Analysis of SOCS-3 Promoter Responses to Interferon γ (2025)

The Stat1 Binding Motif of the Interferon-gamma Receptor Is Sufficient to Mediate Stat5 Activation and Its Repression by SOCS3

Journal of Biological Chemistry, 2001

Signal transduction via the interferon-␥ (IFN-␥) receptor requires the tyrosine phosphorylation of signal transducers and activators of transcription (Stats). Whereas tyrosine phosphorylation of Stat1 occurs in all cells, activation of Stat5 by IFN-␥ is cell type-restricted. Here we investigated the mechanism of Stat5 activation by the IFN-␥ receptor. In transfection assays both Stat5 isoforms, Stat5a and Stat5b, were phosphorylated on tyrosine in response to IFN-␥. Stat5 activation required the presence of tyrosine 420 (Tyr-420) in the murine IFNGR1 receptor chain, which also serves as the Stat1 binding site. Moreover, a peptide including Tyr-440, the Stat1 binding site of the human IFNGR1 chain, conferred the ability upon a synthetic receptor to activate Stat5. Suppressor of cytokine signaling 3 (SOCS3) inhibited the activation of Stat5 by the IFN-␥ receptor, and the Tyr-440-containing peptide stretch was sufficient for repression. SOCS3 expression had little effect on the activity of Jak kinases not associated with cytokine receptors. In IFN-␥-treated, Stat1-deficient fibroblasts Stat5 was inefficient in inducing transcription of a Statdependent reporter gene, suggesting it does not per se make a major contribution to the expression of IFN-␥responsive genes.

Stat1-independent induction of SOCS-3 by interferon-γ is mediated by sustained activation of Stat3 in mouse embryonic fibroblasts

Biochemical and Biophysical Research Communications, 2005

Using microarray technology, we previously demonstrated that IFN-c induces suppressor of cytokine signaling-3 (SOCS-3) in Stat1À/À mouse embryonic fibroblasts and bone marrow-derived macrophages. In this study, we have investigated the mechanism by which SOCS-3 is induced by Stat1-independent signal transduction pathway. Tyrosine kinases Jak1 and Jak2 are required for SOCS-3 induction by IFN-c in mouse embryonic fibroblasts. IFN-c stimulated strong and sustained activation of Stat1 whereas Stat3 activation was weak and transient in wild-type fibroblasts. In contrast, Stat3 is activated strongly and in a sustained manner in Stat1À/À fibroblasts. The Src kinase inhibitor SU6656 suppressed IFN-c activation of Stat3 in both wild-type and Stat1À/À fibroblasts. However, SU6656 inhibited IFN-c induction of SOCS-3 completely in Stat1À/À but not in wild-type fibroblasts. Knock down of Stat3 by short interfering RNA abrogated Stat3 activation and SOCS-3 induction by IFN-c in Stat1À/À fibroblasts. In human fibrosarcoma cell line 2fTGH, IFN-c activated Stat1 but not Stat3. SOCS-3 induction by IFN-c is strictly Stat1-dependent. The Stat1 docking site is required for SOCS-3 induction by IFN-c in human lung adenocarcinoma cells. We propose a model in which sustained activation of Stat1 or Stat3 mediates SOCS-3 induction by IFN-c in wild-type and Stat1À/À mouse embryonic fibroblasts, respectively.

SOCS1 Is a Critical Inhibitor of Interferon γ Signaling and Prevents the Potentially Fatal Neonatal Actions of this Cytokine

Cell, 1999

IFN␥ acts by binding to and inducing the multimerization of † The Cooperative Research Centre for Cellular Growth Factors a cell surface receptor composed of the IFNGR1 and IFNGR2 chains (Hemmi et al., 1994; Novick et al., 1994). PO Royal Melbourne Hospital Victoria 3050 This results in juxtaposition of janus kinase 1 (JAK1) bound to IFNGR1 and JAK2 bound to IFNGR2, which Australia ‡ Center For Functional Genomics And Human Disease cross-phosphorylate and activate each other (Muller et al., 1993a; Watling et al., 1993; Igarashi et al., 1994). Institute of Reproduction and Development Monash Medical Centre Activated JAKs phosphorylate tyrosine residues within the cytoplasmic domains of the receptor subunits, which Victoria 3165 Australia act as docking sites for signal transducer and activator of transcription 1 (STAT1; Greenlund et al., 1994; Heim et al., 1995). Phosphorylation of a C-terminal tyrosine (Y701) in STAT1 facilitates interaction with the SH2 do-Summary main of a second STAT1 molecule, mediating dimerization (Shuai et al., 1994). STAT1 dimers subsequently Mice lacking suppressor of cytokine signaling-1 (SOCS1) migrate to the nucleus, where they bind to gamma-actidevelop a complex fatal neonatal disease. In this study, vated sequence (GAS) elements contained within the SOCS1 Ϫ/Ϫ mice were shown to exhibit excessive repromoters of IFN␥-inducible genes (Shuai et al., 1992; sponses typical of those induced by interferon ␥ (IFN␥), Muller et al., 1993b), including the genes for inducible were hyperresponsive to viral infection, and yielded nitric oxide synthase (iNOS) and the transcription factor macrophages with an enhanced IFN␥-dependent cainterferon regulatory factor 1 (IRF1). The increased suspacity to kill L. major parasites. The complex disease ceptibility to Mycobacteria, Leishmania major, and in SOCS1 Ϫ/Ϫ mice was prevented by administration of some viruses in mice or humans harboring mutations in anti-IFN␥ antibodies and did not occur in SOCS1 Ϫ/Ϫ the genes for IFN␥, its receptor, IRF1, or iNOS highlights mice also lacking the IFN␥ gene. Although IFN␥ is the importance of this pathway in resistance to infection essential for resistance to a variety of infections, the (Huang et al., 1993; Matsuyama et al., 1993; Kamijo et potential toxic action of IFN␥, particularly in neonatal al., 1994; MacMicking et al., 1995; Durbin et al., 1996; mice, appears to require regulation. Our data indicate Meraz et al., 1996; Newport et al., 1996; Lu et al., 1998). that SOCS1 is a key modulator of IFN␥ action, allowing The actions of IFN␥ are not always beneficial, since the protective effects of this cytokine to occur without infections may elicit a host response of sufficient magnithe risk of associated pathological responses. tude to become life threatening, for example, morbidity associated with Staphylococcus aureus infection and Introduction the hepatotoxicity associated with hepatitis B infection (Billiau and Vandekerckhove, 1991; Ando et al., 1993; Resistance to infections is dependent on the coordi-Matthys et al., 1995). The potentially toxic effects of nated action of the cytokine network. Key contributions IFN␥, including fatty degeneration of the liver, have also are made by interferons (IFN; Billiau, 1996; Boehm et been demonstrated in experiments in which circulating al., 1997; De Maeyer and De Maeyer-Guignard, 1998), IFN␥ levels are experimentally elevated in neonatal mice which comprise two broad groups. Type I interferons (Gresser, 1982; Toyonaga et al., 1994). Regulatory mechinclude the closely related forms of IFN␣ and a single anisms must therefore exist to maintain the fine balance form of IFN␤, whereas IFN␥ is the sole type II interferon between beneficial and detrimental responses to IFN␥. (De Maeyer and De Maeyer-Guignard, 1998). The IFN␣ This is achieved in part through the production of inproteins and IFN␤ are produced by many cell types in terleukin-4 (IL-4), IL-10, and IL-13, which counteract the response to viral infection (De Maeyer and De Maeyereffects of IFN␥ (Paul, 1991; Moore et al., 1993; Zurawski Guignard, 1998). In contrast, IFN␥ is produced excluand de Vries, 1994), and by negative regulation of IFN␥ sively by activated T cells and natural killer (NK) cells signal transduction, for example, by the SH2 domain-(Billiau, 1996; Boehm et al., 1997; De Maeyer and De containing phosphatase SHP1 (Massa and Wu, 1996). Maeyer-Guignard, 1998). IFN␥ serves to upregulate ex-In vitro studies have also implicated a family of SH2pression of a wide variety of genes involved in antigen containing proteins, the suppressors of cytokine signaling (SOCS) proteins, in the negative regulation of cyto- § To whom correspondence should be addressed (e-mail: hilton@ kine signal transduction. Of the eight SOCS proteins wehi.edu.au). These authors contributed equally to this work. (SOCS1 to 7 and CIS), SOCS1 and SOCS3 appear to be Cell 598 EMBO J. 13, 1591-1600.

Cells Previously Desensitized to Type 1 Interferons Display Different Mechanisms of Activation of Stat-dependent Gene Expression from Naive Cells

Journal of Biological Chemistry, 2003

Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons (IFNs) and other cytokines activate or down-regulate immediate early genes via the Jak/Stat pathway. In contrast, little information is available on interferonactivated gene expression in naïve cells compared with cells that have been desensitized and subsequently resensitized to the actions of these cytokines. In naïve cells, the ISG54 gene is activated via IFN␤-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2. In contrast, in previously desensitized cells IFN␤ weakly stimulates the assembly of an ISGF3-like complex that lacks Stat1, even though ISG54 mRNA induction is the same as in naïve cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of a protein-tyrosine phosphatase. In cells that do not express the tyrosine phosphatase Tc-PTP, the rate of Stat1 dephosphorylation is the same in naïve and previously desensitized cells. These results implicate Tc-PTP in a novel role in the regulation of type 1 interferon-stimulated gene expression.

Interferons up-regulate STAT1, STAT2, and IRF family transcription factor gene expression in human peripheral blood mononuclear cells and macrophages

Journal of Immunology, 1997

IFN signaling is mediated by binding of IFNs to their receptors and subsequent activation of Janus tyrosine kinase (JAK)-STAT signaling pathway. Stimulation of cells with IFN-a leads to the assembly of IFN-stimulated gene factor 3 transcription factor complex formed by STAT1 , STATZ, and p48 protein. IFN-y signaling is mediated by homodimeric STATl protein. Although these signaling molecules are expressed constitutively, there is also evidence of transcriptional regulation by IFNs. We have characterized the expression of STAT and IFN regulatory factor (IRF) family transcription factors in primary human blood mononuclear cells and macrophages in response to IFN-a and IFN-y stimulation. We show that IFN-a and IFN-y rapidly and efficiently enhanced STATI, STAT2, p48, and IRF-1 gene expression. IFN-y induced IRF-1 gene expression more strongly than IFN-a. Stimulation experiments in the presence of protein synthesis inhibitor, cycloheximide, suggested that these genes were activated directly by IFNs. IRF-2 gene was apparently only weakly responsive to IFNs in these cells. When macrophages were pretreated with low doses of IFN-y and then stimulated with IFN-a, clearly enhanced formation of specific transcription factor complexes was detected. This suggests that higher intracellular levels of STAT1, STATZ, and p48 protein may result in enhanced signal transduction for cytokines utilizing these transcription factors. C ytokine signal transduction from the cell surface receptor to the nucleus is mediated by receptor-associated Janus family tyrosine kinases (JAKs)' and STAT proteins (the JAK-STAT pathway). To date, four JAK kinase genes (JAKI, JAK2, JAK3, and TYK2) and seven STAT genes (STATI, 2, 3, 4, 5a, 5b, and 6) have been molecularly cloned. Receptor-associated ligand-activated JAKs phosphorylate STAT proteins on tyrosine residues, which leads to STAT protein activation, dimerization, translocation into the nucleus, and transcriptional activation of target genes. The IFN signaling system has functioned as a model for cytokine signaling in general (for reviews, see Refs. 1-3). IFNs are classified into type I (IFN-a,-p, and-m) and type I1 (IFN-y) IFNs and they have antiviral, antiproliferative, and immunomodulatory functions (4, 5). The two types of IFNs use distinct cell surface receptors, but their signal transduction pathways are partially overlapping. IFN-aP receptor-associated JAKl and TYK2 tyrosine phosphorylate STATl and STAT2 proteins. For full transcriptional activation, STATl also needs to be phosphorylated on a serine residue (6) by a serine-threonine kinase (mitogen-activated protein kinase) (7). Activated STATl and STAT2

Identification of STAT2 Serine 287 as a Novel Regulatory Phosphorylation Site in Type I Interferon-induced Cellular Responses

Journal of Biological Chemistry, 2013

Activation of Different Stat5 Isoforms Contributes to Cell-Type- Restricted Signaling in Response to Interferons

1996

Tyrosine phosphorylation and activation of the transcription factor Stat5 occur in response to stimuli like granulocyte-macrophage colony-stimulating factor, interleukin-3, or erythropoietin that stimulate both proliferation and differentiation of hematopoietic cells. It is unclear whether Stat5 is part of a proliferative response or part of the events leading to cellular differentiation. Here we report that agents promoting differentiation but not proliferation of hematopoietic cells, like phorbol ester or both types of interferons (IFNs), activate Stat5 in promonocytic U937 cells. Both IFN types caused tyrosine phosphorylation and DNA binding of predominantly one Stat5 isoform (Stat5a) despite expression of both Stat5a and Stat5b proteins. Monocytic differentiation of U937 cells led to a strong decrease in IFN-␥-mediated activation of Stat5 but not of Stat1. Transactivation of Stat5-target genes occurred in response to IFN-␥, which activates both Stat5 and Stat1, but not in response to granulocyte-macrophage colony-stimulating factor, which activates only Stat5. Tyrosine phosphorylation of Stat5 is not generally part of the IFN response. IFN-␥ did not cause Stat5 activation in HeLa cells, despite the expression of both Stat5 isoforms at similar levels. By contrast, IFN-␣ caused tyrosine phosphorylation and DNA binding of exclusively the b isoform of Stat5, and activated Stat5b formed a DNA binding activity previously found in HeLa cells and designated IFN-␣ activation factor 2. Taken together, our results demonstrate that ligand binding of IFN receptors leads to an isoform-specific activation of Stat5 in a restricted number of cell lineages. Moreover, they suggest that Stat5 might be part of the differentiation response of myeloid cells.

Phosphorylation of the Stat1 transactivating domain is required for the response to type I interferons

EMBO reports, 2003

Stat1 (signal transducer and activator of transcription 1) regulates transcription in response to the type I interferons IFN-α and IFN-β, either in its dimerized form or as a subunit of the interferon-stimulated gene factor 3 (Isgf3) complex (consisting of Stat1, Stat2 and interferon-regulating factor 9). Full-length Stat1-α and the splice variant Stat1-β, which lacks the carboxyl terminus and the Ser727 phosphorylation site, are found in all cell types. IFN-induced phosphorylation of Stat1-α on Ser727 occurs in the absence of the candidate kinase, protein kinase C-δ. When expressed in Stat1-deficient cells, Stat1-β and a Stat1-S727A mutant both restored the formation of Stat1 dimers and of the Isgf3 complex on treatment with IFN-β. By contrast, only Stat1-α restored the ability of IFN-β to induce high levels of transcription from target genes of Stat1 dimers and Isgf3 and to induce an antiviral state. Our data suggest an important contribution of the Stat1 C terminus and its phosphorylation at Ser727 to the transcriptional activities of the Stat1 dimer and the Isgf3 complex.

Functional Crosstalk between Type I and II Interferon through the Regulated Expression of STAT1

PLoS Biology, 2010

Autocrine priming of cells by small quantities of constitutively produced type I interferon (IFN) is a well-known phenomenon. In the absence of type I IFN priming, cells display attenuated responses to other cytokines, such as anti-viral protection in response to IFNc. This phenomenon was proposed to be because IFNa/b receptor1 (IFNAR1) is a component of the IFNc receptor (IFNGR), but our new data are more consistent with a previously proposed model indicating that regulated expression of STAT1 may also play a critical role in the priming process. Initially, we noticed that DNA binding activity of STAT1 was attenuated in c-Jun 2/2 fibroblasts because they expressed lower levels of STAT1 than wild-type cells. However, expression of STAT1 was rescued by culturing c-Jun 2/2 fibroblasts in media conditioned by wild-type fibroblasts suggesting they secreted a STAT1-inducing factor. The STAT1-inducing factor in fibroblast-conditioned media was IFNb, as it was inhibited by antibodies to IFNAR1, or when IFNb expression was knocked down in wild-type cells. IFNAR1 2/2 fibroblasts, which cannot respond to this priming, also expressed reduced levels of STAT1, which correlated with their poor responses to IFNc. The lack of priming in IFNAR1 2/2 fibroblasts was compensated by over-expression of STAT1, which rescued molecular responses to IFNc and restored the ability of IFNc to induce protective anti-viral immunity. This study provides a comprehensive description of the molecular events involved in priming by type I IFN. Adding to the previous working model that proposed an interaction between type I and II IFN receptors, our work and that of others demonstrates that type I IFN primes IFNc-mediated immune responses by regulating expression of STAT1. This may also explain how type I IFN can additionally prime cells to respond to a range of other cytokines that use STAT1 (e.g., IL-6, M-CSF, IL-10) and suggests a potential mechanism for the changing levels of STAT1 expression observed during viral infection.

Loss of Interferon-Induced Stat1 Phosphorylation in Activated T Cells

Journal of Interferon <html_ent glyph="@amp;" ascii="&amp;"/> Cytokine Research, 2004

Modulation of cytokine responsiveness following T cell activation represents an important mechanism that shapes the fate of T cells after encounters with antigens. We activated T cells in mice with superantigen and assessed their ability to phosphorylate Stat1 in response to interferon-␥ (IFN-␥) and IFN-␣. After 4 h of activation in vivo, T cells became deficient in their ability to phosphorylate Stat1 in response to either cytokine. The loss of IFN sensitivity was accompanied by increased mRNA transcription for multiple suppressors of cytokine signaling (SOCS) genes (SOCS1, SOCS3, and SOCS7). The transcript levels of these SOCS were elevated only during the early hours after activation and were at or below normal levels by 60 h. Likewise, the activation-induced inhibition of IFN-␣ signaling was transient, and sensitivity was restored by 3 days postactivation. The loss of sensitivity to IFN-␥ persisted, however, and was still evident at 3 days. These data suggest that SOCS-independent mechanisms specific for inhibition of IFN-␥ signaling may be present at later stages of the T cell response. The loss of Stat1 signaling may be a factor in differentiation of T cells during and after activation, and it could also represent a protective mechanism against the toxic effects of IFN-␥ during immune responses.