated with post-weaning multi-systemic wasting syndrome (PMWS) in young weaned pigs. Immune stimulation was found to activate the replication of PCV2 and exacerbate the clinical outcome of the infection. Proper amount of interferon-α (IFN-α) is able to enhance PCV2 infection and production in Porcine kidney-15 (PK-15) cells when administered after inoculation.MethodsIn the present study, luciferase reporter assays, construction of mutant viruses, Analysis the replication efficiency and the response to IFN-α treatment in PK-15 cells and animal experiments were carried out to analyze the function of interferon-stimulated response element (ISRE) of PCV2 and its role during viral replication in vitro and in vivo.ResultsA functional viral ISRE sequence, 5′-CTGAAAACGAAAGA-3′, was identified in Rep gene promoter (Prep) of PCV2. PCV2 Prep is composed of two mini promoters, the proximal one span the sequence +1 to -106, containing an ISRE while the distal mini promoter is composed of three tandem GC box like sites locate at -85 to -194. It was demonstrated that viral ISRE is necessary for porcine IFN-α initiated luciferase expression enhancement and it plays an important role in affecting the replication efficiency of PCV2 in vivo and in vitro.ConclusionsThese findings provide a theoretical basis for the Phenomenon of immunostimulation is able to enhance PCV2 infection, and improve the understanding of the complicated mechanisms involved in the host and pathogen interactions of PCV2.

IL‐28B (IFN‐λ3) and IFN‐α synergistically inhibit HCV replication

Genetic variation in the IL-28B (interleukin-28B; interferon lambda 3) region has been associated with sustained virological response (SVR) rates in patients with chronic hepatitis C treated with peginterferon-α and ribavirin. However, the mechanisms by which polymorphisms in the IL-28B gene region affect host antiviral responses are not well understood. Using the HCV 1b and 2a replicon system, we compared the effects of IFN-λs and IFN-α on HCV RNA replication. The anti-HCV effect of IFN-λ3 and IFN-α in combination was also assessed. Changes in gene expression induced by IFN-λ3 and IFN-α were compared using cDNA microarray analysis. IFN-λs at concentrations of 1ng/mL or more exhibited concentration- and time-dependent HCV inhibition. In combination, IFN-λ3 and IFN-α had a synergistic anti-HCV effect; however, no synergistic enhancement was observed for interferon-stimulated response element (ISRE) activity or upregulation of interferon-stimulated genes (ISGs). With respect to the time course of ISG upregulation, the peak of IFN-λ3-induced gene expression occurred later and lasted longer than that induced by IFN-α. In addition, although the genes upregulated by IFN-α and IFN-λ3 were similar to microarray analysis, interferon-stimulated gene expression appeared early and was prolonged by combined administration of these two IFNs. In conclusion, IFN-α and IFN-λ3 in combination showed synergistic anti-HCV activity in vitro. Differences in time-dependent upregulation of these genes might contribute to the synergistic antiviral activity.

A Novel Viral SOCS from Infectious Spleen and Kidney Necrosis Virus: Interacts with Jak1 and Inhibits IFN-α Induced Stat1/3 Activation

Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-α-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations (F18D, S66A, S85A, and R64K) of ISKNV-vSOCS significantly impaired the inhibition of IFN-α-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-α-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.

(-)-Epigallocatechin gallate inhibits the expression of indoleamine 2,3-dioxygenase in human colorectal cancer cells

Immune escape, the ability of tumor cells to avoid tumor-specific immune responses, occurs during the development and progression of several types of human malignancies, including colorectal cancer (CRC). Indoleamine 2,3-dioxygenase (IDO), the tryptophan catabolic enzyme, plays a significant role in regulating the immune response and provides tumor cells with a potent tool to evade the immune system. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), the major catechin in green tea, on the inhibition of IDO expression induced by interferon (IFN)-γ in human CRC cells. We found that IFN-γ increased the expression levels of IDO protein and mRNA in HT29 and SW837 CRC cell lines. Treatment of SW837 cells with EGCG significantly decreased IFN-γ-induced expression of IDO protein and mRNA in a dose-dependent manner. Enzymatic activity of IDO, determined by the concentration of L-kynurenine in the culture medium, was also significantly inhibited by EGCG treatment. Phosphorylation of signal transducer and activator of transcription 1 (STAT1) induced by IFN-γ was also significantly inhibited by EGCG. Reporter assays indicated that EGCG inhibited the transcriptional activities of IDO promoters, IFN-stimulated response element and IFN-γ activation sequence, activated by STAT1 phosphorylation. These findings suggest that EGCG may exert antitumor effects on CRC, at least in part, by inhibiting the expression and function of IDO through the suppression of STAT1 activation. EGCG may, thus, serve as a potential agent for antitumor immunotherapy and be useful in the chemoprevention and/or treatment of CRC.

Hepatitis C Virus NS5A Disrupts STAT1 Phosphorylation and Suppresses Type I Interferon Signaling

Responses to alpha interferon (IFN-α)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs). Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment.

High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery

Most of current strategies for antiviral therapeutics target the virus specifically and directly, but an alternative approach to drug discovery might be to enhance the immune response to a broad range of viruses. Based on clinical observation in humans and successful genetic strategies in experimental models, we reasoned that an improved interferon (IFN) signaling system might better protect against viral infection. Here we aimed to identify small molecular weight compounds that might mimic this beneficial effect and improve antiviral defense. Accordingly, we developed a cell-based high-throughput screening (HTS) assay to identify small molecules that enhance the IFN signaling pathway components. The assay is based on a phenotypic screen for increased IFN-stimulated response element (ISRE) activity in a fully automated and robust format (Z′>0.7). Application of this assay system to a library of 2240 compounds (including 2160 already approved or approvable drugs) led to the identification of 64 compounds with significant ISRE activity. From these, we chose the anthracycline antibiotic, idarubicin, for further validation and mechanism based on activity in the sub-µM range. We found that idarubicin action to increase ISRE activity was manifest by other members of this drug class and was independent of cytotoxic or topoisomerase inhibitory effects as well as endogenous IFN signaling or production. We also observed that this compound conferred a consequent increase in IFN-stimulated gene (ISG) expression and a significant antiviral effect using a similar dose-range in a cell-culture system inoculated with encephalomyocarditis virus (EMCV). The antiviral effect was also found at compound concentrations below the ones observed for cytotoxicity. Taken together, our results provide proof of concept for using activators of components of the IFN signaling pathway to improve IFN efficacy and antiviral immune defense as well as a validated HTS approach to identify small molecules that might achieve this therapeutic benefit.

Regulatory effects of mTORC2 complexes in type I IFN signaling and in the generation of IFN responses

IFNs transduce signals by binding to cell surface receptors and activating cellular pathways and regulatory networks that control transcription of IFN-stimulated genes (ISGs) and mRNA translation, leading to generation of protein products that mediate biological responses. Previous studies have shown that type I IFN receptor-engaged pathways downstream of AKT and mammalian target of rapamycin complex (mTORC) 1 play important roles in mRNA translation of ISGs and the generation of IFN responses, but the roles of mTORC2 complexes in IFN signaling are unknown. We provide evidence that mTORC2 complexes control IFN-induced phosphorylation of AKT on serine 473 and their function is ultimately required for IFN-dependent gene transcription via interferon-stimulated response elements. We also demonstrate that such complexes exhibit regulatory effects on other IFN-dependent mammalian target of rapamycin-mediated signaling events, likely via engagement of the AKT/mTORC1 axis, including IFN-induced phosphorylation of S6 kinase and its effector rpS6, as well as phosphorylation of the translational repressor 4E-binding protein 1. We also show that induction of ISG protein expression and the generation of antiviral responses are defective in Rictor and mLST8-KO cells. Together, our data provide evidence for unique functions of mTORC2 complexes in the induction of type I IFN responses and suggest a critical role for mTORC2-mediated signals in IFN signaling.

Interferon-γ-activated human leukocyte antigen-G gene expression in placenta-derived multipotent cells involves the epigenetic control and transcriptional regulation (167.12)

Abstract Placenta-derived multipotent cells (PDMCs) are progenitor cells isolated from the human term placenta with differentiation capacity for all three germ layers as well as strong immunomodulatory properties. The placenta is central to maintaining fetomaternal tolerance, and one of the most important immunomodulatory molecules involved is the human leukocyte antigen-G (HLA-G). HLA-G is a non-classical major hisotocompatibility complex (MHC) class I molecule which exhibits a restricted tissue-specific expression and exerts multiple immuneregulatory functions for maintaining the tolerance of placenta. We have shown that PDMCs can resist natural killer (NK) cell-mediated cytotoxicity by interferon-γ (IFN-γ)-induced surface HLA-G expression, suggesting that the expression of HLA-G may be involved the cis-acting epigenetic control and transcriptional regulation. Recently, we found that IFN-γ induces the expression of IFN regulatory factor 1 (IRF-1) and phosphorylation of STAT-1 in PDMCs. We speculate that the control of HLA-G in PDMCs involved not only the IFN-γ cis-acting regulatory elements including the IFN-stimulated response element (ISRE) and IFN-γ activation site (GAS), but also DNA-methylation which synergistically render the HLA-G gene promoter highly responsive to IFN-γ. Taken together, our results indicate that IFN-γ-activated HLA-G gene expression in PDMCs is involved the cis-acting epigenetic control and transcriptional regulation through JAK/STAT-1 signaling pathway.

Abstract LB-282: Myxovirus (influenza virus) resistance 1 (MX1) polymorphism in prostate cancer

Abstract PURPOSE: Myxovirus (influenza virus) resistance 1 (MX1) is an interferon regulated gene responsible for a specific antiviral state against viral infection. We have previously shown that MX1 is up-regulated by androgens in the normal prostate epithelial cells; however, there is no known role for MX1 in cancer. Meta-analysis of different expression databases e.g. NCBI GEO and Oncomine suggested a strong inverse association between MX1 expression and prostate cancer. To confirm these studies, we performed Immunohistochemistry on normal prostate and prostate cancer tissues. Our data revealed that MX1 expression was indeed decreased in PCa as compared to normal, indicating that MX1 could be transcriptionally down-regulated. Literature studies indicated that MX1 down-regulation could be due to a specific polymorphism in the proximal MX1 promoter. In the promoter region of MX1 at position -88, a single nucleotide polymorphism G>T (rs2071430) is involved in modifying the gene expression. Interestingly, the rs2071430 harbors an interferon-stimulated response element (ISRE) that is required for MX1 expression in response to interferons. The “T” allele restores whereas the “G” allele attenuates ISRE binding resulting in increased or decreased MX1 expression respectively. Based on these observations we hypothesized that decreased expression of MX1 in prostate cancer could be due to the rs2071430 polymorphism. DESIGN METHODS: We investigated MX1 rs2071430 polymorphism in genomic DNA from equal number of disease free and prostate cancer samples (N=305). The sample size required to achieve statistically significant associations were calculated using the power calculator for case control genetic association studies. Polymorphism was detected by sequencing the PCR amplicon through SNPdetector to ensure that the SNP was due to heterozygous allelic variations and not due to sequencing artifacts. RESULTS: The data suggests that the GG genotype (low promoter activity) is higher in PCa (72%) as compared to normal (58.6%). The TT genotype (high activity) was higher in normal (5.7%) compared to PCa (2.4%). No TT genotype was observed in Caucasian normal samples. The results were statistically significant at P<0.05. Our results, for the first time demonstrate that MX1 is down-regulated in prostate cancer possibly at the transcriptional level due to ISRE polymorphism in the promoter region. ACKNOWLEDGEMENTS: This research is supported by NIH/NCMHD grant 1P20MD002285-01. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-282. doi:1538-7445.AM2012-LB-282

Abstract 4402: The anti-tumor effects of two novel multivalent/multifunctional agents derived from hR1, a humanized anti-insulin-like growth factor receptor-I monoclonal antibody, are enhanced in renal cell carcinoma and synergistic with an mTOR inhibitor

Abstract Among kidney cancer types, approximately 90% are renal cell carcinomas (RCC). Advanced or metastatic RCC, which presents in about one third of the patients, has a poor prognosis as it is resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-α2b (IFN-γ2b) alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only modest improvements in clinical outcomes. One observation made with mTOR inhibitors is that cancer cells can overcome the effects of the inhibitor by activating the insulin-like growth factor-I (IGF-I) signaling pathways. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival, particularly among patients with high-grade tumors. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to multiple tumor types, including RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. To enhance the anti-tumor activity of hR1, we applied the Dock-and-Lock (DNL) platform technology to generate 1R-2b, comprising a conjugate of hR1 IgG with two dimers of interferon-α2b, and Hex-hR1, comprising 6 Fab fragments of hR1 tethered onto a common Fc. There was no loss in cell binding for both 1R-2b and Hex-hR1 when compared to parental hR1 as determined by flow cytometry. Units of activity for 1R-2b as measured by a luciferase reporter gene fused to a promoter containing the interferon-stimulated response element (iLite kit), yielded a specific activity of 3750 U/pmole versus 180 U/pmole and 3255 U/pmole for two different forms of peginterferon alpha-2a (60 and 31 kDa), respectively. An in vitro cytotoxicity assay with1R-2b demonstrated growth inhibition of two different RCC cell lines, 786-0 and ACHN with EC50-values of 0.049 and 0.062 pmole/mL, respectively. In terms of receptor down-regulation, Hex-hR1 could effectively down-regulate IGF-IR at concentrations 10-fold lower than parental hR1 IgG. In soft-agar growth assays, all three agents (hR1, Hex-hR1 and 1R-2b) could significantly inhibit colony formation of 786-0 and ACHN (P<0.038 and P<0.0022, respectively). When combined with temsirolimus, an mTOR inhibitor, in vitro cytotoxicity assays demonstrated a synergistic interaction with hR1, Hex-hR1, and 1R-2b. This synergy occurred at concentrations as low as 10 nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b. Experimental therapeutic efficacy studies are currently underway to determine if these in vitro observations will translate to the in vivo setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4402. doi:1538-7445.AM2012-4402

The complement C1qA enhances retinoic acid‐inducible gene‐I‐mediated immune signalling

The cellular innate immune response is essential for recognizing and defending against viral infection. Retinoic acid-inducible gene-I (RIG-I) and virus-induced signaling adaptor (VISA) mediated immune signalling is critically involved in RNA-virus-induced innate immune responses. Here we demonstrate that the complement C1qA interacts with different RIG-I pathway components and enhances RIG-I-VISA-mediated signalling pathway as well as TBK1-mediated activation of interferon-β (IFN-β) promoter. Our data show that over-expression of C1qA up-regulates RIG-I-mediated activation of IFN-stimulated responsive element (ISRE) and nuclear factor-κB reporters and IFN-β transcription, but not IFN regulatory factor-3-mediated and inhibitor of κB kinase-mediated activation of ISRE and nuclear factor-κB promoter. In addition, C1qA can counteract the function of the C1q receptor gC1qR in RIG-I-mediated signalling. Our results reveal the important role of complement C1qA in the innate immune response.

Regulation of interferon pathway in 2-methoxyestradiol-treated osteosarcoma cells

BackgroundOsteosarcoma is a bone tumor that often affects children and young adults. Although a combination of surgery and chemotherapy has improved the survival rate in the past decades, local recurrence and metastases still develop in 40% of patients. A definite therapy is yet to be determined for osteosarcoma. Anti- tumor compound and a metabolite of estrogen, 2-methoxyestradiol (2-ME) induces cell death in osteosarcoma cells. In this report, we have investigated whether interferon (IFN) pathway is involved in 2-ME-induced anti-tumor effects in osteosarcoma cells.Methods2-ME effects on IFN mRNA levels were determined by Real time PCR analysis. Transient transfections followed by reporter assays were used for investigating 2-ME effects on IFN-pathway. Western blot analyses were used to measure protein and phosphorylation levels of IFN-regulated eukaryotic initiation factor-2 alpha (eIF-2α).Results2-ME regulates IFN and IFN-mediated effects in osteosarcoma cells. 2 -ME induces IFN gene activity and expression in osteosarcoma cells. 2-ME treatment induced IFN-stimulated response element (ISRE) sequence-dependent transcription and gamma-activated sequence (GAS)-dependent transcription in several osteosarcoma cells. Whereas, 2-ME did not affect IFN gene and IFN pathways in normal primary human osteoblasts (HOB). 2-ME treatment increased the phosphorylation of eIF-2α in osteosarcoma cells. Furthermore, analysis of osteosarcoma tissues shows that the levels of phosphorylated form of eIF-2α are decreased in tumor compared to normal controls.Conclusions2-ME treatment triggers the induction and activity of IFN and IFN pathway genes in 2-ME-sensitive osteosarcoma tumor cells but not in 2-ME-resistant normal osteoblasts. In addition, IFN-signaling is inhibited in osteosarcoma patients. Thus, IFN pathways play a role in osteosarcoma and in 2-ME-mediated anti-proliferative effects, and therefore targeted induction of IFN signaling could lead to effective treatment strategies in the control of osteosarcoma.

Suppression of IP-10/CXCL10 gene expression in LPS- and/or IFN-γ-stimulated macrophages by parasite-secreted products

T helper (Th)2 polarized immune responses are characteristically dominant in helminth infections. The gene expression of interferon (IFN)-γ-inducible protein 10 (IP-10/CXCL10), which promotes Th1 responses, in mouse macrophages stimulated with lipopolysaccharide (LPS) and/or IFN-γ was suppressed by excretory/secretory (ES) products of Spirometra erinaceieuropaei plerocercoids. ES products suppressed LPS- and/or IFN-γ-induced transcriptional activities of a luciferase reporter gene under the control of a 243-bp fragment of the IP-10 gene promoter/enhancer, which contains an IFN-stimulated response element (ISRE) and two κB elements. Consistent with this result, ES products inhibited ISRE-dependent heterologous promoter activities and LPS- or IFN-γ-induced ISRE-binding activity. ES products also suppressed LPS-induced IFN-β gene expression. Furthermore, ES products suppressed nuclear factor (NF)-κB RelA (p65)-dependent transcriptional activity, whereas ES products had no effect on the κB-binding activity. These results suggest that ES products suppress the IP-10 gene expression by inhibiting the ISRE- and RelA-dependent transcriptional activities in mouse macrophages.

Significant induction of apoptosis in renal cell carcinoma cells transfected with cationic multilamellar liposomes containing the human interferon-β gene through activation of the intracellular type 1 interferon signal pathway

We previously reported that cationic multilamellar liposome containing the human interferon-β (huIFN-β) gene (IAB-1) demonstrated significant cytotoxic effect in the NC65 human renal cell carcinoma (RCC) cell line. In this study, we investigated the molecular mechanisms of IAB-1-induced apoptosis and cytotoxicity in RCC cells. Remarkable in vitro cytotoxic and apoptosis-inducing effects of IAB-1 against NC65 cells were observed by a colorimetric method and TUNEL staining, respectively. In contrast, treatment of NC65 cells with exogenously added huIFN-β protein induced low-level cytotoxicity without apoptosis. Neutralizing antibodies against huIFN-β significantly suppressed the cytotoxic effect of huIFN-β protein, but they were unable to block the effect of IAB-1. Cytotoxicity assays using transwell plates revealed that NC65 cells treated with IAB-1 did not secrete cytotoxic soluble factors other than IFN-β. Substantial enhancement of interferon-stimulated response element (ISRE) activity of NC65 cells by IAB-1 was demonstrated by promoter reporter assays. In addition, immunofluorescence using confocal microscopy revealed the intracellular expression of IFN-β and its receptor induced by IAB-1. The induction of c-Myc by IAB-1 was suggested by a cDNA macroarray and was confirmed by western blot analysis. These findings indicate that IAB-1 induces significant cytotoxicity and apoptosis in NC65 cells, possibly through enhanced ISRE activity, that is associated with increased intracellular localization of huIFN-β and IFN-receptor. Our data support the potential clinical application of IAB-1 gene therapy for RCC resistant to IFN.

Activation of Oas1a gene expression by type I IFN requires both STAT1 and STAT2 while only STAT2 is required for Oas1b activation

The murine 2′–5′ oligoadenylate synthetase 1a (Oas1a) and Oas1b genes are type 1 IFN responsive genes. Oas1a is an active synthetase with broad antiviral activity mediated through RNase L. Oas1b is inactive but can inhibit Oas1a synthetase activity and mediate a flavivirus-specific antiviral activity through an unknown RNase L-independent mechanism. Analysis of promoter elements regulating gene transcription confirmed that an IFN-stimulated response element (ISRE) is required for IFN beta-activation but neither the overlapping IRF binding site present in both promoters nor the adjacent Oas1b NF-kappa B site is required. Mutation of the overlapping STAT site negatively affected IFN beta-induction of Oas1a but not of Oas1b. Also, IFN beta induction of Oas1a was STAT1- and STAT2-dependent, while induction of Oas1b was STAT1-independent but STAT2-dependent. The two promoters differ at a single nucleotide in the STAT site. The data indicate that these two duplicated genes can be differentially regulated by IFN beta.

IFN-α inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome

HBV infection remains a leading cause of death worldwide. IFN-α inhibits viral replication in vitro and in vivo, and pegylated IFN-α is a commonly administered treatment for individuals infected with HBV. The HBV genome contains a typical IFN-stimulated response element (ISRE), but the molecular mechanisms by which IFN-α suppresses HBV replication have not been established in relevant experimental systems. Here, we show that IFN-α inhibits HBV replication by decreasing the transcription of pregenomic RNA (pgRNA) and subgenomic RNA from the HBV covalently closed circular DNA (cccDNA) minichromosome, both in cultured cells in which HBV is replicating and in mice whose livers have been repopulated with human hepatocytes and infected with HBV. Administration of IFN-α resulted in cccDNA-bound histone hypoacetylation as well as active recruitment to the cccDNA of transcriptional corepressors. IFN-α treatment also reduced binding of the STAT1 and STAT2 transcription factors to active cccDNA. The inhibitory activity of IFN-α was linked to the IRSE, as IRSE-mutant HBV transcribed less pgRNA and could not be repressed by IFN-α treatment. Our results identify a molecular mechanism whereby IFN-α mediates epigenetic repression of HBV cccDNA transcriptional activity, which may assist in the development of novel effective therapeutics.