NO and prostaglandin pathways share numerous similarities and the two molecules can be produced simultaneously in the same tissues, as described in a number of models of inflammation such as endotoxin induced septic shock, carrageenan-induced pouch or paw inflammation () [117
]. Hence numerous studies have explored the potential crosstalk between NO and prostaglandin pathways and increasing evidence supports this idea. However, the detailed mechanisms by which NO regulates prostaglandin production or vice versa is still controversial, partly because the interaction between these two pathways occurs at multiple levels along with the complexity of NO redox chemistry [117
]. An initial report detailing the cross talk between NO and prostaglandin was made by Needleman’s group showing that NO can activate cyclooxygenase [119
]. In this study, NOS and COX2 activity were induced in a macrophage cell line, RAW264.7, treated with LPS to produce both NO and prostaglandin. The production of both was attenuated by NOS inhibitors. As the NOS inhibitors used in this study do not have any NSAID characteristics, the inhibition of prostaglandin production was likely due to the decrease in NO production and not as a direct effect on COX2 [119
]. This study was further corroborated by in vitro
studies showing that even exogenous NO gas or chemical donors can induce COX1 activity [120
]. Moreover, NO-mediated activation of COX does not seem to be limited to its enzyme activity. Lie et al
showed that NO can increase COX2 mRNA levels via the β-catenin/TCF pathway leading to activation of the polyoma enhancer activator 3 (PEA3) transcription factor [122
]. Furthermore, NO also interacts with various other pathways which can influence COX expression such as the cAMP/PKA/CRE and JNK/Jun/ATF2 signaling cascades [122
The role of Nitric oxide Synthase (NOS) and Cyclooxygenase
In addition to this body of evidence showing an up-regulation of COX activity by NO, a large number of reports supporting the idea that NO can inactivate COX under certain conditions also exists. Levi’s group showed that LPS-induced COX2 expression and production of prostaglandin in microglia cells are augmented by NOS inhibitors [124
]. Indeed, similar findings were reported in various other cells types including a J774 macrophage cell line and the rat Kupper cells [125
]. Interestingly, the interaction between NO and prostaglandin is not uni-directional, as it has been reported that NSAIDs such as aspirin and indomethacin can significantly reduced NOS activity [127
The mechanism by which the NOS and COX pathways interact is complicated by conflicting reports in the field. For instance, while most of the actions by NO are mediated by its interaction with Fe in heme prosthetic group of soluble guanylate cyclase (sGC), Salvemini et al
has shown that inhibition of sGC with the sGC-specific inhibitor, methylene blue, does not attenuate NO-mediated activation of prostaglandin biosynthesis [119
] and has proposed that NO may directly activate COX enzyme. This theory is further supported by numerous reports demonstrating that exogenous NO donors can activate recombinant COX. However, Tsai et al
reported that it is unlikely that NO directly binds to heme group in COX to activate under anaerobic condition [128
]. One reason for this disagreement may be due to the complexity of NO chemistry with the result that various NO species are generated, as described earlier.
Currently, there are several proposed mechanisms by which NO mediates prostaglandin synthesis (). One is that NO and superoxide can be produced simultaneously and can react together to form peroxynitrite, which can feedback to modify COX. Another possible mechanism is the direct S-nitrosylation of COX enzymes. These two post-translational modifications can alter the function of these enzymes and are further discussed below.
Proposed mechanisms of NO-mediated regulation of prostaglandin production
Peroxynitrite-mediated regulation of prostaglandin synthesis
Superoxide and nitric oxide are readily produced in the cells under both physiologic and pathological conditions [37
]. Upon production these two compounds, peroxynitrite can be easily formed. Many laboratories have reported that peroxynitrite can interact with COX enzymes but the consequence of the interaction differs depending on where this interaction occurs. So far, two peroxynitrite binding sites in COX enzymes have been identified: within the endogenous heme moiety and at tyrosine residues on the protein.
Peroxynitrite has been shown to activate both COX1 and COX2 by several groups. In particular, treatment of cells with SIN-1, an exogenous peroxynitrite generator, enhances prostaglandin synthesis and this activation is inhibited by oxygen radical scavengers, strongly implicating peroxynitrite as an activator of COX [129
]. The mechanism by which peroxynitrite activates COX activity is still unclear, however, it has been speculated that peroxynitrite may be interacting with Fe in the heme group of COX and forms a radical intermediate product which can accelerate the enzyme reaction [130
]. This hypothesis is supported by the fact that peroxynitrite is known to interact with the heme group of certain proteins such as myeloperoxidase and horseradish peroxidase to generate an intermediate product [133
Most of the studies about peroxynitrite and COX enzymes have been focused on tyrosine residue modification in the enzyme and the formation of a nitrotyrosine residue. It appears that this modification, especially at Tyr385, leads to an inactivation of enzyme [134
], as demonstrated by inhibition of COX1 enzyme activity when either purified enzyme COX1 or smooth muscle cells are treated with NO donors and arachidonic acid simultaneously [138
]. It is interesting to note that pure NO donors such as NOC-7 does not lead to formation of nitrotyrosine in the absence of arachidonic acid, while peroxynitrite, SIN-1 or tetranitromethane (TMN) can readily carry out this reaction.
It appears that peroxynitrite has dual actions on COX depending on where it interacts in the enzyme. Even though nitrotyrosine modification of COXs is detected in many pathological conditions and clearly inactivates its enzyme activity in vitro,
the exact mechanism of nitration remains elusive. The levels of peroxynitrite markedly increase under pathological conditions, as inflammation induces NO production from iNOS and superoxide production from by NAD(P)H and xanthine oxidase and correlates to formation of nitrotyrosine in COXs. However, further investigation is required to identify different conditions that determine the target selectivity of peroxynitrite: to choose between interacting with tyrosine to inactivate an enzyme versus interaction with heme in COXs to activate it. Interestingly, Deeb et al
showed that holoCOX1 (heme-containing) can be nitrated at Tyr 385 by peroxynitrite, even in the absence of arachidonic acid, while apoCOX1 (heme-deficient) is modified at the different site, thus indicating a heme requirement for peroxynitrite-mediated Tyr385 nitration [139
]. The identity of the peroxynitrated tyrosine residue in apoCOX1 and its downstream biological function are yet to be determined.
It has been proposed that peroxynitrite may interact with Fe in the heme moiety of COX to generate a short-lived intermediate which can facilitate the formation of nitrotyrosine [140
]. While heme is essential for this reaction within COX, the role of this co-factor is unclear, as there are other proteins which can be nitrated without heme moiety in the protein [141
]. It is possible that Fe binding in the heme molecule in COX may expose the Tyr385 residue, which resides just below the heme moiety, making it more readily accessible to peroxynitrite. It will be interesting to examine whether recombinant COX with non-Fe protoporphyrin such as Zn-protoporphyrin or Sn-protoporphyrin can be nitrated by peroxynitrite. This theory, however, conflicts in part with the aforementioned evidence that peroxynitrite binding to the heme moiety in COX leads to activation of the enzyme while nitration of Tyr385 leads to enzyme inactivation. This may be partially explained by differing concentrations of arachidonic acid, which can be oxidized by peroxynitrite, with this oxidized substrate having different impacts on COX activities. Deeb et al
has indeed shown that higher arachidonic acid reduces formation nitrotyrosine in COX1 [142
Most importantly, this peroxynitration modification of COX enzymes has been detected in many pathological or chronic inflammatory conditions such as atherosclerosis, Parkinson’s disease or Alzheimer’s disease. However, it is not clear whether an inactivation of this enzyme by nitrotyrosine formation contributes to the pathology of these diseases or simply an indication of nitrossative stress.
Finally, COXs are not the only target which can be modified by Peroxynitrite. Prostacyclin synthase (PGCI) is selectively inhibited by peroxynitrite at low concentration (IC50=50 nM) while thromboxane A2 (TxA2)-synthase is activated [143
]. In particular, PGCI contains one Cys at position 469 which is involved in heme binding and spectrophotometry study showed that this thiolate ligand at the fifth coordination position of the heme iron is not affected by peroxynitrite. It appears that nitration of tyrosine residue 430 in PGCI interferes the metal center of the active site and inhibits its activity [144
S-nitrosylation of COX
The recent development of a simple method to detect S-nitrosylation flourished identification of new targets and its functional consequence since this modification was first reported with albumin by Stamler et al.
COX has also been identified as a target of this modification [5
]. It was first proposed by Hajjar et al
showing that S-nitrosothiol may be responsible for NO-mediated activation of COX as it occurs in an heme-independent manner [147
]. On the other hand, Marnett’s group investigated the role of free cysteines in COX1 and showed that Cys313 and Cys540 mutation into serine alone inhibited its enzyme activity implying that NO-mediated Cys modification may interfere with COX1 activity [148
]. Interestingly, Marnett’s and Hajjar’s groups independently reported that SNAP, an NO chemical donor, cannot activate COX1 [138
], thus confusion exists over whether S-nitrosylation of COX1 leads to its activation.
Our group has shown that iNOS can specifically bind to COX2, leading to direct S-nitrosylation of cysteine residues and activate COX2 [150
]. This interaction and modification is specific for COX2 as iNOS does not interact with COX1. In particular, we observed more than one S-nitrosylated cysteine residues in LPS/IFN-γ activated RAW264.7 cells and individually mutated each cysteine to serine. We have identified Cys523, located near the catalytic domain, as the only cysteine which does not affect base level enzyme activity and when mutated, NO can no longer activate COX2. This cysteine is located in near the catalytic site of COX2 and we speculated that this modification leads to a slight conformational change in COX2 to affect its enzyme kinetics. Indeed, we performed viscosity studies examining enzyme activity in various concentrations of sucrose and showed that release of product is accelerated in the presence of NO donor. However, it is not possible to exclude a potential involvement of any other cysteines for NO-mediated activation of COX2 as mutation of other cysteines alone affected the basal level enzyme activity of COX2. Given that NO has a high affinity for heme, it is unclear how NO can specifically S-nitrosylate cysteines of COX2 rather than interacting with heme moiety. This may be partially explained by the fact that S-nitrosylation of COX2 by iNOS requires physical interaction between the two proteins, specifically between the subunit binding site of iNOS and catalytic domain of COX2. Hence, we speculated that NO generated from iNOS is simply in direct proximity of the S-nitrosylation target residue in COX2 for immediate delivery and modification. Indeed, disruption of iNOS/COX2 binding diminishes S-nitrosylation and activation of COX2 in LPS-induced RAW264.7 cells. Interestingly, Goodwin et al
demonstrated that removal of superoxide in LPS-activated RAW264.7 cells inhibited prostaglandin formation to the same extent as NOS inhibitors [137
]. While the immediate speculation will be peroxynitrite-mediated nitrotyrosine formation of COX2, Cross’ group showed that peroxynitrite can also generate S-nitrosothiol [151
] and therefore, further studies are necessary to identify the form of NO-mediated modification of COX2 in activated macrophages.
iNOS-mediated regulation of prostaglandin production is not limited to COX enzymes. Xu et al
showed that iNOS activates cytolosic phospholipase A2a (cPLA2α) by S-nitrosylation [152
]. In this case, cPLA2α does not directly interact with iNOS, but rather forms ternary complex with COX2 and iNOS, and hence induction of COX2 is a crucial step for S-nitroyslation and activation of cPLA2α [152
S-nitrosylation of COX2 does not seem to be limited to macrophages. Unlike other tissues, COX2 in the brain is constitutively expressed. We showed that COX2 is S-nitrosylated in neuronal cells by nNOS [87
]. To our surprise, nNOS binding is mediated by its PDZ domain, differing from the domain near the catalytic region that mediates iNOS binding to COX-2. We also showed that a physical interaction between nNOS and COX2 is required for S-nitrosylation of COX2 and that disruption of binding attenuated NMDA-mediated excitotoxicity, as did a selective COX2 inhibitor.