Abstract
In our previous studies, we have found that the prepubertal increase in thyroid hormone levels induces osterix (Osx) signaling in hypertrophic chondrocytes to transdifferentiate them into osteoblasts. To test if Osx expressed in chondrocytes directly contributes to transdifferentiation and secondary ossification, we generated Osx flox/flox ; Col2-Cre-ERT2 mice and knocked out Osx with a single injection of tamoxifen at postnatal day (P) 3 prior to evaluation of the epiphyseal bone phenotype by µCT, histology, and immunohistochemistry (IHC) at P21. Vehicle (oil)-treated Osx flox/flox ; Col2-Cre-ERT2 and tamoxifen-treated, Cre-negative Osx flox/flox mice were used as controls. µCT analysis of tibial epiphyses revealed that trabecular bone mass was reduced by 23% in the Osx conditional knockout (cKO) compared with control mice. Trabecular number and thickness were reduced by 28% and 8%, respectively, while trabecular separation was increased by 24% in the cKO mice. Trichrome staining of longitudinal sections of tibial epiphyses showed that bone area and bone area adjusted for total area were decreased by 22% and 18%, respectively. IHC studies revealed the presence of abundant Osx-expressing prehypertrophic chondrocytes in the epiphyses of control mice at P10, but not in the cKO mice. Furthermore, expression levels of MMP13, COL10, ALP, and BSP were considerably reduced in the epiphyses of cKO mice. We also found that Osx overexpression in ATDC5 chondrocytes increased expression of Col10, Mmp13, Alp, and Bsp. Our data indicate that Osx expressed in chondrocytes plays a significant role in secondary ossification by regulating expression of genes involved in chondrocyte hypertrophy and osteoblast transdifferentiation.