Runt-related transcription factor-2 (Runx2) is required for bone matrix protein gene expression in committed osteoblasts in mice

Runt-related transcription factor-2 (Runx2) is an essential transcription factor for osteoblast differentiation. However, its functions after the commitment into osteoblasts are controversial and remain to be clarified. We generated enhanced green fluorescent protein (EGFP)-Cre transgenic mice driven by the 2.3-kilobase (kb) Col1a1 promoter, and Runx2 was deleted in osteoblasts and odontoblasts in Runx2fl/flCre mice. The sutures and fontanelles were more widely opened in Runx2fl/flCre newborns than in Runx2fl/fl newborns. Runx2fl/flCre mice exhibited dwarfism with shorter incisors and 37% had irregularly aligned incisors. The volume of trabecular bone in femurs and vertebrae and their bone mineral density (BMD), in addition to the cortical thickness and BMD were reduced in Runx2fl/flCre mice compared with Runx2fl/fl mice in both sexes. The bone formation of both trabecular and cortical bone, osteoblast number, osteoclast surface, osteoblast proliferation, and the serum levels of procollagen type 1 N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase 5b (TRAP5b), and C-terminal cross-linked telopeptide of type 1 collagen (CTX1) were reduced in Runx2fl/flCre mice. The expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap&Bglap2, and of Tnfsf11 was lower in Runx2fl/flCre mice than in Runx2fl/fl mice. The expression of Runx2 target genes, including Ihh, Fgfr1, Fgfr2, Fgfr3, Tcf7, Wnt10b, Pth1r, Sp7, and Dlx5, was also reduced. Osteoblasts in Runx2fl/fl mice were cuboidal and contained abundant type I collagen α1 (Col1a1), whereas those in Runx2fl/flCre mice were deflated and contained a small amount of Col1a1. Runx2 activated the reporter activity of the 2.3-kb Col1a1 promoter and bound the region around the Col1a1 transcription start site. The deletion of Runx2 by Cre-expressing adenovirus in Runx2fl/fl primary osteoblasts impaired osteoblast differentiation and the expression of genes encoding major bone matrix proteins, and osteoclastogenesis was inhibited due to the reduction of Tnfsf11 expression in the osteoblasts. This study demonstrated that Runx2 is required for the expression of the major bone matrix protein genes and Tnfsf11 after commitment into osteoblasts in mice. © 2021 American Society for Bone and Mineral Research (ASBMR).