GDF11 activates AMPK-dependent mitophagy to drive osteogenic differentiation of rat bone marrow mesenchymal stem cells

BACKGROUND: Growth differentiation factor 11 (GDF11) has emerged as a potential regulator of bone regeneration; however, the molecular mechanisms through which it influences osteogenic differentiation, particularly in relation to mitochondrial quality control, remain unclear. This study aimed to elucidate the role of adenosine monophosphate-activated protein kinase (AMPK)-dependent mitophagy in GDF11-mediated osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMMSCs). METHODS: rBMMSCs were induced toward osteogenic differentiation with or without GDF11 treatment. To specifically inhibit AMPK-dependent mitophagy, Compound C, an AMPK inhibitor, was employed. Osteogenic differentiation was evaluated using alkaline phosphatase (ALP) staining and activity assays, while Alizarin Red S (ARS) staining was performed to assess matrix mineralization. The expression of Mitophagy- and osteogenesis-associated markers was analyzed through immunofluorescence staining, quantitative real-time PCR, and western blotting. RESULTS: GDF11 significantly enhanced the osteogenic differentiation of rBMMSCs, as evidenced by increased ALP activity, more intense ALP staining, enhanced calcium nodule formation, and elevated expression of ALP and RUNX2. GDF11 activated mitochondrial function by promoting AMPK phosphorylation and inducing Mitophagy. Inhibition of AMPK significantly impaired Mitophagy, while Compound C-mediated blockade of AMPK-dependent mitophagy not only suppressed basal osteogenic differentiation but also abolished the pro-osteogenic effects of GDF11. This was reflected by a pronounced reduction in GDF11-induced ALP activity, mineralization, and the expression of key osteogenic genes at both the mRNA and protein levels. CONCLUSION: GDF11 enhances the osteogenic differentiation of rBMMSCs by activating AMPK-dependent mitophagy. These findings identify AMPK-dependent Mitophagy as a pivotal mechanism mediating the osteogenic actions of GDF11, providing new mechanistic insights that may guide the development of novel strategies for bone regeneration.