IRF-3-dependent and augmented target genes during viral infection.

Activation of the transcription factor interferon regulatory factor-3 (IRF-3) is an essential event in the innate immune response to viral infection. To understand the contribution of IRF-3 to host defense, we used a systems biology approach to analyze global gene expression dependent on IRF-3. Comparison of expression profiles in cells from IRF-3 knockout animals or wild-type siblings following viral infection revealed three sets of induced genes, those that are strictly dependent on IRF-3, augmented with IRF-3, or not responsive to IRF-3. Products of identified IRF-3 target genes are involved in innate or acquired immunity, or in the regulation of cell cycle, apoptosis and proliferation. These results reveal the global effects of one transcription factor in the immune response and provide information to evaluate the integrated response to viral infection.

Identification of STAT3 as a specific substrate of breast tumor kinase.

Breast tumor kinase (Brk) is a non-receptor tyrosine kinase distantly related to the Src family kinase. It is expressed in more than 60% of breast tumors, but the biological role of this kinase remains to be determined. Only a limited number of substates have been identified for Brk, and the link of Brk to tumorigenesis remains largely unknown. In this study, we provide evidence that the signal transducer and activator of transcription 3, STAT3, is a physiological target of Brk. Activation of STAT3 previously has been linked to oncogenesis, and results in this study demonstrate that STAT3 is tyrosine phosphorylated and transcriptionally activated in cells expressing endogenous Brk. Signal transducer and activator of transcription 3 is specifically targeted since other STAT members are not responsive to Brk expression. Signal transducer and activator of transcription 3 activation requires the catalytic activity of Brk, and expression of both STAT3 and Brk stimulate cellular proliferation. In addition, we have identified a negative regulator of Brk, the suppressor of cytokine signaling, SOCS3. The SOCS3 protein is known to block signaling mediated by cytokine receptors, and here we find that SOCS3 is able to repress the activity of the Brk non-receptor tyrosine kinase.

Involvement of protein phosphatase 2A in the interleukin-3-stimulated Jak2-Stat5 signaling pathway.

In this study, we report that the tyrosine kinase, Janus kinase 2 (Jak2), associates with the serine/threonine protein phosphatase 2A (PP2A) in 32Dcl3 myeloid progenitor cells. The association between Jak2 and PP2A transiently increases following interleukin-3 (IL-3) stimulation and activation of Jak2. The catalytic subunit of PP2A is tyrosine phosphorylated by Jak2 in vitro and in vivo, resulting in inhibition of phosphatase activity. PP2A also associates with Stat5 in 32Dcl3 cells in an IL-3-dependent manner. Pretreatment of 32Dcl3 cells with okadaic acid (OA), an inhibitor of PP2A, resulted in increased tyrosine phosphorylation and nuclear translocation of Stat5. Our results suggest that PP2A plays a negative regulatory role in regulating the IL-3 signaling pathway via formation of complexes with Jak2 and Stat5.

Rac1 mediates STAT3 activation by autocrine IL-6.

The activity of the small GTPase, Rac1, plays a role in various cellular processes including cytoskeletal rearrangement, gene transcription, and malignant transformation. In this report constitutively active Rac1 (Rac V12) is shown to stimulate the activation of STAT3, a member of the family of signal transducers and activators of transcription (STATs). The activity of Rac1 leads to STAT3 translocation to the nucleus coincident with STAT3-dependent gene expression. The expression of Vav (Delta1-187), a constitutively active guanine nucleotide exchange factor for the Rho GTPases, or activated forms of Ras or Rho family members, leads to STAT3-specific activation. The activation of STAT3 requires tyrosine phosphorylation at residue 705, but is not dependent on phosphorylation of Ser-727. Our studies indicate that Rac1 induces STAT3 activation through an indirect mechanism that involves the autocrine production and action of IL-6, a known mediator of STAT3 response. Rac V12 expression results in the induction of the IL-6 and IL-6 receptor genes and neutralizing antibodies directed against the IL-6 receptor block Rac1-induced STAT3 activation. Furthermore, inhibition of the nuclear factor-kappaB activation or disruption of IL-6-mediated signaling through the expression of IkappaBalpha S32AS36A and suppressor of cytokine signaling 3, respectively, blocks Rac1-induced STAT3 activation. These findings elucidate a mechanism dependent on the induction of an autocrine IL-6 activation loop through which Rac1 mediates STAT3 activation establishing a link between oncogenic GTPase activity and Janus kinase/STAT signaling.

Apoptosis is promoted by the dsRNA-activated factor (DRAF1) during viral infection independent of the action of interferon or p53.

An apoptotic cellular defense mechanism is triggered in response to viral dsRNA generated during the course of infection by many DNA and RNA viruses. We demonstrate that apoptosis induced by dsRNA or a paramyxovirus is independent of the action of interferon as it can proceed in a variety of cell lines and primary cells deficient in an interferon response. Initiation of apoptosis appears to be triggered by activation of a cellular transcription factor, the dsRNA-activated factor (DRAF1). DRAF1 is composed of interferon regulatory factor 3 (IRF-3) and the transcriptional coactivators CREB binding protein (CBP) or p300. We find that activation of IRF-3 in the absence of viral infection stimulates apoptosis. In addition, a negative interfering mutant blocks both target gene induction and apoptosis, demonstrating a requirement for gene expression by IRF-3/DRAF1 to promote apoptosis. IRF-3/DRAF1 target gene expression is also induced in response to a distinct apoptotic stimulus, the DNA damaging agent etoposide. The activity of the p53 tumor suppressor does not appear to be required for IRF-3/DRAF1-mediated apoptosis.

Nuclear export signal located within theDNA-binding domain of the STAT1transcription factor.

Latent signal transducers and activators of transcription (STATs) reside in the cytoplasm but rapidly accumulate in the nucleus following cytokine stimulation. Nuclear accumulation requires specific tyrosine phosphorylation and STAT dimerization. The presence of STATs in the nucleus is transient, however, and within hours the STATs reappear in the cytoplasm. Results indicate that STAT1 can be dephosphorylated in the nucleus and actively exported by the chromosome region maintenance 1 (CRM1) export receptor. CRM1 recognizes a specific amino acid sequence located within the DNA-binding domain of STAT1. This region shares sequence and functional properties of characterized nuclear export signals. The location of this sequence within STAT1 suggests that it is not accessible to CRM1 when STAT1 is bound to DNA. Evidence is presented to support a model in which STAT1 is tyrosine dephosphorylated in the nucleus and dissociates from DNA, allowing recognition by CRM1 and nuclear export. The regulated export of STAT1 may contribute to silencing of the signal pathway and/or to re-establish STAT1 in the cytoplasm to monitor activity of receptor-kinase signals.

Regulated nuclear-cytoplasmic localization of interferon regulatory factor 3, a subunit of double-stranded RNA-activated factor 1.

Viral double-stranded RNA (dsRNA) generated during the course of infection leads to the activation of a latent transcription factor, dsRNA-activated factor 1 (DRAF1). DRAF1 binds to a DNA target containing the type I interferon-stimulated response element and induces transcription of responsive genes. DRAF1 is a multimeric transcription factor containing the interferon regulatory factor 3 (IRF-3) protein and one of the histone acetyl transferases, CREB binding protein (CBP) or p300 (CBP/p300). In uninfected cells, the IRF-3 component of DRAF1 resides in the cytoplasm. The cytoplasmic localization of IRF-3 is dependent on a nuclear export signal, and we demonstrate IRF-3 recognition by the chromosome region maintenance 1 (CRM1) (also known as exportin 1) shuttling receptor. Following infection and specific phosphorylation, IRF-3 accumulates in the nucleus where it associates with CBP and p300. We identify a nuclear localization signal (NLS) in IRF-3 that is critical for nuclear accumulation. Mutation of the NLS abrogates nuclear localization even following infection. The NLS appears to be active constitutively, but it is recognized by only a subset of importin-alpha shuttling receptors. Evidence is presented to support a model in which IRF-3 normally shuttles between the nucleus and the cytoplasm but cytoplasmic localization is dominant prior to infection. Following infection, phosphorylated IRF-3 can bind to the CBP/p300 proteins resident in the nucleus. We provide the evidence of a role for CBP/p300 binding in the nuclear sequestration of a transcription factor that normally resides in the cytoplasm.

The epithelial cell response to rotavirus infection.

Rotavirus is the most important worldwide cause of severe gastroenteritis in infants and young children. Intestinal epithelial cells are the principal targets of rotavirus infection, but the response of enterocytes to rotavirus infection is largely unknown. We determined that rotavirus infection of HT-29 intestinal epithelial cells results in prompt activation of NF-kappaB (<2 h), STAT1, and ISG F3 (3 h). Genetically inactivated rotavirus and virus-like particles assembled from baculovirus-expressed viral proteins also activated NF-kappaB. Rotavirus infection of HT-29 cells induced mRNA for several C-C and C-X-C chemokines as well as IFNs and GM-CSF. Mice infected with simian rotavirus or murine rotavirus responded similarly with the enhanced expression of a profile of C-C and C-X-C chemokines. The rotavirus-stimulated increase in chemokine mRNA was undiminished in mice lacking mast cells or lymphocytes. Rotavirus induced chemokines only in mice <15 days of age despite documented infection in older mice. Macrophage inflammatory protein-1beta and IFN-stimulated protein 10 mRNA responses occurred, but were reduced in p50-/- mice. Macrophage inflammatory protein-1beta expression during rotavirus infection localized to the intestinal epithelial cell in murine intestine. These results show that the intestinal epithelial cell is an active component of the host response to rotavirus infection.

Cooperation of the transcriptional coactivators CBP and p300 with Stat6.

Interleukin-4 (IL-4) functions as a critical regulatory cytokine of the immune response. A major effect of IL-4 is the induction of specific gene expression mediated by activation of a latent transcription factor, Stat6. To understand the mechanism by which Stat6 induces gene transcription, the effects of two histone acetylase coactivators, CREB binding protein (CBP) and p300, were evaluated. Both CBP and p300 were found to cooperate with Stat6 for induction of Stat6-dependent transcription. This cooperation does not appear to be due to acetylation of Stat6. The adenoviral E1A oncoprotein, known to bind CBP and p300, can inhibit the ability of CBP and p300 to function as coactivators of Stat6. The cooperative effect of CBP and p300 depends on the presence of a carboxyl-terminal region of Stat6. Stat6 molecules lacking this region behave as negative interfering molecules for Stat6-dependent transcription. Point mutations within this region also affect transcription by Stat6 in response to IL-4, identifying a motif that appears to be required for transcription, possibly through functional cooperation with CBP/p300.

Interferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1.

Cells respond to viral infection or double-stranded RNA with the transcriptional induction of a subset of alpha/beta interferon-stimulated genes by a pathway distinct from the interferon signal pathway. The transcriptional induction is mediated through a DNA sequence containing the alpha/beta interferon-stimulated response element (ISRE). We previously identified a novel transcription factor, designated double-stranded RNA-activated factor 1 (DRAF1), that recognizes this response element. The DNA-binding specificity of DRAF1 correlates with transcriptional induction, thereby distinguishing it as a positive regulator of alpha/beta interferon-stimulated genes. Two of the components of DRAF1 have now been identified as interferon regulatory factor 3 (IRF-3) and the transcriptional coactivator CREB-binding protein (CBP)/p300. We demonstrate that IRF-3 preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection. Translocation of IRF-3 is accompanied by an increase in serine and threonine phosphorylation. Coimmunoprecipitation analyses of endogenous proteins demonstrate an association of IRF-3 with the transcriptional coactivators CBP and p300 only subsequent to infection. In addition, antibodies to the IRF-3, CBP, and p300 molecules react with DRAF1 bound to the ISRE target site of induced genes. The cellular response that leads to DRAF1 activation and specific gene expression may serve to increase host survival during viral infection.

Cloning and characterization of soluble and transmembrane isoforms of a novel component of the murine type I interferon receptor, IFNAR 2.

This report describes the cloning of cDNAs encoding transmembrane and soluble isoforms of a novel chain of the murine type I interferon (IFN) receptor and characterization of its capability to bind ligand and transduce signals. The transmembrane receptor (murine IFNAR 2c) has an extracellular domain of 215 amino acids and an intracellular domain of 250 amino acids, with 48% amino acid and 71% nucleotide identity with human IFNAR 2c. The cDNA for the soluble murine receptor (IFNAR 2a) encodes a 221-amino acid polypeptide identical to the first 210 amino acids of IFNAR 2c plus a novel 11 amino acids. Northern blot analyses show that murine IFNAR 2 is expressed as two transcripts of 4 kilobases encoding the transmembrane isoform and 1.5 kilobases encoding the more abundant soluble isoform. Studies using primary murine cells that lack IFNAR 1 show that IFNAR 2 is expressed, and cells bind type I IFN ligand, but do not transduce signals as detected by electrophoretic mobility shift assays of ISGF3 or GAF complexes binding to their cognate oligonucleotides. These cells show no effects on the ability of IFNgamma to activate these complexes. These studies demonstrate that the IFNAR 2 transmembrane (2c) and soluble (2a) isoforms are conserved between the human and mouse and that IFNAR 2c has intrinsic ligand binding activity, but no intrinsic signal transducing activity as measured in this study.

Distinct STAT structure promotes interaction of STAT2 with the p48 subunit of the interferon-alpha-stimulated transcription factor ISGF3.

Cells express a variety of STAT (signal transducer and activator of transcription) transcription factors that are structurally homologous and yet function specifically in response to particular cytokines. The functions of the individual STATs are dependent on distinct protein-protein interactions. STAT1 and STAT2 are activated by tyrosine phosphorylation in response to type I interferons-alpha/beta (IFN-alpha/beta) and subsequently form a multimeric transcription factor designated the IFN-alpha-stimulated gene factor 3 (ISGF3). ISGF3 is a unique STAT complex because it also contains a non-STAT molecule, p48, which is a critical DNA-binding component. We provide evidence that STAT2 specifically interacts with p48 in vivo before and after IFN-alpha stimulation. The specificity of ISGF3 formation is therefore a result of the distinct nature of the STAT2 molecule. Coimmunoprecipitation assays demonstrate p48 association with STAT2 but not STAT1. Hybrid STAT2. STAT1 molecules were used to identify a region of STAT2 which specifically associates with p48. The region of STAT2 interaction spans an amino-terminal region of two predicted coiled coils. The studies demonstrate the in vivo existence of a STAT2.p48 complex and a distinct STAT2.STAT1 complex after IFN-alpha stimulation. Data suggest that distinct bipartite complexes STAT2.p48 and STAT2.STAT1 translocate to the nucleus and associate on the DNA target site as ISGF3.

Interleukin-4-induced STAT6 recognizes and activates a target site in the promoter of the interleukin-4 receptor gene.

This study addresses the function of STAT6 in interleukin-4-stimulated gene expression. A specific STAT6 DNA-binding target site has been identified in the promoter of the interleukin-4 receptor gene, and STAT6 is shown to be involved in mediating activation of gene expression via this site. STAT6 can mediate transcription of a heterologous reporter gene construct containing the interleukin-4 receptor STAT6 binding site. In addition, evidence is provided that demonstrates a distinct effect of STAT6 DNA binding specificity on transcriptional regulation since transcription was not stimulated from a competent but different DNA binding site. To confirm the role of STAT6 in gene activation, STAT6 mutant proteins were generated and analyzed for their ability to function in interleukin-4-induced transcription. Although the interleukin-2 gamma chain receptor subunit has been demonstrated to be a component of the interleukin-4 receptor, it is not required for specific gene activation.

Interferon signal transduction.

The interferon signal transduction pathway initiates at a cell surface receptor and mediates the activation of target genes in the nucleus. The binding of interferon to a transmembrane receptor stimulates the activation of associated tyrosine kinases of the Janus kinase (JAK) family. Subsequently, latent cytoplasmic transcription factors are activated by tyrosine phosphorylation and function as signal transducers and activators of transcription (STATs). Advances in the field of interferon research have contributed to our understanding of signal transduction induced by many cytokines that also use JAK/STAT signaling pathways to activate early response genes. The specificity of signal activation by distinct cytokines that share these signaling components, and the molecular interaction of the signaling components with each other and their respective cytokine receptors represent major areas of research that are beginning to be elucidated. Signaling molecules other than the JAKs and STATs have also been found to be activated following interferon binding. In addition, the induction of type I interferon stimulated genes by double-stranded RNA in the absence of interferon provides another pathway of specific gene activation.

Interleukin 2 activates STAT5 transcription factor (mammary gland factor) and specific gene expression in T lymphocytes.

Although prolactin and interleukin 2 (IL-2) can elicit distinct physiological responses, we have found that their signal pathways share a common signal transducer and activator of transcription, STAT5. STAT5 was originally identified as a mammary gland factor induced by prolactin in lactating breast cells. Here we demonstrate that STAT5 is activated after IL-2 stimulation of two responsive lymphocyte cell lines, Nb2 and YT. Activation of STAT5 is measured both by IL-2-induced tyrosine phosphorylation and by IL-2-induced DNA binding. The STAT5 DNA recognition site is the same as the interferon gamma-activated site (GAS) in the interferon regulatory factor 1 gene. We demonstrate that the GAS element is necessary and sufficient for transcriptional induction by both IL-2 and prolactin in T lymphocytes. These results indicate that the role of STAT5 in the regulation of gene expression is not restricted to mammary cells or to prolactin, but is an integral part of the signal pathway of a critical immunomodulatory cytokine, IL-2.

Characterization of specific DNA-binding factors activated by double-stranded RNA as positive regulators of interferon alpha/beta-stimulated genes.

Viral infection results in transcriptional activation of the cellular interferon alpha/beta-stimulated genes (ISGs) independent of the autocrine action of interferon alpha/beta (IFN-alpha/beta). Induction of ISG expression by virus appears to be mediated through production of viral double-stranded RNA (dsRNA). Previously, we identified two novel dsRNA-activated factors (DRAFs) that bind to the interferon-stimulated response element (ISRE), the DNA sequence that mediates transcriptional activation by IFN-alpha/beta. In this report we define sequences that flank the classical ISRE to be necessary for DRAF1 binding. More significantly, it is shown that the sequences required to bind DRAF1 correlate with the ability to mediate ISG induction by virus. These results strongly suggest that DRAF1 is a positive regulator of ISG transcription. DRAF1 is shown to bind selectively to the promoters of those ISGs which are strongly induced by viral infection, again suggesting the functional significance of this factor. UV cross-linking experiments indicate that DRAF1 and DRAF2 share a common DNA-binding subunit of approximately 70 kDa which is referred to as the DRAF binding component (DRAFB). DRAFB is shown to preexist in the cytoplasm of unstimulated cells. Consistent with this observation, both DRAF1 and DRAF2 are activated in the cytoplasm prior to nuclear translocation.

Characterization of the interleukin-4 nuclear activated factor/STAT and its activation independent of the insulin receptor substrate proteins.

The activation of a latent DNA binding factor by interleukin-4 (IL-4), the IL-4 nuclear activated factor (IL-4 NAF), occurs within minutes of IL-4 binding to its receptor. Molecular characterization of IL-4NAF by ultraviolet light cross-linking experiments revealed a single protein of 120-130 kDa in contact with the DNA target site. Glycerol gradient sedimentation analysis indicated a molecular mass of IL-4 NAF consistent with a monomer that is capable of binding DNA. The IL-4 NAF target site is a palindromic sequence that is also recognized by the interferon-induced transcription factor, p91/STAT1 alpha. However, IL-4 NAF and p91/STAT1 alpha display distinguishable DNA binding specificities that may generate one level of specificity in the expression of target genes. Previous studies suggested the involvement of the insulin receptor substrate-1 (IRS-1) in the IL-4 signal transduction pathway. Although IRS-1 is involved in the stimulation of mitogenesis, our results demonstrate that activation of IL-4 NAF is independent of IRS-signaling proteins. The results of this study indicate that IL-4 stimulates bifurcating signal pathways that can direct mitogenesis via the IRS-signaling proteins and specific gene expression via the IL-4 NAF.

Signal transduction and activation of gene transcription by interferons.

Advances in the field of interferon research have identified a signal transduction pathway that initiates at a cell surface receptor and culminates at target genes in the nucleus. The binding of interferon to a transmembrane receptor stimulates the concomitant activation of tyrosine kinases of the Janus kinase (JAK) family. Subsequently, latent cytoplasmic transcription factors are activated by tyrosine phosphorylation and function as signal transducers and activators of transcription (STATs). The STATs form homomeric or heteromeric protein complexes that translocate to the nucleus to bind to specific DNA sequences in the promoters of stimulated genes. The discovery of this regulated pathway in the interferon system served as a paradigm for receptor to nucleus signal transmission by a variety of cytokines.

Receptor to nucleus signaling by prolactin and interleukin 2 via activation of latent DNA-binding factors.

The mechanism of action of prolactin (PRL), a lactogenic and immunoregulatory hormone, has remained undetermined despite its critical role in development. This study identifies a DNA-binding factor induced by PRL that appears to mediate a signal from the cell surface receptor to specific gene expression in the nucleus. PRL stimulates the proliferation of Nb2 T-lymphoma cells and activates transcription of the interferon-regulatory factor 1 (IRF-1) gene. Within minutes of PRL stimulation, a PRL-induced factor (PRLIF) is activated and binds to a target site in the promoter of the IRF-1 gene. The PRLIF-binding site contains an inverted GAAA repeat that is also functional in the hormone-responsive beta-casein gene. The PRL-receptor complex signals tyrosine phosphorylation of JAK2, a nonreceptor tyrosine kinase, which may lead to activation of PRLIF. T-cell proliferation and transcriptional activation of the IRF-1 gene is also induced by the cytokine interleukin 2 (IL-2). This report demonstrates the rapid activation of an IL-2 nuclear-activated factor that recognizes the same GAAA inverted repeat in the IRF-1 promoter. PRLIF and IL-2 nuclear-activated factor are newly identified factors that appear to serve fundamental roles in the signal transduction pathways of PRL and IL-2, respectively, leading to the transcriptional regulation of responsive genes.

Receptor to nucleus signaling via tyrosine phosphorylation of the p91 transcription factor.

Recent studies on signal transduction stimulated by interferons have defined pathways that link cell surface receptors to target genes in the nucleus. After interferon binding, nonreceptor tyrosine kinases are activated that phosphorylate components of latent DNA-binding factors in the cytoplasm. These phosphorylated factors form multimeric complexes that translocate to the nucleus and bind to specific DNA sequences in the promoters of induced genes. A 91-kD factor (p91 or Stat 91) is activated by interferons and serves as a subunit partner in the composition of diverse transcription factors.