| Summary: | 碩士 === 長庚大學 === 天然藥物研究所 === 95 === Pro-inflammatory cytokines including tumor necrosis factor- (TNF-) and interleukin-1(IL-1) have been reported to up-regulate multiple inflammatory genes in airway inflammation. The cytosolic phospholipase A2 (cPLA2) plays a critical role in catalyzing the hydrolysis of glycerophospholipids to liberate arachidonic acid (AA) which is further converted into prostaglandins (PG) catalyzed by cyclooxygenase (COX). In human tracheal smooth muscle cells (HTSMCs), the amount of de novo synthesis of cPLA2 protein and PGE2 production was induced by cytokines (TNF- and IL-1. Therefore, in present study, we investigated whether different mechanisms participating in cytokines-induced expression of cPLA2 and enhanced synthesis of PGE2 analyzed by Western blotting, RT-PCR, promoter activity and ELISA assay. Our results demonstrated that cytokines significantly increased cPLA2 protein, mRNA, luciferase activity and PGE2 productions, and these effects were regulated by several of signaling transduction molecules including, MAPKs (ERK1/2, p38, JNK1/2), Src/EGFR,PDGFR/P13K/Akt, PLC/Ca2+, and PKC isoforms. Pretreatment with their respective inhibitors (U0126, SB202190, SP600125, PP1, AG1296, AG1478, LY294002, SH-5, U73122, BAPTA/AM, GÖ6976, Ro-318220 and rottlerin) or transfection with siRNAs (MEK1, p42, p38, JNK2, Src, Akt) significantly attenuated phosphorylation protein kinases, cPLA2 expression and PGE2 synthesis induced by cytokines. In addition, expression of cPLA2 by cytokines was also inhibited by a NF-B inhibitor (helenalin) or by over-expression of dominant negative mutants of NIK and IKK-. Cytokines-induced NF-B translocation was blocked by transfection with dominant negative mutants of NIK, IKK- and IKK-, but not by these MAPKs inhibitors, suggesting that MAPKs and NF-B independently regulated cPLA2 expression and PGE2 synthesis. Furthermore, down-regulation of p300 by p300 siRNA transfection or pretreatment of cells with p300 inhibitor curcumin almost completely blocked cytokines-dependent cPLA2 expression. Most surprisingly, p300 was associated with the cPLA2 promoter (-595 to +75), which was dynamically linked to histone H4 acetylation stimulated by cytokines, as determined by chromatin immunoprecipitation assay (ChIP). Furthermore, an enrichment of p300 and histone H4-associated cPLA2 DNA complexes appeared in cytokines-treated HTSMCs. Furthermore, activation of CBP/p300 mediated through MAPK or Akt phosphorylation has been reported. Herein, we found that inhibition of JNK, p38 and Akt pathway by their specific inhibitors causes a decrease recruitment of p300 and histone H4 interacted with cPLA2 promoter. On the contrary, phosphorylation of CaMKII by cytokines may lead to nucleus export of HDACs and induce genes repression. However, KT5926 (a CaMKII inhibitor) and siRNA for CaMKII had no effect on cPLA2 expression, this complicated mechanism remains to be investigated. Thus, these results provide a new insight into the molecular mechanisms that activation of NIK/IKK, MAPKs (ERK1/2, p38, JNK1/2) Src/EGFR/P13K/Akt, PLC/Ca2+ and PKC isoforms signaling pathways may eventually stimulate p300 activity, assemble transcription factors, and lead to cPLA2 expression induced by cytokines in HTSMCs. Increased understanding of mechanisms underlying cPLA2 gene expression induced by cytokines will create opportunities for the development of anti-airway inflammation therapeutic strategies.
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