The expression of prostaglandin E receptors EP2 and EP4 and their different regulation by lipopolysaccharide in C3H/HeN peritoneal macrophages.

The expression and regulation of the PGE receptors, EP(2) and EP(4), both of which are coupled to the stimulation of adenylate cyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP(4) but not EP(2) was found in nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNA expression of EP(4) was down-regulated by LPS but not by medium change. PGE(2) increased the cAMP content of both LPS-treated and nontreated cells. ONO-604, an EP(4) agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPS for 3 h, but not for 6 h. Butaprost, an EP(2) agonist, was effective only in the cells treated with LPS for 6 h. The inhibitory effects of ONO-604 on TNF-alpha and IL-12 production were equipotent with PGE(2) at any time point, but the inhibitory effects of butaprost were only seen from 14 h after stimulation. PGE(2) or dibutyryl cAMP alone, but not butaprost, reduced EP(4) expression, and indomethacin reversed the LPS-induced down-regulation of EP(4), indicating that the down-regulation of EP(4) is mediated by LPS-induced PG synthesis and EP(4) activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in EP(4) expression was found in the cells treated with PGE(2) alone, but not in LPS-treated cells. In contrast, up-regulation of EP(2) expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP(4) is involved mainly in the inhibition of cytokine release, and that the gene expression of EP(2) and EP(4) is differentially regulated during macrophage activation.

[A study for functions of prostaglandin E receptor EP4 subtype by analysing knockout mice].

The physiological role of the prostaglandin (PG) E2 receptor EP4 subtype was investigated by the generation of EP4-deficient mice by gene targeting. Loss of the EP4 receptor was not lethal in utero, but most EP4 (-/-) neonates became pale and lethargic approximately 24 h after birth, and died within 72 h. Less than 5% of the EP4 (-/-) mice survived and grew normally more than a year. Marked congestion in the pulmonary capillaries were observed before death, suggesting that EP4 (-/-) neonates had left-sided heart failure. Histological examination revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization study showed that EP4 mRNA was strongly expressed in the ductus. The treatment of indomethacin, an inhibitor of PG synthesis, on wild-type fetus induced constriction of ductus arteriosus, while the ductus in EP4 (-/-) fetus was insensitive to indomethacin. These results suggest that neonatal death is at least partly due to patent ductus arteriosus, and that the EP4 receptor plays a role in the regulation of the patency of this vessel. They also indicate that the normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.

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 1alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), 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-1alpha, TNF-alpha, 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.

Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2).

Female mice lacking the gene encoding the prostaglandin (PG) E(2) receptor subtype EP(2) (EP(2)(-/-)) become pregnant and deliver their pups at term, but with a much reduced litter size. A decrease in ovulation number and a much reduced fertilization rate were observed in EP(2)(-/-) females without difference of the uterus to support implantation of wild-type embryos. Treatment with gonadotropins induced EP(2) mRNA expression in the cumulus cells of ovarian follicles of wild-type mice. The immature cumuli oophori from wild-type mice expanded in vitro in response to both follicle-stimulating hormone and PGE(2), but the response to PGE(2) was absent in those from EP(2)(-/-) mice. Cumulus expansion proceeded normally in preovulatory follicles but became abortive in a number of ovulated complexes in EP(2)(-/-) mice, indicating that EP(2) is involved in cumulus expansion in the oviduct in vivo. No difference in the fertilization rate between wild-type and EP(2)(-/-) mice was found in in vitro studies using cumulus-free oocytes. These results indicate that PGE(2) cooperates with gonadotropin to complete cumulus expansion for successful fertilization.

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)-1beta, 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-1beta or LPS. Our results establish that PGE2 mediates fever generation in response to both exogenous and endogenous pyrogens by acting at the EP3 receptor.

Characterization of the gene for the mouse prostaglandin E receptor subtype EP2: tissue-specific initiation of transcription in the macrophage and the uterus.

Genomic DNA clones for the mouse prostaglandin (PG) E receptor subtype EP2 were isolated and characterized. The mouse EP2 gene is composed of 2 exons and 1 intron, and spans 16 kb. The intron which is approx. 12 kb in length is located at the end of the sixth transmembrane domain, as with other prostanoid receptor genes. Based on this structure, transcripts were analysed in endotoxin-treated macrophages and pseudopregnant uteri, in which abundant expression of EP2 mRNA was observed. Sequence analysis of cDNA clones from these origins and Northern hybridization of these RNAs revealed that the uterine EP2 mRNA (U-type) has a longer 5'-untranslated region than the macrophage EP2 transcript (M-type). The major transcription initiation sites for M-type and U-type EP2 are located 124 and 769 bp upstream of the translation start site, respectively. The M-type was expressed in various tissues, whereas the U-type was found only in the uterus. The 2 kb segment containing the immediate 5'-flanking and 5'-noncoding regions contain three consensus sequences for the NF-IL6 binding site, one consensus sequence for the NF-kappaB binding site, four AP-2 consensus sequences, one AP-4 consensus sequence, one potential cAMP response element, and one potential progesterone response element. These results suggest that EP2 gene expression in the macrophage and uterus is under the control of distinct mechanisms involving alternative promoters.

Patent ductus arteriosus and neonatal death in prostaglandin receptor EP4-deficient mice.

The physiological role of the prostaglandin E2 receptor EP4 subtype was investigated by generation of EP4-deficient-mice by gene targeting. Loss of the EP4 receptor was not lethal in utero, but most EP4 (-/-) neonates became pale and lethargic approximately 24 h after birth and died within 72 h. Less than 5% of the EP4 (-/-) mice survived and grew normally more than a year. Histological examination revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization study showed that EP4 mRNA was strongly expressed in the ductus. These results suggest that neonatal death is at least partly due to patent ductus arteriosus and that the EP4 receptor plays a role in regulation of the patency of this vessel. They also indicate that normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.

Female reproduction in mice lacking the prostaglandin F receptor. Roles of prostaglandin and oxytocin receptors in parturition.

Female mice lacking the receptor for prostaglandin F2 alpha (FP) do not deliver fetuses at term, although these can be successfully rescued by cesarean section. No induction of oxytocin receptor mRNA is found in the uterus of these mice, and they show no uterine contraction on intravenous administration of oxytocin. Furthermore, a decline in serum progesterone levels during the periparturition period is not observed in these animals. Ovariectomy at day 19 of pregnancy restored induction of the oxytocin receptor and caused successful delivery in these animals. These results indicate not only the essential role of luteolytic PGF 2 alpha action in natural parturition but also the importance of oxytocin receptor induction in this process.

Characterization of the mouse prostaglandin F receptor gene: a transgenic mouse study of a regulatory region that controls its expression in the stomach and kidney but not in the ovary.

BACKGROUND: The actions of prostaglandin F2alpha are mediated by a cell-surface receptor (FP), but little is known about the regulation of FP gene expression. To clarify the mechanisms underlying tissue specific transcription of the mouse FP gene, we isolated and characterized mouse genomic DNA clones encoding FP. RESULTS: Structural analysis revealed that the mouse FP gene is composed of three exons and two introns, and spans more than 11 kilobases. By primer extension and PCR analyses, the major transcription start site was identified as a cytosine nucleotide, but additional sites of transcription initiation were found in the ovary. There was no apparent difference in the FP gene transcription initiation site between the ovary, kidney and stomach. Sequence analysis of the putative promoter region showed only two potential SP-1 binding sites, but no other typical well-known consensus sequences. We generated transgenic mice with the potential promoter region of the FP gene connected to the lacZ reporter gene. Northern blot analysis showed that the pattern of expression of the transgene corresponded to that of FP expression, except in the ovary. Upon analysis by in situ hybridization, the lacZ gene transcripts were found to be expressed in the fundic glands in the stomach, and the cortical tubules in the kidney, in which endogenous FP transcripts were also expressed. On the contrary, expression of lacZ transcripts was not detected in the corpora lutea, where the highest expression of FP mRNA was observed. CONCLUSIONS: These studies suggest that a separate control mechanism exists for FP expression in the ovary, distinct from the expression in the stomach and kidney.

Failure of parturition in mice lacking the prostaglandin F receptor.

Mice lacking the gene encoding the receptor for prostaglandin F2alpha (FP) developed normally but were unable to deliver normal fetuses at term. Although these FP-deficient mice showed no abnormality in the estrous cycle, ovulation, fertilization, or implantation, they did not respond to exogenous oxytocin because of the lack of induction of oxytocin receptor (a proposed triggering event in parturition), and they did not show the normal decline of serum progesterone concentrations that precedes parturition. Ovariectomy at day 19 of pregnancy restored induction of the oxytocin receptor and permitted successful delivery in the FP-deficient mice. These results indicate that parturition is initiated when prostaglandin F2alpha interacts with FP in ovarian luteal cells of the pregnant mice to induce luteolysis.

Mouse prostaglandin E receptor EP3 subtype mediates calcium signals via Gi in cDNA-transfected Chinese hamster ovary cells.

We recently cloned the mouse prostaglandin (PG) E receptor EP3 subtype that is coupled to adenylate cyclase inhibition through Gi and identified three isoforms which are produced through alternative splicing. In Chinese hamster ovary cells expressing each EP3 isoform, PGE2 induced an immediate increase in the intracellular Ca2+ concentration ([Ca2+]i) due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. This increase was abolished by prior treatment with pertussis toxin (PT). PGE2 also stimulated an accumulation of inositol trisphosphate (IP3) in a PT-sensitive manner. Both the PGE2-induced increase in [Ca2+]i and accumulation of IP3 were blocked by the phospholipase C inhibitor U-73122. Thus, EP3 is linked to phospholipase C activation via Gi, and this activation leads to Ca2+ mobilization from internal stores and influx from the extracellular medium.