Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5' distal enhancers: role of reactive oxygen intermediates and AP-1

Am J Respir Cell Mol Biol. 1998 Feb;18(2):226-34. doi: 10.1165/ajrcmb.18.2.2910.


Heme oxygenase-1 (HO-1) is a stress-response protein, the expression of which is transcriptionally regulated by agents that cause oxidative stress. We have previously shown that lipopolysaccharide (LPS)-induced HO-1 gene transcription in RAW 264.7 macrophage cells is mediated by a distal enhancer called SX2, located 4 kb upstream from the HO-1 transcription initiation site (Am. J. Respir. Cell Mol. Biol. 1995;13:387-398). We have recently identified a second distal enhancer, called AB1, located 6 kb upstream from the SX2 distal enhancer (J. Biol. Chem. 1995;270:11977-11984). Here we report the extension of our studies to investigate whether the AB1 distal enhancer and/or other potential regulatory elements in the entire 5' distal flanking sequences (11-kb region) of the HO-1 gene may also mediate HO-1 gene transcription in response to LPS. Using deletional analysis, we found that the AB1 enhancer also mediates LPS-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer and electrophoretic-mobility-shift assays of nuclear extracts from LPS-treated cells further demonstrated that the transcription factor activator protein-1 (AP-1) is critical for AB1-mediated HO-1 gene activation by LPS. We also found increased expression of AP-1 family members c-fos and c-jun by Northern blot analyses after treatment with LPS. Further, we observed that LPS-treated RAW 264.7 cells produced high levels of reactive oxygen intermediates (ROI) as measured through flow-cytometric analysis of dichlorofluoroscein (DCF)-stained cells. Treatment of cells with the antioxidants N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide (DMSO) not only blunts LPS-induced production of ROI, but also significantly attenuates LPS-induced HO-1 messenger RNA (mRNA) expression and gene transcription. Taken together, these data suggest that LPS regulates HO-1 gene transcription in part by inducing the production of ROI, which initiate signal-transduction pathway(s) leading to the activation of AP-1-dependent HO-1 gene transcription.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcysteine / pharmacology
  • Animals
  • Antioxidants / pharmacology
  • Cell Extracts
  • Cell Line
  • Cell Nucleus
  • Dimethyl Sulfoxide / pharmacology
  • Enhancer Elements, Genetic / genetics*
  • Gene Expression Regulation, Enzymologic / genetics
  • Genes, fos / genetics
  • Genes, jun / genetics
  • Heme Oxygenase (Decyclizing) / genetics*
  • Heme Oxygenase-1
  • Lipopolysaccharides / pharmacology*
  • Macrophages
  • Membrane Proteins
  • Mice
  • RNA, Messenger / analysis
  • Reactive Oxygen Species / physiology*
  • Regulatory Sequences, Nucleic Acid / genetics
  • Sequence Deletion
  • Transcription Factor AP-1 / physiology
  • Transcriptional Activation / drug effects
  • Transcriptional Activation / genetics*


  • Antioxidants
  • Cell Extracts
  • Lipopolysaccharides
  • Membrane Proteins
  • RNA, Messenger
  • Reactive Oxygen Species
  • Transcription Factor AP-1
  • Heme Oxygenase (Decyclizing)
  • Heme Oxygenase-1
  • Hmox1 protein, mouse
  • Acetylcysteine
  • Dimethyl Sulfoxide