Mitotic transcription factor circuits control the phenotype of mesenchymal cells

Objectives: Stringent control of proliferation in mesenchymal stem cells, osteoprogenitors, and preosteoblasts is essential for the normal growth and development of bone. Progression of differentiation along the osteogenic lineage requires the complex genetic and biochemical interplay of gene regulatory signaling pathways involving several key transcription factors and distinct growth factor-dependent kinase cascades. Down regulation of Runx2 in late G1 during the cell cycle may be mediated by CDK-related and ubiquitin/ proteasome dependent protein degradation. However, Runx2 gene expression during the cell cycle is also controlled at least in part at the transcriptional level. The main findings of this study are that transmission of mRNAs for lineage specific transcription factors during mitosis as a novel epigenetic mechanism.

Methods: MC3T3-E1 cells were maintained in a-MEM supplemented with10% FBS. Cells grown in medium plus FBS were treated with 100 ng/ml nocodazole for 18 h, followed by shake-off of mitotic cells. MC3T3-E1 were seeded in 6-well plates and transfected the next day at 30-40% confluency with different oligonucleotides using Oligofectamine in Opti-MEM according to the manufacturer's instructions. Cells were harvested 72 h after transfection for Western blot analysis and RT-qPCR.

Results: Nocodazole treated MC3T3-E1 cells showed four transcription factor (TF) genes (Runx2, Runx3, Satb2 and Msx2) are mitosis-related. In MC3T3-E1 cells that were arrested in mitosis using nocodazole, we find that Runx2 and Satb2 exhibits reduced mobility in SDS-PAGE as compared to Runx2 and Satb2 isolated from asynchronous cells, nearly confluent cultures which are almost devoid of mitotic cells. Runx2 and Satb2 are phosphorylated during mitosis. When we used to siMix (siRunx2, siRunx3, siSatb2 and siMsx2) at MC3T3-E1 cells, siMix suppresses mRNA for Sox4, Sp7/Osx and Sox2, activates mRNA for Dlx3.

Conclusions: These findings suggest that osteoblast induced to preosteoblast by inhibition of osteoblast specific TFs.