Active mitochondria support osteogenic differentiation by stimulating β-catenin acetylation

Abstract

Bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) can differentiate into osteoblasts (OBs), adipocytes, or chondrocytes. As BMSCs undergo OB differentiation, they up-regulate mitochondrial oxidative phosphorylation (OxPhos). Here, we investigated the mechanism(s) connecting mitochondrial OxPhos to OB differentiation. First, we found that treating BMSC-like C3H10T1/2 cells with an OxPhos inhibitor reduces their osteogenic potential. Interestingly, ATP levels were not reduced, as glycolysis compensated for the decreased OxPhos. Thus, mitochondria support OB differentiation not only by supplying ATP, but also by other mechanisms. To uncover these mechanisms, we stimulated OxPhos in C3H10T1/2 cells by replacing media glucose with galactose and observed that this substitution increases both OxPhos and osteogenesis even in the absence of osteoinducers. β-Catenin, an important signaling pathway in osteogenesis, was found to be responsive to OxPhos stimulation. β-Catenin activity is maintained by acetylation, and mitochondria generate the acetyl donor acetyl-CoA, which upon entering the Krebs cycle is converted to citrate capable of exiting mitochondria. Cytosolic citrate is converted back to acetyl-CoA by ATP citrate lyase (ACLY). We found that inhibiting ACLY with SB204990 (SB) reverses the galactose-induced β-catenin activity and OB differentiation. This suggested that acetylation is involved in β-catenin activation after forced OxPhos stimulation, and using immunoprecipitation, we indeed detected SB-sensitive β-catenin acetylation. Both β-catenin acetylation and activity increased during osteoinduction coincident with OxPhos activation. These findings suggest that active mitochondria support OB differentiation by promoting β-catenin acetylation and thus activity.

Mitochondria transfer enhances bone marrow mesenchymal stem cell functions and promotes bone defect healing

“…Cell differentiation is also associated with an increase in mitochondrial content and activity, according to previous studies [45][46][47]. The activation of mitochondrial OXPHOS in BMSCs is known to trigger osteogenic differentiation via acetylation and activation of β-catenin signaling [48].…”

Concise Review: The Regulatory Mechanism of Lysine Acetylation in Mesenchymal Stem Cell Differentiation

“…Differentiation of MSCs ( Table 3). The degree of histone acetylation of related regulatory genes may reflect the maintenance and differentiation status of MSCs [79]. The acetylation of H3K9 and H3K14 (H3K9ac, H3K14ac) is a marker of gene activation [80].…”

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

“…The addition of 50 μg/mL ascorbate (Sigma A4544) and 2.5 mM β-glycerol phosphate (USB Corp Cleveland, OH, 21655), 25 ng/mL BMP2 (R&D systems 355-BM-050/CF), 25 ng/mL Wnt3a (R&D systems 5036-WN-010), 5 ng/mL IGF1 (Sigma 13769-50UG), 0.2 ng/mL TGFβ (R&D systems 240-B), or 1 ng/mL PTH aa1-34 (R&D systems 3011/1) to αMEM media induced osteogenesis. To confirm osteogenesis, cells were stained with OB-specific ALP-specific stain (Thermo NBT/BCIP 1-step 34042) and with 0.5 % CV (Sigma C3886) to determine total cell count as previously described (13). For metabolic inhibitory studies, cells were incubated with either 0.1 μg/mL Oligomycin (Oligo, Sigma 75351), 0.1 μM Antimycin A (AA, Sigma A8674), 0.1 μM Rotenone (ROT, Sigma R-8875), or 0.2 μM Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, Sigma C2920) for 48 hours prior to osteoinduction, and inhibitors were present during the entire time course of induction.…”

“…Currently, it is unclear what role metabolic plasticity plays in OB differentiation and development. Increases in OxPhos in BMSCs (10,12,13) and calvarial osteoblasts (11) have been reported; however, there remains the unanswered possibility that the observed increase in oxygen consumption is a result of decreased coupling efficiency or increases in nonmitochondrial oxygen consumption. There is also the possibility that long bone and calvarial bone OB metabolism are biologically distinct (14).…”