Tsonwin Hai

Systems biology approaches identify ATF3 as a negative regulator of Toll-like receptor 4

The innate immune system is absolutely required for host defence, but, uncontrolled, it leads to inflammatory disease. This control is mediated, in part, by cytokines that are secreted by macrophages. Immune regulation is extraordinarily complex, and can be best investigated with systems approaches (that is, using computational tools to predict regulatory networks arising from global, high-throughput data sets). Here we use cluster analysis of a comprehensive set of transcriptomic data derived from Toll-like receptor (TLR)-activated macrophages to identify a prominent group of genes that appear to be regulated by activating transcription factor 3 (ATF3), a member of the CREB/ATF family of transcription factors. Network analysis predicted that ATF3 is part of a transcriptional complex that also contains members of the nuclear factor (NF)-κB family of transcription factors. Promoter analysis of the putative ATF3-regulated gene cluster demonstrated an over-representation of closely apposed ATF3 and NF-κB binding sites, which was verified by chromatin immunoprecipitation and hybridization to a DNA microarray. This cluster included important cytokines such as interleukin (IL)-6 and IL-12b. ATF3 and Rel (a component of NF-κB) were shown to bind to the regulatory regions of these genes upon macrophage activation. A kinetic model of Il6 and Il12b messenger RNA expression as a function of ATF3 and NF-κB promoter binding predicted that ATF3 is a negative regulator of Il6 and Il12b transcription, and this hypothesis was validated using Atf3-null mice. ATF3 seems to inhibit Il6 and Il12b transcription by altering chromatin structure, thereby restricting access to transcription factors. Because ATF3 is itself induced by lipopolysaccharide, it seems to regulate TLR-stimulated inflammatory responses as part of a negative-feedback loop.

Initiation and execution of lipotoxic ER stress in pancreatic beta-cells.

Free fatty acids (FFA) cause apoptosis of pancreatic β-cells and might contribute to β-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary β-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca2+ stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor β-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary β-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced β-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca2+ depletion. The IRE1 and resulting JNK activation contribute to β-cell apoptosis. PERK activation by palmitate also contributes to β-cell apoptosis via CHOP.

Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response

ABSTRACT In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.

Tissue-specific Pattern of Stress Kinase Activation in Ischemic/Reperfused Heart and Kidney

In this report we investigate the molecular mechanisms that contribute to tissue damage following ischemia and ischemia coupled with reperfusion (ischemia/reperfusion) in the rat heart and kidney. We observe the activation of three stress-inducible mitogen-activated protein (MAP) kinases in these tissues: p38 MAP kinase and the 46- and 55-kDa isoforms of Jun N-terminal kinase (JNK46 and JNK55). The heart and kidney show distinct time courses in the activation of p38 MAP kinase during ischemia but no activation of either JNK46 or JNK55. These two tissues also respond differently to ischemia/reperfusion. In the heart we observe activation of JNK55 and p38 MAP kinase, whereas in the kidney all three kinases are active. We also examined the expression pattern of two stress-responsive genes, c-Jun and ATF3. Our results indicate that in the heart both genes are induced by ischemia and ischemia/reperfusion. However, in the kidney c-Jun and ATF3 expression is induced only by ischemia/reperfusion. To correlate these molecular events with tissue damage we examined DNA laddering, a common marker of apoptosis. A significant increase in DNA laddering was evident in both heart and kidney following ischemia/reperfusion and correlated with the pattern of kinase activation, supporting a link between stress kinase activation and apoptotic cell death in these tissues.

Analysis of ATF3, a Transcription Factor Induced by Physiological Stresses and Modulated by gadd153/Chop10

We demonstrate that ATF3, a member of the ATF/CREB family of transcription factors, is induced in a variety of stressed tissues: mechanically injured liver, toxin-injured liver, blood-deprived heart, and postseizure brain. We also demonstrate that an ATF3-interacting protein, gadd153/Chop10, forms a nonfunctional heterodimer with ATF3: the heterodimer, in contrast to the ATF3 homodimer, does not bind to the ATF/cyclic AMP response element consensus site and does not repress transcription. Interestingly, ATF3 and gadd153/Chop10 are expressed in inverse but overlapping manners during the livers response to carbon tetrachloride (CCl4): the level of gadd153/Chop10 mRNA is high in the normal liver and greatly decreases upon CCl4 treatment; the level of ATF3 mRNA, on the other hand, is low in the normal liver and greatly increases upon CCl4 treatment. We hypothesize that in nonstressed liver, gadd153/Chop10 inhibits the limited amount of ATF3 by forming an inactive heterodimer with it, whereas in CCl4-injured liver, the synthesis of gadd153/Chop10 is repressed, allowing the induced ATF3 to function.

Role for Activating Transcription Factor 3 in Stress-Induced β-Cell Apoptosis

ABSTRACT Activating transcription factor 3 (ATF3) is a stress-inducible gene and encodes a member of the ATF/CREB family of transcription factors. However, the physiological significance of ATF3 induction by stress signals is not clear. In this report, we describe several lines of evidence supporting a role of ATF3 in stress-induced β-cell apoptosis. First, ATF3 is induced in β cells by signals relevant to β-cell destruction: proinflammatory cytokines, nitric oxide, and high concentrations of glucose and palmitate. Second, induction of ATF3 is mediated in part by the NF-κB and Jun N-terminal kinase/stress-activated protein kinase signaling pathways, two stress-induced pathways implicated in both type 1 and type 2 diabetes. Third, transgenic mice expressing ATF3 in β cells develop abnormal islets and defects secondary to β-cell deficiency. Fourth, ATF3 knockout islets are partially protected from cytokine- or nitric oxide-induced apoptosis. Fifth, ATF3 is expressed in the islets of patients with type 1 or type 2 diabetes, and in the islets of nonobese diabetic mice that have developed insulitis or diabetes. Taken together, our results suggest ATF3 to be a novel regulator of stress-induced β-cell apoptosis.

ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in cancer cells

The ubiquitin-proteasome system has recently emerged as a major target for drug development in cancer therapy. The proteasome inhibitor bortezomib has clinical activity in multiple myeloma and mantle cell lymphoma. Here we report that Eeyarestatin I (EerI), a chemical inhibitor that blocks endoplasmic reticulum (ER)-associated protein degradation, has antitumor and biologic activities similar to bortezomib and can synergize with bortezomib. Like bortezomib, EerI-induced cytotoxicity requires the up-regulation of the Bcl-2 homology3 (BH3)-only pro-apoptotic protein NOXA. We further demonstrate that both EerI and bortezomib activate NOXA via an unanticipated mechanism that requires cooperation between two processes. First, these agents elicit an integrated stress response program at the ER to activate the CREB/ATF transcription factors ATF3 and ATF4. We show that ATF3 and ATF4 form a complex capable of binding to the NOXA promoter, which is required for NOXA activation. Second, EerI and bortezomib also block ubiquitination of histone H2A to relieve its inhibition on NOXA transcription. Our results identify a class of anticancer agents that integrate ER stress response with an epigenetic mechanism to induce cell death.

ATF4 degradation relies on a phosphorylation-dependent interaction with the SCF(betaTrCP) ubiquitin ligase.

ABSTRACT The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein βTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IκBα and β-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of βTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of βTrCP. The F-box-deleted βTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCFβTrCP complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.

ATF3 Gene GENOMIC ORGANIZATION, PROMOTER, AND REGULATION

ATF3 gene, which encodes a member of the activating transcription factor/cAMP responsive element binding protein (ATF/CREB) family of transcription factors, is induced by many physiological stresses. As a step toward understanding the induction mechanisms, we isolated the human ATF3 gene and analyzed its genome organization and 5′-flanking region. We found that the human ATF3 mRNA is derived from four exons distributed over 15 kilobases. Sequence analysis of the 5′-flanking region revealed a consensus TATA box and a number of transcription factor binding sites including the AP-1, ATF/CRE, NF-κB, E2F, and Myc/Max binding sites. As another approach to understanding the mechanisms by which the ATF3 gene is induced by stress signals, we studied the regulation of the ATF3 gene in tissue culture cells by anisomycin, an approach that has been used to study the stress responses in tissue culture cells. We showed that anisomycin at a low concentration activates the ATF3 promoter and stabilizes the ATF3 mRNA. Significantly, co-transfection of DNAs expressing ATF2 and c-Jun activates the ATF3 promoter. A possible mechanism implicating the C-Jun NH-terminal kinase/stress-activated protein kinase (JNK/SAPK) stress-inducible signaling pathway in the induction of the ATF3 gene is discussed.

Activating Transcription Factor 3, a Stress-inducible Gene, Suppresses Ras-stimulated Tumorigenesis *

ATF3 is a stress-inducible gene that encodes a member of the ATF/CREB family of transcription factors. Current literature indicates that ATF3 affects cell death and cell cycle progression. However, controversies exist, because it has been demonstrated to be a negative or positive regulator of these processes. We sought to study the roles of ATF3 in both cell death and cell cycle regulation in the same cell type using mouse fibroblasts. We show that ATF3 promotes apoptosis and cell cycle arrest. Fibroblasts deficient in ATF3 (ATF3-/-) were partially protected from UV-induced apoptosis, and fibroblasts ectopically expressing ATF3-/- under the tet-off system exhibited features characteristic of apoptosis upon ATF3 induction. Furthermore, ATF3-/- fibroblasts transitioned from G2 to S phase more efficiently than the ATF3+/+ fibroblasts, suggesting a growth arrest role of ATF3. Consistent with the growth arrest and pro-apoptotic roles of ATF3, ATF3- fibroblasts upon Ras transformation exhibited higher growth rate, produced more colonies in soft agar, and formed larger tumor upon xenograft injection than the ATF3+/+ counterparts. ATF3-/- cells, either with or without Ras transformation, had increased Rb phosphorylation and higher levels of various cyclins. Significantly, ATF3 bound to the cyclin D1 promoter as shown by chromatin immunoprecipitation (ChIP) assay and repressed its transcription by a transcription assay. Taken together, our results indicate that ATF3 promotes cell death and cell arrest, and suppresses Ras-mediated tumorigenesis. Potential explanations for the controversy about the roles of ATF3 in cell cycle and cell death are discussed.