Epigenic Control of ENaC Transcription and Sodium Transport

描述（由申请人提供）：本项目的主要目标是以ENaC（上皮钠通道）为模型，确定控制mIMCD 3细胞和小鼠肾脏中Na+稳态的新型表观遗传机制。ENaC突变与Liddle综合征和PHA-1（假性醛固酮减少症1型）的相关性以及醛固酮对ENaC的严格和复杂调节表明ENaC在Na+平衡和血压调节中的重要性。醛固酮是Na+吸收的主要调节剂，主要通过在多个水平控制ENaC功能起作用，包括转录和细胞表面表达。在经典模型中，醛固酮通过激活两种不同但相似的NR（核受体）来增强转录：MR和GR（盐皮质激素和糖皮质激素受体）。小鼠中的MR破坏导致PHA-1和出生后约10天的动物死亡。最近，我们报道了另一种信号通路，通过一系列事件将醛固酮作用与染色质修饰和ENaC 1转录激活偶联，包括组蛋白H3 K79甲基转移酶Dot 1a的减少（端粒沉默的破坏物1）和推定的转录因子AF 9（来自9号染色体的ALL 1融合基因），SGK 1（血清和糖皮质激素调节激酶）介导的AF 9磷酸化，从而下调Dot 1a-AF 9相互作用，并在mIMCD 3细胞中靶向ENaC 1启动子处的组蛋白H3 K79低甲基化。尽管有这些发现，NR/醛固酮和Dot 1a/AF 9复合物之间的假定拮抗作用仍然没有得到解决;醛固酮刺激的ENaC转录和ENaC活性之间的相关性仍然没有得到很好的定义;并且导致PHA-1的MR破坏的分子缺陷仍然难以捉摸。在本提案中，我们打算填补这些空白。在目的1中，我们将检验NR-醛固酮和Dot 1a-AF 9复合物通过MR-AF 9和/或GR-AF 9相互作用相互抑制其对手的DNA结合活性以动态控制mIMCD 3细胞中ENaC 1转录的假设;目的2将检验mIMCD 3细胞中ENaC转录的变化转化为ENaC活性变化的假设;目标3将更全面地检查MR-/-小鼠，以检验我们的新的醛固酮网络和其他ENaC调节因子中的组分因MR缺乏而失调的假设，这有助于PHA-1的发展，并证实两种复合物对ENaC转录和活性的相互拮抗作用，如目标1和2所定义的。因此，拟议的研究将1）整合醛固酮作用的两种模式：NR的激活和Dot 1a-AF 9介导的抑制的缓解; 2）将醛固酮作用与染色质介导的ENaC转录激活，增强ENaC活性和Na+转运联系起来; 3）揭示PHA-1发育的分子机制。 公共卫生相关性 Na+平衡对于血压控制是重要的，并且主要通过类固醇激素醛固酮对ENaC的分子作用来实现，ENaC是一种将Na+通过细胞膜转运到细胞中的离子通道。拟议的研究旨在使用一种现有的基因工程小鼠模型，定义将醛固酮作用与染色质修饰、ENaC的转录激活和小鼠肾脏中Na+摄取增强联系起来的新机制。

DESCRIPTION (provided by applicant): The broad goal of this project is to define novel epigenetic mechanisms controlling Na+ homeostasis in mIMCD3 cells and in mouse kidney, with ENaC (epithelial sodium channel) as the model. The association of ENaC mutations with Liddle's syndrome and PHA-1 (pseudohypoaldosteronism type 1) as well as the tight and complex regulation of ENaC by aldosterone indicates the importance of ENaC in the regulation of Na+ balance and blood pressure. Aldosterone is a major regulator of Na+ absorption and acts primarily by controlling ENaC function at multiple levels, including transcription and cell surface expression. In the classical model, aldosterone enhances transcription by activating two distinct but similar types of NR (nuclear receptors): MR and GR (mineralocorticoid and glucocorticoid receptors). MR disruption in mouse leads to PHA-1 and animal death around day 10 after birth. Recently, we reported an alternative signaling pathway that couples aldosterone action to chromatin modifications and ENaC1 transcriptional activation through a series of events including reduction of histone H3 K79 methyltransferase Dot1a (disruptor of telomeric silencing 1) and putative transcription factor AF9 (ALL1-fused gene from chromosome 9), SGK1 (serum and glucocorticoid regulated kinase)-mediated phosphorylation of AF9 and thus downregulation of Dot1a-AF9 interaction, and targeted histone H3 K79 hypomethylation at the ENaC1 promoter in mIMCD3 cells. Despite these findings, the putative antagonism between the NR/aldosterone and Dot1a/AF9 complexes remains unaddressed; the correlation between aldosterone-stimulated ENaC transcription and ENaC activity is still not well defined; and the molecular defects derived from MR disruption that result in PHA-1 remain elusive. In this proposal, we intend to fill these gaps. In Aim 1, we will test the hypothesis that the NR-aldosterone and Dot1a-AF9 complexes mutually inhibit the DNA binding activity of their opponent through MR-AF9 and/or GR-AF9 interactions to dynamically control ENaC1 transcription in mIMCD3 cells; Aim 2 will test the hypothesis that changes in ENaC transcription translate into changes in ENaC activity in mIMCD3 cells; and Aim 3 will examine MR-/- mice more fully to test the hypothesis that the components in our new aldosterone network and other ENaC regulatory factors are deregulated by MR deficiency, which contributes to the development of PHA-1, and to confirm the mutual antagonism of the two complexes on ENaC transcription and activity as defined in Aim 1 and 2. Therefore, the proposed studies will 1) integrate the two modes of aldosterone action: activation of NR and relief of Dot1a-AF9-mediated repression; 2) link aldosterone action to chromatin-mediated ENaC transcriptional activation, enhanced ENaC activity and Na+ transport; and 3) shed new light on molecular mechanisms of PHA-1 development. PUBLIC HEALTH RELEVANCE Na+ balance is important for blood pressure control and is primarily achieved by the molecular action of the steroid hormone aldosterone on ENaC, an ion channel that transports Na+ through the cell membrane into the cells. The proposed studies are aimed to define novel mechanisms linking aldosterone action to chromatin modifications, transcriptional activation of ENaC, and enhanced Na+ uptake in mouse kidney, using one existing genetically engineered mouse model.