O45-regulation of osteoblast differentiation and ECM remodeling by Bmp2/4 in vitro

Both of bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4) are two closely related members of the transforming growth factor beta superfamily and play diverse roles in normal and pathological processes. However, detail understandings of mechanisms through which Bmp2 and Bmp4 exert their effects remain elusive due to their functional compensations each other. To study roles of Bmp2/Bmp4 in osteoblast differentiation and extracellular matrix (ECM) remodeling, calvarial osteoblasts from Bmp2/4 conditional mice with Cre recombinase recognition site (loxP) were isolated and transfected with simian virus 40 large T antigen to generate immortalized BMP2C/C4C/C (iBMP2 C/C/4C/C) osteoblast lines. The BMP2/4 genes in the iBMP2 C/C/4C/C cells were double knocked out by Ad-Cre recombinase infection. Differentiation and mineralization of iBMP2C/C/4C/C knock-out (iBmp2C/C/4C/C KO) cells were detected by alkaline phosphatase (ALP) and alizarin (ALZ) red S staining analyses. ECM remodeling was also observed in fluorescent microscope. Cell differentiation was dramatically decreased in the iBMP2C/C/4C/C KO cells compared to that of the iBMP2C/C/4C/C osteoblasts. Mineralization was also reduced in these KO cells by ALZ staining. Furthermore, Bmp2/4 double knock-out cells have major defects in remodeling the ECM as reflected by changes in collagen type I processing. Here we for the first time demonstrate the establishment of iBmp2C/C/4C/C KO osteoblasts. Cell differentiation and mineralization in the iBmp2C/C/4C/C KO cells were decreased. Furthermore, ECM processing in these KO cells was impaired. This indicates that BMP2/4 play important roles in osteoblast differentiation and ECM remodeling.