Heme Oxygenase

Until 1997, two HO isoforms were described: an inducible isoform, HO-1 and a constitutive isoform HO-2. These isoforms are the products of different genes and share only 43% homology. A 24-amino-acid segment, which forms a hydrophobic pocket within the tertiary structure of the protein, is common to both the isoforms and is considered the active center of the enzyme (67). Metalloporphyrins such as Zn-protoporphyrin-IX and Sn-protoporphyrin-IX (Zn-PP-IX and Sn-PP-IX, respectively) bind to the hydrophobic pocket of HO but do not catalyze hydrolysis (or they do so at a much lower rate than heme), thereby inhibiting HO activity (68,69). In fact, Zn-PP-IX may be considered as an endogenous inhibitor, since it is synthesized instead of heme in the case of iron deficiency (14). Apart from the identity between the active centers of the enzyme, HO-1 and HO-2 broadly differ in cell and tissue regulation, and distribution.

Heme oxygenase-1, also referred as Hsp-32, is induced by various stimuli including ROS, RNS, ischemia, heat shock, LPS, hemin and the neuroprotective agent Neotrofin (67,70,71). Furthermore, in cultured human cells, HO-1 expression can be repressed by hypoxia or by the treatment with interferon-y or desferrioxamine (72). On the contrary, HO-2, the constitutive form, is responsive to developmental factors and adrenal glucocor-ticoids (67,70). Although HO-1 and HO-2 catalyze the same reaction, they play different roles in protecting tissues against injuries. Based on several lines of evidence, the current hypothesis suggests that HO-1 induction is one of the earlier cellular response to tissue damage and is responsible for the rapid transformation of the pro-oxidant heme into CO and BR, two molecules with anti-inflammatory and antioxidant activity. On the contrary, HO-2, constitutively expressed, is primarily involved in maintaining cell heme homeosta-sis and recent evidence proposed, for this isoform, a new role as an endogenous sensor of gaseous molecules such as oxygen, CO and NO (73,74). This characteristic inducibility of ho-1 gene strictly relies on its configuration: the 6.8-kb gene is organized into 4 introns and 5 exons. A promoter sequence is located approximately 28 bp upstream from the transcriptional site of initiation. In addition, different transcriptional enhancer elements, such as heat shock element and metal regulatory element reside in the flanking 5' region. Also, inducer-responsive sequences have been identified in the proximal enhancer located upstream the promoter and, more distally, in two enhancers located 4 and 10 kb upstream the initiation site (75). The molecular mechanism that confers inducible expression of ho-1 gene in response to numerous and diverse conditions has remained elusive. One important clue has recently emerged from a detailed analysis of the transcriptional regulatory mechanisms controlling the mouse and human ho-1 genes. The induction of ho-1 is regulated principally by two upstream enhancers, E1 and E2 (76). Both enhancer regions contain multiple stress (or antioxidant) responsive elements (StRE, also called ARE) that also conform to the sequence of the Maf-recognition element (MARE) (77) with a consensus sequence (GCnnnGTA) similar to that of other antioxidant enzymes (78). There is now evidence to suggest that heterodimers of NF-E2-related factor 2 (Nrf2) and one or another of the small Maf proteins (i.e. MafK, MafF and MafG) are directly involved in the induction of ho-1 gene through these MAREs (77). A possible model, centered on Nrf2 activity, suggests that the ho-1 gene locus is situated in a chromatin environment that is permissive for activation. Since the MARE can be bound by various heterodimeric basic leucine zipper (bZip) factors including NF-E2, as well as several other NF-E2-related factors (Nrfl, Nrf2 and Nrf3), Bach, Maf and AP-1 families (79), random interaction of activators with the ho-1 gene enhancers would be expected to cause spurious expression. This raises a paradox as to how cells reduce transcriptional noise from the ho-1 locus in the absence of metabolic or environmental stimulation. This problem could be reconciled by the activity of repressors that prevent non-specific activation. One possible candidate is the heme protein Bachl, a transcriptional repressor endowed with DNA-binding activity, which is negatively regulated upon binding with heme. Bachl-heme interaction is mediated by evolutionarily conserved heme regulatory motifs (HRMs), including the cysteine-proline dipeptide sequence in Bachl. Hence, a plausible model accounting for the regulation of ho-1 gene expression by Bachl and heme, is that expression of ho-1 gene is regulated through antagonism between transcription activators and the repressor Bachl. Under normal physiological conditions, expression of HO-1 is repressed by Bachl-Maf complex, while increased levels of heme displace Bachl from the enhancers and allow activators, such as heterodimer of Maf with NF-E2-related activators (Nrf2), to interact with the tran-scriptional promotion of ho-1 (79). To our knowledge, the Bachl/ho-1 system is the first example in higher eukaryotes that involves a direct regulation of a transcription factor for an enzyme gene by its substrate. Thus, regulation of ho-1 gene involves a direct sensing of heme levels by Bachl (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate affects repressor-activator antagonism. However, depending on the cell type HO-1 induction may not be always beneficial for cells, in particular for human cells. This idea is sustained by the evidence that in cultured human cells, hypoxia, interferon-y and desferrioxamine repress HO-1 expression via the activation of Bachl, whereas the same stimuli cause HO-1 induction in rodent cells (72,80). The reason why an antioxidant enzyme such as HO-1 is repressed in humans under conditions of oxidative stress is still under debate. The current hypothesis is that HO-1 repression may be useful in situations where either the mitochondrial heme availability, or the energy expenditure necessary for heme degradation is diverted to prevent local accumulation of CO, iron and BR; in addition, HO-1 repression may decrease iron supply to cancer cells or pathogens, such as bacteria and protozoa, which require iron as an essential cofactor for cell proliferation (72).

Heme oxygenase activity is also regulated by BVR because the latter reduces BV, the inhibitory product of the oxygenase activity, into BR (81). The molecular mass of BVR ranges between 41-42 kDa (human) and 33-34 kDa (rat); it is a dual cofactor and dual pH-dependent and requires free SH groups (82). Until now, BVR was considered a noninducible protein but recent data showed that the reductase can be induced by LPS and bromobenzene at a post-transcriptional level whereas heat shock has no effect (83,84). In the rat brain, BVR is co-expressed in cells that display HO-1 and/or HO-2 under normal conditions, as well as in regions and cell types that have the potential to express heat shock-inducible HO-1 protein (83). Further evidence demonstrated that BVR exhibited developmental changes with the activity increasing after birth and reaching an adult level by day 28 postpartum. Immunohistochemical analysis revealed age-related pattern of the expression of BVR in select rat brain areas such as the cortex, substantia nigra, hippocampus and cerebellum (81).

HO-1 is ubiquitary and particularly abundant in reticuloendothelial organs such as liver and spleen, whereas HO-2 is localized in specific organs such as brain, kidney and testis (67). The CNS is endowed with very high HO activity under basal conditions, mostly accounted for by HO-2, the latter being expressed in neuronal populations in fore-brain, hippocampus, hypothalamus, midbrain, basal ganglia, thalamus, cerebellum and brainstem. The inducible isoform is instead present in very small amounts and is localized in sparse groups of neurons, including the ventromedial and paraventricular nuclei of the hypothalamus (67). This finding indicates that the activation of HO-1 and the following formation of CO can be induced by many noxious stimuli within the nuclei that are primarily involved in the central regulation of the stress response. In fact, neurons located within the parvicellular part of the paraventricular nucleus release both CRH and arginin-vasopressin (AVP), the neuropeptides that initiate the endocrine response to a stressor stimulating the release of pituitary ACTH (14). HO-1 is also found within cells of glial lineage, where its gene expression can be induced by oxidative stress (85).

In 1997, Mahin Maines and her group described a third HO isoform called HO-3. It is a protein of about 33 kDa encoded by a single transcript of 24 kb and constitutively expressed in rat liver, spleen, kidney and brain (86). In a recent article, Scapagnini et al. investigated the regional brain expression of HO-3 and they found that this isoform is expressed mainly in astrocytes of hippocampus, cerebellum and cortex (78). The regulation of ho-3 gene expression and its synthesis is poorly understood and its possible role in the physiology and pathology remains to be further clarified.

Nitrosative stress and heme oxygenase

With regard to the modulation of HO by nitrosative stress, it is important to distinguish between the two HO isoforms and the tissues where this interaction occurs.

It is well established that NO and RNS induce ho-1 gene and protein in different conditions with a mechanism not fully understood (87). However, taking into consideration the strong pro-oxidant activity of NO and RNS it is plausible to conclude that HO-1 induction has to be considered as a mechanism by which cells can react to stressful conditions. In fact, HO-1 induction by NO is very important in selected cells, such as macrophages for two reasons: first, because HO-1 activity depletes cells from heme, toxic if in excess and second, because the production of BR and CO through the HO activity ensures an efficient scavenging of ROS and RNS and a further inhibition of NADPH-oxidase and iNOS, thus contributing to the resolution of oxidative conditions (88). In addition, peroxynitrite and nitroxyl anion have been shown to increase, in a dose-dependent manner, HO-1 expression in endothelial cells and human colo-rectal adenocarcinoma cells (89-92). In brain cells, NO has been shown to induce HO-1 expression in rat astrocytes and microglia (93,94) as well as in rat hippocampus (95).

NO has been shown to inhibit or stimulate HO activity and this differential modulation depends on the tissue or cell line. In particular, studies carried out on endothelial or smooth muscle cells have shown that NO is able to increase HO activity (96-98), whereas Willis et al. (99) demonstrated that NO (released by sodium nitroprusside) reduced HO activity in rat brain and spleen homogenates. The reason for this dual effect of NO on HO activity was clearly explained by Maines (67) on the basis of the chemical structure of NO: due to its free radical nature NO can reduce HO activity either by inactivating proteins, in particular those rich in thiol groups such as HO-2, or by forming nitrosyl-heme that prevent the oxygen binding to HO which is mandatory for its activation (67). By virtue of these actions NO can reduce HO activity, this effect being particularly relevant in brain because of the abundance of neuronal HO-2. Meanwhile, the free radical nature of NO can induce HO-1 protein and HO activity, and this biochemical event is very important in those cells (endothelial and smooth muscle cells) in which HO-1 is predominant. Furthermore, NO can regulate HO activity by modulating the activity of S-aminolevulinic acid synthase, the rate-limiting enzyme in heme synthesis, or ferritin, the iron-storage protein (67). Moreover, peroxynitrite and nitroxyl anion share with NO the dual effect on HO activity because the first has been shown to decrease HO activity in rat brain or spleen microsomal preparation (100) and increase the oxygenase activity in endothelial cells (89) while the second increased HO activity in vascular cells (92) but there is no evidence of HO modulation on other cell lines. Taken together, these data demonstrated that the role of NO and RNS in regulating the HO activity strictly depends on the cell type and HO isoform.

An interesting corollary emerges by these studies: it has been demonstrated that BR is able to interact with NO and as a result of this interaction the formation of an N-nitrosated product of BR or BV occurs (15-17). Biliverdin shares with BR this scavenging effect, even if the biological importance of the BV-NO interaction is limited due to the rapid transformation of BV into BR by BVR. Furthermore, even CO, the gaseous product of HO activity, inhibits NO-mediated vasodilation in the adult rat cerebral microcirculation and this effect is probably due to the photo-reversible gas binding to the prosthetic heme of NOS (101). Therefore it is possible to hypothesize a negative feedback between HO products and NO: in this framework CO, BV and BR could act in concert to reduce the unnecessary stimulation of HO by NO.

Heme oxygenase and brain aging

Heme oxygenase has the unique feature to be involved either in the antioxidant machinery of cells or to produce molecules involved in the signal transduction pathways. This characteristic is very important for brain. In fact, HO-1 exerts a strong neuroprotective function by degrading pro-oxidant heme and producing biliverdin, the precursor of the antioxidant and antinitrosative molecule BR; in the meantime, CO, a gaseous neuromodu-lator involved in neuronal long-term potentiation (LTP), and therefore synaptic plasticity, is derived from HO activity (102). Considering that during aging both a decrease in antiox-idant defense as well as cognitive impairment happen, it has been recently investigated whether HO-1/HO-2 dysfunction could be responsible for these phenomena. A significant reduction in HO-1 and HO-2 expression has been documented in hippocampus and substantia nigra of 20-month-old rat brain compared to young rats and was paralleled by a concomitant decrease in NOS expression in neurons of hypothalamic paraventricular and mammillary nuclei as well as hippocampal neurons (102). A first consequence of this reduction in HO-1 and HO-2 levels is the significant reduction in the capacity of brain to react to heat shock stress as demonstrated by the lack of induction of ho-1 gene in cerebellum and aqueductal cells (102). This result implies that aged brain is much more sensible to oxidant conditions, such as heat shock, due to the inability of HO-1 to be induced. The importance of HO-1 and HO-2 in cognitive functions was demonstrated by the evidence that Neotrofin, a cognitive enhancing and neuroprotective drug, was able to increase both HO-1 and HO-2 expression in adult rat brain (71,102). Based on the previous statements, it is plausible to argue that the marked induction of HO-1 and HO-2 by Neotrofin is responsible for a significant increase in CO production which, in turn, enhances cognitive processes.

Heme oxygenase and neurodegenerative disorders

The role of ROS and RNS in the pathogenesis of neurodegenerative disorders has been clearly demonstrated (3,53,98,103-106). With regard to the contribution of HO-1 in neurodegeneration, there is no consensus in the literature. In fact, there is no doubt that HO-1 is neuroprotective, but there is evidence of a detrimental effect of this enzyme in neural tissues probably due to the possible toxic effects of CO and iron (107). Due to its strong antioxidant properties and wide distribution within the CNS, HO-1 has been proposed as a key enzyme in the prevention of brain damage (14,67,70). In a very interesting study, Panahian et al. (108) using transgenic mice overexpressing HO-1 in neurons, demonstrated the neuroprotective effect of this enzyme in a model of ischemic brain damage and attributed the HO-1 beneficial effects to an increase of pro-survival molecules such as cGMP, bcl-2 and the iron-sequestering protein as well as to a reduction of pro-apoptotic p53.

Up-regulation of HO-1 in the substantia nigra of PD patients has been demonstrated. In these patients, nigral neurons containing cytoplasmic Lewy bodies exhibited in their proximity maximum HO-1 immunoreactivity (109). As with AD (110,111) up-regulation of HO-1 in the nigral dopaminergic neurons by oxidative stress was shown (112).

Hemin, an inducer of HO-1, inhibited effectively experimental autoimmune encephalomyelitis (EAE), an animal model of the human disease MS (113). In contrast, tin-mesoporphyrin-IX, an inhibitor of HO activity, markedly exacerbated EAE (113). These results suggest that endogenous HO-1 plays an important protective role in EAE and MS.

The role played by HO-1 in AD, a neurodegenerative disorder which involves a chronic inflammatory response associated with both oxidative brain injury and beta-amyloid associated pathology, is particularly interesting. Significant increases in the levels of HO-1 have been observed in AD brains in association with neurofibrillary tangles and also HO-1 mRNA was found increased in AD neocortex and cerebral vessels (114,115). HO-1 increase was not only in association with neurofibrillary tangles, but also co-localized with senile plaques and glial fibrillary acidic protein-positive astrocytes in AD brains (116). In addition, Takeda et al. explored the relationship between HO-1 and tau protein, the latter being the major component of intraneuronal neurofibrillary tangles in AD. In transfected neuroblastoma cells overexpressing HO-1, the activity of this enzyme was increased, and conversely, the level of tau protein was significantly decreased when compared with antisense HO-1 or vector-transfected cells (115). The suppression of tau protein expression was almost completely counteracted by zinc-deuteroporphyrin, a specific inhibitor of HO activity (115). The activated forms of extracellular signal-regulated kinases (ERKs) were also decreased in cells overexpressing HO-1 although no changes in the expression of total ERKs were observed (115).

Taken together, all these findings do not allow singling out a product of HO activity as the main neuroprotective factor; rather a complex puzzle of regulatory interactions between heme degradation products and cellular pathways involved in cell death/survival is hypothesized.

The protective role played by HO-1 in AD raised new possibilities regarding the possible use of natural substances, which are able to increase HO-1 levels, as potential drugs for the treatment of AD. In this light, the phenolic compounds contained in some herbs and spices, e.g. curcumin are very promising (117-119). Curcumin is the active antioxidant principle in Curcuma longa, a coloring agent and food additive commonly used in Indian culinary preparations. This polyphenolic substance has the potential to inhibit lipid peroxidation and to effectively intercept and neutralize ROS and RNS (120). In addition, curcumin has been shown to significantly increase HO-1 in astrocytes and vascular endothelial cells (119,121). The latter effect on HO-1 can explain, at least in part, the strong antioxidant properties of curcumin, in particular keeping in mind that HO-1-derived BR has the ability to efficiently scavenge both ROS and RNS (15-17,122,123). Epidemi-ological studies suggested that curcumin, as one of the most prevalent nutritional and medicinal compounds used by the Indian population, is responsible for the significantly reduced (4.4-fold) prevalence of AD in India compared to United States (124). Based on these findings, Lim and colleagues have provided convincing evidence that dietary curcumin, given to an Alzheimer transgenic APPSw mouse model (Tg2576) for 6 months, resulted in the suppression of indices of inflammation and oxidative damage in the brain of these mice (125). Furthermore, in a human neuroblastoma cell line it has recently been shown that curcumin inhibits NFkB activation, efficiently preventing neuronal cell death (120).

Ferulic acid (FA) is another phenolic compound and a major constituent of fruits and vegetables with strong antioxidant and anti-inflammatory properties. Recently, it has been demonstrated that FA ethyl ester (FAEE), naturally occurring and a more hydrophobic form of FA, protects synaptosomal membrane system and neuronal cell culture systems against hydroxyl and peroxyl radical oxidation (126,127) as well as mice against beta-amyloid-induced microglial activation (128). Other than this direct antioxidant property, FAEE has been shown to increase HO activity either in rat astrocytes or neurons (118,129) thus corroborating the hypothesis that HO activation is a common pathway through which phenolic compounds can exert neuroprotective effects.

Acetyl-L-carnitine (LAC), is a compound of great interest for its wide clinical application in various neurological disorders: it may be of benefit in treating Alzheimer's dementia, chronic fatigue syndrome, depression in the elderly, HIV infection, diabetic neuropathies, ischemia and reperfusion of the brain, cognitive impairment of alcoholism and aging (12,13). It is promoted as a nutritional agent producing cognitive benefits for middle-aged and elderly people, is involved in cellular energy production and in maintenance and repair processes in neurons (3,117,120,130). In addition to its principal function, acetylcarnitine, and the carnitine system, buffer potentially toxic acyl-CoA metabolites and modulates the ratio of acyl-CoA:CoA (13,130). The latter regulates the activity of many mitochon-drial enzymes involved in the citric acid cycle, gluconeogenesis, the urea cycle and fatty oxidation (130). Modifications in cardiolipin composition are recognized to accompany functional changes in brain mitochondria which include all proteins of the inner mitochon-drial membrane that generally require interaction with cardiolipin for optimal catalytic activity (120,130). Acetylcarnitine fed to old rats increased cardiolipin levels compared to that of young rats and also restored protein synthesis in the inner mitochondrial membrane, as well as cellular oxidant : antioxidant balance (12,13,130) suggesting that administration of this compounds may improve cellular bioenergetics in aged rats. Interestingly, caloric restriction, a dietary regimen that extends life span in rodents, maintains the levels of 18:2 acyl side chains and inhibits the cardiolipin composition changes (130). In addition, caloric restriction showed to retard the aging-associated changes in oxida-tive damage, mitochondrial oxidant generation and antioxidant defenses observed during aging (130). Recently, by using suppressive subtractive hybridization (SSH) strategy, a PCR-based cDNA subtraction procedure particularly efficient for obtaining expressed transcripts often obscured by more abundant ones, it was reported that LAC modulates specific genes in the rat CNS, such as the hsp72 gene, the gene for the isoform of 14-3-3 protein and that encoding for the precursor mitochondrial P3 of ATP synthase lipid-binding protein (130).

Recent data from our laboratory have provided experimental evidence that acetylcar-nitine is cytoprotective against oxidative insults in astrocytes through up-regulation of stress responsive genes (Fig. 1). These results have shown for the first time that acetyl-carnitine induces HO-1 and Hsp60 heat shock proteins with a mechanism involving activation and nuclear translocation of the transcription factor Nrf2 (131). In addition, changes in the redox status of glutathione were also observed (130,131). It is conceivable that acetylcarnitine alone, in unstressed conditions, by promoting acetylation of DNA-binding proteins, may modulate ARE-mediated expression of stress-inducible genes, such as ho-1, y -glutamylcysteine synthetase, Mn-SOD and glutathione S-transferase.

HO-1 Hsp 60 Hsp 72 iNOS ROS
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