Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor‐β (TGF‐β) superfamily. BMPs are initially identified by their capability to induce bone formation when implanted subcutaneously or intramuscularly in rodents (Urist, 1965; Wozney et al., 1988). To date, about 20 unique BMP ligands have been identified and compose at least four subgroups based on their amino acid sequence similarity (Sakou, 1998; Shi and Massague, 2003; Kishigami and Mishina, 2005). BMP2 and BMP4 are most similar to decapentaplgic (Dpp) in Drosophila melonogaster and belong to the BMP2/4 subclass as both of the two ligands exhibit a high affinity for the extracellular ligand binding domains of the type I BMP receptor (Hayward et al., 2002; Shi and Massague, 2003). The capacity of BMP2 to induce osteoblast differentiation has been rigorously demonstrated (Takuwa et al., 1991; Yamaguchi et al., 1991; Kubler et al., 1998; Welch et al., 1998; Bax et al., 1999; Chung et al., 1999; Wu et al., 2011). Moreover, BMP4 also plays an important role in osteogenesis (Martinovic et al., 2006; Wang et al., 2006; Luppen et al., 2008; Miyazaki et al., 2008). However, it is difficult to decipher unique roles of BMP2 and/or BMP4 during osteogenesis because of their functional redundancy each other (Selever et al., 2004). BMP2/4 are involved in organ development (Reversade et al., 2005; Cejalvo et al., 2007; Goldman et al., 2009; Uchimura et al., 2009). Mice with BMP2/4 conditional knock‐out (cKO) exhibited severe impairments of osteogenesis and displayed different genotypic and phenotypic characteristics compared to that of BMP2 or BMP4 null mice (Bandyopadhyay et al., 2006). Furthermore, clinical investigations showed that variants in BMP2/4 genes are susceptible to otosclerosis and other diseases (Schrauwen et al., 2008; Tomlinson et al., 2011; Mu et al., 2012). Otosclerosis is a common form of adult‐onset conductive hearing loss resulting from abnormal bone remodeling of the bony labyrinth that surrounds the inner ears. Genotyping pups bred between BMP2 and BMP4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for BMP2/4 is much low (Uchimura et al., 2009). Therefore, generation of a dual BMP2/4ko/ko osteoblastic cell line would be a valuable asset for studying the modulatory effects of BMP2/4 on osteoblast differentiation and relevant molecular events involved in bone‐relate gene expression and extracellular matrix remodeling. In the present study, we established an immortalized mouse deleted BMP2/4 osteoblast cell line using Cre‐recombinase to simultaneously knock‐out BMP2 and BMP4 genes in immortalized mouse floxed BMP2/4 osteoblastic cells and observed these cell behaviors. We further examined cell growth as well as their genotypic and phenotypic characteristics. Finally, we tested whether biological functions of these BMP2/4ko/ko cells were rescued by exogenous BMP2 and/or BMP4.