TLR4 activation alters labile heme levels to regulate BACH1 and heme oxygenase-1 expression in macrophages

Free Radic Biol Med. 2019 Jun:137:131-142. doi: 10.1016/j.freeradbiomed.2019.04.024. Epub 2019 Apr 24.

Abstract

Heme oxygenase (HO)-1, a stress-inducible enzyme that converts heme into carbon monoxide (CO), iron and biliverdin, exerts important anti-inflammatory effects in activated macrophages. HO-1 expression is mainly governed by a mutual interplay between the transcriptional factor NRF2 and the nuclear repressor BTB and CNC homology 1 (BACH1), a heme sensor protein. In the current study we hypothesized that alterations in the levels of intracellular labile heme in macrophages stimulated by lipopolysaccharide (LPS), a prototypical pro-inflammatory Toll-like receptor (TLR)4 agonist, are responsible for BACH1-dependent HO-1 expression. To this end, labile heme was determined in both mouse bone marrow-derived macrophages (mBMDMs) and human monocyte-derived macrophages (hMDMs) using an apo-horseradish peroxidase-based assay. We found that LPS raised the levels of labile heme, depressed BACH1 protein and up-regulated HO-1 in mBMDMs. In contrast, in hMDMs LPS decreased labile heme levels while increasing BACH1 expression and down-regulating HO-1. These effects were abolished by the TLR4 antagonist TAK-242, suggesting that TLR4 activation triggers the signaling cascade leading to changes in the labile heme pool. Studies using mBMDMs from BACH1-/- and NRF2-/- mice revealed that regulation of HO-1 and levels of labile heme after LPS stimulation are strictly dependent on BACH1, but not NRF2. A strong interplay between BACH1-mediated HO-1 expression and intracellular levels of labile heme was also confirmed in hMDMs with siRNA knockdown studies and following inhibition of de novo heme synthesis with succinylacetone. Finally, CORM-401, a compound that liberates CO, counteracted LPS-dependent down-regulation of HO-1 and restored levels of labile heme in hMDMs. In conclusion, alterations of labile heme levels in macrophages following TLR4 stimulation play a crucial role in BACH1-mediated regulation of HO-1 expression.

Keywords: BACH1; Carbon monoxide; Heme; Heme oxygenase-1; Inflammation; Lipopolysaccharide; Macrophages.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Basic-Leucine Zipper Transcription Factors / genetics
  • Basic-Leucine Zipper Transcription Factors / metabolism*
  • Cells, Cultured
  • Gene Expression Regulation
  • Heme / metabolism
  • Heme Oxygenase-1 / genetics
  • Heme Oxygenase-1 / metabolism*
  • Humans
  • Inflammation / metabolism*
  • Lipopolysaccharides / immunology
  • Macrophages / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism*
  • Signal Transduction
  • Sulfonamides / pharmacology
  • Toll-Like Receptor 4 / antagonists & inhibitors
  • Toll-Like Receptor 4 / metabolism*

Substances

  • BACH1 protein, human
  • Bach1 protein, mouse
  • Basic-Leucine Zipper Transcription Factors
  • Lipopolysaccharides
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • Sulfonamides
  • Toll-Like Receptor 4
  • ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate
  • Heme
  • Heme Oxygenase-1