新しい機能性遺伝子同定技術の創出と成人病性血管障害関連遺伝子探索への応用

金沢大学医学部本研究の目的は、任意の表現形質を担う遺伝子を機能面から系統的かつ簡便に同定する技術として代表者らが独自に考案したAntisense Display法を新しい機能性遺伝子スクリーニング技術として確立し、成人病性血管障害関連遺伝子、特に血管新生抑制性遺伝子と糖尿病で起こる血管周皮細胞喪失の原因となる遺伝子の分離に適用することである。平成12年度の研究は、当初計画どおりに遂行され、以下成果を得た。1.種々のホスホロチオエート型アンチセンス(AS)プールを調製してヒト微小血管内皮細胞の培養培地に添加し、細胞増殖変化を指標にスクリーニング条件の検討を行なった。この結果、16種の配列を含む鎖長10merのASプールが最も良好に作用したことから、以後このレパートリーをスクリーニングに用いることに決定した。2.1プールあたり16種のA3からなる計512のプールを各3μmolスケールで合成・精製し、スクリーニング用の新規アンチセンスレパートリーの調製を完了した。3.各ASプールをヒト微小血管内皮細胞の培養培地に添加し、内皮細胞の増殖促進を指標に血管新生抑制性遺伝子の一次スクリーニングを開始した。4.後期糖化反応生成物(advanced glycation endproducts,AGE)による周皮細胞喪失(pericyte loss)に関与する遺伝子のスクリーニングのためのアッセイ条件を決定した。高次スクリーニングの過程では、AS配列の鎖長を順次長くし標的配列分離の特異性を上げる等の改良を図る予定である。研究課題/領域番号:12204050, 研究期間(年度):2000出典：研究課題「新しい機能性遺伝子同定技術の創出と成人病性血管障害関連遺伝子探索への応用」課題番号12204050（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-12204050/）を加工して作

インスリン産生細胞活性化因子の精製とその性質

金沢大学医学部インスリンを産生する膵ランゲルハンス(ラ)島β細胞はきわめて再生・増殖能に乏しく、これが糖尿病発症の大きな要因の一つとされている。膵β細胞増殖を活性化する因子を明らかにできれば、これまでほとんど不明であった膵β細胞の増殖機構の解明および糖尿病患者自身の膵β細胞を再生・増殖させ治癒させるという新しい治療法開発が期待できる。本研究の目的は、申請者らが最近新しく見出したインスリン産生細胞活性化因子を精製するとともにその性質を明らかにすることである。平成8年度の研究は、以下のとおりに進行した。1.申請者らが膵β細胞増殖を刺激する因子を分泌することを見出した細菌の培養液より、(1)イオン交換クロマトグラフィー(Q Sepharose)、(2)ヒドロキシアッパタイトクロマトグラフィー、(3)疎水相互作用クロマトグラフィー(Phenyl Superose)、(4)ゲルろ過(Superdex 75)を用いて、膵ラ島DNAへの^3H-チミジン取込を指標に活性化因子を精製した。2.上記の最終精製標品(Superdex 75画分)をSDS-ポリアクリルアミドゲル電気泳動法で解析を行ったところ、分子量26,000の蛋白が検出された。3.Superdex 75画分を単離ランゲルハンス島の培養に添加してDNAへの^3H-チミジン取込を解析したところ、用量依存的に取込が増大し約1ng/mlの濃度で最大となった。4.Superdex 75画分をSDS-ポリアクリルアミドゲル電気泳動後PVDF膜に転写し、26kDa蛋白のアミノ酸配列を決定したところ、N-末端から11アミノ酸の配列が得られた。当該アミノ酸配列を用いてデータベース検索を行ったが、有意な相同性を有する蛋白は存在せず、本蛋白はこれまで報告されていない新規の蛋白と考えられた。研究課題/領域番号:08680645, 研究期間(年度):1996出典：研究課題「インスリン産生細胞活性化因子の精製とその性質」課題番号08680645（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-08680645/）を加工して作

Regulation of alternative splicing of the receptor for advanced glycation endproducts (RAGE) through G-rich cis-elements and heterogenous nuclear ribonucleoprotein H

金沢大学医薬保健研究域医学系Receptor for advanced glycation endproducts (RAGE) is a cell-surface receptor. The binding of ligands to membrane-bound RAGE (mRAGE) evokes cellular responses involved in various pathological processes. Previously, we identified a novel soluble form, endogenous secretory RAGE (esRAGE) generated by alternative 5′ splice site selection in intron 9 that leads to extension of exon 9 (exon 9B). Because esRAGE works as an antagonistic decoy receptor, the elucidation of regulatory mechanism of the alternative splicing is important to understand RAGE-related pathological processes. Here, we identified G-rich cis-elements within exon 9B for regulation of the alternative splicing using a RAGE minigene. Mutagenesis of the G-rich cis-elements caused a drastic increase in the esRAGE/mRAGE ratio in the minigene-transfected cells and in loss of binding of the RNA motif to heterogenous nuclear ribonucleoprotein (hnRNP) H. On the other hand, the artificial introduction of a G-stretch in exon 9B caused a drastic decrease in the esRAGE/mRAGE ratio accompanied by the binding of hnRNP H to the RNA motif. Thus, the G-stretches within exon 9B regulate RAGE alternative splicing via interaction with hnRNP H. The findings should provide a molecular basis for the development of medicines for RAGE-related disorders that could modulate esRAGE/mRAGE ratio. © The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Low molecular weight heparin suppresses receptor for advanced glycation end products-mediated expression of malignant phenotype in human fibrosarcoma cells

医薬保健研究域医学系The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor and its engagement by ligands such as high mobility group box 1 (HMGB1) is implicated in tumor growth and metastasis. Low molecular weight heparin (LMWH) has an antagonistic effect on the RAGE axis and is also reported to exert an antitumor effect beyond the known activity of anticoagulation. However, the link between the anti-RAGE and antitumor activities of LMWH has not yet to be fully elucidated. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, invasion, and metastasis by blocking the RAGE axis using in vitro and in vivo assay systems. Stably transformed HT1080 human fibrosarcoma cell lines were obtained, including human full-length RAGE-overexpressing (HT1080RAGE), RAGE dominant-negative, intracellular tail-deleted RAGE-overexpressing (HT1080dnRAGE), and mock-transfected control (HT1080mock) cells. Confocal microscopy showed the expression of HMGB1 and RAGE in HT1080 cells. The LMWH significantly inhibited HMGB1-induced NFκB activation through RAGE using an NFκB-dependent luciferase reporter assay and the HT1080 cell lines. Overexpression of RAGE significantly accelerated, but dnRAGE expression attenuated HT1080 cell proliferation and invasion in vitro, along with similar effects on local tumor mass growth and lung metastasis in vivo. Treatment with LMWH significantly inhibited the migration, invasion, tumor formation, and lung metastasis of HT1080RAGE cells, but not of HT1080mock or HT1080dnRAGE cells. In conclusion, this study revealed that RAGE exacerbated the malignant phenotype of human fibrosarcoma cells, and that this exacerbation could be ameliorated by LMWH. It is suggested that LMWH has therapeutic potential in patients with certain types of malignant tumors. © 2013 Japanese Cancer Association

Low-molecular weight fractions of Japanese soy sauce act as a RAGE antagonist via inhibition of RAGE trafficking to lipid rafts

Advanced glycation end-products (AGE) have been implicated in aging and the pathogenesis of diabetic complications, inflammation, Alzheimer\u27s disease, and cancer. AGE engage the cell surface receptor for AGE (RAGE), which in turn elicits intracellular signaling, leading to activation of NF-κB to cause deterioration of tissue homeostasis. AGE are not only formed within our bodies but are also derived from foods, endowing them with flavor. In the present study, we assessed the agonistic/antagonistic effects of food-derived AGE on RAGE signaling in a reporter assay system and found that low-molecular weight AGE can antagonize the action of AGE-BSA. Foods tested were Japanese soy sauce, coffee, cola, and red wine, all of which showed fluorescence characteristics of AGE. Soy sauce and coffee contained Nε-carboxymethyl-lysine (CML). Soy sauce, coffee, and red wine inhibited the RAGE ligand-induced activation of NF-κB, whereas cola had no effect on the ligand induction of NF-κB. The liquids were then fractionated into high-molecular weight (HMW) fractions and low-molecular weight (LMW) fractions. Soy sauce-, coffee-, and red wine-derived LMW fractions consistently inhibited the RAGE ligand induction of NF-κB, whereas the HMW fractions of these foods activated RAGE signaling. Using the LMW fraction of soy sauce as a model food-derived RAGE antagonist, we performed a plate-binding assay and found that the soy sauce LMW fractions competitively inhibited AGE-RAGE association. Further, this fraction significantly reduced AGE-dependent monocyte chemoattractant protein-1 (MCP-1) secretion from murine peritoneal macrophages. The LMF from soy sauce suppressed the AGE-induced RAGE trafficking to lipid rafts. These results indicate that small components in some, if not all, foods antagonize RAGE signaling and could exhibit beneficial effects on RAGE-related diseases. © 2013 The Royal Society of Chemistry

Development and prevention of advanced diabetic nephropathy in RAGE-overexpressing mice

金沢大学医薬保健研究域医学系Vascular complications arising from multiple environmental and genetic factors are responsible for many of the disabilities and short life expectancy associated with diabetes mellitus. Here we provide the first direct in vivo evidence that interactions between advanced glycation end products (AGEs; nonenzymatically glycosylated protein derivatives formed during prolonged hyperglycemic exposure) and their receptor, RAGE, lead to diabetic vascular derangement. We created transgenic mice that overexpress human RAGE in vascular cells and crossbred them with another transgenic line that develops insulin-dependent diabetes shortly after birth. The resultant double transgenic mice exhibited increased hemoglobin A1c and serum AGE levels, as did the diabetic controls. The double transgenic mice demonstrated enlargement of the kidney, glomerular hypertrophy, increased albuminuria, mesangial expansion, advanced glomerulosclerosis, and increased serum creatinine compared with diabetic littermates lacking the RAGE transgene. To our knowledge, the development of this double transgenic mouse provides the first animal model that exhibits the renal changes seen in humans. Furthermore, the phenotypes of advanced diabetic nephropathy were prevented by administering an AGE inhibitor, (±)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195), thus establishing the AGE-RAGE system as a promising target for overcoming this aspect of diabetic pathogenesis

Nurture vs. nature in diabetic vasculopathy: roles of advanced glycation endproducts and the receptor for them

金沢大学大学院医学部医学系研究科As is diabetes itself, diabetic vasculopathy is a multifactor disease. Studies conducted in this lab revealed advanced glycation endproducts (AGE) as the major environmental account for vascular cell derangement characteristic of diabetes, and the receptor for AGE (RAGE) as the major genetic factor that responds to them. AGE fractions that caused the vascular derangement were proven to be RAGE ligands. When made diabetic, RAGE-overexpressing transgenic mice exhibited the exacerbation of the indices of nephropathy, and this was prevented by the inhibition of AGE formation. We also created RAGE-deficient mice. They showed marked amelioration of diabetic nephropathy. Extracellular signals and nuclear factors that induce the transcription of human RAGE gene were also identified, which would be regarded as risk factors of diabetic complications. Through an analysis of vascular polysomal poly(A)+ RNA, we came across a novel splice variant coding for a soluble RAGE protein, and named it endogenous secretory RAGE (esRAGE). esRAGE was able to capture AGE ligands and neutralize the AGE action on endothelial cells, suggesting that this variant has a potential to protect blood vessels from diabetes-induced injury. The AGE–RAGE system should thus be regarded as a candidate molecular target for overcoming this life- and quality of life (QOL)-threatening disease

Endogenous secretory receptor for advanced glycation end-products inhibits amyloid-β 1-42 uptake into mouse brain

The cell-surface receptor for advanced glycation end-products (RAGE) has been implicated in the development of diabetic vascular complications and Alzheimer\u27s disease. RAGE has been considered to be involved in amyloid-β 1-42 (Aβ 1-42) uptake into brain. In the present study, we demonstrate that endogenous secretory RAGE (esRAGE), a decoy form of RAGE generated by alternative RNA processing, is able to inhibit Aβ 1-42 influx into mouse brain. Surface plasmon resonance and competitive binding assays revealed that human Aβ 1-42 interacted with human esRAGE within the immunoglobulin V type region. We next examined the uptake and distribution of 125I-labeled human Aβ 1-42 in various organs and body fluids of newly created mice overexpressing human esRAGE as well as RAGE-null and wild-type (WT) mice. The transition of the 125I-labeled Aβ 1-42 from circulation to brain parenchyma peaked at 30 min after the injection into WT mice, but this was significantly blunted in esRAGE-overexpressing and RAGE-null mice. Significant reduction in 125I-labeled Aβ 1-42-derived photo-stimulated luminescence were marked in ventricles, cerebral cortex, hippocampus, especially CA1 and CA3 regions, putamen, and thalamus. The results thus suggest the potential of esRAGE in protection against the development of Alzheimer\u27s disease. © 2012 - IOS Press and the authors. All rights reserved.Thesis of Sugihara Takahiro / 学位論文 医学甲第2227 杉原 崇

The role of AGE-Rage system in the development of diabetic nephropathy in vivo

金沢大学大学院医学部医学系研究科Vascular complications are what eventually threaten the lives of diabetic patients. Here we show direct in vivo evidence that the interaction between advanced glycation end products (AGE), the formation of which is accelerated during prolonged hyperglycemic exposure, and a cell surface receptor for AGE (RAGE) is the major cause of such complications. We created transgenic mice that overexpress human RAGE in vascular cells and crossbred them with another transgenic line which develops insulin-dependent diabetes early after birth. The resultant double transgenic mice exhibited accelerated kidney changes compared with single transgenic littermates, and the nephropathy was ameliorated by an inhibitor of AGE formation. The AGE–RAGE system will thus be a promising target for overcoming diabetic complications