In this work, we found that sub-toxic Pb concentrations suppressed bone nodule formation and inhibited differentiation in MC3T3-E1 subclone 14 cells, as shown by decreased expression levels of the differentiation markers alkaline phosphatase (ALP), type 1 collagen (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2).
Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67.
The efficacy of gene silencing against Runt-related transcription factor 2 ( Runx2) expression and the long-term assessment of mineralized nodule formation in osteoblast cultures were evaluated.
The overexpression of CREB enhanced mineralized nodule formation and up-regulated (P < 0.05) the mRNA levels of odonto/osteogenic-related markers, including ALP, Col I, RUNX2, OSX and OCN, and also increased (P < 0.05) the protein expression of DSP and RUNX2.
Interestingly, rapamycin in rapamycin plus lipopolysaccharide (LPS)‑treated BMSCs significantly increased the gene expression levels of Sp7 transcription factor, runt related transcription factor 2, alkaline phosphatase (ALP) and collagen I (Col I), ALP activity, and calcium nodule at different time points in vitro, indicating that osteoblast differentiation occurs by rapamycin when BMSCs are exposed to LPS simultaneously.
Over-expression of miR-495 significantly inhibited ALP activity and mineralized nodule formation as well as the expression of RUNX-2, BMP-2, and Osterix.
Co-treatment with chemical inhibitor of c-Met and osteogenic inducing media enhanced osteoblast-specific genes expression and calcium nodule formation accompanied by increased Runx2 expression via c-Met receptor-dependent but Erk-Smad signaling independent pathway.