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Physiol. Rev. 86: 583-650, 2006; doi:10.1152/physrev.00011.2005
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Heme Oxygenase-1/Carbon Monoxide: From Basic Science to Therapeutic Applications

Stefan W. Ryter, Jawed Alam and Augustine M. K. Choi

Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Department of Molecular Genetics, Alton Ochsner Medical Foundation, Louisiana State University Medical Center, New Orleans, Louisiana

ABSTRACT
I. PERSPECTIVE
II. HEME OXYGENASE ISOZYMES
    A. Enzymatic Activity and Its Measurement
    B. Biochemical Properties
    C. Genetics
    D. Tissue Distribution
    E. Subcellular Localization
    F. Phylogeny
III. REGULATION OF HEME OXYGENASES
    A. Induction of HO-1 by Chemical and Physical Stress
    B. Signal Transduction
    C. Gene/Promoter Regulation
IV. FUNCTIONAL PROPERTIES OF HEME OXYGENASE-DERIVED IRON
    A. HO-Derived Iron and Gene Regulation
    B. Antiapoptotic Roles of HO-Derived Iron and Ferritin
V. FUNCTIONAL PROPERTIES OF HEME OXYGENASE-DERIVED BILE PIGMENTS
    A. Metabolism of Biliverdin and Bilirubin
    B. Protective Effects In Vitro and In Vivo
VI. CARBON MONOXIDE
    A. Properties, Environmental or Endogenous Sources, and Toxicity
    B. Mechanisms of CO-Dependent Cell Signaling
    C. CO Releasing Molecules: Biochemical Properties
    D. Vasodilation
    E. Antiapoptotic, Anti-inflammatory, and Antiproliferative Mechanisms
        1. Antiapoptotic effects
        2. Anti-inflammatory mechanisms
        3. Antiproliferative mechanisms
    F. Neurotransmission
VII. PROTECTIVE ROLES OF HEME OXYGENASE-1/CARBON MONOXIDE IN DISEASE MODELS
    A. Inflammatory Diseases
        1. Sepsis
        2. Asthma
    B. Lung Injury Models
        1. Oxidative lung injury
        2. Bleomycin-induced pulmonary fibrosis
        3. Ventilator-induced lung injury
    C. Cardiovascular Injury/Disease
        1. Myocardial infarction
        2. Systemic hypertension
        3. Pulmonary hypertension
        4. Atherosclerosis
        5. Vascular injury
    D. Ischemia/Reperfusion
    E. Organ Transplantation
VIII. CONCLUSIONS AND FUTURE DIRECTIONS
    A. Summary
    B. Conclusions and Future Directions
GRANTS
ACKNOWLEDGMENTS
REFERENCES

   ABSTRACT
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The heme oxygenases, which consist of constitutive and inducible isozymes (HO-1, HO-2), catalyze the rate-limiting step in the metabolic conversion of heme to the bile pigments (i.e., biliverdin and bilirubin) and thus constitute a major intracellular source of iron and carbon monoxide (CO). In recent years, endogenously produced CO has been shown to possess intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. The era of gaseous molecules in biomedical research and human diseases initiated with the discovery that the endothelial cell-derived relaxing factor was identical to the gaseous molecule nitric oxide (NO). The discovery that endogenously produced gaseous molecules such as NO and now CO can impart potent physiological and biological effector functions truly represented a paradigm shift and unraveled new avenues of intense investigations. This review covers the molecular and biochemical characterization of HOs, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by environmental stress. Furthermore, the current understanding of the functional significance of HO shall be discussed from the perspective of each of the metabolic by-products, with a special emphasis on CO. Finally, this presentation aspires to lay a foundation for potential future clinical applications of these systems.


   I. PERSPECTIVE
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The race towards the identification and characterization of a potent vasodilating substance produced by endothelial cells in the 1980s resulted in a new paradigm in biomedical research and human diseases. Research during this era raised the provocative and intriguing notion that the endothelial cell-derived relaxing factor (EDRF) was not a peptide, protein, lipid mediator, or nucleic acid as originally speculated but rather a soluble gaseous molecule. The era of gaseous molecules officially began with the reports of endothelium-dependent vasorelaxation in 1980, which led to the unequivocal identification of EDRF as nitric oxide (NO) (197, 253, 254, 451). The impact of NO in biomedical research and applications to human diseases since the formal discovery of NO has been extraordinary and has arguably generated unrivaled intense interest and passion greater than any other biomolecule in this century. Ironically, we have known for a longer time, some two decades before the discovery of NO, that cells can produce another endogenous gaseous molecule by an enzymatic reaction initially described by Tenhunen and Schmid in 1968: the catalytic breakdown of heme by the microsomal heme oxygenase (HO) enzyme system which releases carbon monoxide (CO) (672, 674). Although HO-derived CO production represents the major intracellular pathway that generates endogenous CO, this process has gone relatively unnoticed by the scientific community during the first 25 years after the discovery of HO. The fact that HO is the rate-limiting enzyme in the breakdown of heme led the drive to better understand this reaction, resulting in a wealth of information on protein structure and reaction mechanism (501). The regulation of its inducible isozyme HO-1, by a broad spectrum of chemical and physical agents, led to a similar intense research effort to understand the mechanisms of gene regulation by environmental stress (19). However, the by-products of this reaction iron, bilirubin, and CO were for many years viewed as obscure waste products, with potential toxicological implications. With regard to CO, the known physiological fact that the administration at high enough concentrations to increase blood carboxyhemoglobin to critical levels can result in tissue hypoxia and subsequent lethality, undoubtedly did not attract many investigators into this field other than those interested in the epidemiology and toxicology of CO poisoning.

In the past decade, the interest in HO isozymes has shifted from their well-defined metabolic function of heme catabolism and erythrocyte turnover to another critical physiological function as a cytoprotective mechanism in numerous models of cellular stress and organ pathology. Even more interestingly, the HO field has recently been absorbed with a passion for understanding the intriguing biological functions of the catalytic by-products of HO-1, in particular CO. Thus the scientific verdict by the jury of investigations on the importance of HO and especially CO has not been rendered as of now. This review presents our current understanding of HO and CO in various physiological and pathophysiological states and their potential for therapeutic applications. We hope that this comprehensive review will prepare the scientific community to address the question of whether the biological importance of CO will attain the same far-reaching proportions as NO and provide a convincing argument that it is worth waiting for the jury to issue its final verdict.


   II. HEME OXYGENASE ISOZYMES
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A. Enzymatic Activity and Its Measurement

The origin of the colored pigments of the bile, biliverdin and bilirubin, from the degradation of hemoglobin heme, has been known long before the identification of the enzymatic reaction involved (397, 503). The correlation of endogenous CO found in the blood with hemoglobin heme degradation, and the {alpha}-carbon selectivity of this process, predates the discovery of heme oxygenase by several decades (116–118, 613–614). Tenhunen et al. (672–674) initially characterized heme oxygenase (HO) (EC 1.14.99.3) as a distinct enzyme system responsible for heme degradation in hepatic microsomes. Their work and the initial debate regarding the involvement of cytochrome P-450 in the heme degradation process (671, 583) was resolved when Maines and Kappas (412, 413) demonstrated that heme degradation occurred independently of cytochrome P-450.

HO catalyzes the first and rate-limiting step in the oxidative degradation of heme b (Fe-protoporphyrin-IX) to form the open-chain tetrapyrrole biliverdin-IX{alpha} (672) (Fig. 1). The HO reaction displays regiospecificity for the heme molecule, such that only the {alpha}-isomer of biliverdin is produced (489). Biliverdin-IX{alpha} (BV) is subsequently converted to bilirubin-IX{alpha} (BR) by an NAD(P)H-dependent reductase (675). HO catalyzed heme cleavage releases the heme iron in the ferrous form Fe (II) and eliminates the {alpha}-methene bridge carbon of the heme as CO (672). The HO enzymatic activity requires three moles of molecular oxygen (O2) per heme molecule oxidized, and reducing equivalents from NADPH:cytochrome P-450 (cytochrome c) reductase (NADPH: hemoprotein reductase, EC 1.6.2.4)(491, 672, 674, 764). Despite early reports of an exclusive role for NADPH in this process (672, 759), NADH can also serve as an electron-donating cofactor for HO activity in vitro (415). The ability of NADH to support heme degradation was demonstrated in reconstituted HO reactions consisting of NADPH:cytochrome P-450 reductase, biliverdin reductase, and partially purified, solubilized preparations of HO from human or rat liver (9, 411), as well as in microsomes derived from rat spleen, liver, or bone marrow (252, 415). Using reconstitution experiments, Maines et al. (411) initially proposed a role for NADH:cytochrome b5 reductase in NADH-dependent heme degradation. Subsequent studies provided immunochemical and biochemical evidence for the principle role of NADPH:cytochrome P-450 reductase in heme degradation in the presence of either reducing cofactor and demonstrated against the specific involvement of NADH:cytochrome b5 reductase (239, 489, 765).


Figure 1
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  		FIG. 1. The pathway of heme metabolism. Heme oxygenase enzymes (HO; E.C. 1.14.99.3; heme-hydrogen donor: oxygen oxidoreductase) catalyze the rate-limiting step in heme metabolism. Both HO isozymes (HO-1 and HO-2) oxidize heme (ferriprotoporphyrin IX) to the bile pigment biliverdin-IX{alpha}. The reaction requires 3 mol of molecular oxygen and NADPH:cytochrome P-450 reductase as a source of electrons. The cleavage of the heme ring releases the coordinated iron, as well as the {alpha}-methene bridge carbon as carbon monoxide (CO). The principal HO reaction product, biliverdin-IX{alpha}, is further metabolized to bilirubin-IX{alpha} by NAD(P)H:biliverdin reductase (BVR). M, methyl; V, vinyl; P, propionate.

 
Upon binding to the HO apoprotein, the heme molecule serves as both substrate and catalytic cofactor in its own degradation (672). Several current reviews describe the understanding of the HO reaction mechanism and its intermediates (501, 554, 727, 762). The reaction initiates with the NADPH-dependent reduction of the ferric heme-iron in the HO-heme complex, which binds O2 to form an oxyferrous intermediate that in turn accepts a second electron from NADPH (672, 756, 763–764, 768). The resulting ferric hydroperoxide (Fe III-OOH) intermediate hydroxylates the heme ring at the {alpha}-methene bridge carbon, forming {alpha}-hydroxy heme (544, 728, 762). Two further oxidation cycles involve the elimination of the {alpha}-methene bridge carbon as CO during the formation of verdoheme, and the subsequent formation of a ferribiliverdin-IX{alpha} complex (BV-Fe III) (325, 441, 566, 763, 766). The utilization and number of reducing equivalents required during the intermediate steps in the reaction cycle has been a matter of controversy (392, 425, 440, 501, 762). In contrast to the first step in the oxidation of heme, the oxidation of {alpha}-hydroxy-heme may not require activation of O2 at the heme iron center. Instead, the reaction of O2 appears to occur at the meso-edge of the porphyrin molecule (566, 762). An additional reduction step releases ferrous iron from the biliverdin complex (756, 762). HO activity displays a preference for heme b, with activity toward heme c and hematoheme also observed (351, 406, 419, 769). Intact hemoproteins such as cytochrome c and hemoglobin do not serve as enzymatic substrates (406, 419, 687, 769). Both NO and CO, small gaseous heme ligands, can bind to the heme oxygenase-heme complex (441, 716).

Synthetic metalloporphyrins such as cobalt-protoporphyrin-IX (CoPPIX) (403, 770), tin-protoporphyrin (SnPPIX) (151–152, 770), zinc- or manganese-protoporphyrin-IX (ZnPPIX, MnPPIX) (151, 770), tin and chromium mesoporphyrins (SnMP, CrMP) (150), and iron or zinc deuteroporphyrin-2,4-bis-glycol (FeDPBG, ZnDPBG) (99, 449), can act as competitive inhibitors of HO activity in vitro. It should be noted, however, that CoPPIX, albeit an inhibitor of HO activity in vitro, is a potent inducer of HO activity in vivo, and care must be taken in extrapolating in vitro results to in vivo conditions (153). In addition, dual control mechanisms exist for metalloporphyrins as exemplified by SnPPIX, which potently inhibits HO activity while increasing the content of HO protein in the liver (573).

A number of analytical methods have been published for the relative determination of HO activity in protein extracts of cells and tissues. The most common method for determination of HO activity depends on the detection of BR formation by spectroscopy. Essentially, in vitro HO reactions consist of a protein extract incubated with an excess of heme substrate and NADPH in a physiological buffer. BR is quantified by chloroform extraction from the reaction mixture, followed by visible spectroscopy at 464 nm (251–252). Earlier versions of this assay describe NADPH difference spectroscopy on protein reaction mixtures (579, 674, 672). Microsomal fractions can be used as a source of enzyme protein (104,000 g pellet), although commonly low-speed supernatants (whole cell extracts) may also be used (556, 581, 672). Because BV is difficult to assay by spectrophotometric methods, the quantification of HO activity depends on the complete conversion of BV to BR by BV reductase (BVR) (581). BVR may be supplied from crude extracts (rat liver 105,000 g supernatant fraction) or in partially purified form (350, 675). Protein extracts from some cell types contain sufficient BVR to obviate the need for exogenous supplementation, which may also coprecipitate with membrane preparations, presumably in association with HO-1 (327, 554).

HO assays using isotopically labeled 14C-heme have been described, which rely on the detection of [14C]bilirubin by recrystallization from chloroform extracts (674, 671) or thin-layer chromatographic separation (608). High-performance liquid chromatography (HPLC) has also been implemented for the separation and detection of heme metabolites and for the quantification of HO activity (Fig. 2) (62, 366, 387, 554–556, 560). This method allows the simultaneous detection of BV and BR and heme on a single chromatogram, simplifying quantification and eliminating the need for BVR supplementation (554). Alternatively, CO has been used as an end point in HO activity assays by measuring CO evolution in headspace gas, using a gas chromatograph coupled to a reduction gas detector (88, 710). More sensitive methods for the detection of CO in headspace gas utilizing gas chromatography/mass spectroscopy (GC-MS) have recently been described (3, 297).


Figure 2
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  		FIG. 2. Analysis of bile pigments by HPLC. HPLC chromatograph showing the resolution of tetrapyrrole standards in an alcohol extract of sonicated microsomal protein solution. Final concentrations were 2 µM biliverdin (BV), 2 µM Bilirubin-IX{alpha} (BRIX{alpha}), 0.35 µM BRIII{alpha}, 0.27 µM BRXIII{alpha}, 12.5 µM hemin, 0.4 µM mesoporphyrin (MP), corresponding to injected amounts of 240 pmol BV, 240 pmol BRIX{alpha}, and 48 pmol MP. The tetrapyrroles eluted with the following typical retention times: 1) biliverdin (BV) 5.29 min, 2) hemin 9.8 min, 3) bilirubin isomer (III{alpha} or XIII{alpha}) 10.39 min, 4) bilirubin-IX{alpha} (BRIX{alpha}) 10.9 min, 5) bilirubin isomer (III{alpha} or XIII{alpha}) 11.38 min, and 6) mesoporphyrin (MP) 12.54 min. Optical density was detected at 405 nm. [From Ryter et al. (554).]

 
A limitation of HO activity assays in the present form includes their inability to discriminate between HO-1 and HO-2 activity in a protein extract, although inducible activity is typically assumed to represent HO-1. Furthermore, these enzymatic assays provide a relative quantification of the active enzyme protein in a cellular protein extract, in the presence of an excess of exogenous substrate and cofactors; thus they do not necessarily represent activity profiles in vivo. The field may benefit from the development of analytical methods to measure the intracellular formation of heme metabolites in situ, including BR, and CO. In one example of such an attempt, a novel assay measuring endogenous CO production in vascular cells by mid-infrared laser absorption spectroscopy has been described (452).

B. Biochemical Properties

Two genetically distinct isozymes of HO have been characterized: an inducible form, heme oxygenase-1 (HO-1), and a constitutively expressed form, heme oxygenase-2 (HO-2) (407, 419, 687). HO-1 proteins (~32 kDa) were first purified to homogeneity from the livers of CoCl2 or heme-induced rats and from porcine spleen (411, 757, 758). The resulting HO-1 proteins bind heme in a 1:1 complex and display hemoprotein characteristics similar to methemoglobin, with characteristic absorption maxima at 405 nm for the oxidized form of the heme-HO complex (758). CO, as well as the heme ligands azide and cyanide, form complexes with the ferrous and ferric forms, respectively. The purified rat HO-1 protein displays functional HO activity in reconstituted microsomal systems in the presence of heme and NADPH:cytochrome P-450 reductase (758). Other reports have described the purification of HO-1 (~31–33 kDa) from the hepatic microsomes of the human (5, 6, 9), bovine (770), chicken (61), and rabbit (689).

The resolution of the crystal structure of human HO-1 has revealed a flexible bihelical structure surrounding the heme pocket (357, 591). A conserved histidine (His-25) imidazole acts as the heme iron ligand. The bound heme additionally contacts two glycine residues (Gly-139/Gly-143) in the distal helix domain (591). Biochemical evidence supports the formation of HO complexes with BVR and NADPH:cytochrome P-450 reductase (357, 717, 771). From predicted polypeptide sequences, the mouse HO-1 contains one cysteine (free thiol group), while HO-1 proteins from other species (pig, human, and rat) do not contain cysteine (296, 465, 601, 651, 761). The bovine HO-1 was initially reported to contain four titratable cysteines, but nevertheless was not itself subject to redox regulation of activity by sulfhydryl reagents or metal ions (770). Cysteine does not appear in current bovine HO-1 sequence data on record (accession no AAX08985; XB_590873; and Ref 582). Reports of apparent inhibition of HO activity by sulfhydryl reagents refer to reconstituted microsomal systems and apparently result from the inhibition of BVR and/or cytochrome P-450 reductase, which have critical sulfhydryl groups susceptible to modification by these compounds (411, 770).

A distinct isozyme of heme oxygenase, HO-2, has been identified in rat liver, spleen, brain, and testes (65, 418, 687–689). HO-2 was initially purified to homogeneity from rat testes and has an apparent molecular mass of 36 kDa (687). Human HO-2 displays 88% amino acid homology to rat HO-2 (429). A larger form of HO-2 (~42 kDa) was partially purified from rabbit testes (689).

Both HO-1 and HO-2 catalyze the identical biochemical reaction, in that they efficiently transform heme into biliverdin-IX{alpha} with similar substrate specificity and cofactor requirements. However, in a comparative analysis of rat HO-1 and HO-2, differential properties with respect to enzyme kinetics and substrate Km values have been reported (0.24 and 0.67 µM, respectively), as well as differences in apparent thermostability and immunoreactivity (407, 419, 687). In HO-2, the His-45 serves as the proximal heme ligand, with an accessory role for His-151 (265, 431).

The rat HO-1 and HO-2 share 43% amino acid homology (548). A highly conserved sequence of 24 amino acid residues has been identified in common to both HO-2 (rabbit and rat) and HO-1 (rat, mouse, human) (549). Furthermore, both HO-1 and HO-2 share similar hydrophobic regions at the extreme COOH terminus that serve to anchor the proteins in cellular membranes (266, 431, 601).

HO-2 contains functional domains not present in HO-1. These domains, termed heme regulatory domains, which contain a conserved Cys-Pro motif, provide additional heme binding sites distinct from the heme catalytic domain (429). The functions of these additional heme centers of HO-2 suggest a heme-dependent function for HO-2 distinct from heme degradation. The additional heme centers of HO-2 have been proposed to act as a sink for both NO and CO (142, 247, 416, 429).

The second step in heme metabolism, NAD(P)H:biliverdin reductase (EC 1.3.1.24) (BVR), subsequently converts the water-soluble biliverdin-IX{alpha} into lipophillic bilirubin-IX{alpha} by reduction of the {gamma}-methene bridge carbon (672, 674, 675). BVR was originally purified from rat liver and kidney (350, 675). In addition to the major alpha form (BV{alpha}R) found in human liver (34 kDa), a second isotype (21 kDa; BVbetaR) exists that produces bilirubin-beta or fetal bilirubin (176, 337, 338, 741). BV{alpha}R may use both NADH and NADPH for activity, while BV-betaR displays preference for NADPH. The rat liver BV{alpha}R displays differential pH optima for NADH and NADPH cofactors (i.e., ~7.0 and ~8.7, respectively) (350). BVR contains critical -SH groups rendering it susceptible to inhibition by heavy metals and thiol reagents (350, 770). Recent evidence also suggests that human BV{alpha}R naturally contains zinc (417).

C. Genetics

HO-1 and HO-2 represent the products of distinct genes (ho-1, ho-2, also specified as hmox1, hmox2) (127, 407, 419, 428, 686). The human, mouse, and rat ho-1 genes (~14 kb) and the rat ho-2 gene (~12 kb) share similar organization into five exons and four introns (17, 432, 465, 602). The entire genome sequence of the rat BVR gene (~12 kb) has also been described, which also displays an organization of five exons and four introns (427).

Fluorescence in situ hybridization studies show that the human ho-1 and ho-2 genes localize to separate chromatin regions (ho-1: 22q12; ho-2: 16p13.3) (349). Human BV{alpha}R localizes to chromosome 7 (511), while human BV-betaR to chromosome 19q13.13->q13.2 (565).

A number of HO-1 corresponding cDNA clones have been described (169, 296, 319, 601, 651). Human HO-1 cDNA clones (1.5 kb) were isolated from heme-induced macrophages, and from sodium arsenite-induced fibroblasts, encoding a 288-amino acid protein with a predicted molecular mass of 32 kDa (319, 761). HO-1 cDNA has been cloned from the rat spleen (1.5 kb) (285 amino acids, 33 kDa) (601). Murine HO-1 cDNA was cloned from sodium arsenite-induced fibroblasts (289 amino acids, 33 kDa) (296). A porcine clone (1.5 kb; 288 amino acids, 33 kDa) and an avian clone (1.25 kb; 296 amino acids, 33.5 kDa) have also been described (169, 651). Human HO-1 cDNA displays a high degree of homology (80%) with the rodent cDNAs, while the avian form displays the lowest sequence homology (62%) (169, 761).

Likewise, several HO-2 cDNA clones have also been described (428, 548–549, 687). Two forms of HO-2 cDNA have been cloned from human and rat representing the products of two alternatively polyadenylated homologous transcripts (1.3/1.7 kb in human and 1.3/1.9 kb in rat). The resulting predicted polypeptides of 313 and 315 amino acids for the human and the rat, respectively, share an 88% amino acid homology (428, 548). Additional HO-2 cDNAs corresponding to single transcripts have been isolated from the rabbit (1.3 kb) (549) and the monkey (1.7 kb) (686).

McCoubrey et al. (430) isolated a cDNA (2.4 kb) encoding a putative third HO isozyme (HO-3) in rat tissues. The predicted polypeptide has a high amino acid sequence homology to HO-2 (~90%) and thus shares many common sequence motifs, including putative heme regulatory domains (430).

The ho-3 mRNA transcripts corresponding to the cDNA clone reported by McCoubrey et al. (430) were detected in a number of rat tissues, including the spleen, liver, thymus, prostate, heart, kidney, testis, and brain (430). A recent study reported a distribution pattern of ho-3 mRNA in the brain in the hippocampus, cerebellum, and cortex (579). When expressed in E. coli, the resulting HO-3 protein (~33 kDa) had very little heme degrading activity, and therefore, the function of this protein remains enigmatic (430).

A recent study raises uncertainty on the existence and functional relevance of HO-3 in vivo. Hayashi et al. (231) identified two HO-3-related genes (ho-3a/b) in the rat in an attempt to clone the ho-3 gene corresponding to the cDNA described by McCoubrey et al. (430). The ho-3a/b genes are nearly homologous to HO-2 exons 2–5 and contain no introns. Furthermore, Hayashi et al. (231) reported a lack of expression of corresponding ho-3 mRNA and HO-3 protein in rat tissues, and due to high homology with ho-2 derived sequences, questioned the previous analysis of ho-3 mRNA distribution. To address this issue, another investigation utilized the ho-2–/– mice to avoid potential homology overlaps and reported no transcripts corresponding to ho-3 in the mouse brain (789). The presence of stop codons within the coding regions, as well as the lack of detectable mRNA or protein product in rat tissues, led Hayashi et al. (231) to conclude that HO-3a/b represent pseudogenes originating from HO-2 transcripts.

D. Tissue Distribution

The inducible form of heme oxygenase, HO-1, occurs at a high level of expression in the spleen and other tissues that degrade senescent red blood cells, including specialized reticuloendothelial cells of the liver and bone marrow. High levels of HO activity are detected in these tissues (672, 673). HO is present in hematopoietic stem cells of the bone marrow (1, 10, 72), where it may inhibit cellular differentiation by lowering the intracellular concentration of heme, a differentiation factor for these cells (1, 10). HO is also present in the liver parenchyma, which is the site of uptake and degradation of plasma heme, hemoglobin, and methemalbumin. Under conditions of hemolysis, HO activity dramatically increases in the liver parenchyma, kidney, and macrophages in response to increased levels of circulating hemoglobin (519, 520, 673).

In most other tissues not directly involved in erythrocyte or hemoglobin metabolism, HO-1 typically occurs at low to undetectable levels under basal conditions but responds to rapid transcriptional activation by diverse chemical and physical stimuli.

The highest expression of HO-2, the constitutively expressed isozyme, occurs in the testes, but the protein is also found abundantly and ubiquitously in other systemic tissues including, but not limited to, the brain and central nervous system, vasculature, liver, kidney, and gut (407, 408, 419, 687, 773). HO-2 does not respond to transcriptional activation by environmental stress but may respond to developmental regulation by adrenal glucocorticoids in the brain (409, 535).

E. Subcellular Localization

Since their initial discovery in 1968, HO-1 enzymes have been characterized as endoplasmic reticulum (ER) associated proteins, due to the abundant detection of HO activity in microsomal (104,000 g) fractions. Both HO-1 and HO-2 contain a COOH-terminal hydrophobic domain segment that suggests a general membrane compartmentalization (266, 601, 761). Constitutive expression of the rat HO-1 cDNA in monkey kidney cells indicated an ER localization of the expression product (266, 601). Recent studies have raised the possibility of the functional compartmentalization of HO-1 in other subcellular domains beside the ER, including but not limited to the nucleus and plasma membrane (Fig. 3). The potential functional subcellular compartmentalization of HO enzymes raises an intriguing issue of organelle specific function of HO metabolites, for example, CO, which is not yet fully characterized.


Figure 3
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  		FIG. 3. Subcellular localization of heme metabolic enzymes. The scheme shows the possible subcellular compartmentalization of the principle enzymes in heme degradation based on limited studies. The ER localization of HO-1 has been known for many years (266, 601). The caveolar localization of HO-1 is supported by our recent publication (327). See text for other citations. BVR, biliverdin reductase; HO-1, heme oxygenase-1; HO-2, heme oxygenase-2; RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum.

 
A recent study from this laboratory has described a functional association of HO-1 with plasma membrane caveolae in endothelial cells (327). Mouse lung endothelial cells (MLEC) were exposed to several known inducers of HO-1, including lipopolysaccharide (LPS), heme, and hypoxia, and their lysates were subjected to sucrose density gradient fractionation. In the absence of chemical stimulation, the detectable HO-1 in MLEC localized to a single fraction with a density consistent with the predicted ER localization (Fig. 4). Interestingly, the treatment of MLEC with inducers of HO-1 expression not only induced the quantity of HO-1 expressed, but dramatically altered the subcellular distribution of HO-1 among the fractions analyzed. Moreover, some variation in this distribution occurred in an inducer-specific fashion. The inducers heme and LPS caused a dramatic translocation of HO-1 to cytochrome c-containing fractions, suggesting a possible mitochondrial localization of HO-1. After stimulation with all three inducers, endothelial HO-1 was shown to localize, in part, to low-density or detergent-resistant fractions that contained caveolin-1, a marker and principle structural component of caveolae. Consistent with the requirement of these factors to constitute the complete heme metabolic pathway, NADPH:cytochrome P-450 reductase and BVR also colocalized with HO-1 to the caveolin-1-containing fractions. The combined caveolae fractions from LPS stimulated MLEC contained functionally active HO, by enzymatic activity assays. Interestingly, the downregulation of caveolin-1 by antisense expression resulted in a dramatic increase in HO enzyme activity despite stable protein expression, indicating a possible negative regulatory role of caveolin-1 toward HO activity. Conversely, overexpression of caveolin-1 resulted in a downregulation of LPS-inducible HO activity. These observations were supported by coimmunoprecipitation studies that indicate a physical interaction between HO-1 and caveolin-1. The negative regulation of endothelial NO synthase (eNOS) activity by caveolin-1 has also been demonstrated (180). These observations suggest a possible functional role of HO enzymes in caveolae and also suggest that caveolin-1 may serve as a molecular brake on signaling mechanisms involving small gaseous second messengers that originate in the caveolae (327).


Figure 4
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  		FIG. 4. Caveolae compartmentalization of HO-1 in rat pulmonary aortic endothelial cells (PAEC). Rat PAEC were exposed to stimuli such as hemin (20 µM, for 18 h), LPS (100 ng/ml, for 18 h), or hypoxia (1% O2, 5% CO2 mixture, 8 h). Total cell lysates were loaded on the discontinuous sucrose gradients for 18 h (39,000 rpm, SW 41 rotor). Twelve fractions of each were obtained and subjected to Western blot analysis for HO-1, caveolin-1, and eNOS. Note that caveolin-1 was concentrated in fractions 4–5. [From Kim et al. (327).]

 
Relatively little is known about the possible functional compartmentalization of HOs to the nucleus. Preliminary studies indicate that heme stimulates the nuclear translocation of HO-1. Furthermore, HO-2 was detected constitutively in the nucleus of NIH3T3 cells and was proposed to facilitate the entry of HO-1 (P. A. Dennery; see Ref. 485). We have observed diffuse nuclear staining of HO-1 in endothelial cell preparations stimulated with LPS, which coincides with the appearance of HO-1 in high-density sucrose gradient fractions (H. P. Kim, X. Wang, F. Galbiati, S. W. Ryter, and A. M. Choi, personal communication). Nuclear localization of HO-1 was also observed in astroglial cells stimulated with glutamate (393a). Electron microscopy studies of monkey retinal ganglion cells revealed localization of HO-2 to the outer nuclear membrane, in addition to ER localization (403). Interestingly, nuclear localization of BVR has been reported in response to endotoxin stress in the rat kidney (410). Aside from the generally accepted principle function of BVR as a soluble metabolic enzyme, possible functional roles of the BVR in other cellular and nuclear processes have been suggested. These potential activities of the BVR include the regulation of protein phosphorylation, DNA binding activity, and nuclear transcriptional regulation (14, 344, 567).

F. Phylogeny

Recent studies indicate a high degree of evolutionary conservation of the heme degrading enzymes among animal, plant, and fungal kingdoms. HO-1 appears ubiquitous in higher animals with a high degree of structural similarity and functional identity between enzymes from humans, large mammals, rodents, and birds. Interestingly, a number of HO-1-like proteins and activities have been described in lower organisms including flies, bacteria, fungi, plants, and algae (53, 121, 362, 442, 467, 468, 514, 537, 570, 587, 615, 724, 727, 729, 733, 779, 780, 784, 785).

Among insects, plants, fungi, and bacteria, the basic mechanism of HO-catalyzed heme cleavage appears to resemble that described for mammalian isozymes, in that BV and CO are produced from heme. In most cases, the {alpha}-isoform of biliverdin is exclusively produced, with exceptions as noted below. Some differences also arise in the cofactor requirements of HO and BVR homologs, in the processing of BV, as well as in the overall functional significance of heme metabolism among various organisms.

A HO-1 homolog recently isolated from Drosophila melanogaster lacks a proximal heme ligand and displays a broader selectivity for heme catalysis ({alpha}, beta, {delta}) (780). The pathogenic yeast Candida albicans contains a homolog of HO (CaHMX1) with a high degree of similarity to mammalian isoforms, which plays a critical role in iron acquisition from heme derived from the host organism (514, 570). No functional HO homolog has been described for the "budding yeast" Saccharomyces cerevisiae, although several heme-binding proteins have been characterized (38, 529). The product of the HMX1 gene facilitates heme utilization in this organism (529).

In higher plants, the Arabidopsis thaliana HY1 gene, for example, encodes a plant homolog of HO-1 that utilizes ferredoxin as the reducing partner instead of NADPH:cytochrome P-450 reductase (467, 468). In general, plants, which lack the equivalent of mammalian BVR, do not produce bilirubin. In Arabidopsis thaliana, a ferredoxin-dependent BVR activity (HY2) reduces biliverdin-IX{alpha} to phytochromobilin (334), which in turn is utilized as a precursor for phytochrome chromophore, a critical component of plant photoreceptors (132, 467, 468). Similarly, the HO homolog of the unicellular red algae Cyanidium caldarium generates biliverdin-IX{alpha}, which is utilized for phycobillin synthesis instead of bilirubin production (53).

Several soluble HO homologs have been described in various bacterial strains (reviewed in Refs. 189, 727). Cyanobacteria, as exemplified by Synechocystis sp. PCC 6803, contains two heme oxygenases (Syn ho-1, Syn ho-2). Syn ho-1 displays structural and functional similarity with mammalian forms of HO-1 (121, 442, 779). Interestingly the cyanobacterium ho-1 can complement HO-deficient Arabidopsis thaliana mutants (hy1) to restore phytochrome biosynthesis (731). Unlike higher plants, Synechocystis sp. PCC 6803 contains a mammalian-like BVR activity (bvdR) that generates bilirubin-IX{alpha} that is utilized as a phycobillin precursor (586).

In the Gram-positive bacterium Corynebacterium diphtheriae, the HmuO gene product produces biliverdin-IX{alpha} and CO from heme and requires a bacterial NADPH-dependent reductase (597, 729). Staphylococcus aureus contains two HOs (IsdG and IsdI) of perhaps the smallest known molecular size (13 kDa) (615, 733).

HO enzymes have also been described in Gram-negative bacterial strains including Neisseria meningitides (HemO) (784, 785) and Pseudomonas aeruginosa (PigA, BphO) (537, 724). The PigA gene product differs from mammalian and other bacterial HOs in that it displays an altered regiospecificity for heme cleavage, producing beta,{delta}-biliverdins. The second HO-like isozyme BphO of Pseudomonas aeruginosa produces exclusively biliverdin-{alpha} for the synthesis of bacterial phytochrome. This organism may have segregated its heme-degrading activities related to iron acquisition and pigment biosynthesis (189). In summary, the HOs of bacterial and fungal pathogens serve a principle functional role as a mechanism for the acquisition of iron from exogenous heme or hemoglobin as an essential nutrient for growth and pathogenesis (189, 587). In plants, photosynthetic, and nonphotosynthetic bacteria however, the HO enzymes also provide the first step in metabolic pathways for the synthesis of pigments: phytochromobillins, phycobillins, and bacterial phytochromes (189).


   III. REGULATION OF HEME OXYGENASES
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A. Induction of HO-1 by Chemical and Physical Stress

The increased synthesis of the HO-1 protein occurs as a general response to stress in biological systems. The response appears to occur ubiquitously among most tissues tested and also among higher organisms including humans, mammals, marsupials, birds, and fish. While variants of HO have been described in many organisms from bacteria to plants (see sect. IIF), a discussion of inducible gene regulation in lower phyla is omitted here in a focus on mammalian systems. The general nature of the response and the vast literature now surrounding the phenomenon render it difficult to provide a comprehensive list of all inducing compounds in all cell types and tissues tested. Nevertheless, Table 1 references a selection of classical and recent examples, in favor of omitting cell types in the text. With few exceptions, as described for the response to NO below, the response depends largely on transcriptional activation of the ho-1 gene and the de novo synthesis of mRNA, regardless of cell type or inducing chemical. This has been verified under a number of conditions using nuclear run-off assays for gene transcription (22, 317, 386, 546) and/or experiments with the transcriptional inhibitor actinomycin D (445, 446, 574, 600). This section will provide an introduction to the classes of stress that may elicit the ho-1 response. Attention to the mechanisms involved in signaling (see sect. IIIB) and transcriptional regulation (see sect. IIIC) associated with the response to discrete inducers shall be given in the following sections.


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  		TABLE 1. Induction of HO-1 by chemical and physical stress

 
HO-1 belongs to a larger family of stress proteins whose transcriptional regulation also responds to adverse environmental conditions. Of the known mammalian stress protein families, the expression of the heat shock proteins (HSPs) (20–30 kDa, 70–73 kDa, 90 kDa, 104–110 kDa) constitutes a global cellular response to protein denaturation associated with hyperthermia (642). The HSPs collectively participate in the subcellular trafficking of unfolded, nascent, or denatured protein and, furthermore, provide an underlying molecular mechanism for the development of acquired cellular thermoresistance (125). HO-1 shares no apparent amino acid homology with HSPs nor displays protein chaperone activity. However, the promoter regions of various ho-1 genes contain heat shock elements similar to those originally identified in the regulatory regions of various hsp genes. HO-1 has sometimes been classified as a heat shock protein (Hsp32) due to its transcriptional responsiveness to hyperthermia, which, however, is apparently somewhat restricted to rodent systems (600, 761). Hyperthermic shock causes accumulation of HO-1 protein and mRNA in various rat organs including liver, heart, and brain (170–172, 174, 533).

A second general class of stress proteins, the glucose regulated proteins (GRPs), constitutes a general response to ER-associated stress, also known as the "ER stress response," or "malfolded protein response." The GRPs, multiple members of several molecular mass classes (75, 78–80, and 94–100 kDa), respond to transcriptional upregulation after glucose starvation, disruption of intracellular calcium homeostasis, protein glycosylation interference, and expression of malfolded proteins (361). Interestingly, HO-1, which normally resides in the ER, also can respond to transcriptional activation in some cell types by nutrient depletion (90, 91, 663). The overlap of the HO-1 response with the GRPs in hepatoma cultures appeared to be restricted to an inducible response to glucose depletion (91). However, induction of HO-1 by a broader spectrum of ER stress-inducing agents has been noted in several studies, such as the Ca2+-ATPase inhibitor thapsigargin, which depletes ER Ca2+ stores, or brefeldin A, which interferes with the translocation of glycosylated proteins (391, 203). In addition to interference with the folding and posttranslational processing of nascent polypeptides, interference with the proteolytic degradation of proteins also provokes a stress response. Chemical inhibitors of the proteosome join the list of metabolic inhibitors that can activate ho-1 in parallel with an ER-stress response (736).

Preceding its unequivocal identification as HO-1 by molecular cloning methods, a distinct low-molecular-weight protein of 32-kDa stress protein (p32) was found to be synthesized following cellular stimulation with a number of cytotoxic agents including sodium arsenite, ultraviolet A (UVA; 320–380 nm) radiation, quinones, sulfhydryl reagents, heat shock, hydrogen peroxide, tumor promoters, and heavy metal salts (78, 296, 318, 319, 664, 665). The term HO-1 will be used hereafter in this review to represent and supercede all references to p32 or Hsp32.

In early studies of ho-1 gene activation, various tumor promoters including 12-O-tetradecanoylphorbol-13-acetate (TPA), its structural analog phorbol-12,13-didecanoate, as well as structurally distinct indole alkaloid and polyacetate tumor promoters induced HO-1 protein and/or mRNA in cell culture (241–243, 296, 347) (Table 1). TPA mimics the effects of diacylglycerol as an agonist of protein kinase C (PKC) and exerts multiple effects including the stimulation of cellular growth and differentiation programs, associated with the activation of early response genes (324). TPA treatment induced ho-1 mRNA levels in human and mouse myelomonocytic cell lines in parallel with their TPA-induced differentiation to macrophages (347, 469). In contrast to TPA-induced differentiation programs, HO-1 expression and activity appear to be negatively correlated with the heme-dependent differentiation of erythroid precursors (1, 4, 10, 373).

The ho-1 gene is also commonly induced by agents and chemicals that produce an oxidative cellular stress involving the generation of reactive oxygen species (ROS). In this regard, HO-1 has been recognized as a general response to oxidative stress. Known examples of ROS generating systems that activate ho-1 include hydrogen peroxide (H2O2), quinones, and related compounds (i.e., menadione) which can generate superoxide anion radical (O2–) and/or H2O2 through redox cycling, photosensitizers, and UVA radiation (207, 318, 319). UVA radiation generates ROS by photoexcitation of endogenous chromophores. Studies with chemical quenching compounds implied a predominant role for singlet molecular oxygen (1O2) in the activation of ho-1 mRNA by UVA treatment (52). Photodynamic therapy generates a similar response from the photoexcitation of synthetic chromophores most commonly based on porphyrin or chlorin structures by application of visible light at discrete wavelengths (148). Phototherapy with dihematoporphyrin ether/ester induced HO-1 expression in rodent tissue culture (207). Exposure to high oxygen tension (hyperoxia) also generates an oxidative stress, by enhancing the leakage of partially reduced forms of oxygen from the mitochondrial electron transport chain (190). Hyperoxic stress strongly induces ho-1 mRNA and protein expression in macrophages and lung-derived cell lines (137, 363, 659).

HO-1 regulation responds in several models to diminished oxygen (O2) tension (hypoxia), which can induce its mRNA or protein in several in vivo and in vitro systems that include various animal cell types of vascular or pulmonary vascular origin (i.e., endothelial and smooth muscle) (228, 365, 470, 552, 559, 563). Despite the common end point of ho-1 activation, the molecular pathway leading to activation may vary in a cell-type specific manner (228). Hypoxia causes transcriptional repression, rather than activation, in several human cell types (331, 477).

Metabolites of bio-oxidation reactions have also been studied as potential inducers of HO-1, including oxidized lipids (238, 345), 4-hydroxy-2-nonenal (4-HNE) (51, 263), oxidized low-density lipoprotein (13, 264, 740), and advanced glycation end products (646, 744). HO-1 has been implicated in the pathologies of a number of diseases associated with oxidative stress including Alzheimer’s disease (89, 584, 585, 617, 618, 656, 661), diabetes (11, 131, 141, 390, 530, 696), and atherosclerosis (267, 291, 612, 718).

A separate but related group of inducing chemicals include the thiol-reactive substances, which have the ability to modify reactive -SH groups in protein and in nonprotein thiols such as reduced glutathione (GSH). The formation of mixed disulfides of glutathione, GSSR, by these agents effectively depletes GSH, as such adducts are not reversible by glutathione reductase. Compounds in this family include sodium-meta-arsenite (34, 164, 296, 319) and diethylmaleate (DEM) (577). The induction of HO-1 by sodium arsenite represents a general response reproducible in a broad selection of animal cell types (34).

The heavy metal salts potently activate the gene in cell culture systems and produce tissue-specific effects in vivo depending on the compound and route of administration (78, 303, 424, 574, 664, 665). These compound include salts of cadmium (Cd2+), cobalt (Co2+), zinc (Zn2+), tin (Sn2+), lead (Pb2+), and mercury (Hg2+). For example, in injection of metal salts into rats, most metals tested produced a hepatic HO-1 induction, with Cd2+ and Co2+ the most potent. On the other hand, Sn2+ or Ni2+ and Hg2+ displayed a selective induction response in the kidney and heart, respectively (414). Heavy metals form complexes with thiols, such as reduced glutathione (GSH). In vivo, heavy metals cause a characteristic depletion of hepatic GSH followed by a rebound increase from de novo synthesis (414). Prior complexation of metals with thiol complexes diminished their effectiveness at eliciting the HO-1 response in vivo (414).

The complexation of metals with protoporphyrin IX (PPIX), the natural precursor of heme, generates metalloporphyrins. Several metals such as Co2+, Zn2+, Cu2+, and Fe2+ can serve as substrates for ferrochelatase, the enzyme that incorporates iron into PPIX in the final step of heme synthesis. The substrate and catalytic cofactor of HO-1, heme (iron-protoporphyrin-IX), acts as an inducer of ho-1 gene expression and activity (22, 673, 761).

Several metalloporphyrins, including SnPPIX and ZnPPIX, can induce HO-1 transcription while paradoxically acting as competitive inhibitors of HO activity both in vitro and in vivo (573). In contrast, CoPPIX, which is a potent inhibitor in vitro, is a powerful inducer of HO activity in vivo (153, 573).

Free protoporphyrin, in the absence of a central metal chelate, can also activate ho-1, albeit only in the context of its photoactivation by intense visible light at discrete wavelengths (561, 562).

Since the discovery that NO, a gaseous free radical molecule, acts as a potent physiological regulator of many processes, including vascular tone, neurotransmission, inflammation, as well as a bactericidal agent in the macrophage respiratory burst, not surprisingly, intensive investigation has followed on the role(s) of NO in inducible gene regulation. NO produces potent HO-1 induction in many cell types tested, including fibroblasts and many vascular cell types. In addition to direct administration in gaseous form (422), HO-1 induction also follows the application of a number of chemical NO donor compounds in vitro, including sodium nitroprusside and spermine NONOate, nitrosating agents or nitrosonium cation generators such as S-nitroso-N-acetylpenicillamine, and the peroxynitrite generator 3-morpholinosydnonimine (74, 94, 157, 185, 226, 229, 462, 655, 749). Similarly, various chemical derivatives of NO also elicit the HO-1 response when directly applied to cell culture, including peroxynitrite (ONOO–), S-nitrosothiols, nitrosohemes, and the nitroxyl anion (Angeli’s salt) (94, 188, 479, 480). A major mechanism for NO-mediated signaling effects involves the activation of soluble guanylate cyclase (sGC), leading to enhanced cGMP formation. The majority of studies to date, however, have demonstrated a general ineffectiveness of cGMP analogs in inducing ho-1, in cell types where NO promoted the response (94, 157, 229, 422, 655). Nevertheless 8-BrcGMP can induce ho-1 in several cell types including hepatocytes and endothelial cells (257, 322). In addition to direct activation of guanylate cyclase, NO and its derivative reactive nitrogen species (RNS) can participate in a multiplicity of redox reactions (732). NO reacts with O2– to form the oxidant peroxynitrite (ONOO–). RNS such as nitrosonium cation (NO+) can modify reactive thiol groups in protein. Thus the generation of metabolites of NO, including peroxynitrite, S-nitrosothiols, or nitrosohemes, have been implicated as potential secondary mechanisms underlying ho-1 activation by NO donor compounds (185, 188, 463, 480). A second generalization from these studies suggests a largely transcriptional activation of ho-1 by NO and its metabolites (157, 185, 229, 422, 462). However, in human cell types, a major posttranscriptional component has also been observed, whereby NO treatment promoted the stabilization of ho-1 mRNA (64, 422). The regulation of ho-1 by NO and its derivative reactive nitrogen species (RNS) has recently been reviewed (463).

A broad class of electrophilic polyphenolic compounds, which also are classified as antioxidants, has recently emerged as potent ho-1 inducing agents, including caffeic acid phenethyl ester, carnosol, curcumin, and resveratrol (46, 290, 423, 461, 580). Many of these substances are plant-derived antioxidants. For example, the phenolic compounds curcumin and carnosol are derived from the spices turmeric and rosemary, respectively. Resveratrol, from grape skin and seeds, is a common constituent of wine (290). Quercetin is a ubiquitous bioflavonoid found in many plant-derived foods. The latest additions to this list of natural inducers of ho-1 include the plant-derived chalcones, rosolic acid, and the garlic organosulfur compounds (93, 187). Ginko biloba extractum (EGb 761), a complex mixture of phenolic and terpenoid compounds, induces HO-1 expression in neuronal cell culture in association with its cytoprotective activity (788). In general, many phenolic compounds activate ho-1 through the Nrf-2/Keap1 axis, as discussed in section IIIC, which plays a major role in the transcriptional activation of the ho-1 gene by electrophillic compounds.

In addition to that of pro-oxidant states and other xenobiotic stress, HO-1 can represent a general molecular marker of proinflammatory states. Thus the role of HO-1 as a general cytoprotectant is not limited to stress from exogenous chemical and physical agents but may also be important during systemic stress caused by injury or infection. HO induction occurs as a component of the hepatic acute phase response (APR), a systemic reaction to injury or infection in the liver characterized by physiological changes including global alterations in hepatic protein synthesis (235). The hepatic APR is strongly induced by bacterial endotoxins (LPS), which stimulates cytokine production from monocytes and macrophages (200). The physiological changes characteristic of the APR are regulated by cytokines, including interleukin (IL)-6, IL-1, and tumor necrosis factor (TNF)-{alpha}. Thus a number of proinflammatory agents are potent activators of the HO-1 response. The most notable example is bacterial LPS, which typically produces a robust ho-1 activation in cell culture (79, 80, 348) and in vivo following injection (545). The intraperitoneal injection of LPS induces HO activity in rat peritoneal macrophages and in hepatic parenchyma and sinusoidal cells (56, 546). The induction of hepatic HO expression (i.e., mRNA or activity) by LPS and other cytokines, in general, could be inhibited by glucocorticoids (82) or thiol antioxidants (545) and augmented by GSH depleting agents (545). More recent studies have reaffirmed that LPS injection induces HO-1 mRNA, protein, and enzyme activity in the liver, lung, and kidney (652).

In addition to LPS, a model of exogenous inflammatory stress caused by infections, HO-1 responds to exogenous stimulation with cell-derived inflammatory mediators, such as the interleukins (IL-1{alpha}, IL-1beta, IL-6, IL-11) and cytokines such as TNF-{alpha} (157, 446, 484, 677, 678). The intraperitoneal injection of TNF-{alpha} and IL-1 also induces hepatic HO mRNA expression and/or enzymatic activity in mice (546). In cell culture models, induction of ho-1 mRNA by TNF-{alpha} could be blocked by a variety of inhibitors, including downregulation of PKC by prolonged TPA treatment, Ca2+ ionophores, phospholipase A2 inhibitors, and thiol antioxidants (678). Interestingly, the anti-inflammatory cytokine IL-10 can also stimulate ho-1 in macrophages (367). HO-1 has been proposed as a downstream effector of the anti-inflammatory action of IL-10 (367). Conversely, IL-10 upregulation has been implicated in the anti-inflammatory effects of CO (504).

Several growth factors also elicit a HO-1 response in a cell type or tissue specific fashion, including transforming growth factor (TGF)-beta (in lung and ocular tissues) (352, 484, 488), platelet-derived growth factor (PDGF) (158) (in vascular cells), nerve growth factor (NGF) (in discrete neural cells) (388, 568), and erythropoietin in human bone marrow stem cells (10). In contrast, interferon-{gamma} has been shown in several cell culture models to repress, rather than activate, HO-1 transcription (698).

Early studies on the regulation of HO-1 by hormone-induced systemic stress demonstrated that hypoglycemic shock induced by insulin injection as well as glucagon and cAMP administration induced hepatic HO activity in rats (41). Hormones, eicosanoids, and other circulating mediators have been studied as inducers of HO-1 in discrete cell types (Table 1). These include cyclopentone prostaglandins of the J series such as prostaglandin J2 (PGJ2) and its sequential metabolites {Delta}12-prostaglandin J2 (12-PGJ2) (335, 336, 483) and 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) (27, 389, 787, 789), which potently induce cellular differentiation and exert antiproliferative activity. Other prostaglandins implicated in ho-1 activation include prostaglandin D2 (PGD2), the precursor of the J-series prostaglandins as well as prostaglandin A1 (PGA1) (389) and prostaglandin E2 (PGE2) in distinct cell types (96).

Atrial natriuretic peptide (ANP), a heart-derived cardiovascular hormone, plays a role in the regulation of blood pressure and can stimulate ho-1 activation in human vascular endothelial cells (322). The induction of hypertension in rats by angiotensin II, a hormone with hemodynamic effects, was associated with increased HO-1 expression in the aorta (269), and also in the heart, liver, and kidney of rats (15, 268, 377). In vitro this hormone produced induction of ho-1 in rat kidney cell models including renal proximal tubule epithelial cells, although this has not yet been established in human cell models (55, 609).

The HO-1 induction response may occur as a general consequence of exposure to environmental or industrial pollutants, which include some of the aforementioned agents such as heavy metal salts and solar ultraviolet radiation. Observations of ho-1 induction phenomena have been reported in vitro and during inhalation studies involving cigarette smoke (466), air-borne particulates including diesel exhaust (100), mineral fibers (chrysotile and crocidolite) (650), organic solvents such as benzene and bromobenzene (8, 221), nitrogen dioxide (288), zinc oxide from welding exhaust, (122) and ozone exposure (660, 240, 700). A recent study shows that continuous exposure to ozone in rats (0.8 ppm; 6 days) induces HO-1 predominantly in the skin by direct contact and to a lesser extent in the lung by inhalation (700). Thus HO-1 protein expression could be utilized as a general molecular marker of adverse environmental conditions in fish, birds, wildlife, and humans (66).

The compounds and agents described above represent only a partial list, and due to the generality of the ho-1 response to any conditions that cause cellular stress, certainly many more will be described. Since the discovery of the ho-1 transcriptional response, a number of hypotheses have emerged that have attempted to unify the mechanisms between the stimulus (treatment with exogenous inducing chemical) and the response of ho-1 gene activation. It has become evident that due to the diversity of stimuli and model systems studied, no unifying mechanism can be provided and that likely multiple mechanisms operate with cell-type and inducer-specific variations. One of the earliest of these hypotheses states that a transient increase in intracellular heme content may mediate the induction of HO-1 by certain inducers of the response. This hypothesis rests in part on the fact that heme itself is a potent inducer of the gene in vitro and in vivo, whether applied in free form or in protein complexes. Several inducing chemicals including insulin, epinephrine, diethylmaleate, CS2, and endotoxin are associated with transient increases in the hepatic heme pool (325). Furthermore, a limited number of hepatotoxins that induce HO-1 in hepatic systems are also associated with cytochrome P-450 loss or degradation. For example, CS2 induces hepatic HO-1 in conjunction with its acceleration of heme release from cytochrome P-450 as a consequence of irreversible apoprotein modification (278, 325). In one case, hepatic HO-1 induction coincided with loss of cytochrome P-450 as the result of partial surgical hepatectomy (760). Hepatic induction of HO-1 by heavy metals was dissociated from degradation of cytochrome P-450 (154). There is only limited evidence in extrahepatic tissues that a transient increase in intracellular free heme or increase in hemoprotein degradation precedes the induction of ho-1. In one example, the accelerated degradation of cytochrome P-420 was noted in parallel with the UVA-induced activation of HO-1 in human skin fibroblasts (354). This hypothesis is limited by the fact that many ho-1 inducing chemicals, such as heavy metals and thiol reagents, may not cause appreciable hemoprotein degradation and/or elevations in intracellular heme (325). Although not necessarily a universal intermediate mechanism in the response to xenobiotic inducers of the gene, heme levels play an integral part in ho-1 regulation by activating the translocation of Nrf2, and concurrently relieving transcriptional repression of ho-1, as detailed in section IIIC.

A second general hypothesis that has emerged states that a transient increase in intracellular reactive oxygen intermediates (ROS) mediates the induction of HO-1 in a redox-regulated pathway. This hypothesis is based in part on the fact that a large number of inducing agents are oxidants themselves or are associated with the intracellular production of ROS. This hypothesis is also supported by evidence that the induction of HO-1 by many, but not all, inducing chemicals can be inhibited by millimolar concentrations of N-acetyl-L-cysteine (NAC), an antioxidant and precursor for GSH (Table 2). For example, the transcriptional upregulation of ho-1 by H2O2 but not that of heme is inhibitable by NAC (18). This hypothesis is compromised by the fact that ROS production by certain agents, especially hypoxia and various cytokines, remains controversial. Furthermore, treatment of cell cultures with nonthiol antioxidants does not universally inhibit ho-1 activation by various inducers. For example, beta-caroteine inhibits ho-1 activation by UVA radiation, whereas the membrane antioxidant {alpha}-tocopherol augments the response in the same cell model (51, 690). The antioxidant caroteinoid lycopene potentiated UVA-induced DNA damage and induction of ho-1 activation, as a result of the photodegradation of the lycopene to oxidized metabolites (750). Furthermore, many plant-derived phenolic compounds such as quercetin, curcumin, that are naturally occurring antioxidants can induce ho-1, rather than inhibit ho-1 in cell culture models. Thus ROS generating systems produce a strong ho-1 activation, yet the generation of ROS is not necessarily an intermediate event in the pathway elicited by all classes of inducing chemicals.


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  		TABLE 2. Inhibition of HO-1 expression by N-acetyl-L-cysteine

 
The activation of ho-1 can be inhibited by metal chelating agents such as desferrioxamine (DFO), in a variety of cell culture systems in an inducer-specific fashion. For example, the induction of HO-1 by UVA radiation and H2O2 are inhibited by DFO treatment in vitro (320). Other examples of this phenomenon in the context of numerous cellular stress conditions are recapitulated in Table 3. While iron essentially does not exist in biological systems in "free" form, it is thought that potentially reactive iron exists in an intracellular pool in complexes with low-molecular-weight organic molecules (543). The original interpretation of the phenomenon was that metal chelating compounds act by removing a pool of potentially reactive iron, which amplifies the effect of oxidative stress conditions (320). In support of this hypothesis, iron loading has been shown to sensitize endothelial cells to peroxide stress (43). Iron loading potently synergizes the HO-1 activation by hypoxia in endothelial cells (559). A second interpretation of this phenomenon is that the chelating compounds remove a pool of iron that is required for gene transcription. Paradoxically DFO potently activates HIF-1 under normoxic conditions, which is among the factors that govern the transcriptional activation of ho-1 under hypoxia (365, 593). The prolyl hydroxylase involved in HIF-1{alpha} degradation requires iron (275). Recently, it has also