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Cellular stress responses regulate the mammalian circadian clock

今村, 聖路 東京大学 DOI:10.15083/0002001409

2021.09.08

概要

ほぼすべての生物は概日時計と呼ばれる自律的な計時システムを備えており、睡眠覚醒リズムやホルモンリズムなど、さまざまな生理現象における約24時間のリズムが制御されている。哺乳類における概日時計の発振系は、CLOCKやBMAL1などの時計タンパク質から構成される転写・翻訳を介した負のフィードバックループが基本骨格となる。この発振系に加えて概日時計は、その振動位相を外界の環境変化に同調するための入力系も兼ね備えている。さらに、概日時計がもつ自律振動性および位相同調能という重要な特性の発揮には、時計関連タンパク質のリン酸化制御が極めて重要な役割を担うことが明らかになってきた。これまでに当研究室において、各種タンパク質キナーゼの阻害剤スクリーニングによりCLOCKおよびBMAL1のリン酸化を担う責任キナーゼが探索された。その結果、ストレス応答性のMAPKであるc-JunN-terminal Kinase(JNK)の阻害剤として知られるSP600125を培養細胞に投与すると、BMAL1のリン酸化レベルが低下し、細胞の概日性転写リズムが長周期化することが明らかにされた。そこで私は、JNKがBMAL1のリン酸化を介して時計機構へと入力するという作業仮説を立て研究を開始した。

JNKの活性化がBMAL1のリン酸化レベルに与える影響を解析するために、種々のマウスJNKアイソフォームをクローニングした。これらをBMAL1と共に繊維芽細胞に発現させてウェスタンブロット解析に供したところ、導入した各種JNKが高浸透圧刺激に依存して活性化すると共に、BMAL1のリン酸化レベルが増強した。続いて、JNKの機能阻害がBMAL1と時計発振に与える影響について解析した。SP600125は非常に標的範囲の広い薬剤であり、複数のキナーゼを同時に阻害する。そこで培養細胞に発現しているJNKアイソフォームを全て抑制できるshRNAを作製し、RNAiによりJNKをノックダウンした。shRNAを繊維芽細胞に発現させ、共免疫沈降およびウェスタンブロット解析に供したところ、細胞内在のJNK量が低下し、同時にBMAL1のリン酸化レベルが有意に低下した。さらにこのとき、繊維芽細胞の示すリズムが顕著に長周期化することを明らかにした。以上の結果より、JNKはBMAL1をリン酸化する主要なキナーゼであり、時計の発振速度を調節することが判明した。ここまでの研究成果を総合すると、細胞へのストレス刺激はリン酸化シグナルを介して概日時計機構へと入力する可能性が強く示唆された。細胞ストレス応答と概日時計機構はどちらも細胞が有する最も基礎的な生命現象であるが、これまでに両者の連関の有無や、両者を仲介する分子実体についてはほとんど知られていなかった。そこで私は、細胞へのストレス刺激とそれに惹起される細胞内ストレス応答シグナルが如何にして概日時計を制御するのかを引き続き追究した。

興味深いことに、慢性的な高あるいは低浸透圧刺激を培養細胞に与えると、細胞の示す概日リズムがそれぞれ長周期あるいは短周期化することを見出した。さらに、一過的な高あるいは低浸透圧刺激は細胞時計をリセットした。数あるストレス応答性MAPKの中でも特に、Apoptosis signal-regulating kinase(ASK)ファミリーは低あるいは高浸透圧刺激の両方に応答し、それぞれ活性化あるいは不活性化するというユニークな特性をもつ。また。ASKはJNKよりも直接的に細胞ストレスを感受する上流MAP3Kに位置する。ASK欠損マウスより胎児線維芽細胞を採取し、その概日リズムを観察したところ、浸透圧ストレスに依存した細胞リズム周期の変化やリズムのリセットが完全に抑制されることが明らかになった。続いて胎児繊維芽細胞に浸透圧刺激を与えた際の時計遺伝子群の応答プロファイルを網羅的に解析し、時計機構への入力作用点を調べた。その結果、既知の入力刺激を与えた場合とは異なった時計遺伝子群が浸透圧刺激に応答して30分以内に転写誘導(初期応答)されることを見出した。さらに、ASK欠損細胞においては浸透圧刺激に依存した時計遺伝子の発現誘導が完全に阻害されることが明らかになった。続いて、光照射がASK-KOマウスの行動リズムに与える影響を観察することにより、ASKが時計機構に対して果たす役割を個体レベルで検証した。その結果、ASK-KOマウスは光パルス照射による行動リズムの位相シフトや、恒明条件下での行動リズム周期に異常が認められた。これらの結果より、ASKは細胞外からのストレス(同調)刺激を受容して細胞内キナーゼシグナルへと変換するメディエーターとして機能し、概日時計の自律振動や位相同調に重要な役割を果たすことが示された。

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