Spironolactone-induced degradation of the TFIIH core complex XPB subunit suppresses NF-κB and AP-1 signalling

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

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Anti-Inflammatory Agents / pharmacology*
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Disease Models, Animal
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism
  • Eplerenone / pharmacology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • HEK293 Cells
  • Humans
  • Hypertension, Pulmonary / drug therapy*
  • Hypertension, Pulmonary / genetics
  • Hypertension, Pulmonary / metabolism
  • Hypertension, Pulmonary / physiopathology
  • Inflammation Mediators / metabolism*
  • Lung / drug effects
  • Lung / metabolism
  • Mineralocorticoid Receptor Antagonists / pharmacology*
  • Mutation
  • NF-kappa B / genetics
  • NF-kappa B / metabolism*
  • Proteasome Endopeptidase Complex / metabolism
  • Proteolysis
  • Pulmonary Artery / drug effects*
  • Pulmonary Artery / metabolism
  • Pulmonary Artery / physiopathology
  • RNA Polymerase II / metabolism
  • Rats, Sprague-Dawley
  • Retrospective Studies
  • Signal Transduction / drug effects*
  • Spironolactone / pharmacology*
  • Transcription Factor AP-1 / genetics
  • Transcription Factor AP-1 / metabolism*
  • Transcription Factor TFIIH / genetics
  • Transcription Factor TFIIH / metabolism*

Substances

  • Anti-Inflammatory Agents
  • DNA-Binding Proteins
  • Inflammation Mediators
  • Mineralocorticoid Receptor Antagonists
  • NF-kappa B
  • Transcription Factor AP-1
  • XPBC-ERCC-3 protein
  • Transcription Factor TFIIH
  • Spironolactone
  • Eplerenone
  • RNA Polymerase II
  • Proteasome Endopeptidase Complex
  • DNA Helicases