296 Proc. Jpn. Acad., Ser. B 83 (2007) [Vol. 83,

Review Physiology and pathophysiology of prostanoid receptors

By Shuh NARUMIYA1,2;y

(Communicated by Tasuku HONJO, M.J.A.)

Abstract: Prostanoids, consisting of prostaglandins (PGs) and thromboxanes (TXs), are oxygenated products of C20 unsaturated fatty acids. They include PGD2,PGE2,PGF2,PGI2, and TXA2. Given that aspirin-like nonsteroidal anti-inflammatory drugs exert their actions by suppressing prostanoid production, prostanoids have been implicated in processes inhibited by these drugs, including inflammation, fever, and pain. Prostanoids also contribute to vascular homeostasis, reproduction, and regulation of kidney and gastrointestinal functions. How prostanoids exert such a variety of actions had remained unclear, however. Prostanoids are released outside of cells immediately after their synthesis and exert their actions by binding to receptors on target cells. We have identified a family of eight types or subtypes of G protein– coupled receptors that mediate prostanoid actions. Another G protein–coupled receptor was also identified as an additional receptor for PGD2. Genes for these receptors have been individually disrupted in mice, and analyses of these knockoutmicehavenotonlyelucidatedthemolecular and cellular mechanisms of known prostanoid actions but also revealed previously unknown actions. In this article, I review the physiological and pathophysiological roles of prostanoids and their receptors revealed by these studies.

Keywords: prostaglandin, thromboxane, cyclooxygenase, G protein–coupled receptor

Prostanoids, consisting of prostaglandins the 5-cis, 13-trans, and 17-cis double bonds, re- (PGs) and thromboxanes (TXs), are oxygenated spectively. Given that arachidonic acid is the most products derived from C20 unsaturated fatty acids abundant among these precursor fatty acids in most (Fig. 1). -Homolinolenic acid, arachidonic acid, mammals, including humans, the series 2 prosta- and 5,8,11,14,17-eicosapentaenoic acid are the noids predominate in these organisms. These pre- precursor fatty acids for the series 1, 2, and 3 cursor fatty acids are esterified to the sn-2 position prostanoids, which contain the 13-trans double of glycerophospholipids in cell membranes and are bond, the 5-cis and 13-trans double bonds, and liberated from the membrane phospholipids by the action of phospholipase A2 in response to various 1) 1 Department of Pharmacology, Kyoto University Faculty physiological or pathological stimuli. Arachidonic of Medicine, Kyoto, Japan. acid thus liberated is metabolized by the action of 2 Recipient of the Imperial Prize and the Japan Academy cyclooxygenase (COX) to PGH ,whichisthen Prize in 2006. 2 y Department of Pharmacology, Kyoto University Faculty converted to various PGs by the respective PG of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan (e-mail: synthases (Fig. 1). There are two isoforms of COX; [email protected]). one is COX-1 that is constitutively expressed in Abbreviations: PG: prostaglandin; TX: thromboxane; COX: cyclooxygenase; NSAID: nonsteroidal anti-inflammatory many tissues, and the other is COX-2, expression of drug; GPCR: G protein–coupled receptor; cAMP: cyclic adeno- which is induced by various inflammatory as well as sine monophosphate; Th: T helper; ACTH: adrenocorticotropic other stimuli and suppressed by glucocorticoids hormone; LPS: lipopolysaccharide; IL: interleukin; TNF: tumor 2) necrosis factor; CRF: corticotropin-releasing factor; DRG: dorsal such as dexamethasone. Similarly, there are two root ganglion; Ig: immunoglobulin; OVA: ovalbumin; SNP: major isoforms of PGE synthase, cytosolic PGE single nucleotide polymorphism; DNFB: dinitrofluorobenzene; synthase (cPGES) that is constitutively expressed IBD: inflammatory bowel disease; DSS: dextran sodium sulfate; PTH: parathyroid hormone; RANKL: receptor activator of and membrane-bound PGE synthase-1 (mPGES-1) NF-B ligand. that, like COX-2, is induced by various proinflam- doi: 10.2183/pjab/83.296 #2007 The Japan Academy Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 297

COOH

Arachidonic acid

Cyclooxygenase 2O2

7 4 2 O 9 8 6 5 3 COOH 10 14 1516 18 20 O 11 12 13 17 19 OOH PGG2

COOH

PGI synthase TX synthase O COOH COOH O O O O OH OH

OH PGH2 TXA2 OH PGD synthase PGF synthase PGI2 PGE synthase OH OH O COOH COOH COOH

O OH OH OH OH OH PGD 2 PGE2 PGF2 α

Fig. 1. Pathway of prostanoid biosynthesis. Arachidonic acid is metabolized by the action of cyclooxygenase (COX) first to prostaglandin endoperoxide (PGG2) and then to PGH2, which is subsequently converted to various PGs and TXA2 by respective synthases. Aspirin-like nonsteroidal anti-inflammatory drugs (NSAIDs) exert their effects by inhibiting COX and thereby suppressing prostanoid biosynthesis. matory stimuli and suppressed by glucocorticoids.3) they exert central actions such as induction of fever Prostanoids thus formed are released outside of or sleep. They regulate secretion and motility in cells immediately after their synthesis. PGH2, the gastrointestinal tract as well as transport of PGI2,andTXA2 are chemically unstable and are ions and water in the kidney. They play roles in degraded to inactive products under physiological apoptosis, cell differentiation, and oncogenesis. conditions, with half-lives of 30 s to a few minutes. They also regulate the activity of blood platelets Other PGs, although chemically stable, are metab- both positively and negatively, and they contribute olized rapidly. For example, they are inactivated to vascular homeostasis and hemostasis. Prosta- during a single passage through the lung. Prosta- noids thus exert a wide variety of actions in various noids are therefore thought to act locally, in the cells and tissues and the body. However, the vicinity of the site of their production. mechanisms underlying such actions remained a Aspirin-like nonsteroidal anti-inflammatory mystery until the receptors responsible were iden- drugs (NSAIDs) exert their pharmacological ac- tified, their cDNAs cloned, and their roles analyzed. tions by inhibiting COX and suppressing prosta- Furthermore, whereas methods for the chemical noid production. The actions of prostanoids have synthesis of natural prostanoids and related com- therefore been examined either by the addition of pounds have been developed for reproduction of or exogenous prostanoids to cells, tissues, or the body interference with the various bioactivities of pros- or by analysis of the effects of aspirin-like drugs on tanoids, they have been of limited use without various physiological or pathophysiological proc- knowledge of the prostanoid receptors and their esses. The typical actions of prostanoids revealed by properties. these studies include induction of the relaxation or contraction of various types of smooth muscle. Structure, signal transduction mechanisms, anddistributionofprostanoidreceptors Prostanoids also modulate neuronal activity by inhibiting or stimulating neurotransmitter release Given that prostanoids are produced from fatty or sensitizing sensory fibers to noxious stimuli, and acids and were therefore generally regarded as 298 S. NARUMIYA [Vol. 83, hydrophobic compounds, it was initially thought 29 amino acids, respectively, that do not share any that they were incorporated into the cell membrane structural motifs or hydrophobic features.13)–15) and exerted their actions by perturbing lipid fluid- These isoforms show similar ligand binding charac- ity. However, prostanoids are not as hydrophobic as teristics but distinct signal transduction properties, originally thought and do not incorporate into or as described below. Such multiple splice isoforms permeate the cell membrane.4) Furthermore, com- for EP3 have also been identified in other species parison of the potencies of various prostanoids as including rat, rabbit, cow, and human.11) In addi- determined by bioassays in various tissues revealed tion to this family of prostanoid receptors, there is a 16) that each prostanoid possesses a unique activity distinct type of receptor for PGD2. This receptor, profile, indicating that each compound has a designated CRTH2, was originally cloned as an specific site of action. Moreover, various synthetic orphan receptor expressed in T helper (Th) 2 TXA2 agonists and antagonists were developed in lymphocytes and has recently been shown to bind the late 1970s to early 80s, and these compounds PGD2 with an affinity as high as that of DP, facilitated the pharmacological characterization of although its binding profile for other PGD analogs 5) a receptor for TXA2. Biochemical evidence also differs from that of DP. CRTH2 belongs to the supported the existence of prostanoid receptors. family of chemokine receptors and mediates chemo- Beginning in the mid-1960s, the actions of prosta- taxisofTh2lymphocytesaswellasofeosinophils noids were repeatedly found to be associated with and basophils toward PGD2. changes in the levels of intracellular second mes- Signal transduction pathways linked to pros- sengers such as cyclic adenosine monophosphate tanoid receptors have been studied by examination (cAMP), phosphoinositides, and Ca2þ. In addition, of the effects of agonists on the intracellular the availability of radioisotopically labeled prosta- concentrations of second messengers such as cAMP, noid derivatives in the early 1970s allowed the Ca2þ, and inositol phosphates in cells transfected demonstration that many tissues and cells possess with expression vectors for each receptor. These specific high-affinity binding sites for prosta- studies revealed that the eight types or subtypes of noids.6)–8) prostanoid receptors can be divided into three These various studies culminated in the pro- groups on the basis of their modes of signal trans- posal of a pharmacological classification of prosta- duction (Fig. 2B): IP, DP, EP2, and EP4 mediate noid receptors into those specific for TX, PGI, PGE, an increase in the intracellular level of cAMP and PGF, or PGD, designated TP, IP, EP, FP, and have been designated ‘‘relaxant’’ receptors; TP, FP, DP receptors, respectively, with EP receptors and EP1 induce elevation of intracellular Ca2þ and being further divided into four subtypes designated have been denoted as ‘‘contractile’’ receptors; and EP1, EP2, EP3, and EP4.9),10) However, none of EP3 elicits a decrease in the intracellular concen- these receptors had been isolated or cloned until tration of cAMP and has been termed an ‘‘inhib- 13) we purified the TXA2 receptor from human blood itory’’ receptor. This functional classification of platelets in 198911) and cloned its cDNA in 1991.12) prostanoid receptors is consistent with the phylo- The TXA2 receptor thus revealed is a G protein– genetic tree derived from the nucleotide sequences coupled, rhodopsin-type receptor with seven trans- of the corresponding genes.17) These studies indi- membrane domains (Fig. 2A). Homology screening cate that the prostanoid receptors originated from a of cDNA libraries prepared from mouse and other primitive PGE receptor; this primitive receptor mammals subsequently resulted in the identifica- gave rise to the PGE receptor subtypes, and the tion of the primary structures of the remaining receptors for other PGs and TX subsequently seven types or subtypes of prostanoid receptors.13) evolved from functionally related PGE receptor All are G protein–coupled, rhodopsin-type recep- subtypes by gene duplication. It should be noted, tors with seven transmembrane domains and each is however, that this functional grouping of prosta- encoded by a distinct gene. Several variants of EP3 noid receptors is based on the coupling of each that are generated by alternative splicing of exons receptor to only one of three signaling pathways, encoding the carboxyl-terminal tail were also iden- increase or decrease in the intracellular cAMP level tified. The three EP3 splice isoforms in mouse, , , or elevation in the intracellular Ca2þ concentration; and , contain carboxyl-terminal tails of 30, 26, or it does not exclude the possibility that these Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 299

A

CHO CHO

T I N T N W M S P A L P R F C P G L S S G P M G E A Q E L A V D P P S R H G L G A E S T G P W L R R E C D N F R L F R T Extracellular S CPY V L I L L T W G Q A V C S S E A A F T R V G Q K S A F T L A E Y L L P H Q M R I W S F F L F A V G G V S V A V I V V G M L L L I S F T M L L G P Y G T I F P G L L R C V L L S W C V V V L F G L G V A Plasma G L L V G S A T W L A G S P L A L L F L L S V N Q N L L A F L V M I L D T L G A A I L A L L N G L D P membrane L S V A A W V T L G M L V L Q W V V L C A G S V A Y A L F S E V T A M I L G A T A T L M F L F R W E R A C V R S Y R H R Q L E S R V S A G R G G H T Q Y D V S I S H R L T A G P R Intracellular R V Q R R P E F A A A Q Q L S R P Q P Q L L R G S R S R T S Q T L Q P Q L L S R P B

TP cAMP IP Relaxant EP1 2+ DP Ca Contractile EP2 FP EP4

EP3

cAMP Inhibitory

Fig. 2. (A) Membrane topology model for the human TXA2 receptor (modified from ref. 118 with permission). Each amino acid is represented by the single-letter code within a circle. White letters in black circles indicate residues that are conserved among members of the prostanoid receptor family. The model is based on hydrophilicity analysis of the amino acid sequence. Asparagine residues at positions 4 and 16 are glycosylated. (B) Phylogenetic tree and functional grouping of prostanoid receptors (modified from ref. 118 with permission). receptors are able to couple to more than one G receptor gene shows a specific pattern of expression protein and signal transduction pathway. in the mouse body, with expression levels differing The wide range of prostanoid actions had among tissues (Fig. 3A).18)–25) In situ hybridization suggested that prostanoid receptors might be ubiq- further revealed that, even within the same organ, uitously distributed throughout the body. The transcripts of different receptor genes were dis- cloning of the receptor cDNAs made it possible to tributed differentially. In the kidney, for example, examine the tissue distribution and cellular local- the EP3 gene is expressed in the tubular epithelium ization of each member of the prostanoid receptor of thick ascending limb of the Henle loop, and family. Northern blot analysis revealed that each collecting ducts in the outer medulla, the EP1 gene 300 S. NARUMIYA [Vol. 83,

AB

Fig. 3. (A) Northern blot analysis of transcripts derived from the eight types or subtypes of prostanoid receptor in mouse tissues (from ref. 119 with permission). (B) In situ hybridization of mRNAs for the EP1, EP3, and EP4 subtypes of the PGE receptor in mouse kidney (from ref. 26 with permission). Scale bar, 1 mm. in the papillary collecting ducts, the EP4 gene in analogs, iloprost and carbacyclin, indeed bound to the glomerulus26) (Fig. 3B), the IP gene predom- IP with relatively high affinity, but they also bound inantly in blood vessels such as the interlobular to EP3 (both drugs) or EP1 (iloprost) with similar 27) arteries and afferent glomerular arterioles, and or higher affinities (Table 1). 17-Phenyl-PGE2, the FP gene in the distal convoluted tubule and which has been used as an EP1 agonist in many cortical collecting ducts.28) Some of the results of analyses, was found to bind to EP3 with a higher these analyses are consistent with known functions affinity and to FP with a similar affinity relative of prostanoids in corresponding tissues. For exam- to that with which it binds EP1. Such cross- ple, the distributions of EP3, EP1, and EP4 in the reactivity of conventional PG analogs likely reflects kidney correlate with the PGE2-mediated regula- the fact that, during their development, they tion of ion transport, water reabsorption, and were screened with bioassays based on native glomerular filtration, respectively, and that of IP tissues and cells that express more than one type in the glomerular arterioles may be related to of prostanoid receptor. A new generation of com- prostanoid-induced renin secretion associated with pounds has since been developed that was screened reduced renal perfusion. However, other results, with a panel of cloned prostanoid receptors such as the observed enrichment of TP and IP in (Table 1).30)–32) These agents are highly selective the thymus, were unexpected and are indicative of for each receptor type or subtype. Indeed, they have functions of prostanoids yet to be clarified. been used in combination with knockout mice deficient in each receptor type or subtype to Development of prostanoid receptor type- or elucidate the physiological roles of the correspond- subtype-selective agonists and antagonists ing receptor, and they have been shown to exert The cloning of eight members of the prostanoid pharmacological actions consistent with the pheno- receptor family has facilitated both the reassess- types of these knockout mice. ment of previously developed receptor-active com- pounds and the development of new drugs. Exami- Generation and analysis of knockout mice nation of the binding properties of a series of PGs deficient in each type or subtype of prostanoid receptor and available PG analogs with a panel of CHO cells stably expressing each cloned receptor re- The roles of prostanoids in various physiolog- vealed that most of these compounds exhibited no ical and pathophysiological processes were initially 29) absolute selectivity. For example, two PGI2 suggested by comparison of the effects of aspirin- Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 301

Table 1. Binding specificity of prostanoid analogs developed before and after cDNA cloning of the prostanoid receptor family. Dissociation constants (nM) for binding of each compound to the eight types or subtypes of mouse prostanoid receptors are shown. The lower values for each compound are shown in bold letters (from ref. 117 with permission).

Compound DP EP1 EP2 EP3 EP4 FP IP TP Compounds developed before receptor cloning Iloprost >10;000 21 1,600 22 2,300 >10;000 10 >10;000 Carbacyclin >10;000 >10;000 16,000 31 2,300 1,200 110 >10;000 17-Phenyl >10;000 14 >10;000 4 1,000 60 >10;000 >10;000

-PGE2 Compounds developed after receptor cloning DP agonist L-644,698 0.9 >25;400 267 3,730 9,280 >25;400 >25;400 >25;400 EP1 antagonist ONO-8713 >10;000 0.3 3,000 1,000 >10;000 1,400 10,000 10,000 EP2 agonist ONO-AE1-259 >10;000 >10;000 3 >10;000 >10;000 >10;000 >10;000 >10;000 EP3 agonist ONO-AE-248 >10;000 >10;000 3,700 8 4,200 >10;000 >10;000 >10;000 EP4 agonist ONO-AE1-329 >10;000 >10;000 2,100 1,200 10 >10;000 >10;000 >10;000 like drugs with the actions of each prostanoid added prostanoids in these phenomena and have increased exogenously. However, such studies did not clearly our understanding of their molecular and neuronal indicatewhichtypeofprostanoidandwhichtypeof mechanisms. prostanoid receptor mediate a given action or how Fever. Fever is a prominent component of important such prostanoid actions are. These issues sickness behavior. Cellular components of infectious were addressed by the generation of mice deficient organisms, such as bacterial LPS, as well as in each of the prostanoid receptors as a result of cytokines such as interleukin (IL)-1, IL-6, and disruption of the corresponding gene by homologous tumor necrosis factor (TNF)- serve as exogenous recombination. The phenotypes of these knockout and endogenous pyrogens, respectively, and stim- mice are summarized in Table 2, and the physio- ulate the neural pathways responsible for increasing logical significance and clinical implications of some body temperature.34) Fever is suppressed by of these findings are addressed in the remainder of NSAIDs, indicating that PGs are important medi- this review. ators of fever generation. PGE2 has been implicated Prostanoid receptors and brain function. as such a mediator on the basis of the observation Systemic disease is associated with general and that injection of PGE2 into the brain induces fever characteristic central nervous system symptoms in many species. Examination of mice lacking each and signs including fever, release of adrenocortico- subtype of PGE receptor (EP1, EP2, EP3, or EP4) tropic hormone (ACTH), reduced locomotion and revealed that the EP3 knockout animals failed to 33) social contact, anorexia, and increased sleep. show a febrile response to PGE2,IL-1,orLPS These phenomena, collectively referred to as ‘‘sick- (Fig. 4A).35) This finding thus demonstrated that ness behavior,’’ can be reproduced experimentally PGE2 mediates fever generation in response to both in animals by administration of noxious substances exogenous and endogenous pyrogens by acting at such as lipopolysaccharide (LPS) and inflammatory EP3, a conclusion that was supported further by a cytokines. Given that treatment with NSAIDs study based on telemetry.36) Fever induction occurs alleviates most of these behaviors as typically seen inthepreopticareaoftheorganosumvasculatum in fever generation, it had been thought, but not lamina terminalis, a region of the brain with a definitively proven, that prostanoids contribute to relatively ineffective blood-brain barrier, and it is their generation. Studies of knockout mice have now known that LPS and IL-1 each induce expres- provided direct evidence for the participation of sion of both COX-2 and mPGES-1 in brain micro- 302 S. NARUMIYA [Vol. 83,

Table 2. Phenotypes of mice deficient in prostanoid receptors.

Receptor Phenotypes DP Decreased allergic responses in OVA-induced bronchial asthma77Þ 46Þ Impaired PGD2-induced sleep Facilitated mobilization of dendritic cells in skin immune responses88Þ;89Þ CRTH2 Suppression of allergic inflammation in IgE-induced dermatitis81Þ Augmentation of airway inflammation in OVA-induced asthma82Þ EP1 Decreased aberrant foci formation to azoxymethane98Þ 61Þ Deceased PGE2-induced mechanical allodynia Impaired ACTH response to systemic endotoxin administration42Þ Impulsive behavior in response to environmental or social stress43Þ EP2 Impaired ovulation and fertilization62Þ{64Þ Salt-sensitive hypertension63Þ 106Þ;107Þ Vasopressor or impaired vasodepressor response to PGE2 108Þ Loss of bronchodilation in response to PGE2 Impaired osteoclastogenesis in vitro104Þ Impaired amplification of COX and angiogenesis of intestinal polyps in Apc716 mice97Þ EP3 Impaired febrile response to pyrogens35Þ Impaired duodenal bicarbonate secretion and mucosal integrity109Þ 106Þ Enhanced vasodepressor response to PGE2 Loss of indomethacin-sensitive urine-diluting function110Þ Decreased acetic acid–induced writhing after endotoxin treatment54Þ 73Þ;74Þ Impaired PGE2-induced potentiation of platelet activation Impaired angiogenesis to transplanted cancer and chronic inflammation111Þ Enhanced allergic response in OVA-induced bronchial asthma85Þ EP4 Patent ductus arteriosus66Þ;67Þ 106Þ Impaired vasodepressor response to PGE2 Decreased inflammatory bone resorption102Þ;103Þ 105Þ Lack of PGE2-induced bone formation in vivo Exaggerated DSS-induced colitis94Þ Impaired Langerhans cell migration in skin immune responses87Þ Decreased aberrant foci formation in response to azoxymethane99Þ FP Loss of parturition65Þ Impaired generation of tachycardia in response to inflammatory stimuli71Þ IP Thrombotic tendency53Þ Decreased inflammatory swelling53Þ Decreased acetic acid–induced writhing53Þ Enhanced cardiac ischemia-reperfusion injury112Þ Impaired adaptive gastric cytoprotection113Þ Impaired capsaicin-induced gastric cytoprotection114Þ Enhanced pulmonary hypertension and vascular remodeling under chronic hypoxic conditions115Þ Enhanced atherosclerosis75Þ TP Bleeding tendency and resistance to thromboembolism68Þ Enhanced immune response due to facilitation of dendritic cell–T cell interaction69Þ Suppression of initiation and progression of atherosclerosis75Þ Impaired generation of tachycardia in response to inflammatory stimuli71Þ Reduced endotoxin-induced microcirculatory dysfunction in the liver116Þ Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 303

B CNS Brain PGE2 A LPS micro- EP3-containing IL-1 vessels EP3 neurons in POA in OVLT GABA C) ° Dorsomedial hypothalamus Wild-type EP1–/– Sympathetic premotor neurons in rostral raphe EP3–/– pallidus nucleus Vehicle Sympathetic Body temperature ( preganglionic neurons Time (min) Heat production Vasoconstriction in adipose tissue C

Fig. 4. (A) Impaired febrile response to LPS in mice deficient in EP3 (from ref. 35 with permission). Wild-type (closed circles), EP1= (open circles), or EP3= (open triangles) mice were injected with LPS at time zero; control wild-type mice were injected with vehicle (closed triangles). (B) Current model for the neural pathway that underlies fever generation. OVLT, organosum vasculatum lamina terminalis; POA, preoptic area. (C) Impaired ACTH response to LPS in mice deficient in EP1 or EP3 (from ref. 42 with permission). Wild-type (BL6) or the indicatedEPknockoutmicewereinjectedwithLPSorvehicle()andthe plasma concentration of ACTH was measured 1 h thereafter. P<0:01. vessels and that both of these enzymes are essential induced fever generation deduced from these find- for fever generation.37)–39) Identification of the role ings is shown in Fig. 4B. of EP3 was not only consistent with this afferent ACTH release. Another prominent aspect of pathway but also helped to identify the efferent sickness behavior is activation of the hypothalamic- pathway in fever generation. Examination of EP3- pituitary-adrenal axis. In this response, a popula- expressing neurons in the preoptic area revealed tion of neurons in the paraventricular nucleus of the that they contain the inhibitory neurotransmitter hypothalamus is activated to secrete corticotropin- -aminobutyric acid (GABA) and project to neu- releasing factor (CRF), which then triggers the rons in the dorsomedial hypothalamus that, in turn, release of ACTH from the pituitary into the blood- send their projections to the medullary raphe stream and the consequent stimulation of cortico- region, where sympathetic premotor neurons re- steroid secretion from the adrenal gland. A role for sponsible for febrile and thermoregulatory respons- prostanoids in this response had been suggested but es are localized.40),41) The pathway of pyrogen- the evidence was inconclusive before the issue was 304 S. NARUMIYA [Vol. 83, examined in EP knockout mice. Injection of wild- mice, another PG-mediated pathway may oppose type mice with LPS revealed that the increase in EP1 action in the brain. The balance between such the plasma concentration of ACTH consisted of two opposing PG actions may set a point for impulsivity components, an NSAID-resistant transient response control during exposure to various types of stressful that peaked 2 h after LPS administration and stimuli. an NSAID-sensitive response that was sustained Sleep. PGD2 is abundantly produced in the for 3 h.42) Examination of this latter response in brain and is a potent endogenous promoter of sleep mice deficient in each EP subtype revealed it to in rats and other mammals including humans.44) be impaired in mice lacking either EP1 or EP3 The lipocalin-type PGD synthase is present in high (Fig. 4C). Further analysis revealed that the EP1- amounts in the leptomeninges of the brain and is mediated pathway and the EP3-mediated pathway thought to be responsible for the production of converge at activation of the CRF-containing PGD2 in this organ. Consistently, when stimulation neurons in the hypothalamus. Given that activation by tail clipping of transgenic mice that overexpress of EP1 induces a rise in intracellular Ca2þ concen- this type of PGD synthase caused increase in brain tration, the detection of EP1 immunoreactivity in PGD2 content and concomitantly induced a marked 45) the presynaptic terminals of neurons that form increase in slow-wave sleep. This PGD2-induced synapses on the somas and axons of CRF-contain- sleep is apparently mediated by the DP receptor ing neurons indicates that EP1 contributes directly also present in the leptomeninges. Infusion of PGD2 to synaptic activation of the CRF neurons. into the lateral ventricle was thus found to increase Stress behavior. Sickness behavior can be slow-wave sleep in wild-type mice but not in DP- viewed as a stress response to stimuli associated deficient mice,46) showing that DP is required for with sickness. Phenomena such as fever generation PGD2-induced slow-wave sleep. and ACTH release that are components of sickness PGD2-induced sleep has been shown to be behavior thus also occur in other stress responses mediated by adenosine acting at the A2A subtype of including those to psychological stimuli. However, adenosine receptor.47) Consistent with this finding, prostanoids do not contribute to either fever gen- the extracellular adenosine content of the subar- eration or ACTH release in response to psycholog- achnoid space was found to be increased in a DP- 46) ical stressors such as hyperthermia to restraint dependent manner after PGD2 infusion. Al- stress.35) On the other hand, we recently showed though baseline sleep-wake patterns were essential- that EP1 participates in the control of impulsive ly identical between wild-type animals and either behavior of mice in response to environmental or DP-deficient mice or mice deficient in the lipocalin- social stress.43) For example, EP1-deficient mice type PGD synthase, intracerebroventricular injec- exhibited impaired cliff avoidance and jumped from tion of selenium tetrachloride, an inhibitor of this the raised platform in this test. They also showed PGD synthase, suppressed both non-rapid eye impulsive aggression to younger conspecifics in the movement sleep and rapid eye movement sleep in resident-intruder test as well as a markedly shorter wild-type mice but not in mice deficient in either lag time to and more episodes of fighting induced DP or the PGD synthase,48) indicating that sele- by electric stimuli compared with wild-type ani- nium tetrachloride acts on the PGD synthase to mals. Conversely, injection of wild-type mice with suppress sleep. Furthermore, administration of a an EP1-selective agonist attenuated their fighting specific DP antagonist, ONO-4127Na, also mark- episodes in the latter test. EP1-deficient mice edly reduced the sleep period in rats.48) These exhibited increased dopamine turnover in several findings indicate that the PGD synthase–PGD2-DP brain regions compared with wild-type mice, and pathway regulates physiological sleep, and that the their impulsive behavior was suppressed by injec- deficiency of a component of this pathway may be tion with an antagonist of the D1 subtype of compensated for by other sleep-regulating systems dopamine receptor, indicating that PGE2-EP1 during development of the knockout animals. On signaling modulates the activity of dopaminergic the other hand, this conclusion does not exclude the neurons to control behavior. Given that adminis- possibility that the PGD2-DP system is also in- tration of NSAIDs to wild-type mice did not induce volved in generation of pathological sleep. Injection the behavioral phenotype observed in EP1-deficient of IL-1 or TNF- into the PGD2-sensitive region of Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 305 the rat brain induces sleep in a COX-2–dependent model, a hyperalgesic response can be induced by 49) manner. Overproduction of PGD2 has also been either of these PGs and that whether endogenous observed in certain sleep disorders, including sys- PGE2 or PGI2 (or both) mediates this response temic mastocytosis and African sleeping sickness, in depends on which of these PGs is produced in a humans.44) Furthermore, the human malarial para- given context. site Plasmodium falciparum produces PGs, includ- The TRPV1 cation channel is the receptor for ing PGD2, that may contribute to sleepiness and capsaicin and detects heat and pH in pain sensation. other symptoms of this disease.50) Moriyama et al. examined how prostanoid signaling Prostanoid receptors and pain sensa- might be integrated with the action of this channel tion. The role of PGs in inflammatory pain has in pain sensation and found that both PGE2 and been suggested by the antinociceptive effects of PGI2 augment thermal hyperalgesia mediated by aspirin-like drugs. Consistently, studies in various TRPV1 and that such augmentation is attenuated model systems have shown that exogenous PGs by deficiency of EP1 and IP, respectively.55) Fur- induce hyperalgesia (an increased sensitivity to a ther examination of the effects of prostanoid signal- painful stimulus) or allodynia (a pain response to a ingonTRPV1-mediatedcurrentsinisolatedDRG usually nonpainful stimulus). These studies also neurons as well as in cultured cell lines expressing showed that PGE2,PGE1,andPGI2 exert stronger the recombinant prostanoid receptors revealed effects than the other PGs, implicating EP and IP that EP1 or IP signaling lowers the threshold for in the induction of inflammatory pain.51) activation of the TRPV1 channel by temperature or Peripheral hyperalgesia. Although a spinal ac- pH in a manner dependent on protein kinase C, and tion is also suggested (see below), the main site of that the channel can also be activated in a protein the hyperalgesic action of prostanoids lies in the kinase A-dependent manner by IP or EP4 signaling. periphery, where PGs are thought to sensitize the The involvement of EP4 seems consistent with the free ends of sensory neurons. The cell bodies of finding that a selective EP4 antagonist attenuated primary sensory afferents are located in dorsal root the thermal hyperalgesia apparent with peripheral ganglia (DRG), and several prostanoid receptor inflammation induced by injection of Freund’s mRNAs, including those for IP, EP1, EP3, and complete adjuvant.56) Signaling by prostanoids EP4, have been detected in DRG neurons.27),52) and other inflammatory molecules thus appears to Subjection of mice deficient in these receptors to be integrated at the level of DRG neurons. How- various pain models revealed that they all contrib- ever, whereas the TRPV1 channel is a pain receptor ute to mediation of pain sensationinacontext- for heat and pH, it does not mediate the pain dependent manner. For example, we found that the response to mechanical stimuli57); given that pros- pain response of IP-deficient mice in the acetic acid tanoids also augment mechanical pain sensation, a writhing test was reduced to a level similar to that separate integration mechanism for PG-mediated observed in wild-type mice treated with the COX hyperalgesia likely exists. inhibitor indomethacin,53) indicating that the hy- Central hyperalgesia. In addition to their hy- peralgesic response in this model is evoked by peralgesic actions in the periphery, PGs augment endogenous PGI2 acting at IP. On the other hand, the processing of pain information in the spinal = = = = treatment of EP1 , EP2 , EP3 , EP4 , cord. Administration of PGE2 into the dorsal horn IP=, or wild-type mice with LPS before examina- of the rat spinal cord was found to reduce in an tion of acetic acid–induced writhing subsequently EP2-dependent manner the extent of neurotrans- revealed that not only IP= mice but also EP3= mission mediated by the inhibitory transmitter mice showed a reduced response.54) These findings glycine during pain sensation.58) This finding sug- thus indicated that nociception in the writhing gested that PGE2 might facilitate by this mecha- response is mediated predominantly by IP in un- nism the transmission of nociceptive input through treated mice, whereas it is mediated by both IP and the dorsal horn of the spinal cord to higher brain EP3 in LPS-treated mice. The observation that areas where pain becomes conscious. Consistent intraperitoneal injection of either PGE2 or PGI2 with this notion, EP2-deficient mice exhibited only induced moderate writhing responses in wild-type short-lasting hyperalgesia after a peripheral inflam- mice further suggested that, even in the same matory stimulus, failing to manifest a second 306 S. NARUMIYA [Vol. 83, sustained hyperalgesic phase of spinal origin that is several previous estrous cycles. Sugimoto et al.65) apparent in wild-type animals.59) Together, the found that, despite the lack of apparent defects in available evidence thus indicates that prostanoids the estrous cycle, FP-deficient mothers do not acting at various receptor types or subtypes regu- undergo parturition, apparently because of the late pain sensation at multiple levels and in a absence of labor. They further showed that FP- redundant manner. deficient mice do not undergo parturition even Allodynia. Intrathecal injection of PGE2 in- when administered exogenous oxytocin and that duces allodynia in mice, as manifested by squeak- such dams show no prepartum decline in progester- ing, biting, and scratching in response to low- one levels. A reduction in progesterone levels threshold stimuli.60) This response was also appa- induced by ovariectomy 24 h before term resulted rent in EP3-deficient mice but was not observed in in up-regulation of uterine receptors for oxytocin EP1-deficient mice,61) suggesting that EP1 medi- and normal parturition in FP-deficient mice. These ates PGE2-induced allodynia. observations indicate that the luteolytic action of Prostanoid receptors and reproduction. PGF2 is required in mice to reduce progesterone Various reproductive effects of NSAIDs have long levels and thereby to allow the initiation of labor indicated that prostanoids function at multiple (Fig. 5B). steps in pregnancy and parturition. Studies of Prostanoid receptors and closure of the knockout mice deficient in prostanoid receptors ductus arteriosus. At birth, with the commence- confirmed such functions and provided greater ment of respiration, humans and other mammals insight into their underlying mechanisms. undergo a marked change in their circulation. The Three groups demonstrated failure of pregnan- fetal pattern of circulation, in which blood is cy at an early stage in EP2-deficient mice.62)–64) shunted from the main pulmonary artery directly They found that EP2-deficient females consistently to the aorta via the ductus arteriosus, is thus deliver fewer pups than do their wild-type counter- transformed into the pulmonary circulation system parts irrespective of the genotypes of the mating of the neonate. This adaptive change is achieved by males. Ovulation was slightly impaired and the rate closure of the ductus arteriosus. Maternal admin- of fertilization was greatly reduced in the EP2- istration of NSAIDs induces contraction of the deficient female mice. Hizaki et al.62) further showed ductus in late-term fetuses, and administration of a that this phenotype is due to impaired expansion of vasodilatory PG such as PGE1 maintains the the cumulus oophorus. Given that EP2 and COX-2 patency of the ductus in neonates. The patency of are induced in the cumulus in response to gonado- the ductus during the fetal period has thus been tropins, and that PGE2 induces cumulus expansion thought to be maintained predominantly by the by increasing the intracellular concentration of dilatory effect of a PG, with its closure being cAMP, these researchers proposed that PGE2 and induced by withdrawal of the dilatory PG as well as EP2 form a positive feedback loop to induce the resulting from active contraction elicited by an oophorus maturation required for fertilization dur- increased oxygen tension. Dilatory prostanoid re- ing and after ovulation (Fig. 5A). Indeed, the ceptors such as IP and EP4 are present in the proportion of unovulated eggs in the ovary was ductus, suggesting that they function to maintain found to higher for EP2-deficient mice than for its patency. Disruption of the mouse IP gene, wild-type controls. however, did not appear to result in any abnormal- 53) PGF2 is recognized as an inducer of luteolysis ity of the ductus. On the other hand, most EP4- in domesticated animals such as sheep and cows, deficient mice die within 3 days after birth as a and it has been implicated in parturition through its result of marked pulmonary congestion and heart action as a potent uterotonic agent. However, FP- failure.66),67) Administration of indomethacin to deficient mice were found not to show any abnor- maternal mice during late pregnancy did not induce malities in early pregnancy or in the estrous closure of the ductus in EP4-deficient fetuses, 65) cycle. This apparent discrepancy might be ex- indicating that the dilatory effect of PGE2 on this plained by the facts that luteolysisisnotrequired vessel is mediated by EP4. These results thus for entrance into a new estrous cycle in mice, and indicate that EP4 plays an important role in the that mouse ovaries contain corpora lutea from ductus arteriosus, and they suggest that, in the Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 307

A B

Lutenizing hormone Unknown signal(s) (LH) COX-2

Cumulus EP2 cell PGF2α COX-2 FP PGE2 in corpus luteum

Cumulus Luteolysis expansion Oxytocin Oxytocin Parturition Fertilization receptor

Fig. 5. Roles and mechanisms of action of the PGE2-EP2 pathway in expansion of the cumulus oophorus and fertilization (A)and of the PGF2-FP pathway in induction of parturition (B).

absence of EP4, a compensatory mechanism main- comitant increase in urinary excretion of PGE2. tains ductus patency not only in the fetal period These observations indicated that PGE2 is pro- but also after birth. duced in the body in response to a high-salt diet and Prostanoid receptors and cardiovascular functions to reduce blood pressure via the relaxant homeostasis. EP2 receptor, suggesting that dysfunction of this Blood pressure. Most PGs exert contractile or pathway may contribute to the development of salt- relaxant effects (or both) on vascular smooth sensitive hypertension. muscle in vitro and in vivo.PGI2 and TXA2,which Hypertension is also induced by a reduction in are produced in large amounts by vascular endo- renal perfusion, which is associated with an increase thelial cells and platelets, respectively, exhibit in the plasma concentration of renin and mobiliza- potent vasodilatory and vasoconstricting actions, tion of the renin-angiotensin-aldosterone system. 70) respectively. PGE2 also induces either contraction Fujino et al. examined the contribution of pros- or relaxation of blood vessels dependent on the EP tanoids to the development of this condition by subtype expressed. Although mice deficient in IP or subjecting mice deficient in either IP or each of the TP do not show abnormalities of blood pressure four EP subtypes to a two-kidney, one-clip model of under basal conditions,53),68),69) the vasodilatory and renovascular hypertension. They found that hyper- vasocontractile actions of PGE2 become evident in tensioninthismodelwasamelioratedintheIP- mice deficient in EP subtypes. Kennedy et al.63) deficient mice but not in any of the EP-deficient administered PGE2 or PGE analogs intravenously animals. Consistent with these observations, the = to EP2 mice and found that PGE2 or a mixed plasma renin activity, the abundance of renin EP1/3 agonist, sulprostone, evoked pronounced mRNA in the kidney, and the plasma concentration hypertension, whereas an EP2 agonist, butaprost, of aldosterone were all substantially reduced in the failed to induce the transient hypotension seen in IP knockout animals compared with those in the wild-type mice. The researchers suggested that the wild type. Given that IP is expressed in the afferent absence of EP2 abolishes the ability of the mouse glomerular arterioles, that expression of the renin vasculature to undergo vasodilation in response to gene is expanded to these arterioles in response to PGE2 and unmasks the contractile response medi- reduced perfusion of the kidney, and that PGI2 ated by the vasoconstrictor EP subtype (or sub- induces renin release from cultured juxtaglomerular types). When fed a high-salt diet, the EP2-deficient cells in vitro, these researchers suggested that PGI2- mice developed marked hypertension with a con- IP signaling directly stimulates renin release. Al- 308 S. NARUMIYA [Vol. 83,

ternatively, such signaling may regulate the perfu- the effects of such injury. In contrast to PGI2, sion pressure of the juxtaglomerular apparatus TXA2 has been implicated in thrombosis and locally and induce renin release indirectly. hemostasis on the basis of its ability to induce Heart rate. Prostanoids have been thought not platelet aggregation and vasoconstriction. Indeed, to contribute to the regulation of heart rate, given TP-deficient mice showed increased bleeding ten- that ingestion of NSAIDs does not affect the heart dencies and were resistant to cardiovascular shock rate of animals under basal conditions. However, induced by intravenous infusion of arachidonic acid animals subjected to systemic inflammation such as or the TP agonist U-46619.68),69) Cheng et al.72) septic shock exhibit an increased heart rate, known examined the interplay between IP and TP signal- as inflammatory tachycardia, that is often sensitive ing in cardiovascular homeostasis. They subjected to NSAID treatment. Given that systemic inflam- IP= mice and TP= mice to vascular injury by a mation is associated with many symptoms and signs balloon catheter and found that IP deficiency of sickness behavior, including fever and ACTH increased, whereas TP deficiency decreased, in- release, in addition to tachycardia, and that fever is jury-induced vascular proliferation and platelet generated at least in part by elevated sympathetic activation. They further showed that the augment- discharge, inflammatory tachycardia has been ed response apparent in IP= mice was abolished thought to be induced by enhanced sympathetic by ablation of TP. The researchers thus concluded activity at the sinoatrial node. However, by sub- that PGI2, through its action at IP, modulates jecting prostanoid receptor-deficient mice to a platelet-vasculature interactions in vivo and specif- model of inflammatory tachycardia, Takayama et ically limits the response to TXA2. 71) al. found that EP3-deficient animals, which do In addition to PGI2 and TXA2,PGE2 acts on not develop fever, manifested a level of inflamma- blood platelets and promotes their aggregation. The tory tachycardia similar to that observed in wild- identity of the receptor mediating this action of type mice, thus ruling out a role for fever generation PGE2 and its pathophysiological significance had in this process. They further showed that prosta- remained unknown, however. By studying mice noids directly increased heart rate in a TP- and FP- deficient in each EP subtype, Fabre et al.73) and Ma dependent manner in vitro, and that the inflamma- et al.74) identified EP3 as a receptor that mediates tory tachycardia induced by either cytokines or potentiation of platelet aggregation by PGE2. LPSwassuppressedinmicedeficientinTPorFP EP3= mice thus exhibited a decreased response and was almost completely abolished in mice in an arachidonic acid–induced thrombosis model. deficient in both TP and FP. Moreover, injection In addition, Ma et al.74) found that bleeding time of the -blocker propranolol failed to prevent the was markedly prolonged in EP3 knockout mice. LPS-induced increase in heart rate under the same These observations suggest that this action of PGE2 conditions in wild-type mice. These results thus may play a role in vascular homeostasis in vivo. suggestthatprostanoidsare produced locally in the Atherosclerosis. Whereas PGI2 and TXA2,in heart during systemic inflammation and are able to addition to their roles in acute vascular accidents, induce tachycardia directly. have long been implicated in chronic vascular Bleeding, hemostasis, and thrombosis. Given diseases such as atherosclerosis and diabetic vas- that PGI2 and TXA2 have opposing effects on culopathy, the contribution of these prostanoids platelets and blood vessels, a balance between these was only recently assessed experimentally. We two prostanoids has been thought to be important addressed this issue by mating atherosclerosis- for prevention of thrombosis and vasospasm and for prone ApoE-deficient mice with mice deficient hemostasis. Indeed, a study of IP= mice53) showed in either IP or TP and then examining progression that, whereas these animals develop and age of atherosclerosis in the ApoE=IP= or normally, they manifest an increased thrombotic ApoE=TP= double knockouts.75) We found that tendency in the presence of endothelial damage. atherosclerosis was accelerated or delayed, respec- These findings confirmed the long-held view of PGI2 tively, in these mutant animals, despite the fact as an endogenous antithrombotic agent and sug- that they manifested similar plasma cholesterol and gested that this antithrombotic system is activated triglyceride concentrations. The lumen of the in- in response to vascular injury in order to attenuate nominate artery was almost completely occluded Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 309

= = in 45-week-old ApoE IP mice (Fig. 6). These ApoE-/-TP-/- ApoE-/- ApoE-/-IP-/- observations suggest the requirement for preserva- tion of vascular PGI2-IP signaling in maintenance of vascular homeostasis, and they underline the importance of selective lifelong manipulation of vascular IP and TP signaling. This view has been clinically confirmed in patients treated with selec- tive COX-2 inhibitors. Since COX-2 is rapidly induced by inflammatory cytokines and mitogens Fig. 6. Atherosclerosis in the innominate artery of ApoE=, and accounts largely for PGs produced in inflam- ApoE=TP=,andApoE=IP= mice (from ref. 75 with mation and cancer, and COX-1 was speculated to permission). Arterial cross-sections prepared from 45-week-old mice were stained with hematoxylin-eosin. function to produce protective PGs in gastric mucosa, selective COX-2 inhibitors have been developed as new types of NSAIDs with potent the initial activation of mast cells and subsequent anti-inflammatory activity and minimal gastroin- disease development. For example, PGD2 is a major testinal toxicity. Indeed, these drugs have been prostanoid produced by activated mast cells and is widely used, for example, in arthritis patients, released in large amounts during asthmatic attacks without gastrointestinal intolerance. However, a in certain patients. The role of PGD2 in allergic selective COX-2 inhibitor, rofecoxib, was recently asthma long remained unclear, however. We exam- withdrawn from the market because of an increased ined this issue by applying the ovalbumin (OVA)- rate of cardiovascular accidents in patients taking induced asthma model to DP-deficient mice.77) this drug. This cardiovascular hazard has been Sensitization and aerosol challenge of DP= mice suggested to be due to preferential inhibition by this with OVA induced increases in the serum concen- type of drug of COX-2 that is induced by shear tration of IgE similar to those observed in wild-type stress in the endothelium and mediates vascular mice. However, the DP-deficient animals developed PGI2 production combined with preservation of substantially reduced asthmatic responses in this 76) platelet COX-1 that catalyzes TXA2 production. model; the concentrations of Th2 cytokines and the Prostanoid receptors, allergy, and immuni- extents of lymphocyte accumulation and eosinophil ty Given that NSAIDs are mostly without effects infiltration in the lungs of the mutant animals after on allergy and immunity, it has been generally OVA challenge were greatly reduced compared with thought that prostanoids contribute little, if any, to those apparent in the wild type. These observations these processes, although substantial amounts of thus indicated that PGD2 is a mast cell–derived prostanoids are produced during their course. mediator that serves to mediate asthmatic respons- Analysis of the receptor knockout mice has re- es. This conclusion was supported recently by single vealed, however, that prostanoids function at var- nucleotide polymorphism (SNP) analysis of the DP ious steps in both allergy and immunity, and that gene (PTGDR) in humans. This gene is located different prostanoids often act antagonistically. at chromosome 14q22.1, a region that has been Allergy. The type I allergic reaction underlies associated with asthma and atopy. Oguma et al.78) the pathogenesis of bronchial asthma, atopic der- examined SNPs of PTGDR in Caucasian and black matitis, and anaphylactic shock. Affected individ- individuals with asthma and control subjects. They uals produce immunoglobulin (Ig) E antibodies to identified three haplotypes that consisted of four allergens such as those derived from house dust SNPs in the promoter region of the gene and found mites and plant pollen. Exposure to those allergens that each haplotype manifested a different promot- results in cross-linking of IgE receptors on the er activity. Furthermore, the promoter activity was surface of mast cells, the consequent activation of shown to be significantly correlated with suscepti- these cells, and the development of an allergic bility to asthma. reaction. The Th2 subset of T lymphocytes and PGD2 may also function in allergy by acting at their cytokines are important mediators of IgE the receptor CRTH2. CRTH2 is expressed in cells production as well as the development of allergic important in allergy such as Th2 lymphocytes, disease. Various prostanoids are produced during eosinophils, and basophils. Given that stimulation 310 S. NARUMIYA [Vol. 83,

of CRTH2 induces chemotaxis of these cells in vitro, reaction. These results thus implicated PGE2-EP3 it has been suggested that CRTH2 facilitates signaling in suppression of mast cell activation. allergic inflammation.16) Indeed, administration of Consistent with this conclusion, an EP3-selective selective agonists for CRTH2 to the airway or agonist was found to inhibit the antigen-induced painting of these substances on the skin of sensi- release of histamine and leukotrienes from sensi- tized animals was found to augment infiltration of tized lung tissue in vitro and to suppress airway inflammatory cells into the lungs and skin, respec- inflammation in OVA-challenged mice in vivo. tively.79),80) The generation of CRTH2 knockout With regard to the latter observation, the EP3 mice allowed further examination of the importance agonist was most effective when administered sub- of the PGD2-CRTH2 pathway in the natural course cutaneously 3 h after OVA challenge, indicating of allergy. Allergic inflammation associated with that the major site of EP3 action is at a step (or IgE-induced dermatitis was found to be suppressed steps) downstream of mast cell activation. Further in CRTH2-deficient mice,81) whereas airway inflam- analysis revealed that administration of the EP3 mation and eosinophil infiltration were augmented agonist suppressed induction of the expression of in the knockout mice subjected to the OVA-induced various asthma-related genes, including those for asthma model.82) The latter study also showed that chemokines and tissue remodeling factors, in the IL-5 production by activated T cells from CRTH2- lung, and that EP3 is coexpressed with some of deficient mice in vitro was increased compared with these molecules in the airway epithelium. On the that observed with wild-type cells. These results basis of these findings, we suggested that PGE2 indicated that CRTH2 indeed functions to facilitate produced during allergy acts at EP3 on both mast allergy in situ at the site of inflammation, but that cells and airway epithelial cells, thereby blunting this receptor also regulates cytokine production in activation of mast cells and impeding progression of the early phase of allergy development, raising the the allergic reaction by inducing down-regulation question as to whether suppression of this pathway of the expression of relevant genes in the airway would result in an overall beneficial effect in epithelium (Fig. 7). DP is also expressed in the patients. airway epithelium, and, given its opposing mecha- These various observations suggest that PGD2 nism of signal transduction relative to that of EP3, signaling facilitates allergic responses not only in it may facilitate the asthmatic reaction by increas- mice but also in humans. However, if PGD2 is the ing expression of these latter genes. only prostanoid that functions in allergic asthma, Application of the OVA-induced asthma model administration of NSAIDs would be expected to to mice deficient in other prostanoid receptors ameliorate asthmatic symptoms. Instead, NSAIDs revealed that airway inflammation was also aug- are either without effect or induce severe attacks in mented in IP-deficient mice. In contrast to EP3- certain asthmatic patients, a syndrome known as deficient mice, however, IP= mice exhibited aspirin-induced asthma.83) This discrepancy might substantially higher plasma concentrations of anti- be explained by the existence of a prostanoid other gen-specific and total IgE, indicating that PGI2-IP than PGD2 that negatively modulates allergic signaling functions in sensitization to IgE produc- reactions. The most likely candidate for such a tion.86) prostanoid is PGE2,giventhatPGE2 has been Immunity. Whereas prostanoid receptors such known for some time to exert antiallergy effects in as EP, IP, and TP are widely expressed in cells of some contexts.84) We subjected mice deficient in the immune system and the immunomodulatory each EP subtype individually to the OVA-induced actions of PGE2 in vitro were well established, little asthma model and examined their responses.85) was known until recently of the in vivo roles of these Among the four knockout mouse strains, only receptors in immunity. Kabashima et al.69),87) EP3-deficient mice exhibited substantially exagger- examined this issue with the contact hypersensitiv- ated airway inflammation compared with that ity model, in which they painted dinitrofluoroben- observed in their wild-type counterparts while zene (DNFB) on the skin of the mouse abdomen on showing similar plasma concentrations of anti- day 0 for immunization and applied the same OVA IgE. The EP3-deficient animals also mani- hapten to the ear on day 5 for elicitation of immune fested an enhanced passive cutaneous anaphylaxis inflammation, which was assayed by measurement Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 311

Allergens Airway mucosal epithelium

DP IgE PGD2

PGE2 EP3 B Lymphocyte EP3 Amplifi- Expression of cation Activation allergy-associated genes of mast cells

Th2 Eosinophil Early phase of allergic Th2-type cytokine response infiltration Bronchial constriction, Late Phase of secretion allergic response Airway remodeling, hypersensitivity

Fig. 7. A model for functional antagonism between the PGD2-DP pathway and the PGE2-EP3 pathway in development of allergic inflammationassociatedwithasthma.PGD2 is released in response to mast cell activation and facilitates allergic inflammation at least in part by acting at DP in the airway epithelium. On the other hand, PGE2 is produced in the surrounding tissues during mast cell activation and suppresses development of allergic inflammation by inhibiting induction of various asthma-related genes in the epithelium. of ear swelling the next day. They subjected mice TP signaling thus contribute to different steps of deficient in TP or EP4 to this model and examined immunization and functionally antagonize each their phenotypes. Whereas the EP4-deficient mice other with regard to their effects on T cell activa- 87) exhibited impaired hypersensitivity, the TP-de- tion. In addition, PGD2-DP signaling likely directly 69) ficient animals showed an exaggerated response. antagonizes the effect of PGE2-EP4 signaling in Application of antagonists specific for TP or EP4 in Langerhans cells. Trottein and collaborators stud- a phase-specific manner revealed that both PGE2- ied Schistosoma mansoni infection in the skin and EP4 signaling and TXA2-TP signaling function in found that parasite-derived PGD2 acts at DP in the immunization phase of this model; that is, in the Langerhans cells to induce their retention in the period between the initial hapten application and epidermis.88) This study was extended to show that the generation of effector T cells specific for the activation of DP interferes with TNF-–induced applied hapten. Detailed analysis demonstrated migration of Langerhans cells and migration of that PGE2-EP4 signaling facilitates mobilization, pulmonary dendritic cells in response to antigen migration, and maturation of Langerhans cells challenge.89) These prostanoid actions at different after initial antigen application and that TXA2- immunization steps are summarized in Fig. 8. TP signaling modulates the interaction between Prostanoid receptors and inflammation. na€ve T cells and antigen-loaded dendritic cells in Local reddening, heat generation, and swelling are draining lymph nodes and thereby regulates the classic signs of acute inflammation and are caused extent of T cell activation. PGE2-EP4 and TXA2- by increased blood flow and vascular permeability 312 S. NARUMIYA [Vol. 83,

Antigen

Inflammatory Uptake by LCs stimulus to Effector T cells keratinocytes LC mobilization, PGE2/ EP4 maturation, and PGs migration PGD2/ DP

LC conjugation TXA 2 with naïve T cells T cell differentiation Lymph node

Fig. 8. Sites of prostanoid actions in skin immune responses. The PGE2-EP4 pathway and the PGD2-DP pathway regulate dendritic cell migration and maturation in mutually antagonistic manners, whereas the TXA2-TP pathway regulates the interaction between dendritic cells and na€ve T cells in draining lymph nodes. LC, Langerhans cell. with resultant edema. The inhibitory effects of expression in relevant tissues. Examples of these NSAIDs on these inflammatory signs and the vaso- prostanoid actions include those observed in intes- dilatory action of exogenously administered pros- tinal inflammation and collagen-induced arthritis. tanoids such as PGI2 and PGE2 implicated prosta- Human inflammatory bowel disease (IBD), which noidsasmediatorsofthevascularresponsesin includes Crohn’s disease and ulcerative colitis, is a acute inflammation. Indeed, a study of IP-deficient chronic, relapsing, and remitting condition of un- mice subjected to the carrageenin-induced paw knownoriginthatexhibits various features of swelling model showed that inflammatory swelling immunological inflammation and affects at least was reduced by 50% in these animals, an effect one in 1000 people in Western countries.91) IBD is similar in magnitude to that achieved by treatment characterized by inflammation in the large or small of wild-type mice with NSAIDs, indicating that intestine associated with diarrhea, hemoccult, ab- PGI2 and IP constitute the principal PG system dominal pain, weight loss, anemia, and leukocytosis. responsible for mediating vascular changes in this Studies in humans have implicated impaired mu- model of inflammation.53) On the other hand, Yuhki cosal barrier function, pronounced innate immunity, et al.90) showed that EP2 and EP3 as well as IP production of proinflammatory and immunoregula- mediate exudate formation in carrageenin-induced tory cytokines, and the activation of CD4þ Tcells mouse pleurisy, suggesting that PGE2 and PGI2 in the pathogenesis of IBD. One of the major risk elicit inflammatory responses in a context-depend- factors for triggering or worsening the disease is ent manner, that is, one dependent on the type administration of NSAIDs,92) as confirmed by ex- of stimulus and site in the body, and that their periments in which COX-deficient mice were sub- contribution may also change during the course of jected to dextran sodium sulfate (DSS)-induced inflammation. intestinal inflammation, an animal model for IBD.93) The above knockout mouse studies thus sub- Whereas the evidence in humans and animals stantiated the role of prostanoids in acute inflam- indicated that COX-derived prostanoids contribute mation. However, more novel and important find- to the defense against intestinal inflammation, the ings obtained by studies using the knockout mice identity of the prostanoids and receptors involved in were that prostanoids exert both proinflammatory this process and their mode of actions remained and anti-inflammatory actions and that these ac- obscure. We therefore examined the susceptibility of tions are often produced through regulation of gene mice deficient in each of the eight types or subtypes Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 313

in prostanoid receptors to the DBA/1J background +/+ -/- A EP4 EP4 B EP4+/+ and subjected the resulting progeny to collagen- induced arthritis.95) Whereas the incidence of arthritis was unaffected, the extent and progression of this condition were markedly suppressed in IP- deficient mice as well as in mice both deficient in EP2 and treated with an EP4 antagonist. These findings thus indicated that PGI2-IP signaling and -/- EP4 PGE2 signaling at EP2 and EP4 mediate joint inflammation in this model. Further analysis re- vealed that both PGI2 and PGE2 pathways regulate expression of arthritis-related genes, including those for IL-6, vascular endothelial growth factor– A, and RANKL (see below), in synovial fibroblasts and thereby contribute to arthritic inflammation, Fig. 9. Exacerbation of DSS-induced colitis in EP4-deficient bone destruction, and pannus formation. Collagen- mice (from ref. 94 with permission). EP4= mice exhibit induced arthritis thus represents a condition in severe hemorrhagic colitis in response to the administration of which pro-inflammatory actions of prostanoids are 3% DSS in drinking water, which induces only marginal colitis elicited through expression of pro-inflammatory in wild-type mice. The gross appearance (A) and hematoxylin- eosin staining (B) of the intestine from wild-type (EP4þ=þ)or genes.Thesamereceptor,EP4,exertsananti- EP4= mice are shown. inflammatory action in intestinal inflammation and a proinflammatory action in arthritis, emphasizing of prostanoid receptors to DSS treatment.94) We the context-dependent roles of prostanoid signaling. found that only EP4-deficient mice developed severe Intestinal polyposis and colon cancer. A colitis in response to treatment with 3% DSS role for COX isoforms and their reaction products (Fig. 9); wild-type animals require treatment with in the development of colon cancer was first 7% DSS to develop this condition. We further suggested by epidemiological studies in humans confirmed this finding by reproducing the phenotype showing a reduced incidence of colon cancer in users in wild-type mice by administration of an EP4- of aspirin and other NSAIDs. It was subsequently selective antagonist. EP4 deficiency was shown to confirmed by pharmacological experiments with result in impairment of mucosal barrier function rodents administered with these drugs and finally that was associated with epithelial loss, crypt verified by experiments with transgenic mice and damage, and accumulation of neutrophils and two mouse models of human familial adenomatous CD4þ T cells in the colon. DNA microarray analysis polyposis. Both Min mice and Apc716 mice harbor revealed increased expression of genes associated truncation mutations in the APC gene and develop with immune responses and reduced expression of polyposis as a result of loss of heterozygosity in genes associated with mucosal repair and remodel- heterozygous mutants. Taketo and colleagues96) ing in the colon of EP4-deficient mice. On the basis showed that disruption of the COX-2 gene resulted of these findings, we concluded that EP4 maintains in marked decreases in both the number and size of intestinal homeostasis by preserving mucosal integ- intestinal polyps that develop in Apc716 mice. rity and down-regulating immune responses. Inhibition of polyp formation in Apc716 mice was Rheumatoid arthritis is a disease of immuno- also observed in response to treatment with a COX- logical inflammation that is widely treated with 2–selective inhibitor. This group further examined NSAIDs. Collagen-induced arthritis is an animal whichtypeofprostanoidorprostanoidreceptor model of rheumatoid arthritis in which immuniza- contributes to polyp formation by generating tion with type II collagen induces polyarthritis Apc716 mice in which the EP1, EP2, or EP3 gene within 2 to 4 weeks in susceptible animals such as is ablated.97) Homozygous deletion of the EP2 gene DBA/1J mice. This model is sensitive to NSAID in Apc716 mice resulted in substantial decreases in treatment, indicating that prostanoids participate the number and size of intestinal polyps, effects in its pathogenesis. We backcrossed mice deficient similar to those induced by COX-2 gene disruption. 314 S. NARUMIYA [Vol. 83,

Increased PGE2-EP2 signaling was found to in- implicating both COX-2 and PGE2 in this proc- crease COX-2 expression through an increase in the ess.101) Sakuma et al.102) and Miyaura et al.103) intracellular level of cAMP as well as to increase the examined the identity of the EP subtype respon- expression of vascular endothelial growth factor in sible for mediating this action of PGE2.Sakuma the polyp stroma. It remains to be determined, et al. found that PGE2-induced osteoclast formation however, whether these are the only prostanoid was impaired in cultures of osteoblasts from EP4- actions in colon carcinogenesis. deficient mice and osteoclast precursors from Wakabayashi and collaborators addressed this the spleen of wild-type mice. IL-1,TNF-,and question more directly by examining azoxymeth- basic fibroblast growth factor also each failed to ane-induced formation of aberrant cryptic foci in induce osteoclast formation in these cultures. mice deficient in each prostanoid receptor. They Miyaura et al. added PGE2 to cultures of parietal found that the formation of such foci was inhibited bone from mice deficient in each of the EP subtypes in both EP1= mice and EP4= mice but not in as well as from wild-type mice and examined bone those deficient in other receptor types or sub- resorption by measuring the release of Ca2þ into the types.98),99) In both instances, the number of foci medium. They found that the induction of bone was reduced to 50 to 60% of that apparent in wild- resorption by PGE2 was greatly impaired, whereas type mice. These researchers further found that bone resorption in response to dibutyryl cAMP was administration of either an EP1-specific antagonist, unaffected, in bone from EP4-deficient mice. These ONO-8711, or an EP4 antagonist, ONO-AE2-227, studies unequivocally established a role for EP4 in in the diet reduced the number and size of polyps in the induction of osteoclast differentiation factor and Min mice. They also examined whether EP1 in PGE2-dependent bone resorption. deficiency affects the development of colon cancer In contrast, Li et al.104) showed that the 100) induced by azoxymethane. They found that osteoclastogenic response to PGE2,PTH,or1,25- colon cancer incidence and multiplicity as well as dihydroxyvitamin D in vitro was impaired in tumor volume were reduced and that tumor cell cultures of cells derived from EP2-deficient mice. apoptosis was increased in EP1 knockout mice. This apparent discrepancy likely reflects redundant These results thus indicate that EP1 plays a key roles of the two relaxant PGE receptor subtypes. role in colon cancer development and is a potential Sakuma et al.102) and Miyaura et al.103) detected a target for chemoprevention of this condition. small but significant PGE2-dependent osteoclasto- Bone metabolism. Bone undergoes continu- genic response remaining in cells derived from EP4- ous destruction and renewal, a process termed bone deficient mice, and Li et al.104) observed a further remodeling, with bone resorption and formation decrease in osteoclastogenesis in response to the being mediated by osteoclasts and osteoblasts, addition of an EP4-selective antagonist to EP2- respectively. Such remodeling is controlled by deficient cells. Such redundant actions of EP2 and systemic humoral factors such as parathyroid EP4 were confirmed by Suzawa et al.,30) who hormone (PTH), estradiol, and vitamin D as well demonstrated both an additive effect of an EP2- as by local cytokines such as IL-1,IL-6,and selective agonist and an EP4-selective agonist in insulin-like growth factor. Osteoclasts develop from resorption of wild-type bone as well as bone- precursor cells of the macrophage lineage in the resorbing activity of an EP2 agonist in EP4= bone microenvironment. Factors such as PTH, bone. vitamin D, IL-1, and IL-6 act on osteoblasts to In addition to bone resorption, it has long been induce the synthesis of an osteoclast differentiation known that systemic administration of PGE2 in- factor known as receptor activator of NF-Bligand duces bone formation in vivo.Themechanismof (RANKL), which in turn stimulates the formation this action and its relation to the bone-resorbing of mature osteoclasts from hematopoietic precur- activity of this PG had remained obscure, however. 105) sors through cell-cell interaction. These factors Yoshida et al. infused PGE2 into the periosteal induce COX-2 expression in osteoblasts, and their regionofthefemurofwild-typemiceormice- induction of osteoclast differentiation is inhibited, deficient in each EP subtype with the use of a mini– at least in part, by aspirin-like drugs; such inhib- osmotic pump. After 6 weeks, the femur was ition was reversed by the addition of PGE2, isolated and bone formation was examined. Radio- Nos. 9–10] Physiology and pathophysiology of prostanoid receptors 315

in this article, the identification and cDNA cloning control rats Ovx rats of prostanoid receptors and the generation of corresponding knockout mice have provided an- swers to this long-standing question and defined a physiological mechanism for each known prostanoid action. Furthermore, studies with mice deficient in each of these receptors have revealed previously unknown functions of prostanoids that had not been predicted from the effects of aspirin-like drugs. Ovx rats administered ONO-4819 These newly discovered functions include actions in the brain, the immune system, and allergy. These low dose High dose studies have shown how this family of substances delicately regulates various processes in the body and its responses to environmental stimuli. Such knowledge will be exploited to provide insight into pathophysiology in humans and for the develop- ment of new therapeutics that selectively modulate each of these actions.

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Profile

Shuh Narumiya was born in 1949 and started his research career in 1971 with studies on poly-ADP ribosylation and poly-ADP-ribose glycohydrolase in the Department of Medical Chemistry of Professor Osamu Hayaishi, Kyoto University Faculty of Medicine. After studying tryptophan metabolism as a graduate student in the same Department, he spent two years as a postdoctoral fellow in the Wellcome Research Laboratories in the laboratory of Sir John Vane, studying lipoxygenases. He returned to Japan in 1981 and, since then, has been working in Kyoto University, being promoted to Professor in Pharmacology in 1992. During this period, he and his group first purified thromboxane A2 receptor from human blood platelets in 1989, then cloned cDNAs for the family of eight types and subtypes of protsanoid receptors, generated knockout mice deficient in each of these receptors individually and elucidated physiological and pathophysiological roles each receptor plays in the body. Their works stimulated a variety of research efforts to establish a research field for prostanoid receptors. Shuh Narumiya also discovered an ADP-ribosyl transferase in Clostridium botulinum now known as botulinum C3 exoenzyme, and identified small GTPase Rho as its target. C3 exoenzyme was then widely used to dissect functions of Rho protein and these studies revealed that Rho functions as a molecular switch in cell morphogenesis, adhesion and motility by inducing specific types of actin cytoskeleton. Shuh Narumiya then identified and cloned several effector proteins for Rho such as ROCK and mDia, and elucidated the pathway from Rho to the actin cytoskeleton. For these achievements, he was awarded the Osaka Science Prize in 1998, the Takeda Prize for Medicine in 1999, the Erwin von Baelz Prize in 1999, the Lorenzini Giovanni Gold Medal in 2000, the Uehara Prize in 2002, and the Imperial Prize and the Japan Academy Prize in 2006. Between 2005 and 2007, he was the Dean of Kyoto University Faculty of Medicine.