Cellular responses to altered gravity: in vitro an in vivo insights into skeletal dynamics

Since the appearance of life on Earth, approximately 3.8 billion years ago, all organisms have been subjected to the continuous force of gravity, which has significantly influenced biological evolution and the development of organic structures. Gravity affects various tissues, particularly bone, which undergoes remodeling in response to changes in gravitational forces, such as those experienced in microgravity or hypergravity conditions. This study investigates the effects of altered gravity on bone tissue and the role of lipocalin-2 (LCN-2), a protein whose expression is influenced by microgravity condition. In vitro experiments, were conducted using the MLO-Y4 murine osteocyte cell line to assess the impact of LCN-2 on bone metabolism. The addition of LCN-2 at a concentration of 200 ng/µL was found to increase the expression of peroxisome proliferator-activated receptor gamma (Pparγ), sclerostin (Sost), and osteocalcin (Ocn), podoplanin (E11) suggesting a role in bone cell differentiation and homeostasis. However, LCN-2 did not induce apoptosis in osteocytes, as indicated by the unchanged Bax/Bcl2 ratio. Nevertheless, it did increase the expression of senescence-associated genes such as P53, P21, and P16, hinting at a potential role in osteocyte aging. Additionally, the interaction between simulated microgravity, using the Random Positioning Machine, and LCN-2 presence was examined. Simulated microgravity in combination with LCN-2 revealed a synergistic effect on modulating the Bax/Bcl2 ratio, while senescence genes were predominantly influenced by microgravity alone without a synergistic effect with LCN-2. Elevated levels of LCN-2 were detected in the serum of hindlimb unloading (HLU) mice, which simulates microgravity conditions in rodents. A higher concentration of LCN-2 (1000 ng/µL) was then tested, revealing similar impacts on the E11 gene, associated with bone homeostasis, but not on genes related to bone cell differentiation. The influence on apoptosis and senescence was observed, although the changes were not statistically significant, with the exception of P53. More investigations are needed to elucidate the complex role of LCN-2 in bone homeostasis. The in vivo segment of the study assessed the effects of hypergravity through a 14-day preliminary experiment followed by a 27-day extended investigation. Health indicators such as water intake and body weight were monitored, along with gene expression in the femurs and tibiae of the mice. Results suggested that a sustained exposure to hypergravity (3g) for 27 days prompted a shift towards bone deposition, as evidenced by the expression of bone formation markers Col1a1 and Ocn and remodeling markers Rankl and Opg. These findings were supported by MicroCT scans showing increased bone volume and cortical thickness in hypergravity-exposed mice compared to controls. Inversely to the effects seen in simulated microgravity, LCN-2 expression was downregulated by hypergravity, underscoring its potential role in the body's response to changes in gravitational forces.