Activating transcription factor 2 (ATF2) negatively regulates the expression of antimicrobial peptide genes through tumor necrosis factor (TNF) in Macrobrachium nipponense.

Activating transcription factor 2 (ATF2), a member of the bZIP transcription factor family, is involved in multiple physiological and developmental processes, yet its role in the innate immunity remains unclear. In this study, two isoforms (named as MnATF2a and MnATF2b) of ATF2 gene were identified in Macrobrachium nipponense and were produced by exon skipping. The full length of MnATF2a is 2328 bp with an open reading frame of 2079 bp that encode 692 amino acids. MnATF2a has 237 bp nucleotides more than MnATF2b and the extra 237 bp is a complete exon. MnATF2a and MnATF2b proteins contain the same conserved and typical bZIP domain at the C-terminus. MnATF2a has 79 amino acids more than MnATF2b. MnATF2a and MnATF2b are widely distributed in a variety of immune tissues. After Vibrio parahaemolyticus and Staphylococcus aureus infection, the expression levels of MnATF2a and MnATF2b were significant up-regulated in the gills and stomach at 12 h. RNA interference analysis showed that knockdown of the total MnATF2 gene significantly inhibits the transcription of tumor necrosis factor (TNF) and promotes the expression of crustins (including Cru3, Cru4, and Cru7). Further study showed that knockdown of MnTNF evidently increase the expression of Cru3, Cru4, and Cru7. Our research indicates that ATF2 negatively regulate the expression of AMPs by regulating the transcription of TNF in M. nipponense. This study provides valuable information about the function of ATF2 family in the innate immunity in crustacean.

SpATF2 participates in maintaining the homeostasis of hemolymph microbiota by regulating dual oxidase expression in mud crab.

Activating transcription factors 2 (ATF2) is a transcription factor of the members of ATF/CREB family that is phosphorylated and activated by the mitogen-activated protein kinase (MAPK) in responding to the stimulation of stimuli. In present study, SpATF2 from mud crab (Scylla paramamosain) was identified and studied. The open reading frame of SpATF2 with 2136 bp in length encodes a protein with 711 amino acids. The SpATF2 protein includes the putative zinc finger domain in the N-terminus and bZIP type DNA-binding domain in the C-terminal. Tissue distribution of SpATF2 transcripts showed that SpATF2 was ubiquitously expressed in all examined tissues of the untreated mud crabs, with the highest expression levels in muscle and hepatopancreas. The transcriptional level of SpATF2 was up-regulated in the hemocytes after Vibrio parahemolyticus or WSSV infection. Reporter gene assays indicated that SpATF2 could activate the expression of dual oxidase (SpDuox1) in S. paramamosain. The RNA interference (RNAi) of SpATF2 significantly decreased the expression of SpDuox1, and consequently reduced reactive oxygen species production thereby significantly increased the bacterial load in the hemolymph of mud crabs. Similarly, significant reduction in bacterial clearance of hemolymph was observed after the V. parahemolyticus infection in SpATF2 knockdown mud crabs. This study showed that SpATF2 played a vital role in maintaining homeostasis of the hemolymph microbiota through regulating the expression of dual oxidase of mud crab.

Anti-inflammatory effect of Vaccinium oldhamii stems through inhibition of NF-κB and MAPK/ATF2 signaling activation in LPS-stimulated RAW264.7 cells.

BACKGROUND: Vaccinium oldhamii (V. oldhamii) has been reported to exert a variety of the pharmacological properties such as anti-oxidant activity, anti-cancer activity, and inhibitory activity of α-amylase and acetylcholinesterase. However, the anti-inflammatory activity of V. oldhamii has not been studied. In this study, we aimed to investigate anti-inflammatory activity of the stem extracts from V. oldhamii, and to elucidate the potential mechanisms in LPS-stimulated RAW264.7 cells. METHODS: Cell viability was evaluated by MTT assay. The determination of NO and PGE2 production was performed using Griess reagent and Prostaglandin E2 ELISA Kit, respectively. The change of mRNA or protein level was evaluated by RT-PCR and Western blot. RESULTS: Among VOS, VOL and VOF, the inhibitory effect of NO and PGE2 production induced by LPS was highest in VOS treatment. Thus, VOS was selected for the further study. VOS dose-dependently blocked LPS-induced NO and PGE2 production by inhibiting iNOS and COX-2 expression, respectively. VOS inhibited the expression of pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α. In addition, VOS suppressed TRAP activity and attenuated the expression of the osteoclast-specific genes such as NFATc1, c-FOS, TRAP, MMP-9, cathepsin K, CA2, OSCAR and ATPv06d2. VOS inhibited LPS-induced NF-κB signaling activation through blocking IκB-α degradation and p65 nuclear accumulation. VOS inhibited MAPK signaling activation by attenuating the phosphorylation of ERK1/2, p38 and JNK. Furthermore, VOS inhibited ATF2 phosphorylation and blocked ATF2 nuclear accumulation. CONCLUSIONS: These results indicate that VOS may exert anti-inflammatory activity by inhibiting NF-κB and MAPK/ATF2 signaling. From these findings, VOS has potential to be a candidate for the development of chemopreventive or therapeutic agents for the inflammatory diseases.

Elevated Cyclic AMP Inhibits Mycobacterium tuberculosis-Stimulated T-cell IFN-γ Secretion Through Type I Protein Kinase A.

Cyclic adenosine monophosphate (cAMP) is critical in immune regulation, and its role in tuberculosis infection remains unclear. We determined the levels of cAMP in peripheral blood mononuclear cells (PBMC) from tuberculosis patients and the mechanisms for cAMP suppression of IFN-γ production. PBMC from tuberculosis patients contained significantly elevated cAMP than latent tuberculosis infected subjects (LTBI), with an inverse correlation with IFN-γ production. Consistent with this, the expression of cAMP response element binding protein (CREB), activating transcription factor (ATF)-2 and c-Jun were reduced in tuberculosis patients compared with LTBI. PKA type I specific cAMP analogs inhibited Mtb-stimulated IFN-g production by PBMC through suppression of Mtb-induced IFN-γ promoter binding activities of CREB, ATF-2, and c-Jun and also miR155, the target miRNA of these transcription factors. Neutralizing both IL-10 and TGF-ß1 or supplementation of IL-12 restored cAMP-suppressed IFN-g production. We conclude that increased cAMP inhibits IFN-g production through PKA type I pathway in tuberculosis infection.

Generation of recombinant affinity reagents against a two-phosphosite epitope of ATF2.

Activating Transcription Factor 2 (ATF2) plays an important role in mammalian cell proliferation, apoptosis and DNA repair. Its activation is dependent on the sequential phosphorylation of residue threonine 71 (T71) followed by threonine 69 (T69) in its transactivation domain. While these modifications can be directed by a variety of kinases, the time to reach full phosphorylation is dependent on which signaling pathway has been activated, which is thought to be important for proper temporal regulation. To explore this phenomenon further, there have been ongoing efforts to generate affinity reagents for monitoring phosphorylation events in cellular assays. While phospho-specific antibodies have been valuable tools for monitoring cell signaling events, those raised against a peptide containing two or more adjacent phosphosites tend to cross-react with that peptide's various phospho-states, rendering such reagents unusable for studying sequential phosphorylation. As an alternative, we have employed the N-terminal Forkhead-associated 1 (FHA1) domain of yeast Rad53p as a scaffold to generate recombinant affinity reagents via phage display and were successful in generating a set of reagents that can distinguish between the dual-phosphorylated epitope, 63-IVADQpTPpTPTRFLK-77, and the mono-phosphorylated epitope, 63-IVADQpTPTPTRFLK-77, in the human ATF2 transactivation domain.

Vaginal LPS changed gene transcriptional regulation response to ischemic reperfusion and increased vulnerability of fetal brain hemorrhage.

During pregnancy, both ischemic reperfusion and bacterial agent LPS are known risk factors for fetal brain damage. However, there is a lack of evidence to explain whether vaginal LPS affects the fetus response to ischemic reperfusion. Here we reported that there was more than 2 folds higher vulnerability of fetal brain hemorrhage response to ischemic reperfusion when mother mouse was treated with vaginal LPS. As our previously reported, ischemic reperfusion induces P53-dependent fetal brain damage was based on a molecular mechanism: the transcriptional pattern was changed from HIF-1alpha-dependent to P53-dependent immediately. In the present work, only with vaginal LPS precondition, phosphorylation of activated transcriptional factor (ATF) 2 at Thr71 appeared in response to ischemic reperfusion. Moreover, this phosphorylation was completely blocked by pre-treatment with a P53 inhibitor, pifithrin-α. We concluded that vaginal LPS precondition trigged the p53-dependent phosphorylation of ATF2 in response to ischemic reperfusion, which played an important role of increasing vulnerability to hemorrhage in fetus.

Human macrophage SCN5A activates an innate immune signaling pathway for antiviral host defense.

Pattern recognition receptors contain a binding domain for pathogen-associated molecular patterns coupled to a signaling domain that regulates transcription of host immune response genes. Here, a novel mechanism that links pathogen recognition to channel activation and downstream signaling is proposed. We demonstrate that an intracellular sodium channel variant, human macrophage SCN5A, initiates signaling and transcription through a calcium-dependent isoform of adenylate cyclase, ADCY8, and the transcription factor, ATF2. Pharmacological stimulation with a channel agonist or treatment with cytoplasmic poly(I:C), a mimic of viral dsRNA, activates this pathway to regulate expression of SP100-related genes and interferon ß. Electrophysiological analysis reveals that the SCN5A variant mediates nonselective outward currents and a small, but detectable, inward current. Intracellular poly(I:C) markedly augments an inward voltage-sensitive sodium current and inhibits the outward nonselective current. These results suggest human macrophage SCN5A initiates signaling in an innate immune pathway relevant to antiviral host defense. It is postulated that SCN5A is a novel pathogen sensor and that this pathway represents a channel activation-dependent mechanism of transcriptional regulation.

Activating transcription factor 2 in mesenchymal tumors.

Activating transcription factor 2 (ATF2) is a member of activator protein 1 superfamily, which can heterodimerize with other transcription factors regulating cell differentiation and survival. ATF2 assembles into a complex with the synovial sarcoma translocation, chromosome 18 (SS18)-synovial sarcoma, X breakpoint (SSX) fusion oncoprotein, and the transducin-like enhancer of split 1 (TLE1) corepressor, driving oncogenesis in synovial sarcoma. The fusion oncoproteins in many other translocation-associated sarcomas incorporate transcription factors from the ATF/cAMP response element binding or E26 families, which potentially form heterodimers with ATF2 to regulate transcription. ATF2 may therefore play an important role in the oncogenesis of many mesenchymal tumors, but as yet, little is known about its protein expression in patient specimens. Herein we perform immunohistochemical analyses using a validated specific antibody for ATF2 expression and intracellular localization on a cohort of 594 malignant and 207 benign mesenchymal tumors representing 47 diagnostic entities. Melanoma served as a positive control for nuclear and cytoplasmic staining. High nuclear ATF2 expression was mainly observed in translocation-associated and/or spindle cell sarcomas including synovial sarcoma, desmoplastic small round cell tumor, endometrial stromal sarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor, and solitary fibrous tumor. Cytoplasmic ATF2 expression was less frequently seen than nuclear expression in malignant mesenchymal tumors. Benign mesenchymal tumors mostly showed much lower nuclear and cytoplasmic ATF2 expression.

Inducible cAMP early repressor (ICER) is a novel regulator of RIG-I mediated IFN-ß production.

Antiviral responses can be triggered by the cytoplasmic RNA helicase RIG-I that binds to viral RNA. RIG-I-mediated signaling stimulates the transcription factors IRF3 and NF-κB and their activation mechanisms have been intensively studied. Here we examined Sendai virus (SV)-mediated activation of the transcription factor CREB and the role of its feedback repressor ICER in production of endogenous antiviral proteins. We show that SV infection and the mitochondrial adapter protein MAVS promote CREB phosphorylation that is dependent upon p38 MAPK and MK2. ICER is induced by CREB and acts as a feedback repressor of CRE-dependent transcription. We found that SV infection stimulated induction of ICER mRNA and protein expression. Surprisingly, ectopic expression and siRNA-mediated knockdown of ICER revealed that ICER is a positive regulator of the production of antiviral IFN-ß and IP10 during SV infection. In contrast, ICER did not affect SV-elicited phosphorylation of IRF3, NF-κB or ATF2/c-Jun, transcription factors governing IFN-ß and IP10 synthesis. However, expression of ICER increased total IRF3 protein levels during SV infection. These results point to a novel role of ICER in antiviral immune signaling acting to increase levels of antiviral effectors.

Autocrine IL-8 and VEGF mediate epithelial-mesenchymal transition and invasiveness via p38/JNK-ATF-2 signalling in A549 lung cancer cells.

Soluble factors in tumour microenvironment play a major role in modulating the metastatic potential of cancer cells. Herein, we investigated the effect of autocrine cytokines and growth factors in the form of self-conditioned medium (CM) on A549 lung carcinoma cells. We demonstrated that CM induced morphological and molecular changes associated with epithelial-mesenchymal transition viz change in shape from cuboidal to spindle, actin cytoskeleton remodelling, upregulation of vimentin and downregulation of E-cadherin etc. These changes were accompanied with enhanced motility, invasion, anchorage-independent growth and anoikis-resistance. Amongst the different factors of CM, IL-8 and VEGF were found to play a major role in the CM-induced motility and invasion. In the intracellular signalling cascade, CM triggered phosphorylation of JNK and p38 which was associated with the CM-enhanced invasiveness. In CM-treated cells, activated p38 and JNK further activated ATF-2 (Activating Transcription Factor-2) and knock-down of ATF-2 abrogated the CM-induced invasiveness, suggesting the signal transduction along the p38/JNK-ATF-2 axis. Furthermore, neutralising IL-8 and VEGF in CM, significantly abrogated CM-induced phosphorylation of ATF-2. Conversely, exogenous addition of these individual cytokines in plain medium, increased the activation of ATF-2 and invasiveness marginally. However, when added in combination these cytokines (IL-8 and VEGF) resulted in drastic increase in ATF-2 phosphorylation and subsequent invasiveness suggesting their synergetic interplay in the observed phenomenon. Taken together, our results identify IL-8/VEGF induced JNK/p38-ATF-2 as a novel pro-invasive pathway, which may be explored as potential therapeutic target to circumvent the invasiveness of lung malignancies.

HangAmDan-B, an ethnomedicinal herbal mixture, suppresses inflammatory responses by inhibiting Syk/NF-κB and JNK/ATF-2 pathways.

HangAmDan-B (HAD-B) is a powdered mixture of eight ethnopharmacologically characterized folk medicines that is prescribed for solid masses and cancers in Korea. In view of the finding that macrophage-mediated inflammation is a pathophysiologically important phenomenon, we investigated whether HAD-B modulates inflammatory responses and explored the associated molecular mechanisms. The immunomodulatory activity of HAD-B in toll-like receptor-activated macrophages induced by lipopolysaccharide (LPS) was assessed by measuring nitric oxide (NO) and prostaglandin E(2) (PGE(2)) levels. To identify the specific transcription factors (such as nuclear factor [NF]-κB and signaling enzymes) targeted by HAD-B, biochemical approaches, including kinase assays and immunoblot analysis, were additionally employed. HAD-B suppressed the production of PGE(2) and NO in LPS-activated macrophages in a dose-dependent manner. Furthermore, the extract ameliorated HCl/EtOH-induced gastritis symptoms. Moreover, HAD-B significantly inhibited LPS-induced mRNA expression of inducible NO synthase and cyclooxygenase (COX)-2. Interestingly, marked inhibition of NF-κB and activating transcription factor was observed in the presence of HAD-B. Data from direct kinase assays and immunoblot analysis showed that HAD-B suppresses activation of the upstream signaling cascade involving spleen tyrosine kinase, Src, p38, c-Jun N-terminal kinase, and transforming growth factor ß-activated kinase 1. Finally, kaempferol, but not quercetin or resveratrol was identified as a bioactive compound in HAD-B. Therefore, our results suggest that HAD-B possesses anti-inflammatory activity that contributes to its anticancer property.

Autoantibody against activating transcription factor-2 in patients with systemic sclerosis.

OBJECTIVES: To determine the prevalence and clinical correlation of autoantibody to activating transcription factor (ATF)-2, a transcription factor of ATF/CREB family, in patients with systemic sclerosis (SSc). METHODS: Anti-ATF-2 Ab was examined by ELISA and immunoblotting using human recombinant ATF-2. ATF-2 activity to bind target DNA was evaluated by ELISA using a plate coated with oligonucleotide containing the consensus binding site for ATF-2. RESULTS: IgG anti-ATF-2 Ab levels in SSc patients (n=69) were significantly higher than those in normal controls (n=26). SSc patients positive for IgG anti-ATF-2 Ab had significantly longer disease duration, more frequent presence of decreased %VC and %DLco, and elevated levels of serum IgG, serum IgA, and erythrocyte sedimentation rates than those negative. More-over, IgG anti-ATF-2 Ab levels correlated inversely with %VC or %DLco. The presence of anti-ATF-2 Ab in SSc patients was confirmed by immunoblotting analysis. IgG isolated from serum samples of SSc patients positive for IgG anti-ATF-2 Ab by ELISA slightly but significantly inhibited ATF-2 activity compared with normal controls. CONCLUSIONS: These results suggest that anti-ATF-2 Ab is a new autoantibody in SSc and that it serves as a novel serological marker for inflammation and lung involvement in SSc.

AP-1 activated by toll-like receptors regulates expression of IL-23 p19.

Interleukin (IL)-23, a new member of the IL-12 family, plays a central role in the Th17 immune response and in autoimmune diseases. It is clear that activated macrophages and dendritic cells produce IL-23, but the molecular mechanisms whereby inflammatory signals stimulate IL-23 expression are not fully understood. We demonstrate that induction of IL-23 p19 gene expression by LPS depends on the TLR4 and MyD88 pathways. All three MAPK pathways (ERK, JNK, and p38) that are activated by lipopolysaccharide (LPS) stimulation were shown to exert a positive effect on p19 expression. We cloned a 1.3-kb putative p19 promoter and defined its transcription initiation sites by the 5'-rapid amplification of cDNA ends method. By analyzing IL-23 p19 promoter mutants, we have identified a promoter region (-413 to +10) that contains several important elements, including NF-kappaB and AP-1. In addition to NF-kappaB, we have demonstrated that the proximal AP-1 site is important for p19 promoter activation. Mutation of the AP-1 site resulted in the loss of p19 promoter activation. Electrophoretic mobility shift assay (EMSA) analysis showed that c-Jun and c-Fos bind to the AP-1 site, which was confirmed by a chromatin immunoprecipitation assay. Furthermore, co-transfection of c-Jun and ATF2 synergistically induced p19 promoter activation, and c-Jun and ATF2 formed a protein complex, demonstrated by co-immunoprecipitation. Finally, LPS-stimulated peritoneal macrophages from IL-10-deficient mice expressed significantly higher IL-23 p19 than macrophages from wild type mice, and the addition of recombinant IL-10 strongly inhibited LPS-induced p19 expression. Thus, this study suggests that MyD88-dependent Toll-like receptor signaling induces IL-23 p19 gene expression through both MAPKs and NF-kappaB.

House dust mite allergen Der f 2-induced phospholipase D1 activation is critical for the production of interleukin-13 through activating transcription factor-2 activation in human bronchial epithelial cells.

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Der f 2-induced interleukin (IL)-13 production. The major house dust mite allergen, Der f 2, increased PLD activity in human bronchial epithelial cells (BEAS-2B), and dominant negative PLD1 or PLD1 siRNA decreased Der f 2-induced IL-13 expression and production. Treatment of Der f 2 activated the phospholipase Cgamma (PLCgamma)/protein kinase Calpha (PKCalpha)/p38 MAPK pathway. Der f 2-induced PLD activation was attenuated by PLCgamma inhibitors (U73122 and PAO), PKCalpha inhibitors (RO320432 and GO6976), and p38 MAPK inhibitors (SB203580 and SB202190). These results indicate that PLCgamma, PKCalpha, and p38 MAPK act as upstream activators of PLD in Der f 2-treated BEAS-2B cells. Furthermore, expression and production of IL-13 increased by Der f 2 were also blocked by inhibition of PLCgamma, PKCalpha, or p38 MAPK, indicating that IL-13 expression and production are related to a PLCgamma/PKCalpha/p38 MAPK pathway. We found that activating transcription factor-2 (ATF-2) was activated by Der f 2 in BEAS-2B cells and activation of ATF-2 was controlled by PLD1. When ATF-2 activity was blocked with ATF-2 siRNA, Der f 2-induced IL-13 expression and production were decreased. Thus, ATF-2 might be one of the transcriptional factors for the expression of IL-13 in Der f 2-treated BEAS-2B cells. Taken together, PLD1 acts as an important regulator in Der f 2-induced expression and production of IL-13 through activation of ATF-2 in BEAS-2B cells.

IFN-beta production by TLR4-stimulated innate immune cells is negatively regulated by GSK3-beta.

TLR 4 stimulation of innate immune cells induces a MyD88-independent signaling pathway that leads to the production of IFN-beta. In this study, we demonstrate glycogen synthase kinase 3-beta (GSK3-beta) plays a fundamental role in this process. Suppression of GSK3-beta activity by either pharmacological inhibition, small interfering RNA-mediated gene silencing, or ectopic expression of a kinase-dead GSK3-beta mutant enhanced IFN-beta production by TLR4-stimulated macrophages. Conversely, ectopic expression of a constitutively active GSK3-beta mutant severely attenuated IFN-beta production. GSK3-beta was found to negatively control the cellular levels of the transcription factor c-Jun and its nuclear association with ATF-2. Small interfering RNA-mediated knockdown of c-Jun levels abrogated the ability of GSK3-beta inhibition to augment IFN-beta, demonstrating that the ability of GSK3 to control IFN-beta production was due to its ability to regulate c-Jun levels. The ability of GSK3 inhibition to control IFN-beta production was confirmed in vivo as mice treated with a GSK3 inhibitor exhibited enhanced systemic levels of IFN-beta upon LPS challenge. These findings identify a novel regulatory pathway controlling IFN-beta production by TLR4-stimulated innate immune cells.

Type I IFN induction in response to Listeria monocytogenes in human macrophages: evidence for a differential activation of IFN regulatory factor 3 (IRF3).

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-beta and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-kappaB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-beta gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-beta response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-beta response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.

ATF2 impairs glucocorticoid receptor-mediated transactivation in human CD8+ T cells.

Chronic inflammatory diseases often have residual CD8(+) T-cell infiltration despite treatment with systemic corticosteroids, which suggests divergent steroid responses between CD4(+) and CD8(+) cells. To examine steroid sensitivity, dexamethasone (DEX)-induced histone H4 lysine 5 (K5) acetylation and glucocorticoid receptor alpha (GCR alpha) translocation were evaluated. DEX treatment for 6 hours significantly induced histone H4 K5 acetylation in normal CD4(+) cells (P = .001) but not in CD8(+) cells. DEX responses were functionally impaired in CD8(+) compared with CD4(+) cells when using mitogen-activated protein kinase phosphatase (1 hour; P = .02) and interleukin 10 mRNA (24 hours; P = .004) induction as a readout of steroid-induced transactivation. Normal DEX-induced GCR alpha nuclear translocation and no significant difference in GCR alpha and GCR beta mRNA expression were observed in both T-cell types. In addition, no significant difference in SRC-1, p300, or TIP60 expression was found. However, activating transcription factor-2 (ATF2) expression was significantly lower in CD8(+) compared with CD4(+) cells (P = .009). Importantly, inhibition of ATF2 expression by small interfering RNA in CD4(+) cells resulted in inhibition of DEX-induced transactivation in CD4(+) cells. The data indicate refractory steroid-induced transactivation but similar steroid-induced transrepression of CD8(+) cells compared with CD4(+) cells caused by decreased levels of the histone acetyltransferase ATF2.

Development of a microsphere-based p38alpha MAP kinase no-wash assay.

A nonradioactive, microsphere-based, no-wash assay for the measurement of p38alpha mitogen-activated protein (MAP) kinase activity was established. In this assay, a glutathione-S-transferase activating transcription factor 2 (amino acids 19-96) fusion protein (GST-ATF-2) was used as substrate for p38alpha MAP kinase. The assay involves immobilization of GST-ATF-2 on glutathione-microspheres (GSH-microspheres), addition of test solution containing p38alpha MAP kinase and test compounds, and measurement of the respective substrate phosphorylation with the aid of a bi-phospho-specific antibody. The optimization of test conditions is described in this article. With an optimized standard protocol, p38alpha MAP kinase inhibitors were investigated and IC50 values were compared to those derived using known assays. This assay might be useful in testing drug candidates.

Arrested natural killer cell development associated with transgene insertion into the Atf2 locus.

Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1-specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.