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.
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Abbreviations
- Adv:
-
adenovirus
- ALPL:
-
alkaline phosphatase
- BMD:
-
bone mineral density
- BMSC:
-
bone marrow stromal cell
- ChIP:
-
chromatin immunoprecipitation
- cKO:
-
conditional knockout
- CTX-1:
-
carboxy-terminal type I collagen crosslink
- DLX3:
-
distal-less homeobox 3
- EBF:
-
endochondral bone formation
- ECM:
-
extracellular matrix
- H&E:
-
hematoxylin and eosin
- MAR:
-
mineral apposition rate
- M-BMM:
-
M-CSF-dependent bone marrow macrophages
- μCT:
-
micro-computed tomography
- OCN:
-
osteocalcin
- OPG:
-
osteoprotegerin
- RANKL:
-
receptor activator of nuclear factor kappa-B ligand
- TDO:
-
tricho-dento-osseous
- TF:
-
transcription factor
- TRAP:
-
tartrate-resistant acid phosphatase
- WT:
-
wild type
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Acknowledgements
We thank Dr. T Clemens for the OCN-cre mice; S Russell, of the University of Massachusetts MusculoSkeletal Imaging Facility for performing μCT analyses; Dr. K Zaal of the NIAMS Light Imaging Core Facility; G Gutierrez-Cruz of the Genome Analyzer Core Facility; Drs. A Maeda and M Young of the NIDCR, NIH; Dr. B Foster of NIAMS, NIH for assistance with ALPL staining, Audrey Asselin of the INSERM UMRS 1138 (Team Berdal), for assistance with cell culture, and Christophe Klein of the INSERM UMRS 1138 (CICC) and Laura Solomon (UVM graduate student) for osteoclast quantification. The Morasso and Lian laboratories and their funding contributed equally to this project. This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH. JBL is supported by NIH grant R37 DE012528.
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Isaac, J., Erthal, J., Gordon, J. et al. DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo. Cell Death Differ 21, 1365–1376 (2014). https://doi.org/10.1038/cdd.2014.82
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DOI: https://doi.org/10.1038/cdd.2014.82
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