IADR Abstract Archives
TNAP Regulates Mitochondrial Activity and Cell Metabolism in Cranial Preosteoblasts
Objectives: We recently showed that non-specific alkaline phosphatase (TNAP) plays an essential role in promoting cranial bone progenitor cell cycle progression, cytokinesis and proliferation. It is unknown how TNAP mediates these effects. It is well known that mitochondria have a central role in the regulation of cell proliferation and metabolism. Here we provide evidence that diminished TNAP expression and activity lead to mitochondrial dysfunction and changes in cell metabolism. We hypothesize that these metabolic changes may mediate the diminished cell cycle progression of TNAP deficient cells.
Methods: In vitro studies were performed in cranial cells isolated from TNAP-/- and TNAP+/+ mice, and in MC3T3E1(C4) cells which were stably transduced with TNAP or control nontarget shRNA. Mitochondrial location and content were assayed by staining with Mitotracker (Invitrogen) and BCIP/NBT (Sigma). Mitochondria enzyme activity was assayed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Metabolic function was measured by Mito Stress Test and Cell Energy Phenotype Test (Agilent Seahorse Assay).
Results: Mitochondrial staining with BCIP/NBT showed plasma membrane staining in differentiated cranial osteoblasts and cytoplasmic staining in pre-differentiated cranial osteoblast progenitor cells. Mitochondrial staining with mitotracker revealed higher intensity of mitochondrial staining in TNAP deficient as compared to control cells, particularly in the peri-nuclear region. TNAP deficient cells exhibited significantly higher MTT activity and significantly higher levels of basal respiration, ATP production, proton leak, maximal respiration, and non-mitochondrial oxygen consumption (p<0.05). TNAP deficient cells also showed higher levels of aerobic and anaerobic respiration compared to control cells (p<0.05).
Conclusions: These results indicate that TNAP is essential for normal mitochondrial function and cell metabolism in osteoblast progenitor cells, which could explain why TNAP is needed for cell proliferation. If similar results are found in muscle cells, this could account for the muscle weakness that is seen in humans with hypophosphatasia (metabolic disorder caused by TNAP deficiency).
Methods: In vitro studies were performed in cranial cells isolated from TNAP-/- and TNAP+/+ mice, and in MC3T3E1(C4) cells which were stably transduced with TNAP or control nontarget shRNA. Mitochondrial location and content were assayed by staining with Mitotracker (Invitrogen) and BCIP/NBT (Sigma). Mitochondria enzyme activity was assayed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Metabolic function was measured by Mito Stress Test and Cell Energy Phenotype Test (Agilent Seahorse Assay).
Results: Mitochondrial staining with BCIP/NBT showed plasma membrane staining in differentiated cranial osteoblasts and cytoplasmic staining in pre-differentiated cranial osteoblast progenitor cells. Mitochondrial staining with mitotracker revealed higher intensity of mitochondrial staining in TNAP deficient as compared to control cells, particularly in the peri-nuclear region. TNAP deficient cells exhibited significantly higher MTT activity and significantly higher levels of basal respiration, ATP production, proton leak, maximal respiration, and non-mitochondrial oxygen consumption (p<0.05). TNAP deficient cells also showed higher levels of aerobic and anaerobic respiration compared to control cells (p<0.05).
Conclusions: These results indicate that TNAP is essential for normal mitochondrial function and cell metabolism in osteoblast progenitor cells, which could explain why TNAP is needed for cell proliferation. If similar results are found in muscle cells, this could account for the muscle weakness that is seen in humans with hypophosphatasia (metabolic disorder caused by TNAP deficiency).