维生素D_3、9-顺式维甲酸及其相应核受体对hsp90β基因表达的调控

The Regulation of Vitamin D_3 and 9-cis-retinoic Acid and Their Receptors on Human hsp90β Gene

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 study the effects of vitamin D 3(VD 3), 9 cis retinoic acid (9 cis RA)and their receptors on the regulation of human hsp90β gene. Methods We first transfected Jurkat cells with “full length”(-1 039 bp/+1 531 bp)hsp90β reporter plasmid β1.11,then the transfected cells were treated with VD 3 or/and 9 cis RA;or β1.11 was cotransfected with wild type vitamin D 3 receptor (VDR) or/and retinoid X receptor (RXR) expression construction and the reporter activities were assayed.Western blot was carried out to detect the protein level of hsp90β in the Jurkat cells that were transfected by VDR cDNA or treated with VD 3 or/and 9 cis RA.By electrophoresis mobility shift assay (EMSA),we evaluated the DNA binding activity of VDR and RXR in Jurkat cell nuclear extracts. Results With VD 3 or/and 9 cis RA treatment,the expression of hsp90β gene was slightly increased.The induction became prominent when both of the ligands were present. The unliganded VDR or RXRα all slightly repressed the constitutive expression of hsp90β gene,while overexpression of VDR and RXRα further inhibited the gene.In addition,EMSA results showed that unliganded VDR and RXR simultaneously bound to the vitamin D 3 response element (VDRE) of hsp90β gene. Conclusions These results suggest that VD 3/VDR and 9 cis RA/RXR coparticipate in the regulation of hsp90β gene in Jurkat cells.