IFN Signal Transduction Research Articles

Abstract 573: Ionizing radiation activates IFN signaling in tumor cells

Abstract Ionizing radiation (IR) uses multiple pathways to mediate killing in tumor cells. We found that fractionated irradiation leads to the constitutive overexpression of Interferon Stimulated Genes (ISGs) which are controlled by Signal Transducer and Activator of Transcription 1 (Stat1). Stat1 mediated IFN signal transduction and at the same time was involved in the production of Type I and Type II IFNs. IR-induced ISGs represent the following groups: 1) mitochondrial-related genes with pro- and anti-apoptotic functions, 2) cell cycle control and regulators of transcription, 3) protein modification and degradation, 4) cytoplasmic sensors of nucleic acids, and 5) anti-viral defense. Fractionated or single-dose IR induced the IFN pathway mediated by activation of Stat1 in breast, colon, prostate and head and neck tumor xenografts. Depletion of tumor-derived IFNs led to the suppression of IR response indicating that IFN production/signaling is a component of anti-tumor IR action. Cell-autonomous experiments indicated that tumor cells up-regulate Stat1 and ISGs and produce IFNα in response to single dose IR (3Gy). Up-regulated Stat1 in irradiated tumor cells was not phosphorylated at Tyr701 position compared to Stat1 activation following interferon stimulation. In a syngeneic tumor model of B16F1, IR-induced activation of IFN signaling in tumors (assessed both by up-regulation of the Stat1-dependent pathway and induction of Type I and Type II IFNs) was dependent on TNFα signaling in the host and was impaired in TNFR1,2-/- mice. We therefore conclude that IR induces IFN signaling in tumor cells as a component of the cytotoxic response. This interferon induction depends on TNFα signaling in host cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 573. doi:10.1158/1538-7445.AM2011-573

Interferon-β and interferon-λ signaling is not affected by interferon-induced refractoriness to interferon-α in vivo

Therapy of chronic hepatitis C with pegylated interferon α (pegIFN-α) and ribavirin achieves sustained virological responses in approximately half of the patients. Nonresponse to treatment is associated with constitutively increased expression of IFN-stimulated genes in the liver already before therapy. This activation of the endogenous IFN system could prevent cells from responding to therapeutically injected (peg)IFN-α, because prolonged stimulation of cells with IFN-α induces desensitization of the IFN signal transduction pathway. Whether all types of IFNs induce refractoriness in the liver is presently unknown. We therefore treated mice with multiple injections and different combinations of IFN-α, IFN-β, IFN-γ, and IFN-λ. Pretreatment of mice with IFN-α, IFN-β, and IFN-λ induced a strong expression of the negative regulator ubiquitin-specific peptidase 18 in the liver and gut. As a result, IFN-α signaling was significantly reduced when mice where reinjected 16 hours after the first injection. Surprisingly, both IFN-β and IFN-λ could activate the Janus kinase-signal transducer and activator of transcription (STAT) pathway and the expression of IFN-stimulated genes despite high levels of ubiquitin-specific peptidase 18. IFN-λ treatment of human liver biopsies ex vivo resulted in strong and maintained phosphorylation of STAT1, whereas IFN-α-induced STAT1 activation was transient. Contrary to the action of IFN-α, IFN-β, and IFN-λ signaling in the liver does not become refractory during repeated stimulation of the IFN signal transduction pathway. The sustained efficacy of IFN-β and IFN-λ could be an important advantage for the treatment patients who are nonresponders to pegIFN-α, through a preactivated endogenous IFN system.

Bovine Plasmacytoid Dendritic Cells Are the Major Source of Type I Interferon in Response to Foot-and-Mouth Disease Virus In Vitro and In Vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.

S-Adenosyl-Methionine and Betaine Improve Early Virological Response in Chronic Hepatitis C Patients with Previous Nonresponse

Background/AimsTreatment of chronic hepatitis C (CHC) with pegylated interferon α (pegIFNα) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFNα signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFNα signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFNα on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFNα/ribavirin.MethodsIn this open-label pilot study, 29 patients with CHC who failed previous therapy with (peg)IFNα/ribavirin were treated with SAMe, betaine, pegIFNα2b and ribavirin. Treatment duration was 6 or 12 months, depending on genotype, and the protocol comprised a stopping rule at week 12 if early virological response (EVR) was not achieved. Virological and biochemical response and safety were assessed throughout the treatment.Results29 patients were enrolled and treated according to the study protocol. 79% of the patients were infected with genotype 1, 72% had advanced fibrosis, 76% had previously received pegIFNα/ribavirin, and only 14% achieved EVR to the previous treatment. When treated with the study medications, 17 patients (59%) showed an EVR, only 3 (10%) however achieved a sustained virological response (SVR). SAMe and betaine were found to be safe when used with pegIFNα/ribavirin.ConclusionThe addition of SAMe and betaine to pegIFNα/ribavirin improves early virological response in CHC.Trial RegistrationClinicalTrials.gov NCT00310336

Anti-influenza Action of Multinutrient Functional Peptide Complex (MFPC) Grinization ®

Type III interferons (IFN), IFN-λ or IL-28/29, are new members of the IFN super-family. Except for using distinct receptors, type I and type III IFNs share the same major post receptor signaling components to activate the transcription of a similar set of IFN-stimulated genes (ISGs). To examine the antiviral effects of the new type IFNs against West Nile virus (WNV), we compared the antiviral effects of IFN-α and IFN-λ on WNV virus-like particle (VLP) infection and replicon replication in Huh7.5 and Hela cells. The results revealed that (i) both types of IFNs could efficiently prevent the WNV infection, but IFN-α demonstrated a stronger antiviral efficacy; (ii) WNV genome replication in VLP-infected cells and replicon-containing cell lines could only be inhibited by IFN-α, but not IFN-λ; (iii) in agreement with the observed antiviral effects, only IFN-λ-induced activation of JAK-STAT signaling pathway and induction of ISG expression were completely inhibited in WNV replicon-containing cell lines, but IFN-α signal transduction was either unaffected or only partially inhibited in Huh7.5 or Hela cells by the virus. Hence, the differential inhibition of WNV on IFN-α and IFN-λ signal transduction implies that the receptors of the two types of IFNs, but not the common post receptor signaling components, could be selectively targeted either directly by WNV nonstructural proteins or indirectly by the cellular responses induced by the virus infection to inhibit the signal transduction of the cytokines.

A polymorphism associated with STAT3 expression and response of chronic myeloid leukemia to interferon

Interferon alpha (IFN) induces variable responses in chronic myeloid leukemia (CML), with 8-30% of early chronic phase cases achieving a complete cytogenetic response. We hypothesized that polymorphic differences in genes encoding IFN signal transduction components might account for different patient responses. We studied 174 IFN-treated patients, of whom 79 achieved less than 35% Philadelphia-chromosome (Ph) positive metaphases (responders) and 95 failed to show any cytogenetic response (more than 95% Ph-positive metaphases; non-responders). We compared 17 single nucleotide polymorphisms (SNPs) at IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT3 and STAT5a/b between the two groups and found a significant difference for rs6503691, a SNP tightly linked to STAT5a, STAT5b and STAT3 (minor allele frequency 0.16 for non-responders; 0.06 for responders, P=0.007). Levels of STAT3 mRNA correlated with rs6503691 genotype (P<0.001) as assessed by real time quantitative PCR and therefore we conclude that rs6503691 is associated with the STAT3 expression levels and response of CML patients to IFN.

Interferon Tau in the Ovine Uterus

Gwonhwa Song*, Jae Yong Han*, Thomas E. Spencer** and Fuller W. Bazer*, **WCU Biomodulation Major, Department of Agricultural Biotechnology, and Research Institute for Agriculture and Life Sciences, Seoul National University, Korea*, Department of Animal Science, Center for Animal Biotechnology and Genomics, Texas A&M University, 442 Kleberg Center, 2471 TAMU, College Station, Texas 77843-2471, USA**ABSTRACTThe peri-implantation period in mammals is critical with respect to survival of the conceptus(embryo/fetus and associated extraembryonic membranes) and establishment of pregnancy. During this period of pregnancy, reciprocal communication between ovary, conceptus, and endometrium is required for successful implantation and placentation. At this time, interferon tau(IFNT) is synthesized and secreted by the mononuclear trophectodermal cells of the conceptus between days 10 and 21~25. The actions of IFNT to signal pregnancy recognition and induce or increase expression of IFNT-stimulated genes (ISGs), such as ISG15 and OAS, are mediated by the Type I IFN signal transduction pathway. This article reviews the history, signaling pathways of IFNT and the uterine expression of several IFNT-stimulated genes during the peri-implantation period. Collectively, these newly identified genes are believed to be critical to unraveling the mechanism(s) of reciprocal fetal-maternal interactions required for successful implantation and pregnancy. (Key words :Interferon tau, Progesterone, Sheep, Uterus, Implantation)

Antiviral effect of interferon lambda against West Nile virus

Type III interferons (IFN), IFN-λ or IL-28/29, are new members of the IFN super-family. Except for using distinct receptors, type I and type III IFNs share the same major post receptor signaling components to activate the transcription of a similar set of IFN-stimulated genes (ISGs). To examine the antiviral effects of the new type IFNs against West Nile virus (WNV), we compared the antiviral effects of IFN-α and IFN-λ on WNV virus-like particle (VLP) infection and replicon replication in Huh7.5 and Hela cells. The results revealed that (i) both types of IFNs could efficiently prevent the WNV infection, but IFN-α demonstrated a stronger antiviral efficacy; (ii) WNV genome replication in VLP-infected cells and replicon-containing cell lines could only be inhibited by IFN-α, but not IFN-λ; (iii) in agreement with the observed antiviral effects, only IFN-λ-induced activation of JAK-STAT signaling pathway and induction of ISG expression were completely inhibited in WNV replicon-containing cell lines, but IFN-α signal transduction was either unaffected or only partially inhibited in Huh7.5 or Hela cells by the virus. Hence, the differential inhibition of WNV on IFN-α and IFN-λ signal transduction implies that the receptors of the two types of IFNs, but not the common post receptor signaling components, could be selectively targeted either directly by WNV nonstructural proteins or indirectly by the cellular responses induced by the virus infection to inhibit the signal transduction of the cytokines.

Polymorphic Variation at the STAT5 Locus Is Associated with the Response of CML Patients to Interferon Alpha.

Interferon alpha (IFN) induces complete hematologic remission in 70% to 80% of patients with CML in early chronic phase, with up to 8–30% of cases achieving a complete cytogenetic response (CCR). Although patient responses correlate with Hasford and Sokal risk scores, the molecular basis for this heterogeneity remains poorly understood. We hypothesized that polymorphic differences in the IFN signal transduction cascade might account for different patient responses. IFN binds the heterodimeric type I interferon receptor, the two subunits of which are encoded by IFNAR1 and IFNAR2. Binding activates the JAK1 and TYK2 non-receptor tyrosine kinases, which phosphorylate and activate STAT proteins. We studied 187 cases treated with IFN based regimens as part of the German CML studies I-III. Of these, 105 were defined as non-responders (no cytogenetic response) and 82 as responders (achievement of <=34% Philadelphia-chromosome positive metaphases) after a median treatment duration of 23 and 40 months, respectively. There was no significant difference in the incidence of der(9) deletions between the two groups. We genotyped 14 single nucleotide polymorphisms (SNPs) within or close to the genes encoding IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT3 and STAT5a/b and compared allele frequencies between responders and non-responders. Significant differences were found for two SNPs at the STAT5a and STAT5b loci. For rs16967611 the minor allele frequency (maf) was 0.34 for non-responders and 0.28 for responders (P=0.21; chi-squared test) and for rs6503691 the maf was 0.16 for non-responders and 0.07 for responders (P=0.005). The latter SNP remained significant when corrected for multiple testing. Three other SNPs at STAT5a/b (rs9900213, rs17500235 and rs17591972) were also analyzed and yielded P values of 0.87, 0.01 and 0.15 respectively. STAT5a, STAT5b and STAT3 are tightly linked at 17q21 and show strong linkage disequilibrium according to HapMap data. To determine if the genotype at rs6503691 is related to gene expression levels, we measured STAT5a, STAT5b and STAT3 mRNA in 214 pretreatment CML patients by real time quantitative PCR. There was no difference in the median normlised expression of STAT3 and STAT5a in cases with a c/c genotype compared to those with c/t or t/t, however for STAT5b the expression level was higher in cases with a c/c genotype (P=0.02). We conclude that rs6503691, located within STAT5b intron 1, is associated with the expression level of STAT5b and response of CML patients to IFN.

Suppression of the Interferon-Mediated Innate Immune Response by Pseudorabies Virus

Pseudorabies virus (PRV) is an alphaherpesvirus related to the human pathogens herpes simplex virus type 1 (HSV-1) and varicella-zoster virus. PRV is capable of infecting and killing a wide variety of mammals. How it avoids innate immune defenses in so many hosts is not understood. While the anti-interferon (IFN) strategies of HSV-1 have been studied, little is known about how PRV evades the IFN-mediated immune response. In this study, we determined if wild-type PRV infection can overcome the establishment of a beta interferon (IFN-beta)-induced antiviral state in primary rat fibroblasts. Using microarray technology, we found that the expression of a subset of genes normally induced by IFN-beta in these cells was not induced when the cells were simultaneously infected with a wild-type PRV strain. Expression of transcripts associated with major histocompatibility complex class I antigen presentation and NK cell activation was reduced, while transcripts associated with inflammation either were unaffected or were induced by viral infection. This suppression of IFN-stimulated gene expression occurred because IFN signal transduction, in particular the phosphorylation of STAT1, became less effective in PRV-infected cells. At least one virion-associated protein is involved in inhibition of STAT1 tyrosine phosphorylation. This ability to disarm the IFN-beta response offers an explanation for the uniform lethality of virulent PRV infection of nonnatural hosts.

Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines.

Newcastle disease virus (NDV) is a negative-strand RNA virus with oncolytic activity against human tumors. Its effectiveness against tumors and safety in normal tissue have been demonstrated in several clinical studies. Here we show that the spread of NDV infection is drastically different in normal cell lines than in tumor cell lines and that the two cell types respond differently to beta interferon (IFN-beta) treatment. NDV rapidly replicated and killed HT-1080 human fibrosarcoma cells but spread poorly in CCD-1122Sk human skin fibroblast cells. Pretreatment with endogenous or exogenous IFN-beta completely inhibited NDV replication in normal cells but had little or no effect in tumor cells. Thus, the outcome of NDV infection appeared to depend on the response of uninfected cells to IFN-beta. To investigate their differences in IFN responsiveness, we analyzed and compared the expression and activation of components of the IFN signal transduction pathway in these two types of cells. The levels of phosphorylated STAT1 and STAT2 and that of the ISGF3 complex were markedly reduced in IFN-beta-treated tumor cells. Moreover, cDNA microarray analysis revealed significantly fewer IFN-regulated genes in the HT-1080 cells than in the CDD-1122Sk cells. This finding suggests that tumor cells demonstrate a less-than-optimum antiviral response because of a lesion in their IFN signal transduction pathway. The rapid spread of NDV in HT-1080 cells appears to be caused by their deficient expression of anti-NDV proteins upon exposure to IFN-beta.

Evaluation of the anti-hepatitis C virus effects of cyclophilin inhibitors, cyclosporin A, and NIM811

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. We recently discovered that the immunosuppressant cyclosporin A (CsA) and its analogue lacking immunosuppressive function, NIM811, strongly suppress the replication of HCV in cell culture. Inhibition of a cellular replication cofactor, cyclophilin (CyP) B, is critical for its anti-HCV effects. Here, we explored the potential use of CyP inhibitors for HCV treatment by analyzing the HCV replicon system. Treatment with CsA and NIM811 for 7 days reduced HCV RNA levels by 2–3 logs, and treatment for 3 weeks reduced HCV RNA to undetectable levels. NIM811 exerted higher anti-HCV activity than CsA at lower concentrations. Both CyP inhibitors rapidly reduced HCV RNA levels even further in combination with IFNα without modifying the IFNα signal transduction pathway. In conclusion, CyP inhibitors may provide a novel strategy for anti-HCV treatment.

A Role for Mixed Lineage Kinases in Regulating Transcription Factor CCAAT/Enhancer-binding Protein-β-dependent Gene Expression in Response to Interferon-γ

Transcription factor CCAAT/enhancer-binding protein-beta (C/EBP-beta) regulates a variety of cellular functions in response to exogenous stimuli. We have reported earlier that C/EBP-beta induces gene transcription through a novel interferon (IFN)-response element called gamma-IFN-activated transcriptional element. We show here that IFN-gamma-induced, C/EBP-beta/gamma-IFN-activated transcriptional element-dependent gene expression is regulated by mixed lineage kinases (MLKs), members of the mitogen-activated protein kinase kinase kinase family. MLK3 appears to activate C/EBP-beta in response to IFN-gamma by a mechanism involving decreased phosphorylation of a specific phosphoacceptor residue, Ser(64), within the transactivation domain. Decreased phosphorylation of Ser(64) was independent of IFN-gamma-stimulated ERK1/2 activation and did not require the ERK phosphorylation site Thr(189) located in regulatory domain 2 of C/EBP-beta. Together these studies provide the first evidence that MLK3 is involved in IFN-gamma signaling and identify a novel mechanism of transcriptional activation by IFN-gamma.

Interferonα Activates NF-κB in JAK1-deficient Cells through aTYK2-dependentPathway

In addition to activating members of the STAT transcription factor family, interferon alpha/beta (IFNalpha/beta) activates the NF-kappaB transcription factor. To determine the role of the Janus tyrosine kinase (JAK)-STAT pathway in NF-kappaB activation by IFN, we examined NF-kappaB activation in JAK1-deficient mutant human fibrosarcoma cells. In wild-type fibrosarcoma cells (2fTGH), IFN activates STAT1, STAT2, and STAT3, as well as NF-kappaB complexes comprised of p50 and p65. In contrast, in JAK1-deficient cells, IFN induces NF-kappaB activation and NF-kappaB dependent gene transcription but does not activate these STAT proteins and has no effect on STAT-dependent gene transcription. Expression of a catalytically inactive TYK2 tyrosine kinase in JAK1-deficient cells, as well as in the highly IFN-sensitive Daudi lymphoblastoid cell line, abrogates NF-kappaB activation by IFN. Moreover, IFN does not promote NF-kappaB activation in TYK2-deficient mutant fibrosarcoma cells. Our results demonstrate a dichotomy between the classical JAK-STAT pathway and the NF-kappaB signaling pathway. In the IFN signaling pathway leading to STAT activation, both JAK1 and TYK2 are essential, whereas NF-kappaB activation requires only TYK2.

Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

Measles virus suppresses interferon-α signaling pathway: suppression of Jak1 phosphorylation and association of viral accessory proteins, C and V, with interferon-α receptor complex

To establish infections, viruses use various strategies to suppress the host defense mechanism, such as interferon (IFN)-induced antiviral state. We found that cells infected with a wild strain of measles virus (MeV) displayed nearly complete suppression of IFN-α-induced antiviral state, but not IFN-γ-induced state. This phenomenon is due to the suppression of IFN-α-inducible gene expression at a transcriptional level. In the IFN-α signal transduction pathway, Jak1 phosphorylation induced by IFN-α is dramatically suppressed in MeV-infected cells; however, phosphorylation induced by IFN-γ is not. We performed immunoprecipitation experiments using antibodies against type 1 IFN receptor chain 1 (INFAR1) and antibody against RACK1, which is reported to be a scaffold protein interacting with type I IFN receptor chain 2 and STAT1. These experiments indicated that IFNAR1 forms a complex containing the MeV-accessory proteins C and V, RACK1, and STAT1 in MeV-infected cells but not in uninfected cells. Composition of this complex in the infected cells altered little by IFN-α treatment. These results indicate that MeV suppresses the IFN-α, but not IFN-γ, signaling pathway by inhibition of Jak1 phosphorylation. Our data suggest that functional disorder of the type I IFN receptor complex is due to “freezing” of the receptor through its association with the C and/or V proteins of MeV.

Antiapoptotic effect of interferon-α on hepatic stellate cells (HSC): A novel pathway of IFN-α signal transduction via Janus kinase 2 (JAK2) and caspase-8

The hepatic stellate cell (HSC), the pericyte of the liver sinusoids belongs to the mesenchymal cells of the liver. Damaging noxae induce a transformation from the quiescent (vitamin A-storing cell) to the activated (connective tissue-producing cell) state. The balance between proapoptotic and surviving factors decides about the fate of the activated HSC. Interferon-alpha (IFN-alpha) has been shown to elicit antiproliferative and/or antifibrogenic effects in various cell types of mesenchymal origin. We therefore investigated the effect of IFN-alpha on primary cultured rat HSC in their quiescent (day 2) and activated state (day 7). IFN-alpha significantly inhibited spontaneous apoptosis in activated HSC in vitro and simultaneously inhibited cell cycle progression by inducing a G1 arrest. The effect of IFN-a is not accompanied by a modulation of CD95, CD95L, p53, p21(WAF1), p27, bcl-2, bcl-xL, bax, NFkappaB, or IkappaB gene expression. Surprisingly, the IFN-alpha effect could be abolished completely by blocking JAK2 activity or JAK2 translation. The downregulating effect of IFN-alpha on the activity of caspase-8 and caspase-3 could also be neutralized using tyrphostin AG490 or JAK-2 antisense. Taken together IFN-alpha inhibits apoptosis of activated HSC by activation of JAK2 which inhibits the caspase-8 apoptosis pathway.

Functional significance of globotriaosyl ceramide in interferon‐α2/type 1 interferon receptor‐mediated antiviral activity

The N-terminus of the type 1 interferon receptor subunit, IFNAR1, has high amino acid sequence similarity to the receptor binding B subunit of the Escherichia coli-derived verotoxin 1, VT1. The glycolipid, globotriaosyl ceramide (Gb3: Gal α1 → 4 Gal β 1 → 4 Glu β 1 → 1 Cer) is the specific cell receptor for VT1. Gb3-deficient variant cells selected for VT resistance are cross-resistant to interferon-α (IFN-α)–mediated antiproliferative activity. The association of eIFNAR1 with Gal α 1 → 4 Gal containing glycolipids has been previously shown to be important for the receptor-mediated IFN-α signal transduction for growth inhibition. The crucial role of Gb3 for the signal transduction of IFN-α–mediated antiviral activity is now reported. IFN-α–mediated antiviral activity, nuclear translocation of activated Stat1, and increased expression of PKR were defective in Gb3-deficient vero mutant cells, although the surface expression of IFNAR1 was unaltered. The VT1B subunit was found to inhibit IFN-α–mediated antiviral activity, Stat1 nuclear translocation and PKR upregulation. Unlike VT1 cytotoxicity, IFN-α–induced Stat1 nuclear translocation was not inhibited when RME was prevented, suggesting that the accessory function of Gb3 occurs at the plasma membrane. IFN-α antiviral activity was also studied in Gb3-positive MRC-5 cells, which are resistant to IFN-α growth inhibition, partially resistant to VT1 but still remain fully sensitive to IFN-α antiviral activity, and two astrocytoma cell lines expressing different Gb3 fatty acid isoforms. In both systems, long chain fatty acid-containing Gb3 isoforms, which are less effective to mediate VT1 cytotoxicity, were found to correlate with higher IFN-α–mediated antiviral activity. Inhibition of Gb3 synthesis in toto prevented IFN-α antiviral activity in all cells. We propose that the long chain Gb3 fatty isoforms preferentially remain in the plasma membrane, and by associating with IFNAR1, mediate IFN-α antiviral signaling, whereas short chain Gb3 fatty acid isoforms are preferentially internalized to mediate VT1 cytotoxicity and IFNAR1-dependent IFN-α growth inhibition. J. Cell. Physiol. 182:97–108, 2000. © 2000 Wiley-Liss, Inc.

Nonsteroidal anti-inflammatory drug metabolism potentiates interferon alfa signaling by increasing STAT1 phosphorylation.

A sustained response to standard interferon therapy for chronic hepatitis C has been demonstrated in no more than 25% of patients. To improve interferon alfa (IFN-alpha) antiviral effect, a number of combination therapies with IFNs plus other drugs have been proposed for both relapser and nonresponder hepatitis C virus (HCV)-infected patients. Although the causes of IFN resistance in subsets of HCV-infected patients are unknown, both viral and host factors have been involved, including defects in IFN signal transduction and IFN-alpha/beta receptor down-regulation. Here, we report that nonsteroidal anti-inflammatory drugs (NSAIDs), which have been proposed for IFN-alpha combination therapy in nonresponders, potentiate IFN-alpha signaling. We found that, in the hepatoma cell lines, CCL13/Chang and HepG2, indomethacin, a selective cyclo-oxygenase 1 and 2 (COX-1 and COX-2) inhibitor, increases IFN-alpha stimulation of interferon-stimulated response element (ISRE)-dependent transcription in a dose-dependent manner. Interestingly, maximal potentiation was observed with suboptimal IFN-alpha concentrations. Indomethacin exerts its effects by synergizing with IFN-alpha in inducing STAT1 activation by phosphorylation, without affecting concurrent Jak1 phosphorylation. Our data indicate that blockade of arachidonic acid (AA) metabolism by indomethacin activates a signaling pathway that converges on STAT1 activation to potentiate IFN-alpha-dependent gene activation.

Determination of Residues Involved in Ligand Binding and Signal Transmission in the Human IFN-α Receptor 2

AbstractThe human IFN-α receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-α2/α1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-α2/α1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-α8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-α2, but remained responsive to hIFN-β. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-α2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.