Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells
✍ Scribed by Y. Okada; O. Voznesensky; H. Herschman; J. Harrison; C. Pilbeam
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 210 KB
- Volume
- 78
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
The tumor promoter phorbol 13-myristate 12-acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP-1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase-2 (PGHS-2) expression in murine osteoblastic MC3T3-E1 cells, but no functional AP-1 binding motifs in the 5Ј-flanking region have been identified. In MC3T3-E1 cells transfected with Ϫ371/ϩ70 bp of the PGHS-2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS-2 promoter region identified three potential AP-1 elements in the Ϫ371/ϩ70 bp region, and deletion analysis suggested that the sequence 5Ј-aGAGTCA-3Ј at Ϫ69/Ϫ63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP-1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS-2 probe spanning this sequence, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Mutation of this AP-1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3-E1 cells. On EMSA, serum induces binding to a PGHS-2 probe spanning the AP-1 site, binding is blocked by an unlabeled AP-1 canonical sequence, and antibodies specific for c-Jun and c-Fos inhibit binding. Joint mutation of this AP-1 site and the nearby CRE site at Ϫ56/Ϫ52 bp, previously shown to mediate serum, v-src and PDGF induction of PGHS-2 in NIH-3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3-E1 cells. Hence, the AP-1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS-2 promoter activity.