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AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells.

ABSTRACT: Our previous work in 293T cells and AF17(-/-) mice suggests that AF17 upregulates expression and activity of the epithelial Na(+) channel (ENaC), possibly by relieving Dot1a-AF9-mediated repression. However, whether and how AF17 directly regulates Dot1a cellular distribution and ENaC function in renal collecting duct cells remain unaddressed. Here, we report our findings in mouse cortical collecting duct M-1 cells that overexpression of AF17 led to preferential distribution of Dot1a in the cytoplasm. This effect could be blocked by nuclear export inhibitor leptomycin B. siRNA-mediated depletion of AF17 caused nuclear accumulation of Dot1a. AF17 overexpression elicited multiple effects that are reminiscent of aldosterone action. These effects include 1) increased mRNA and protein expression of the three ENaC subunits (?, ? and ?) and serum- and glucocorticoid inducible kinase 1, as revealed by real-time RT-qPCR and immunoblotting analyses; 2) impaired Dot1a-AF9 interaction and H3 K79 methylation at the ?ENaC promoter without affecting AF9 binding to the promoter, as evidenced by chromatin immunoprecipitation; and 3) elevated ENaC-mediated Na(+) transport, as analyzed by measurement of benzamil-sensitive intracellular [Na(+)] and equivalent short circuit current using single-cell fluorescence imaging and an epithelial Volt-ohmmeter, respectively. Knockdown of AF17 elicited opposite effects. However, combination of AF17 overexpression or depletion with aldosterone treatment did not cause an additive effect on mRNA expression of the ENaC subunits. Taken together, we conclude that AF17 promotes Dot1a nuclear export and upregulates basal, but not aldosterone-stimulated ENaC expression, leading to an increase in ENaC-mediated Na(+) transport in renal collecting duct cells.

SUBMITTER: Wu H

PROVIDER: S-EPMC3210795 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:The epithelial Na(+) channel (ENaC) that mediates regulated Na(+) reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (I(Na)) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline I(Na) by approximately 50% and I(Na) could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of alpha and gamma ENaC were sequentially substituted with glycines. This scan yielded two valine residues in gamma ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an approximately 20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P(1) position residue for PE and substitution of alanine 190 in the gamma subunit eliminated I(Na) activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the gamma subunit (gamma(Th)) allowed for I(Na) activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T(176)-S(198)) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the gamma(Th) but not the wild-type gamma subunit. These results demonstrate that gamma subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the gamma subunit results in ENaC activation.

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AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells.

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