Prostacyclin Synthase as an Ambivalent Regulator of Inflammatory Reactions

Abstract

Prostacyclin (PGI₂) synthase (PGIS) and microsomal prostaglandin (PG) E synthase-1 (PGES-1) are PG terminal synthases which functionally couple with inducible cyclooxygenase-2 (COX-2) as their upstream enzymes to produce PGI₂ and PGE₂, respectively. Non-steroidal anti-inflammatory drugs exert their pharmacological effects by the inhibition of COX-2 and thereby suppression of the biosynthesis of these PGs. PGIS is abundantly expressed in vascular endothelial and smooth muscle cells and has been shown to be critical for regulation of platelet aggregation and vascular tone. In addition to its role in vascular regulation, PGIS has been shown to be expressed in inflammatory cells including macrophages, and the proinflammatory roles of PGIS has been demonstrated. On the other hand, several investigators have recently reported that PGIS functions as an anti-inflammatory mediator by macrophage polarization and have indicated that PGIS is an ambivalent regulator of inflammatory reactions. In this review, we summarize the current understanding of proinflammatory and anti-inflammatory functions of PGIS and discuss its potential as a novel anti-inflammatory therapeutic target.

1. INTRODUCTION

Prostanoids are cyclic and oxygenated metabolites of arachidonic acid (AA) and have a broad range of physiological and pathological activities.¹⁾ Biosynthesis of prostanoids from AA is catalyzed sequentially by cyclooxygenase (COX) and specific prostaglandin (PG) terminal synthases.^2–4) Widely used nonsteroidal anti-inflammatory drugs (NSAIDs) exert their pharmacological effects by reducing prostanoid production via inhibition of COX. There are two COX isozymes, COX-1 and COX-2.²⁾ COX-1 is expressed constitutively in most tissues and is generally responsible for the immediate production of prostanoids that control normal physiological functions such as maintenance of gastrointestinal or renal homeostasis. On the other hand, COX-2 is induced in response to inflammatory or mitogenic stimuli and mediates delayed prostanoid generation related to inflammatory reactions and carcinogenesis. NSAIDs exert their effects by inhibiting COX-2, but they also inhibit COX-1; as a result, the long-term use of classical NSAIDs is associated with severe side effects, mainly gastrointestinal irritations and renal injury. Novel types of NSAIDS that specifically inhibit COX-2 showed reduced gastrointestinal complications as expected, but several clinical trials indicated a significantly increased cardiovascular risk of COX-2-specific NSAIDs.^5,6)

A COX metabolite, PGH₂, is converted to different PG species by specific PG terminal synthases.^3,4) To date, multiple PG terminal synthases have been identified (Fig. 1). While NSAIDs suppress the production of all PG species, the inhibition of a species-specific PG terminal synthase could theoretically suppress only one PG species. Because of the potential of PG terminal synthases for more selective modulation of PG production, they are now being investigated as a novel target for NSAIDs. In vitro analysis has revealed that each PG terminal synthase preferentially couples with COX-1 or COX-2 as its upstream enzymes.^7,8) Among these PG terminal synthases, microsomal PGE synthase-1 (mPGES-1) and PGI synthase (PGIS) especially coupled with COX-2, suggesting that these two enzymes are involved in COX-2-related diseases. Thus, these two enzymes have gained attention as potential new drug targets for COX-2-related diseases such as inflammatory diseases and cancer. Of these two enzymes, mPGES-1 is shown to be induced by various types of stimuli as well as COX-2^7,9–11); other research groups as well as our own have used mPGES-1 knockout (KO) mice to show that mPGES-1 plays a critical role in inflammatory reactions.^12–15) It has also been reported that mPGES-1 deficiency mitigates the symptoms of neurological inflammatory diseases such as Alzheimer’s disease.^16–18) Several mPGES-1-selective inhibitors were recently developed and shown to suppress inflammatory reactions in experimental animal models.^4,19,20) Our studies using mPGES-1 KO mice have further shown that mPGES-1 contributes to tumor growth, invasion and metastasis.^21,22) Chemical-induced carcinogenesis in mice was also suppressed by mPGES-1 deletion.^23–26) mPGES-1-selective inhibitors are also expected as novel anti-cancer drugs.

Fig. 1. Biosynthetic Pathways of Prostanoids

Arachidonic acid (AA) released from membrane phospholipids by the action of phospholipase A₂ (PLA₂) is converted to prostaglandin H₂ (PGH₂) by cyclooxygenase (COX), COX-1 and 2, and this PGH₂ is then isomerized to different prostanoids by their PG-specific terminal enzymes. PGDS, PGD synthase; L-PGDS, lipocalin-type PGDS; H-PGDS, hematopoietic PGDS; PGES, PGE synthase; cPGES; cytosolic PGES; mPGES-1, microsomal PGES-1; mPGES-2, microsomal PGES-2; PGFS, PGF synthase; AKRs, aldo–keto reductases; PGIS, prostacyclin synthase; TXS, thromboxane A synthase.

Whereas mPGES-1 is an inducible enzyme like COX-2, PGIS is highly and constitutively expressed in vascular endothelial and smooth muscle cells.^27–29) Its product, PGI₂, is a strong vasodilator and the most potent endogenous inhibitor of platelet aggregation; PGI₂ also inhibits the growth of vascular smooth muscle cells.³⁰⁾ PGIS is coupled with COX-2 as described above,⁸⁾ and COX-2/PGIS-derived PGI₂ has been shown to be critical for the regulation of platelet aggregation and vascular tone. Yokoyama et al. demonstrated that PGIS gene deletion impaired vascular homeostasis in mice.³¹⁾ The blood pressure of the PGIS KO mice was significantly higher than that of WT mice. It has been indicated that the specific inhibition of COX-2 alters the balance between platelet-derived thromboxane A₂ (TXA₂) and endothelium-derived PGI₂, leading to increases in the risk of thrombosis due to altered vascular tone.^5,6)

Importantly, our in situ hybridization analysis revealed that PGIS is expressed not only in vascular cells but also in other cells such as fibroblasts in the heart myocardium and lung parenchyma cells.²⁹⁾ Peritoneal macrophages (MΦs), dendritic cells, and T cells also express PGIS.^29,32) We found the constitutive expression of PGIS protein in thioglycolate-induced murine peritoneal MΦs.³³⁾ Treatment of MΦs with lipopolysaccharide (LPS) upregulated COX-2 and mPGES-1 protein levels, but the PGIS protein level was not altered by LPS stimulation. The expression of PGIS in MΦs suggested the role of PGIS in inflammatory reactions. Moreover, since PGI₂ has the ability to enhance vascular permeability, this implies the involvement of PGIS-derived PGI₂ in inflammatory reactions. Indeed, studies using PGI₂ receptor (IP) KO mice revealed that PGI₂ is involved in inflammatory and pain responses as well as the regulation of vascular tone.³⁴⁾ On the other hand, several investigators have recently reported that PGIS-derived PGI₂ functions as an anti-inflammatory mediator by MΦ polarization.^35,36) These findings have indicated that PGIS is an ambivalent regulator of inflammatory reactions. In this review, we summarize the current understanding of proinflammatory and anti-inflammatory functions of PGIS and discuss its possibility as a novel anti-inflammatory therapeutic target.

In addition to its roles in vascular regulation and inflammatory reactions, it has been recently reported that PGIS and PGIS-derived PGI₂ play an important role in preventing tumor growth and progression. We found that PGIS deficiency enhanced azoxymethane-induced colon carcinogenesis in mice.³³⁾ Keith et al. reported that in a smoke-exposure model, pulmonary-specific PGIS-overexpressing mice were protected from developing lung tumors.^37,38) The roles of PGIS in carcinogenesis are summarized in our current review.³⁹⁾

2. PROINFLAMMATORY FUNCTIONS OF PGIS

As described above, gene deletion of IP reduced inflammatory and pain responses.³⁴⁾ It was also reported that the disease severity of collagen-induced arthritis was reduced by IP deficiency in mice.⁴⁰⁾ We recently showed that inflammatory reactions such as exudation of leukocytes and inflammatory pain hypersensitivity were significantly suppressed in PGIS KO mice.³³⁾ In thioglycolate-induced peritonitis, the peritoneal leukocyte number was increased steadily after the injection of thioglycolate, accompanied by an increase in the 6-keto-PGF_1α level (a stable metabolite of PGI₂) in the peritoneal cavity. PGIS deficiency did not significantly affect the number of exudate leukocytes at the early stage of peritonitis, but significantly decreased their number at the late stage of inflammatory reaction. Among the exudate leukocytes, the exudation of MΦ was especially suppressed by PGIS deficiency. It was also found that the migration of MΦ into the peritoneal cavity was suppressed more effectively in PGIS/mPGES-1 double-KO (DKO) mice than in PGIS KO mice. We further investigated the effect of PGIS deficiency on inflammatory pain hypersensitivity, as assessed by the LPS-primed acetic acid-induced writhing reaction. As a result, we found that the writhing reaction was reduced in both PGIS KO and mPGES-1 KO mice, and was also suppressed more effectively in PGIS/mPGES-1 DKO mice compared to PGIS KO and mPGES-1 KO mice. These results suggested that PGIS-derived PGI₂ acts on IP at inflamed sites and exacerbates inflammatory reactions in cooperation with mPGES-1-derived PGE₂.

We recently found that in an allergic contact dermatitis model, PGIS and mPGES-1 coordinately promote cutaneous immune responses.⁴¹⁾ Allergic contact dermatitis is a common chronic inflammatory skin disease and consists of two phases, the sensitization phase and the elicitation phase. In the sensitization phase, in response to antigen sensitization, antigen-presenting cells such as dendritic cells capture antigens and migrate to regional lymph nodes, and T lymphocytes are activated and differentiate to effector T cells. In the elicitation phase, the repeated exposure of the skin to the antigen results in the activation of several immune cells, including T cell recruitment; then inflammation is developed in the skin area exposed to the antigen. By using pharmacologic and genetic approaches, the critical roles of PG receptors in contact hypersensitivity (CHS) responses have been demonstrated in a murine 1-fluoro-2,4-dinitorobenzene (DNFB)-induced CHS model. PGI₂-IP signaling and PGE₂-EP1/EP2/EP4 signaling contribute to the progression of inflammation in the elicitation phase of allergic contact dermatitis,^42–45) whereas PGE₂-EP3 signaling has an anti-inflammatory effect in the elicitation phase.⁴⁶⁾ We investigated the involvement of two PG terminal synthases, PGIS and mPGES-1, using both PGIS KO and mPGES-1 KO mice, which both showed significantly attenuated CHS compared with that seen in WT mice during the elicitation phase. The CHS response was suppressed more effectively in PGIS/mPGES-1 DKO mice compared to PGIS KO and mPGES-1 KO mice. Furthermore, we made WT bone marrow (BM) mouse chimeras and found that the transplantation of WT mouse-derived BM cells restored the impaired CHS response in mPGES-1 KO mice but did not restore the response in PGIS KO mice. These results suggested that PGIS expressed in MΦs might be not involved in the DNFB-induced CHS response and that PGIS in non-BM-derived cells might play a critical role in allergic contact dermatitis CHS. Our immunohistochemical analysis revealed that PGIS was expressed in fibroblast-like cells around the cartilage in inflamed ear tissues. The PGIS-derived PGI₂ produced in these cells might act on IP receptors to promote CHS responses by Th1 activation in cooperation with mPGES-1-derived PGE₂ produced in infiltrated leukocytes including dendritic cells.

Moreover, we recently reported the involvement of PGIS-derived PGI₂ in cyclophosphamide (CP)-induced cystitis.⁴⁷⁾ CP has been widely used in the treatment of various malignancies and autoimmune diseases, but acrolein, a byproduct of CP, is known to cause severe hemorrhagic cystitis. In the bladder tissues of CP-treated WT mice, the levels of PGE₂, 6-keto-PGF_1α and other PGs were increased in association with hemorrhagic cystitis. We found that the bladder bleeding and intense hyperemia induced by CP was significantly attenuated in PGIS KO mice compared to WT mice. On the other hand, mPGES-1 KO mice developed bladder flare reactions similarly to WT mice. These results suggested that PGIS-derived PGI₂ is involved in hemorrhagic cystitis, but mPGES-1-derived PGE₂ is not. Gene deletion of PGIS not only decreased bladder edema but also downregulated the expression of vascular endothelial growth factor and accompanying angiogenesis, and then attenuated CP-induced vascular leakage. It was also found that myeloperoxidase activity (an indicator of the presence of neutrophils) and gene expression of chemokines such as Cxcl1, Cxcl2 and Cxcl5 were suppressed by PGIS deficiency. Among the immune cells, neutrophils play major roles in exacerbating bladder inflammation in hemorrhagic cystitis.⁴⁸⁾ The promotion of neutrophil migration into bladder tissues might also be mediated by PGIS-derived PGI₂ via the upregulation of chemokine expression. In addition, PGIS deficiency and administration of the IP receptor-selective antagonist RO1138452 suppressed not only bladder hemorrhage but also bladder pain-like nociceptive behavior. It was also reported that the EP1 antagonist ONO-8130 reduced bladder pain caused by hemorrhagic cystitis.⁴⁹⁾ PGI₂-IP and PGE₂-EP1 signaling might enhance bladder pain in cystitis in a cooperative mechanism. It was noteworthy that our experiments using BM chimeras revealed that transplantation of WT mouse-derived BM cells did not restore the impaired hemorrhagic cystitis in PGIS KO mice. The PGI₂ involved in hemorrhagic cystitis might be produced by the PGIS expressed in the lamina propria of bladder tissue, rather than by the PGIS expressed in some type of BM-derived cells such as MΦs. Figure 2 summarizes the involvement of PGIS-derived PGI₂ in CP-induced hemorrhagic cystitis.

Fig. 2. Involvement of PGIS in CP-Induced Hemorrhagic Cystitis

Cyclophosphamide (CP) has been widely used in the treatment of various malignancies and autoimmune diseases, but acrolein, a byproduct of CP, causes severe hemorrhagic cystitis. PGIS-derived PGI₂ acts on IP and then exacerbates CP-induced cystitis by increasing vascular permeability and by promoting neutrophil migration. Cystitis-related nociceptive behavior is also mediated by PGIS-derived PGI₂-IP signaling.

Fig. 3. Regulation of MФ Polarization by PGIS

Downregulation of PGIS promoted MФ polarization to the M1 subtype, and these MФs exhibited excessive production of proinflammatory cytokines such as IL-1β and TNF-α. Conversely, upregulation of PGIS promoted the polarization to the M2 subtype, which exhibited excessive production of anti-inflammatory cytokines such IL-10 and TGF-β.

3. ANTI-INFLAMMATORY FUNCTIONS OF PGIS

As we mentioned above, PGIS-derived PGI₂ has proinflammatory functions, but PGIS expressed in BM-derived cells such as MΦs might be not involved in some of PGI₂-mediated proinflammatory reactions. Very recently, several investigators have reported that PGIS-derived PGI₂ produced in MΦs functions as not a proinflammatory but an anti-inflammatory mediator by MΦ polarization.^35,36) Ipseiz et al. reported that the transcription factor Gata6 controlled the expression of PGIS, and PGIS-derived PGI₂ suppressed the LPS-induced production of proinflammatory interleukin (IL)-1β via IL-10 induction in murine resident peritoneal MФs.³⁵⁾ IL-1β production requires two steps, production of immature pro-IL-1β and subsequent IL-1β maturation from its pro-form by inflammasome activation. Gata6-KO resident peritoneal MФs exhibited aberrant production of IL-1β upon LPS stimulation by the upregulation of IL-1β processing. Ipseiz et al. found that a soluble molecule secreted by WT resident peritoneal MФs actively inhibited IL-1β processing in the Gata6-KO MФs, and that PGIS expression and the subsequent production of PGI₂ were reduced in Gata6-KO MФs. They further showed that PGI₂ analogues iloprost, beraprost and cicaprost, significantly increased IL-10 production in Gata6-KO MФs. Moreover, beraprost and IL-10 significantly reduced IL-1β production from Gata6-KO MФs. These results indicated that a Gata6-PGIS-derived PGI₂-IL-10-axis functions as a major regulator of IL-1β processing in resident peritoneal MФs and of the related inflammatory reactions. It was noteworthy that Gata6 deficiency had a great effect on the expression pattern of PG terminal synthases and the subsequent production of prostanoids in resident peritoneal MФs. In Gata6-KO resident peritoneal MФs, PGIS expression and PGI₂ production were downregulated and conversely, thromboxane synthase (TXS) expression and thromboxane A₂ production were upregulated. A quarter-century ago, Kuwamoto et al. investigated that the gene expression of PGIS and TXS in murine resident MФs or in MФs elicited with casein or bacillus Calmette-Guérin (BCG), and found the expression of PGIS and TXS mRNAs was inversely regulated in MФs.³²⁾ Their Northern blot analyses showed that the PGIS mRNA was expressed in a decreasing order in the resident, and casein- and BCG-elicited MФs. In contrast, the TXS mRNA was expressed in an increasing order in the resident, and casein- and BCG-elicited MФs. These findings indicate that switching of prostanoid production is closely involved in MФ polarization and suggest the possibility that the prostanoid switching of MФ regulates inflammatory responses.

Very recently, Pan et al. reported that PGIS played a pivotal role in alcoholic liver disease (ALD) by regulating MФ polarization.³⁶⁾ The M1 phenotype of MФ promotes a proinflammatory response, whereas the M2 phenotype plays a key role in the resolution of inflammation. Pan et al. demonstrated that PGIS was downregulated in ALD and that liver-specific overexpression of PGIS alleviated ethanol-induced liver injury and suppressed macrophage M1 polarization in mice. They further discovered that overexpression of PGIS in RAW 264.7 cells suppressed LPS-plus-interferon-γ-induced MФ polarization to the M1 subtype, which showed excessive production of proinflammatory cytokines such as IL-1β and tumor necrosis factor (TNF)-α, and promoted IL-4-induced polarization of MФ to M2 subtype; these M2 MФs exhibited excessive production of anti-inflammatory cytokines such as IL-10 and transforming growth factor (TGF)-β. To the contrary, PGIS knockdown promoted M1 polarization and suppressed M2 polarization. It was also shown that the expression of PGIS was regulated by microRNA miR-140-3p.1 and that the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway was involved in PGIS-mediated M2 polarization. These findings suggest that PGIS has therapeutic effects on ALD by inhibiting M1 polarization and promoting M2 polarization in liver tissues.

Bergqvist et al. investigated the expression of PGIS in human diseased tendon tissues and found that biopsies from resolved (pain-free) patients showed increased PGIS mRNA expression compared to patients with persistent tendon disease.⁵⁰⁾ They proposed that PGIS-mediated PGI₂ production might be a protective response to promote vascularization and minimize thrombosis at the inflamed site. MФ polarization at the resolution phase of inflammatory reactions might be also regulated by PGIS-derived PGI₂.

4. CONCLUSION

Since the use of NSAIDs that suppress prostanoid production by the inhibition of COX activity is associated with severe side effects, COX downstream PG terminal synthases have become attractive therapeutic targets as alternatives to COX inhibition. Among these PG terminal synthases, PGIS has been garnering particular attention as well as mPGES-1. As summarized in this review, PGIS is mainly a promoter of the inflammatory reaction, and PGIS-derived PGI₂ promotes and exacerbates inflammatory reactions, like mPGES-1-derived PGE₂. In the future, PGIS inhibitors will be developed as novel NSAIDs without the adverse effects associated with current PG-related therapies, such as induction of gastrointestinal irritations.

It should also be noted that PGIS-derived PGI₂ also seems to function as an anti-inflammatory mediator at the steady-state and the resolution phase of inflammatory reactions. In addition, although it was reported that PGIS is an anti-inflammatory regulator which promotes MФ polarization to the M2 phenotype, the molecular mechanisms by which PGIS-mediated PGI₂ production regulates MФ polarization have not been fully elucidated. Further studies on PGIS will lead to the development of novel drugs which prevent and resolve inflammatory diseases.

Acknowledgments

This work was supported by JSPS KAKENHI Grants for Scientific Research (B) (Nos. 16H05108 and 19H03375 to S.H.), for Scientific Research (C) (No. 21K06549 to Y.S.) and for Early Career Scientists (No. 22K15283 to T.O.) from the Japan Society for the Promotion of Science, and Grants-in-Aid for Scientific Research on Innovative Areas (Nos. 23116515 and 25116720 to S.H.) from the Ministry of Education, Sports, Science, Culture and Technology of Japan.

Conflict of Interest

The authors declare no conflict of interest.

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