Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation

Bone remodeling is a continuous physiological process that requires constant generation of new osteoblasts from mesenchymal stem cells (MSCs). Differentiation of MSCs to osteoblast requires a metabolic switch from glycolysis to increased mitochondrial respiration to ensure the sufficient energy supply to complete this process. As a consequence of this increased mitochondrial metabolism, the levels of endogenous reactive oxygen species (ROS) rise. In the current study we analyzed the role of forkhead box O3 (FOXO3) in the control of ROS levels in human MSCs (hMSCs) during osteogenic differentiation. Treatment of hMSCs with H₂O₂ induced FOXO3 phosphorylation at Ser294 and nuclear translocation. This ROS-mediated activation of FOXO3 was dependent on mitogen-activated protein kinase 8 (MAPK8/JNK) activity. Upon FOXO3 downregulation, osteoblastic differentiation was impaired and hMSCs lost their ability to control elevated ROS levels. Our results also demonstrate that in response to elevated ROS levels, FOXO3 induces autophagy in hMSCs. In line with this, impairment of autophagy by autophagy-related 7 (ATG7) knockdown resulted in a reduced capacity of hMSCs to regulate elevated ROS levels, together with a reduced osteoblast differentiation. Taken together our findings are consistent with a model where in hMSCs, FOXO3 is required to induce autophagy and thereby reduce elevated ROS levels resulting from the increased mitochondrial respiration during osteoblast differentiation. These new molecular insights provide an important contribution to our better understanding of bone physiology.

骨重建（Bone remodeling）是一种持续的生理过程，需要不断由间充质干细胞（mesenchymal stem cells, MSCs）分化生成新的成骨细胞。间充质干细胞向成骨细胞分化的过程，需要发生从糖酵解到线粒体呼吸增强的代谢转换，以提供充足能量完成该过程。这种线粒体代谢增强的后果，会导致内源性活性氧（endogenous reactive oxygen species, ROS）水平升高。在本研究中，我们分析了叉头框转录因子O3（forkhead box O3, FOXO3）在人源间充质干细胞（human MSCs, hMSCs）成骨分化过程中对ROS水平的调控作用。用H₂O₂处理hMSCs可诱导FOXO3在Ser294位点发生磷酸化，并使其发生核转位。这种由ROS介导的FOXO3激活依赖于丝裂原活化蛋白激酶8（mitogen-activated protein kinase 8, MAPK8/JNK）的活性。当FOXO3表达被下调后，成骨细胞分化过程受到损伤，hMSCs也丧失了调控ROS水平升高的能力。我们的研究结果还表明，在ROS水平升高的情况下，FOXO3可诱导hMSCs发生细胞自噬。与此一致的是，通过敲低自噬相关蛋白7（autophagy-related 7, ATG7）抑制细胞自噬，会降低hMSCs调控ROS水平升高的能力，同时也会削弱成骨细胞分化能力。综上，我们的研究结果支持如下模型：在hMSCs中，FOXO3可通过诱导细胞自噬，降低成骨细胞分化过程中因线粒体呼吸增强而升高的ROS水平。这些新的分子机制认知，为我们更好地理解骨生理过程提供了重要的理论贡献。