研究等業績 - その他 - 沼田 朋大
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Short TRPM2 prevents the targeting of full-length TRPM2 to the surface transmembrane by hijacking to ER associated degradation.
Yamamoto S, Ishii T, Mikami R, Numata T, Shimizu S
Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE ) 520 ( 3 ) 520 - 525 2019年12月
Membrane proteins are targeted to the surface transmembrane after folding and assembling in the endoplasmic reticulum (ER). Misfolded- and unassembled-proteins are degraded by proteasomes following ubiquitination, termed ER-associated degradation (ERAD). Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive channel. One of the TRPM2 splicing variants, short TRPM2 (TRPM2-S) having only the N-terminus and first two transmembrane domains, was reported to prevent full-length TRPM2 (TRPM2-L) activation. Although TRPM2-S interacts with TRPM2-L, the inhibitory mechanisms of TRPM2-S are unclear. We found that TRPM2-S prevents transmembrane expression of TRPM2-L by targeting ERAD. TRPM2-S expression was lower than that of TRPM2-L, and was increased by an ERAD inhibitor. TRPM2-S was not expressed at the transmembrane. This suggests that TRPM2-S is a substrate for ERAD. Upon the simultaneous expression of TRPM2-S, the transmembrane expression of TRPM2-L was attenuated and the poly-ubiquitination of TRPM2-L was facilitated. Our study may clarify why TRPM2-S inhibits oxidative stress-induced TRPM2-L activation.
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SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation.
Komatsu S, Nomiyama T, Numata T, Kawanami T, Hamaguchi Y, Iwaya C, Horikawa T, Fujimura-Tanaka Y, Hamanoue N, Motonaga R, Tanabe M, Inoue R, Yanase T, Kawanami D
Endocrine journal 67 ( 1 ) 99 - 106 2019年11月
Cancer is currently one of the major causes of death in patients with type 2 diabetes mellitus. We previously reported the beneficial effects of the glucagon-like peptide-1 receptor agonist exendin-4 against prostate and breast cancer. In the present study, we examined the anti-cancer effect of the sodium-glucose cotransporter 2 (SGLT2) inhibitor ipragliflozin using a breast cancer model. In human breast cancer MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. Ipragliflozin at 1-50 μM significantly and dose-dependently suppressed the growth of MCF-7 cells. BrdU assay also revealed that ipragliflozin attenuated the proliferation of MCF-7 cells in a dose-dependent manner. Because the effect of ipragliflozin against breast cancer cells was completely canceled by knocking down SGLT2, ipragliflozin could act via inhibiting SGLT2. We next measured membrane potential and whole-cell current using the patch clamp technique. When we treated MCF-7 cells with ipragliflozin or glucose-free medium, membrane hyperpolarization was observed. In addition, glucose-free medium and knockdown of SGLT2 by siRNA suppressed the glucose-induced whole-cell current of MCF-7 cells, suggesting that ipragliflozin inhibits sodium and glucose cotransport through SGLT2. Furthermore, JC-1 green fluorescence was significantly increased by ipragliflozin, suggesting the change of mitochondrial membrane potential. These findings suggest that the SGLT2 inhibitor ipragliflozin attenuates breast cancer cell proliferation via membrane hyperpolarization and mitochondrial membrane instability.
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Numata T, Sato-Numata K, Okada Y
Scientific reports ( NATURE PUBLISHING GROUP ) 9 ( 1 ) 15554 - 15554 2019年10月
Japanese Kampo medicines Junchoto and Mashiningan are mixtures of numerous herbal plant extracts and empirically known to exert laxative actions by stimulating fluid secretion in the colonic epithelium. However, it is unknown which and how the herbal components of these crude Kampo drugs are effective to stimulate ion effluxes causing fluid secretion. Here, we selected four herbal components of Junchoto and Mashiningan, Mashinin (MSN), Kyonin (KYN), Tonin (TON), and Daio (DIO), which are putatively laxatives, and examined their effects on the ion channel activity of human colonic epithelial Caco-2 cells. Patch clamp analyses revealed that MSN activated whole-cell current characteristics of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, whereas KYN, TON, and DIO activated the large-conductance and voltage-activated K+ (BK) channel. Furthermore, electronic cell sizing showed that MSN induced secretory volume decrease (SVD) sensitivity to a CFTR blocker, whereas TON, KYN, and DIO induced SVD sensitivity to a K+ channel blocker. In conclusion, MSN and TON, KYN, and DIO promote fluid secretion from colonic epithelial cells by activating CFTR and BK channels. Thus, Japanese Kampo medicines, Junchoto and Mashiningan, exert anti-constipation actions by inducing KCl efflux through the combined actions of CFTR- and BK-stimulating herbal components.
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Efficacy of mepolizumab for patients with severe asthma and eosinophilic chronic rhinosinusitis
Numata T.
BMC Pulmonary Medicine ( BMC Pulmonary Medicine ) 19 ( 1 ) 2019年10月
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Construction of a Fluorescent Screening System of Allosteric Modulators for the GABA(A) Receptor Using a Turn-On Probe
Seiji Sakamoto, Kei Yamaura, Tomohiro Numata, Fumio Harada, Kazuma Amaike, Ryuji Inoue, Shigeki Kiyonaka, Itaru Hamachi
ACS CENTRAL SCIENCE ( AMER CHEMICAL SOC ) 5 ( 9 ) 1541 - 1553 2019年09月
gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABA(A) receptors (GABA(A)Rs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABA A Rs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABA(A)Rs. We here developed the first turn-on fluorescent imaging probe for GABA(A)Rs, which can be used to quantitatively evaluate ligand-receptor interactions under live cell conditions. Using noncovalent labeling of GABA(A)Rs with this turn-on probe, a new imaging-based ligand assay system, which allows discovery of positive allosteric modulators (PAMs) for the GABA(A)R, was successfully constructed. Our system is applicable to high-throughput ligand screening, and we discovered new small molecules that function as PAMs for GABA(A)Rs. These results highlight the power of the use of a turn-on fluorescent probe to screen drugs for complicated membrane proteins that cannot be addressed by conventional methods.
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井上隆司, 胡耀鵬, 沼田朋大
医学のあゆみ ( 医歯薬出版 ) 270 ( 10 ) 954 - 960 2019年09月
TRPM4チャネルはTRP蛋白質スーパーファミリーのなかにあって、一価陽イオンを選択的に透過させるユニークな性質をもつ。全身に広く発現し、種々の生体機能の制御に関与するカルシウム(Ca2+)依存性陽イオンチャネル(CAN)の分子実体と考えられている。最近、TRPM4蛋白質の分子立体構造が解明され、そのイオン選択性や活性化機構についての理解が大きく進んだ。また、TRPM4ゲーティングの数理化により、細胞内Ca2+動態に連結した実用性のある細胞興奮モデルが構築されている。本稿では、これらの新しい知見を紹介しつつ、このチャネルが心血管系の生理機能や病態生理に果たす役割について述べる。(著者抄録)
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Numata T, Sato-Numata K, Okada Y
Physiological reports ( Wiley ) 7 ( 13 ) e14157 2019年07月
Because intravaginal pH is strongly acidic, it is important to investigate the effects of acidosis on cervical cancer cells. Recently, in response to strong acidosis, human cervical cancer HeLa cells were shown to exhibit necrosis after showing persistent cell swelling induced by Cl- influx. Since cation influx should be accompanied with Cl- influx to drive water inflow causing cell swelling, we here studied on the nature of acidotoxic cation conductance. The mRNA/protein expression was assessed by RT-PCR and Western blotting. Ionic currents were measured by patch-clamping techniques. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and caspase 3/7 activity, respectively. Cell volume and size were measured by electronic sizing and video-microscopic measurements, respectively. Acid exposure enhanced TRPM7 activity endogenously expressed in HeLa cells and exogenously overexpressed in HEK293T cells. Gene silencing of TRPM7 abolished acid-induced cell swelling and necrosis but rather induced activation of apoptotic caspase 3/7 in HeLa cells. Overexpression with the pore charge-neutralizing D1054A mutant suppressed acid-enhanced cation currents, acid-induced cell swelling, and acidotoxic necrosis in HEK293T cells. Progesterone treatment was surprisingly found to suppress molecular and functional expression of TRPM7 and cell proliferation in HeLa cells. Furthermore, in the progesterone-treated cells, acid exposure did not induce persistent cell swelling followed by necrosis but induced persistent cell shrinkage and apoptotic cell death. These results indicate that in the human cervical cancer cells, TRPM7 is essentially involved in acidotoxic necrotic cell death, and progesterone inhibits TRPM7 expression thereby inhibiting acidotoxic necrosis by switching to apoptosis.
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Nianchao Qian, Atsuhiko Ichimura, Daisuke Takei, Reiko Sakaguchi, Akihiro Kitani, Ryohei Nagaoka, Masato Tomizawa, Yuu Miyazaki, Hitoshi Miyachi, Tomohiro Numata, Sho Kakizawa, Miyuki Nishi, Yasuo Mori, Hiroshi Takeshima
Science signaling ( American Association for the Advancement of Science ({AAAS}) ) 12 ( 576 ) 2019年04月
During endochondral ossification of long bones, the proliferation and differentiation of chondrocytes cause them to be arranged into layered structures constituting the epiphyseal growth plate, where they secrete the cartilage matrix that is subsequently converted into trabecular bone. Ca2+ signaling has been implicated in chondrogenesis in vitro. Through fluorometric imaging of bone slices from embryonic mice, we demonstrated that live growth plate chondrocytes generated small, cell-autonomous Ca2+ fluctuations that were associated with weak and intermittent Ca2+ influx. Several genes encoding Ca2+-permeable channels were expressed in growth plate chondrocytes, but only pharmacological inhibitors of transient receptor potential cation channel subfamily M member 7 (TRPM7) reduced the spontaneous Ca2+ fluctuations. The TRPM7-mediated Ca2+ influx was likely activated downstream of basal phospholipase C activity and was potentiated upon cell hyperpolarization induced by big-conductance Ca2+-dependent K+ channels. Bones from embryos in which Trpm7 was conditionally knocked out during ex vivo culture exhibited reduced outgrowth and displayed histological abnormalities accompanied by insufficient autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the growth plate. The link between TRPM7-mediated Ca2+ fluctuations and CaMKII-dependent chondrogenesis was further supported by experiments with chondrocyte-specific Trpm7 knockout mice. Thus, growth plate chondrocytes generate spontaneous, TRPM7-mediated Ca2+ fluctuations that promote self-maturation and bone development.
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徐脈性不整脈遺伝子変異TRPM4-E7Kチャネルの数理モデルシミュレーション
胡 耀鵬, 平石 敬三, 倉原 琳, 沼田 朋大, 井上 隆司
日本生理学雑誌 ( (一社)日本生理学会 ) 81 ( 1 ) 4 - 4 2019年02月
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Cell Volume-Activated and Volume-Correlated Anion Channels in Mammalian Cells: Their Biophysical, Molecular, and Pharmacological Properties.
Okada Y, Okada T, Sato-Numata K, Islam MR, Ando-Akatsuka Y, Numata T, Kubo M, Shimizu T, Kurbannazarova RS, Marunaka Y, Sabirov RZ
Pharmacological reviews 71 ( 1 ) 49 - 88 2019年01月
There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca2+-activated Cl- channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs.
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Roles of volume-regulatory anion channels, VSOR and Maxi-Cl, in apoptosis, cisplatin resistance, necrosis, ischemic cell death, stroke and myocardial infarction.
Okada Y, Numata T, Sato-Numata K, Sabirov RZ, Liu H, Mori SI, Morishima S
Current topics in membranes ( ELSEVIER ACADEMIC PRESS INC ) 83 205 - 283 2019年
Two types of anion channels are directly activated by osmotic swelling and are involved in the regulatory volume decrease (RVD) in most types of mammalian cells, and they include the volume-sensitive outwardly rectifying anion channel (VSOR), also called the volume-regulated anion channel (VRAC), and the large-conductance maxi-anion channel (Maxi-Cl). In cardiomyocytes, a splice variant of cystic fibrosis transmembrane conductance regulator anion channel (cardiac CFTR) participates in the RVD mechanism under β-adrenergic stimulation. VSOR and Maxi-Cl are also involved in facilitation of the RVD process by releasing extracellular autocrine/paracrine signals, glutamate and ATP. Apoptotic cell death starts with cell shrinkage, called the apoptotic volume decrease (AVD), which is also caused by activation of VSOR. Since VSOR is implicated not only in the AVD induction but also in the uptake of an anti-cancer drug, cisplatin, downregulation of VSOR activity is causatively involved in acquisition of cisplatin resistance in cancer cells. Necrotic cell death exhibits persistent cell swelling, called the necrotic volume increase (NVI), which is coupled to RVD dysfunction due to impaired VSOR function. Acidotoxic and lactacidosis-induced necrotic cell death is induced both by glutamate release mediated by astroglial VSOR and Maxi-Cl and by exaggerated Cl- influx mediated by neuronal VSOR under prolonged depolarization caused by activation of ionotropic glutamate receptor (iGluR) cation channels. Both VSOR and Maxi-Cl are elaborately involved, in a manner as double-edged swords, in ischemia- and ischemia-reperfusion-induced apoptotic or necrotic cell death in cerebral and myocardial cells by mediating not only Cl- transport but also release of glutamate and/or ATP. Cardiac CFTR exerts a protective action against ischemia(-reperfusion)-induced cardiac injury, called myocardial infarction (MI), which is largely necrotic. Cardiac Maxi-Cl activity may participate in protection against ischemia(-reperfusion) injury by mediating ATP release.
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がん SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する
小松 志保, 野見山 崇, 沼田 朋大, 堀川 剛, 川波 賢子, 濱口 百合子, 高橋 貴理子, 岩屋 智加予, 田中 智子, 井上 隆司, 柳瀬 敏彦
糖尿病合併症 ( (一社)日本糖尿病合併症学会 ) 32 ( Suppl.1 ) 224 - 224 2018年10月
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【消化器病漢方治療-理論と実践】理論 漢方薬作用機序の科学的解明 潤腸湯/麻子仁丸
沼田 朋大, 井上 隆司
臨床消化器内科 ( (株)日本メディカルセンター ) 33 ( 11 ) 1371 - 1376 2018年09月
＜文献概要＞便秘は日常生活に大きなストレスをもたらす.便秘の症状を緩和するためにいくつかの作用機序の異なる薬物が使用されているが,漢方薬をはじめそれらの分子作用基盤はよく理解されていないことが多い.最近,慢性便秘漢方薬である潤腸湯/麻子仁丸の効果機序が分子生理学的手法を用いた機能解析によって明らかにされた.興味深いことに潤腸湯の投与は,細胞内cAMP上昇によってCFTRチャネルの活性化を介したクロライドイオン流出を誘発し,腸管内への水分泌を促進することが明らかとなった.本稿では,この話題を中心に,類似の作用機序を有し,近年新しい範疇の緩下剤として注目されている小腸刺激性下剤,ルビプロストンについても議論したい.
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TRPM7-mediated spontaneous Ca<sup>2+</sup> entry regulates the proliferation and differentiation of human leukemia cell line K562.
Takahashi K, Umebayashi C, Numata T, Honda A, Ichikawa J, Hu Y, Yamaura K, Inoue R
Physiological reports ( WILEY ) 6 ( 14 ) e13796 2018年07月
Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real-time RT-PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+ ]i ) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+ ]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2-APB, Gd3+ , and FTY720 dose dependently decreased basal [Ca2+ ]i . A spontaneously active inward current (Ispont ) contributive to basal [Ca2+ ]i was identified by the nystatin-perforated whole-cell recording. Ispont permeated Ca2+ comparably to Na+ , and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA-mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced γ-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.
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Tomohiro Numata, Kaori Sato-Numata, Yasunobu Okada, Ryuji Inoue
Journal of Natural Medicines ( Springer Nature ) 72 ( 3 ) 694 - 705 2018年06月
Constipation is a common symptom frequently compromising the quality of daily life. Several mechanistically different drugs have been used to mitigate constipation, including Japanese herbal (Kampo) medicines. However, the mechanisms of their actions are often not well understood. Here we aimed to investigate the molecular mechanisms underlying the effects of Junchoto (JCT), a Kampo medicine empirically prescribed for chronic constipation. Cl− channel activity was measured by the patch-clamp method in human cystic fibrosis transmembrane conductance regulator (CFTR)-expressing HEK293T cells and human intestinal Caco-2 cells. cAMP was measured by a luciferase-based assay. Cell volume change was measured by a particle-sizing and particle-counting analyzer and video-microscopic measurement. In both CFTR-expressing HEK293T and Caco-2 cells, JCT dose-dependently induced whole-cell currents showing typical biophysical and pharmacological features of CFTR. Robust expression of CFTR was confirmed by RT-PCR and Western blotting in Caco-2 cells. Luciferase-based measurement revealed that JCT increases intracellular cAMP levels. Administration of the adenylate cyclase inhibitor SQ22536 or CFTR inhibitor-172, or treatment with small interfering RNAs (siRNA) targeting CFTR, abolished JCT-induced whole-cell currents, suggesting that elevated intracellular cAMP by JCT causes activation of CFTR in Caco-2 cells. Finally, blockade of CFTR activity by CFTR inhibitor-172 or siRNA-knockdown of CFTR or application of SQ22536 markedly reduced the degree of cell volume decrease induced by JCT. JCT can induce a Cl− efflux through the CFTR channel to promote water secretion, and this effect is likely mediated by increased cAMP production.
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高橋 貴理子, 沼田 朋大, 山浦 健, 井上 隆司
日本生理学雑誌 ( (一社)日本生理学会 ) 80 ( 2 ) 31 - 32 2018年05月
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沼田 朋大, 佐藤 かお理[沼田], 井上 隆司
日本生理学雑誌 ( (一社)日本生理学会 ) 80 ( 2 ) 32 - 33 2018年05月
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SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する
小松 志保, 野見山 崇, 沼田 朋大, 岩屋 智加予, 川波 賢子, 濱口 百合子, 田中 智子, 田邉 真紀人, 井上 隆司, 柳瀬 敏彦
糖尿病 ( (一社)日本糖尿病学会 ) 61 ( 4 ) 291 - 291 2018年04月
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SGLT2阻害薬イプラグリフロジンは乳癌細胞増殖を抑制する
小松 志保, 野見山 崇, 沼田 朋大, 堀川 剛, 川波 賢子, 濱口 百合子, 高橋 貴理子, 岩屋 智加予, 田中 智子, 井上 隆司, 柳瀬 敏彦
糖尿病 ( (一社)日本糖尿病学会 ) 61 ( Suppl.1 ) S - 232 2018年04月