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

航天飞行中的微重力环境会影响人体健康，如骨质流失。通过使用三维回转器模拟模拟航空航天微重力的模拟微重力（SMG），我们首次发现SMG降低粘着斑激酶（FAK）/ras同源家族成员-A（RhoA）活性，并证明SMG通过调节FAK/RhoA调节的mTORC 1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丢失与Wnt调节的成骨细胞分化的抑制有关。但是，控制Wnt的上游信号仍然难以捉摸。 假说.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。 目标.通过消除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抑制。 提议为了检查SMG的作用，将收获在地面条件（1g）下或在模拟SMG条件（μg）的三维旋转回转器上在T25培养瓶或腔室培养载玻片中生长3天的永生成骨细胞MC 3 T3细胞用于各种细胞和分子表征。这些包括通过共聚焦显微镜评估SMG诱导的细胞骨架和粘着斑改变，通过真实的时间聚合酶链反应（RT-PCR）和蛋白质印迹分析测量SMG诱导的FAK/RhoA和Wnt信号转导以及Wnt调节的成骨细胞分子（β-连环蛋白、LEF 1、Runx 2、OPG和BMP-2）下调。这些还包括通过光学显微镜评估用茜素红染料染色的MC 3 T3成骨细胞的SMG减少的矿化。为了评估FAK/RhoA是否是Wnt的上游信号，我们将重复上述实验以测量在具有或不具有FAK-和RhoA-特异性抑制剂以敲低FAK和RhoA的培养基中在Ig下培养的MC 3 T3细胞中成骨细胞蛋白的表达和成骨细胞矿化。为了进一步证实这一点，我们将使用在含有或不含有细胞毒性坏死因子-1（CNF 1）（FAK/RhoA的激活剂）的培养基中以μg培养的MC 3 T3细胞重复上述实验，以评估CNF 1是否转化SMG诱导的FAK/RhoA/Wnt调节分子的抑制和成骨细胞矿化。此外，我们还将评估CNF 1是否在后肢卸载（HU）模型中转化SMG诱导的成骨细胞骨形成抑制，这是一种耐受性良好的模拟体内微重力的方法，其中用或不用CFN 1喂养的小鼠的尾巴将悬浮2周。将通过组织学和显微计算机断层扫描（micr-CT）分析测量小鼠股骨和胫骨的骨密度。冲击FAK/RhoA-Wnt网络的新发现可能会为具有骨丢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.