Heme oxygenase-1 (HO-1) provides an inducible defense mechanism that can be activated ubiquitously in cells and tissues in response to noxious stimuli, conferring cellular protection against injury inflicted by such stimuli [1
]. HO-1 serves a vital metabolic function as the rate-limiting step in the oxidative catabolism of heme-b
, to generate equimolar carbon monoxide (CO), biliverdin-IXα (BV), and ferrous iron; the BV generated is subsequently converted to bilirubin-IXα (BR) by NADPH biliverdin reductase (BVR)[4
]. The essential role of HO-1 in stress adaptation has been demonstrated by the phenotype of ho-1−/−
) mice and in a unique case of human HO-1 deficiency. The ho-1−/−
mice exhibit altered tissue iron distribution, increased susceptibility to pulmonary ischemia/reperfusion (I/R) injury, but paradoxical resistance to hyperoxic lung injury [5
]. The HO-1 deficient child exhibited extensive endothelial cell damage, anemia, hypobilirubinemia, aberrant tissue iron deposition, and increased inflammatory indices [9
]. Furthermore, vascular endothelial cells derived from ho-1−/−
mice or the HO-1 deficient child exhibited enhanced susceptibility to oxidative stress in vitro
HO-1 expression can confer cytoprotection in many lung and vascular injury and disease models [1
]. The cytoprotective effects of HO-1 are related to end-product formation () [1
], though non-catalytic functions of the protein have also been proposed [12
]. The pharmacological application of CO and BV/BR can mimic the HO-1 dependent cytoprotection in many injury models [1
]. Tissue protection generally involves inhibition of apoptosis and related cell death pathways, although inhibition of inflammation and/or cell proliferation may also be involved, depending on the specific injury model [1
]. In recent years, intensive investigation has focused on potential therapeutic tools to manipulate apoptosis in order to alter the outcome of pulmonary or vascular diseases. Such approaches have involved the use of agonists or inhibitors of specific signal transduction components, (eg.
, mitogen activated protein kinases, MAPK), or antioxidants, thus far with limited clinical efficacy [13
]. In this regard, the targeted manipulation of HO-1 or of its end-products remains a promising experimental and translational strategy. This review will focus on the role of HO-1/CO in modulating cell death mechanisms in tissue injury and pathology models. The specific roles of HO-1 in inflammation and carcinogenesis have been reviewed elsewhere [15
]. Recent works examining the therapeutic potential of HO-1/CO in models of oxidative stress, I/R injury, cigarette smoke exposure, and other forms of acute and chronic lung or vascular injuries will be described.
Mechanisms of Cell Death
Just like the multi-cellular organisms they constitute, cells must die. The method of cell death may be traumatic, resulting from acute, accidental, or non-physiological injury (ie.
, necrosis), or may arise as the consequence of genetic programs, (ie.
, apoptosis or Type I programmed cell death). Apoptosis provides essential homeostatic functions in regulating growth and development of organs, and in tissue responses to injurious stimuli, such as exposure to xenobiotics or adverse environmental conditions [17
]. Disruption of apoptosis may lead to tumorigenesis or autoimmune disease, whereas excessive apoptosis may cause organ failure [18
]. The role of apoptosis in the pathogenesis of specific diseases remains controversial, but has been implicated in cigarette smoke (CS)-induced chronic obstructive pulmonary disease (COPD), oxidative lung injury, pulmonary I/R injury, transplant-associated I/R injury, and others [20
Apoptosis is distinguished from necrosis on the basis of morphological and biochemical features [17
]. Apoptosis requires the activation of proteases (eg.
, caspases) and nucleases within an intact plasma membrane, resulting in organelle decomposition [25
]. On the other hand, necrosis is characterized by membrane damage and leakage of cytosol into the extracellular space, which may promote local inflammation and damage to surrounding tissues [24
]. The cardinal biochemical features of apoptosis include DNA fragmentation, mitochondrial dysfunction, and increased expression or activation of pro-apoptotic (eg.
, Bax, Bid) relative to anti-apoptotic (eg.
, Bcl-2, Bcl-Xl
) proteins of the Bcl-2 family [25
Cells can initiate apoptosis by two distinct pathways, a receptor-dependent “extrinsic” pathway and an “intrinsic” pathway involving mitochondrial dysfunction [28
] (). In response to stimuli, (ie.
, nutrient deprivation, DNA-damaging agents), proapoptotic Bcl-2 family proteins such as Bax initiate the intrinsic pathway by permeabilizing the outer mitochondrial membrane, thereby releasing cytochrome c
) from the inter-membrane space [31
]. Cytosolic Cyt-c
forms a complex with apoptotic protease-activating factor-1, and dATP which activates caspase-9, and its downstream caspase-3, resulting in the activation of the apoptosis [3
Inhibition of apoptosis by HO-1 and its enzymatic products
In contrast, the extrinsic pathway initiates when a death ligand, (ie.
, FasL) interacts with its cell surface receptor (i.e.
, Fas), forming a death-inducing signal complex (DISC)[28
]. Activation of Fas triggers the rapid recruitment of FADD (Fas-associated death domain protein) and caspase-8, resulting in the activation of caspase-8. Active caspase-8 cleaves Bid into truncated Bid (tBid), which translocates to the mitochondrial membrane, assists in Bax activation, and triggers Cyt-c
]. Thus, the extrinsic and intrinsic pathways converge at the mitochondria, and share similar features downstream of Bax activation and Cyt-c
Recent studies indicate that autophagy, a regulated pathway for internal organelle or protein degradation influences the outcome of cell death pathways [17
]. During autophagy, cytoplasmic double membrane-bound vesicles (ie.
, autophagosomes) sequester damaged organelles for delivery to the lysosome where they are degraded and recycled [35
]. The Bcl-2 interacting protein Beclin 1 and the microtubule-associated protein-1 light chain-3 (LC3), represent major regulators of autophagosome formation in human cells [39
]. Autophagy plays a central role in cellular adaptation to environmental stress, such as oxidative stress, serum starvation, endoplasmic reticulum stress, hypoxia, and infection [34
]. Activation of autophagy is regarded as a survival mechanism during nutrient deprivation, whereas excessive autophagy may promote cell death under certain conditions [37
]. Recent studies suggest that autophagic proteins may cross-regulate the initiation of the apoptotic program in response to stress [42
]. Though increased autophagosome formation is frequently observed in dying cells, the casual relationship between autophagy and cell death remains a matter of current controversy [45
]. The role of HO-1 in regulating specific pathway elements of autophagy and apoptosis will be discussed in subsequent sections.
Heme Oxygenase-1: biochemical properties and subcellular localization
HO-1, the inducible HO isozyme, is the major inducible low molecular weight (32–34 kDa) stress protein of mammalian cells and tissues [46
]. Heme oxygenase has a major constitutively expressed isozyme, heme oxygenase-2 (HO-2) [47
]. HO-1 and HO-2 represent the products of two distinct genes [48
]. Although HO-1 and HO-2 both catalyze heme-b
to biliverdin, the two enzymes differ in primary structure and biochemical/biophysical properties [48
]. A highly conserved sequence of 24 amino acid residues corresponding to the heme catalytic domain has been identified in common to both HO-1 and HO-2 [50
]. Furthermore, both HO-1 and HO-2 hare similar hydrophobic regions at the carboxyl-terminus that anchor the proteins in cellular membranes [51
]. A gene potentially encoding a third HO isozyme, ho-3
was cloned in the rat, though further analysis has suggested that the rat ho-3
genes represent processed pseudogenes, and to date, no natural protein has been found [54
The expression of HO-1 occurs at very low levels in most tissues under physiological conditions, with the exception of the spleen, the site of erythrocyte hemoglobin turnover, where high levels of HO-1 are constitutively present. In contrast, HO-2 is expressed under basal conditions in most tissues, including testes, spleen, liver, kidney, brain, and vasculature (Reviewed
HO-1 is an integral membrane component of the smooth endoplasmic reticulum (ER), evidenced by its abundance in microsomal membrane preparations. Recent studies have identified possible subcellular compartmentalization of HO-1 beyond ER membranes [56
]. For example, a nuclear localization of HO-1 has been described [56
]. These studies have suggested that HO-1 undergoes a proteolytic truncation of the carboxyl terminal hydrophobic region to facilitate nuclear entry. Despite apparent lack of heme catalytic activity of the truncated nuclear HO-1, recent work suggests that HO-1 may have a non-catalytic function in regulating nuclear transcription factor activities [12
]. Increased nuclear localization of HO-1 has been associated with several clinical disease conditions, as highlighted by recent studies in prostate cancer [57
], suggesting a possible role for nuclear HO-1 accumulation in malignancy.
Furthermore, HO-1 and functional HO activity can be detected in detergent-resistant fractions (lipid rafts) of plasma membrane. Sucrose density gradient fractionation revealed the presence of HO-1 in low-density caveolin-1 containing fractions (caveolae). Within this compartment, caveolin-1 was shown to bind to HO-1 and negatively regulate HO activity [58
]. Caveolin-1 regulates a number of signaling-related molecules in caveolae, (ie.
, endothelial nitric oxide synthase)[59
]. The regulation of HO activity in this compartment may represent a mechanism for the controlled production of CO for signaling purposes [58
]. However, the functional significance of HO-1 in plasma membrane lipid rafts remains unclear at present.
HO-1 also localizes in part to the mitochondria as shown in pulmonary epithelial cells [60
] and in rat hepatocytes [61
]. In epithelial cells, the expression of HO-1 protein and activity in the mitochondria increases in response to toxic stimuli, including cigarette smoke, bacterial lipopolysaccharide, and hemin [60
]. While the mitochondria play central roles in the regulation of apoptosis, it remains unclear whether mitochondria-localized HO-1 modulates the apoptotic program by acting on specific components of the mitochondria. In conclusion, the specificity and function of the novel compartmentalization (ie.
, nucleus, mitochondria, lipid raft) of HO-1 remains unclear, but further study may reveal additional mechanisms relevant to the anti-apoptotic or cytoprotective potential of HO-1.
Regulation of HO-1 transcription
The transcriptional upregulation of the ho-1
gene, and subsequent de novo
synthesis of the corresponding protein occurs in response to elevated levels of its natural substrate heme, and to a multiplicity of endogenous factors including NO, cytokines, heavy metals, hormones, and growth factors (Reviewed
]). Many agents that induce HO-1 are associated with oxidative stress in that they (I) directly or indirectly promote the intracellular generation of reactive oxygen species (ROS), (II) fall into a class of electrophilic antioxidant compounds which includes plant-derived polyphenolic substances [62
], or (III) complex intracellular reduced glutathione and other thiols [65
]. HO-1 expression responds to pro-inflammatory stress, associated with the production of ROS from activated inflammatory cells [67
]. Furthermore, HO-1 expression responds to changes in ambient O2
tension, such as hyperoxia (increased pO2
) or hypoxia (decreased pO2
), both which can modulate mitochondrial ROS production. The regulation of HO-1 by hypoxia can vary in a cell type and species-specific manner, with transcriptional repression rather than induction reported in human cell types [68
Because expression of HO-1 is regulated by a wide array of compounds in a cell-type and inducer-specific fashion, defining unifying transcriptional regulatory mechanisms has been challenging [69
]. Two enhancer regions located at approximately −4kb and −10kb relative to the ho-1
transcriptional start site have been identified in the mouse gene [70
]. The dominant sequence element of the enhancers is the stress-responsive element (StRE), which is structurally and functionally similar to Maf response element (MARE) and the antioxidant response element (ARE)[69
]. Several transcriptional regulators bind these sequences, including nuclear factor erythroid 2-related factor-2 (Nrf2) and Bach1. These are both members of the Cap’n’collar/basic-leucine zipper family capable of heterodimerizing with small Maf proteins and regulating MARE-driven genes. Nrf2 contains a transcription activation domain and positively regulates HO-1 transcription, whereas Bach1 competes with Nrf2 and represses transcription [73
]. Mice lacking the gene for Bach1 have dramatic increases in HO-1 expression in the heart, lung and liver, indicating a role for Bach1 in tonic suppression of HO-1 transcription. Conversely, inhibition of Nrf2 gene function results in impairment of transcriptional responses to known inducers of HO-1 [73
]. Thus, HO-1 expression depends upon changes in the balance of activity of Nrf2 and Bach1. Heme can relieve Bach1 of DNA binding activity through complex formation, promote the nuclear export of Bach1 and inhibit the proteasomal degradation of Nrf2, thus enhancing HO-1 expression [73
]. The Kelch-like ECH-associated protein (Keap1) inhibits HO-1 expression by sequestering Nrf2 in the cytoplasm. Keap1 facilitates the targeting of Nrf2 by Cullin 3-based E3 ubiquitin ligase complex, which marks Nrf2 for proteasomal degradation [83
]. Under basal conditions, Keap1 binds to Nrf2 and sequesters it in the cytoplasm, which results in a lower accumulation of Nrf2 in nucleus and reduced transcription of the HO-1 gene [85
]. When cells are exposed to electrophiles or oxidants, Nrf2 dissociates from Keap1, translocates to the nucleus, and binds to target genes [89
]. In Keap1 null mice, Nrf2 constitutively increases in the nucleus and inappropriately stimulates its target genes, leading to growth retardation and early death. However, the phenotypic abnormalities can be reversed by reduction of Nrf2 abundance in the Keap1-null mutant background [91
]. Nrf-2 knockout mice were more susceptible to tobacco smoke-induced lung apoptosis and emphysema relative to wild type mice, illustrating a critical role for Nrf2 in the regulation of adaptive responses to stress [92
While the Nrf2 system has been characterized extensively in the mouse as the major stress inducible operator of the ho-1
gene, many additional cis
-acting promoter elements have been identified in the context of the mouse, rat, and human ho-1
upstream regulatory regions which may also contribute to ho-1
regulation in a cell type an inducer-specific fashion. Examples of elements that respond in an inducer-specific or selective fashion include those responsive to hyperthermia [93
] or hypoxia [94
] which represent the targets of distinct DNA-binding proteins such as heat shock factors (HSF) and hypoxia-inducible factor-1 (HIF-1), respectively.
While the human ho-1
promoter contains regions analogous to the upstream enhancer regions described in the mouse, further studies have revealed additional complexities specific to the human ho-1
promoter. The human ho-1
gene contains an additional 10 bp sequence element, termed the cadmium responsive element (CdRE), occurring within the –4kB enhancer region but extrinsic to the StRE sequence homologies, which mediates CdCl2
induction of the gene [95
]. Furthermore, the human −4kb enhancer region contains a binding site preferentially targeted by cyclic AMP responsive element binding factor (CREB) [96
]. A region at −9.5 kb corresponds to a binding site for the early growth response-1 (Egr-1) factor, which mediates the response to metalloporphyrin induction [97
]. A number of proximal promoter elements have also been identified as potentially important in human ho-1
gene regulation [98
]. These include E-box motifs potentially recognized by the upsteam stimulatory factor (USF), and/or basic helix loop helix (bHLH) proteins [101
], ETS binding sites (EBS) [103
], as well as binding sites for nuclear factor-kappa B (NF-κB) [104
], STAT-3 [105
], activator protein-2 (AP-2) [104
], and HSF-1[99
]. An upstream region contains potential binding sites for hepatocyte nuclear factors (HNE1 and HNE4), AP-1, STAT-x, c-Rel, and GATA-X [106
]. These studies reveal the complex nature of ho-1
gene regulation, which potentially involves the coordinated interaction of multiple transcription factors. The functional significance of all of these factors however, remains incompletely understood [10
A microsatellite (GT)n
dinucleotide length polymorphism has been described in the promoter region of the human ho-1
gene, which can result in the impaired transcriptional regulation and decreased expression of HO-1 in individuals that carry the long (L) allele [ie
., (GT)n ≥30] of this polymorphism [108
]. Multiple human genetic epidemiology studies have examined the association between this length polymorphism in the human ho-1
gene, and COPD-related traits. The “Long” allele of the GT repeat (≥25, ≥30, ≥32, or ≥33 repeats, depending on study) has been associated with COPD [109
], emphysema [108
], COPD severity [110
], lung function decline in COPD [111
] and lung function decline in the general population [112
]. For example, in a retrospective study of French smokers the L allele [(GT)n ≥ 33] was associated with decreased lung function parameters relative to non-carriers. The greatest decline in lung function was observed in heavy smokers that carried the L allele [112
]. However, the association with lung function decline was not replicated in COPD subjects in the National Heart, Lung, and Blood Institute Lung Health Study (115), while other studies have demonstrated associations with different alleles [ie
., (GT)n ≥30] of the GT repeat [116
]. Thus additional studies are required before a consensus can be realized. Interestingly, lymphoblastoid cells cultured from homozygous carriers of the L allele displayed increased susceptibility to oxidant (H2
)-mediated apoptosis in vitro
]. These studies suggest that a genetically-dependent downregulation of HO-1 expression may arise in subpopulations, possibly linked to increased susceptibility to oxidative stress-related disease.