Cytokine Induced Cyclooxygenase-2 Expression in Human Alveolar Epithelial Cells: Involvement of PKC and MAPKs Pathways

• Main Authors: Yi-Tao Sun, 孫逸濤
• Other Authors: Ching-Chow Chen
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/34067900074832696052

碩士 === 國立臺灣大學 === 藥理學研究所 === 87 === The signaling pathway for tumor necrosis factor-a (TNF-a) induced cycloxygenase-2 (COX-2) expression was studied in human NCI-H292 epithelial cells. TNF-a induced a dose- and time-dependent increase in COX-2 expression PGE2 production was induced in parallel. Induction of COX-2 promoter activity by TNF-a was blocked by PDTC, a NF-kB inhibitor, indicating the involvement of NF-kB in mediating COX-2 expression in NCI-H292 cells. The enhancement of NF-kB specific DNA-protein binding activity in the nuclear extracts was observed by 10 min, 1 hr or 24 hr treatment with TNF-a. Supershift assays demonstrated the activation of p65/p50 NF-kB heterodimer after TNF-a stimulation. Analysis of the proteins involved in NF-kB binding showed translocation of p65 from cytosol to the nucleus after 10 min treatment with TNF-a. The onset of NF-kB activation correlated with the cytosolic degradation of IkB-a which was rapidly resynthesized after 1 hr TNF-a treatment. The PI-PLC inhibitor, U73122 attenuated the TNF-a-induced COX-2 expression, NF-kB activation and COX-2 promoter activity, as did the tyrosine kinase inhibitor genistein. TNF-a-stimulated IP formation and PKC activity were inhibited by both U73122 and genistein. The PKC inhibitor, staurosporine also resulted in the inhibition of TNF-a-induced COX-2 expression, NF-kB activation and COX-2 promoter activity. TPA, a PKC activator stimulated COX-2 expression, NF-kB activation and COX-2 promoter activity; these effects were inhibited by genistein. These results indicate that TNF-a might act through PI-PLCg to elicit PKC activation, protein tyrosine kinase activation and eventually COX-2 expression. Ten-minute treatment of cells with TNF-a resulted in the activation of p44/42 MAPK and p38. TNF-a-induced COX-2 expression and COX-2 promoter activity were inhibited by both MEK inhibitor, PD 98059 which did inhibit the activation of p44/42 and p38 inhibitor, SB 203580 which did inhibit the activation of p38. Overexpression of dominant negative mutant for ERK2, p38 or JNK inhibited the activation of COX-2 promoter activity by TNF-a. Sphingomyelin hydrolysis produced ceramide is another possible signal pathway for TNF-a. C2-ceramide, a cell permeable ceramide analog, SMase and OE all induced activation of p44/42 MAPK and p38. They also elicited COX-2 expression and COX-2 promoter activity which were inhibited by PD 98059 or SB 203580. Glutathione, a neutral SMase inhibitor attenuated TNF-a-, C2-ceramide- or OE-induced COX-2 expression, COX-2 promoter activity and MAPKs activation. These data suggest that TNF-a acts through PI-PLCg to induce PKC activation, then activates tyrosine kinase, or acts through sphingomyelinase pathway, to produce ceramide, then induces MAPKs activation. Both pathways result in the stimulation of NF-kB DNA-protein binding, NF-kB transactivation, and finally, COX-2 expression.