Zhang Wenzheng, Kone Bruce C
Departments of Internal Medicine and of Integrative Biology, Pharmacology, and Physiology, The University of Texas Medical School at Houston, Houston, Texas 77030, USA.
Am J Physiol Renal Physiol. 2002 Nov;283(5):F904-11. doi: 10.1152/ajprenal.00156.2002.
The H(+)-K(+)-ATPase alpha(2) (HKalpha(2)) gene plays a central role in potassium homeostasis, yet little is known about its transcriptional control. We recently demonstrated that the proximal promoter confers basal transcriptional activity in mouse inner medullary collecting duct 3 cells. We sought to determine whether the kappaB DNA binding element at -104 to -94 influences basal HKalpha(2) gene transcription in these cells. Recombinant NF-kappaB p50 footprinted the region -116/-94 in vitro. Gel shift and supershift analysis revealed NF-kappaB p50- and p65-containing DNA-protein complexes in nuclear extracts of mouse inner medullary collecting duct 3 cells. A promoter-luciferase construct with a mutated -104/-94 NF-kappaB element exhibited higher activity than the wild-type promoter in transfection assays. Overexpression of NF-kappaB p50, p65, or their combination trans-repressed the HKalpha(2) promoter. The histone deacetylase (HDAC) inhibitor trichostatin A partially reversed NF-kappaB-mediated trans-repression of the HKalpha(2) promoter. HDAC6 overexpression inhibited HKalpha(2) promoter activity, and HDAC6 coimmunoprecipitated with NF-kappaB p50 and p65. These results suggest that HDAC6, recruited to the DNA protein complex, acts with NF-kappaB to suppress HKalpha(2) transcription and identify NF-kappaB p50 and p65 as novel binding partners for HDAC6.
H(+)-K(+)-ATP酶α(2)（HKα(2)）基因在钾离子稳态中起核心作用，但其转录调控机制却鲜为人知。我们最近发现近端启动子赋予小鼠髓质内集合管3细胞基础转录活性。我们试图确定位于-104至-94的κB DNA结合元件是否影响这些细胞中HKα(2)基因的基础转录。重组核因子κB p50在体外与-116/-94区域结合。凝胶迁移和超迁移分析显示，在小鼠髓质内集合管3细胞核提取物中存在含核因子κB p50和p65的DNA-蛋白质复合物。在转染实验中，具有突变的-104/-94核因子κB元件的启动子-荧光素酶构建体比野生型启动子表现出更高的活性。核因子κB p50、p65或它们的组合的过表达反式抑制HKα(2)启动子。组蛋白脱乙酰酶（HDAC）抑制剂曲古抑菌素A部分逆转了核因子κB介导的HKα(2)启动子的反式抑制。HDAC6的过表达抑制HKα(2)启动子活性，并且HDAC6与核因子κB p50和p65共免疫沉淀。这些结果表明，募集到DNA-蛋白质复合物的HDAC6与核因子κB共同作用抑制HKα(2)转录，并确定核因子κB p50和p65为HDAC6的新型结合伴侣。
