研究等業績 - 原著論文 - 石井 聡
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Janovicz A., Majer A., Kosztelnik M., Geiszt M., Chun J., Ishii S., Tigyi G.J., Benyó Z., Ruisanchez É.
Experimental Biology and Medicine ( Experimental Biology and Medicine ) 248 1887 - 1894 2023年10月 [査読有り]
研究論文（学術雑誌） 国際共著
Lysophosphatidylcholine (LPC) is a bioactive lipid that has been shown to attenuate endothelium-dependent vasorelaxation contributing to endothelial dysfunction; however, the underlying mechanisms are not well understood. In this study, we investigated the molecular mechanisms involved in the development of LPC-evoked impairment of endothelium-dependent vasorelaxation. In aortic rings isolated from wild-type (WT) mice, a 20-min exposure to LPC significantly reduced the acetylcholine chloride (ACh)–induced vasorelaxation indicating the impairment of normal endothelial function. Interestingly, pharmacological inhibition of autotaxin (ATX) by GLPG1690 partially reversed the endothelial dysfunction, suggesting that lysophosphatidic acid (LPA) derived from LPC may be involved in the effect. Therefore, the effect of LPC was also tested in aortic rings isolated from different LPA receptor knock-out (KO) mice. LPC evoked a marked reduction in ACh-dependent vasorelaxation in Lpar1, Lpar2, and Lpar4 KO, but its effect was significantly attenuated in Lpar5 KO vessels. Furthermore, addition of superoxide dismutase reduced the LPC-induced endothelial dysfunction in WT but not in the Lpar5 KO mice. In addition, LPC increased H<sub>2</sub>O<sub>2</sub> release from WT vessels, which was significantly reduced in Lpar5 KO vessels. Our findings indicate that the ATX–LPA–LPA<sub>5</sub> receptor axis is involved in the development of LPC-induced impairment of endothelium-dependent vasorelaxation via LPA<sub>5</sub> receptor–mediated reactive oxygen species production. Taken together, in this study, we identified a new pathway contributing to the development of LPC-induced endothelial dysfunction.
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Inverse agonism of lysophospholipids with cationic head groups at Gi-coupled receptor GPR82
Yasuda D., Hamano F., Masuda K., Dahlström M., Kobayashi D., Sato N., Hamakubo T., Shimizu T., Ishii S.
European Journal of Pharmacology ( European Journal of Pharmacology ) 954 175893 2023年09月 [査読有り]
研究論文（学術雑誌） 国際共著
GPR82 is an orphan G protein-coupled receptor (GPCR) that has been implicated in lipid storage in mouse adipocytes. However, the intracellular signaling as well as the specific ligands of GPR82 remain unknown. GPR82 is closely related to GPR34, a GPCR for the bioactive lipid molecule lysophosphatidylserine. In this study, we screened a lipid library using GPR82-transfected cells to search for ligands that act on GPR82. By measuring cyclic adenosine monophosphate levels, we found that GPR82 is an apparently constitutively active GPCR that leads to Gi protein activation. In addition, edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine), an artificial lysophospholipid with a cationic head group that exerts antitumor activity, inhibited the Gi protein activation by GPR82. Two endogenous lysophospholipids with cationic head groups, lysophosphatidylcholine (1-oleoyl-sn-glycero-3-phosphocholine) and lysophosphatidylethanolamine (1-oleoyl-sn-glycero-3-phosphoethanolamine), also exhibited GPR82 inhibitory activity, albeit weaker than edelfosine. Förster resonance energy transfer imaging analysis consistently demonstrated that Gi protein-coupled GPR82 has an apparent constitutive activity that is edelfosine-sensitive. Consistent data were obtained from GPR82-mediated binding analysis of guanosine-5'-O-(3-thiotriphosphate) to cell membranes. Furthermore, in GPR82-transfected cells, edelfosine inhibited insulin-induced extracellular signal-regulated kinase activation, like compounds that function as inverse agonists at other GPCRs. Therefore, edelfosine is likely to act as an inverse agonist of GPR82. Finally, GPR82 expression inhibited adipocyte lipolysis, which was abrogated by edelfosine. Our findings suggested that the cationic lysophospholipids edelfosine, lysophosphatidylcholine and lysophosphatidylethanolamine are novel inverse agonists for Gi-coupled GPR82, which is apparently constitutively active, and has the potential to exert lipolytic effects through GPR82.
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Spencer S.A., Suárez-Pozos E., Verdugo J.S., Wang H., Afshari F.S., Guo L., Manam S., Yasuda D., Ortega A., Lister J.A., Ishii S., Zhang Y., Fuss B.
Journal of Neurochemistry 163 ( 6 ) 478 - 499 2023年02月 [査読有り]
研究論文（学術雑誌） 国際共著
The developmental process of central nervous system (CNS) myelin sheath formation is characterized by well-coordinated cellular activities ultimately ensuring rapid and synchronized neural communication. During this process, myelinating CNS cells, namely oligodendrocytes (OLGs), undergo distinct steps of differentiation, whereby the progression of earlier maturation stages of OLGs represents a critical step toward the timely establishment of myelinated axonal circuits. Given the complexity of functional integration, it is not surprising that OLG maturation is controlled by a yet fully to be defined set of both negative and positive modulators. In this context, we provide here first evidence for a role of lysophosphatidic acid (LPA) signaling via the G protein-coupled receptor LPA6 as a negative modulatory regulator of myelination-associated gene expression in OLGs. More specifically, cell surface accessibility of LPA6 was found to be restricted to the earlier maturation stages of differentiating OLGs, and OLG maturation was found to occur precociously in Lpar6 knockout mice. To further substantiate these findings, a novel small molecule ligand with selectivity for preferentially LPA6 and LPA6 agonist characteristics was functionally characterized in vitro in primary cultures of rat OLGs and in vivo in the developing zebrafish. Utilizing this approach, a negative modulatory role of LPA6 signaling in OLG maturation could be corroborated. During development, such a functional role of LPA6 signaling likely serves to ensure timely coordination of circuit formation and myelination. Under pathological conditions as seen in the major human demyelinating disease multiple sclerosis (MS), however, persistent LPA6 expression and signaling in OLGs can be seen as an inhibitor of myelin repair. Thus, it is of interest that LPA6 protein levels appear elevated in MS brain samples, thereby suggesting that LPA6 signaling may represent a potential new druggable pathway suitable to promote myelin repair in MS.
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Yanagida, K., Masago, K., Yasuda, D., Hamano, F., Kurikawa, Y., Shimizu, T. ,Ishii, S.
Human Molecular Genetics 32 ( 5 ) 825 - 834 2022年09月 [査読有り]
研究論文（学術雑誌） 国内共著
In human autosomal recessive woolly hair/hypotrichosis (ARWH/HT), many mutations have been identified in a gene encoding LPA6, a G protein-coupled receptor (GPCR) for lysophosphatidic acid (LPA). However, information regarding the effects of such mutations on receptor function is limited. In this study, we examined functional impacts of selected amino acid changes in LPA6 identified in ARWH/HT patients. In our exogenous expression experiments, all mutants except S3T failed to respond to LPA, indicating that they are loss-of-function mutants. Among the nine mutants, five (D63V, G146R, N246D, L277P, and C278Y) displayed impaired expression at the cell surface due to endoplasmic reticulum (ER) retention, indicating that these mutants are trafficking-defective, as reported in other disease-associated GPCRs. Notably, alkyl-OMPT, a potent synthetic agonist for LPA6 restored the defective cell surface expression of two of the ER-retained mutants, D63V and N246D, possibly by its chaperoning function that allows them to escape intracellular retention as well as proteasomal degradation. Furthermore, the alkyl-OMPT-rescued N246D mutant was shown be functional. Our findings encourage future application of pharmacoperone therapy for ARWH/HT patients with specific LPA6 mutations.
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Yasuda, D., Kobayashi, D., Akahoshi, N., Ohto-Nakanishi, T., Yoshioka, K., Takuwa, Y., Mizuno, S., Takahashi, S., and Ishii, S.
Journal of Clinical Investigation 129 ( 10 ) 4332 - 4349 2019年10月 [査読有り]
研究論文（学術雑誌） 国内共著
Lysophosphatidic acid (LPA) is a potent lipid mediator with various biological functions mediated through six G protein-coupled receptors (GPCRs), LPA1-6. Previous studies have demonstrated that LPA-Gα12/Gα13 signaling plays an important role in embryonic vascular development. However, the responsible LPA receptors and underlying mechanisms are poorly understood. Here, we show a critical role of LPA4 and LPA6 in developmental angiogenesis. In mice, Lpa4;Lpa6 double knockout (DKO) embryos were lethal due to global vascular deficiencies, and endothelial cell (EC)-specific Lpa4;Lpa6 DKO retinas had impaired sprouting angiogenesis. Mechanistically, LPA activated the transcriptional regulators YAP and TAZ through LPA4/LPA6-mediated Gα12/Gα13-Rho-ROCK signaling in ECs. YAP/TAZ knockdown increased β-catenin- and Notch intracellular domain (NICD)-mediated endothelial expression of the Notch ligand delta-like 4 (DLL4). Fibrin gel sprouting assay revealed that LPA4/LPA6, Gα12/Gα13, or YAP/TAZ knockdown consistently blocked EC sprouting, which was rescued by a Notch inhibitor. Of note, the inhibition of Notch signaling also ameliorated impaired retinal angiogenesis in EC-specific Lpa4;Lpa6 DKO mice. Overall, these results suggest that the Gα12/Gα13-coupled receptors LPA4 and LPA6 synergistically regulate endothelial Dll4 expression through YAP/TAZ activation. This could in part account for the mechanism of YAP/TAZ-mediated developmental angiogenesis. Our findings provide a novel insight into the biology of GPCR-activated YAP/TAZ.
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Molecular mechanism of lysophosphatidic acid-induced hypertensive response
Kano K.
Scientific Reports ( Scientific Reports ) 9 ( 1 ) 2019年02月 [査読有り]
研究論文（学術雑誌） 国内共著
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Yanagida K.
JCI insight ( JCI insight ) 3 ( 24 ) e97293 2018年12月 [査読有り]
研究論文（学術雑誌） 国内共著
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Tsukahara R.
Journal of Pharmacological Sciences ( Journal of Pharmacological Sciences ) 136 ( 2 ) 93 - 96 2018年02月 [査読有り]
研究論文（学術雑誌） 国内共著
Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.
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Tanishima M.
Journal of Biological Chemistry ( Journal of Biological Chemistry ) 292 ( 42 ) 17250 - 17257 2017年10月 [査読有り]
研究論文（学術雑誌） 国内共著
Upon stimulation of toll-like receptors with various microbial ligands, induction of a variety of inflammatory genes is elicited by activation of a myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathway. Interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) plays an essential role in this pathway by activating nuclear factor kappa B (NF-kappa B) and mitogen-activated kinases (MAPKs). Here, we identified optineurin (OPTN) as an IRAK1-binding protein by yeast two-hybrid screening using IRAK1 as bait. A C-terminal fragment of OPTN harboring a ubiquitin-binding domain was co-immunoprecipitated with IRAK1. In reporter analyses, overexpression of OPTN inhibited IL-1 beta-, IRAK1-, and LPS-induced NF-kappa B activation. Consistently, OPTN deficiency resulted in increased NF-kappa B activation in response to IL-1 beta/LPS stimulation. To address the mechanisms underlying the inhibitory effect of OPTN on NF-kappa B signaling, we focused on tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), which is an adaptor protein of IRAK1 and upon polyubiquitination plays a crucial role during NF-kappa Bactivation. Overexpression of OPTN prevented TRAF6 polyubiquitination. Furthermore, OPTN H486R mutant, which is unable to recruit the deubiquitinase CYLD, failed to inhibit IRAK1-induced NF-kappa Bactivation. These results suggest that the IRAK1-binding protein OPTN negatively regulates IL-1 beta/LPS-induced NF-kappa Bactivation by preventing polyubiquitination of TRAF6.
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Takara K.
Cell Reports ( Cell Reports ) 20 ( 9 ) 2072 - 2086 2017年08月 [査読有り]
研究論文（学術雑誌） 国内共著
Vascular normalization in tumors may improve drug delivery and anti-tumor immunity. Angiogenesis inhibitors induce hypoxia, which may facilitate malignant progression; therefore, we investigated other methods to promote vascular maturation. Here, we show that lysophosphatidic acid (LPA) enhances blood flow by promoting fine vascular networks, thereby improving vascular permeability and suppressing tumor growth when combined with anti-cancer drug treatment. Six different G protein-coupled receptors have been identified as LPA receptors (LPA1-6). In studies using mutant mice, we found that LPA4 is involved in vascular network formation. LPA4 activation induces circumferential actin bundling beneath the cell membrane and enhances linear adherens junction formation by VE-cadherin in endothelial cells. Therefore, we conclude that activation of LPA4 is a promising approach for vascular regulation.
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TDAG8 involved in initiating inflammatory hyperalgesia and establishing hyperalgesic priming in mice
Dai S.
Scientific Reports ( Scientific Reports ) 7 41415 2017年02月 [査読有り]
研究論文（学術雑誌） 国際共著
Chronic pain, resulting from injury, arthritis, and cancer, is often accompanied by inflammation. High concentrations of protons found in inflamed tissues results in tissue acidosis, a major cause of pain and hyperalgesia. Acidosis signals may mediate a transition from acute to chronic hyperalgesia (hyperalgesic priming) via proton-sensing G-protein-coupled receptors (GPCRs). The expression of T-cell death-associated gene 8 (TDAG8), a proton-sensing GPCR, is increased during inflammatory hyperalgesia. Attenuating TDAG8 expression in the spinal cord inhibits bone cancer pain, but whether TDAG8 is involved in inflammatory hyperalgesia or hyperalgesic priming remains unclear. In this study, we used TDAG8-knockout or -knockdown to explore the role of TDAG8 in pain. Suppressed TDAG8 expression delayed the onset of inflammatory hyperalgesia and shortened hyperalgesic time in mice. In a dual acid-injection model (acid [pH 5.0] injected twice, 5 days apart), shRNA inhibition of TDAG8 shortened the duration of the second hyperalgesia. Similar results were found in TDAG8-deficient mice. The dual administration of TDAG8 agonist also confirmed that TDAG8 is involved in hyperalgsic priming. Accordingly, TDAG8 may mediate acidosis signals to initiate inflammatory hyperalgesia and establish hyperalgesic priming.
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Involvement of TRPV1 and TDAG8 in Pruriception Associated with Noxious Acidosis
Lin S.H.
Journal of Investigative Dermatology ( Journal of Investigative Dermatology ) 137 ( 1 ) 170 - 178 2017年01月 [査読有り]
研究論文（学術雑誌） 国際共著
Itch and pain are closely related but are distinct sensations. Intradermal injection of acid generates pain in both rodents and humans; however, few studies have addressed the intriguing question of whether acid (protons) can evoke itch like other algogens by spatial contrast activation of single nociceptors. Here, we report that (i) citric acid (0.2 mol/L) pH-dependently induced a scratching response in mice when applied intradermally to nape or cheek skin, (ii) acidified buffer elevated intracellular calcium levels in dorsal root ganglion pruriceptors, and (iii) injection of intradermal citric acid (pH 3.0) into the nape induced a pruritogen-like but not algogen-like c-Fos immunoreactivity pattern in the cervical spinal cord. Using pharmacological and genetic approaches, we identified potential acid-sensing channels/receptors involved in acidic citrate-evoked itch. Results indicate that TRPV1, but neither ASIC3 nor TRPA1, is involved in the acidic citrate-induced scratching response. Furthermore, one of the proton-sensing G-protein-coupled receptors, TDAG8, was highly (similar to 71%) expressed in Nppb(+) dorsal root ganglion pruriceptors. Itch induced by acidic citrate, but not alpha-methyl-5-hydroxytryptamine, chloroquine, compound 48/80, or bile acid, was markedly decreased in TDAG8(-/-) mice. In a heterologous expression system, TDAG8 potentiated the acid-induced calcium response by regulating TRPV1. Thus, protons could evoke pruriception by acting on TDAG8 to regulate TRPV1 activation with its mechanism of future therapeutic relevance.
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LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis.
Sumida, H., Noguchi, K., Kihara, Y., Abe, M., Yanagida, K., Hamano, F., Sato, S., Tamaki, K., Morishita. Y., Kano, M.R., Iwata, C., Miyazono, K., Sakimura, K., Shimizu, T., and Ishii, S.
Blood 116 5060 - 5070 2010年04月
研究論文（学術雑誌） 単著
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The leukotriene B(4) receptor, BLT1, is required for the induction of experimental autoimmune encephalomyelitis.
Kihara, Y., Yokomizo, T., Kunita, A., Morishita, Y., Fukayama, M., Ishii, S., and Shimizu, T.
Biochem. Biophys. Res. Commun. 394 673 - 678 2010年01月
研究論文（学術雑誌）
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Adiponectin and AdipoR1 regulate PGC-1alpha and mitochondria by Ca(2+) and AMPK/SIRT1.
Iwabu, M., Yamauchi, T., Okada-Iwabu, M., Sato, K., Nakagawa, T., Funata, M., Yamaguchi, M., Namiki, S., Nakayama, R., Tabata, M., Ogata, H., Kubota, N., Takamoto, I., Hayashi, Y.K., Yamauchi, N., Waki, H., Fukayama, M., Nishino, I., Tokuyama, K., Ueki, K., Oike, Y., Ishii, S., Hirose, K., Shimizu, T., Touhara, K., and Kadowaki, T.
Nature 464 1313 - 1319 2010年01月
研究論文（学術雑誌）
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Role of PAF receptor in proinflammatory cytokine expression in the dorsal root ganglion and tactile allodynia in a rodent model of neuropathic pain.
Hasegawa, S., Kohro, Y., Shiratori, M., Ishii, S., Shimizu, T., Tsuda, M., and Inoue, K.
PLoS One 5 e10467 2010年01月
研究論文（学術雑誌）
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The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor. (Direct submission).
Ihara, Y., Kihara, Y., Hamano, F., Yanagida, K., Morishita, Y., Kunita, A., Yamori, T., Fukayama, M., Aburatani, H., Shimizu, T., and *Ishii, S.
Proc. Natl. Acad. Sci. U. S. A. 107 17309–17314 2010年01月
研究論文（学術雑誌）
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Opposing effects of platelet-activating factor and lyso-platelet activating factor on neutrophil and platelet activation.
Welch, E.J., Naikawadi, R.P., Li, Z., Lin, P., Ishii, S., Shimizu, T., Tiruppathi, C., Du, X., Subbaiah, P.V., and Ye, R.D.
Mol. Pharmacol. 75 227 - 234 2009年01月
研究論文（学術雑誌）
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Involvement of proton-sensing TDAG8 in extracellular acidification-induced inhibition of pro-inflammatory cytokine production in peritoneal macrophages.
Mogi, C., Tobo, M., Tomura, H., Murata, N., He, X.-d., Sato, K., Kimura, T., Ishizuka, T., Sasaki, T., Sato, T., Kihara, Y., Ishii, S., Harada, A., and Okajima, F.
J. Immunol. 182 3243–3251 2009年01月
研究論文（学術雑誌）
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Identification and characterization of a novel lysophosphatidic acid receptor, p2y5/LPA6.
Yanagida, K., Masago, K., Nakanishi, H., Kihara, Y., Hamano, F., Tajima, Y., Taguchi, R., Shimizu T., and *Ishii, S.
J. Biol. Chem. 284 17731 - 17741 2009年01月
研究論文（学術雑誌）