Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of
heme degradation and regulates the cellular
heme content. Gene expression of the inducible
isoform of HO, HO-1, is upregulated in response to various oxidative stress stimuli. To investigate the regulatory role of
anoxia and reoxygenation (A/R) on hepatic HO-1 gene expression, primary cultures of rat hepatocytes were exposed after an
anoxia of 4 hr to normal
oxygen tension for various lengths of time. For comparison, gene expression of the noninducible HO
isoform, HO-2, and that of the
heat-shock protein 70 (HSP70) were determined. During reoxygenation, a marked increase of HO-1 and HSP70 steady-state
mRNA levels was observed, whereas no alteration of HO-2
mRNA levels occurred. Corresponding to HO-1
mRNA, an increase of HO-1
protein expression was determined by Western blot analysis. The
anoxia-dependent induction of HO-1 was prevented by pretreatment with the transcription inhibitor,
actinomycin D, but not by the
protein synthesis inhibitor,
cycloheximide, suggesting a transcriptional regulatory mechanism. After exposure of hepatocytes to
anoxia, the relative levels of
oxidized glutathione increased within the first 40 min of reoxygenation. Pretreament of cell cultures with the
antioxidant agents,
beta-carotene and
allopurinol, before exposure to A/R led to a marked decrease of HO-1 and HSP70
mRNA expression during reoxygenation. An even more pronounced reduction of
mRNA expression was observed after exposure to
desferrioxamine. Taken together, the data demonstrate that HO-1 gene expression in rat hepatocyte cultures after A/R is upregulated by a transcriptional mechanism that may be, in part, mediated via the generation of ROS and the
glutathione system.