Interleukin-6 modulates interferon-regulated gene expression by inducing the ISGF3 gamma gene using CCAAT/enhancer binding protein-beta(C/EBP-beta).

Although interleukin-6 (IL-6) alone does not induce the expression of IFN stimulated genes (ISG), a low dose priming of cells with IL-6 strongly enhances the cellular responses to interferon-alpha (IFN-alpha). This effect of IL-6 is not due to superstimulation of the JAK-STAT pathway. Rather, IL-6 induces expression of ISGF3 gamma (p48), a subunit of the multimeric transcription factor ISGF3. As a result IFN-alpha robustly activates gene transcription in IL-6 primed cells. We have shown earlier that the transcription of ISGF3 gamma gene is regulated through a novel element GATE (gamma-IFN activated transcriptional element). We show here IL-6 induces the ISGF3 gamma gene through GATE. Transcription factor C/EBP-beta is required for inducing ISGF3 gamma gene expression through GATE. A mutant C/EBP-beta inhibits the IL-6 inducible ISGF3 gamma gene expression through GATE. Together, these results establish a molecular basis for the synergy between IFNs and IL-6.

CCAAT/enhancer-binding protein-beta regulates interferon-induced transcription through a novel element.

We have described previously a novel interferon (IFN)-responsive cis-acting enhancer element called gamma-IFN-activated transcriptional element (GATE). GATE is distinct from the known IFN-stimulated elements and binds to novel transacting factors. To identify the gamma-IFN-responsive transacting factors that interact with GATE, we have screened a cDNA expression library derived from IFN-gamma-stimulated murine macrophage cell line and isolated three different cDNAs. Among these is a gene coding for the pleiotropic transcription factor, CCAAT/enhancer-binding protein-beta (C/EBP-beta). We report here that the gene for C/EBP-beta binds to GATE and induces gene expression. A mutant C/EBP-beta interferes with the IFN-gamma-stimulated transcription of the ISGF3gamma (p48) promoter. Other members of the C/EBP family do not cause these effects. Interestingly, the expression of C/EBP-beta, not the other members of its family, is induced by IFN-gamma. These studies thus identify a novel role for C/EBP-beta in the IFN-signaling pathways.

Regulation of interferon-alpha/beta-stimulated gene expression through the gamma-activated transcriptional element.

Interferons (IFNs) stimulate gene expression to mediate their biological actions. A multimeric transcription factor consisting of STAT1, STAT2 and p48, a DNA binding protein, regulates IFN-alpha/beta stimulated gene expression. Since the cellular level of p48 is also increased by pre-treatment of cells with IFN-gamma, it is also known as ISGF3gamma. To understand how IFN-gamma regulates the expression of the p48 gene, we have previously isolated and characterized the promoter of murine p48 gene and identified a novel gamma-IFN activated transcriptional element (GATE). In this study using several mutant constructs of p48 promoter we have determined that the same element responds to IFN-alpha/beta treatment. Relatively high doses of IFN-alpha/beta compared to IFN-gamma are required for the induction of p48 promoter. This ability of IFN-alpha/beta to regulate GATE dependent gene expression is linked to the activation of a factor induced by IFN-alpha. However, IFN-gamma induces the binding of two gamma-IFN inducible factors (GIFs) to GATE. The IFN-alpha inducible GATE binding factor is not recognized by specific antibodies raised against the known IFN-regulated factors. It is likely IFN-gamma is a stronger inducer of this gene because it activates two GIFs. GATE-like elements present in hither to undefined IFN-stimulated genes may control IFN-responses in a unique manner.

Gamma interferon augments macrophage activation by lipopolysaccharide by two distinct mechanisms, at the signal transduction level and via an autocrine mechanism involving tumor necrosis factor alpha and interleukin-1.

When given in the presence of gamma interferon (IFN-gamma), otherwise nontoxic doses of lipopolysaccharide (LPS or endotoxin) become highly lethal for mice. The mechanisms of this synergistic toxicity are not known. We considered the possibility that an interaction between the LPS-induced NF-kappaB and IFN-gamma-induced JAK-STAT pathways at the pretranscriptional level may enhance the LPS-induced signals. To test this hypothesis, we incubated murine macrophage RAW 264.7 cells with IFN-gamma for 2 h before addition of different doses of LPS. Consistent with the synergistic induction of inducible nitric oxide synthase mRNA and nitric oxide production by a combination of LPS and IFN-gamma, IFN-gamma strongly augmented LPS-induced NF-kappaB activation and accelerated the binding of NF-kappaB to DNA to as early as 5 min. In agreement with this, IFN-gamma pretreatment promoted rapid degradation of IkappaB-alpha but not that of IkappaB-beta. Inhibition of protein synthesis during IFN-gamma treatment suppressed LPS-initiated NF-kappaB binding. A rapidly induced protein appeared to be involved in IFN-gamma priming. Preincubation of cells with antibodies to tumor necrosis factor alpha or the interleukin-1 receptor partially reduced the priming effect of IFN-gamma. In a complementary manner, LPS enhanced the activation of signal-transducing activator of transcription 1 by IFN-gamma. These data suggest novel mechanisms for the synergy between IFN-gamma and LPS by which they cross-regulate the signal-transducing molecules. Through this mechanism, IFN-gamma may transform a given dose of LPS into a lethal stimulus capable of causing sepsis. It may also serve a beneficial purpose by enabling the host to respond quickly to relatively low doses of LPS and thereby activating antibacterial defenses.

Induction of interferon synthesis and activation of interferon-stimulated genes by liposomal transfection reagents.

Liposome-mediated transfection is a widely used technique for the introduction of exogenous DNA into mammalian cells. We observed a significant induction of endogenous interferon (IFN)-stimulated genes (ISGs) in cells treated with the liposomal reagents, lipofectamine and DOSPER, in the absence of DNA. Liposome treatment induced expression of reporter constructs driven by IFN-responsive promoter elements, demonstrating a generalized effect on ISG expression. The kinetics of ISG induction were markedly delayed in response to liposome as compared with IFN or double-stranded RNA. ISG induction could be transferred to naive cells with conditioned medium from liposome-treated cells, suggesting that a secreted factor was responsible for this activity. A cell line defective in IFN signaling was refractory to liposome-induced ISG expression, indicating a role for IFN in this induction. Indeed, liposome treatment directly induced IFN-beta gene expression and, thus, represents a novel IFN inducer. IFN induction by liposomal reagents and its potential effects on transgene expression should be considered in the choice of transfection reagent. The ability of liposomal gene delivery reagents to induce IFN synthesis in the host may prove useful in gene therapy approaches to viral and neoplastic diseases.

Thioredoxin reductase mediates cell death effects of the combination of beta interferon and retinoic acid.

Interferons (IFNs) and retinoids are potent biological response modifiers. By using JAK-STAT pathways, IFNs regulate the expression of genes involved in antiviral, antitumor, and immunomodulatory actions. Retinoids exert their cell growth-regulatory effects via nuclear receptors, which also function as transcription factors. Although these ligands act through distinct mechanisms, several studies have shown that the combination of IFNs and retinoids synergistically inhibits cell growth. We have previously reported that IFN-beta-all-trans-retinoic acid (RA) combination is a more potent growth suppressor of human tumor xenografts in vivo than either agent alone. Furthermore, the IFN-RA combination causes cell death in several tumor cell lines in vitro. However, the molecular basis for these growth-suppressive actions is unknown. It has been suggested that certain gene products, which mediate the antiviral actions of IFNs, are also responsible for the antitumor actions of the IFN-RA combination. However, we did not find a correlation between their activities and cell death. Therefore, we have used an antisense knockout approach to directly identify the gene products that mediate cell death and have isolated several genes associated with retinoid-IFN-induced mortality (GRIM). In this investigation, we characterized one of the GRIM cDNAs, GRIM-12. Sequence analysis suggests that the GRIM-12 product is identical to human thioredoxin reductase (TR). TR is posttranscriptionally induced by the IFN-RA combination in human breast carcinoma cells. Overexpression of GRIM-12 causes a small amount of cell death and further enhances the susceptibility of cells to IFN-RA-induced death. Dominant negative inhibitors directed against TR inhibit its cell death-inducing functions. Interference with TR enzymatic activity led to growth promotion in the presence of the IFN-RA combination. Thus, these studies identify a novel function for TR in cell growth regulation.

The polyoma virus T antigen interferes with interferon-inducible gene expression.

Murine polyoma virus (MPyV) is a small DNA virus that induces tumors in multiple tissues of infected host. In this investigation, we show that cell lines derived from wild type virus-induced breast tumors are resistant to the growth inhibitory action of interferon beta (IFN-beta). Furthermore, replication of heterologous viruses such as vesicular stomatitis virus and encephalomyocarditis virus was not inhibited by IFN-beta in these cells. This effect was due to inhibition of IFN-stimulated gene expression by viral T antigen. Activation of IFN-stimulated gene factor 3 was inhibited in cells derived from a tumor induced by wild-type MPyV but not those from a mutant that lacks the pRB binding site of the large T antigen. Similarly IFN-gamma-inducible gene expression was also inhibited in cells transformed by wild-type virus. The levels of components of IFN-stimulated gene factor 3 and signal transducing Janus tyrosine kinases were comparable between the cells transformed by the wild-type and mutant viruses. The viral large T antigen bound to Janus tyrosine kinase 1 and inactivated signaling through IFN receptors. Thus, these studies identify a mechanism of viral resistance to IFN action.

The interferon-inducible murine p48 (ISGF3gamma) gene is regulated by protooncogene c-myc.

p48 protein is an integral component of the multimeric interferon (IFN)-regulated transcription factor, ISGF3. We have shown earlier that this gene is regulated by a novel IFN-gamma-regulated element. In addition to the IFN-regulated element, a myc-max binding site is also present in this promoter. In this investigation we have studied the role of this site in the regulation of the p48 gene. In serum-induced quiescent cells Myc up-regulated the expression of p48 mRNA. We show that the protooncogene Myc regulates the expression of p48 through the element CACGTG. Mutations in this motif abolish Myc-inducibility of the reporter genes carrying p48 promoter elements. Purified Myc and Max proteins interact with the Myc-stimulated element of the p48 promoter. We also show that cells lacking p48 expression are highly susceptible to the cytocidal action of anticancer drugs. Taken together these data suggest that p48 may function as an anti-stress cell survival factor.

Modulation of interferon action by retinoids. Induction of murine STAT1 gene expression by retinoic acid.

We have previously demonstrated that up-regulation of STAT1 protein by all-trans-retinoic acid (RA) in interferon (IFN)-unresponsive cells permits growth inhibition by IFNs. Here, we show that the promoter of STAT1 directly responds to retinoic acid treatment. Sequence and functional analysis of the murine STAT1 promoter have identified a direct repeat motif that serves as a retinoic acid response element. Mutagenesis of this element resulted in a loss of response to RA. This element is activated by RA receptors alpha, beta, and gamma. In vivo, RA receptor beta and retinoid X receptor alpha preferentially interacted with this element. Thus, these data define a molecular basis for the synergy between IFNs and retinoids in tumor growth inhibition.

Interferon gamma-induced transcription of the murine ISGF3gamma (p48) gene is mediated by novel factors.

In this investigation, we show that the gene encoding p48, a subunit of transcription factor ISGF3, is transcriptionally induced by interferon gamma (IFN-gamma). We have identified a novel IFN-gamma-activated response element in the p48 gene promoter. This motif, notated as gamma-activated transcriptional element (GATE), has no significant resemblance to either pIRE (palindromic IFN-response element) or GAS (the IFN-gamma-activated sequence) but has partial homology to ISRE (IFN-stimulated response element). When fused to a neutral promoter, GATE, a 24-bp element, induced the expression of reporter genes following IFN-gamma treatment. In murine RAW cells, two IFN-gamma-inducible factors (GIF) bind to GATE. Binding of these factors to GATE is inhibited by cycloheximide and staurosporine. Although p48 gene induction is dependent on STAT1 and JAK1, activated STAT1 does not bind to GATE. Thus, GIFs appear to be novel trans-acting factors in the IFN-signaling pathway.