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航空航天微重力，我们首次发现SMG降低了粘着斑激酶(FAK)/ras同源家族成员A(RhoA)的活性，并证明SMG通过调节FAK/RhoA调节的mTORC1途径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出现的骨丢失已被发现与抑制Wnt调节的成骨细胞分化有关。但是，控制WNT的上游信号仍然难以捉摸。 假设。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。通过取消FAK/RhoA调控的Wnt通路，评价SMG对体外成骨细胞分化和体内骨丢失的抑制作用。 求婚。为了验证SMG的作用，我们将收集在T25培养瓶或室内培养玻片中生长的永生成骨细胞MC3T3，在地面条件下(1g)或在模拟SMG条件的三维旋转回转器(µg)上生长3天，以进行各种细胞和分子特征的研究。包括通过共聚焦显微镜评估SMG诱导的细胞骨架和局部粘连的改变，通过实时聚合酶链式反应(RT-PCR)和Western blotting分析SMG诱导的FAK/RhoA和Wnt信号的下调以及Wnt调控的成骨细胞分子(-catenin、LEF1、Runx2、OPG和BMP-2)。这也包括在光学显微镜下评估用茜素红染色的MC3T3成骨细胞SMG减少矿化的情况。为了评估FAK/RhoA是否是Wnt的上游信号，我们将重复上述实验，在1g培养的MC3T3细胞中检测成骨蛋白的表达和成骨细胞的矿化，在加入或不加入FAK和RhoA特异性抑制剂的培养液中下调FAK和RhoA。为了进一步证实这一点，我们将利用微克培养的MC3T3细胞在加或不加细胞毒坏死因子-1(FAK/RhoA的激活剂)的培养液中重复上述实验，以评估CNF1是否能逆转SMG对FAK/RhoA/Wnt调节的分子的抑制和成骨细胞的矿化。此外，我们还将评估CNF1是否能在后肢卸载(HU)模型中逆转SMG对成骨细胞骨形成的抑制作用，HU是一种耐受性良好的体内模拟微重力的方法，在该模型中，喂食或不喂食CFN1的小鼠的尾巴将被悬吊2周。小鼠股骨和胫骨的骨密度将通过组织学和微型计算机断层扫描(MICR-CT)分析进行测量。冲击力。FAK/RhoA-Wnt网络的新发现可能为有骨质丢失风险的宇航员和骨质疏松患者的治疗开发带来新的靶点。

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.