Discoidin Receptor 2 Controls Mesenchymal Cell Lineage and Bone Formation

Methods: Mice with a spontaneous deletion in Ddr2 (Ddr2^slie/slie mice) were used as a source of calvarial osteoblasts, marrow stromal cells (MSCs) or ear mesenchymal stem cells (EMSCs). Runx2 and PPARg phosphorylation was measured using antibodies specific to MAPK phosphorylation sites (RUNX2, S319; PPARg, S112). Bone structure was assessed by microCT. RUNX2 and PPARg transcriptional activity were measured using 6OSE2-Luc and ARE-luc reporters.

Results: Ddr2^slie/slie mice had multiple skeletal defects. Skulls had widened calvarial sutures and increased mandibular porosity. A progressive decrease in tibial trabecular BV/TV was observed when wild type, Ddr2^+/slie and Ddr2^slie/slie mice were compared. These changes were accompanied by drops in trabecular number and thickness and increased trabecular spacing in both males and females. Interestingly, cortical thickness was not affected in males, but was lower in Ddr2^slie/slie females. To examine the role of DDR2 signaling in cell differentiation, we prepared calvarial osteoblasts,  BMSCs and EMSCs from the 3 genotypes and grew cells under osteogenic and adipogenic conditions. Regardless of cell source, Ddr2^slie/slie cells exhibited defective osteoblast differentiation and accelerated adipogenesis. Furthermore, both RUNX2-S319 phosphorylation and PPARg-S112 phosphorylation were decreased in Ddr2^slie/slie cells, resulting in decreased RUNX2 and increased PPARg transcriptional activity.

Conclusion: Ddr2 is necessary for normal bone development and MSC differentiation to osteoblasts. This regulation requires MAPK-dependent phosphorylation of RUNX2 and PPARg transcription factors.