Inhibition Of Stat1 Tyrosine Phosphorylation Research Articles

Shp-2 contributes to anti-RSV activity in human pulmonary alveolar epithelial cells by interfering with the IFN-α-induced Jak/Stat1 pathway.

Src homology phosphotyrosyl phosphatase 2 (Shp-2) is a ubiquitously expressed protein that is involved in a variety of cellular processes, including antiviral interferon signalling pathways. In this study, we investigated the role of Shp-2 in the host cell interactions of human respiratory syncytial virus (RSV). We report significant changes in the expression of Shp-2 in human pulmonary alveolar epithelial cells (A549) upon RSV infection. We also report that blocking Shp-2 does not affect viral replication or virus-induced interferon-alpha (IFN-α) production. Interestingly, whereas A549 cells were activated by IFN-α, the blocking of Shp-2 resulted in increased viral replication that was associated with the reduced expression of the IFN-stimulated genes of 2′,5′-oligoadenylate synthetases and Mx1, and the concomitant inhibition of Stat1 tyrosine phosphorylation. Our findings suggest that Shp-2 contributes to the control of RSV replication and progeny production in pulmonary alveolar epithelial cells by interfering with IFN-α-induced Jak/Stat1 pathway activation rather than by affecting the production of IFN-α itself.

Enterohemorrhagic Escherichia coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system.

Identifying Mechanisms by Which Escherichia coli O157:H7 Subverts Interferon-γ Mediated Signal Transducer and Activator of Transcription-1 Activation

Enterohemorrhagic Escherichia coli serotype O157:H7 is a food borne enteric bacterial pathogen that causes significant morbidity and mortality in both developing and industrialized nations. E. coli O157:H7 infection of host epithelial cells inhibits the interferon gamma pro-inflammatory signaling pathway, which is important for host defense against microbial pathogens, through the inhibition of Stat-1 tyrosine phosphorylation. The aim of this study was to determine which bacterial factors are involved in the inhibition of Stat-1 tyrosine phosphorylation. Human epithelial cells were challenged with either live bacteria or bacterial-derived culture supernatants, stimulated with interferon-gamma, and epithelial cell protein extracts were then analyzed by immunoblotting. The results show that Stat-1 tyrosine phosphorylation was inhibited by E. coli O157:H7 secreted proteins. Using sequential anion exchange and size exclusion chromatography, YodA was identified, but not confirmed to mediate subversion of the Stat-1 signaling pathway using isogenic mutants. We conclude that E. coli O157:H7 subverts Stat-1 tyrosine phosphorylation in response to interferon-gamma through a still as yet unidentified secreted bacterial protein.

Activation of STAT1 is required for interferon-alpha-mediated cell death

Interferon-alpha (IFNα)-induced cell death of tumor cells is likely mediated through several signaling pathways. We previously demonstrated that blocking the activation of phosphoinositide-3-kinase, PI3K, or mammalian target of rapamycin, mTOR, partially inhibited apoptosis induced by IFNα. Here, we postulate using pharmacological inhibition and dominant negative mutants that activation of signal transducer and activator of transcription-1, STAT1, is also required for the cell death induced by IFNα. Inhibition of STAT1 tyrosine phosphorylation and DNA binding by a naturally occurring rotenoid deguelin also rescued U266 myeloma cell lines from IFNα-induced apoptosis. Deguelin had no effect on upstream Jak kinases or STAT2 phosphorylation suggesting the involvement of a yet unknown mechanism. Inhibition of STAT1 tyrosine phosphorylation and activity was independent of the known effects of deguelin on PI3K, Akt or mTOR as shown using selective pharmacological inhibitors against these kinases. The combination of deguelin and PI3K or mTOR antagonists further inhibited apoptosis suggesting that both the Jak–STAT and the PI3K/mTOR pathways contribute to the induction of apoptosis by IFNα in these cells. Over-expression of STAT1-Y701A or K410/413A mutants in Rhek-1 keratinocytes largely inhibited apoptosis further supporting the importance of STAT1 phosphorylation and activity for IFNα-induced cell death. Thus, at least two signaling pathways, one of which requires STAT1 activation, cooperate to mediate IFNα-induced apoptosis.

Cross-Inhibition of Interferon-Induced Signals by GM-CSF Through a Block in Stat1 Activation

We investigated the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) on biologic signals induced by interferon-alpha (IFN-alpha) and IFN-gamma. In hematopoietic cell lines, IFN-induced signaling was investigated by Western blotting, electrophoretic mobility shift assays (EMSA), flow cytometry, protein-tyrosine phosphatase (PTP) assays, and RT-PCR. GM-CSF inhibited IFN-alpha-induced and IFN-gamma-induced Stat1 tyrosine phosphorylation in a time-dependent manner. EMSA showed that GM-CSF inhibited IFN-alpha-induced and IFN-gamma-induced IFN-gamma activator sequence (GAS) binding activity. As a consequence, IFN-induced transcription of the early response gene, IFN-stimulated gene 54 (ISG54), was inhibited. The expression of IFN regulatory factor-1 (IRF-1) and MHC class I antigens was downregulated at protein levels in hematopoietic cell lines (U937, THP1). In contrast to GM-CSF, granulocyte colony-stimulating factor (G-CSF) and interleukin-3 (IL-3) did not influence the IFN-induced Stat1 activation. To explore the molecular mechanism of suppression of Stat1 tyrosine phosphorylation, we investigated the induction and activation of cytokine-inducible SH2-containing protein/suppressor of cytokine signaling (CIS/SOCS) molecules and phosphatases on GM-CSF treatment. In contrast to G-CSF and IL-3, GM-CSF strongly induced the expression of CIS1 and SOCS2 at mRNA levels, but overexpression of CIS1 or SOCS2 in HEK293 cells did not show inhibition of Stat1 tyrosine phosphorylation upon IFN treatment. In PTP assays, on GM-CSF incubation, no enhanced src homology 2 domain tyrosine phosphatase 1 and 2 (SHP1 and SHP2) activity was detectable. However, GM-CSF-induced downregulation of Tyk2 and Jak1 tyrosine phosphorylation as well as Tyk2 protein levels likely contributed to the reduced Stat1 tyrosine phosphorylation. In hematopoietic cells, GM-CSF antagonizes IFN-induced signals by a block in Stat1 activation.

Multiple seropathotypes of verotoxin-producing Escherichia coli (VTEC) disrupt interferon- γ-induced tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1

Verotoxin-producing Escherichia coli (VTEC) O157:H7 inhibits interferon- γ-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1 in epithelial cells, independent of Verotoxins and the locus of enterocyte effacement pathogenicity island. Although E. coli O157:H7 is the major cause of disease in humans, non-O157:H7 VTEC also cause human disease. However, the virulence properties of non-O157:H7 VTEC are less well characterized. The aims of this study were to define the ability of VTEC strains of differing seropathotypes (classified as A–E) to inhibit interferon- γ stimulated Stat1-phosphorylation and to further characterize the bacterial-derived inhibitory factor. Confluent T84 and HEp-2 cells were infected with VTEC strains (MOI 100:1, 6 h, 37 °C), and then stimulated with interferon- γ (50 ng/mL) for 0.5 h at 37 °C. Whole-cell protein extracts of infected cells were collected and prepared for immunoblotting to detect tyrosine phosphorylation of Stat1. The effects of E. coli O55 strains, the evolutionary precursors of VTEC, on Stat1-tyrosine phosphorylation were also determined. The effects of isogenic mutants of O-islands 47 and 122 were tested to determine the role of genes encoded on these putative pathogenicity islands in mediating VTEC inhibition of the interferon- γ-Stat1 signaling cascade. To evaluate potential mechanism(s) of inhibition, VTEC O157:H7-infected cells were treated with pharmacological inhibitors, including, wortmannin and LY294002. Relative to uninfected cells, Stat1-tyrosine phosphorylation was significantly reduced after 6 h infection of both T84 and HEp-2 cells by VTEC strains of all five seropathotypes. E. coli O55 strains, but not enteropathogenic E. coli (EPEC), also caused inhibition of Stat1-tyrosine phosphorylation, suggesting that this effect was acquired early in the evolution of VTEC. Stat1-activation did not recover in epithelial cells infected with isogenic mutants of O-islands 47 and 122, indicating that the inhibitory factor was not contained in these genomic regions. Stat1-phosphorylation remained intact when VTEC-infected cells were treated with wortmannin (0–100 nM), but not by treatment with the more specific PI3-kinase inhibitor, LY294002. Inhibition of interferon- γ stimulated Stat1-tyrosine phosphorylation by VTEC of multiple seropathotypes indicates the presence of a common inhibitory factor that is independent of bacterial virulence in humans. The results of treatment with wortmannin suggest that the bacterial-derived inhibitory factor employs host cell signal transduction to mediate inhibition of Stat1-activation.

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.

Conditioned Medium from EnterohemorrhagicEscherichia coli-Infected T84 Cells Inhibits Signal Transducer and Activator of Transcription 1 Activation by Gamma Interferon

Gamma interferon (IFN-gamma) is a cytokine important to host defense which can signal through signal transducer and activator of transcription 1 (Stat1). Enterohemorrhagic Escherichia coli (EHEC) modulates host cell signal transduction to establish infection, and EHEC serotypes O113:H21 and O157:H7 both inhibit IFN-gamma-induced Stat1 tyrosine phosphorylation in vitro. The aim of this study was to delineate both bacterial and host cell factors involved in the inhibition of Stat1 tyrosine phosphorylation. Human T84 colonic epithelial cells were challenged with direct infection, viable EHEC separated from T84 cells by a filter, sodium orthovanadate, isolated flagellin, bacterial culture supernatants, and conditioned medium treated with proteinase K, trypsin, or heat inactivation. Epithelial cells were then stimulated with IFN-gamma and protein extracts were analyzed by immunoblotting. The data showed that IFN-gamma-inducible Stat1 tyrosine phosphorylation was inhibited when EHEC adhered to T84 cells, but not by bacterial culture supernatants or bacteria separated from the epithelial monolayer. Conditioned medium from T84 cells infected with EHEC O157:H7 suppressed Stat1 activation, and this was not reversed by treatment with proteinases or heat inactivation. Use of pharmacological inhibitors showed that time-dependent bacterial, but not epithelial, protein synthesis was involved. Stat1 inhibition was also independent of bacterial flagellin, host proteasome activity, and protein tyrosine phosphatases. Infection led to altered IFN-gamma receptor domain 1 subcellular distribution and decreased expression in cholesterol-enriched membrane microdomains. Thus, suppression of host cell IFN-gamma signaling by production of a contact-dependent, soluble EHEC factor may represent a novel mechanism for this pathogen to evade the host immune system.

Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization.

Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.

IFN-alpha antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3.

Eighty percent of patients newly infected with the hepatitis C virus (HCV) develop chronic infection, suggesting that HCV can develop effective strategies to escape the unspecific and specific immune response of the host. Because SOCS molecules have been recognized to be powerful inhibitors of cytokine signaling via the Jak/STAT pathway, virus-induced expression of these molecules should be an efficient instrument to counteract the cellular response toward interferons (IFNs), an essential part of first line antiviral immune response. This study shows that overexpression of HCV core protein inhibits IFN-alpha-induced tyrosine phosphorylation and activation of STAT1 in hepatic cells. With the use of a STAT1-YFP fusion protein, further evidence is given that HCV core is capable to inhibit nuclear translocation of STAT1. Inhibition of STAT1-tyrosine phosphorylation was accompanied by the induction of SOCS3-mRNA expression, suggesting that the HCV core protein impairs IFN-alpha-induced signal transduction via induction of SOCS3 expression. HCV core protein was competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist. These IFN-antagonistic properties of the HCV core protein may be part of the molecular basis of IFN-alpha unresponsiveness in about one-half of chronically infected HCV-patients.

Protein kinase A-mediated inhibition of gamma interferon-induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic transcription factors in human monocytes by Ehrlichia chaffeensis.

Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis. Our previous study showed that gamma interferon (IFN-gamma) added prior to or at early stage of infection inhibited infection of human monocytes with E. chaffeensis; however, after 24 h of infection, IFN-gamma had no antiehrlichial effect. To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-gamma, tyrosine phosphorylation of Stat1, Jak1, and Jak2 in E. chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis. Viable E. chaffeensis organisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-gamma within 30 min of infection. Similar results were obtained with human peripheral blood monocytes infected with E. chaffeensis. Heat or proteinase K treatment but not periodate treatment of E. chaffeensis abrogated the inhibitory effect, suggesting that protein factor(s) of E. chaffeensis is responsible for the inhibition of IFN-gamma-induced tyrosine phosphorylation. Preincubation of E. chaffeensis with the Fab fragment of dog anti-E. chaffeensis immunoglobulin G also abrogated the inhibitory effect. On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation. These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-gamma. Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection with E. chaffeensis. In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3',5'-cyclic phosphorothioate, E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated. These results suggest that E. chaffeensis blocks IFN-gamma-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell.

The Bmx Tyrosine Kinase Induces Activation of the Stat Signaling Pathway, Which Is Specifically Inhibited by Protein Kinase Cδ

Members of the hematopoietically expressed Tec tyrosine kinase family have an important role in hematopoietic signal transduction, as exemplified by the crucial role of Btk for B-cell differentiation and activation. Although a variety of cell surface receptors have been found to activate Tec tyrosine kinases, the specific signaling pathways and substrate molecules used by Tec kinases are still largely unknown. In this study a Tec family kinase, Bmx, was found to induce activation of the Stat signaling pathway. Bmx induced the tyrosine phosphorylation and DNA binding activity of all the Stat factors tested, including Stat1, Stat3, and Stat5, both in mammalian and insect cells. Bmx also induced transcriptional activation of Stat1- and Stat5-dependent reporter genes. Other cytoplasmic tyrosine kinases, Syk, Fyn, and c-Src, showed no or only weak ability to activate Stat proteins. Expression of Bmx in mammalian cells was found to induce activation of endogenous Stat proteins without activation of endogenous Jak kinases. We further analyzed the Bmx-mediated activation of Stat1, which was found to be regulated by protein kinase C δ (PKCδ) isoform, but not β 1, ε, or ζ isoforms, leading to inhibition of Stat1 tyrosine phosphorylation. In conclusion, these studies show that Bmx, a Tec family kinase, can function as an activator of the Stat signaling pathway and identify a role for PKCδ in the regulation of Bmx signaling.

The Bmx Tyrosine Kinase Induces Activation of the Stat Signaling Pathway, Which Is Specifically Inhibited by Protein Kinase Cδ

AbstractMembers of the hematopoietically expressed Tec tyrosine kinase family have an important role in hematopoietic signal transduction, as exemplified by the crucial role of Btk for B-cell differentiation and activation. Although a variety of cell surface receptors have been found to activate Tec tyrosine kinases, the specific signaling pathways and substrate molecules used by Tec kinases are still largely unknown. In this study a Tec family kinase, Bmx, was found to induce activation of the Stat signaling pathway. Bmx induced the tyrosine phosphorylation and DNA binding activity of all the Stat factors tested, including Stat1, Stat3, and Stat5, both in mammalian and insect cells. Bmx also induced transcriptional activation of Stat1- and Stat5-dependent reporter genes. Other cytoplasmic tyrosine kinases, Syk, Fyn, and c-Src, showed no or only weak ability to activate Stat proteins. Expression of Bmx in mammalian cells was found to induce activation of endogenous Stat proteins without activation of endogenous Jak kinases. We further analyzed the Bmx-mediated activation of Stat1, which was found to be regulated by protein kinase C δ (PKCδ) isoform, but not β 1, ε, or ζ isoforms, leading to inhibition of Stat1 tyrosine phosphorylation. In conclusion, these studies show that Bmx, a Tec family kinase, can function as an activator of the Stat signaling pathway and identify a role for PKCδ in the regulation of Bmx signaling.