Although human mutations and in vitro studies suggest that DLX3 is involved in bone formation, its in vivo role has not been elucidated. To address the functions of DLX3 in the appendicular skeleton, we generated and analysed mice carrying conditional loss-of-function mutation of DLX3 in osteoblasts (Dlx3OCN-cKO). Using dynamic bone formation, histological and micro-computed tomography analyses, we demonstrated that in vivo DLX3 deletion in osteoblasts results in significant increase in bone mass throughout the lifespan. In absence of DLX3, endochondral bone formation still takes place at the growth plate but we observed more trabeculae that extend deeper into the medullary cavity. Furthermore Dlx3OCN-cKO cortical bone is thicker with higher mineral apposition rate, decreased bone mineral density and increased cortical porosity. By combining in vivo site-specific gene profiling, TRAP staining and ex vivo culture of M-CSF-dependent mononuclear cells, we showed that the increase in trabecular bone mass in Dlx3OCN-cKO mice does not arise from impaired osteoclastic activity but from direct enhancement of bone-forming osteoblast activity with an imbalance in bone homeostasis in favour of bone apposition. In vivo RNA-seq analysis on Dlx3OCN-cKO metaphysis demonstrated that DLX3 deletion in osteoblasts results in up-regulation of genes encoding transcription factors essential for osteoblastogenesis as well as genes important to mineral deposition and bone turnover. Finally, using DLX3-deleted bone marrow stromal cells and ChIP-seq analysis, we demonstrated that DLX3 removal results in increased osteoblast differentiation associated with enhanced occupancy of key transcriptional activators of osteogenesis on the bone-specific osteocalcin (OCN) promoter. In conclusion, these results demonstrate for the first time that DLX3 plays a central role in the maintenance of bone homeostasis and skeletal integrity by attenuating bone mass accrual in the appendicular skeleton.
Disclosure: The authors declared no competing interests. This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH. JB Lian is supported by NIH grant R37 DE012528.