Activation of mitochondria is an acute Akt-dependent response during osteogenic differentiation

Osteogenic differentiation, the process by which bone marrow mesenchymal stem/stromal (a.k.a. skeletal stem) cells and osteoprogenitors form osteoblasts, is a critical event for bone formation during development, fracture repair, and tissue maintenance. Extra- and intracellular signaling pathways triggering osteogenic differentiation are relatively well known; however, the ensuing change in cell energy metabolism is less clearly defined. Here we tested the effect of osteogenic media containing ascorbate and β-glycerol phosphate, or various osteogenic hormones and growth factors on energy metabolism in long bone (ST2)- and calvarial bone (MC3T3-E1)-derived osteoprogenitors. We show that osteogenic media, and differentiation factors, Wnt3a and BMP2, stimulate mitochondrial oxidative phosphorylation (OxPhos) with little effect on glycolysis. The activation of OxPhos occurs acutely, suggesting a metabolic signaling change rather than protein expression change. To this end, we found that the observed mitochondrial activation is Akt-dependent. Akt is activated by osteogenic media, Wnt3a, and BMP2, leading to increased phosphorylation of various mitochondrial Akt targets, a phenomenon known to stimulate OxPhos. In sum, our data provide comprehensive analysis of cellular bioenergetics during osteoinduction in cells of two different origins (mesenchyme vs neural crest) and identify Wnt3a and BMP2 as physiological stimulators of mitochondrial respiration via Akt activation. * AA : Antimycin A ALP : Alkaline phosphatase BMP2 : Bone Morphogenic Protein 2 CV : Crystal Violate ECAR : Extracellular Acidification Rate FCCP : Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone OBM : Osteoblastic media a.k.a. Ascorbic acid and beta-gycero phosphate OBs : Osteoblasts OCR : Oxygen Consumption Rate Oligo : Oligomycin OxPhos : Oxidative Phosphorylation ROS : Reactive Oxygen Species ROT : Rotenone Wnt3a : Wingless protein 3a