摘要： Background: Coordinated chondrocyte proliferation and differentiation are required for normal endochondral bone growth. Transcription factors binding to the cyclicAMP response element (CRE) are known to regulate these processes. One member of this family, Activating Tanscription Factor 3 (ATF3), is expressed during skeletogenesis and acts as a transcriptional repressor, but the function of this protein in chondrogenesis is unknown. Results: Here we demonstrate that Atf3 mRNA levels increase during mouse chondrocyte differentiation in vitro and in vivo. In addition, Atf3 mRNA levels are increased in response to cytochalasin D treatment, an inducer of chondrocyte maturation. This is accompanied by increased Atf3 promoter activity in cytochalasin D-treated chondrocytes. We had shown earlier that transcription of the cell cycle genes cyclin D1 and cyclin A in chondrocytes is dependent on CREs. Here we demonstrate that overexpression of ATF3 in primary mouse chondrocytes results in reduced transcription of both genes, as well as decreased activity of a CRE reporter plasmid. Repression of cyclin A transcription by ATF3 required the CRE in the cyclin A promoter. In parallel, ATF3 overexpression reduces the activity of a SOX9-dependent promoter and increases the activity of a RUNX2-dependent promoter. Conclusion: Our data suggest that transcriptional induction of the Atf3 gene in maturing chondrocytes results in down-regulation of cyclin D1 and cyclin A expression as well as activation of RUNX2-dependent transcription. Therefore, ATF3 induction appears to facilitate cell cycle exit and terminal differentiation of chondrocytes.

关键词： shockwave   analgesic effect   nerve fiber   injury   regeneration  

摘要： Although extracorporeal shockwave has been applied in the treatment of various diseases, the biological basis for its analgesic effect remains unclear. Therefore, we investigated the dorsal root ganglion neurons of rats following shockwave exposure to the footpad to elucidate its effect on the peripheral nervous system. We used activating transcription factor 3 (ATF3) and growth-associated phosphoprotein (GAP-43) as markers for nerve injury and axonal regeneration, respectively. The average number of neurons inummoreactive for ATF3 increased significantly in the treated rats at all experimental time points, with 78.3% of those neurons also exhibiting immunoreactivity for GAP-43. Shockwave exposure induced injury of the sensory nerve fibers within the exposed area. This phenomenon may be linked to the desensitization of the exposure area, not the cause of pain, considering clinical research with a particular absence of painful adverse effect. Subsequent active axonal regeneration may account for the reinnervation of exposed area and the amelioration of the desensitization. (c) 2006 Elsevier B.V. All rights reserved.

关键词： intervertebral disc   immunohistochemistry   ATF3   GAP-43   dorsal root ganglion (DRG)   low back pain   nerve ingrowth  

摘要： Study Design. Using a retrograde tracing method and immunohistochemistry, we assessed the expression of activating transcription factor 3 (ATF3), a marker of nerve injury, and growth-associated protein 43 (GAP-43), a marker of axonal growth, in dorsal root ganglion (DRG) neurons innervating the lumbar intervertebral discs in rats. Objectives. To investigate ATF3 and GAP-43 expression in DRGs innervating the intervertebral discs after exposure of the nucleus pulposus to the outside of the anulus fibrosus. Summary of Background Data. Degeneration of lumbar intervertebral discs is considered as a cause of low back pain. We speculated that exposure of the nucleus pulposus to the outside of the anulus fibrosus may induce nerve injury and ingrowth into the disc. Methods. A neurotracer, Fluoro-Gold (F-G), was applied to the ventral aspect of L5-L6 intervertebral discs in 20 rats. The rats were classified into 2 groups: an NP group whose disc was punctured to expose the nucleus pulposus (n = 10) and a sham-operated group whose anulus fibrosus surface was scratched superficially (n = 10). Ten days after surgery, bilateral L1-L5 DRGs were processed for staining of ATF3 and GAP-43. Results. In the NP group, 13.9% +/- 2.9% of the F-G-labeled neurons innervating the discs were positive for ATF3, while 19.3% +/- 2.7% were positive for GAP-43. In contrast, in the sham-operated group, only 0.8% +/- 0.4% of the F-G-labeled neurons were positive for ATF3 while 7.4% +/- 1.7% were positive for GAP-43. The percentage of both ATF3-immunoreactive (IR) and GAP-43-IR neurons in the NP group was significantly higher than in the sham-operated group (P < 0.05). Conclusions. ATF3-IR and GAP-43-IR neurons were significantly increased in the NP group. These results suggested that exposure of the nucleus pulposus to the outside of the anulus fibrosus induced nerve injury and ingrowth growth into the discs. These findings may explain discogenic lower back pain in patients with lumbar disc degenerat

关键词： Molecular biology  

摘要： 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)-kappa B family of transcription factors. Promoter analysis of the putative ATF3-regulated gene cluster demonstrated an over-representation of closely apposed ATF3 and NF-kappa B 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-kappa B) 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-kappa B 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.

关键词： aTF3   DNA damage   p53   protein interaction   stress response   tumorigenesis   tumor suppressor  

摘要： ATF3 is a member of the ATF/CREB family of transcription factors involved in the cellular response to a large variety of stresses including DNA damage. However, neither the signaling leading to nor the biological significance of its induction upon stress is well understood. Although it is generally believed that ATF3 exerts its function in the stress response by regulating transcription, to date, only a limited number of target genes have been identified. We recently reported that ATF3 interacts with the tumor suppressor p53 to increase its stability in the genotoxic response. While providing the cell a general means of responding to diverse adverse environment cues, this mechanism confers ATF3 with an ability to promote tumor suppressor functions. Conversely, dysfunction of ATF3 impairs the p53-mediated cellular response to DNA damage, allowing cells to be readily transformed by oncogenes. Consistent with this notion is the observation of downregulated ATF3 expression in most human cancers. Therefore, our findings indicate that ATF3 intersects with p53-associated pathways ensuring genomic integrity. The ability of ATF3 to stabilize p53-induced pathways thus represents a means of effectively countering DNA damage caused by environmental insult the latter leading to oncogene activation and ultimately malignant transformation.

关键词： AP-1, activator protein-1   ATF3, activating transcription factor 3   C/EBP, CCAAT/enhancer-binding protein   ChIP, chromatin immunoprecipitation   CsA, cyclosporin A   EMSA, electrophoretic mobility shift assay   HDAC, histone deacetylase  

摘要： Expression of adiponectin decreases with obesity and insulin resistance. At present, the mechanisms responsible for negatively regulating adiponectin expression in adipocytes are poorly understood. In this investigation, we analyzed the effects of 5' serial deletion constructs on the murine adiponectin promoter. Here, we identified the repressor region located between -472 and -313 bp of the promoter. Removal of the putative nuclear factor of activated T-cells (NFATs) binding site increased the promoter activity, and overexpression of NFATc4 reduced the promoter activity. Treatment with the calcium ionophore A23187, an activator of NFAT, reduced mRNA as well as promoter activity. The binding of NFATc4 to the promoter was associated with increased recruitment of histone deacetylase 1 and reduced acetylation of histone H3 at the promoter site. In addition, binding of activating transcription factor 3 (ATF3) to the putative activator protein-1 site located adjacent to the NFAT binding site also repressed the promoter activity. Treatment with thapsigargin, an inducer of ATF3, reduced both mRNA and promoter activity. Importantly, the binding activities of NFATc4 and ATF3, increased significantly in white adipose tissues of ob/ob and db/db mice compared with controls. Taken together, this study demonstrates for the first time that NFATc4 and ATF3 function as negative regulators of adiponectin gene expression, which may play critical roles in downregulating adiponectin expression in obesity and type 2 diabetes.

关键词： AHPN   6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid   ATF3   activating transcription factor3   c9   t11-CLA   cis -9   trans -11 isomer of CLA   CHX   cycloheximide   CLA   conjugated linoleic acid   CRC   colorectal cancer   CREB   cAMP response element binding protein   GSK-3   glycogen synthase kinase-3   IGF-1   insulin-like growth factor-1   LA   linoleic acid   LBD   ligand binding domain   NAG-1   NSAID-activated gene-1   NSAIDs   non-steroidal anti-inflammatory drugs   PI3K   phosphatidylinositol 3-kinase   PPAR   peroxisome proliferator-activated receptor   t10   c12-CLA   trans -10   cis -12 isomer of CLA  

摘要： Conjugated linoleic acids (CLAs), naturally occurring fatty acids in ruminant food products, have anti-tumorigenic and pro-apoptotic properties in animal as well as in vitro models of cancer. However, the cellular mechanism has not been fully understood. NAG-1 (non-steroidal anti-inflammatory drug-activated gene-1) is induced by several dietary compounds and belongs to a TGF-beta superfamily gene associated with pro-apoptotic and anti-tumorigenic activities. The present study was performed to elucidate the molecular mechanism by which CLA stimulates anti-tumorigenic activity in human colorectal cancer (CRC) cells. The trans-10, cis-12-CLA (t10,c12-CLA) repressed cell proliferation and induced apoptosis, whereas linoleic acid or c9,t11-CLA showed no effect on cell proliferation and apoptosis. We also found that t10,c12-CLA induced the expression of a pro-apoptotic gene, NAG-1, in human CRC cells. Inhibition of NAG-1 expression by small interference RNA (siRNA) results in repression of t10,c12-CLA-induced apoptosis. Microarray analysis using t10,c12-CLA-treated HCT-116 cells revealed that activating transcription factor 3 (ATF3) was induced and its expression was confirmed by western analysis. The t10,c12-CLA treatment followed by the overexpression of ATF3 increased NAG-1 promoter activity in HCT-116 cells. We further provide the evidence that t10,c12-CLA inhibited the phosphorylation of AKT and the blockage of GSK-3 by siRNA abolished t10,c12-CLA-induced ATF3 and NAG-1 expression. The current study demonstrates that t10,c12-CLA stimulates ATF3/NAG-1 expression and subsequently induces apoptosis in an isomer specific manner. These effects may be through inhibition of AKT/GSK-3 beta pathway in human CRC cells.

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