| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 87 === Human heat stable alkaline phosphatases are encoded by two closely related genes: the placental alkaline phosphatase (PAP), which specifies the term placental isozyme, and the germ cell alkaline phosphatase GCAP, which is expressed primarily in germ cells. In the human gastric cancer cell line TMK-1 cells, glucocorticoid exhibits an marked induction in the enzyme activity and accumulation of the mRNA of PAP. Analysis of mRNA stability and run on transcription demonstrated that glucocorticoid induction of PAP gene epxression occurs primarily at the level of transcription. In order to study the molecular mechanism underlying the glucocorticoid induction, PAP and GCAP promoters were fused to luciferase gene and transfection were carried out in TMK-1 cells. Dexamethasone confers about 6 to 10 fold of induction in luciferase activity in PAP promoter. Despite that the PAP and GCAP promoters share about 86% in sequence identity, the GCAP promoter directs a very low level of response to dexamethasone. The dexamethasone response region in PAP promoter was identified to be located in between -280 to -375. Insertion of this region into GCAP, CMV promoters, and TATA-luciferase reporter enabled these promoter responsive to dexamethasone when cotransfected with the glucocorticoid receptor (GR) expression plasmid pRS-hGR. Further studies indicated that the region from -296 to -331 is critical for glucocorticoid induction in all four different human cell lines assayed. Sequence analysis of the PAP promoter identified several potential half-glucocorticoid response elements (GRE) at -374, -307, -302, -292, and +33, respectively. In order to identify which of these potential GREs that is required for PAP promoter activity, DNA oligonucleotides encompass various length in between -375 to -280 were inserted into TATA-luciferase reporter. Transfection studies indicated that any one of the half-GREs alone is not enough to confer full glucocortcoid response and other regions may be required in the glucocorticoid-mediated induciton. In gel mobility shift assays using rat GR DNA binding domain (GR-DBD), only the GREs at -374, -302, and +33 were shown to bind GR-DBD efficiently. These results demonstrated that in TMK-1 cells, the increased transcription of PAP gene in the presence of glucocorticoid is mediated in part by interaction of the GR with the imperfect half-GREs in the gene promoter. In addition, other potential elements in PAP promoter may be involved in conferring the full and specific transcription activity mediated by glucocorticoid.
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