Sp1 trans-activates and is required for maximal aldosterone induction of the αENaC gene in collecting duct cells

Abstract

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.

Keywords: chromatin; epigenetic; gene expression; transcription factor.

Fig. 1.

Map of the R3 subregion of the αENaC 5′-flanking region. The nucleotide positions of the Sp1 and Af9 elements are schematized. The wild-type and mutated +222/+229 sequences used for EMSA and trans-activation assays are also indicated.

Fig. 2.

Sp1 protein binds to +222/+229 of the αENaC promoter. A: EMSA in which a biotin-labeled duplex probe corresponding to +208 to +240 of the mouse αENaC promoter was incubated with 10 μg of mouse inner medullary collecting duct (mIMCD)3 cell nuclear extracts alone (lane 1) or in the presence of a 40-fold molar excess of wild-type unlabeled probe (lane 2) or mutant unlabeled probe (lane 3). Gel is representative of 4 experiments. B: antibody interference assays were performed using 2 μg of nonimmune IgG or rabbit polyclonal antibodies against Sp1 or Sp3 (as a specificity control). Sp1 antibody decreased formation of complex A, whereas Sp3 antibody had no effect. Gel is representative of 4 experiments. C: mutations of the +222/+229 site as in Fig. 1 were introduced into pcDNA3.1-Zeo-1.3ENaCα-Luc to create pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc as described under materials and methods. Stable mIMCD3 cell lines harboring the pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (Δ+222/+229) constructs were established. Chromatin immunoprecipitation (ChIP)/qPCR experiments with anti-Sp1 antibody and primers to amplify the R3 subregion were conducted. The value for the final amplicon/input DNA obtained from the WT cells was set as 1, and that of the Δ+222/+229 was normalized to it. Error bars indicate ± SE. *P < 0.05 vs. WT; n = 3.

Fig. 3.

Mutation of the +222/+229 Sp1 binding site in the αENaC gene inhibits promoter activity. The stable mIMCD3 cell lines harboring the pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (Δ+222/+229) constructs established in Fig. 2 were assayed for luciferase activities and normalized to cell protein content. The value obtained for WT-transfected cells was set to 1. The values shown are the mean of triplicate determinations or 4 independent experiments. Error bars indicate ± SE. *P < 0.05 vs. WT.

Fig. 4.

Sp1 trans-activates αENaC via the +222/+229 Sp1 element. A: stable mIMCD3 cell lines harboring the pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) constructs in Fig. 2 were transiently transfected with an Sp1 expression vector or its gutless vector. After 24 h, the cells were treated with vehicle or aldosterone for 24 h, and then the firefly activities were measured and normalized to cell protein content. The value obtained for vehicle-treated, empty vector-transfected cells harboring pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) was set to 1, and the other conditions were normalized to it. The values shown are the mean of triplicate determinations or 4 independent experiments. Error bars indicate ± SE. *P < 0.05 vs. vehicle-treated, empty vector-transfected WT controls. **P < 0.05 vs. aldosterone-treated, empty vector-transfected WT controls. B: pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) and pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) cell lines were transiently transfected with control siRNA or Sp1-specific siRNA. After 24 h, the cells were treated with vehicle or aldosterone for 24 h, and then the firefly activities were measured and normalized to cell protein content. The value obtained for control siRNA-transfected cells harboring pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) was set to 1, and the other conditions were normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. *P < 0.05 vs. vehicle-treated, control siRNA-transfected. **P < 0.05 vs. aldosterone-treated, control siRNA-transfected.

Fig. 5.

Sp1 knockdown reduces αENaC mRNA expression in mIMCD3 cells. The cells were transfected with control siRNA or Sp1-specific siRNA. After 24 h, the cells were treated with vehicle or aldosterone for 24 h, and then total RNA was harvested, and αENaC and GAPDH mRNA levels were measured by qRT-PCR. The normalized ENaCα/GAPDH mRNA levels are presented. *P < 0.05 vs. vehicle-treated, control siRNA-transfected. **P < 0.05 vs. aldosterone-treated, control siRNA-transfected; n = 3.

Fig. 6.

Sequential ChIP analysis showing co-occupancy of Af9 and Sp1 at the R3 (−57/+438) subregion of the αENaC promoter. Chromatin from mIMCD3 cells was sequentially immunoprecipitated with the Af9 antibody or IgG (“First ChIP”), followed by re-ChIP with anti-Sp1 antibody or IgG (as a negative control). Reciprocal ChIP with Sp1 or IgG, followed by re-ChIP with Af9 or IgG, was also performed. Precipitated R3 subregion segments were detected using agarose gels (A: representative of 3 independent experiments) and quantified by qPCR (B: n = 3). Relative chromatin enrichment was calculated as described in materials and methods.

Fig. 7.

Reciprocal effects of Sp1 and Dot1a/Af9 on αENaC promoter occupancy and activity are independent. A: chromatin from mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Sp1 binding site mutant pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) was immunoprecipitated with the Af9 antibody or IgG. Precipitated R3 subregion segments were quantified by qPCR (n = 3). The value for Af9-IP amplicon/input DNA obtained from pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) was set to 1, and the value from pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected. B: chromatin from mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Sp1 binding site mutant pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) was immunoprecipitated with the Dot1a antibody or IgG. Precipitated R3 subregion segments were quantified by qPCR (n = 3). The value for Dot1a-IP amplicon/input DNA obtained from pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) was set to 1, and the value from pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected. C: mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Sp1 binding site mutant pcDNA3.1-Zeo-1.3ENaCα^{ΔSp1+222/+229}-Luc (ΔSp1^+222/+229) were transiently transfected with a EGFP-mDot1a expression vector or EGFP vector. After 24 h, the firefly activities were measured and normalized to cell protein content. The value obtained for EGFP-transfected cells was set to 1, and that of the pEGFP-mDOt1a-transfected cells was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected. D: mIMCD3 cells stably transfected with pcDNA3.1-Zeo-1.3ENaCα-Luc (WT) or the Af9 binding site mutant pcDNA3.1-Zeo-1.3ENaCα^ΔAf9+78/+92 were transiently transfected with empty vector or the Sp1 expression vector pBS-Sp1. After 24 h, the firefly activities were measured and normalized to cell protein content. The value obtained for vector-transfected cells was set to 1, and that of the pBS-Sp1-transfected cells was normalized to it. The values shown are the mean of triplicate determinations of 3 independent experiments. Error bars indicate ± SE. No statistically significant differences between group means were detected.

Fig. 8.

Aldosterone promotes Sp1 recruitment to the R3 (−57/+438) subregion of the αENaC promoter without altering Sp1 protein levels. A: mIMCD3 cells were cultured in DMEM/F12 plus 10% charcoal-stripped serum and then treated with vehicle, 1 μM aldosterone, 1 μM spironolactone, or 1 μM aldosterone and 1 μM spironolactone for 2 h as indicated. The cells were harvested and chromatin was prepared. ChIP/qPCR assays (n = 3) with the anti-Sp1 antibody and primers to amplify the R3 subregion. The value for the final amplicon/input DNA obtained from the vehicle-treated cells was set as 1, and the aldosterone treatment values were normalized to it. Error bars indicate ± SE. *P < 0.05 vs. corresponding vehicle control. B: immunoblots of nuclear and cytoplasmic extracts from the vehicle- and aldosterone-treated cells were probed with antibodies directed against Sp1 or P84 (as a nuclear protein marker). Densitometric analysis was performed and the Sp1/P84 ratio was calculated. The value for vehicle-treated cells was set as 1, and the aldosterone treatment values were normalized to it (n = 3).