Sirtuin 1 functionally and physically interacts with disruptor of telomeric silencing-1 to regulate alpha-ENaC transcription in collecting duct

Abstract

Aldosterone increases renal tubular Na+ absorption in large part by increasing transcription of the epithelial Na(+) channel alpha-subunit (alpha-ENaC) expressed in the apical membrane of collecting duct principal cells. We recently reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the alpha-ENaC promoter in mouse inner medullary collecting duct mIMCD3 cells, and that aldosterone acts to disrupt this complex and its inhibitory effects (Zhang, W., Xia, X., Reisenauer, M. R., Rieg, T., Lang, F., Kuhl, D., Vallon, V., and Kone, B. C. (2007) J. Clin. Invest. 117, 773-783). Here we demonstrate that the NAD(+)-dependent deacetylase sirtuin 1 (Sirt1) functionally and physically interacts with Dot1 to enhance the distributive activity of Dot1 on H3K79 methylation and thereby represses alpha-ENaC transcription in mIMCD3 cells. Sirt1 overexpression inhibited basal alpha-ENaC mRNA expression and alpha-ENaC promoter activity, surprisingly in a deacetylase-independent manner. The ability of Sirt1 to inhibit alpha-ENaC transcription was retained in a truncated Sirt1 construct expressing only its N-terminal domain. Conversely, Sirt1 knockdown enhanced alpha-ENaC mRNA levels and alpha-ENaC promoter activity, and inhibited global H3K79 methylation, particularly H3K79 trimethylation, in chromatin associated with the alpha-ENaC promoter. Sirt1 and Dot1 co-immunoprecipitated from mIMCD3 cells and colocalized in the nucleus. Sirt1 immunoprecipitated from chromatin associated with regions of the alpha-ENaC promoter known to associate with Dot1. Aldosterone inhibited Sirt1 association at two of these regions, as well as Sirt1 mRNA expression, in a coordinate manner with induction of alpha-ENaC transcription. Overexpressed Sirt1 inhibited aldosterone induction of alpha-ENaC transcription independent of effects on mineralocorticoid receptor trans-activation. These data identify Sirt1 as a novel modulator of alpha-ENaC, Dot1, and the aldosterone signaling pathway.

FIGURE 1.

Overexpressed Sirt1 down-regulates basal α-ENaC mRNA expression and α-ENaC promoter activity in collecting duct cells. A, representative immunoblot showing expression of endogenous Sirt1 in mIMCD3 cells, and Sirt1 overexpression by transient transfection of mIMCD3 with pcDNA3.1-Sirt1. B, qRT-PCR showing that transient overexpression of Sirt1 inhibits the expression of endogenous α-ENaC mRNA in mIMCD3 cells. β-Actin levels were invariant under the different conditions. Data are presented as mean ± S.D. of four independent experiments. *, p < 0.05 versus pcDNA3.1-transfected cells. C, luciferase assay demonstrating that transient Sirt1 overexpression suppresses basal expression of a stably incorporated α-ENaC promoter-luciferase construct (α-ENaC-Luc) in mIMCD3 cells. The cells were transiently transfected with empty vector pcDNA3.1 or pcDNA3.1-Sirt1 along with the Renilla luciferase reporter pRL-SV40 as an internal control. The firefly luciferase activity of each sample was normalized to its Renilla luciferase activity to generate the “relative luciferase activity.” The relative luciferase activity of the vector-transfected cells was designated as 1 and utilized to determine the relative level and the significance of the other samples. Data represent the mean ± S.D. of five independent experiments. *, p < 0.05 versus corresponding values for α-ENaC-Luc + pcDNA3.1. D, luciferase assay demonstrating that co-overexpression of Sirt1 and Dot1 repress luciferase activity in mIMCD3 cell stable lines expressing the α-ENaC promoter-luciferase minigene to a greater degree than individual overexpression of Sirt1 or Dot1 (α-ENaC-Luc). Data are the mean ± S.D. of three independent experiments each performed in triplicate. *, p < 0.05 versus vector; **, p < 0.05 versus Dot1 or Sirt1.

FIGURE 2.

siRNA knockdown of Sirt1 up-regulates basal α-ENaC mRNA expression and α-ENaC promoter activity in collecting duct cells. A, qRT-PCR analysis (left) and representative immunoblot (IB) (right) demonstrating RNA interference-mediated knockdown of Sirt1 expression in mIMCD3 cells. Cells were transfected with a scrambled control siRNA or a Sirt1-specific siRNA. qRT-PCR data are mean ± S.D. *, p < 0.05 versus control siRNA, n = 5. B, qRT-PCR showing that siRNA knockdown of Sirt1 augments the expression of endogenous α-ENaC mRNA. mIMCD3 cells were transfected as in A. *, p < 0.05 versus control siRNA, n = 4. C, luciferase assay in mIMCD3 cell stable lines expressing the α-ENaC promoter-luciferase minigene (α-ENaC-Luc) that had been transiently transfected with control or Sirt1-specific siRNAs. Data are presented as mean ± S.D. of four independent experiments. *, p < 0.05 versus control siRNA.

FIGURE 3.

Sirt1 associates and colocalizes with Dot1. A, pEGFP-Dot1 or empty vector (Vec) was transiently transfected into HEK293 cells, and 36 h later, the cells were harvested for immunoprecipitation (IP) with anti-Sirt1 antibody or isotype control IgG. The precipitated immune complex was further analyzed by immunoblot (IB) with anti-EGFP to determine the expression level of Dot1. The blot is representative of three independent experiments (IgG_H, IgG heavy chain; IgG_L, IgG light chain). B, pRFP-Sirt1 and pEGFP-Dot1 were transiently co-transfected into mIMCD3 cells. Cells were fixed 24 h after transfection, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to localize nuclei. The expression and intracellular localization of red (Sirt1) and green (Dot1) fluorescence-tagged proteins were examined by laser scanning confocal microscopy. Images are representative of three independent experiments. Magnification, ×40.

FIGURE 4.

Sirt1 knockdown inhibits distributive H3K79 methylation in mIMCD3 cell chromatin associated with subregions of the α-ENaC 5′-flanking region. The subregions are schematized in the top left panel. mIMCD3 cells were transfected with a scrambled control siRNA or a Sirt1-specific siRNA. ChIP-qPCR analysis of mono- (K79me1), di- (K79me2), and tri-methylated (K79me3) H3K79 in mIMCD3 cells was then performed. Data are presented as mean ± S.D. (n = 4).

FIGURE 5.

Deacetylase independence of Sirt1-mediated inhibition of α-ENaC transcription in mIMCD3 cells. A, representative (n = 3) immunoblot showing that trichostatin A (TSA) treatment of mIMCD3 cells resulted in robust acetylation of p53, and that this was significantly abrogated by transfection with wild type Sirt1, but not the deacetylase-dead Sirt1H355Y mutant. This result established in vivo that the Sirt1H355Y mutant is indeed incompetent to deacetylate target substrate. B, the effects of transfection of Sirt1H355Y on the activity of an α-ENaC promoter-luciferase minigene stably integrated into the mIMCD3 cell genome was compared with that of wild type Sirt1 in the presence of vehicle or 1 μm aldosterone. *, p < 0.05 versus corresponding α-ENaC-Luc control, n = 4. C, nicotinamide, which inhibits both the deacetylase and ADP-ribosyltransferase activities of Sirt1, does not alter the ability of Sirt1 to inhibit α-ENaC-Luc activity. mIMCD3 cell lines expressing a stably incorporated α-ENaC promoter-Luc minigene were treated with or without 5 mm nicotinamide in the presence or absence of 1 μm aldosterone for 7 h. α-ENaC-Luc activity was then measured. Data are mean ± S.D., n = 4. D, luciferase activity (means ± S.D.) was measured in the stable α-ENaC promoter-Luc mIMCD3 cells that had been transiently transfected with empty vector or expression plasmids encoding full-length Sirt1, amino acids 1–240 of Sirt1, or amino acids 210–500 of Sirt1, and then treated with vehicle or aldosterone for 24 h. The value for the vector-transfected, vehicle-treated samples was set as 1 and utilized to determine the relative level and the significance of the other samples. *, p < 0.05 versus corresponding vector control, n = 4. The diagram above the graph depicts the protein domain structure of murine Sirt1.

FIGURE 6.

Aldosterone down-regulates Sirt1 mRNA expression and its association with chromatin at the α-ENaC promoter as it up-regulates α-ENaC promoter activity and endogenous α-ENaC mRNA expression. A, luciferase assay demonstrating time course of aldosterone-induced expression of a stably incorporated α-ENaC promoter-luciferase construct in mIMCD3 cells. The mIMCD3 cell lines harboring stably transfected pGL3Zeocin-1.3α-ENaC (α-ENaC-Luc) or its empty parent vector (pGL3-Basic) were grown in charcoal-stripped serum for 24 h, and then treated with or without aldosterone (1 μm) or vehicle for the indicated times. The relative luciferase activity of the vector-transfected cells was designated as 1 and utilized to determine the relative level and the significance of the other samples. *, p < 0.05 versus α-ENaC-Luc, n = 5. B, time course experiments showing the effects of treatment with 1 μm aldosterone for the indicated intervals on the expression of Sirt1 and α-ENaC mRNAs in mIMCD3 cells. The mRNAs for the two genes were measured from the same samples by qRT-PCR. *, p < 0.05 versus time = 0, n = 6. The representative immunoblot (IB) (n = 3), probed first with anti-Sirt1 antibody and then with anti-α-tubulin antibody as a loading and housekeeping reference, shows a similar down-regulation with aldosterone treatment. The Sirt1/tubulin ratio represents the results of scanning densitometry of the blots. C, aldosterone down-regulates Sirt1 association with specific regions of the α-ENaC promoter in vivo. mIMCD3 cells were treated with vehicle or 1 μm aldosterone for the indicated time periods, and ChIP assays were performed with anti-Sirt1 antibody or nonimmune IgG and primer sets designed to amplify sequential subregions of the α-ENaC 5′-flanking region (see Fig. 4). Representative (n = 3) agarose gels of the final PCR products are shown. IP, immunoprecipitation.

FIGURE 7.

MR independence of Sirt1 inhibition of aldosterone-induced α-ENaC transcription in mIMCD3 cells. A, the effect of transfection of Sirt1 on the activity of an α-ENaC promoter-luciferase minigene (α-ENaC-Luc) stably integrated into the mIMCD3 cell genome was measured in the presence and absence of vehicle, 1 μm aldosterone (Aldo), and/or 1 μm spironolactone as indicated. *, p < 0.05 versus corresponding aldosterone-treated α-ENaC promoter-luc control; **, p < 0.05 versus corresponding aldosterone-treated α-ENaC-Luc+pcDNA3.1-Sirt1, n = 6. B, Sirt1 overexpression does not affect the aldosterone (Aldo) (1 μm) inducibility of a co-transfected GRE-luciferase construct (GRE-Luc) in mIMCD3 cells. n = 4.