1,25-dihydroxyvitamin D3 down-regulation of PHEX gene expression is mediated by apparent repression of a 110 kDa transfactor that binds to a polyadenine element in the promoter
1,25-dihydroxyvitamin D3 down-regulation of PHEX gene expression is mediated by apparent repression of a 110 kDa transfactor that binds to a polyadenine element in the promoter
复制标题
DOI：
10.1074/jbc.m404278200
复制
发表时间：
2004-11-05
影响因子：
4.8
通讯作者：
Ghishan, FK
中科院分区：
文献类型：
--
作者：
Hines, ER;Kolek, OI;Ghishan, FK
PHEX基因编码成骨细胞中表达的内肽酶，其使未表征的肽激素磷酸激素失活，磷酸激素抑制骨矿化以及肾磷酸盐重吸收和维生素D生物活化。我们证明，1 α-25-二羟维生素D-3（1，25（OH）（2）D-3），活性肾维生素D代谢产物，减少PHEX mRNA在大鼠成骨细胞系，UMR-106，以及在小鼠颅骨。在转染的UMR-106细胞中鼠PHEX基因的启动子/报告基因构建体分析将1，25（OH）（2）D-3的抑制作用定位于-133至-74 bp区域，并且凝胶迁移率变动实验显示1，25（OH）（2）D-3处理细胞减少了核蛋白与近端PHEX启动子中bp-116至-100的17个腺嘌呤片段的结合。显性负性维生素D受体（VDR）的过表达或17个A-T碱基对的该序列的缺失通过减弱基础启动子活性而消除了1，25（OH）（2）D-3的抑制作用，表明该区域介导1，25（OH）（2）D-3反应并参与基础转录。Southwestern印迹分析和DNA亲和纯化表明，一个身份不明的110 kDa的核蛋白结合到聚（A）元件。由于1，25（OH）（2）D-3配体的VDR既不与PHEX启动子的多聚腺嘌呤区域结合，也不直接影响110 kDa转因子的结合，因此我们得出结论，1，25（OH）（2）D-3通过VDR介导的这种新型反式激活因子的抑制（或修饰）间接降低PHEX表达。因此，我们已经确定了PHEX基因转录所需的顺式元件，参与PHEX表达的负反馈控制，从而调节磷酸激素的作用。
The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3), the active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)(2)D-3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)(2)D-3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)(2)D-3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)(2)D-3 response and is involved in basal transcription. Southwestern blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)(2)D-3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)(2)D-3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.