| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 98 === Human pulmonary alveolar epithelial cells play an important role in the proliferation, migration, and inflammatory processes in the respiratory system. Several factors have been shown to trigger the mechanisms for the pathologenesis of airway diseases including asthma and chronic obstructive pulmonary disease (COPD). Although the factors concerning about the increased incidence of inflammatory responses are well known, however, the intracellular signaling pathways involved in the expression of inflammatory proteins are not completely recognized. Elevated levels of pro-inflammatory cytokines such as tumor necrosis factor- (TNF-) have been found in the airway fluids, which may induce up-regulation of cytosolic phospholipase A2 (cPLA2) implicated in the pathogenesis of inflammatory diseases.
Although TNF- has been reported to activate all of mitogen-activated protein kinases (MAPKs) including p42/p44 MAPK, p38 MAPK, and JNK/SAPK, and transactivation of growth factor receptors and PI3K/Akt, the relationship between the activation of these signaling pathways and expression of cPLA2 or other genes remains largely unknown in human alveolar epithelial cells (HPAEpiCs). Therefore, whether activation of these MAPKs, growth factor receptors and PI3K/Akt pathways by TNF- linked to cPLA2 expression is needed determining in HPAEpiCs. In addition, it is of interest that many of the genes regulated by MAPKs are dependent on NF-B, AP-1, and p300 for transcription. These transcription factors have also been shown to be involved in cPLA2 gene expression at the transcriptional level in various cell types. Western blot and Real-time RT-PCR showed that in HPAEpiCs, TNF-α induced cPLA2 mRNA and protein expression in a time-dependent manner, which were attenuated by pretreatment with the inhibitors of ROS (NAC, APO, DPI), PDGF receptor (AG1296), PI3K (Wortmannine), and MEK1/2 (PD98059) or transfection with siRNA of p42. These results suggest that PDGFR transactivation participates in cPLA2 expression induced by TNF-α. Accordingly, TNF-α-stimulated phosphorylation of p38 MAPK and JNK were inhibited by pretreatment with NAC, APO, or DPI. TNF- induced cPLA2 expression was blocked by the selective inhibitors of AP-1 (Tanshinone IIA) and NF-B (Bay11-7082). Moreover, TNF-α-stimulated cPLA2 promoter activity was blocked by these selective inhibitors.
In this study we investigated the effect of TNF-α induced cPLA2 expression at the transcriptional and translational levels, which were mediated through three independent pathways. First, TNF-α activated Jak2-dependent PDGFR transactivation, PI3K/Akt, p42/p44 MAPK, and p300/c-Jun/c-Fos/ATF2/AP-1 signalung pathway in HPAEpiCs. Secand, TNF-α-stimulated TNFR1 induced association of TRAF2, ASK-1 and p47phox, which promoted recruitment with ROS production, p38 MAPK phosphorylation, JNK1/2 phosphorylation resulting in AP-1 activation and cPLA2 expression and PGE2 release. In addation, TNF-α-induced ROS production which promoted recrument with NIK, IKK resulting in NF-B activation and cPLA2 expression and PGE2 release. These results provide new insights into the mechanisms of TNF-α action which may be therapeutic value in lung diseases.
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