Attallah Kappas

Heme oxygenase (HO) is the rate-limiting enzyme in heme catabolism and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. Heme treatment increased both HO activity and HO-1 mRNA in the human erythroleukemic cell line K562. Electrophoretic mobility-shift assays of nuclear protein extracts from heme-treated and control cells with specific oligonucleotide probes containing binding sites for known transcription factors, including AP-1, AP-2, Sp1, NF-kappa B, CTF/NF1, TFIID, OKT1, and CREB, and oligonucleotides containing serum-, metal-, and glucocorticoid-responsive elements demonstrated a specific and marked increase in the NF-kappa B and AP-2 transcription factors and, to a lesser extent, an increase in AP-1. No significant increase in other transcription factors over the control, untreated cells was observed. DNase I footprint assays using purified transcription factors revealed the presence of NF-kappa B and AP-2 binding sites in the proximal part of the promoter region of the human HO-1 gene. Moreover, nucleotide sequence analysis of the HO-1 promoter region showed that the protected regions encompassed NF-kappa B and AP-2 consensus binding sites. The presence of regulatory sequences for the binding of transcription factors such as NF-kappa B and AP-2, whose activation is associated with the immediate response of the cell to an injury, may be an indication of the important role which HO-1 may play in defense mechanisms against tissue injury.

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an approximately 3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with > 85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Heme is essential for cell respiration, energy generation, and oxidative biotransformations. The latter function is exemplified by the oxidative metabolism of various endogenous and exogenous chemicals catalyzed by the heme protein cytochrome P-450. Recent studies have established that metal ions directly regulate cellular content of heme, and thus of heme proteins by controlling production of delta-aminolevulinate synthetase and heme oxygenase, the rate-limiting enzymes for heme synthesis and degradation, respectively. Metal ions also alter cellular content of glutathione. In excess amounts, metal ions greatly accelerate the turnover and degradation of heme and substantially impair the oxidative functions of cells--particularly those dependent on cytochrone P-450. As a result, the biological impact of chemicals which are detoxified or metabolically transformed by the P-450 system is greatly altered.