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The heme oxygenase (HO) isozymes catalyze oxidation of the heme molecule to biliverdin and carbon monoxide (CO) with the release of chelated iron. Presently, we have defined, for the first time, propensity for site of injury-directed induction of isozymes – the stress-inducible isozyme, HO-1, responds distal (below) and the glucocorticoid (GC)-inducible HO-2 responds proximal (above) to the site of injury. We have also shown that reactive iron (Fe3+) and cGMP staining spatially resemble that of HO-1; which, in turn, colocalizes in motor neurons with transcription factors: Fas-associated protein containing death domain (FADD), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and p53. Spinal cord injury (SCI) was inflicted by clip compression for 30 min, and analyses were carried out after 4 h or 16 h. When compared with spinal cord segments proximal to the site of injury, northern blot analysis showed remarkably higher levels of HO-1 mRNA distal (below) to the site of injury at both time points. In contrast, HO-2 mRNA levels were elevated proximal (above) to the site of injury and more prominently at 16 h post SCI. Immunohistochemical analyses were carried out using 2 × 5 mm segments above and below the compression site. When compared with segments above the site of injury, the intensity of HO-1 immunostaining and the number of HO-1 positive neurons in the ventral horn motor neurons were prominently increased in segments below the injury. Western blot analysis confirmed the observations. HO-2 protein was mapped to the ventral horn motor neurons, oligodendrocytes, the Clarke's nucleus neurons and the ependymal cells. When compared with segments below the site of injury, neuronal HO-2 staining intensity was increased above the site of injury, and most notably at 16 h. These observations were also confirmed by western blotting and HO activity measurements. Tissue Fe3+ and cGMP staining were increased and prominently mapped below the site of injury, where cGMP colocalized with HO-1 in the nucleus of the motor neurons. Also, a site of injury-directed pattern of induction of FADD, TRAIL, and p53 immunoreactivity, and a widespread colocalization of the oncogenes with HO-1 protein, were found within motor neurons below the level of injury.
We forward the hypothesis that HO-1 and HO-2 have different roles in the defense mechanisms of the injured nervous system. We hypothesize that HO-1 protects against further damage by contributing to controlled cell death through their intrinsic suicide program, while HO-2 is involved in suppression of inflammatory response by NO derived radicals.
The heme oxygenase (HO; EC188.8.131.52) system is the most effective mechanism for heme (heme b, Fe-protoporphyrin) degradation (reviewed by Maines 1992). To date, three isozymes: HO-1, HO-2, and HO-3 (Maines et al. 1986; McCoubrey and Maines 1994, 1997a, 1997b) have been described. HO-1 and HO-2 are the catalytically active forms and have been fully characterized. HO-3 was more recently described and has marginal activity (McCoubrey et al. 1997b). HO-1 and HO-2 represent different gene products (Cruse and Maines 1988) and, except for the heme-binding domain, share little similarity in primary structure, gene organization or regulation (Rotenberg and Maines 1990).
Traditionally the HO system was considered only in the context of heme catalytic activity. In the past few years this view has changed by finding that the products of HO activity: biliverdin, carbon monoxide (CO) and iron, are all biologically active molecules (reviewed by Maines 1997). Biliverdin and its reduction product, bilirubin, display antioxidant properties (Stocker et al. 1987; Panahian et al. 1999b). CO is suspected to function as a signaling molecule and a gaseous modulator for the second messenger, cGMP production in neurons (reviewed by Maines 1997; Snyder et al. 1998). cGMP effects ion channels (Nathanson et al. 1995; Finn et al. 1996), activity of phosphodiesterases (Degerman et al. 1996), protein phosphorylation by cGMP-dependent kinases (Vaandrager and De Jonge 1996), and effects output of excitatory amino acids (Matsuoka et al. 1999). Because, an increased HO activity leads to increased production of CO (Rodgers et al. 1994) and cGMP levels (Maines et al. 1993; Xue et al. 2000), it is likely that increased HO activity would modulate the output of excitatory amino acids. On the other hand, iron released in the course of tetrapyrrole cleavage is a potent catalyst for lipid peroxidation and oxygen free-radical formation (Aust 1995).
The oxygen radicals activate signaling pathways that result in activation of many genes, including those of oncogenes that are effectors in apoptotic cell suicide and tissue homeostasis. p53 is among the set of regulatory molecules whose expression is influenced by the redox state of the cell (Arrigo 1999). p53 has been implicated in a variety of cellular functions, including maintenance of genomic integrity, with its role in apoptosis being undisputed (reviewed by Gottlieb and Oren 1998). A recent study showed that p53 colocalizes with HO-1 in neurons in penumbra after ischemic injury to the brain (Panahian et al. 1999a). It has been reported that p53 and HO-1 concomitantly respond to cytotoxic prostaglandins (Ikai et al. 1998).
p53-associated apoptosis is suspected to be a common mechanism of cell death in neurodegenerative diseases. Upregulation of the gene has been reported in patients with amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease (de LaMonte et al. 1998), as well as in stroke (Panahian et al. 1999a). p53 transcriptionally activates a number of target genes that are involved in apoptosis including Fas/APO-1 and DR-4/5, which are receptors for Fas-associated protein containing death domain (FADD) and TNF-related apoptosis-inducing ligand (TRAIL), respectively (Sheikh et al. 1998). The cytotoxic death domain ligands, FADD and TRAIL (also known as APO-2 L), are members of the TNF ligand family. They transmit the cell suicide response by the different axes triggered in response to cytokines (Chinnaiyan et al. 1995; Pan et al. 1997).
For the most part, published reports suggest a role for the HO system in cellular defense mechanisms. However, it is reasonable to suspect that differences that exist in the primary structure of HO-1 and HO-2, and regulation of their gene expression, may lend to their differential function in defense mechanisms. HO-2 is among a select group of six proteins that have a heme binding motif known as the ‘heme regulatory motif’ (McCoubrey et al. 1997a) and has been suggested to function as a ‘sink’ for NO and gaseous heme ligands (Maines 1997; Ding et al. 1999). Moreover, in the central nervous system, the bulk of HO activity is attributed to the glucocorticoid (GC)-responsive HO-2 isozyme (Sun et al. 1990; Weber et al. 1994), which is expressed at high levels in neurons (Ewing and Maines 1992). As it happens, the adrenal GCs are among the less-than-a-handful of stimuli that induce HO-2 (Chen and Maines 2000; Li and Clark 2000) and the only therapeutic agents proven effective in reducing neurological damage after SCI (Bracken et al. 1990; Young et al. 1994). On the other hand, HO-1, which is an immediate early gene, is readily induced in the central nervous system by a multitude of stimuli, including hypoxia/hyperthermia, cerebral ischemia, change in intracellular redox status, subarachnoid hemorrhage and trauma (Ewing and Maines 1991, 1993; Maines et al. 1995; Geddes et al. 1996; Bergeron et al. 1997; Mautes et al. 1998; Turner et al. 1998; Panahian et al. 1999a).
To gain insight into the possibility of differential functions of HO-1 and HO-2 in defense mechanisms, we have compared HO-1 and HO-2 gene expression relative to the site of SCI and, have examined the site of injury-directed increases in the redox active heme degradation product, Fe3+, and the second messenger, cGMP, after SCI. Also, the induction of transcription factors, that are activated by changes in redox state of the cell and their colocalization in the spinal cord neurons with HO-1, were examined. Based on the present findings, the previous report on the mechanism of regulation of HO isozymes, and the known changes that occur in the cellular homeostasis after SCI, we are presenting an argument for activity of both isozymes in the defense mechanism of the organism, albeit in different capacities.
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With respect to the site of injury, this study has defined the temporal and directional patterns of HO-1 and HO-2 response to SCI; and has described colocalization of HO-1 with cGMP and transcriptional factors, p53, FADD, and TRAIL. The study has also shown injury site-directed increased tissue levels of redox active Fe3+, which is like that of HO-1. To our knowledge, this is the first demonstration of these phenomena, that together with what is known about the primary structure of HO isozymes and regulation of their expression, have permitted us to propose that HO-1 and HO-2 play differential roles in neuronal response to injury and protection of the neural tissue. We propose that HO-1 protects against further destruction of the damaged tissue by promoting autodestruction of neuronal cells, rather than by inflammatory cascade events. It is further proposed that HO-2 protects against the latter events by acting as a ‘sink’ for nitric oxide derived free radical species (Ding et al. 1999). The reasoning for our proposal is provided in the following.
As assessed by the rate of degradation of denatured hemoproteins by the purified preparations of HO isozymes (Kutty et al. 1988), HO-1 is by far the more catalytically active form among the two isozymes. HO-1 being a stress-responsive gene, has been shown to be induced by nitrogen-derived molecules (Foresti et al. 1997; Hartsfield et al. 1997; Ding et al. 1999). A component of the factors that influence HO-1 gene transcription in SCI is likely the high output of NO produced by inducible NO synthase (iNOS). NO derivatives induce HO-1 by activating MAP kinases, ERK (1 and 2) and p38 pathways (Chen and Maines 2000). Increased synthesis of NO is important not only to cell killing, i.e. host defense, but also to immune response (reviewed by MacMicking et al. 1997). Increased expression of HO-1 modulates inflammatory immune response and immune effector functions (Willis et al. 1996; Iyer et al. 1998; Woo et al. 1998). In turn, the immune modulation has been attributed, in part, to the inhibition of protein phosphorylation and kinase activity by heme degradation product bilirubin (Woo et al. 1998). Inhibition of protein phosphorylation has been recently shown to result in a significant decrease in infarct volume after stroke in mice expressing dysfunctional CD95 ligand as well as postischemic expression of TRAIL within brain apoptotic tissue (Martin-Villalba et al. 1999). Activation, i.e. increased phosphorylation of ERK MAP kinase (Alessandrini et al. 1999), as well as induction of HO-1 in cerebral ischemia (Geddes et al. 1996; Panahian et al. 1999a), have been reported. MAP kinase pathway activation also triggers pro-apoptotic gene activation.
Our data, showing colocalization of the induced HO-1 with pro-apoptotic gene expression, allows for suggestion that the inductions are related. Based on the observed increase in redox active Fe3+ staining of the spinal cord below the site of compression, together with the fact that Fe3+ is a potent catalyst for oxygen derived radicals that can signal the activation of pro-apoptotic genes, it is reasonable to suggest that increased expression of HO-1 is causally linked to the expression of p53, FADD, and TRAIL. Indeed a recent study has shown activation of Caspase 1 by CO and inhibition of cell death by apoptosis when Caspase 1 activity was inhibited (Thom et al. 2000). We argue that this would constitute a protective mechanism, because an important dimension of secondary injury to the spinal cord can be necrotic cell death (Anderson and Hall 1993), which promotes inflammation. Unlike necrotic cells, cells killed by apoptosis will be cleared in situ by autodestruction, without inducing inflammation and damage to the surrounding tissue. Indeed, using in situ nick-end labeling, the presence of apoptotic neuronal and glial cells in the spinal cord of rats with compression injury has been documented (Li et al. 1996; Crowe et al. 1997; Wada et al. 1999). A recent study suggests that HO-1 induction inhibits TNF-α-mediated apoptosis (Petrache et al. 2000). However, because TNF-α can be considered both anti- and pro-apoptotic (Hu et al. 2000), and that activation of TNF is one of the pathways that promote apoptosis, the present suggestion and the published report would not be inconsistent.
According to the study by Wada et al. (1999), apoptosis appears to be linked to the activation of the post synaptic receptors for excitatory amino acids such as glutamate. It has been shown that the levels of excitatory amino acids increase after SCI (Panter et al. 1990; Yanase et al. 1995). Interestingly, the mechanism of CO signaling, generated by HO activity, is thought to involve activation of soluble guanylyl cyclase with ultimate activation of glutamate receptors (Stevens and Wang 1993; Verma et al. 1993; Zhou et al. 1993; Dawson and Snyder 1994). Accordingly, it is reasonable to suggest that a marked increase in HO-1 would alter the output of excitatory amino acids, which in turn could influence autodestruction of the neural cells and upregulation of pro-apoptotic genes (Ellis et al. 1991).
The finding that cGMP localizes to the nucleus of neurons that express high levels of HO-1 is noteworthy because cGMP is an intracellular second messenger that mediates signal transduction for various gene transcription (Gudi et al. 1997), as well as actions of a large number of hormones and neurotransmitters (de Vles et al. 2000). cGMP binds to regulatory domain of kinases and promotes conformational changes that activate their catalytic functions (Gudi et al. 1997) and translocation via the nuclear pore complex (Gorlich and Mattaj 1996) into the nucleus. Accordingly, the present findings could be consistent with the possibility that nuclear localization of cGMP reflects on increased intracellular signaling and gene transcription.
Aside from its catalytic activity-related functions, a possible mechanism by which HO-2 induction would be neuroprotective can be explained on the basis of the primary structure of the protein, which contains cysteine residues, its hemoprotein nature (McCoubrey et al. 1997a) and the interaction of its heme moiety with NO derivatives (Ding et al. 1999). Up to now, very few stimuli have been described that induce HO-2: glucocorticoids (GCs) and opiates are among them (Weber et al. 1994; Li and Clark 2000). The adrenal GCs are inducers of HO-2 and enhance HO-2 expression at transcriptional and translational levels (Weber et al. 1994; Liu et al. 2000). GCs are also the only neuroprotective agents in SCI (Bracken et al. 1990; Young et al. 1994). The mechanism by which GCs control gene expression requires binding to the GcR.
Recently it was reported (Yan et al. 1999) that, subsequent to SCI, there is a marked increase in expression of GcR protein and that the protein is localized to the ventral horn neurons. GcR protein was detected in oligodendrocytes and astrocytes. In the present study, HO-2 was also mapped to the ventral horn motor neurons and oligodendrocytes. The increased expression of HO-2 is then likely to reflect increased expression of the receptor, which in turn has been considered to involve increased expression of the endogenous cytokines (Yan et al. 1999). It is known that cytokines such as TNF-α, interleukin-1β and interleukin 6 are induced systemically after SCI (Yakovlev and Faden 1994), and may enhance GC production by increasing ACTH and/or corticotropin-releasing hormone levels (Hermus and Sweep 1990).
A mechanism by which pro-inflammatory cytokines cause tissue injury involves activation of nuclear factor-κB (NF-κB), which has been shown to be activated by traumatic SCI (Bethea et al. 1998). Overexpression of FADD which, along with caspase-8-related protein and caspase-8, is one of the components of the tumor necrosis factor receptor type-1 (TNF-R1), specifically activates NF-κB (Hu et al. 2000). Activation of NF-κB promotes the expression of inflammatory mediators such as NO synthase (Hall et al. 1994). As a result of this activation, nitrogen derived molecules that can directly cause cell killing are produced. The interaction of HO-2 bound heme with NO derivatives has been postulated to buffer activity of NO and provide a ‘sink’ for the free radical (Maines 1997; Ding et al. 1999). The physiological basis for preferential increase in HO-2 expression above the site of injury is not evident, however, it can be considered that severance of neuronal signaling to the spinal cord segment below the site of injury may blunt HO-2 gene response below the site of injury. In other words, HO-2 induction may depend on factors generated in neuronal tissue above the site of injury. That such factor(s) may include ACTH was discussed above.
To achieve functional recovery after SCI, neurons in both ascending and descending pathways must be preserved (Yick et al. 1999). As shown here and established previously (Ewing et al. 1992), there is a robust expression of HO-2 in the ascending spinocerebellar tract formed by Clarke's nucleus, which projects to the cerebellum, as well as the descending rubrospinal motor tract formed by red nucleus. Cerebellar Purkinje neurons and layer V cortical neurons were also previously shown to contain high levels of HO-2 (Ewing and Maines 1992). Since Clarke's nucleus, red nucleus, and Purkinje neurons are selectively vulnerable to an array of traumatic, as well as ischemic and hypoxic conditions (Sanner et al. 1994; Radovsky et al. 1997; Yick et al. 1999), and ventral horn motor neurons and cortical layer V motor neurons are selectively vulnerable to neurodegenerative conditions (Tandan and Bradley 1985), induction of HO-2 by adrenal GCs may be a plausible mechanism by which these neurons are protected after SCI.
In conclusion, the propensity for site of injury-directed induction of HO-1 and HO-2 may point to their distinct function in neural cell protection following SCI. This may reflect their primary protein structure and gene regulation. These findings may represent a previously unrecognized target for development of therapeutic approaches for treatment of spinal cord injury.