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Shuh Narumiya - One of the best experts on this subject based on the ideXlab platform.
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Thromboxane A2 Receptor signaling in endothelial cells attenuates monocrotaline induced liver injury
Toxicology and Applied Pharmacology, 2019Co-Authors: Fumisato Otaka, Shuh Narumiya, Yoshiya Ito, Tomoyoshi Inoue, Hirotoki Ohkubo, Nobuyuki Nishizawa, Ken Kojo, Tomohiro Betto, Sakiko Yamane, Wasaburo KoizumiAbstract:Sinusoidal obstruction syndrome (SOS) is a major complication of chemotherapy and hematopoietic stem cell transplantation. The early stage of SOS is characterized by liver sinusoidal endothelial cell (LSEC) injury accompanied by platelet aggregation. Thromboxane A2 (TxA2) induces platelet aggregation through the Thromboxane prostanoid (TP) Receptor. In this study, we explored the role of TP signaling in a monocrotaline (MCT)-induced mouse model of SOS. Relative to wild-type (WT) mice, TP-deficient (TP-/-) mice exhibited more severe MCT-liver injury, as indicated by elevated levels of alanine aminotransferase (ALT) and coagulative necrosis. Extensive accumulation of platelets in the liver was observed in both WT and TP-/- mice. TP expression co-localized with CD31-positive LSECs. MCT treatment caused LSEC destruction, concomitant with elevated expression of matrix metalloproteinases (MMPs) and adhesion molecules in WT mice, and LSEC damage was further exacerbated in TP-/- mice. Viability of isolated LSECs was lower in cells from TP-/- mice, whereas mRNA levels of MMPs and adhesion molecules were higher; U46619, a TxA2 agonist, reduced these levels in WT mice. These data suggest that TP signaling has no effect on platelet accumulation during MCT-induced liver injury, but instead prevents injury by suppressing LSEC damage.
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adhesion of platelets through Thromboxane A2 Receptor signaling facilitates liver repair during acute chemical induced hepatotoxicity
Life Sciences, 2015Co-Authors: Tsutomu Minamino, Shuh Narumiya, Yoshiya Ito, Hirotoki Ohkubo, Ken Kojo, Wasaburo Koizumi, Yuki Shimuzu, Nobuyuki Nishizwa, Hideki Amano, Masataka MajimaAbstract:Abstract Aims Platelets have been suggested to play an important role in liver regeneration and repair after hepatic resection and acute liver injury. However, the underlying mechanisms of liver repair remain elusive. Signaling through Thromboxane prostanoid (TP) Receptor participates in inflammation and tissue injury through platelet aggregation. On the other hand, TP Receptor signaling also is involved in tissue repair and tumor growth through angiogenesis. The present study was examined whether or not TP Receptor signaling contributes to liver repair and sinusoidal restoration from acute liver injury through platelet adhesion to the hepatic sinusoids. Main methods Carbon tetrachrolide (CCl 4 ) was used to induce acute liver injury in TP Receptor knockout mice (TP −/− mice) and their wild-type littermates (WT mice). Key findings Compared with WT mice, TP −/− mice exhibited delayed in liver repair and sinusoidal restoration after CCl 4 treatment, which were associated with attenuated hepatic expression of pro-angiogenic factors. Intravital microscopic observation revealed that adhering platelets to the sinusoids was increased in WT livers during the repair phase as compared with TP −/− livers, and platelet adhesion was dependent on TP Receptor signaling. The levels of hepatocyte growth factor (HGF) in platelets from WT mice treated with CCl 4 for 48 h were greater than those form TP −/− mice, and HGF enhanced the expression of angiogenic factors in cultured human umbilical vein endothelial cells (HUVECs). Significance These results suggested that TP Receptor signaling facilitates liver repair and sinusoidal restoration from acute liver injury through HGF release from platelets adhering to the sinusoids.
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effects of salt loading on blood pressure in mice lacking the prostanoid Receptor gene
Circulation, 2005Co-Authors: Hidetsuna Watanabe, Fumitaka Ushikubi, Shuh Narumiya, Tetsuo Katoh, Masaaki Eiro, Masaya Iwamoto, Tsuyoshi WatanabeAbstract:Background This study examined whether targeted disruption of the genes for the prostacyclin Receptor (IP) or the Thromboxane A2 Receptor (TP) confers a susceptibility to salt-dependent hypertension. Methods and Results Eight female IP- or TP-deficient mice were examined. Baseline systolic blood pressure (SBP) did not differ between TP(-/-) and TP(+/+), but was significantly lower in the IP(-/-) group than in the IP(+/+). With a high salt diet, SBP in IP(-/-) gradually increased. In contrast, SBP in the IP(+/+), TP(-/-), or TP(+/+) groups remained unchanged. Conclusions The prostacyclin Receptor may participate in the maintenance of baseline BP. With salt loading, BP adaptation may take place, at least in part, via IP mediated signals. (Circ J 2005; 69: 124 -126)
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arg60 to leu mutation of the human Thromboxane A2 Receptor in a dominantly inherited bleeding disorder
Journal of Clinical Investigation, 1994Co-Authors: Takako Hirata, Akira Kakizuka, Minoru Okuma, Ichiro Fuse, Fumitaka Ushikubi, Shuh NarumiyaAbstract: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 ofG 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 (Arg4-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 Arg'0 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. (J. Clin. Invest.
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arg60 to leu mutation of the human Thromboxane A2 Receptor in a dominantly inherited bleeding disorder
Journal of Clinical Investigation, 1994Co-Authors: Takako Hirata, Akira Kakizuka, Minoru Okuma, Ichiro Fuse, Fumitaka Ushikubi, Shuh NarumiyaAbstract:Abstract 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.
Hsinhsiung Tai - One of the best experts on this subject based on the ideXlab platform.
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Thromboxane A2 Receptor mediated release of matrix metalloproteinase 1 mmp 1 induces expression of monocyte chemoattractant protein 1 mcp 1 by activation of protease activated Receptor 2 par2 in a549 human lung adenocarcinoma cells
Molecular Carcinogenesis, 2014Co-Authors: Hsinhsiung TaiAbstract:Matrix metalloproteinases (MMPs) and monocyte chemoattractant protein-1 (MCP-1, CCL2) are known to be upregulated in many tumors. Their roles in tumor invasion and metastasis are being uncovered. How they are related to each other and involved in tumor progression remains to be determined. Earlier it was reported that I-BOP-initiated activation of Thromboxane A2 Receptor (TP) induced the release of MMP-1, MMP-3, and MMP-9 from lung cancer A549 cells overexpressing TPα (A549-TPα). Herein it was found that MMP-1, but not MMP-3 or MMP-9, induced the expression of MCP-1 in A549 cells. Conditioned medium (CM) from I-BOP activated, MMP-1 siRNA pretreated A549-TPα cells induced greatly attenuated expression of MCP-1 in A549 cells indicating that MMP-1 in the CM contributed significantly to the expression of MCP-1. MMP-1 was shown to activate protease-activated Receptor 2 (PAR2) instead of commonly assumed PAR1 to increase the expression of MCP-1 in A549 cells. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, and also PAR2 agonist peptide, was inhibited by a PAR2 antagonist; (2) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was attenuated significantly by pretreatment of cells with PAR2-siRNA. These results suggest that PAR2 is a novel MMP-1 target mediating MMP-1-induced signals in A549 lung cancer cells.
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activation of Thromboxane A2 Receptor tp increases the expression of monocyte chemoattractant protein 1 mcp 1 chemokine c c motif ligand 2 ccl2 and recruits macrophages to promote invasion of lung cancer cells
PLOS ONE, 2013Co-Authors: Hsinhsiung TaiAbstract:Thromboxane synthase (TXAS) and Thromboxane A2 Receptor (TP), two critical components for Thromboxane A2 (TXA2) signaling, have been suggested to be involved in cancer invasion and metastasis. However, the mechanisms by which TXA2 promotes these processes are still unclear. Here we show that TXA2 mimetic, I-BOP, induced monocyte chemoattractant protein -1(MCP-1)/chemokine (C-C motif) ligand 2 (CCL2) expression at both mRNA and protein levels in human lung adenocarcinoma A549 cells stably over-expressing TP Receptor α isoform (A549-TPα). The induction of MCP-1 was also found in other lung cancer cells H157 and H460 that express relatively high levels of endogenous TP. Using specific inhibitors of several signaling molecules and promoter/luciferase assay, we identified that transcription factor SP1 mediates I-BOP-induced MCP-1 expression. Furthermore, supernatants from I-BOP-treated A549-TPα cells enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Moreover, co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. These findings suggest that TXA2 may stimulate invasion of cancer cells through MCP-1-mediated macrophage recruitment.
Therese B Kinsella - One of the best experts on this subject based on the ideXlab platform.
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the α but not the β isoform of the human Thromboxane A2 Receptor is a target for nitric oxide mediated desensitization independent modulation of tpα signaling by nitric oxide and prostacyclin
Journal of Biological Chemistry, 2016Co-Authors: Helen M Reid, Therese B KinsellaAbstract:In humans, Thromboxane A2 signals through two Thromboxane A2 Receptor (TP) isoforms termed TPα and TPβ. Signaling by TPα, but not TPβ, is subject to prostacyclin-induced desensitization mediated by direct protein kinase (PK) A phosphorylation where Ser329 represents the phosphotarget (Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412-20423). In the current study, the effect of the vasodilator nitric oxide (NO) on intracellular signaling by the TP isoforms was investigated. The NO donor 3-morpholinosydnonimine, HCl (SIN-1) and 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) functionally desensitized U46619-mediated calcium mobilization and inositol 1,4,5-trisphosphate generation by TPα whereas signaling by TPβ was unaffected by either agent. NO-mediated desensitization of TPα signaling occurred through a PKG-dependent, PKA- and PKC-independent mechanism. TPα, but not TPβ, was efficiently phosphorylated by PKG in vitro and underwent NO/PKG-mediated phosphorylation in whole cells. Deletion/site-directed mutagenesis and metabolic labeling studies identified Ser331 as the target residue of NO-induced PKG phosphorylation of TPα. Although TPαS331A was insensitive to NO/PKG-desensitization, similar to wild type TPα its signaling was fully desensitized by the prostacyclin Receptor agonist cicaprost occurring through a PKA-dependent mechanism. Conversely, signaling by TPαS329A was insensitive to cicaprost stimulation whereas it was fully desensitized by NO/PKG signaling. In conclusion, TPα undergoes both NO- and prostacyclin-mediated desensitization that occur through entirely independent mechanisms involving direct PKG phosphorylation of Ser331, in response to NO, and PKA phosphorylation of Ser329, in response to prostacyclin, within the unique carboxyl-terminal tail domain of TPα. On the other hand, signaling by TPβ is unaffected by either NO or prostacyclin.
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the wilms tumour suppressor protein wt1 acts as a key transcriptional repressor of the human Thromboxane A2 Receptor gene in megakaryocytes
Journal of Cellular and Molecular Medicine, 2009Co-Authors: Annemarie M Gannon, Therese B KinsellaAbstract:In humans, the TPα and TPβ isoforms of the Thromboxane A2 Receptor are transcriptionally regulated by distinct promoters, designated Prm1 and Prm3. Previous investigations identified two upstream repressor regions (URR) 1 and URR2 within Prm1. Herein, it was sought to characterize Prm1, identifying the factor(s) regulating URR1 and URR2 in human erythroleukaemia (HEL) 92.1.7 cells. Genetic reporter assays and 5′ deletions confirmed the presence of URR1 and URR2 but also identified a third repressor, designated RR3, within the proximal ‘core’ promoter. Bioinformatic analysis revealed several GC elements representing putative sites for Egr1/Sp1/Wilms tumour (WT)1 within URR1, URR2 and RR3. While mutation of three GC elements within URR1 and of an adjacent GC element suggested that repressor binding occurs through a cooperative mechanism, repressors binding to the single GC elements within URR2 and RR3 act independently to regulate Prm1. While electrophoretic mobility shift assays and supershift assays demonstrated that each of the GC elements can bind Egr1 and WT1 in vitro, chromatin immunoprecipitations established that WT1 is the factor predominantly bound to each of the repressor regions in vivo. Additionally, ectopic expression of –KTS isoforms of WT1 decreased Prm1-directed gene expression and TPα mRNA expression. Collectively, these data establish WT1 as a critical repressor of Prm1, suppressing TPα expression in the platelet progenitor megakaryoblastic HEL cells.
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homologous desensitization of signalling by the beta β isoform of the human Thromboxane A2 Receptor
Biochimica et Biophysica Acta, 2006Co-Authors: Leanne P Kelleyhickie, Therese B KinsellaAbstract:Abstract Thromboxane (TX) A2 is a potent stimulator of platelet activation/aggregation and smooth muscle contraction and contributes to a variety of pathologies within the vasculature. In this study, we investigated the mechanism whereby the cellular responses to TXA2 mediated through the TPβ isoform of the human TXA2 Receptor (TP) are dynamically regulated by examining the mechanism of agonist-induced desensitization of intracellular signalling and second messenger generation by TPβ. It was established that TPβ is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPβ undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 has been identified as the key phospho-target. In addition, TPβ also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled Receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and β-arrestin association, thereby blocking TPβ internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs. TPβ signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes β-arrestin association and, in turn, facilitates TPβ internalization. Thromboxane (TX) A2 is a potent stimulator of platelet aggregation and smooth muscle contraction and contributes to a variety of vascular pathologies. Herein the mechanism whereby the cellular responses to TXA2 mediated through the TPβ isoform of the human TXA2 Receptor (TP) are dynamically regulated was investigated by examining the mechanism of its agonist-induced desensitization of intracellular signalling and second messenger generation. TPβ is subject to profound agonist-induced homologous desensitization of signalling (intracellular calcium mobilization and inositol 1,3,5 trisphosphate generation) in response to stimulation with the TXA2 mimetic U46619 and this occurs through two key mechanisms: TPβ undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, protein kinase (PK)C mechanism whereby Ser145 within intracellular domain (IC)2 was identified as the key phospho-target. In addition, TPβ also undergoes more profound and sustained agonist-induced desensitization involving G protein-coupled Receptor kinase (GRK)2/3-phosphorylation of both Ser239 and Ser357 within its IC3 and carboxyl-terminal C-tail domains, respectively. Inhibition of phosphorylation of either Ser239 or Ser357, through site directed mutagenesis, impaired desensitization while mutation of both Ser239 and Ser357 almost completely abolished desensitization of signalling, GRK phosphorylation and β-arrestin association, thereby blocking TPβ internalization. These data suggest a model whereby agonist-induced PKC phosphorylation of Ser145 partially impairs TPβ signalling while GRK2/3 phosphorylation at both Ser239 and Ser357 within its IC3 and C-tail domains, respectively, sterically inhibits G-protein coupling, profoundly desensitizing signalling, and promotes β-arrestin association and, in turn, facilitates TPβ internalization.
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the α but not the β isoform of the human Thromboxane A2 Receptor is a target for prostacyclin mediated desensitization
Journal of Biological Chemistry, 2000Co-Authors: Marietherese Walsh, John F Foley, Therese B KinsellaAbstract:In this study, we examined the effects the prostacyclin Receptor (IP) agonist cicaprost exhibited on U46619-mediated Thromboxane A2 Receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (HEK) 293 cells stably overexpressing the individual TPα or TPβ isoforms. Consistent with previous studies, cicaprost abrogated U46619-mediated platelet aggregation and mobilization of intracellular calcium ([CA2+]i). In HEK 293 cells, signaling by TPα, but not TPβ, was subject to IP-mediated desensitization in a protein kinase A-dependent, protein kinase C-independent manner. Desensitization of TPα signaling was independent of the nature of the IP agonist used, the level of IP expression, or the subtype of Gq protein. Signaling by TPΔ328, a truncated variant of TP devoid of the divergent residues of the TPs, or by TPαS329A, a site-directed mutant of TPα, were insensitive to IP agonist activation. Whole cell phosphorylations established that TPα, but not TPβ or TPαS329A, is subject to IP-mediated phosphorylation and that TPα phosphorylation is inhibited by H-89. Thus, we conclude that TPα, but not TPβ, is subject to cross-desensitization by IP mediated through direct protein kinase A phosphorylation at Ser329 and propose that TPα may be the isoform physiologically relevant to TP:IP-mediated vascular hemostasis.
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expression and tissue distribution of the mrnas encoding the human Thromboxane A2 Receptor tp alpha and beta isoforms
Biochimica et Biophysica Acta, 1998Co-Authors: Sinead M Miggin, Therese B KinsellaAbstract:The human Thromboxane A2 Receptor (TP), a G protein-coupled Receptor, exists as two isoforms, TPalpha and TPbeta, which arise by alternative mRNA splicing and differ exclusively in their carboxyl terminal cytoplasmic regions. In this study, a reverse transcriptase-polymerase chain reaction (RT-PCR)-based strategy was developed to examine the expression of the TPs in tissues of physiologic relevance to TXA2. Although most of the 17 different cell/tissue types examined expressed both TP isoforms, the liver hepatoblastoma HepG2 cell line was found to exclusively express TPalpha mRNA. In most cell types, TPalpha mRNA predominated over TPbeta mRNA. Moreover, although the levels of TPalpha mRNA expression were similar in most of the cell/tissue types examined, extensive differences in the levels of TPbeta mRNA were observed. Consequently, the relative expression of TPalpha: TPbeta mRNA varied considerably due to extensive differences in TPbeta mRNA expression. Most strikingly, primary HUVECs were found to express: (i) low levels of TPbeta and (ii) approximately 6-fold greater levels of TPalpha than TPbeta. These data were confirmed in the spontaneously transformed HUVEC derived ECV304 cell line. Expression of TP mRNAs in the various tissue/cells correlated with protein expression, as assessed by radioligand binding using the selective TP antagonist [3H]SQ29,548.
Fumitaka Ushikubi - One of the best experts on this subject based on the ideXlab platform.
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effects of salt loading on blood pressure in mice lacking the prostanoid Receptor gene
Circulation, 2005Co-Authors: Hidetsuna Watanabe, Fumitaka Ushikubi, Shuh Narumiya, Tetsuo Katoh, Masaaki Eiro, Masaya Iwamoto, Tsuyoshi WatanabeAbstract:Background This study examined whether targeted disruption of the genes for the prostacyclin Receptor (IP) or the Thromboxane A2 Receptor (TP) confers a susceptibility to salt-dependent hypertension. Methods and Results Eight female IP- or TP-deficient mice were examined. Baseline systolic blood pressure (SBP) did not differ between TP(-/-) and TP(+/+), but was significantly lower in the IP(-/-) group than in the IP(+/+). With a high salt diet, SBP in IP(-/-) gradually increased. In contrast, SBP in the IP(+/+), TP(-/-), or TP(+/+) groups remained unchanged. Conclusions The prostacyclin Receptor may participate in the maintenance of baseline BP. With salt loading, BP adaptation may take place, at least in part, via IP mediated signals. (Circ J 2005; 69: 124 -126)
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arg60 to leu mutation of the human Thromboxane A2 Receptor in a dominantly inherited bleeding disorder
Journal of Clinical Investigation, 1994Co-Authors: Takako Hirata, Akira Kakizuka, Minoru Okuma, Ichiro Fuse, Fumitaka Ushikubi, Shuh NarumiyaAbstract: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 ofG 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 (Arg4-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 Arg'0 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. (J. Clin. Invest.
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arg60 to leu mutation of the human Thromboxane A2 Receptor in a dominantly inherited bleeding disorder
Journal of Clinical Investigation, 1994Co-Authors: Takako Hirata, Akira Kakizuka, Minoru Okuma, Ichiro Fuse, Fumitaka Ushikubi, Shuh NarumiyaAbstract:Abstract 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.
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Thromboxane A2 Receptor is highly expressed in mouse immature thymocytes and mediates dna fragmentation and apoptosis
Journal of Experimental Medicine, 1993Co-Authors: Fumitaka Ushikubi, Tsunehisa Namba, Masakazu Hirata, Yu Ichi Aiba, Ken Ichi Nakamura, Osam Mazda, Yoshimoto Katsura, Shuh NarumiyaAbstract:We have recently revealed that the thymus is the organ showing the highest expression of Thromboxane (TX) A2 Receptor in mice. In this study, thymic cell populations expressing the Receptor were identified, and the effects of a TXA2 agonist on these cells were examined. Radioligand binding using a TXA2 Receptor-specific radioligand revealed a single class of binding sites in the thymocytes with an affinity and specificity identical to those reported for the TXA2 Receptor. The Receptor density in these cells was comparable to that seen in blood platelets. This Receptor is most highly expressed in CD4-8- and CD4+8+ immature thymocytes, followed by CD4+8- and CD4-8+ cells. The Receptor density in splenic T cells was less than one fifth of that in CD4+8+ cells and no binding activity was detectable in splenic B cells. The addition of a TXA2 agonist, STA2, to thymocytes induced the disappearance of the CD4+8+ cells in a time- and concentration-dependent manner and caused DNA fragmentation. These changes were blocked by a specific TXA2 antagonist, S-145. These results demonstrate that TXA2 induces apoptotic cell death in immature thymocytes by acting on the TXA2 Receptor on their cell surface and suggest a role for the TXA2/TXA2 Receptor system in the thymic micro-environment.
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cloning and expression of cdna for a human Thromboxane A2 Receptor
Nature, 1991Co-Authors: Masakazu Hirata, Fumitaka Ushikubi, Yasunori Hayashi, Yoshifumi Yokota, Ryoichiro Kageyama, Shigetada Nakanishi, Shuh NarumiyaAbstract:Thromboxane A2 is a very unstable arachidonate metabolite, yet a potent stimulator of platelet aggregation and a constrictor of vascular and respiratory smooth muscles1. It has been implicated as a mediator in diseases such as myocardial infarction, stroke and bronchial asthmA2. Using a stable analogue of this compound we recently purified the human platelet Thromboxane A2 Receptor to apparent homogeneity3. Using an oligonucleotide probe corresponding to its partial amino-acid sequence, we have obtained a complementary DNA clone encoding this Receptor from human placenta and a partial clone from cultured human megakaryocytic leukaemia cells. The placenta cDNA encodes a protein of 343 amino acids with seven putative transmembrane domains. The protein expressed in COS-7 cells binds drugs with affinities identical to those of the platelet Receptor, and that in Xenopus oocytes opens CA2+-activated Cl- channel on agonist stimulation. Northern blot analysis and nucleotide sequences of the two clones suggest that an identical species of the Thromboxane A2 Receptor is present in platelets and vascular tissues. This first report on the molecular structure of an eicosanoid Receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.
Weiliang Xia - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for ligand recognition of the human Thromboxane A2 Receptor.
Nature Chemical Biology, 2018Co-Authors: Hengxin Fan, Shuanghong Chen, Xiaojing Yuan, Shuo Han, Hui Zhang, Weiliang Xia, Qiang ZhaoAbstract:Stimulated by Thromboxane A2, an endogenous arachidonic acid metabolite, the Thromboxane A2 Receptor (TP) plays a pivotal role in cardiovascular homeostasis, and thus is considered as an important drug target for cardiovascular disease. Here, we report crystal structures of the human TP bound to two nonprostanoid antagonists, ramatroban and daltroban, at 2.5 A and 3.0 A resolution, respectively. The TP structures reveal a ligand-binding pocket capped by two layers of extracellular loops that are stabilized by two disulfide bonds, limiting ligand access from the extracellular milieu. These structures provide details of interactions between the Receptor and antagonists, which help to integrate previous mutagenesis and SAR data. Molecular docking of prostanoid-like ligands, combined with mutagenesis, ligand-binding and functional assays, suggests a prostanoid binding mode that may also be adopted by other prostanoid Receptors. These insights into TP deepen our understanding about ligand recognition and selectivity mechanisms of this physiologically important Receptor. Structures of the human Thromboxane A2 Receptor, a member of the prostanoid family of G-protein-coupled Receptors, in complex with two synthetic antagonists reveal that ligands access the ligand-binding pocket from the plane of the lipid bilayer.
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Thromboxane A2 Receptor antagonist sq29548 suppresses the lps induced release of inflammatory cytokines in bv2 microglia cells via suppressing mapk and nf κb signaling pathways
Molecular Medicine Reports, 2017Co-Authors: Aijuan Yan, Gaoyu Cai, Weiliang XiaAbstract:Inflammation in the brain, characterized by the activation of microglia, is hypothesized to participate in the pathogenesis of neuronal disorders. It is proposed that Thromboxane A2 Receptor (TXA2R) activation is involved in thrombosis/hemostasis and inflammation responses. In the present study, the anti-inflammatory effects of SQ29548 on lipopolysaccharide (LPS)-stimulated BV2 microglial cells and its molecular mechanisms were investigated. In the BV2 cell line, LPS-stimulated nitric oxide (NO) and inflammatory cytokine release, and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor (NF)-κB were assessed using an NO assay kit, reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In vitro studies demonstrated that SQ29548 inhibited LPS-stimulated BV2 activation and reduced the mRNA expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α and inducible NO synthase via inhibition of MAPKs and the NF-κB signaling pathway. SQ29548 inhibited the LPS-induced inflammatory response by blocking MAPKs and NF-κB activation in BV2 microglial cells.
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Thromboxane A2 Receptor antagonist sq29548 reduces ischemic stroke induced microglia macrophages activation and enrichment and ameliorates brain injury
Scientific Reports, 2016Co-Authors: Aijuan Yan, Tingting Zhang, Xiao Yang, Jiaxiang Shao, Fanxia Shen, Weiliang XiaAbstract:Thromboxane A2 Receptor (TXA2R) activation is thought to be involved in thrombosis/hemostasis and inflammation responses. We have previously shown that TXA2R antagonist SQ29548 attenuates BV2 microglia activation by suppression of ERK pathway, but its effect is not tested in vivo. The present study aims to explore the role of TXA2R on microglia/macrophages activation after ischemia/reperfusion brain injury in mice. Adult male ICR mice underwent 90-min transient middle cerebral artery occlusion (tMCAO). Immediately and 24 h after reperfusion, SQ29548 was administered twice to the ipsilateral ventricle (10 μl, 2.6 μmol/ml, per dose). Cerebral infarction volume, inflammatory cytokines release and microglia/macrophages activation were measured using the cresyl violet method, quantitative polymerase chain reaction (qPCR), and immunofluorescence double staining, respectively. Expression of TXA2R was significantly increased in the ipsilateral brain tissue after ischemia/reperfusion, which was also found to co-localize with activated microglia/macrophages in the infarct area. Administration of SQ29548 inhibited microglia/macrophages activation and enrichment, including both M1 and M2 phenotypes, and attenuated ischemia-induced IL-1s, IL-6, and TNF-α up-regulation and iNOS release. TXA2R antagonist SQ29548 inhibited ischemia-induced inflammatory response and furthermore reduced microglia/macrophages activation and ischemic/reperfusion brain injury.
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Thromboxane A2 Receptor polymorphism in association with cerebral infarction and its regulation on platelet function
Current Neurovascular Research, 2015Co-Authors: Jiaxiang Shao, Jing Zhao, Wanlin Yang, Jing Yan, Shengdi Chen, Weiliang XiaAbstract:Binding of Thromboxane A 2 (TXA 2 ) to its Receptor TXA 2 R modulates thrombosis/hemostasis and plays a vital role in the pathogenesis of cerebral infarction (CI). In this study, we investigated the relationship between TXA 2 R polymorphisms and CI in Chinese Han population and the effect on platelet function by these polymorphisms. Polymerase chain reaction and ligase detection reaction (PCR-LDR) was performed in 230 CI patients and 143 healthy volunteers to examine four single nucleotide polymorphisms (SNPs) in human TXA 2 R gene (C795T, T924C and G1686A in the exon region, and rs768963 in the promoter region). We found that rs768963 polymorphism was significantly more frequent in the CI group than in the non-CI group and the T-T-G-T haplotype of C795T-T924C-G1686A-rs768963 was significantly less frequent in the CI subjects (0.238 versus 0.339; OR 0.617 [95%CI 0.444-0.856]). In the meantime, we constructed wild-type and mutant (C795T, G910A and T924C) eukaryotic expression plasmids, and transfected these plasmids into human embryonic kidney (HEK) 293T cells or Chinese hamster ovary (CHO) cells stably expressing human TXA 2 R (GPb/a- CHO). C795T and T924C variants of TXA2R led to increased ligand binding-induced intracellular calcium influx and fibrinogen-integrin conjugation, while dominant negative mutant G910A abolished the signal enhancement. Together these data show that TXA2R polymorphisms may affect platelet function and the risk of developing cerebral ischemia.