IRF-1 mRNA Research Articles

The serine/threonine phosphatase inhibitor, calyculin A, inhibits and dissociates macrophage responses to lipopolysaccharide.

LPS-stimulated macrophages (M phi) produce inflammatory mediators that are largely responsible for the pathophysiology associated with septic shock. M phi respond to LPS with rapid protein phosphorylation and dephosphorylation on serine, threonine, and tyrosine residues. If these events are critical for the cellular response to LPS, the kinases and/or phosphatases involved may be vulnerable targets for pharmacologic intervention. Recent studies demonstrated that tyrosine kinase inhibitors block LPS-induced tyrosine phosphorylation of MAP kinases as well as TNF-alpha and IL-1 beta production. To investigate a role for serine/threonine phosphatases, we evaluated the effect of calyculin A, a potent serine/threonine phosphatase inhibitor, on LPS stimulation of murine M phi. Pretreatment of M phi with calyculin A inhibited LPS-induced expression of six immediate-early genes: TNF-alpha, IL-1 beta, IFN-beta, IP-10, IRF-1, and TNFR-2. Calyculin A added 1.5 h after LPS treatment greatly reduced accumulation of IP-10, IRF-1, and TNFR-2 mRNA, but not TNF-alpha, IL-1 beta, and IFN-beta mRNA. Calyculin A, in the absence or presence of LPS, resulted in sustained tyrosine phosphorylation of the MAP kinases. These findings suggest that an "early" serine/threonine phosphatase activity is essential for LPS stimulation of M phi and that the activation of MAP kinases is not sufficient for the induction of these immediate-early genes. The requirement for a "late" phosphatase activity for expression of a subset of LPS-inducible genes dissociates at least two regulatory pathways in LPS signal transduction.

The Growth-dependent Expression of Angiotensin II Type 2 Receptor Is Regulated by Transcription Factors Interferon Regulatory Factor-1 and −2

Angiotensin II type 2 (AT2) receptor is abundantly and widely expressed in fetal tissues but present only in restricted tissues in the adult such as brain and atretic ovary. This receptor is speculated to be involved in tissue growth and/or differentiation. To elucidate the molecular mechanism of growth-regulated AT2 receptor expression, we cloned the mouse AT2 receptor genomic DNA and studied its promoter function in mouse fibroblast-derived R3T3 cells, which express AT2 receptor in the confluent, quiescent state but very low levels of the receptor in actively growing state. Promoter/luciferase reporter deletion analysis of AT2 receptor in R3T3 cells showed that the putative negative regulatory region is located between the positions -453 and -225, which plays an important role in the transcriptional control of AT2 receptor gene expression along with the cell growth. We identified the interferon regulatory factor (IRF) binding motif in this region using DNase foot-printing analysis and demonstrated that IRF binding oligonucleotide treatment increased the AT2 receptor expression in growing R3T3 cells but not in confluent cells. Furthermore, by antisense treatment, we demonstrated that IRF-2 attenuated the AT2 receptor expression in both growing and confluent R3T3 cells, whereas IRF-1 enhanced AT2 receptor expression in the confluent cells only. Consistent with this result, gel mobility shift assay demonstrated that growing R3T3 cells exhibited only IRF-2 binding, whereas confluent cells exhibited both IRF-1 and IRF-2 binding. Furthermore, we observed using reverse transcription-polymerase chain reaction that the IRF-1 mRNA expression was more abundant in confluent cells than growing cells, whereas IRF-2 expression did not change with R3T3 cell growth. We conclude that, in confluent cells, the enhanced expression of IRF-1 antagonizes the IRF-2 effect and increases the AT2 receptor expression. We speculate that these transcriptional factors influence cell growth in part by regulating AT2 receptor expression.

A major role for the protein tyrosine kinase JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6.

The protein tyrosine kinases JAK1, JAK2 and Tyk2 and STATs (signal transducers and activators of transcription) 1 and 3 are activated in response to interleukin-6 (IL-6) in human fibrosarcoma cells. In mutant cells lacking JAK1, JAK2 or Tyk2, the absence of one kinase does not prevent activation of the others; activation does not, therefore, involve a sequential three-kinase cascade. In the absence of JAK1, the phosphorylation of the gp130 subunit of the IL-6 receptor and the activation of STATs 1 and 3 are greatly reduced. JAK1 is also necessary for the induction of IRF1 mRNA, thus establishing a requirement for the JAK/STAT pathway in the IL-6 response. JAK2 and Tyk2 although activated cannot, in the absence of JAK1, efficiently mediate activation of STATs 1 and 3. A kinase-negative mutant of JAK2 can, however, inhibit such activation, and ancillary roles for JAK2 and Tyk2 are not excluded. A major role for JAK1 and the nonequivalence of JAK1 and JAK2 in the IL-6 response pathway are, nevertheless, clearly established for these cells.

Activation of IFN-alpha, IFN-gamma, MxA, and IFN regulatory factor 1 genes in influenza A virus-infected human peripheral blood mononuclear cells.

Mononuclear blood cells have an important role in immunity as they produce different cytokines in response to microbial infections. We infected primary human mononuclear blood cells with a pathogenic influenza A virus (A/Beijing/353/89) and studied the activation of IFN-alpha, IFN-gamma, IRF-1, and MxA genes. IFN-alpha and IFN-gamma steady state mRNA levels peaked at 6 to 9 h after infection and declined rapidly thereafter. Only a modest (twofold) increase in IRF-1 mRNA was seen. MxA gene expression, normally strictly regulated by IFN-alpha/beta, had expression kinetics similar to those of IFN mRNA. Infection experiments done in the presence of cycloheximide showed that influenza virus infection could induce all genes studied in the absence of detectable protein synthesis. Pretreatment with IFN-alpha, but not with IFN-gamma, caused a dose-dependent inhibition of influenza virus replication in PBMC, and this inhibition correlated with increasing levels of MxA protein. Influenza virus replication was also inhibited in a stably transfected, MxA-expressing promonocytic U937 cell line. The results suggest that MxA protein significantly contributes to IFN-mediated host defence mechanisms against influenza A virus.

Differential expression of interferon regulatory factor 1 (IRF-1), IRF-2, and interferon consensus sequence binding protein genes in lipopolysaccharide (LPS)-responsive and LPS-hyporesponsive macrophages.

Macrophages secrete interferon (IFN), as well as other cytokines, following lipopolysaccharide (LPS) stimulation. The interferon regulatory factors (IRFs) comprise a family of DNA-binding proteins that have been implicated in the transcriptional regulation of IFN and certain IFN-inducible genes. We therefore characterized basal and LPS-inducible levels of IRF-1, IRF-2, and interferon consensus sequence binding protein (ICSBP) mRNA in LPS-responsive macrophages and compared the expression of these genes in macrophages that typify two murine models of LPS hyporesponsiveness. In the first model, the LPS-hyporesponsive phenotype of the C3H/HeJ mouse is genetically determined and maps to the Lps locus on mouse chromosome 4. In the second model, normally LPS-responsive macrophages acquire a transient LPS-hyporesponsive phenotype following a prior exposure to LPS, a phenomenon referred to as "endotoxin tolerance." Using reverse transcription PCR, we detected basal levels of IRF-1 mRNA in LPS-responsive (Lpsn) macrophages that were approximately 15 times higher than those found in LPS-hyporesponsive (Lpsd) macrophages. Conversely, Lpsd macrophages expressed basal levels of IRF-2 mRNA that were approximately 18 times higher than those expressed in Lpsn macrophages. LPS stimulation resulted in a dose- and time-dependent accumulation of IRF-1, IRF-2, and ICSBP mRNA only in Lpsn macrophages. Cycloheximide inhibited the accumulation of LPS-stimulated IRF-2 and ICSBP mRNA, but not IRF-1 mRNA, thus designating IRF-1 an immediate-early, LPS-inducible gene. Finally, macrophages rendered tolerant to endotoxin expressed elevated but nonmaximal mRNA levels for all three transcription factors that are not reinduced upon secondary challenge with LPS. Thus, the IRFs may represent yet an additional molecular pathway in the complex response to LPS.

Reversal of interferon-gamma-resistant phenotype by poly(I:C): possible involvement of ISGF2 (IRF1) in interferon-gamma-mediated induction of the IDO gene.

Indoleamine 2,3-dioxygenase (IDO) is induced in many cell lines by interferon-gamma (IFN-gamma) treatment. IDO mRNA increases rapidly from 4 h after IFN-gamma treatment to at least 24 h after treatment in ME180 cells. The IFN-gamma-resistant mutant of ME180, IR3B6B, expresses only one-sixth the amount of IDO message after IFN-gamma treatment and very low levels of IDO. However, pretreatment of these mutants with poly(I:C) restores normal levels of IDO mRNAs and IDO activity. Since IRF1 mRNA induction is also low in IR3B6B cells after IFN-gamma treatment, we examined whether there was any relationship between IRF1 induction and IDO induction by IFN-gamma. The steady-state level of IRF1 mRNA was elevated by treating IR3B6B cells with poly(I:C) and IFN-gamma. Poly(I:C)-mediated reversal of IFN-gamma-resistant phenotype and induction of IDO and IRF1 messages are inhibited by 2-aminopurine. Transient transfection of IRF1 cDNA in ME180 cells resulted in activation of IDO transcription. Nuclear extracts prepared from IFN-gamma-treated ME180 and IR3B6B cells affected differently the mobility of a 80-bp DNA fragment of the 5' regulatory region of IDO gene. Pretreatment of IR3B6B cells with poly(I:C) and addition of IFN-gamma resulted in increased DNA binding of nuclear proteins to the DNA. Pre- and post-treatment of nuclear extract of IFN-gamma-treated ME180 cells with anti-IRF1 antibody resulted in a super shift in mobility of the probe with the abolishment of normal gel-shift pattern.(ABSTRACT TRUNCATED AT 250 WORDS)

Priming of human monocytes for enhanced lipopolysaccharide responses: expression of alpha interferon, interferon regulatory factors, and tumor necrosis factor.

Culture of human monocytes with either granulocyte-macrophage colony-stimulating factor or gamma interferon (IFN-gamma) results in a primed state, during which these cells express heightened responses to bacterial lipopolysaccharide (LPS). The production of IFN-alpha in response to LPS by human monocytes has an absolute requirement for priming. Tumor necrosis factor (TNF) expression is also greatly enhanced in primed monocytes after LPS stimulation, but unlike IFN-alpha, TNF is readily expressed in unprimed monocytes as well. In an effort to determine the molecular events associated with IFN-alpha induction in this system, freshly isolated human monocytes were primed by culture with either IFN-gamma or granulocyte-macrophage colony-stimulating factor and then treated with LPS; expression of IFN-alpha subtype 2 (IFN-alpha 2), IFN regulatory factors (IRFs), and TNF was assessed by Northern (RNA blot) analysis. IRF-1 mRNA is expressed at high levels in monocytes and is regulated by both LPS and priming cytokines, but its expression alone does not correlate with the induction of IFN-alpha 2 expression. IRF-2 mRNA is expressed in a more gradual manner following LPS stimulation, implying a possible feedback mechanism for inhibiting IFN-alpha expression. However, nuclear run-on analysis indicates that IFN-alpha 2 is not transcriptionally modulated in this system, in striking contrast to TNF, which is clearly regulated at the transcriptional level. In addition, IFN-alpha 2 mRNA accumulation is superinduced when primed monocytes are treated with LPS plus cycloheximide, while TNF mRNA is relatively unaffected. The results demonstrate that priming can affect subsequent LPS-induced gene expression at different levels in human monocytes.

Interleukin-6 activates and regulates transcription factors of the interferon regulatory factor family in M1 cells.

Activation of (2'-5') A synthetase gene expression in interleukin-6 (IL-6)-treated myeloleukemic M1 cells correlates with protein binding to the interferon response sequence enhancer (IRS). A new interferon response sequence complex, F6, is induced by IL-6 independently of interferon and is identified here as comprising the interferon regulatory factor-1 (IRF-1) and IRF-2, by use of specific antibodies in DNA mobility shift assays with probes containing IRF binding sites. IRF-1 and IRF-2 have, respectively, positive and negative transcriptional effects on interferon-beta and interferon-inducible genes. In the IL-6-treated M1or cells, IRF-1 binding is activated early and maximally at 1 h, whereas the onset of IRF-2 binding is delayed. In a cell variant M1res, where (2'-5') A synthetase is no more induced, IRF-2 binding is constitutive, and IRF-1 binding is not seen before or after IL-6 treatment. In sensitive M1or cells, IL-6 rapidly induces IRF-1 mRNA, but in M1res cells, IRF-1 mRNA is constitutively high and not changed by IL-6. IRF-2 mRNA levels are also constitutive and not inducible by IL-6 even in M1or cells. The dissociation between induction of mRNAs and of protein binding observed suggests that the activity of the IRF proteins is regulated by IL-6. Transcripts of a third member of the IRF gene family, ICSBP, encoding a protein known to act as repressor, were found to be strongly down-regulated by IL-6. The rapid activation of IRF-1 and the modulation of the other transcription factors of this family may play a role in the early phase of IL-6 action on the M1 cells.