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]     

Oxysterol-induced osteoblastic differentiation of pluripotent mesenchymal cells is mediated through a PKC- and PKA-dependent pathway

Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in circulation, and in human and animal tissues. The discovery of osteoinductive molecules that can induce the lineage-specific differentiation of cells into osteoblastic cells and therefore enhance bone formation is crucial for better management of bone fractures and osteoporosis. We previously reported that specific oxysterols have potent osteoinductive properties and induce the osteoblastic differentiation of pluripotent mesenchymal cells. In the present report we demonstrate that the induction of osteoblastic differentiation by oxysterols is mediated through a protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanism(s). Furthermore, oxysterol-induced-osteoblastic differentiation is marked by the prolonged DNA-binding activity of Runx2 in M2-10B4 bone marrow stromal cells (MSCs) and C3H10T1/2 embryonic fibroblastic cells. This increased activity of Runx2 is almost completely inhibited by PKC inhibitors Bisindolylmaleimide and Rottlerin, and only minimally inhibited by PKA inihibitor H-89. PKC- and PKA-dependent mechanisms appear to also regulate other markers of osteoblastic differentiation including alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in response to oxysterols. Finally, osteogenic oxysterols induce osteoblastic differentiation with BMP7 and BMP14 in a synergistic manner as demonstrated by the enhanced Runx2 DNA-binding activity, ALP activity, and osteocalcin mRNA expression. Since Runx2 is an indispensable factor that regulates the differentiation of osteoblastic cells and bone formation in vitro and in vivo, its increased activity in oxysterol-treated cells further validates the potential role of oxysterols in lineage-specific differentiation of pluripotent mesenchymal cells and their potential therapeutic use as bone anabolic factors.     

Bone loss induced by Runx2 Over‐expression in mice is blunted by osteoblastic over‐expression of TIMP‐1

The Runx2 gene is essential for osteoblast differentiation and function. In vivo over‐expression of Runx2 in osteoblasts increases bone resorption, and blocks terminal osteoblast differentiation. Several lines of evidence suggest that osteoblastic matrix metalloproteinases (MMPs) could contribute to the increased bone resorption observed in mice over‐expressing Runx2 (Runx2 mice). The goal of our study was to use a transgenic approach to find out whether the inhibition of osteoblastic MMPs can reduce the bone loss induced by the over‐expression of Runx2. We analyzed the effect of the in vivo over‐expression of the TIMP‐1 in osteoblasts on the severe osteopenic phenotype in Runx2 mice. Females with the different genotypes (WT, Runx2, TIMP‐1 and TIMP‐1/Runx2) were analyzed for bone density, architecture, osteoblastic and osteoclastic activity and gene expression using qPCR. TIMP‐1 over‐expression reduces the bone loss in adult Runx2 mice. The prevention of the bone loss in TIMP‐1/Runx2 mice was due to a combination of reduced bone resorption and sustained bone formation. We present evidence that the ability of osteoblastic cells to induce osteoclastic differentiation is lower in TIMP‐1/Runx2 mice than in Runx2 mice, probably due to a reduction in the expression of RANK‐L and of the Runx2 transgene. Osteoblast primary cells from TIMP‐1/Runx2 mice, but not from Runx2 mice, were able to differentiate into fully mature osteoblasts producing high osteocalcin levels. In conclusion, our findings suggest that osteoblastic MMPs can affect osteoblast differentiation. Our work also indicates that osteoblastic MMPs are partly responsible for the bone loss observed in Runx2 transgenic mice. J. Cell. Physiol. 222:219–229, 2010. © 2009 Wiley‐Liss, Inc.