Padmaja Gade

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.

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.

Regulation of the Death-Associated Protein Kinase 1 Expression and Autophagy via ATF6 Requires Apoptosis Signal-Regulating Kinase 1

ABSTRACT The death-associated protein kinase 1 (DAPK1) is an important regulator of cell death and autophagy. Recently, we have identified that ATF6, an endoplasmic reticulum-resident transcription factor, in association with the transcription factor CEBP-β, regulates the gamma interferon (IFN-γ)-induced expression of Dapk1 (P. Gade et al., Proc. Natl. Acad. Sci. U. S. A. 109:10316–10321, 2012, doi.org/10.1073/pnas.1119273109). IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β were essential for the formation of a novel transcriptional complex that regulates DAPK1. Here, we report that IFN-γ activates the ASK1-MKK3/MKK6–p38 mitogen-activated protein kinase (MAPK) pathway for controlling the activity of ATF6. The terminal enzyme in this pathway, p38 MAPK, phosphorylates a critical threonine residue in ATF6 upstream of its DNA binding domain. ATF6 mutants defective for p38 MAPK phosphorylation fail to undergo proteolytic processing in the Golgi apparatus and drive IFN-γ-induced gene expression and autophagy. We also show that mice lacking Ask1 are highly susceptible to lethal bacterial infection owing to defective autophagy. Together, these results identify a novel host defense pathway controlled by IFN-γ signaling.

Nicotine promotes apoptosis resistance of breast cancer cells and enrichment of side population cells with cancer stem cell-like properties via a signaling cascade involving galectin-3, α9 nicotinic acetylcholine receptor and STAT3

Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here, we show that nicotine induces the expression of galectin-3 (an anti-apoptotic β-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine-induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer.

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.

A Noncanonical Flt3ITD/NF-κB Signaling Pathway Represses DAPK1 in Acute Myeloid Leukemia

Purpose: Death-associated protein kinase 1 (DAPK1), a tumor suppressor, is a rate-limiting effector in an endoplasmic reticulum (ER) stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of acute myeloid leukemia (AML) with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design: Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB–and c-Jun–responsive genes, was studied. RNA interference knockdown studies were carried out in an Flt3ITD+ cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD–TAK1–DAPK1 repression, and chromatin immunoprecipitations were carried out to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results: AMLs characterized by normal karyotype with Flt3ITD were found to have 10- to 100-fold lower DAPK1 transcripts normalized to the expression of c-Jun, a transcriptional activator of DAPK1, as compared with a heterogeneous cytogenetic category. In addition, Meis1, a c-Jun-responsive adverse AML prognostic gene signature was measured as control. These Flt3ITD+ AMLs overexpress relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter together with histone deacetylase 2 (HDAC2) and HDAC6 in the Flt3ITD+ human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NF-κB–inducing kinase (NIK), de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ AMLs had selective nuclear activation of p52NF-κB. Conclusions: Flt3ITD promotes a noncanonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ AML. Clin Cancer Res; 18(2); 360–9. ©2011 AACR.

Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.

Differential gene expression is facilitated by transcriptional regulatory mechanisms and chromatin modifications through DNA-protein interactions. One of the widely used assays to study this is chromatin immunoprecipitation (ChIP) assay, which enables analysis of association of regulatory molecules to specific promoters and histone modifications in vivo. This is of immense value as ChIP assays can provide glimpse of the regulatory mechanisms involved in gene expression in vivo. This article outlines the general strategies and protocols to study ChIP assays in differential recruitment of transcriptional factors (TFs) and also global analysis of transcription factor recruitment is discussed. Further, the applications of ChIP assays for discovering novel genes that are dependent on specific transcription factors were addressed.

IFNG and autophagy: A critical role for the ER-stress mediator ATF6 in controlling bacterial infections

IFNG/IFNγ plays a critical role in driving innate and acquired defenses against infectious pathogens. The death-associated protein kinase 1 (DAPK1), originally identified as an activator of IFNG-induced cell death, controls autophagy. Previously, we have shown that transcription factor CEBPB (C/EBP-β) regulates IFNG-induced expression of Dapk1 through a CRE/ATF motif in its enhancer. In this paper we have shown that ATF6, an ER-resident transcription factor regulates IFNG-induced Dapk1 expression through the CRE/ATF site, in association with CEBPB. IFNG-stimulated proteolytic cleavage of ATF6, and MAPK1/3 (ERK2/1)-dependent phosphorylation of CEBPB together control the expression of Dapk1. Consistent with their requirement for DAPK1 expression, IFNG fails to induce autophagy in cells lacking either Atf6 or Cebpb. More importantly, the Atf6−/− mice are highly susceptible to lethal bacterial infections due to a loss of autophagy. This study reported a connection between ER stress and autophagy in mediating antibacterial defenses.

A Central Role for Transcription Factor C/EBP-β in Regulating CD1d Gene Expression in Human Keratinocytes

CD1d is a nonclassical Ag-presenting molecule that presents glycolipid Ags to NKT cells that are involved in immune defense and tumor rejection. It also plays a role in immunoregulatory functions in the epidermis. The mechanisms controlling the expression of CD1d are not well understood. Therefore, we cloned the CD1d gene promoter and characterized its activities in primary human keratinocytes and other cell lines of epithelial origin. We found that a CCAAT box in the CD1d promoter is required for its expression in keratinocytes. We show here that transcription factor C/EBP-β binds to the CCAAT box in the CD1d promoter in vitro and in vivo. Consistent with these observations, deletion of the gene encoding for C/EBP-β caused a loss of CD1d expression. The in vivo regulation of CD1d has significant implications for the pathologic mechanisms of certain immunologic skin diseases in which NKT cells play a role, such as allergic contact dermatitis and psoriasis. Together, these data show a central role for C/EBP-β in regulating CD1d transcription.

Potential role of autophagy in the bactericidal activity of human PMNs for Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax, is acquired by mammalian hosts from the environment, as quiescent endospores. These endospores must germinate inside host cells, forming vegetative bacilli, before they can express the virulence factors that enable them to evade host defenses and disseminate throughout the body. While the role of macrophages and dendritic cells in this initial interaction has been established, the role of polymorphonuclear leukocytes (PMNs) has not been adequately defined. We discovered that while B. anthracis 34F2 Sterne endospores germinate poorly within non-activated human PMNs, these phagocytes exhibit rapid microbicidal activity toward the outgrown vegetative bacilli, independent of superoxide and nitric oxide. These findings suggest that a non-free radical pathway kills B. anthracis bacilli. We also find in PMNs an autophagic mechanism of bacterial killing based on the rapid induction of LC-3 conversion, beclin-1 expression, sequestosome 1 (SQSTM1) degradation and inhibition of bactericidal activity by the inhibitor, 3-methyladenine. These findings extend to PMNs an autophagic bactericidal mechanism previously described for other phagocytes.