RORβ Deficiency Inhibits Endochondral Ossification in Mice by Mediating the HIF ‐1α/ VEGFA Signaling Pathway

ABSTRACT Retinoic acid receptor‐related orphan receptor beta (RORβ) is a ligand‐dependent transcription factor essential for bone metabolism. While RORβ negatively regulates osteoblast differentiation and contributes to age‐related or postmenopausal osteoporosis, its role in early skeletal development remains unclear. This study developed a Rorβ gene knockout (KO) mouse model using CRISPR/Cas9 to investigate its effects on endochondral ossification. At 4 weeks, Rorβ KO mice exhibited dwarfism and early‐onset osteoporosis, with reduced femur length (−8.84%), lower cortical and trabecular bone mass, and impaired bone quality. The trabecular bone was fragile, with reduced surface osteoblasts and impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Chondrogenesis was also disrupted, evidenced by a thinner growth plate, fewer chondrocytes, and a disorganized hypertrophic zone (HZ). The expressions of proliferation (Ki67) and hypertrophic differentiation (Collagen X) markers were significantly reduced. Mechanistically, RORβ deficiency downregulated hypoxia‐inducible factor 1‐alpha (HIF‐1α) and its downstream target vascular endothelial growth factor A (VEGFA) in both the pre‐hypertrophic zone (PHZ) of the growth plate and BMSCs. These findings identify RORβ as a critical regulator of endochondral ossification, linking its loss to skeletal defects via impaired HIF‐1α/VEGFA signaling. This study provides insights into potential therapeutic strategies for skeletal diseases such as skeletal dysplasia.