Staurosporine Effect on AP-2 in MC3T3-E1

• Main Authors: Chiu-Ya Wang, 王秋雅
• Other Authors: Chiun-Jye Yuan
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/16969510104812734977

碩士 === 國立交通大學 === 生物科技研究所 === 88 === Cyclooxygenase 2 (COX-2) or Prostaglandin H synthase 2 (PGHS-2) is the key enzyme in the biosynthesis of the prostaglandins (PGs). COX-2 is inducible in response to cytokines, bacterial endotoxin and tumor promoters. In previous study, we have found that phospholipase A2 (PLA2) induced COX-2 overexpression could be enhanced by staurosporine (Stsp). In this study, we try to delineate the stimulatory effect of Stsp on COX-2 expression in mouse osteoblast like cell line, MC3T3-E1. In an attempt to characterize the signaling pathway of Stsp leading to the overexpression of COX-2, we investigated cis- and trans-acting factors required for COX-2 expression. Two regions, i.e., AP-2 and NF-IL6 corresponding elements, in COX-2 promoter were identified as the positive cis-regulatory elements through luciferase reporter vectors containing various 5’-flanking regions of COX-2 gene. The site-directed mutagenesis of the AP-2 and NF-IL6 recognition sequences of COX-2 promoter also confirmed the role of NF-IL6 and AP-2 in the up-regulation of COX-2 gene. The relative luciferase activities of the cells transfected with GLB/NA or GLB/A reached maximum at eight hours post-treatment. The activation was rapidly declined at ten hours. Gel shift assays showed that AP-2 bound to the AP-2 site. GF 109203X effect experiments found PKC signaling pathway did not involved in COX-2 expression stimulated by Stsp. Moreover, cell death or overexpression of COX-2 did not influence the varies of the relative luciferase activities. We also found that autophagic cell death of MC3T3-E1 caused by Stsp and possibly have relation with AP-2.