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

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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.