Kazushi Morimoto

Prostaglandin E2–EP3 Signaling Induces Inflammatory Swelling by Mast Cell Activation

PGE2 has long been known as a potentiator of acute inflammation, but its mechanisms of action still remain to be defined. In this study, we employed inflammatory swelling induced in mice by arachidonate and PGE2 as models and dissected the role and mechanisms of action of each EP receptor at the molecular level. Arachidonate- or PGE2-induced vascular permeability was significantly reduced in EP3-deficient mice. Intriguingly, the PGE2-induced response was suppressed by histamine H1 antagonist treatment, histidine decarboxylase deficiency, and mast cell deficiency. The impaired PGE2-induced response in mast cell–deficient mice was rescued upon reconstitution with wild-type mast cells but not with EP3-deficient mast cells. Although the number of mast cells, protease activity, and histamine contents in ear tissues in EP3-deficient mice were comparable to those in wild-type mice, the histamine contents in ear tissues were attenuated upon PGE2 treatment in wild-type but not in EP3-deficient mice. Consistently, PGE2–EP3 signaling elicited histamine release in mouse peritoneal and bone marrow–derived mast cells, and it exerted degranulation and IL-6 production in a manner sensitive to pertussis toxin and a PI3K inhibitor and dependent on extracellular Ca2+ ions. These results demonstrate that PGE2 triggers mast cell activation via an EP3–Gi/o–Ca2+ influx/PI3K pathway, and this mechanism underlies PGE2-induced vascular permeability and consequent edema formation.

RhoA/Rho Kinase Signaling in the Cumulus Mediates Extracellular Matrix Assembly

Cumulus cells surround the oocyte and regulate the production and assembly of the extracellular matrix (ECM) around the cumulus-oocyte complex for its timely interaction with sperm in the oviduct. We recently found that C-C chemokines such as CCL2, CCL7, and CCL9 are produced and stimulate integrin-mediated ECM assembly in the postovulatory cumulus to protect eggs and that prostaglandin E(2)-EP2 signaling in the cumulus cells facilitates fertilization by suppressing this chemokine signaling, which otherwise results in fertilization failure by preventing sperm penetration through the cumulus ECM. However, it remains unknown as to what mechanisms underlie chemokine-induced cumulus ECM assembly. Here we report that inhibition of EP2 signaling or addition of CCL7 augments RhoA activation and induces the surface accumulation of integrin and the contraction of cumulus cells. Enhanced surface accumulation of integrin then stimulates the formation and assembly of fibronectin fibrils as well as induces cumulus ECM resistance to hyaluronidase and sperm penetration. These changes in the cumulus ECM as well as cell contraction are relieved by the addition of Y27632 or blebbistatin. These results suggest that chemokines induce integrin engagement to the ECM and consequent ECM remodeling through the RhoA/Rho kinase/actomyosin pathway, making the cumulus ECM barrier resistant to sperm penetration. Based on these results, we propose that prostaglandin E(2)-EP2 signaling negatively regulates chemokine-induced Rho/ROCK signaling in cumulus cells for successful fertilization.

Prostaglandin E2-EP4 signaling suppresses adipocyte differentiation in mouse embryonic fibroblasts via an autocrine mechanism

The prostaglandin (PG) receptors EP4 and FP have the potential to exert negative effects on adipogenesis, but the exact contribution of endogenous PG-driven receptor signaling to this process is not fully understood. In this study, we employed an adipocyte differentiation system from mouse embryonic fibroblasts (MEF) and compared the effects of each PG receptor-deficiency on adipocyte differentiation. In wild-type (WT) MEF cells, inhibition of endogenous PG synthesis by indomethacin augmented the differentiation, whereas exogenous PGE2, as well as an FP agonist, reversed the effect of indomethacin. In EP4-deficient cells, basal differentiation was upregulated to the levels in indomethacin-treated WT cells, and indomethacin did not further enhance differentiation. Differentiation in FP-deficient cells was equivalent to WT and was still sensitive to indomethacin. PGE2 or indomethacin treatment of WT MEF cells for the first two days was enough to suppress or enhance transcription of the Pparg2 gene as well as the subsequent differentiation, respectively. Differentiation stimuli induced COX-2 gene and protein expression, as well as PGE2 production, in WT MEF cells. These results suggest that PGE2-EP4 signaling suppresses adipocyte differentiation by affecting Pparg2 expression in an autocrine manner and that FP-mediated inhibition is not directly involved in adipocyte differentiation in the MEF system.

Expression profiling of cumulus cells reveals functional changes during ovulation and central roles of prostaglandin EP2 receptor in cAMP signaling

To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2(-/-) cumuli before and after ovulation by using microarrays. We prepared cumulus cells from mice just before and 3, 9 and 14 h after human chorionic gonadotropin injection. Key genes including cAMP-related and epidermal growth factor (EGF) genes, as well as extracellular matrix- (ECM-) related and chemokine genes were up-regulated in WT cumuli at 3 h and 14 h, respectively. Ptger2 deficiency differently affected the expression of many of the key genes at 3 h and 14 h. These results indicate that the gene expression profile of cumulus cells greatly differs before and after ovulation, and in each situation, PGE(2)-EP2 signaling plays a critical role in cAMP-regulated gene expression in the cumulus cells under physiological conditions.

Involvement of CD44 in mast cell proliferation during terminal differentiation

By using the recently established culture system that reproduces the terminal differentiation process of connective tissue-type mast cells, we found significant transcriptional induction of CD44. As CD44 is a primary receptor for hyaluronan (HA), which is one of the major extracellular matrix components, we investigated the role of CD44 in cutaneous mast cells. When co-cultured with fibroblasts, mouse bone marrow-derived cultured mast cells (BMMCs) were found to form clusters in an HA-dependent manner. As compared with BMMCs derived from the wild-type mice, those from the CD44−/− mice exhibited impaired growth during the co-cultured period. Furthermore, in the peritoneal cavities and ear tissues, mature mast cells were fewer in number in the CD44−/− mice than in the wild-type mice. We investigated roles of CD44 in mast cell proliferation by reconstituting BMMCs into the tissues of mast cell-deficient, KitW/KitW-v mice, and found that the number of metachromatic cells upon acidic toluidine blue staining in the tissues transplanted with CD44−/− BMMCs was not significantly changed for 10 weeks, whereas that in the tissues transplanted with the CD44+/+ BMMCs was significantly increased. These results suggest that CD44 plays a crucial role in the regulation of the cutaneous mast cell number.

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the Gq/PLC/Ca2+ pathway in a lipid raft-dependent manner.

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G(i/o)-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca(2+)-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP(3) formation and intracellular Ca(2+) mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca(2+) ionophore and the activation of a typical G(q)-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G(q)/phospholipase C activation and intracellular Ca(2+) mobilization in a lipid raft microdomain-dependent manner.

Hot-Spot Residues to be Mutated Common in G Protein-Coupled Receptors of Class A: Identification of Thermostabilizing Mutations Followed by Determination of Three-Dimensional Structures for Two Example Receptors

G protein-coupled receptors (GPCRs), which are indispensable to life and also implicated in a number of diseases, construct important drug targets. For the efficient structure-guided drug design, however, their structural stabilities must be enhanced. An amino-acid mutation is known to possibly lead to the enhancement, but currently available experimental and theoretical methods for identifying stabilizing mutations suffer such drawbacks as the incapability of exploring the whole mutational space with minor effort and the unambiguous physical origin of the enhanced or lowered stability. In general, after the identification is successfully made for a GPCR, the whole procedure must be followed all over again for the identification for another GPCR. Here we report a theoretical strategy by which many different GPCRs can be considered at the same time. The strategy is illustrated for three GPCRs of Class A in the inactive state. We argue that a mutation of the residue at a position of NBW = 3.39 (NBW is the Ballesteros-Weinstein number), a hot-spot residue, leads to substantially higher stability for significantly many GPCRs of Class A in the inactive state. The most stabilizing mutations of the residues with NBW = 3.39 are then identified for two of the three GPCRs, using the improved version of our free-energy function. These identifications are experimentally corroborated, which is followed by the determination of new three-dimensional (3D) structures for the two GPCRs. We expect that on the basis of the strategy, the 3D structures of many GPCRs of Class A can be solved for the first time in succession.

Expanding the synthesizable multisubstituted benzo[b]thiophenes via 6,7-thienobenzynes generated from o-silylaryl triflate-type precursors

Various 2,3-disubstituted 6,7-thienobenzynes have been efficiently generated from the corresponding o-silylaryl triflate-type precursors by activation with fluoride ions. The method has expanded the scope of synthesizable multisubstituted benzothiophenes, including those with various heteroatom substituents, and can be applied to the synthesis of EP4 antagonist analogs.