Osteoblast differentiation is functionally associated with decreased AMP kinase activity

Abstract

Osteoblasts, originating from mesenchymal stem cells, play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (Runx2) have been reported to be essential for osteoblast differentiation, whereas the cytoplasmic signal transduction pathways controlling the differentiation process have not been fully elucidated. AMP-activated protein kinase (AMPK) is a serine-threonine kinase generally regarded as a key regulator of cellular energy homeostasis, polarity, and division. Recent lines of evidence have indicated that the activity of the catalytic alpha subunit of AMPK is regulated through its phosphorylation by upstream AMPK kinases (AMPKKs) including LKB1. Here, we explored the role of AMPK in osteoblast differentiation using in vitro culture models. Phosphorylation of AMPKalpha was significantly decreased during osteoblastic differentiation in both primary osteoblasts and MC3T3-E1, a mouse osteoblastic cell line. Conversely, the terminal differentiation of primary osteoblasts and MC3T3-E1 cells, represented by matrix mineralization, was significantly inhibited by glucose restriction and stimulation with metformin, both of which are known activators of AMPK. Matrix mineralization of MC3T3-E1 cells was also inhibited by the forced expression of a constitutively active form of AMPKalpha. Metformin significantly inhibited gene expression of Runx2 along with osteoblast differentiation markers including osteocalcin (Ocn), bone sialo protein (Bsp), and osteopontin (Opn). Thus, our present data indicate that differentiation of osteoblasts is functionally associated with decreased AMPK activity.

AMP-activated protein kinase pathway and bone metabolism

“…When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation, supporting a role for AMPK activation in bone formation. However, these results are in contrast to the study showing that osteoblast differentiation is functionally associated with decreased AMPK activity, measured by phosphorylation levels of AMPKa subunit (Kasai et al 2009). We found a similar decrease in baseline AMPK activity with time during osteoblast differentiation.…”

“…It causes a large and sustained increase in both AMPKa phosphorylation at Thr-172 and AMPK activation measured by an in vitro peptide phosphorylation assay. Results on osteoblastic MC3T3-E1 cell line are however divergent as AICAR was reported to either phosphorylate AMPK in those cells (Kanazawa et al 2007) or to fail to induce sustained phosphorylation of AMPKa (Kasai et al 2009). Another AMPK agonist, metformin, has uniformly been shown to be a potent stimulator of AMPK activation in bone marrow progenitor cells (BMPC; Molinuevo et al 2010), primary osteoblasts (Shah et al 2010), MC3T3-E1 cells , Kasai et al 2009), ROS 17/2.8 cells (Shah et al 2010), and primary bone marrow macrophages .…”

“…In contrast with skeletal muscle, heart, and liver, and similar to adipose tissue, a1 subunit is the dominant catalytic isoform expressed in bone, suggesting that it may play a major function in skeletal metabolism. Using both RT-PCR and western blot analysis, it was shown that a1 subunit is highly expressed in bone tissue, primary osteoblasts and osteoclasts as well as in osteoblastic cell lines (Kim et al 2008, Kasai et al 2009, Quinn et al 2010, Shah et al 2010. In contrast, the a2 subunit has a very low expression in osteoblasts and bone tissue and is not expressed in ROS 17/2.8 cells and osteoclasts (Quinn et al 2010, Shah et al 2010.…”

Genetic Deletion of Catalytic Subunits of AMP-activated Protein Kinase Increases Osteoclasts and Reduces Bone Mass in Young Adult Mice

“…Interestingly, there has been contradictory evidence among published results regarding AMPK regulation of osteoblast differentiation. A study in primary osteoblasts and MC3T3-E1 osteoblastic cells found that phosphorylation of AMPK is significantly decreased during osteoblastic differentiation and that matrix mineralization is significantly inhibited by activated AMPK (13). Two other independent studies showed that metformin administration enhances osteogenic differentiation of bone marrow progenitor cells and stimulates bone lesion regeneration, whereas aminoimidazole carboxamide ribonucleotide (AICAR) treatment causes significant bone loss (14,15).…”

Metformin Enhances Osteogenesis and Suppresses Adipogenesis of Human Chorionic Villous Mesenchymal Stem Cells

“…Cortizo et al (2006) also found that metformin had no effect on ALP activity in UMR106 rat osteosarcoma cells. At the concentration of 2 mM, metformin reduced mineralization of osteoblasts (Kasai et al 2009). These conflicting results may be attributed to the different culture conditions and cell sources.…”