AMP-activated protein kinase (AMPK) positively regulates osteoblast differentiation via induction of Dlx5-dependent Runx2 expression in MC3T3E1 cells

This study examined the role of AMPK activation in osteoblast differentiation and the underlining mechanism. An AMPK activator (AICAR or metformin) stimulated osteoblast differentiation with increases in ALP and OC protein production as well as the induction of AMPK phosphorylation in MC3T3E1 cells. In addition, metformin induced the phosphorylation of Smad1/5/8 and expression of Dlx5 and Runx2, whereas compound C or dominant negative AMPK inhibited these effects. Transient transfection studies also showed that metformin increased the BRE-Luc and Runx2-Luc activities, which were inhibited by DN-AMPK or compound C. Down-regulation of Dlx5 expression by siRNA suppressed metformin-induced Runx2 expression. These results suggest that the activation of AMPK stimulates osteoblast differentiation via the regulation of Smad1/5/8-Dlx5-Runx2 signaling pathway.     

GATA4 negatively regulates osteoblast differentiation by downregulation of Runx2

Osteoblasts are specialized mesenchymal cells that are responsible for bone formation. In this study, we examine the role of GATA4 in osteoblast differentiation. GATA4 was abundantly expressed in preosteoblast cells and gradually down-regulated during osteoblast differentiation. Overexpression of GATA4 in osteoblastic cells inhibited alkaline phosphatase activity and nodule formation in osteogenic conditioned cell culture system. In addition, overexpression of GATA4 attenuated expression of osteogenic marker genes, including Runx2, alkaline phosphatase, bone sialoprotein, and osteocalcin, all of which are important for osteoblast differentiation and function. Overexpression of GATA4 attenuated Runx2 promoter activity, whereas silencing of GATA4 increased Runx2 induction. We found that GATA4 interacted with Dlx5 and subsequently decreased Dlx5 binding activity to Runx2 promoter region. Our data suggest that GATA4 acts as a negative regulator in osteoblast differentiation by downregulation of Runx2. [BMB Reports 2014; 47(8): 463-468]     

Ghrelin inhibits early osteogenic differentiation of C3H10T1/2 cells by suppressing Runx2 expression and enhancing PPARγ and C/EBPα expression

Ghrelin is a 28‐residue peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor that is expressed in a variety of peripheral tissues, as well as in the brain. In previous studies, ghrelin has been shown to stimulate both adipogenic differentiation from preadipocytes and osteogenic differentiation from preosteoblasts or primary osteoblasts. This study was undertaken to investigate the direct effect of ghrelin on the lineage allocation of mesenchymal stem cells (MSCs). We identified ghrelin receptor mRNA in C3H10T1/2 cells, and we found the levels of this mRNA to be attenuated during osteogenic differentiation. Treatment of cells with ghrelin resulted in both proliferation and inhibition of caspase‐3 activity. In addition, ghrelin decreased serum deprivation‐induced bax protein expression and release of cytochrome c from the mitochondria, whereas it increased bcl‐2 protein expression. Moreover, ghrelin inhibited early osteogenic differentiation, as shown by alkaline phosphatase activity and staining, and inhibited osteoblast‐specific genes expression by altering Runx2, PPARγ, and C/EBPα protein expression. J. Cell. Biochem. 106: 626–632, 2009. © 2009 Wiley‐Liss, Inc.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.