Dataset Information

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

ABSTRACT: Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

SUBMITTER: Zhang W

PROVIDER: S-EPMC1804379 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Zhang Wenzheng W Xia Xuefeng X Reisenauer Mary Rose MR Rieg Timo T Lang Florian F Kuhl Dietmar D Vallon Volker V Kone Bruce C BC

The Journal of clinical investigation 20070301 3

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing th ...[more]

PMID: 17332896

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Project description:We have previously reported that Dot1a is located in the cytoplasm and nucleus (Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. J Biol Chem 284: 35659-35669, 2009), widely expressed in the kidney as detected by its histone H3K79 methyltransferase activity (Zhang W, Hayashizaki Y, Kone BC. Biochem J 377: 641-651, 2004), and involved in transcriptional control of the epithelial Na(+) channel subunit-alpha gene (alphaENaC) (Zhang W, Xia X, Jalal DI, Kuncewicz T, Xu W, Lesage GD, Kone BC. Am J Physiol Cell Physiol 290: C936-C946, 2006). Aldosterone releases repression of alphaENaC by reducing expression of Dot1a and its partner AF9 (Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC. J Biol Chem 281: 18059-18068, 2006) and by impairing Dot1a-AF9 interaction via Sgk1-mediated AF9 phosphorylation (Zhang W, Xia X, Reisenauer MR, Rieg T, Lang F, Kuhl D, Vallon V, Kone BC. J Clin Invest 117: 773-783, 2007). This network also appears to regulate transcription of several other aldosterone target genes. Here, we provide evidence showing that Dot1a contains at least three potential nuclear localization signals (NLSs). Deletion of these NLSs causes green fluorescent protein-fused Dot1a fusions to localize almost exclusively in the cytoplasm of 293T cells as revealed by confocal microscopy. Deletion of NLSs abolished Dot1a-mediated repression of alphaENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to alphaENaC, the mRNA levels of betaENaC, gammaENaC, and Sgk1 are also downregulated by Dot1a and AF9 overexpression. Small interference RNA-mediated knockdown of Dot1a and AF9 or aldosterone treatment leads to an opposite effect. Using single-cell fluorescence imaging or equivalent short-circuit current in IMCD3 and M1 cells, we show that observed transcriptional alterations correspond to changes in ENaC and Sgk1 protein levels as well as benzamil-sensitive Na(+) transport. In brief, Dot1a and AF9 downregulate Na(+) transport, most likely by regulating ENaC mRNA and subsequent protein expression and ENaC activity.

Project description:Primary hyperaldosteronism (PA) is characterized by aldosterone excess and hypertension. This may be linked to increased renal Na+ reabsorption via the epithelial Na+ channel (ENaC) and the NaCl cotransporter (NCC). The majority of PA patients have normal plasma K+ levels, but a subset of cases are associated with hypokalemia. High NCC levels observed in long-term studies with aldosterone-infused rodents have been attributed to direct effects of aldosterone. Aldosterone can also increase active phosphorylated NCC (pT58-NCC) acutely. However, direct effects of aldosterone on NCC have been contested by recent studies indicating that it is rather an indirect effect of hypokalemia. We therefore set out to determine isolated long-term aldosterone and K+ effects on ENaC and NCC using various in vivo and ex vivo approaches. In mice, aldosterone-induced hypokalemia was prevented by simultaneous amiloride infusion, coupled to increased cleavage of α- and γENaC but no effect on NCC. Regression analyses of in vivo data showed a positive correlation between aldosterone/K+ and αENaC but a negative correlation with NCC and pT58-NCC. Ex vivo, exposure of kidney tubules for 21 h to aldosterone increased cleavage of αENaC and γENaC, but no effects were observed on NCC or pT58-NCC. Exposure of tubules to low K+ media reduced αENaC but increased NCC and pT58-NCC. As hypokalemia can enhance cell proliferation markers in the distal convoluted tubule (DCT), we hypothesized that aldosterone infusion would increase proliferating cell nuclear antigen (PCNA) expression. Infusion of aldosterone in mice for 6 days greatly increased PCNA expression in the DCT. Collectively, in vivo and ex vivo data suggest that both aldosterone and K+ can increase ENaC directly. In contrast, the observed increase in abundance and phosphorylation of NCC in aldosterone-infused mice is likely an indirect effect of enhanced ENaC-mediated K+ secretion and subsequent hypokalemia. Thus, it is possible that NCC may only be increased in PA when the condition is associated with hypokalemia.

Dataset Information

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Publications

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

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