| Summary: | 博士 === 長庚大學 === 生物醫學研究所 === 99 === Cigarette smoke (CS) promotes several inflammatory responses in various cell types. In the respiratory system, long-time exposure of CS increases the risk of allergy, asthma, chronic obstructive pulmonary disease (COPD), and even lung cancer. During the airway inflammation, the proinflammatory enzyme-cytosolic phospholipase A2 (cPLA2) catalyzed hydrolysis of membrance phospholipid to release arachiodonic acid which was converted into prostaglandins (PGs) in the airway resident cells and contributed to the development of inflammation. Several factors including cytokines and chemokines have been shown to regulate the expression of cPLA2 and hence, mediate the pathogenesis of airway diseases. CS has been known as a potetial oxidation sources that induces expression of inflammatory proteins through various signaling patways and thus accelerates airway inflammation. During chronic airway inflammation, noxious, infectious, and allergic insults lead to cellular damage and/or bronchial remodelling were also up-regulate heme oxygenase (HO-1) expression against oxidation stress. Although, CS has been reported to induce cPLA2 and HO-1 expression in epithelial cells or macrophages through MAPKs, NF-B and other signaling molecules, the mechanisms underlying CS-induced cPLA2 and HO-1 expression in HTSMCs remain unknown. This study was to investigate the signaling pathways underlying CS-induced cPLA2 and HO-1 expression in HTSMCs.
First, we found that cigarette smoke extract (CSE)-induced cPLA2 protein and mRNA expression, and ROS generation was attenuated by pretreatment with a reactive oxygen species (ROS) scavenger (N-acetylcysteine), or inhibitors of NADPH oxidase (diphenyleneiodonium chloride), (apocynin) and transfection with p47phox siRNA, suggesting that CSE-induced cPLA2 expression was mediated through NADPH oxidase activation and ROS production in HTSMCS. Furthermore, CSE-induced cPLA2 expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), which were further confirmed by transfection with siRNAs of JNK1, p42 and p38 to down-regulate the expression of respective proteins and reduce cPLA2 expression. Induction of cPLA2 by CSE was attenuated by selective inhibitors of NF-kB (helenalin) and AP-1 (curcumin). Moreover, promoter assays revealed that increases of cPLA2, NF-kB and AP-1 luciferase activities stimulated by CSE were attenuated by these inhibitors. These results suggest that in HTSMCs, CSE induced NADPH oxidase activation leading to phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK. These reactions induced nuclear transcription NF-B and AP-1 activities which were essential for CSE-induced cPLA2 gene expression.
Next, our results showed that CSE-induced HO-1 protein, mRNA expression, and promoter activity were attenuated by pretreatment with the ROS scavenger (N-acetyl-L-cysteine) and the inhibitors of c-Src (PP1), NADPH oxidase (DPI) and (APO), MEK1/2 (U0126), p38 MAPK (SB202190), and JNK1/2 (SP600125) or transfection with siRNA of Src, p47phox, NOX2, p42, p38, JNK2, or NF-E2-related factor 2 (Nrf2). CSE-stimulated translocation of p47phox and Nrf2, translocation and NADPH oxidase activity were attenuated by transfection with siRNAs of Src, p47phox, and NOX2 or pretreatment with PP1, DPI, or APO. Furthermore, CSE-induced NOX2, c-Src, and p47phox complex formation was revealed by immunoprecipitation using an anti-NOX2, anti-p47phox, or anti-c-Src Ab followed by Western blot against an anti-NOX2, anti-p47phox, or anti-c-Src Ab. These results demonstrated that CSE-induced ROS generation was mediated through a c-Src/NADPH oxidase/MAPKs pathway, in turn initiated the activation of Nrf2, and ultimately induced HO-1 expression in HTSMCs.
Finally, we demonstrated that CSE-induced cPLA2 protein and mRNA expression was inhibited by pretreatment with the inhibitors of AP-1 (tanshinone IIA) and p300 (garcinol) or transfection with siRNAs of c-Jun, c-Fos, and p300. Moreover, CSE also induced c-Jun and c-Fos expression, which were inhibited by pretreatment with the inhibitors of NADPH oxidase (APO and DPI) and the ROS scavenger (N-acetyl-L-cysteine) or transfection with siRNAs of p47phox and NADPH oxidase (NOX)2. CSE-induced c-Fos expression was inhibited by pretreatment with the inhibitors of MEK1 (U0126) and p38 MAPK (SB202190) or transfection with siRNAs of p42 and p38. CSE-induced c-Jun expression and phosphorylation were inhibited by pretreatment with the inhibitor of JNK1/2 (SP600125) or transfection with JNK2 siRNA. CSE-stimulated p300 phosphorylation was inhibited by pretreatment with the inhibitors of NADPH oxidase and JNK1/2. Furthermore, CSE-induced p300 and c-Jun complex formation was inhibited by pretreatment with APO, DPI, NAC or SP600125. These results demonstrated that CSE-induced cPLA2 expression was mediated through NOX2-dependent p42/p44 MAPK and p38 MAPK/c-Fos and JNK1/2/c-Jun/p300 pathways in HTSMCs.
These results provide the insight into the mechanisms of CSE action in HTSMCs, supporting the hypothesis that CSE contributed to HTSMCs and promoted inflammatory responses involving in the development of airway diseases. Increased understanding of signal transduction mechanisms underlying cPLA2 and HO-1 gene regulation will create opportunities for the development of anti-inflammation therapeutic strategies.
|
|---|
