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目的 研究维生素 D3(VD3)、9-顺式维甲酸 (9- cis- RA)及其相应核受体对 hsp90 β基因表达的调控作用。方法 采用 DEAE- Dextran方法将人 hsp90 β基因调控片段 (- 10 39bp/ +15 31bp)介导的荧光素酶 (L uc)报告基因质粒 β1.11转染 Jurkat细胞 ,并用 VD3和 9- cis- RA分别或同时刺激细胞 ,或将质粒 β1.11及野生型维生素D3受体 (VDR)或 /和维甲酸 X受体 α(RXRα)真核表达质粒共转染 Jurkat细胞 ,检测细胞裂解液中荧光素酶活性 ;用 Western印迹分析方法检测经 VDR真核表达质粒转染或用 VD3和 9- cis- RA刺激的 Jurkat细胞中内源性Hsp90β蛋白的改变 ;通过电泳迁移率变更分析 (EMSA)实验 ,检测 VDR和 RXR能否与含有 hsp90β基因第一内含子中维生素 D3应答元件 (i VDRE)的双链寡核苷酸片段发生特异结合。结果 VD3和 9- cis- RA可分别低水平诱导hsp90β基因表达 ,而两种配体同时加入有协同作用。 VDR或 RXRα分别转染只能较弱地抑制 hsp90β基因表达 ,二者共转染能增强其抑制作用。 EMSA实验证实 ,VDR和 RXR都可特异结合于 i VDRE上。结论 VD3和 9- cis- RA这两条信号途径共同参与 hsp90β基因的启动子活性调节。
Objective To investigate the regulation of hsp90 β gene expression by vitamin D3 (VD3), 9-cis-RA and its corresponding nuclear receptors. Methods The Jurkat cells were transfected with luciferase reporter gene plasmid (L113) mediated by human hsp90 gene (-109bp / +15 31bp) by DEAE-Dextran method. RA were used to stimulate cells separately or simultaneously, and Jurkat cells were cotransfected with plasmid β1.11 and wild-type vitamin D3 receptor (VDR) or / and retinoic acid X receptor α (RXRα) eukaryotic expression plasmid Luciferase activity. The changes of endogenous Hsp90β protein in Jurkat cells transfected with VDR eukaryotic expression plasmids or stimulated with VD3 and 9-cis-RA were detected by Western blot analysis. By electrophoretic mobility shift assay (EMSA) Experiments to examine whether VDR and RXR specifically bind to double stranded oligonucleotide fragments containing the vitamin D3 response element (i VDRE) in the first intron of the hsp90β gene. Results VD3 and 9-cis-RA induced hsp90β gene expression at low level respectively, and synergistic effect was observed when both ligands were added simultaneously. VDR or RXRα transfection could only weakly inhibit the expression of hsp90β gene, the two transfected can enhance its inhibition. EMSA experiments confirmed that both VDR and RXR bind specifically to i VDRE. Conclusion The two signal pathways VD3 and 9-cis-RA are involved in the regulation of promoter activity of hsp90β gene.