Sanjit K. Roy

The cell death regulator GRIM-19 is an inhibitor of signal transducer and activator of transcription 3

GRIM-19 (gene associated with retinoid-IFN-induced mortality 19), isolated as a cell death activator in a genetic screen used to define mechanisms involved in IFN-β- and retinoic acid-induced cell death, codes for a ≈16-kDa protein that induces apoptosis in a number of cell lines. Antisense ablation of GRIM-19 caused resistance to cell death induced by IFN plus retinoic acid and conferred a growth advantage to cells. To understand the molecular bases for its cell death regulatory activity, we used a yeast two-hybrid screen and identified that the transcription factor STAT3 (signal transducer and activator of transcription 3) binds to GRIM-19. GRIM-19 inhibits transcription driven by activation of STAT3, but not STAT1. It neither inhibits the ligand-induced activation of STAT3 nor blocks its ability to bind to DNA. Mutational analysis indicates that the transactivation domain of STAT3, especially residue S727, is required for GRIM-19 binding. Because GRIM-19 does not bind significantly to other STATs, our studies identify a specific inhibitor of STAT3. Because constitutively active STAT3 up-regulates antiapoptotic genes to promote tumor survival, its inhibition by GRIM-19 also demonstrates an antioncogenic effect exerted by biological therapeutics.

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.

Critical role for transcription factor C/EBP-beta in regulating the expression of death-associated protein kinase 1

ABSTRACT Transcription factor C/EBP-β regulates a number of physiological responses. During an investigation of the growth-suppressive effects of interferons (IFNs), we noticed that cebpb−/− cells fail to undergo apoptosis upon gamma IFN (IFN-γ) treatment, compared to wild-type controls. To examine the basis for this response, we have performed gene expression profiling of isogenic wild-type and cebpb−/− bone marrow macrophages and identified a number of IFN-γ-regulated genes that are dependent on C/EBP-β for their expression. These genes are distinct from those regulated by the JAK-STAT pathways. Genes identified in this screen appear to participate in various cellular pathways. Thus, we identify a new pathway through which the IFNs exert their effects on cellular genes through C/EBP-β. One of these genes is death-associated protein kinase 1 (dapk1). DAPK1 is critical for regulating the cell cycle, apoptosis, and metastasis. Using site-directed mutagenesis, RNA interference, and chromatin immunoprecipitation assays, we show that C/EBP-β binds to the promoter of dapk1 and is required for the regulation of dapk1. Both mouse dapk1 and human dapk1 exhibited similar dependences on C/EBP-β for their expression. The expression of the other members of the DAPK family occurred independently of C/EBP-β. Members of the C/EBP family of transcription factors other than C/EBP-β did not significantly affect dapk1 expression. We identified two elements in this promoter that respond to C/EBP-β. One of these is a consensus C/EBP-β-binding site that constitutively binds to C/EBP-β. The other element exhibits homology to the cyclic AMP response element/activating transcription factor binding sites. C/EBP-β binds to this site in an IFN-γ-dependent manner. Inhibition of ERK1/2 or mutation of an ERK1/2 site in the C/EBP-β protein suppressed the IFN-γ-induced response of this promoter. Together, our data show a critical role for C/EBP-β in a novel IFN-induced cell growth-suppressive pathway via DAPK1.

Protein Kinase Cβ Deficiency Increases Fatty Acid Oxidation and Reduces Fat Storage

Metabolic syndrome is common in the general population, but there is little information available on the underlying signaling mechanisms regulating triglyceride (TG) content in the body. In the current study, we have uncovered a role for protein kinase Cβ (PKCβ) in TG homeostasis by studying the consequences of a targeted disruption of this kinase. PKCβ-/- mutant mice were considerably leaner and the size of white fat depots was markedly decreased compared with wild-type littermates. TG content in the liver and skeletal muscle of PKCβ-/- mice was also significantly low. Interestingly, mutant animals were hyperphagic and exhibited higher food intake and reduced feed efficiency versus wild type. The protection from obesity involves elevated oxygen consumption/energy expenditure and increased fatty acid oxidation in adipose tissue with concurrent increased mitochondria genesis, up-regulation of PGC-1α and UCP-2, and down-regulation of perilipin. The ability of PKCβ deficiency to promote fat burning in adipocytes may suggest novel therapeutic strategies for obesity and obesity-related disorders.

The Med1 Subunit of Transcriptional Mediator Plays a Central Role in Regulating CCAAT/Enhancer-binding Protein-β-driven Transcription in Response to Interferon-γ

Transcription factor CCAAT/enhancer-binding protein (C/EBP)-β is crucial for regulating transcription of genes involved in a number of diverse cellular processes, including those involved in some cytokine-induced responses. However, the mechanisms that contribute to its diverse transcriptional activity are not yet fully understood. To gain an understanding into its mechanisms of action, we took a proteomic approach and identified cellular proteins that associate with C/EBP-β in an interferon (IFN)-γ-dependent manner. Transcriptional mediator (Mediator) is a multisubunit protein complex that regulates signal-induced cellular gene transcription from enhancer-bound transcription factor(s). Here, we report that the Med1 subunit of the Mediator as a C/EBP-β-interacting protein. Using gene knock-out cells and mutational and RNA interference approaches, we show that Med1 is critical for IFN-induced expression of certain genes. Med1 associates with C/EBP-β through a domain located between amino acids 125 and 155 of its N terminus. We also show that the MAPK, ERK1/2, and an ERK phosphorylation site within regulatory domain 2, more specifically the Thr189 residue, of C/EBP-β are essential for it to bind to Med1. Last, an ERK-regulated site in Med1 protein is also essential for up-regulating IFN-induced transcription although not critical for binding to C/EBP-β.

Deoxycholyltaurine rescues human colon cancer cells from apoptosis by activating EGFR-dependent PI3K/Akt signaling.

Recent studies indicate that secondary bile acids promote colon cancer cell proliferation but their role in maintaining cell survival has not been explored. We found that deoxycholyltaurine (DCT) markedly attenuated both unstimulated and TNF‐α‐stimulated programmed cell death in colon cancer cells by a phosphatidylinositol 3‐kinase (PI3K)‐dependent mechanism. To examine the role of bile acids and PI3K signaling in maintaining colon cancer cell survival, we explored the role of signaling downstream of bile acid‐induced activation of the epidermal growth factor receptor (EGFR) in regulating both apoptosis and proliferation of HT‐29 and H508 human colon cancer cells. DCT caused dose‐ and time‐dependent Akt (Ser473) phosphorylation, a commonly used marker of activated PI3K/Akt signaling. Both EGFR kinase and PI3K inhibitors attenuated DCT‐induced Akt phosphorylation and Akt activation, as demonstrated by reduced phosphorylation of a GSK‐3‐paramyosin substrate. Transfection of HT‐29 cells with kinase‐dead EGFR (K721M) reduced DCT‐induced Akt phosphorylation. In HT‐29 cells, EGFR and PI3K inhibitors as well as transfection with dominant negative AKT attenuated DCT‐induced cell proliferation. DCT‐induced PI3K/Akt activation resulted in downstream phosphorylation of GSK‐3 (Ser21/9) and BAD (Ser136), and nuclear translocation (activation) of NF‐κB, thereby confirming that DCT‐induced activation of PI3K/Akt signaling regulates both proproliferative and prosurvival signals. Collectively, these results indicate that DCT‐induced activation of post‐EGFR PI3K/Akt signaling stimulates both colon cancer cell survival and proliferation. J. Cell. Physiol. 215: 538–549, 2008.

Down regulation of the transcriptional mediator subunit Med1 contributes to the loss of expression of Metastasis associated dapk1 in human cancers and cancer cells

DAPK1, a ca+2/calmodulin regulated serine/threonine kinase, is a major tumor suppressor, whose expression is lost in multiple tumor types. However, the mechanisms contributing to it are unclear. We have recently shown that CCAAT/Enhancer binding protein‐β (C/EBP‐β) is required for the basal and interferon γ (IFN‐γ)‐induced expression of dapk1 in many cell types. C/EBP‐β interacts with the transcriptional Mediator, a multisubunit complex that couples enhancer bound transcription factors to the basal transcriptional machinery in an IFN‐γ dependent manner for regulating dapk1 expression. Specifically, the Med1 (TRAP220/PBP/DRIP220/CRSP220) subunit associates with the enhancer bound C/EBP‐β at the CRE/ATF site of dapk1 in an IFN‐γ dependent manner for stimulating gene expression. Therefore, we investigated if the mechanism responsible for the loss of dapk1 expression in human cancers involves a failure to recruit C/EBP‐β and/or Med1 to the dapk1 promoter. We compared the relative occupancy of these factors at the dapk1 promoter at CRE/ATF sites in normal and cancer cell lines. A significantly lower binding of these factors to the CRE/ATF site of dapk1 promoter occurred in human cancer cell lines than in normal cells. We show that loss of Med1 expression correlates with a corresponding loss of dapk1 expression in a number of primary human lung carcinomas. Med1 levels were significantly lower in cancer cell lines than in normal controls. Importantly, we show that restoration of Med1 induces the expression of dapk1 in these cancer cells and also attenuates their metastatic potential in vivo. Our studies reveal a critical parameter limiting dapk1 expression in cancer cell lines.

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.