Simulated microgravity blocks osteoblastic differentiation and mineralization leading to bone loss via suppressing the FAK/RhoA-regulated Wnt pathway

太空飞行中的微重力环境会影响人体健康，如骨质流失。我们利用三维倾斜仪模拟航天微重力环境，首次发现SMG可降低FAK/ ras同源家族成员- a （RhoA）活性，并通过调节FAK/RhoA调控的mTORC1通路抑制细胞增殖和转移（Sci Reports 8:3769, 2018）。骨质疏松症患者或宇航员在微重力下的骨质流失已被发现与抑制wnt调节的成骨细胞分化有关。但是，控制Wnt的上游信号仍然是难以捉摸的。上游信号FAK/ rhoa控制的Wnt通路调节smg诱导的成骨细胞分化抑制。* * *的目标。通过取消FAK/ rhoa控制的Wnt通路，评估smg诱导的体外成骨细胞分化和体内骨丢失的抑制作用。为了检验SMG的作用，在T25培养瓶或Chamber culture载玻片中，在地面条件下（1g）或在模拟SMG条件的三维旋转旋转恒温器（µg）上生长3天，收集不朽的成骨细胞MC3T3细胞，进行各种细胞和分子表征。这些包括通过共聚焦显微镜评估smg诱导的细胞骨架和局灶粘附的改变，以及通过实时聚合酶链反应（RT-PCR）和Western blotting分析测量smg诱导的FAK/RhoA和Wnt信号下调以及Wnt调控的成骨细胞分子（-catenin, LEF1, Runx2， OPG和BMP-2）。这些还包括光镜下用茜素红染色的MC3T3成骨细胞smg减少矿化的评估。为了评估FAK/RhoA是否为Wnt的上游信号，我们将重复上述实验，在含有或不含FAK- & RhoA特异性抑制剂以敲除FAK & RhoA的培养基中，在1g培养的MC3T3细胞中测量成骨蛋白表达和成骨矿化。为了进一步证实这一点，我们将在含有或不含细胞毒性坏死性因子-1 （FAK/RhoA的激活剂）的培养基中使用µg培养的MC3T3细胞重复上述实验，以评估CNF1是否转化smg诱导的FAK/RhoA/ wnt调节分子的抑制和成骨细胞的矿化度。此外，我们还将在后肢卸载（HU）模型中评估CNF1是否转化smg诱导的成骨细胞骨形成的抑制，这是一种在体内模拟微重力的耐受良好的方法，在该模型中，喂食或不喂食CFN1的小鼠尾巴将暂停2周。小鼠股骨和胫骨的骨密度将通过组织学和微计算机断层扫描（micr-CT）分析来测量。的影响。FAK/RhoA-Wnt网络的新发现可能为有骨质流失风险的宇航员以及骨质疏松症患者的治疗开发提供新的靶点。

The microgravity environment in spaceflights affects human health such as bone loss. By using a three-dimensional clinostat to model simulated microgravity (SMG) mimicking the aerospace microgravity, we, for the first time, discovered that SMG reduces focal adhesion kinase (FAK)/ras homology family member-A (RhoA) activity, and demonstrated that SMG inhibits cell proliferation and metastasis via modulating FAK/RhoA-regulated mTORC1 pathway (Sci Reports 8:3769, 2018). Bone loss seen in osteoporosis patients or astronauts under microgravity has been found to be related to inhibition of Wnt-regulated osteoblastic differentiation. But, the up-stream signal controlling Wnt is still elusive.***Hypothesis. The up-stream signal FAK/RhoA-controlled Wnt pathway regulates SMG-induced inhibition of osteoblastic differentiation. ***Objectives. To assess SMG-induced inhibition of in vitro osteoblastic differentiation and in vivo bone loss via abrogation of FAK/RhoA-controlled Wnt pathway.***Proposal. To examine SMG's effect, an immortal osteoblast MC3T3 cells growing in T25 culture flasks or Chamber Culture slides under ground conditions (1g) or on a three dimensionally rotated clinostat mimicking SMG conditions (µg) for 3 days will be harvested for various cellular and molecular characterizations. These include assessment of SMG-induced alteration of cytoskeleton and focal adhesions by confocal microscopy, and measurement of SMG-induced down-regulation of FAK/RhoA and Wnt signaling as well as Wnt-regulated osteoblastic molecules (-catenin, LEF1, Runx2, OPG and BMP-2) by real time-polymerase chain reaction (RT-PCR) and Western blotting analyses. These also include assessment of SMG-reduced mineralization of MC3T3 osteoblastic cells stained with Alizarin red dye by light microscopy. To assess whether FAK/RhoA is the up-stream signal for Wnt, we will repeat the above experiments to measure expression of osteoblastic proteins and osteoblastic mineralization in MC3T3 cells cultured under 1g in medium with or without FAK- & RhoA-specific inhibitors to knock-down FAK & RhoA. To further confirm it, we will repeat above experiments using MC3T3 cells cultured under µg in medium with or without cytotoxic necrotizing factor-1 (CNF1) (an activator of FAK/RhoA) to assess whether CNF1 converts SMG-induced suppression of FAK/RhoA/Wnt-regulated molecules and osteoblastic mineralization. In addition, we will also assess whether CNF1 converts SMG-induced suppression of osteoblastic bone formation in the hindlimb unloading (HU) model, a well-tolerated method mimicking microgravity in vivo, in which tails of the mice fed with or without CFN1 will be suspended for 2 weeks. The mouse bone density of femurs and tibiae will be measured by histological and micro-computed tomography (micr-CT) analyses. Impact. The novel finding of the FAK/RhoA-Wnt network may lead to a new target for therapeutic development for astronauts with risk of bone loss as well as for patients with osteoporosis.