Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.     

Heat shock induction of heme oxygenase mRNA in human Hep 3B hepatoma cells

Heat shock treatment of human Hep 3B hepatoma cells led to the induction of mRNA for microsomal heme oxygenase. The maximum induction of heme oxygenase mRNA (5----7-fold) was observed with treatment of cells at 43.5 degrees C, for 60 min. The heat-mediated induction of heme oxygenase mRNA was blocked by simultaneous treatment of cells with actinomycin D or cycloheximide. In contrast to Hep 3B cells, cells of another human hepatoma line, Hep G2, showed little induction of heme oxygenase mRNA by heat treatment. These findings suggest that heat shock treatment induces heme oxygenase mRNA in certain human hepatoma cells, but not in others.     

Human heme oxygenase cDNA and induction of its mRNA by hemin

Hemin treatment increased both activity and mRNA level of heme oxygenase in human macrophages. Using poly(A)-rich RNA prepared from human macrophages treated with hemin, we have constructed a cDNA library in the Okayama-Berg vector. The human heme oxygenase cDNA was isolated by screening this library with a rat cDNA and was subjected to nucleotide sequence analysis. The deduced human heme oxygenase is composed of 288 amino acids with a molecular mass of 32,800 Da. The homology in amino acid sequences between rat and human heme oxygenase is 80%. Like rat heme oxygenase, human enzyme has a putative membrane segment at its carboxyl terminus, which is probably essential for the insertion of heme oxygenase into endoplasmic reticulum. Both rat and human heme oxygenase have no cysteine residues. Recently we have shown that rat heme oxygenase is a heat-shock protein [J. Biol. Chem. 262, 12889-12892 (1987)], and therefore we examined the effects of heat treatment on the induction of heme oxygenase in human macrophages and glioma cells. In contrast to hemin treatment, heat treatment had no apparent effects in either human cell line on the activity of heme oxygenase and its mRNA levels. These results suggest that human heme oxygenase may not be a heat-shock protein.     

Induction of heme oxygenase mRNA by cobalt protoporphyrin in rat liver

The effect of cobaltic(III)-protoporphyrin on heme oxygenase activity and mRNA content was examined in vivo in the adult male rat liver. The activity of heme oxygenase, the rate-limiting enzyme in the degradation of heme, was enhanced, as expected, by cobalt protoporphyrin (25 mumol/kg body weight) in a time-dependent manner. Levels of enzyme activity were increased 2-fold by 8-16 h following treatment and were 6-fold higher than baseline values by 48 h. Administration of cobalt protoporphyrin resulted in a marked increase in heme oxygenase mRNA in the liver. Within 2 h of treatment, mRNA levels had increased 7.9-fold. The induction of heme oxygenase mRNA was maximal at 8 h when the levels were 58.5-fold above baseline. At every time point tested, the increase in heme oxygenase mRNA was several fold greater than that of enzyme activity.     

Regulation of heme metabolism in rat hepatocytes and hepatocyte cell lines: delta-aminolevulinic acid synthase and heme oxygenase are regulated by different heme-dependent mechanisms

Regulation of delta-aminolevulinic acid (ALA) synthase and heme oxygenase was analyzed in primary rat hepatocytes and in two immortalized cell lines, CWSV16 and CWSV17 cells. ALA synthase was induced by 4,6-dioxohepatnoic acid (4,6-DHA), a specific inhibitor of ALA dehydratase, in all three systems; however, the induction in CWSV17 cells was greater than in either of the other two systems. Therefore, CWSV17 cells were used to explore the regulation of both enzymes by heme and 4,6-DHA. Data obtained from detailed concentration curves demonstrated that 4,6-DHA induced the activity of ALA synthase once ALA dehydratase activity became rate-limiting for heme biosynthesis. Heme induced heme oxygenase activity with increases occurring at concentrations of 10 microM or greater. Heme blocked the 4,6-DHA-dependent induction of ALA synthase with an EC50 of 1.25 microM. Heme-dependent decreases of ALA synthase mRNA levels occurred more quickly and at lower concentrations than heme-dependent increases of heme oxygenase mRNA levels. ALA synthase mRNA remained at reduced levels for extended periods of time, while the increases in heme oxygenase mRNA were much more transient. The drastic differences in concentrations and times at which heme-dependent effects were observed strongly suggest that two-different heme-dependent mechanisms control the ALA synthase and heme oxygenase mRNAs. In CWSV17 cells, heme decreased the stability of ALA synthase mRNA from 2.5 to 1.3 h, while 4,6-DHA increased the stability of the mRNA to 5.2 h. These studies demonstrate that regulation of ALA synthase mRNA levels by heme in a mammalian system is mediated by a change in ALA synthase mRNA stability. The results reported here demonstrate the function of the regulatory heme pool on both ALA synthase and heme oxygenase in a mammalian hepatocyte system.     

Induction of heme oxygenase in rat liver. Increase of the specific mRNA by treatment with various chemicals and immunological identity of the enzymes in various tissues as well as the induced enzymes

A specific antibody was prepared against rat liver heme oxygenase which had been induced by bromobenzene treatment. Immunochemical studies with this antibody (IgG) revealed that heme oxygenases from livers of rats treated with hemin, Cd2+, Co2+, or bromobenzene from rat spleen and also from kidney of Sn2+-treated rats were all immunochemically identical. Cell-free synthesis of heme oxygenase was performed in a rabbit reticulocyte lysate system using polysomes isolated from livers of rats treated with either hemin, Cd2+, or bromobenzene, and it was found that translatable mRNA specific for heme oxygenase was actually increased in the liver of rats treated with any of those inducers. Also, the ability of liver polysomes to direct cell-free synthesis of heme oxygenase was apparently proportional to the activity of heme oxygenase in the liver from which polysomes were prepared. The heme oxygenase protein synthesized either in vivo or in vitro showed a molecular weight of 31,000 when examined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. This value is essentially identical with the molecular weight of heme oxygenase purified from rat liver and indicates that a precursor form of heme oxygenase may not be involved in the heme oxygenase synthesis.     

Expression of heme oxygenase and its RNA in mouse liver after injection of heme and splenectomy.

Heme is known to activate the HO (heme oxygenase) gene in cultured cells, but little is known about the effect of heme on the HO gene in intact organisms. The expressions of HO and its RNA in mouse liver were measured using mouse HO cDNA and HO antibody after injection of heme or splenectomy. The antibody was prepared against a beta-galactosidase-HO hybrid protein made in Escherichia coli. The HO mRNA level increased to a maximum 15 h after heme injection. In contrast, expression of HO was maximal about 45 h after heme injection. Essentially the same results were obtained in mice after splenectomy. These results suggest that the HO gene in mouse liver was activated by the injection of heme and splenectomy.     

Selenium antagonizes the induction of human heme oxygenase by arsenite and cadmium ions.

Effects of selenium compounds on the induction of heme oxygenase in human cells exposed to sodium arsenite or cadmium chloride have been investigated by an immunoblotting technique. Exposure of HeLa cells to arsenite or cadmium ions caused a marked increase in the synthesis of heme oxygenase, and the presence of sodium selenite suppressed the induction. DL-Selenocystine was an effective suppressor, and sodium selenate was less effective. DL-Selenomethionine had no effect. Northern blot analysis showed that selenite abolished the induction of heme oxygenase mRNA in the cells exposed to arsenite or cadmium ions. These results indicated that selenium antagonizes the induction of heme oxygenase by heavy metals ions.     

Fractionation of erytroblasts with affinitymediated modifications of their electrical properties using counter-current distribution

Enterally administered, heme is a good source of iron in humans and other animals, but the metabolism of heme by enterocytes has not been fully characterized. Caco-2 cells in culture provide a useful model for studying cells that resemble small intestinal epithelium, both morphologically and functionally. In this paper we show that heme oxygenase, the rate-controlling enzyme of heme catabolism, is present in abundance in Caco-2 cells, and that levels of its mRNA and activity can be increased by exposure of the cells to heme or metal ions (cadmium, cobalt). Caco-2 cells also contain biliverdin reductase activity which, in the basal state, is similar to that of heme oxygenase (approximately 40 pmole of product per mg protein per minute); however, when heme oxygenase is induced, biliverdin reductase may become rate-limiting for bilirubin production.Abbreviations BVR biliverdin reductase - DMEM Dulbecco's modified Eagles medium - DMSO dimethyl sulfoxide - HO heme oxygenase - 1xSSC a solution of 0.015 M sodium citrate/0.15 sodium chloride     

Activation of heme oxygenase and heat shock protein 70 genes by stress in human hepatoma cells

Effects of various stresses were examined on the accumulation of mRNA for microsomal heme oxygenase and a heat shock protein, hsp70, in three human hepatoma cell lines. By heat shock, hsp70 mRNA was induced in all three hepatoma lines, Hep G2, Hep 3B and Hep G2f, while heme oxygenase mRNA was increased only in Hep 3B. Time-courses of the heat shock induction of both mRNAs in Hep 3B were similar. Arsenite caused induction of both mRNAs in all three cell lines, while cadmium increased them in Hep G2 and Hep 3B, but not in Hep G2f cells. These findings suggest that, although both hsp70 and heme oxygenase are heat shock proteins, the mode of induction of mRNAs for these proteins is different.