BMP‐2 promotes differentiation of osteoblasts and chondroblasts in Runx2‐deficient cell lines

Abstract

To investigate the molecular mechanism underlying the differentiation of osteoblasts and chondroblasts, we established a clonal cell lines, RD-C6, from Runx2-deficient mouse embryos. RD-C6 cells expressed almost undetectable levels of phenotypes related to osteoblast and chondroblast differentiation at basal culture condition, whereas treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2) or transduction of BMP-2 by adenovirus effectively induced this cell line to express mRNA related to the differentiation of osteoblasts and chondroblasts including alkaline phosphatase, osteocalcin, and osterix. Transduction of Runx2 also induced the expression of these mRNA in RD-C6 cells. BMP-2 transduction increased expression levels of mRNA for Msx2 and Dlx5, but Runx2 transduction induced no significant increases in expression levels of these mRNA. Microarray analysis using RD-C6 cells with or without rhBMP-2 treatment demonstrated that BMP-2 upregulated 66 genes including 13 transcription-related molecules such as Id1, Id2, Id4, Hey1, Smad6, Smad7, and Msx2. To confirm bone and cartilage formation ability of RD-C6 cells, we transplanted RD-C6 cells into the peritoneal cavity of athymic mice using diffusion chambers with rhBMP-2. RD-C6 cells generated unmineralized cartilage but not bone. These results indicate that BMP-2 induces Runx2-deficient cells to express markers related to osteoblast and chondroblast differentiation using a Runx2-independent pathway, but it failed to induce these cells to differentiate into bone-forming osteoblasts and mature chondrocytes.

Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation

“…Other factors, including Sox9, may play a role in Runx2-independent chondrocyte differentiation, although additional work is necessary to clearly identify these factors. 40 Thus, our data support the hypothesis that SMC-specific removal of Runx2 prevents both endochondral and intramembranous ossification in atherosclerotic lesions. In contrast to our studies, Sun et al 21 reported a role for SMC autonomous Runx2 in macrophage accumulation and atheromatous lesion formation using a very different Runx2 targeting construct.…”

BMP2 Regulates Osterix through Msx2 and Runx2 during Osteoblast Differentiation

“…The cells showed ALP activity. Runx2-deficient mesenchymal cells were isolated from the calvariae of Runx2-deficient embryos as described previously (17). In brief, the anterior region of calvariae from an embryo at embryonic day 18.5 was minced and cultured for 10 -14 days in three-dimensional collagen gel (Cellmatrix, Nitta Gelatin Co.) with ␣-modified minimum Eagle's medium containing 10% fetal bovine serum.…”

“…In brief, the anterior region of calvariae from an embryo at embryonic day 18.5 was minced and cultured for 10 -14 days in three-dimensional collagen gel (Cellmatrix, Nitta Gelatin Co.) with ␣-modified minimum Eagle's medium containing 10% fetal bovine serum. The cells outgrowing from the explants were retrieved by incubation for 30 min with 0.2% collagenase in phosphate-buffered saline (PBS) at 37°C and then cultured with ␣-modified minimum Eagle's medium containing 10% fetal bovine serum (17).…”

miR-199a*, a Bone Morphogenic Protein 2-responsive MicroRNA, Regulates Chondrogenesis via Direct Targeting to Smad1

“…A modified micromass culture technique was carried out, as previously described (21)(22)(23). Transfections were carried out using Lipofectamine 2000 (Invitrogen) in 6-well plates as per the manufacturer's instructions.…”