Cooperative Interaction Between iNos And Ho1

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Biological function of HO system, the nonheme enzymes that oxidize heme to generate CO and the function of NOS system, the heme-containing enzymes that use heme to produce NO, is related to heme. HO degrades heme, but NOS needs heme. However, there are certain similarities and differences between the HO and NOS systems. As for the similarities, both the HO and NOS enzyme systems have constitutive and inducible isoforms. Expression of the inducible form of NOS (iNOS) is enhanced by some of the oxidative stimuli such as bacterial endotoxins (LPS), cytokines, and ROS, which also induce HO-1 (38). Also, expression of both HO-1 and iNOS can be induced in many of the same organs and tissues subjected to stressful conditions, and their induction involves gene activation and de novo synthesis of HO-1 and iNOS enzyme proteins. During inflammation, both iNOS and HO-1 are induced highly in macrophages, neutrophils, and mononuclear cells.

Extending the similarities, products of HO and NOS systems, respectively the CO and NO, are both gaseous ligands that bind to the heme-iron contained in hemoglobin, NADPH oxidase, NOSs, cytochromes and various other hemoprotein enzymes which catalyze several essential biological processes like Og and NO production, oxygen transport, electron transfer, energy production, and biotransformation of xenobio-tics. The Fe++ form of heme, but not the Fe+++ form of heme, contained in these hemoproteins reacts with ^-bonding diatomic gaseous ligands like O2, CO, and NO at physiological pH (46). However, the relative affinity of heme for binding the O2, CO, and NO differs; CO has stronger affinity for hemoglobin than O2 and thus, CO can displace O2 readily from heme. However, NO binds more firmly to hemoglobin heme than CO and thus, NO will replace both O2 and CO from heme (87,88). Alternatively, the NO bound to hemoglobin will undergo oxidation or reduction and will be released, thus delivering NO to tissues.

There are some differences between the HO and NOS systems. As mentioned above, products of HO and NOS systems, respectively CO and NO, share affinity for the heme-iron and they resemble one another in activating the heme-containing GC toward markedly increased production of cGMP (89). Guanyl cyclase is a dimeric hemoprotein and each dimer contains two hemes. Ignarro et al. (90) have demonstrated that NO binds to the heme-iron present in GC and increase its Vmax for enhanced conversion of GTP to cGMP. Furthermore, it was demonstrated that CO generated in vivo by HO activity also binds to the heme in GC to stimulate the production of cGMP, however, much less effectively (only 1/30) than that enhanced by the NO binding (89,91). Furthermore, unlike NO, which is a free radical causing damage to invading pathogens as well as to surrounding host cells, CO is not a free radical and cannot kill invading pathogens or produce tissue damage. In fact, the ability of an organism to mount the Og - and NO-producing inflammatory immune response to invading pathogen is suppressed when the background HO-1 activity is increased. This may be linked to the HO-derived overproduction of CO, which can inhibit the NADPH oxidase catalyzed oxidative burst. Carbon monoxide also binds to the heme-iron contained in iNOS and can inhibit NO production. Thus, CO can inhibit additional production of both Og and NO. Thus, unlike the NO, which is proinflammatory and cytotoxic, CO provides both anti-inflammatory and cytoprotective activity (83,92,93).

In this connection, Willis et al. (92) observed that HO activity in various tissues is increased markedly in response to inflammatory stimuli and the tissues with elevated HO activity is protected from both oxidative and nitrosative injuries. Conversely, when the elevated HO activity is inhibited by administration of Zn-protoporphyrin (Zn-PP), inflammatory response is enhanced further and oxidative or nitrosative injury is potentiated. This observation suggested that elevated HO activity plays a significant role in suppressing host-cell injury caused by excessive inflammatory immune response. Thus, unlike the iNOS, HO-1 may play a key role in protecting host cells against toxicity caused by activation of inflammatory responses, such as those caused by excessive and prolonged induction of iNOS.

Heme oxygenase-1 is also known as heat-shock protein-32 (HSP32) and is induced not only by the heme itself (native physiological substrate) but also by all kinds of stimuli and agents that cause oxidative stress and pathological conditions (e.g., heat shock, ischemia, GSH oxidation and depletion, irradiation, glucose deprivation, hyperoxia or hypoxia, cellular transformation, and disease states) (36,45,80,94-98). Markedly increased level of HO-1 mRNA and protein was detected in the stressed cardiovascular and gastrointestinal systems as well as in the activated immune cells. Specifically, using an ischemia-reperfusion model, oxidative stress applied to kidney, heart, and aorta was shown to cause prominent and sustained increase of HO-1 mRNA and protein expression (94). Also, during the first weeks of life, when the liver of newborn is actively engaged in removing or degrading the fetal hemoglobin, there is a surge of HO activity and this is due mainly to marked increase of HO-1 expression (99,100). However, the hepatic expression of HO-1 declines to an unde-tectable level when removal of fetal hemoglobin is completed in few weeks. In the normal adult liver, HO-2 is the predominant form expressed constitutively. However, when the adult liver is stressed by an administration of heavy metal (e.g., CdCl2), hematin (derived from hemolyzed RBC), bromoben-zene (a free radical generator), or phenylhydrazine (a GSH-depleting drug), there is massive induction of HO-1 mRNA

(up to 100-fold). This induction of HO-1 occurs without any increase of HO-2 expression (101).

The physiological sources of heme, to be used both as the native substrate of HO and also as the inducer of HO-1 expression, includes hemoglobin in RBC, myoglobin in muscles, cytochromes involved in microsomal drug oxidation (P-450), and mitochondrial respiratory chain (cytochrome c) as well as the newly synthesized free heme (i.e., before incorporation into apohemoproteins). There are several reasons why such heme molecules may become available for oxidative degradation by HO. (a) Free heme is being synthesized constantly for ready incorporation into apohemoproteins and the excess of this newly synthesized heme not incorporated into heme proteins is available for degradation. This may be catalyzed by HO-2, the constitutively expressed HO isoform (102). (b) Under oxidative stress, demand for additional synthesis of heme enzymes which generate ROS is halted and hence, more of the unincorporated newly synthesized heme may become available to be used not only as the substrate for degradation by HO but also as the inducer for enhancing expression of HO-1. (c) Most importantly, under the ROS-producing oxidative stress conditions, heme-containing proteins undergo denaturation, fragmentation, and enhanced proteolysis, all promoted by the ROS produced by heme enzymes themselves (6). This releases heme. This liberated heme then undergoes degradation by the HO system and also enhances the expression of HO-1 (103,104). Thus, induction of HO-1 may serve as a primary cellular defense in oxidatively stressed cells to degrade the pro-oxidant heme liberated from denatured hemoproteins and at the same time, to produce the biologically active antioxidant components like bile pigments and CO.

Under oxidatively stressed conditions, expression of iNOS is also enhanced and large amount of NO is produced. This NO, being produced continuously and abundantly by the newly induced iNOS, can act as an effective scavenger of ROS and also as an additional source for further stress. The overproduced NO derived from iNOS can move freely in and out of the cell and can bind to the heme-iron present in iNOS itself and other hemoproteins. Thus, overproduced NO can inhibit not only the additional production of NO catalyzed by iNOS, but also the energy production catalyzed by mitochondrial respiratory cytochromes and the Og production catalyzed by NADPH oxidases (105). Also, as with the binding of Og, the binding of NO to these heme-containing enzymes can also promote destabilization, fragmentation, and proteolysis by proteasomes. Thus, NO can also promote the release of heme from iNOS and NADPH oxidase, and this released heme can undergo degradation and removal by the HO-1 activity, which is enhanced by the released heme (106). This would create a situation in which the CO production increases at the expense of NO production. This CO can move freely in and out of the cell and bind to the heme-iron present in the remaining and functional iNOS. As the result, iNOS activity is inhibited and additional production of NO is blocked. With such "double-punch" inhibitions on NO production (first by NO itself and second by CO), further production of NO will be "turned off,'' allowing the cells to survive from both oxida-tive and nitrosative stresses.

In this manner, the CO-producing HO activity and the NO-producing NOS activity are intimately linked in protecting cells, initially against the oxidative injury caused by Og and subsequently against the nitrosative stress caused by NO. Inhibiting the elevated HO activity by exposing oxida-tively stressed tissues to Zn-PP has been demonstrated to enhance the synthesis of iNOS protein to an even higher level and to enhance the rate of NO production even further, causing markedly increased cytotoxicity (107). Then, a question arises as to why should there be such an inhibitory regulation on the NO-producing iNOS induction by the CO-producing HO-1 induction. NO and the NO-derived RNS (like ONOO~ and NO+) can damage cells by interacting with DNA, -SH groups, aromatic amino acids, and transition metals such as iron in heme-containing proteins and iron-sulfur centers (108,109). Thus, if the iNOS-derived continuous overproduction of NO is not stopped, cells with elevated iNOS activity will be damaged. Therefore, continuing overproduction of NO and the resulting RNS-derived toxicity must be checked by another biological means; current evidence suggests that induction of HO-1 can fulfill this role both by degrading the heme in iNOS and by generating the CO at the same time. Thus, induction of HO-1 expression in aerobic cells plays very effective bifunctional role in cellular defense against the toxi-city caused by enhanced expression of iNOS and continuing overproduction of NO, which occurs in response to the oxida-tive stress initiated by LPS (Fig. 4).

Induction of HO-1 is likely to modulate the overproduction of NO in many ways and several of these modulation mechanisms reflect the fact that iNOS is a hemoprotein. These include the following. (a) Elevated HO-1 activity would accelerate the degradation of newly synthesized heme and would impair de novo synthesis of functional iNOS by limiting the availability of heme needed for incorporation into newly synthesized apo-iNOS protein (active site of iNOS requires two heme molecules) (110). (b) When the NO overproduced by the iNOS itself binds to the heme contained in the iNOS protein, iNOS activity is inhibited (111). In addition, the NO-bound iNOS undergoes destabilization and enhanced pro-teolytic digestion by proteasomes and releases heme (47). This liberated heme can now be degraded to produce and release CO by the elevated HO-1 activity. (c) This CO moves freely within the cell and binds to the heme-iron present in the remaining iNOS, which may still be functioning. This CO binding to the iNOS-heme would inhibit further production of NO by blocking the iNOS activity. In fact, CO has been reported to bind iNOS and to inhibit NO production (12,13). (d) Iron released from the degraded heme can inhibit additional transcription of iNOS gene and can suppress additional synthesis of iNOS protein (61). Based on these inhibitory effects of HO-1 induction on iNOS protein and activity, proposed mechanisms involved in this inhibitory interaction between HO-1 and iNOS is presented schematically (Fig. 5).

Conversely, NO has been shown to modulate the HO activity, both to inhibit as well as to activate the HO activity. Willis et al. (93) demonstrated that HO-2 activity in various tissues is inhibited by an in vivo administration of NO-donors. However, Motterlini et al. (112) have demonstrated that HO-1

Nadph Heme

Figure 5 Schematic diagram showing the pathway leading to iNOS and HO-1 induction. Upon induction of HO-1, further synthesis of iNOS is inhibited due to lack of heme available to be incorporated into the newly synthesized apo-iNOS protein and further production of NO by iNOS is inhibited by the CO generated from the degraded heme.

Figure 5 Schematic diagram showing the pathway leading to iNOS and HO-1 induction. Upon induction of HO-1, further synthesis of iNOS is inhibited due to lack of heme available to be incorporated into the newly synthesized apo-iNOS protein and further production of NO by iNOS is inhibited by the CO generated from the degraded heme.

activity in endothelial cells is increased by exposure to NO donors. This apparent discrepancy may reflect the chemical reactivity of NO with the cysteine-SH groups present in HO-2, but not in HO-1 (113). NO, by being a free radical, can bind to the -SH groups present in HO-2 which has three cysteine residues (114), and can inhibit the HO-2 activity (113). Furthermore, NO can also inhibit both HO-1 and HO-2 activities by binding to the heme substrate with high affinity and prevent the required O2-binding, a process required for the heme substrate to be oxidatively degraded by both HO isoforms (47). This may explain the observation made by

Willis et al. (93). At the same time, Motterlini's observation (112) can be explained by the fact that NO, being a free radical and serving to oxidize GSH, induces the expression of HO-1 via activation of redox-sensitive transcription factors. Majority of stimuli that upregulate the expression of HO-1 do so by lowering cellular sulfhydryl-disulfide (SH/SS) redox ratio, which triggers the activation of redox-sensitive transcription factors involved in HO-1 induction. In the promoter sequence of HO-1 gene, there are AP-1, Nrf2, and NF-kB responsive elements (nucleotide sequences) (1,32,67,115).

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