Rapamycin inhibits osteoblast proliferation and differentiation in MC3T3‐E1 cells and primary mouse bone marrow stromal cells

Abstract

While the roles of the mammalian target of rapamycin (mTOR) signaling in regulation of cell growth, proliferation, and survival have been well documented in various cell types, its actions in osteoblasts are poorly understood. In this study, we determined the effects of rapamycin, a specific inhibitor of mTOR, on osteoblast proliferation and differentiation using MC3T3-E1 preosteoblastic cells (MC-4) and primary mouse bone marrow stromal cells (BMSCs). Rapamycin significantly inhibited proliferation in both MC-4 cells and BMSCs at a concentration as low as 0.1 nM. Western blot analysis shows that rapamycin treatment markedly reduced levels of cyclin A and D1 protein in both cell types. In differentiating osteoblasts, rapamycin dramatically reduced osteoblast-specific osteocalcin (Ocn), bone sialoprotein (Bsp), and osterix (Osx) mRNA expression, ALP activity, and mineralization capacity. However, the drug treatment had no effect on osteoblast differentiation parameters when the cells were completely differentiated. Importantly, rapamycin markedly reduced levels of Runx2 protein in both proliferating and differentiating but not differentiated osteoblasts. Finally, overexpression of S6K in COS-7 cells significantly increased levels of Runx2 protein and Runx2 activity. Taken together, our studies demonstrate that mTOR signaling affects osteoblast functions by targeting osteoblast proliferation and the early stage of osteoblast differentiation.

Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

“…As described in the Introduction section, mTORC1 inhibition by rapamycin was shown to reduce the proliferation of the MC3T3E1 murine osteoblastic cell line and murine bone marrow stromal cells 24. Our data showed that the RANKL‐binding peptides did not stimulate the proliferation of primary osteoblastic cells and that proliferation was inhibited by rapamycin (an mTORC1 inhibitor) to a similar extent in all of the groups (Fig.…”

“…S1). We also showed that the mechanism under which the both RANKL‐binding peptides stimulated bone formation could be explained by the activation of mTORC1 signaling, since rapamycin, an mTORC1 inhibitor, is known to inhibit Runx2 activation and ALP expression 24 (Figs 4 and 5). …”

“…Since mTORC1 signaling has been shown to be one of the important signaling targets for bone formation 24, 25, we investigated whether rapamycin, an inhibitor of mTORC1 activity, could reduce the peptide‐induced acceleration of ALP activity, one of the markers of osteoblast differentiation. Before starting this experiment, we first determined a dose of rapamycin which would not show cytotoxicity in the osteoblast cultures.…”

“…Signaling through the BMP 21, PGE2 22, or Wnt receptors 23 eventually results in the stimulation of Runx2 expression, an early marker of osteoblast differentiation. It was recently demonstrated that rapamycin, which is an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1), a serine/threonine kinase, inhibited osteoblast differentiation through the reduction of Runx2 expression in an osteoblast cell line and bone marrow‐derived stromal cells 24. Another report demonstrated that the osteoblast‐specific deletion of Raptor, one of the components of mTORC1, showed the osteopenic phenotype with a reduction of both Col1a and Bglap (osteocalcin) expression, suggesting that the mTORC1 activation could be one of the pathways for stimulating bone formation 25.…”

Transforming Growth Factor β Suppresses Osteoblast Differentiation via the Vimentin Activating Transcription Factor 4 (ATF4) Axis

“…4). At odds with these results, a low concentration (0.1 nM) of rapamycin has been shown to inhibit Ocn expression and osteoblast differentiation by suppressing the expression of Runx2, in primary mouse bone marrow stromal cells and MC3T3-E1 mouse osteoblastic cells (57). It is currently unknown whether these conflicting observations are a reflection of difference in cell lines, i.e.…”