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Comparative Study
. 2018 Jan 1;114(1):65-76.
doi: 10.1093/cvr/cvx198.

Spironolactone-induced Degradation of the TFIIH Core Complex XPB Subunit Suppresses NF-κB and AP-1 Signalling

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Free PMC article
Comparative Study

Spironolactone-induced Degradation of the TFIIH Core Complex XPB Subunit Suppresses NF-κB and AP-1 Signalling

Jason M Elinoff et al. Cardiovasc Res. .
Free PMC article

Abstract

Aims: Spironolactone (SPL) improves endothelial dysfunction and survival in heart failure. Immune modulation, including poorly understood mineralocorticoid receptor (MR)-independent effects of SPL might contribute to these benefits and possibly be useful in other inflammatory cardiovascular diseases such as pulmonary arterial hypertension.

Methods and results: Using human embryonic kidney cells (HEK 293) expressing specific nuclear receptors, SPL suppressed NF-κB and AP-1 reporter activity independent of MR and other recognized nuclear receptor partners. NF-κB and AP-1 DNA binding were not affected by SPL and protein synthesis blockade did not interfere with SPL-induced suppression of inflammatory signalling. In contrast, proteasome blockade to inhibit degradation of xeroderma pigmentosum group B complementing protein (XPB), a subunit of the general transcription factor TFIIH, or XPB overexpression both prevented SPL-mediated suppression of inflammation. Similar to HEK 293 cells, a proteasome inhibitor blocked XPB loss and SPL suppression of AP-1 induced target genes in human pulmonary artery endothelial cells (PAECs). Unlike SPL, eplerenone (EPL) did not cause XPB degradation and failed to similarly suppress inflammatory signalling. SPL combined with siRNA XPB knockdown further reduced XPB protein levels and had the greatest effect on PAEC inflammatory gene transcription. Using chromatin-immunoprecipitation, PAEC target gene susceptibility to SPL was associated with low basal RNA polymerase II (RNAPII) occupancy and TNFα-induced RNAPII and XPB recruitment. XP patient-derived fibroblasts carrying an N-terminal but not C-terminal XPB mutations were insensitive to both SPL-mediated XPB degradation and TNFα-induced target gene suppression. Importantly, SPL treatment decreased whole lung XPB protein levels in a monocrotaline rat model of pulmonary hypertension and reduced inflammatory markers in an observational cohort of PAH patients.

Conclusion: SPL has important anti-inflammatory effects independent of aldosterone and MR, not shared with EPL. Drug-induced, proteasome-dependent XPB degradation may be a useful therapeutic approach in cardiovascular diseases driven by inflammation.

Keywords: Endothelial dysfunction; Inflammation; Proteasome; Pulmonary arterial hypertension; Xeroderma pigmentosum.

Figures

Figure 1
Figure 1
SPL suppresses NF-κB and AP-1 reporters independent of MR, AR, and GR in HEK293 cells. (A) Western blots of total cell lysate demonstrate human MR, AR, and GR overexpression in HEK293 cells 24 h post-transfection. Only GR had low, but detectable baseline expression. (B) SPL dose-dependently suppressed NF-κB and AP-1 reporter activity in the absence and presence of MR, AR, and GR (P < 0.0001 for the downward trend of slopes). Expression of AR and MR suppressed overall NF-κB signalling (P < 0.0001 and P = 0.004, respectively, for main effects without significant interactions with SPL). For AP-1 signalling, MR, AR and GR expression variably shifted SPL dose-response curves and altered their shapes, particularly at low concentrations (P ≤ 0.0004 for evidence of MR-, AR-, and GR-SPL interactions), but nevertheless SPL dose-dependent suppression of AP-1 was the overall predominant effect. (C) Unlike SPL, increasing doses of EPL in the absence and presence of MR had no effect on NF-κB reporter activity (P = 0.15) and only modestly suppressed AP-1 activity at concentrations ≤5 μM (P = 0.006), but not 10 μM (P = 0.42). Independent of EPL, MR expression alone modestly shifted NF-κB activity downward (P = 0.0006 for main effect) and AP-1 upward (P = 0.0003 for main effect). Twenty-four hours following transfection, cells were treated for 1 h with vehicle control or MR antagonist followed by stimulation with either TNFα (10 ng/ml; NF-κB activation) or PMA (100 nM; AP-1 activation) for 5 h. LUC activity was normalized to the renilla (REN) control. Luciferase results from five (NF-κB) and four (AP-1) independent experiments are presented relative to unstimulated control (mean ± SE).
Figure 2
Figure 2
SPL suppression of NF-κB and AP-1 is reversed by XPB overexpression. XPB overexpression (five independent experiments for each pathway) blocked the ability of SPL to suppress (A) TNFα-induced NF-κB reporter activation and (B) PMA-induced AP-1 reporter activation. LUC activity was normalized to REN control and results are presented as the geometric mean LUC/REN ratio (×100) ± geometric SE plotted on a log10 scale. Total cell lysates were collected concurrent with the timing of luciferase assay experiments, resolved by SDS-PAGE and immunoblotted for XPB and β-actin to demonstrate XPB overexpression in HEK293 cells. Representative Western blots are shown below the graphs. ****P < 0.0001 (ANOVA with post hoc tests, as indicated).
Figure 3
Figure 3
SPL suppression of inflammation in PAECs and the role of XPB degradation. (A) XPB protein expression, localized predominantly to the nucleus, was reduced following treatment with SPL. The effect of SPL on XPB (green) appeared similar in the absence or presence of TNFα stimulation for 2 h and was blocked by proteasome inhibition (MG132; 10 µM). Cell cytoplasm was stained with phalloidin (red) and nuclei were counterstained with DAPI (blue). Fluorescence images are representative of results from three different PAEC donors. Scale bars: 10 μm. (B) In PAECs, SPL (10 µM) significantly suppressed TNFα-induced secretion of IL8, IL6, and CCL2, while EPL (10 µM) had no effect on any of these cytokines (P ≥ 0.77 for the EPL main effect across all three time points). Data are presented as the log10-transformed mean concentration ± SE of four independent experiments using different donors. Multiplex cytokine analysis was performed on cell supernatant collected at 4, 8, and 24 h after TNFα (5 ng/ml) stimulation. **P < 0.01; ****P < 0.0001 for the effect of SPL at 4, 8, and 24 h, respectively; #P ≤ 0.0002 for the SPL main effect, when similar across all three time points. See Supplementary material online, Table S1 for the results of all cytokines analysed. Consistent with findings by immunofluorescence, (C) MG132 blocked SPL-induced XPB degradation in PAECs as determined by total cell lysate Western blots. In contrast to SPL, EPL had no effect on XPB protein levels in the absence or presence of MG132 (P ≥ 0.30 for both). Densitometric quantification of XPB protein expression relative to β-actin is presented as the geometric mean ratio ± geometric SE on log10 scale of four independent experiments using different donors. A representative Western blot is shown below the graph. (D) MG132 significantly blocked the effect of SPL on PMA-induced PTGS2 and INHBA mRNA expression. PAECs were treated with SPL for 1 h followed by stimulation with PMA (10 nM) for 4 h. Expression of mRNA measured by quantitative real-time PCR is presented as fold-change relative to unstimulated cells (geometric mean ± geometric SE) of five independent experiments, each with a different donor, plotted on log10 scale. *P < 0.05; **P < 0.01; ****P < 0.0001 (ANOVA with post hoc tests, as indicated).
Figure 4
Figure 4
SPL in combination with XPB knockdown further suppresses TNFα target genes in PAECs. (A) XPB knockdown and SPL treatment both significantly reduced XPB protein levels, but the impact of SPL was significantly greater and similar in control siRNA (siCTRL) and XPB siRNA transfected PAECs (P = 0.27 for the interaction between SPL and XPB knockdown). Densitometric quantification of XPB protein expression relative to β-actin is presented as the geometric mean ratio ± geometric SE on log10 scale of four independent experiments, each with a different donor. A representative Western blot is shown below the graph. (B) SPL suppressed TNFα-induced IL8, IL6, and CCL2, but not NFKBIA (P = 0.39) mRNA expression in PAECs transfected with siCTRL. XPB knockdown alone in PAECs did not suppress, but rather modestly increased TNFα-induced IL8 (P = 0.03), IL6 (P = 0.003), CCL2 (P = 0.005), and NFKBIA (P = 0.008) expression levels compared with siCTRL. However, compared with SPL alone, the combination of XPB knockdown and SPL had the strongest suppressive effect on TNFα-induced IL8, IL6, CCL2, and NFKBIA. Expression of mRNA measured by quantitative real-time PCR is presented as the fold-change relative to unstimulated cells transfected with siCTRL (geometric mean ± geometric SE) of four independent experiments, each with a different donor, plotted on log10 scale. **P < 0.01; ***P < 0.001; ****P ≤ 0.0001 (ANOVA with post hoc tests, as indicated).
Figure 5
Figure 5
Target gene susceptibility to SPL-mediated suppression is associated with low basal levels of RNAPII occupancy and greater TNFα-induced recruitment. (A) TNFα stimulation significantly increased total RNAPII recruitment to the IL8 promoter region in PAECs, an effect that was suppressed by SPL treatment. In contrast, TNFα stimulation only modestly and non-significantly (P = 0.14) increased total RNAPII occupancy at the NFKBIA promoter, where basal RNAPII occupancy was already significantly higher compared with basal RNAPII occupancy at the IL8 promoter (inset). (B) Similar to total RNAPII, active pSer5-RNAPII occupancy at the IL8 promoter region was significantly increased following TNFα stimulation and suppressed by SPL treatment. However again, TNFα stimulation did not significantly increase pSer5-RNAPII occupancy at the NFKBIA promoter (P = 0.78) where basal pSer5-RNAPII occupancy was already significantly higher compared with the IL8 promoter (inset). (C) Although basal XPB occupancy was similar at the IL8 and NFKBIA promoter regions (P = 0.80; inset), TNFα stimulation significantly increased XPB recruitment to the IL8 but not the NFKBIA promoter region (P = 0.17). Similar to RNAPII and pSer5-RNAPII, XPB recruitment to the IL8, but not the NFKBIA promoter region was significantly suppressed by SPL. PAECs were treated with SPL (10 μM) or vehicle control (CTRL) for 2 h and then stimulated with TNFα (5 ng/ml) or vehicle control for 1 h. Precipitated protein/DNA complexes were eluted and crosslinks reversed for DNA purification, followed by quantitative real-time PCR analysis. RNAPII, pSer5-RNAPII, and XPB promoter enrichment from five independent experiments, each with a different donor were calculated as a percentage of input DNA normalized to unstimulated cells (mean ± SE). RNAPII, pSer5-RNAPII, and XPB basal NFKBIA promoter occupancy (inset) from five independent experiments, each with a different donor was normalized to CTRL IL8 occupancy (mean ± SE). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Figure 6
Figure 6
XP patient-derived fibroblasts harbouring an N-terminal, but not C-terminal XPB mutations were insensitive to SPL. (A) F99S and Q545X + Q739insX42 mutant fibroblasts expressed lower levels of XPB compared with control fibroblasts from an unaffected parent of the F99S patient. SPL induced XPB degradation in both normal fibroblasts and those expressing C-terminal mutations in XPB. In contrast, F99S mutant fibroblasts were resistant to SPL-mediated XPB degradation. Densitometric quantification of XPB protein expression relative to β-actin is presented as the geometric mean ratio ± geometric SE on log10 scale of five independent experiments. A representative Western blot is shown below the graph. (B) In contrast to the unaffected parent, F99S mutant fibroblasts were also resistant to SPL-mediated suppression of IL8 and IL6. TNFα-induced CCL2 and NFKBIA gene transcription was not significantly suppressed by SPL in either normal or F99S mutant fibroblasts. However, in fibroblasts heterozygous for the compound C-terminal mutations (Q545X + Q739insX42), SPL synergistically suppressed TNFα-induced IL8, IL6, CCL2, and NFKBIA. Fibroblasts were treated with SPL for 1 h followed by stimulation with TNFα (10 ng/ml) for 4 h. Expression of mRNA measured by quantitative real-time PCR is presented as the fold-change relative to unstimulated normal fibroblasts (geometric mean ± geometric SE) of five independent experiments, plotted on log10 scale. *P < 0.05; **P < 0.01; ***P < 0.001; ****P ≤ 0.0001 (ANOVA with post hoc tests, as indicated).
Figure 7
Figure 7
SPL treatment decreases XPB in the MCT-PH rat lung and is associated with lower serum concentrations of inflammatory markers in PAH patients. (A) Western blots of homogenized whole lung tissue demonstrated reduced XPB protein levels in MCT-PH rats treated with SPL (40 mg/kg/d) for 14 days (n = 9) compared with either control rats (n = 6) or MCT-PH rats treated with placebo (n = 8). Densitometric quantification of XPB protein expression relative to GAPDH is presented on log10 scale for each animal as well as the geometric mean ± geometric SE. A representative Western blot is shown below. (B) In a multivariate analysis, treatment with SPL was associated with an overall trend toward reduced serum cytokine concentrations in an observational cohort of PAH patients [mean (95% CI) across all cytokines: −29.3% (−50.8, 1.5); P = 0.06]. In contrast, treatment with ET-1 receptor antagonists [24.3% (−11.4, 74.4); P = 0.21], and PDE5 inhibitors [−10.5 (−40.2, 34.0); P = 0.59] did not alter cytokine levels. Discordant effects on individual cytokines precluded a meaningful aggregate estimate of effect size for PGI2 infusions. (C) Effects of SPL, ET-1 receptor antagonists, PGI2 infusions, and PDE5 inhibitors on individual cytokines are depicted as a heatmap. Orange indicates percent increase and blue percent decrease in serum cytokine concentrations. (D) Directional effects of SPL treatment on measured cytokines in PAH patients compared with TNFα-stimulated PAECs in vitro revealed a substantial degree of concordance. ↓:≤15% decrease; ↓↓: 16–30% decrease; ↓↓↓: 31–50% decrease; ↓↓↓↓:>50% decrease; ↑:≤15% increase; *P < 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. For significant interactions between SPL treatment and time in vitro, the largest effect is reported, otherwise main effects are used. Analytes not depicted were either not induced by TNFα in vitro (FGFb and IL10) or were otherwise not detected in vitro (Eotaxin, IL2, IL5) or in vivo (IL1β, IL5, and IL15). See Supplementary material online for exclusion criteria. aUsed to stimulate cells. bComponent of cell culture media.

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