Cell cycle related modulations in Runx2 protein levels are independent of lymphocyte enhancer-binding factor 1 (Lef1) in proliferating osteoblasts.

Runt-related transcription factor Runx2 regulates osteogenic phenotype commitment and attenuates osteoblast growth. Runx2 levels are cell cycle regulated and maximal in the G1 phase of proliferating osteoblasts and during quiescence. The Wnt/Lrp5-Frizzled/beta-catenin/Lef-Tcf signaling cascade also controls progression along the osteogenic lineage with a net anabolic effect that promotes bone formation. However, Lef1 opposes the osteoblast maturation promoting activity of Runx2. Here we examined whether Lef1 controls Runx2 expression during the cell cycle or onset of quiescence in osteoblasts. We inhibited Lef1 expression using short hairpin (sh) RNA interference in stably transfected MC3T3-E1 cells. In asynchronously growing osteoblasts, expression of Lef1 shRNA diminishes Lef1 protein levels, but does not affect Runx2 levels. Cells arrested in different cell cycle stages using mimosine (late G1), hydroxyurea or aphidicolin (S phase) or nocodazole (mitosis) exhibit expected reductions in Runx2 protein levels despite reductions in Lef1. Serum deprived MC3T3-E1 cells normally upregulate Runx2 protein regardless of Lef1 deficiency, although loss of Lef1 reduces cyclin A and increases cyclin D1 expression upon serum withdrawal. Thus, Runx2 protein levels during the cell cycle and onset of quiescence are regulated independently of Lef1, one of the major transcriptional inducers of Wnt signaling in proliferating cells.