Yu Zhiyuan, Li Mei, Zhang Dongyu, Xu William, Kone Bruce C
Departments of Medicine and of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Am J Physiol Renal Physiol. 2009 Jul;297(1):F63-70. doi: 10.1152/ajprenal.00039.2009. Epub 2009 May 6.
The H(+)-K(+)-ATPase alpha(2) (HKalpha2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5'-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-kappaB and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the -144/-135 Sp element influences basal HKalpha2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for -154/-127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HKalpha2 gene in mIMCD3 cells in vivo. HKalpha2 minimal promoter-luciferase constructs with point mutations in the -144/-135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HKalpha2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HKalpha2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HKalpha2 promoter without altering the binding or regulatory influence of NF-kappaB p65 or CREB-1 on the proximal HKalpha2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HKalpha2 gene expression in mIMCD3 cells in vivo and in vitro.
肾集合管和远端结肠的H(+)-K(+)-ATP酶α(2)（HKα2）基因在钾和酸碱平衡中起核心作用，但其转录调控仍知之甚少。我们先前证明其5'-侧翼区近端177 bp在小鼠内髓集合管（mIMCD3）细胞中赋予基础转录活性，且NF-κB和CREB-1结合该区域以改变转录。在本研究中，我们试图确定-144/-135 Sp元件是否影响这些细胞中HKα2基因的基础转录。使用-154/-127探针进行的电泳迁移率变动和超迁移分析揭示了mIMCD3细胞核提取物中含Sp1的DNA-蛋白质复合物。染色质免疫沉淀（ChIP）分析表明，在体内mIMCD3细胞中，Sp1而非Sp3与HKα2基因的该启动子区域结合。在瞬时转染分析中，-144/-135 Sp元件发生点突变的HKα2最小启动子-荧光素酶构建体的活性远低于野生型启动子。Sp1而非Sp3的过表达在mIMCD3细胞以及缺乏Sp因子的SL2昆虫细胞中转激活HKα2近端启动子-荧光素酶构建体。相反，Sp1的小干扰RNA敲低抑制内源性HKα2 mRNA表达，且Sp1与HKα2近端启动子相关染色质的结合未改变NF-κB p65或CREB-1对HKα2近端启动子的结合或调节影响。我们得出结论，Sp1在体内和体外控制mIMCD3细胞中HKα2基因基础表达方面发挥重要的正向作用。
