Human Heme Oxygenase-1 Gene Research Articles

Regulation of expression of the human heme oxygenase-1 gene in transfected chick embryo liver cell cultures

Induction of heme oxygenase (HO) has been proposed as a protective cellular mechanism against oxidative damage. In previous work (Tyrrell et al., Carcinogenesis [1993] 14, 761–765), portions of the 5′ promoter region of the human HO-1 gene linked to the reporter gene chloramphenicol acetyl transferase (CAT), had been transiently expressed in HeLa cells. To extend the study of human HO gene expression into primary liver cells, these reporter gene fusion constructs, containing 121 or 1416 base pairs of the untranscribed 5′-upstream sequences of the human HO-1 gene, were used along with pSV β-Gal plasmid to dually transfect primary cultures of chick embryo liver cells (CELC). The transfected cells were treated with selected metals, heme, phorbol ester, and chemical agents that produce oxidative stress (H 2O 2 or sodium arsenite). Reporter gene activities were measured 18–20 h later. Our major findings are: (1) these HO-CAT constructs were expressed in CELC; (2) unlike HeLa cells, the expression of CAT was detected in CELC without the need for the SV40 enhancer; (3) sodium arsenite and cobalt chloride induced the expression of the HO-CAT constructs whereas heme had no effect on or decreased CAT expression for all of the transfected constructs; (4) study of endogenous chick HO-1 gene expression in CELC showed that HO-1 responded to sodium arsenite treatment in a dose-dependent fashion, and the response was rapid and transient. We conclude that, in chick liver cell cultures, induction of the HO-1 gene by heme is fundamentally different from that produced by transition metals or sodium arsenite. Furthermore, the results suggest that expression of the HO-1 gene is highly conserved across species. © 1997 Elsevier Science B.V. All rights reserved.

Induction of Heme Oxygenase 1 by Hyperoxia is Enhanced with Iron in Human Pulmonary Endothelial Cells † 1830

High concentrations of oxygen are often a necessary and lifesaving therapy, however, these high concentrations are also toxic adding to the overall tissue injury. Early lung damage is associated with endothelial leakage with increased pulmonary edema, protein leak, and hemorrhage. Hyperoxia-induced damage is thought to be from an excess production of toxic reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in response to a variety of stressess including oxidants. We and others have recently identified HO-1as a hyperoxia-inducible gene, however, the increase in response to hyperoxia is relatively low as compared to other stimulants for HO-1. In this study we evaluated the response of HO-1 in isolated cultures of human pulmonary artery endothelial cells (HPAEC) in response to hyperoxia alone and in the presence of iron. HPAEC isolated from segments of PA from heart transplant donors grown to confluence were loaded with iron by exposure to 1 μM FeSO4 and 1 μM 8-hyroxyquinolone. Iron loaded and non-loaded HPAEC were exposed to either room air or 95% oxygen for 4 or 24 h. Total RNA was isolated and evaluated by Northern analysis utilizing a radiolabeled human HO-1 cDNA. We observed a 2-3 fold increase in HO-1 mRNA levels upon exposure to hyperoxia, and 2-4 fold in the iron loaded cells as compared to control cells. Upon exposure to hyperoxia in the iron loaded cells, there was an increase in HO-1 mRNA levels of 10 to 15 fold. A 4.5 kb promoter region of the human HO-1 gene construct was generated using the promoterless human growth hormone plasmid (pOGH). Transient transfection of this construct in HPAEC confered basal expression of the promoter region. There was no significant increase in secreted human growth hormone (reporter gene expression) in response to hyperoxia, iron, or iron plus hyperoxia as compared to heme (a potent stimulus for HO-1). This study indicates that hyperoxia induction of HO-1 in human cells is enhanced by the presence of iron, and begins to determine the regulatory mechanisms of HO-1 induction in response to a hyperoxic insult in HPAEC. The greater induction with iron could result from the production of a stronger oxidant in the presence of iron or a synergistic effect on the HO-1 gene with increased expression. Funded by ALA-FL and CMNT

Regulation of Human Heme Oxygenase-1 Gene Expression Under Thermal Stress

Heme oxygenase-1 is an essential enzyme in heme catabolism, and its human gene promoter contains a putative heat shock element (HHO-HSE). This study was designed to analyze the regulation of human heme oxygenase-1 gene expression under thermal stress. The amounts of heme oxygenase-1 protein were not increased by heat shock (incubation at 42 degrees C) in human alveolar macrophages and in a human erythroblastic cell line, YN-1-0-A, whereas heat shock protein 70 (HSP70) was noticeably induced. However, heat shock factor does bind in vitro to HHO-HSE and the synthetic HHO-HSE by itself is sufficient to confer the increase in the transient expression of a reporter gene upon heat shock. The deletion of the sequence, located downstream from HHO-HSE, resulted in the activation of a reporter gene by heat shock. These results suggest that HHO-HSE is potentially functional but is repressed in vivo. Interestingly, heat shock abolished the remarkable increase in the levels of heme oxygenase-1 mRNA in YN-1-0-A cells treated with hemin or cadmium, in which HSP70 mRNA was noticeably induced. Furthermore, transient expression assays showed that heat shock inhibits the cadmium-mediated activation of the heme oxygenase-1 promoter, whereas the HSP70 gene promoter was activated upon heat shock. Such regulation of heme oxygenase-1 under thermal stress may be of physiologic significance in erythroid cells.

Downregulation of the Human Heme Oxygenase Gene by Glucocorticoids and Identification of 56b Regulatory Elements

Several human heme oxygenase-1 promoter-driven chloramphenicol acetyltransferase constructs were examined in order to analyze promoter activity of the heme oxygenase-1 gene in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by interleukin-6. This induction was shown to be down-regulated by glucocorticoids. Chloramphenicol acetyltransferase assays revealed that the promoter region (56 base pair) between −180 and −120 was responsible for up-regulation by growth factors, as well as for glucocorticoid-directed down-regulation. The same DNA fragment was shown to bind nuclear factor(s) from endothelial cells treated with dexamethasone. Formation of DNA protein complexes peaked after a 6-hour treatment. The DNA fragment was found to contain a sequence recognized by the STAT 3/acute phase response factor.

Lipid metabolite involvement in the activation of the human heme oxygenase-1 gene

Cellular effects of ultraviolet A (UVA) radiation include peroxidation of membrane lipids as well as a decrease in intracellular glutathione. We have investigated whether damage to membrane lipids is involved in the activation of the human heme oxygenase-1 gene by UVA. Irradiation of human skin fibroblasts in the presence of the lipophilic antioxidants, butylated hydroxytoluene and a-tocopherol, enhances the UVA-induced HO-1 mRNA accumulation, suggesting that peroxidation of plasma membrane lipids is not involved. Furthermore, sodium ascorbate, which induces lipid peroxidation mainly in the plasma membrane, induces HO-1 mRNA to low levels only. The decrease in GSH by UVA radiation is not affected by the presence of the lipophilic antioxidants while ascorbate treatment increases the intracellular GSH by twofold above controls. These results indicate that peroxidation of internal membrane lipids, a decrease in the intracellular GSH levels and the integrity of the plasma membrane are all important for the UVA-induction of heme oxygenase-1. Both nonenzymatic as well as enzymatic lipid peroxidation metabolites are inducers of heme oxygenase-1. The nonenzymatic lipid peroxidation product 4-hydroxynonenal induces heme oxygenase-1 mRNA up to 40-fold and the phospholipase metabolites diacylglycerol and arachidonic acid induce this mRNA by three-to sixfold above basal levels. We also demonstrate that the cyclooxygenase metabolites of arachidonic acid are important for the UVA-activation of the heme oxygenase-1 gene.

Regulation of human heme oxygenase-1 gene in transiently transfected primary cultures of chicken embryo liver cells

Regulation of human heme oxygenase-1 gene in transiently transfected primary cultures of chicken embryo liver cells

Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity.

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.

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Differential Control of the Metal-Mediated Activation of the Human Heme Oxygenase-1 and Metallothionein IIa Genes

Heme oxygenase-1, an essential enzyme in heme catabolism, and metallothionein IIA, a small metal-binding protein with clusters of cysteins, are remarkably induced in HeLa cells following the treatment with cadmium or zinc. Both proteins are considered to be involved in the defense system against metal toxicity. Here we showed by transient expression assays that the cadmium-responsive element (CdRE) of the human heme oxygenase-1 gene is not responsive to zinc, whereas a metal-regulatory element (MRE) of the human metallothionein IIA gene is able to respond to either cadmium or zinc. The CdRE is recognized by a certain nuclear protein(s) which is however unable to bind to an MRE of the metallothionein IIA gene. These results suggest that the metal-selective activation of each gene promoter is mediated by a separate mechanism.

Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene.

Heme oxygenase is an essential enzyme in heme catabolism and is inducible by various environmental derangements such as cadmium. The activity and mRNA levels of heme oxygenase were remarkably increased in HeLa cells by the treatment with cadmium. As a first step in studying the molecular mechanisms of this induction, we performed transient expression assays in four human cell lines including HeLa to analyze the cadmium-mediated inducibility of the fusion genes, containing the firefly luciferase gene as a reporter under the human heme oxygenase gene promoter. By determining the luciferase activity expressed in the transfected cells, we found the region between about 4.5 and 4 kilobase pairs upstream from the transcriptional initiation site of the heme oxygenase gene which confers cadmium-mediated inducibility on the fusion gene. The region was then subjected to further functional analysis in HeLa cells, which allowed us to localize the cadmium-responsive element to 20 base pairs. Gel mobility shift assays demonstrated that this 20-base pair element is specifically bound by nuclear protein(s) of HeLa cells, the binding activities of which were however unchanged by the treatment with cadmium. Using the synthetic cadmium-responsive elements containing various base changes, we have identified a 10-base pair sequence, TGCTAGATTT, required for the cadmium-mediated inducibility and in vitro protein binding. We thus suggest that this binding protein(s) is involved in the cadmium-mediated activation of the heme oxygenase gene. Incidentally, the consensus sequence of AP-1 binding site, TGAGTCA, is present downstream of this cadmium-responsive element. However, we provide evidence that AP-1 is not directly involved in the cadmium-mediated induction of the human heme oxygenase gene.

Mapping of the human gene for inducible heme oxygenase to chromosome 22q12.

Heme oxygenase is an essential enzyme in heme catabolism that cleaves heme to form biliverdin, releasing carbon monoxide and iron. There are two isozymes of heme oxygenase: heme oxygenase 1 (HO-1) and heme oxygenase 2, each of which is encoded by a separate gene. It is noteworthy that HO-1 is inducible by various environmental factors, while heme oxygenase 2 is not. Here we have localized the human HO-1 gene to chromosome 22 by polymerase chain reaction (PCR) analysis of human-hamster somatic cell hybrids. The precise region of the HO-1 locus was then determined by fluorescence in situ hybridization, revealing that the human HO-1 gene is localized to 22q12.

18] Transient enhancement of heme oxygenase 1 mRNA accumulation: A marker of oxidative stress to eukaryotic cells

Publisher Summary Bacteria respond to oxidative stress by the rapid and transient expression of a large number of genes. This chapter describes two major regulatory pathways—namely, the oxyR and soxR systems. The oxyR system regulates nine genes induced by hydrogen peroxide in both Escherichia coli and Salmonella typhimurium. Several oxyR-controlled genes (for example, catalase, glutathione reductase, and alkyl hydroperoxide reductase) play a role in defense against oxidative stress. The detection of expression of the oxyR regulon and the soxR regulon provides an early marker of oxidative stress in prokaryotes. The binding of several eukaryotic transcription factors, such as NF-кB and the fos–jun heterodimer (AP-1) may be redox regulated. Expression of the c-jun oncogene is stimulated by either UVC radiation (254 nm) or hydrogen peroxide. On the other hand, expression of the human heme oxygenase 1 (HO-1) gene is not induced by UVC radiation, whereas it is strongly induced by oxidizing agents such as UVA (320–380 nm) radiation or hydrogen peroxide. Additionally, the gene is induced by other agents, such as phorbol esters, heavy metals, and sodium arsenite. Although this phenomenon was originally observed in fibroblasts cultured from human skin, induction occurs in most human cell types and all mammalian cell types so far tested. The induction is clearly related to the cellular redox state because lowering cellular glutathione levels strongly enhances HO-1 mRNA accumulation). For these reasons, altered expression of the HO-1 gene appears to be a fairly sensitive marker of oxidative stress.

Identification of a cis-regulatory element and putative trans-acting factors responsible for 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated induction of heme oxygenase expression in myelomonocytic cell lines.

The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with a variety of agents, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that during this process, the expression of heme oxygenase, a rate-limiting enzyme in heme catabolism, is induced. Treatment with TPA increases heme oxygenase mRNA in other myelomonocytic cell lines also, but not in cell lines of other lineages, such as HeLa cells. Increased heme oxygenase activity may represent one of the functions of activated macrophages, which sequestrate senescent erythrocytes and degrade heme derived from hemoglobin. This cell-type-specific induction by TPA treatment further investigated with respect to transcriptional regulation. We defined a cis-regulatory element, 5'-GTCATATGAC-3', located in the 5'-flanking region (positions -156 to -147) of the human heme oxygenase gene, which confers inducibility by TPA in THP-1 cells but not in HeLa cells. Nuclear proteins that bind to this element, which may be responsible for the cell specificity, were identified in THP-1 nuclear extracts. This element contains the consensus motif CANNTG, to which a large family of basic helix-loop-helix proteins binds. Our results suggest a novel mechanism of TPA-mediated transcriptional regulation in myelomonocytic cell lines.

Identification of a cis-Regulatory Element and Putative trans-Acting Factors Responsible for 12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Mediated Induction of Heme Oxygenase Expression in Myelomonocytic Cell Lines

The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with a variety of agents, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We show here that during this process, the expression of heme oxygenase, a rate-limiting enzyme in heme catabolism, is induced. Treatment with TPA increases heme oxygenase mRNA in other myelomonocytic cell lines also, but not in cell lines of other lineages, such as HeLa cells. Increased heme oxygenase activity may represent one of the functions of activated macrophages, which sequestrate senescent erythrocytes and degrade heme derived from hemoglobin. This cell-type-specific induction by TPA treatment further investigated with respect to transcriptional regulation. We defined a cis-regulatory element, 5'-GTCATATGAC-3', located in the 5'-flanking region (positions -156 to -147) of the human heme oxygenase gene, which confers inducibility by TPA in THP-1 cells but not in HeLa cells. Nuclear proteins that bind to this element, which may be responsible for the cell specificity, were identified in THP-1 nuclear extracts. This element contains the consensus motif CANNTG, to which a large family of basic helix-loop-helix proteins binds. Our results suggest a novel mechanism of TPA-mediated transcriptional regulation in myelomonocytic cell lines.

Novel Regulatory Sites of the Human Heme Oxygenase-1 Promoter Region

Heme oxygenase is the rate-limiting enzyme in heme catabolism, the activity of which is induced by several agents including its substrate heme, u.v. radiation and other oxidant injurious conditions. Its biological function is to provide a specific regulatory mechanism for control of the level of many heme proteins. Using a computer-assisted approach, we examined the presence of different regulatory elements on the human heme oxygenase-1 promoter region which could possibly be involved in its regulation. We identified several NFkB and AP-2-like binding sites in the 5′ untranslated region of the human heme oxygenase gene. DNase I footprint analysis confirmed the presence of these two important transcriptional sites. This study is the first to reveal the presence of NFkB and AP-2 binding sites in the heme oxygenase-1 promoter region and thus suggests a role for these transcriptional factors in the regulation of heme oxygenase-1 expression, a stress and heat shock protein, under injury conditions.

The proximal promoter region of the human heme oxygenase gene contains elements involved in stimulation of transcriptional activity by a variety of agents including oxidants.

The rate of transcription of the heme oxygenase gene is enhanced by a variety of agents including oxidants such as hydrogen peroxide and UVA (320-380 nm) radiation and the sulfhydryl reagent, sodium arsenite. To further analyze the inducible response, we have isolated genomic clones of the human heme oxygenase gene. A 1.44 kb fragment corresponding to a region extending from 1416 bp upstream of the mRNA cap site to 24 bp into the 5' untranslated region of the mRNA has been further subcloned and sequenced and used as the basis for the construction of recombinant CAT transient expression vectors. By deleting large portions of this fragment, we have established that elements within 121 bp of sequence immediately upstream of the mRNA cap site respond to various agents (sodium arsenite, hydrogen peroxide, hemin, cadmium chloride and 12-O-tetradecanoyl-phorbol-13-acetate) to give a 3- to 5-fold enhancement in transient expression of the reporter gene. Under the assay conditions employed, induction can only be detected when a SV40 enhancer element is present upstream of the promoter sequence. However, control experiments show that the SV40 sequences serve to amplify the response and are not directly involved in the induction itself. Only a small induction occurs when the entire 1.44 kb fragment is present. The results are consistent with the possibility that additional inducible enhancer elements lie outside of the sequence under study and that a silencer or negative regulatory element occurs upstream of the mRNA cap site within the 1.44 kb fragment.

Heme oxygenase is a positive acute-phase reactant in human Hep3B hepatoma cells

The effects of human interleukin-6 (hIL-6), the major acute-phase inducer, on the level of the transcript of microsomal heme oxygenase (HO) were examined in a human hepatoma cell line, Hep3B. Messenger RNAs (mRNAs) encoding HO and haptoglobin (Hpt) increased after hIL-6 treatment in a time- and dose-dependent manner. hIL-6 had no effect on the induction of heat-shock protein 70 (hsp70) mRNA, suggesting that the induction of HO by hIL-6 is regulated by a different mechanism from that which mediates the heat-shock induction of this enzyme. The hIL-6- mediated induction of HO mRNA was completely abrogated by simultaneous treatment of cells with actinomycin D, but not with cycloheximide, suggesting that the induction occurs at the level of transcription. A nuclear factor was shown both in untreated, and in the hIL-6-treated Hep3B cells that binds specifically to the IL-6-responsive element (IL6- RE) of the human HO gene. These findings suggest that HO is a positive acute-phase reactant in this human liver-derived cell line, and that the nuclear factor specific to the IL6-RE may be involved in the activation of the HO gene after hIL-6 treatment.

Heme Oxygenase Is a Positive Acute-Phase Reactant in Human Hep3B Hepatoma Cells

The effects of human interleukin-6 (hIL-6), the major acute-phase inducer, on the level of the transcript of microsomal heme oxygenase (HO) were examined in a human hepatoma cell line, Hep3B. Messenger RNAs (mRNAs) encoding HO and haptoglobin (Hpt) increased after hIL-6 treatment in a time- and dose-dependent manner. hIL-6 had no effect on the induction of heat-shock protein 70 (hsp70) mRNA, suggesting that the induction of HO by hIL-6 is regulated by a different mechanism from that which mediates the heat-shock induction of this enzyme. The hIL-6-mediated induction of HO mRNA was completely abrogated by simultaneous treatment of cells with actinomycin D, but not with cycloheximide, suggesting that the induction occurs at the level of transcription. A nuclear factor was shown both in untreated, and in the hIL-6-treated Hep3B cells that binds specifically to the IL-6-responsive element (IL6-RE) of the human HO gene. These findings suggest that HO is a positive acute-phase reactant in this human liver-derived cell line, and that the nuclear factor specific to the IL6-RE may be involved in the activation of the HO gene after hIL-6 treatment.

Endogenous glutathione levels modulate both constitutive and UVA radiation/hydrogen peroxide inducible expression of the human heme oxygenase gene

Induction of the expression of the mammalian heme oxygenase gene appears to be a general response to oxidant stress. In view of the role of glutathione in protecting cells against solar UVA radiation and other forms of oxidant stress, we have investigated the relationship between intracellular glutathione levels and the inducibility of the human heme oxygenase gene after treatment of populations of cultured skin fibroblasts with either UVA radiation or hydrogen peroxide. We observe a clear relationship between cellular glutathione status and both the constitutive and oxidant-inducible accumulation of heme oxygenase mRNA. Glutathione depletion may lead to enhanced gene expression either as a result of the potentiated accumulation of active oxygen intermediates or as a result of the direct influence of glutathione on a critical target involved in signal transduction.

Oxidant Stress Leads to Transcriptional Activation of the Human Heme Oxygenase Gene in Cultured Skin Fibroblasts

Treatment of cultured human skin fibroblasts with near-UV radiation, hydrogen peroxide, and sodium arsenite induces accumulation of heme oxygenase mRNA and protein. In this study, these treatments led to a dramatic increase in the rate of RNA transcription from the heme oxygenase gene but had no effect on mRNA stability. Transcriptional activation, therefore, appears to be the major mechanism of stimulation of expression of this gene by either oxidative stress or sulfydryl reagents.