关键词： ELEMENT-BINDING PROTEIN   ACTIVITY-DEPENDENT NEUROPROTECTION   TRANSCRIPTION FACTOR   HUNTINGTONS-DISEASE   ALZHEIMERS-DISEASE   KINASE-IV   HIPPOCAMPAL-NEURONS   CEREBRAL-ISCHEMIA   CREB   CALCIUM  

摘要： Synapse-to-nucleus signaling triggered by synaptic NMDA receptors can lead to the buildup of a neuroprotective shield. Nuclear calcium activating the cAMP response element binding protein (CREB) plays a key role in neuroprotection acquired by synaptic activity. Here we show that in mouse hippocampal neurons, the transcription factor Atf3 (activating transcription factor 3) is a direct target of CREB. Induction of ATF3 expression by CREB in hippocampal neurons was initiated by calcium entry through synaptic NMDA receptors and required nuclear calcium transients and calcium/calmodulin-dependent protein kinase IV activity. Acting as a transcriptional repressor, ATF3 protects cultured hippocampal neurons from apoptosis and extrasynaptic NMDA receptor-induced cell death triggered by bath application of NMDA or oxygen-glucose deprivation. Expression of ATF3 in vivo using stereotaxic delivery of recombinant adeno-associated virus reduces brain damage following a cerebral ischemic insult in mice. Conversion of ATF3 to a transcriptional activator transforms ATF3 into a potent prodeath protein that kills neurons in cell culture and, when expressed in vivo in the hippocampus, ablates the neuronal cell layer. These results link nuclear calcium-CREB signaling to an ATF3-mediated neuroprotective gene repression program, indicating that activity-dependent shutoff of genes is an important process for survival. ATF3 supplementation may counteract age- and disease-related neuronal cell loss caused by a reduction in synaptic activity, malfunctioning of calcium signaling toward and within the nucleus ("nuclear calciopathy"), or increases in death signaling by extrasynaptic NMDA receptors.

关键词： ACTIVATING TRANSCRIPTION FACTOR-3   VIRUS TYPE-1 HTLV-1   RESPONSE GENE ATF3   STRESS-RESPONSE   TAX PROTEIN   P53 GENE   C-JUN   HBZ   CANCER   ELEMENT  

摘要： Background: Adult T-cell leukemia (ATL) is an aggressive malignancy of CD4(+) T-cells caused by human T-cell leukemia virus type 1 (HTLV-1). The HTLV-1 bZIP factor (HBZ) gene, which is encoded by the minus strand of the viral genome, is expressed as an antisense transcript in all ATL cases. By using yeast two-hybrid screening, we identified activating transcription factor 3 (ATF3) as an HBZ interacting protein. ATF3 has been reported to be expressed in ATL cells, but its biological significance is not known. Results: Immunoprecipitation analysis confirmed that ATF3 interacts with HBZ. Expression of ATF3 was upregulated in ATL cell lines and fresh ATL cases. Reporter assay revealed that ATF3 could interfere with the HTLV-1 Tax's transactivation of the 5' proviral long terminal repeat (LTR), doing so by affecting the ATF/CRE site, as well as HBZ. Suppressing ATF3 expression inhibited proliferation and strongly reduced the viability of ATL cells. As mechanisms of growth-promoting activity of ATF3, comparative expression profiling of ATF3 knockdown cells identified candidate genes that are critical for the cell cycle and cell death, including cell division cycle 2 (CDC2) and cyclin E2. ATF3 also enhanced p53 transcriptional activity, but this activity was suppressed by HBZ. Conclusions: Thus, ATF3 expression has positive and negative effects on the proliferation and survival of ATL cells. HBZ impedes its negative effects, leaving ATF3 to promote proliferation of ATL cells via mechanisms including upregulation of CDC2 and cyclin E2. Both HBZ and ATF3 suppress Tax expression, which enables infected cells to escape the host immune system.

关键词： amyotrophic lateral sclerosis   astrocyte   ATF3   brain stem   motoneuron disease   neuroprotection   AMYOTROPHIC-LATERAL-SCLEROSIS   PERSISTENT INWARD CURRENTS   MOTOR-NEURON DISEASE   SPINAL-CORD   S-100 PROTEIN   ALS   ASTROCYTES   TRANSMISSION   RECEPTORS   DAMAGE  

摘要： Excitotoxic damage to motoneurons is thought to be an important contribution to the pathogenesis of amyotrophic lateral sclerosis (ALS), a slowly developing degeneration of motoneurons that, in most cases of sporadic occurrence, is associated with impaired glial glutamate uptake. Riluzole is the only drug licensed for symptomatic ALS treatment and is proposed to delay disease progression. As riluzole is administered only after full ALS manifestation, it is unclear if its early use might actually prevent motoneuron damage. We explored this issue by using, as a simple in vitro model, hypoglossal motoneurons (a primary target of ALS) of the neonatal rat brainstem slice preparation exposed to excitotoxic stress due to glutamate uptake block by DL-threo-beta-benzyloxyaspartate (TBOA). TBOA evoked sustained network bursting, early (1 h) enhancement of the S100B immunostaining of gray matter astrocytes, and activated the motoneuronal stress ATF-3 transcription factor; 4 h later, loss (30%) of motoneuron staining ensued and pyknosis appeared. Riluzole (5 mu m; applied 15 min after TBOA) inhibited bursting, decreased the frequency of spontaneous glutamatergic events, reversed changes in S100B immunostaining and prevented late loss of motoneuron staining. These results show that excitotoxicity induced by glutamate uptake block developed slowly, and was sensed by glia and motoneurons with delayed cell death. Our data provide novel evidence for the neuroprotective action of riluzole on motoneurons and glia when applied early after an excitotoxic stimulus.

关键词： Angiotensin   Signal transduction   ATF3   Left atrium   NATRIURETIC-PEPTIDE GENE   TYPE-2 RECEPTOR   TRANSGENIC MICE   AT(1) RECEPTOR   GROWTH-FACTOR   AT2 RECEPTOR   EXPRESSION   FIBRILLATION   PRESSURE   ACE  

摘要： The atria respond to various pathological stimuli including pressure and volume overload with remodeling and dilatation. Dilatation of the left atrium is associated with atrial fibrillation. The mechanisms involved in chamber-specific hypertrophy are largely unknown. Angiotensin II is hypothesized to take part in mediating this response. ATF3 is an immediate early gene found at the receiving end of multiple stress and growth stimuli. Here we characterize ATF3 as a direct target gene for angiotensin II. ATF3 expression is regulated by angiotensin receptor-mediated signaling in vivo and in vitro at the transcriptional level. ATF3 induction is mediated by cooperation between both the AT(1A) and AT(2) receptor subtypes. While AT(2)R blocker (PD123319) efficiently blocks ATF3 induction in response to angiotensin II injection, it results in an increase in blood pressure indicating that the effect of angiotensin II on ATF3 is independent of its effect on blood pressure. In contrast to adrenergic stimulation that induces ATF3 in all heart chambers, ATF3 induction in response to angiotensin II occurs primarily in the left chambers. We hypothesize that the activation of differential signaling pathways accounts for the chamber-specific induction of ATF3 expression in response to angiotensin II stimulation. Angiotensin II injection rapidly activates the EGFR-dependent pathways including ERK and PI3K-AKT in the left but not the right atrium. EGF receptor inhibitor (Gefitinib/Iressa) as well as the AKT inhibitor (Triciribine) significantly abrogates ATF3 induction by angiotensin II in the left chambers. Collectively, our data strongly place ATF3 as a unique nuclear protein target in response to angiotensin II stimulation in the atria. The spatial expression of ATF3 may add to the understanding of the signaling pathways involved in cardiac response to neuro-hormonal stimulation, and in particular to the understanding of left atrial-generated pathology such as atrial fibrillation.

关键词： ATF3   detrusor   ganglia   growth   obstruction   p-c-Jun   urinary bladder   RAT URINARY-BLADDER   CONNECTIVE-TISSUE   SMOOTH-MUSCLE   TRANSCRIPTION   NERVE   HYPERTROPHY   MECHANISMS  

摘要： Objective. Infravesical obstruction leads to growth of urinary bladder smooth-muscle cells. The ganglion cells innervating the bladder muscle also increase in size. Stretch of detrusor muscle cells rapidly activates c-Jun NH2-terminal kinase (JNK), which phosphorylates the transcription factor c-Jun, and stimulates the synthesis of the cotranscription factor ATF3. The aim of the study was to determine whether ATF3 and p-c-Jun were involved in growth of bladder smooth-muscle and ganglion cells. Material and methods. The urethra was partially obstructed in female rats. After 3 days or 10 weeks bladders were weighed, fixated and cut for immunohistochemistry to demonstrate ATF3 and p-c-Jun. Ganglia were processed separately. Unoperated and sham-operated rats were used as controls. Results. There was no ATF3 or p-c-Jun in control detrusor muscle. After 3 days of obstruction bladder weight had nearly doubled. Almost all nuclei in the detrusor showed immunofiuorescence for ATF3 and p-c-Jun. After 10 weeks bladder weight had increased 10-fold. Almost all detrusor nuclei still showed p-c-Jun, but few had ATF3 activity. In control ganglia there was no ATF3 and only faint nuclear p-c-Jun activity. After 3 days of obstruction the ganglion cells had increased in size and many nuclei showed intense immunofluorescence for ATF3 and p-c-Jun. After 10 weeks the ganglion cell size had increased further. There was no ATF3 activity and no more p-c-Jun than in control ganglia. Conclusion. ATF3 and p-c-Jun seem to be involved in the growth of the detrusor muscle and its motor innervation following infravesical outlet obstruction.

关键词： TRANSCRIPTION FACTOR ATF3   STRESS-INDUCIBLE GENE   BETA-CATENIN   BREAST-CANCER   SIGNALING PATHWAY   INDUCED APOPTOSIS   CYCLIN D1   C-JUN   COLORECTAL-CANCER   EPITHELIAL-CELLS  

摘要： Female transgenic mice that constitutively overexpress the transcription factor ATF3 in the basal epithelium of the mammary gland develop mammary carcinomas with high frequency, but only if allowed to mate and raise pups early in life. This transgenic mouse model system reproduces some features of human breast cancer in that about 20% of human breast tumor specimens exhibit overexpression of ATF3 in the tumor cells. The ATF3-induced mouse tumors are phenotypically similar to mammary tumors induced by overexpression of activating Wnt/beta-catenin pathway genes. We now show that the Wnt/beta-catenin pathway is indeed activated in ATF3-induced tumors. beta-catenin is transcriptionally up-regulated in the tumors, and high levels of nuclear beta-catenin are seen in tumor cells. A reporter gene for Wnt/beta-catenin pathway activity, TOPGAL, is up-regulated in the tumors and several downstream targets of Wnt signaling, including Ccnd1, Jun, Axin2 and Dkk4, are also expressed at higher levels in ATF3-induced tumors compared to mammary glands of transgenic females. Several positive-acting ligands for this pathway, including Wnt3, Wnt3a, Wnt7b, and Wnt5a, are significantly overexpressed in tumor tissue, and mRNA for Wnt3 is about 5-fold more abundant in transgenic mammary tissue than in non-transgenic mammary tissue. Two known transcriptional targets of ATF3, Snai1 and Snai2, are also overexpressed in the tumors, and Snail and Slug proteins are found to be located primarily in the nuclei of tumor cells. In vitro knockdown of Atf3 expression results in significant decreases in expression of Wnt7b, Tcf7, Snai2 and Jun, suggesting that these genes may be direct transcriptional targets of ATF3 protein. By chromatin immunoprecipitation analysis, both ATF3 and JUN proteins appear to bind to a particular subclass of AP-1 sites upstream of the transcriptional start sites of each of these genes.

关键词： Complementary antibody pairs   Hybridoma antibody screening   Biomarker   Akt2   ATF3   NAE beta   AlphaScreen  

摘要： The utility of antibody reagents for the detection of specific cellular targets for both research and diagnostic applications is widespread and continually expanding. Often it is useful to develop specific antibodies as reagent pairs that distinguish different epitopes of the target such that sandwich enzyme-linked immunosorbent assay can be used for selective and specific detection. However, the identification of pairing antibodies is often cumbersome and labor-intensive even with the use of designed peptide-specific epitopes as antigens. We have developed a robust and high-throughput method for identifying pairing complementary antibodies derived either from commercial sources or during a rabbit hybridoma monoclonal screening and selection process using protein A capture with the AlphaScreen bead-based assay format. We demonstrate the value and effectiveness of this assay with three protein targets: Akt2, ATF3, and NAE beta (the beta-subunit of the neddylation activation enzyme). (c) 2010 Elsevier Inc. All rights reserved.

关键词： ATF3   VSMCs   apoptosis   migration   MATRIX METALLOPROTEINASES   RECEPTOR TRANSACTIVATION   REPRESSOR ATF3/LRF1   ATF3   EXPRESSION   DEATH   APOPTOSIS   PROLIFERATION   INHIBITION   INVASION  

摘要： Activating transcription factor 3 (ATF3) is a member of the ATF/CREB (CAMP responsive element binding protein) family of transcription factors. The expression and the function of ATF3 in vascular smooth muscle cells (VSMCs) remain unknown. The aim of this work is to determine the expression and possible function of ATF3 in VSMCs. We found that VSMCs expressed ATF3, and expression of ATF3 in VSMCs was induced by a variety of stimuli including serum, angiotensin II, and H2O2. Knockdown of ATF3 induced apoptosis of VSMCs, caspase-3 cleavage, and cytochrome c release. The results suggest that ATF3 regulates survivability of VSMCs. Moreover, we found that overexpression of ATF3 promoted migration of VSMCs and induced expression of matrix metalloproteinase 1, 3, and 13. These results suggest that ATF3 plays a role in regulating migration of VSMCs. In addition, we found that the expression of ATF3 was upregulated in smooth muscle cells in the injured mouse femoral arteries compared with the uninjured control group. These results suggest that ATF3 is relevant to disease physiology. (C) 2011 IUBMB IUBMB Life, 63(1): 62-69, 2011

关键词： Copper   HNF4 alpha   HepG2 cell   Transcription   p53   Gene expression   ATF3   GENE-EXPRESSION   WILSON-DISEASE   DNA-BINDING   P53   MECHANISMS   TOXICITY   FACTOR-4-ALPHA   INTERACTS   SUPPRESS   METALS  

摘要： Previous global transcriptome and interactome analyses of copper-treated HepG2 cells identified hepatocyte nuclear factor 4 alpha (HNF4 alpha) as a potential master regulator of copper-responsive transcription. Copper exposure caused a decrease in the expression of HNF4 alpha at both mRNA and protein levels, which was accompanied by a decrease in the level of HNF4 alpha binding to its consensus DNA binding sequence. qRT-PCR and RNAi studies demonstrated that changes in HNF4 alpha expression ultimately affected the expressions of its down-stream target genes. Analysis of upstream regulators of HNF4 alpha expression, including p53 and ATF3, showed that copper caused an increase in the steady-state levels of these proteins. These results support a model for copper-responsive transcription in which the metal affects ATF3 expression and stabilizes p53 resulting in the down-regulation of HNF4 alpha expression. In addition, copper may directly affect p53 protein levels. The suppression of HNF4 alpha activity may contribute to the molecular mechanisms underlying the physiological and toxicological consequences of copper toxicity in hepatic-derived cells. Published by Elsevier B.V.

关键词： activating transcription factor 3 ( ATF3 )   ATF3   Copper   Gene expression   hepatocyte nuclear factor 4α ( HNF4α )   HepG2 cell   HNF4α   ingenuity pathway analysis ( IPA )   p53   quantitative real-time PCR ( qRT-PCR )   RNA interference ( RNAi )   small interfering RNA ( siRNA )   sterol regulatory-element binding protein 2 ( SREBP-2 )   Transcription  

摘要： Abstract: Previous global transcriptome and interactome analyses of copper-treated HepG2 cells identified hepatocyte nuclear factor 4α (HNF4α) as a potential master regulator of copper-responsive transcription. Copper exposure caused a decrease in the expression of HNF4α at both mRNA and protein levels, which was accompanied by a decrease in the level of HNF4α binding to its consensus DNA binding sequence. qRT-PCR and RNAi studies demonstrated that changes in HNF4α expression ultimately affected the expressions of its down-stream target genes. Analysis of upstream regulators of HNF4α expression, including p53 and ATF3, showed that copper caused an increase in the steady-state levels of these proteins. These results support a model for copper-responsive transcription in which the metal affects ATF3 expression and stabilizes p53 resulting in the down-regulation of HNF4α expression. In addition, copper may directly affect p53 protein levels. The suppression of HNF4α activity may contribute to the molecular mechanisms underlying the physiological and toxicological consequences of copper toxicity in hepatic-derived cells. [ABSTRACT FROM AUTHOR]