Circadian clock genes directly regulate expression of the Na+/H+ exchanger NHE3 in the kidney
Circadian clock genes directly regulate expression of the Na+/H+ exchanger NHE3 in the kidney
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DOI：
10.1111/j.1523-1755.2005.00218.x
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发表时间：
2005-04-01
影响因子：
19.6
通讯作者：
Yokoyama, M
中科院分区：
文献类型：
--
作者：
Rohman, MS;Emoto, N;Yokoyama, M
背景。哺乳动物生理学中的日常节律是由一组时钟基因精心策划的转录/翻译反馈环路产生的。然而，人们对从时钟基因振荡器到细胞功能的分子级联知之甚少。方法。在小鼠和大鼠肾脏中检查了 NHE3 和时钟基因的 mRNA 表达谱。首先，使用荧光素酶测定和 E-box 序列定点诱变来评估 CLOCK:BMAL1 反式激活的 NHE3 启动子活性。通过电泳迁移率变动测定 (EMSA) 证实了 CLOCK:BMAL1 异二聚体与 NHE3 启动子的 E-box 序列的直接结合。结果。我们提供的证据表明，肾小管 NHE3（对全身电解质和酸碱稳态至关重要的 Na+/H+ 交换器）是一种受肾脏中 CLOCK:BMAL1 异二聚体直接调节的时钟控制基因。大鼠肾脏中的 NHE3 mRNA 水平表现出昼夜节律动力学，并且这种昼夜节律表达在纯合 CRY1/2 双缺陷小鼠中严重减弱，表明肾小管细胞中外周时钟的转录机制直接调节 NHE3 的昼夜节律表达。通过分析 NHE3 基因的 5' 上游区域，我们发现了一个 E 盒，对于通过 CLOCK:BMAL1 驱动的昼夜节律振荡器进行 NHE3 的转录至关重要。 NHE3 mRNA 的昼夜节律表达通过大鼠肾近曲小管中蛋白水平的振荡来反映。结论。 NHE3应该代表肾脏中外周振荡器的输出基因，其直接受CLOCK:BMAL1异二聚体调节。
Background. Daily rhythms in mammalian physiology are generated by a transcription/translation feedback loop orchestrated by a set of clock genes. However, little is known about the molecular cascade from the clock gene oscillators to cellular function.Methods. The mRNA expression profiles of NHE3 and clock genes were examined in mice and rat kidneys. First, luciferase assays followed by a site directed mutagenesis of an E-box sequence were used to assess the CLOCK:BMAL1-transactivated NHE3 promoter activity. A direct binding of CLOCK:BMAL1 heterodimers to an E-box sequences of NHE3 promoter was confirmed by electrophoretic mobility shift assay (EMSA).Results. We present evidence that renal tubular NHE3, the Na+/H+ exchanger critical for systemic electrolyte and acid-base homeostasis, is a clock-controlled gene regulated directly by CLOCK:BMAL1 heterodimers in kidneys. NHE3 mRNA level in rat kidney displayed circadian kinetics, and this circadian expression was severely blunted in homozygous CRY1/2 double-deficient mice, suggesting that the transcriptional machinery of peripheral clocks in renal tubular cells directly regulates the circadian expression of NHE3. By analyzing the 5' upstream region of the NHE3 gene, we found an E box critical for the transcription of NHE3 via the CLOCK:BMAL1-driven circadian oscillator. The circadian expression of NHE3 mRNA was reflected by oscillating protein levels in the proximal tubules of the rat kidney.Conclusion. NHE3 should represent an output gene of the peripheral oscillators in kidney, which is regulated directly by CLOCK:BMAL1 heterodimers.