Aldosterone-induced Sgk1 relieves Dot1a-Af9–mediated transcriptional repression of epithelial Na+ channel α
Wenzheng Zhang, Xuefeng Xia, Mary Rose Reisenauer, Timo Rieg, Florian Lang, Dietmar Kuhl, Volker Vallon, Bruce C. Kone
Wenzheng Zhang, Xuefeng Xia, Mary Rose Reisenauer, Timo Rieg, Florian Lang, Dietmar Kuhl, Volker Vallon, Bruce C. Kone
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Research Article

Aldosterone-induced Sgk1 relieves Dot1a-Af9–mediated transcriptional repression of epithelial Na+ channel α

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 ENaCα 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 ENaCα promoter and derepression of ENaCα transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCα and likely other aldosterone-induced genes.

Authors

Wenzheng Zhang, Xuefeng Xia, Mary Rose Reisenauer, Timo Rieg, Florian Lang, Dietmar Kuhl, Volker Vallon, Bruce C. Kone

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Figure 2

Af9 phosphorylation directly correlates with Sgk1 expression in mIMCD3 cells.

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Af9 phosphorylation directly correlates with Sgk1 expression in mIMCD3 c...
(A) Aldosterone coordinately induces Sgk1 expression and Af9 Ser435 phosphorylation. mIMCD3 cells were treated with ethanol or aldosterone (Aldo; 1 μM) for the indicated times and examined by IB with the indicated antibodies. Af9 phosphorylation was apparently affected by both ethanol and aldosterone. The Sgk1 or phosphorylated Af9 abundance (mean ± SE; n = 6) was normalized to the corresponding actin and expressed relative to vehicle-treated cells at the 1-hour time point (assigned as 1). *P < 0.05 versus corresponding –aldo. (B) RNAi knockdown of Sgk1 decreases Af9 phosphorylation. mIMCD3 cells stably transfected with pSilencer-4.1–CMV–Neo negative control vector (Vec), pSgk1-RNAi7 (RNAi7), or pSgk1-RNAi9 (RNAi9) were examined by IB with the indicated antibodies. The same set of stably transfected cells were repeatedly examined as in A. Data were analyzed essentially as in A (n = 4). (C) Overexpression of WT Sgk1, but not the kinase-dead mutant Sgk1, promotes Af9 phosphorylation. mIMCD3 cells were transiently transfected with pcDNA3.1 vector (Vec) or its derivatives encoding WT or kinase-dead mutant Sgk1 (Lys127Met; Mut) and analyzed by IB probed with the indicated antibodies. Data were analyzed similarly as in A (n = 4). *P < 0.05 versus vector control.