Cells Previously Desensitized to Type 1 Interferons Display Different Mechanisms of Activation of Stat-dependent Gene Expression from Naive Cells (original) (raw)
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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
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.
Loss of Interferon-Induced Stat1 Phosphorylation in Activated T Cells
Journal of Interferon <html_ent glyph="@amp;" ascii="&"/> 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.
A molecular switch from STAT2-IRF9 to ISGF3 underlies interferon-induced gene transcription
Nature Communications, 2019
Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.
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.
Journal of Biological Chemistry, 2000
The p38 mitogen-activated protein (MAP) kinase is activated during engagement of the type I interferon (IFN) receptor and mediates signals essential for IFN␣dependent transcriptional activation via interferonstimulated response elements without affecting formation of the ISGF3 complex. In the present study, we provide evidence that the small GTPase Rac1 is activated in a type I IFN-dependent manner and that its function is required for downstream engagement of the p38 MAP kinase pathway. We also demonstrate that p38 is required for IFN␣-dependent gene transcription via GAS elements and regulates activation of the promoter of the PML gene that mediates growth inhibitory responses. In studies to determine whether the regulatory effects of p38 are mediated by serine phosphorylation of Stat1 or Stat3, we found that the p38 kinase inhibitors SB203580 or SB202190 or overexpression of a dominant negative p38 mutant do not inhibit phosphorylation of Stat1 or Stat3 on Ser-727 in several IFN␣-sensitive cell lines. Altogether these data demonstrate that the Rac1/ p38 MAP kinase signaling cascade plays a critical role in type I IFN signaling, functioning in cooperation with the Stat-pathway, to regulate transcriptional regulation of IFN␣-sensitive genes and generation of growth inhibitory responses.
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.