DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo

Abstract

Human mutations and in vitro studies indicate that DLX3 has a crucial function in bone development, however, the in vivo role of DLX3 in endochondral ossification has not been established. Here, we identify DLX3 as a central attenuator of adult bone mass in the appendicular skeleton. Dynamic bone formation, histologic and micro-computed tomography analyses demonstrate that in vivo DLX3 conditional loss of function in mesenchymal cells (Prx1-Cre) and osteoblasts (OCN-Cre) results in increased bone mass accrual observed as early as 2 weeks that remains elevated throughout the lifespan owing to increased osteoblast activity and increased expression of bone matrix genes. Dlx3OCN-conditional knockout mice have more trabeculae that extend deeper in the medullary cavity and thicker cortical bone with an increased mineral apposition rate, decreased bone mineral density and increased cortical porosity. Trabecular TRAP staining and site-specific Q-PCR demonstrated that osteoclastic resorption remained normal on trabecular bone, whereas cortical bone exhibited altered osteoclast patterning on the periosteal surface associated with high Opg/Rankl ratios. Using RNA sequencing and chromatin immunoprecipitation-Seq analyses, we demonstrate that DLX3 regulates transcription factors crucial for bone formation such as Dlx5, Dlx6, Runx2 and Sp7 as well as genes important to mineral deposition (Ibsp, Enpp1, Mepe) and bone turnover (Opg). Furthermore, with the removal of DLX3, we observe increased occupancy of DLX5, as well as increased and earlier occupancy of RUNX2 on the bone-specific osteocalcin promoter. Together, these findings provide novel insight into mechanisms by which DLX3 attenuates bone mass accrual to support bone homeostasis by osteogenic gene pathway regulation. Endochondral bone formation (EBF) and homeostasis require a regulated program of mesenchymal condensation, chondrocyte differentiation, vascular invasion, cartilage resorption, osteoprogenitor recruitment and differentiation and bone remodeling. These key processes are under the control of essential bone transcription factors (TFs), including RUNX2, SP7 and homeodomain protein families such as HOX, MSX and DLX.1-3 DLX proteins are important regulators of developmental and differentiation processes, including skeletal development. [4][5][6] A missense mutation in DLX5 leads to split hand and foot malformation 7 and DLX5 and DLX6 are positive transcriptional regulators of osteochondroblastic differentiation. 6 DLX3 is defined as an osteogenic regulator, as human mutations in DLX3 lead to tricho-dento-osseous (TDO) syndrome, an ectodermal dysplasia that causes increased bone mineral density (BMD) in intramembranous and endochondral bones. In vitro, osteocalcin (Ocn), Runx2 9,10 and osteoactivin [9][10][11] are directly regulated by DLX3, and overexpression of DLX3 in osteoprogenitors stimulates transcription of osteogenic markers. 9Recently, we investigated the effects of neural crest deletion of Dlx3 in craniofacial bones. 12 The gene signature of craniofacial...

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2

“…A variety of human, mouse and fish studies suggest reduced expression of Grhl3, Dlx3, Dlx5 and Osx1 could explain the defects we found in calcium and phosphate metabolism (Dworkin et al, 2014;Han et al, 2011;Isaac et al, 2014;Lapunzina et al, 2010;Liu et al, 2015;Nguyen et al, 2013;Ting et al, 2003a). Mineralization of the endochondral skeleton is a complex biological process involving the formation of a calcium phosphate hydroxyapatite crystal that is deposited into an organic extracellular matrix composed primarily of type I collagen.…”

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms

“…Dlx3 is a member of the distalless homeobox family that interacts with transcription factors specific to mineralized tissue to regulate craniofacial and postnatal skeletal development (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Our results indicate that there is a cellular requirement for miR-665 to fine-tune the expression of Dlx3 within physiologic limits for maturation of progenitors to odontoblasts.…”

“…Mutations in DLX3 in humans have been associated with tricho-dento-osseous syndrome (TDO; OMIM 190320) and amelogenesis imperfecta with taurodontism (AIHHT; OMIM 104510), both of which are conditions characterized by abnormalities in tooth formation (35-39). During development, Dlx3 expression occurs in cranial neural crest cells, endochondral osteoblasts, odontoblasts, ameloblasts, hypertrophic chondrocytes, and the developing limb (40,41), and Dlx3-knockout mice die from placental failure at embryonic day 9.5 (E9.5), prior to bone and tooth formation (28). Expression of DLX3 in postmigratory neural crest cells (E9.5) helps commit these cells to further differentiate into odontoblasts and cementoblasts, contributing to formation of mature dentin and cementum for proper tooth formation (40).…”

Dlx genes and the maintenance of bone homeostasis and skeletal integrity

“…In this issue of Cell Death Differentiation, Isaac et al 15 perform the first in vivo study of the postnatal role of DLX3 in the appendicular skeleton. They generate and analyse mice carrying conditional loss-of-function mutations of Dlx3 in mesenchymal cells (Dlx3 Prx1-cKO ) or in osteogenic lineage cells (Dlx3 OCN-cKO ).…”

“…In vitro studies have shown that Dlx3 and Dlx5 associate with the Ocn promoter at the onset of transcriptional activation, concomitant with Runx2 occupancy, and, while Dlx3 occupancy on the Ocn promoter decreases from osteoblast maturation to mineralization, Dlx5 occupancy is maximal during the mineralization stage. 12 Isaac et al 15 demonstrate that Dlx3 deletion in calvaria osteoblasts results in increased occupancy of Dlx5 and increased and premature occupancy of Runx2 on regulatory elements on the Ocn promoter. Thus, Dlx3 deletion could directly affect the network of molecular switches and promote osteoblastic differentiation and bone-forming activity via an enhancement of the occupancy of bone-activator TFs such as Dlx5 and Runx2.…”