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AP-1 Binding (ap-1 + binding)
Selected AbstractsOxidized low-density lipoprotein induces matrix metalloproteinase-9 expression via a p42/p44 and JNK-dependent AP-1 pathway in brain astrocytesGLIA, Issue 1 2009Hui-Hsin Wang Abstract Upregulation of matrix metalloproteinases (MMPs), especially MMP-9, by oxidized low-density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP-9 via a MAPK-dependent AP-1 activation in rat brain astrocyte (RBA)-1 cells. Results revealed by gelatin zymography, RT-PCR, and Western blotting analyses showed that oxLDL-induced proMMP-9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA-1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP-1 (i.e., c-Fos/c-Jun) is crucial for oxLDL-induced proMMP-9 expression which was attenuated by pretreatment with AP-1 inhibitor (curcumin). The regulation of MMP-9 gene transcription by AP-1 was confirmed by oxLDL-stimulated MMP-9 luciferase activity which was totally lost in cells transfected with the AP-1 binding site-mutated MMP-9 promoter construct (mt-AP1-MMP-9). These results suggested that oxLDL-induced proMMP-9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP-1 activation. Understanding the regulatory mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases. © 2008 Wiley-Liss, Inc. [source] Intracellular glutathione in stretch-induced cytokine release from alveolar type-2 like cellsRESPIROLOGY, Issue 1 2004Behrouz Jafari Objective: Ventilator-induced lung injury (VILI) is characterized by release of inflammatory cytokines, but the mechanisms are not well understood. We hypothesized that stretch-induced cytokine production is dependent on oxidant release and is regulated by intracellular glutathione (GSH) inhibition of nuclear factor ,B (NF-,B) and activator protein-1 (AP-1) binding. Methodology: Type 2-like alveolar epithelial cells (A549) were exposed to cyclic stretch at 15% strain for 4 h at 20 cycles/min with or without N-acetylcysteine (NAC) or glutathione monoethylester (GSH-e) to increase intracellular GSH, or buthionine sulfoximine (BSO), to deplete intracellular GSH. Results: Cyclic stretch initially caused a decline in intracellular GSH and a rise in the levels of isoprostane, a marker of oxidant injury. This was followed by a significant increase in intracellular GSH and a decrease in isoprostane. Stretch-induced IL-8 and IL-6 production were significantly inhibited when intracellular GSH was further increased by NAC or GSH-e (P < 0.0001). Stretch-induced IL-8 and IL-6 production were augmented when intracellular GSH was depleted by BSO (P < 0.0001). NAC blocked stretch-induced NF-,B and AP-1 binding and inhibited IL-8 mRNA expression. Conclusions: We conclude that oxidant release may play a role in lung cell stretch-induced cytokine release, and antioxidants, which increase intracellular GSH, may protect lung cells against stretch-induced injury. [source] Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: A potential therapeutic benefit for arthritisARTHRITIS & RHEUMATISM, Issue 10 2008Sze-Kwan Lin Objective To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM),induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. Methods CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. Results EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1),CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. Conclusion By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis. [source] Mediation of interleukin-1,,induced transforming growth factor ,1 expression by activator protein 4 transcription factor in primary cultures of bovine articular chondrocytes: Possible cooperation with activator protein 1ARTHRITIS & RHEUMATISM, Issue 6 2003R. Andriamanalijaona Objective Interleukin-1 (IL-1) and transforming growth factor ,1 (TGF,1) play major roles in osteoarticular diseases, exerting opposite effects on both the catabolism and anabolism of cartilage matrix. Previous findings suggest that IL-1 and TGF,1 could function in a feedback interaction. However, the effect exerted by IL-1 on expression of TGF, by articular chondrocytes is, so far, poorly understood. The present study was carried out to determine the influence of IL-1, on the expression of TGF,1 by bovine articular chondrocytes (BACs) in primary culture. Methods BAC primary cultures were treated with IL-1,, and TGF,1 messenger RNA (mRNA) steady-state levels and protein expression were measured by real-time reverse transcription,polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Transient transfection of TGF,1 gene promoter constructs was performed to delineate the DNA sequences that mediate the IL-1, effect. Electrophoretic mobility shift assays (EMSAs) and supershift analysis were used to characterize the transcription factors binding to these sequences. Results Cultured BACs responded to IL-1, exposure by exhibiting an increase of TGF,1 expression at both the mRNA and protein levels. The effect was found to be mediated by a major 80-bp sequence located between ,732 and ,652 upstream of the transcription initiation site. EMSA and supershift analysis revealed that the transcription factors activator protein 4 (AP-4) and AP-1 specifically bound to the ,720/,696 part of this sequence under IL-1, treatment. Overexpression of AP-4 in the BAC cultures resulted in stimulation of the transcriptional activity of the ,732/+11 TGF,1 promoter construct through the same IL-1,,responsive element. Conclusion IL-1, induces an increase of TGF,1 in articular chondrocytes through activation of AP-4 and AP-1 binding to the TGF,1 gene promoter. These findings may help us understand the role of IL-1, in the disease process. Notwithstanding its deleterious effect on cartilage, IL-1 could initiate the repair response displayed by injured cartilage in the early stages of osteoarthritis through its ability to enhance TGF,1 expression by local chondrocytes. [source] | ||||||||||