Junbo Hu

ERK1 and ERK2 Activate CCAAAT/Enhancer-binding Protein-β-dependent Gene Transcription in Response to Interferon-γ

Interferons (IFNs) regulate the expression of a number of cellular genes by activating the JAK-STAT pathway. We have recently discovered that CCAAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through a novel IFN response element called the γ-IFN-activated transcriptional element (Roy, S. K., Wachira, S. J., Weihua, X., Hu, J., and Kalvakolanu, D. V. (2000) J. Biol. Chem. 275, 12626–12632. Here, we describe a new IFN-γ-stimulated pathway that operates C/EBP-β-regulated gene expression independent of JAK1. We show that ERKs are activated by IFN-γ to stimulate C/EBP-β-dependent expression. Sustained ERK activation directly correlated with C/EBP-βdependent gene expression in response to IFN-γ. Mutant MKK1, its inhibitors, and mutant ERK suppressed IFN-γ-stimulated gene induction through the γ-IFN-activated transcriptional element. Ras and Raf activation was not required for this process. Furthermore, Raf-1 phosphorylation negatively correlated with its activity. Interestingly, C/EBP-β-induced gene expression required STAT1, but not JAK1. A C/EBP-β mutant lacking the ERK phosphorylation site failed to promote IFN-stimulated gene expression. Thus, our data link C/EBP-β to IFN-γ signaling through ERKs.

MEKK1 plays a critical role in activating the transcription factor C/EBP-β-dependent gene expression in response to IFN-γ

IFN-γ induces a number of genes to up-regulate cellular responses by using specific transcription factors and the cognate elements. We recently discovered that CCAAT/enhancer-binding protein-β (C/EBP-β) induces gene transcription through an IFN-response element called γ-IFN-activated transcriptional element (GATE). Using mutant cells, chemical inhibitors, and specific dominant negative inhibitors, we show that induction of GATE-driven gene expression depends on MEK1 (mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase kinase) and ERKs (extracellular signal-regulated protein kinases) but is independent of Raf-1. Interestingly in cells lacking the MEKK1 gene or expressing the dominant negative MEKK1, ERK activation, and GATE dependent gene expression is inhibited. A dominant negative MEKK1 blocks C/EBP-β-driven gene expression stimulated by IFN-γ. These studies describe an IFN-γ-stimulated pathway that involves MEKK1-MEK1-ERK1/2 kinases to regulate C/EBP-β-dependent gene expression.

Thioredoxin participates in a cell death pathway induced by interferon and retinoid combination

Interferons (IFNs) and retinoids are potent tumor growth suppressors. We have shown earlier that the IFN-β and all-trans retinoic acid combination, but not the single agents, induces death in several tumor cell lines. Employing a genetic approach we have recently identified several Genes associated with Retinoid-IFN induced Mortality (GRIM) that mediate the cell death effect of IFN/RA combination. One of the GRIMs, GRIM-12, was identical to human thioredoxin reductase (TR), an enzyme that controls intracellular redox state. To define the participants of TR mediated death pathway we have examined the role of thioredoxin (Trx), its downstream substrate, and its influence on IFN/RA-induced death regulation. Inhibition of the thioredoxin expression by antisense RNA suppressed cell death. Similarly, a mutant Trx1 lacking the critical cysteine residues blocked cell death. In contrast, overexpression of wildtype thioredoxin augmented cell death. This effect of Trx1 was in part due to its ability to augment cell death via caspase-8. The redox inactive Trx1 mutant inhibits the cell death induced by caspase-8 but not caspase-3. These studies identify a novel mechanism of cell death regulation by IFN/RA combination involving redox enzymes.

IFN-γ Inhibits Lipopolysaccharide-Induced Interleukin-1β in Primary Murine Macrophages via a Stat1-Dependent Pathway

Interleukin-1 (IL-1) plays an important role in host defenses against microbial pathogens. Excessive production of this cytokine, however, may be responsible in part for the lethality observed during sepsis. Our studies show that interferon-γ (IFN-γ) downregulates lipopolysaccharide (LPS)-induced interleukin-1β (IL-1β) transcription in primary macrophages. This phenomenon does not occur in splenocytes or bone marrow-derived macrophages from signal transducer and activator of transcription (Stat1)-deficient mice, suggesting that Stat1, a transcription factor involved in IFN signaling, plays a critical role in this process. Moreover, nitric oxide (NO) was also involved in the downregulation of LPS-induced IL-1 by IFN, as addition of the inducible nitric oxide synthase (iNOS) inhibitor L-N6-(1-iminoethyl)lysine (NIL) negated the effect. Kinetic analysis of IL-1 and IFN levels in LPS-treated mice in vivo suggests that IFN-mediated inhibition of IL-1 might be an important negative feedback mechanism for limiti...

Interleukin-6 modulates interferon-regulated gene expression by inducing the ISGF3γ gene using CCAAT/enhancer binding protein-beta (C/EBP-β)

Abstract 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-α (IFN-α). This effect of IL-6 is not due to superstimulation of the JAK-STAT pathway. Rather, IL-6 induces expression of ISGF3γ (p48), a subunit of the multimeric transcription factor ISGF3. As a result IFN-α robustly activates gene transcription in IL-6 primed cells. We have shown earlier that the transcription of ISGF3γ gene is regulated through a novel element GATE (gamma-IFN activated transcriptional element). We show here IL-6 induces the ISGF3γ gene through GATE. Transcription factor C/EBP-β is required for inducing ISGF3γ gene expression through GATE. A mutant C/EBP-β inhibits the IL-6 inducible ISGF3γ gene expression through GATE. Together, these results establish a molecular basis for the synergy between IFNs and IL-6.

A Novel Transactivating Factor That Regulates Interferon-γ-dependent Gene Expression

We have previously identified a novel interferon (IFN)-stimulated cis-acting enhancer element, γ-IFN-activated transcriptional element (GATE). GATE differs from the known IFN-stimulated elements in its primary sequence. Preliminary analysis has indicated that the GATE-dependent transcriptional response requires the binding of novel transacting factors. A cDNA expression library derived from an IFN-γ-stimulated murine macrophage cell line was screened with a32P-labeled GATE probe to identify the potential GATE-binding factors. A cDNA coding for a novel transcription-activating factor was identified. Based on its discovery, we named it as GATE-binding factor-1 (GBF-1). GBF-1 homologs are present in mouse, human, monkey, and Drosophila. It activates transcription from reporter genes carrying GATE. It possesses a strong transactivating activity but has a weak DNA binding property. GBF-1 is expressed in most tissues with relatively higher steady-state levels in heart, liver, kidney, and brain. Its expression is induced by IFN-γ treatment. GBF-1 is present in both cytosolic and nuclear compartments. These studies thus identify a novel transactivating factor in IFN signaling pathways.

IFN-γ-Stimulated Transcriptional Activation by IFN-γ-Activated Transcriptional Element-Binding Factor 1 Occurs via an Inducible Interaction with CAAAT/Enhancer-Binding Protein-β

IFN-γ-activated transcriptional element (GATE)-binding factor 1 (GBF1) was identified as a transactivator that induces gene expression through GATE, a novel IFN-inducible element. Although it can induce gene expression, it is an extremely weak DNA-binding protein on its own. GATE also binds another transcription factor, C/EBP-β. Therefore, we explored whether GBF1 physically interacts with C/EBP-β to induce IFN-γ-regulated transcription. In response to IFN-γ, C/EBP-β undergoes phosphorylation at a critical ERK1/2 phosphorylation motif. Mutational inactivation of this motif and/or interference with the ERK1/2 activation prevented the IFN-γ-induced interactions between GBF1 and C/EBP-β. A 37-aa long peptide derived from the GBF1 protein can associate with C/EBP-β in an IFN-inducible manner. These results identify a converging point for two transactivators that exert their effects through a single response element. Together, our studies identify a novel regulatory mechanism that controls IFN-induced transcription.