Withaferin A Induces Heme Oxygenase (HO-1) Expression in Endothelial Cells via Activation of the Keap1/Nrf2 Pathway

Biochem Pharmacol. 2016 Jun 1;109:48-61. doi: 10.1016/j.bcp.2016.03.026. Epub 2016 Apr 1.

Abstract

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.

Keywords: Antioxidant; Endothelial cell; Molecular pharmacology; Phytochemical; Signal transduction; Transcription regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • Cell Survival / drug effects
  • Crystallography, X-Ray
  • Gene Expression Profiling
  • Gene Expression Regulation / drug effects*
  • HEK293 Cells
  • Heme Oxygenase-1 / genetics*
  • Heme Oxygenase-1 / metabolism
  • Human Umbilical Vein Endothelial Cells / cytology
  • Human Umbilical Vein Endothelial Cells / drug effects
  • Human Umbilical Vein Endothelial Cells / metabolism
  • Humans
  • Hybridomas / cytology
  • Hybridomas / drug effects
  • Hybridomas / metabolism
  • Kelch-Like ECH-Associated Protein 1 / chemistry
  • Kelch-Like ECH-Associated Protein 1 / genetics*
  • Kelch-Like ECH-Associated Protein 1 / metabolism
  • Metabolic Detoxication, Phase II / genetics
  • Molecular Docking Simulation
  • NF-E2-Related Factor 2 / agonists
  • NF-E2-Related Factor 2 / antagonists & inhibitors
  • NF-E2-Related Factor 2 / genetics*
  • NF-E2-Related Factor 2 / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Protein Binding
  • Protein Domains
  • Protein Structure, Secondary
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction
  • Transcription, Genetic
  • Withanolides / chemistry
  • Withanolides / pharmacology*

Substances

  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • RNA, Small Interfering
  • Withanolides
  • HMOX1 protein, human
  • Heme Oxygenase-1
  • withaferin A