Simulated microgravity influences circadian rhythm of NIH3T3 cells

Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm of <i>Per1</i> and <i>Per2</i> synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation of <i>Kras</i>, <i>Raf1</i> and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt the circadian rhythm of <i>Per1</i> and <i>Per2</i> synchronized after simulated microgravity treatment. Together, our results unveil that simulated microgravity could act like a zeitgeber to influence the circadian rhythm of NIH3T3 by acting on MEK/ERK pathway, indicating that MEK/ERK pathway may act as a bridge which connects cells mechanotransduction pathway and circadian rhythm regulation.

重力对地球所有生物均存在显著影响，其中包括生物的昼夜节律（circadian rhythm）；昼夜节律对于蓝藻、真菌、人类等各类生物的生理活动协调均具有核心意义。诸多研究表明，外太空的微重力环境会影响宇航员与啮齿类动物的昼夜节律，但其背后的调控机制仍未明确。本研究利用旋转细胞培养系统（rotary cell culture system）模拟微重力环境，探究了模拟微重力对NIH3T3细胞昼夜节律的调控作用。本实验发现，模拟微重力不仅会改变部分核心昼夜节律基因的mRNA表达水平，还会改变经豆蔻酰佛波醇乙酯（phorbol myristate acetate，常简称PMA）处理后同步化的*Per1*与*Per2*的昼夜节律。值得注意的是，经2小时模拟微重力处理后，MEK/ERK信号通路会被瞬时激活，同时*Kras*、*Raf1*以及p-ERK1/ERK2的表达水平显著上调。此外，MEK/ERK通路的选择性抑制剂U0126能够破坏模拟微重力处理后同步化的*Per1*与*Per2*的昼夜节律。综上，本研究结果表明，模拟微重力可通过调控MEK/ERK通路，发挥类似授时因子（zeitgeber）的作用，进而影响NIH3T3细胞的昼夜节律；这提示MEK/ERK通路或可作为连接细胞机械转导通路与昼夜节律调控的桥梁。