Fumitaka Ushikubi

Impaired febrile response in mice lacking the prostaglandin E receptor subtype EP3

Fever, a hallmark of disease, is elicited by exogenous pyrogens, that is, cellular components, such as lipopolysaccharide (LPS), of infectious organisms, as well as by non-infectious inflammatory insults. Both stimulate the production of cytokines, such as interleukin (IL)-1β, that act on the brain as endogenous pyrogens. Fever can be suppressed by aspirin-like anti-inflammatory drugs. As these drugs share the ability to inhibit prostaglandin biosynthesis, it is thought that a prostaglandin is important in fever generation. Prostaglandin E2 (PGE2) may be a neural mediator of fever, but this has been much debated,. PGE2 acts by interacting with four subtypes of PGE receptor, the EP1, EP2, EP3 and EP4 receptors. Here we generate mice lacking each of these receptors by homologous recombination. Only mice lacking the EP3 receptor fail to show a febrile response to PGE2 and to either IL-1β or LPS. Our results establish that PGE2 mediates fever generation in response to both exogenous and endogenous pyrogens by acting at the EP3 receptor.

Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc Δ716 knockout mice

Arachidonic acid is metabolized to prostaglandin H2 (PGH2) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH2, PGE2 is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE2, EP2, causes decreases in number and size of intestinal polyps in ApcΔ716 mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE2 through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE2 receptors, EP1 or EP3, did not affect intestinal polyp formation in ApcΔ716 mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.

Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin (PG)E2 promotes both bone resorption and bone formation. By infusing PGE2 to mice lacking each of four PGE receptor (EP) subtypes, we have identified EP4 as the receptor that mediates bone formation in response to this agent. Consistently, bone formation was induced in wild-type mice by infusion of an EP4-selective agonist and not agonists specific for other EP subtypes. In culture of bone marrow cells from wild-type mice, PGE2 induced expression of core-binding factor α1 (Runx2/Cbfa1) and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggest that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

Two thromboxane A2 receptor isoforms in human platelets: Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation

Thromboxane A2 (TXA2) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR alpha and TXR beta, respectively. These isoforms differ only in their carboxyl-terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR alpha activates adenylyl cyclase, while TXR beta inhibits it. The Arg60 to Leu mutant of TXR alpha, which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR beta retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA2-induced platelet responses such as shape change and phospholipase A2 activation which remain unaffected in the patients with this mutation.

In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs.

1 Expression of prostacyclin receptor (IP receptor) mRNA was examined in various mouse organs, and the cells expressing IP receptor mRNA were identified by in situ hybridization studies. Co‐localization of mRNA for the IP receptor with that for preprotachykinin A (PPTA), a precursor protein for substance P, with mRNA for the prostaglandin E receptor subtypes (EP1, EP3 and EP4), and with renin mRNA, was examined by double in situ hybridization studies in the dorsal root ganglion and kidney, respectively. 2 IP receptor mRNA was expressed in the thymus and spleen. Expression in the thymus was found exclusively in the medulla, where mature thymocytes expressed transcripts for the IP receptor. Expression in the spleen was found as scattered signals over the white pulp and as punctate signals in the red pulp. The former was found in splenic lymphocytes and the latter in megakaryocytes. 3 IP receptor mRNA was also expressed in the vascular tissues of various organs such as the aorta, coronary arteries, pulmonary arteries and the cerebral arteries, where its expression was confined to smooth muscle cells. No expression was found in veins. In the kidney, IP receptor mRNA was detected in the interlobular arteries and glomerular arterioles but not in the juxtaglomerular (JG) cells which were labelled with the renin mRNA probe. 4 IP receptor mRNA was expressed in about 40% of the neurones in the dorsal root ganglion. Both small‐ and large‐sized neurones were labelled but no labelling was found in the glia. Expression of PPT A mRNA was found in about 30% of total neurones. About 70% of these neurones expressed IP receptor mRNA, and about half of the IP receptor‐positive neurones expressed PPTA mRNA. In addition to IP mRNA, mRNAs for EP1, EP3 and EP4 receptors were expressed in about 30%, 50% and 20%, respectively, of the dorsal root ganglion neurones. About 25%, 41% and 24% of the IP receptor‐positive neurones co‐expressed the EP1, EP3 and EP4 receptor, respectively. 5 These results not only verified IP receptor expression in various cells and tissues known to be sensitive to prostacyclin, but also revealed its expression in other systems, which urges the study of the actions of prostacyclin in these tissues. They also indicated that the actions of prostacyclin on blood vessels and platelets are mediated by the same type of receptor. Absence of IP receptor mRNA in the JG cells suggests that the action of prostacyclin on renin release may be indirect.

Characterization of EP receptor subtypes responsible for prostaglandin E2‐induced pain responses by use of EP1 and EP3 receptor knockout mice

Prostaglandin E2 (PGE2) is known to be the principal pro‐inflammatory prostanoid and play an important role in nociception. To identify PGE receptor (EP) subtypes that mediate pain responses to noxious and innocuous stimuli, we studied them by use of EP1 and EP3 knockout (EP1−/− and EP3−/−) mice. PGE2 could induce mechanical allodynia in EP1+/+, EP3+/+ and EP3−/− mice, but not in EP1−/− mice. N‐methyl‐D‐aspartate (NMDA), the substrate of nitric oxide (NO) synthase L‐arginine, or the NO donor sodium nitroprusside administered intrathecal (i.t.) could induce allodynia in EP3−/− and EP1−/− mice. Activation of EP1 receptors appears to be upstream, rather than downstream, of NMDA receptor activation and NO production in the PGE2‐induced allodynia. Although PGE2 produced thermal hyperalgesia over a wide range of dosages from 50 pg to 0.5 μg kg−1 in EP3+/+ mice, it showed a monophasic hyperalgesic action at 5 ng kg−1 or higher doses in EP3−/− mice. The selective EP3 agonist, ONO‐AE‐248, induced hyperalgesia at 500 pg kg−1 in EP3+/+ mice, but not in EP3−/− mice. Saline‐injected EP1−/− mice showed hyperalgesia, which was reversed by i.t. PGE2 in a dose‐dependent manner. There was no significant difference in the formalin‐induced behaviours between EP1−/− or EP3−/− mice and the cognate wild‐type mice. These results demonstrate that spinal EP1 receptors are involved in the PGE2‐induced allodynia and that spinal EP3 receptors are involved in the hyperalgesia induced by low doses of PGE2. However, the formalin‐induced pain cannot be ascribed to a single EP receptor subtype EP1 or EP3.

Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder.

Recent advances in molecular genetics have revealed the mechanisms underlying a variety of inherited human disorders. Among them, mutations in G protein-coupled receptors have clearly demonstrated two types of abnormalities, namely loss of function and constitutive activation of the receptors. Thromboxane A2 (TXA2) receptor is a member of the family of G protein-coupled receptors and performs an essential role in hemostasis by interacting with TXA2 to induce platelet aggregation. Here we identify a single amino acid substitution (Arg60-->Leu) in the first cytoplasmic loop of the TXA2 receptor in a dominantly inherited bleeding disorder characterized by defective platelet response to TXA2. This mutation was found exclusively in affected members of two unrelated families with the disorder. The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. These results suggest that the Arg60 to Leu mutation is responsible for the disorder. Moreover, dominant inheritance of the disorder suggests the possibility that the mutation produces a dominant negative TXA2 receptor.

Regulation of TNFα and interleukin-10 production by prostaglandins I2 and E2: studies with prostaglandin receptor-deficient mice and prostaglandin E-receptor subtype-selective synthetic agonists

To know which receptors of prostaglandins are involved in the regulation of TNFalpha and interleukin 10 (IL-10) production, we examined the production of these cytokines in murine peritoneal macrophages stimulated with zymosan. The presence of PGE(2) or the PGI(2) analog carbacyclin in the medium reduced the TNFalpha production to one-half, whereas IL-10 production increased several fold; and indomethacin caused the reverse effects, suggesting that endogenous prostaglandins may have a regulatory effect on the cytokine production. Among prostaglandin E (EP) receptor-selective synthetic agonists, EP2 and EP4 agonists caused down-regulation of the zymosan-induced TNFalpha production, but up-regulation on the IL-10 production; while EP1 and EP3 agonists showed no effect. Macrophages harvested from prostaglandin I (IP) receptor-deficient mice showed the up- and down-regulatory effects on the cytokine production by the EP2 and EP4 agonists or PGE(2), but no effect was obtained by carbacyclin. On the contrary, macrophages from EP2-deficient mice showed the effect by PGE(2), carbacyclin, and the EP4 agonist, but not by the EP2 agonist; and the cells from EP4-deficient mice showed the effect by PGE(2), carbacyclin, and EP2 agonist, but not by the EP4 agonist. These functional effects of prostaglandins well accorded with the mRNA expression of TNFalpha and IL-10 when such expression was examined by the RT-PCR method. The peritoneal macrophages from normal mice expressed IP, EP2, and EP4 receptors, but not EP1 and EP3, when examined by RT-PCR. Thus the results suggest that PGI(2) and PGE(2) generated simultaneously with cytokines by macrophages treated with zymosan may influence the cytokine production through IP, EP2, and EP4 receptors.

Thromboxane A2 modulates interaction of dendritic cells and T cells and regulates acquired immunity

Physical interaction of T cells and dendritic cells (DCs) is essential for T cell proliferation and differentiation, but it has been unclear how this interaction is regulated physiologically. Here we show that DCs produce thromboxane A2 (TXA2), whereas naive T cells express the thromboxane receptor (TP). In vitro, a TP agonist enhances random cell movement (chemokinesis) of naive but not memory T cells, impairs DC–T cell adhesion, and inhibits DC-dependent proliferation of T cells. In vivo, immune responses to foreign antigens are enhanced in TP-deficient mice, which also develop marked lymphadenopathy with age. Similar immune responses were seen in wild-type mice treated with a TP antagonist during the sensitization period. Thus, TXA2-TP signaling modulates acquired immunity by negatively regulating DC–T cell interactions.

Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide

Prostaglandin E2 (PGE2) exerts its effects through the PGE receptor that consists of four subtypes (EP1, EP2, EP3, and EP4). Osteoclast formation in the coculture of primary osteoblastic cells (POB) and bone marrow cells was enhanced more by 11‐deoxy‐PGE1 (an EP4 and EP2 agonist) than by butaprost (an EP2 agonist) and other agonists, which suggests that EP4 is the main factor in PGE2‐induced osteoclast formation. PGE2‐induced osteoclast formation was not observed in the coculture of POB from EP4‐deficient (EP4 k/o) mice and spleen cells from wild‐type (w/t) mice, whereas osteoclasts were formed in the coculture of POB from w/t mice and spleen cells from EP4‐k/o mice. In situ hybridization (ISH) showed that EP4 messenger RNA (mRNA) was expressed on osteoblastic cells but not on multinucleated cells (MNCs) in w/t mice. These results indicate that PGE2 enhances osteoclast formation through its EP4 subtype on osteoblasts. Osteoclast formation by interleukin 1α (IL‐1α), tumor necrosis factor α (TNF‐α), basic fibroblast growth factor (bFGF), and lipopolysaccharide (LPS) was hardly observed in the coculture of POB and bone marrow cells, both from EP4‐k/o mice, which shows the crucial involvement of PG and the EP4 subtype in osteoclast formation by these molecules. In contrast, osteoclast formation by 1,25‐hydroxyvitamin D3 (1,25(OH)2D3) was not impaired and that by parathyroid hormone (PTH) was only partially impaired in EP4‐k/o mice, which may be related to the fact that EP4‐k/o mice revealed no gross skeletal abnormalities. Because it has been suggested that IL‐1α, TNF‐α, bFGF, and LPS are involved in inflammatory bone loss, our work can be expected to contribute to an understanding of the pathophysiology of these conditions.(J Bone Miner Res 2000;15:218–227)