Lipopolysaccharide activates transcription of the heme oxygenase gene in mouse M1 cells through oxidative activation of nuclear factor κB

It has been known for a long time that heme oxygenase (HO) is a key enzyme in heme catabolism, and it was found to act as an oxidative-stress protein to produce carbon monoxide; which has similar actions to those of nitrogen monoxide. We examined transcriptional control of the HO gene in mouse M1 (myeloleukemia) cells during treatment with lipopolysaccharide (LPS; an oxidative reagent), Since the promoter region of this gene in human cells contains a 12-O-tetradecanoyl-phorbol-13-acetate(TPA)-responsive element (TRE) and a nuclear-factor-κB-responsive element, HO mRNA expression might be regulated by an oxidative activation pathway. We investigated activation of the HO gene after treatment of M1 cells with LPS. Upon treatment with LPS, H₂O₂ was produced, the nuclear proto-oncogenes fos and jun were activated, then the HO gene was activated. The extent of transcriptional activation of the fos, jun and HO genes in M1 cells treated with LPS was strongly reduced by a scavenger of oxygen radicals (N-acetyl-L-cysteine), but a specific inhibitor of protein kinase C only reduced transcriptional activation by 10-20%. These results suggest that LPS may be an oxidative reagent. Some oxidative reagents (e.g., H₂O₂) are strong activators of NF-κB, and therefore we treated M1 cells with H₂O₂. Essentially the same extends of transcriptional activation of the fos, jun and HO genes were observed as those observed after LPS treatment. Super-shift assays with DNA that contained the TRE motif revealed that the Fos and Jun proteins from nuclei of M1 cells treated with LPS and H₂O₂ bound weakly to the TRE motif, and, in assays with DNA that contained the NF-κB motif, nuclear protein from M1 cells treated with H₂O₂ or LPS bound strongly to the NF-κB motif. These results strongly suggest that the HO gene in M1 cells is mainly activated by LPS through oxidative activation of NF-κB due to production of H₂O₂.