Bone Abstracts

Reduced availability of oxygen, i.e. hypoxia, could occur during disuse, bone development, and fracture. Cartilage tissue is avascular in nature and the microenvironment of cartilage is hypoxic. Hypoxic regulation of gene expression generally involves activation of the hypoxia-inducible factor (HIF) transcription pathway. Receptor activator of nuclear factor-kappaB ligand (RANKL) is an osteoblast/stromal cell derived essential factor for osteoclastogenesis. Hypoxia-induced enhanced osteoclastogenesis via increased RANKL expression has been well demonstrated in in vitro system using osteoblasts. However, during bone development, hypoxic signaling regulation mechanism in mediating osteoclastogenesis, that is, osteoclast recruitment mechanisms after cartilage calcification by hypertrophic chondrocytes has remained unclear. In the present study, we investigated whether hypoxia regulates RANKL expression in ATDC5s, murine chondrocytes and HIF-1α mediates hypoxia-induced RANKL expression by transactivating RANKL promoter.

The expression levels of RANKL mRNA and protein, as well as HIF-1α protein, were significantly increased under hypoxic condition in ATDC5s. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression in ATDC5s under normoxic conditions, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL expression. To further explore to find if HIF-1α directly regulates RANKL transcription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity, whereas mutations of putative HIF-1α-binding elements in RANKL promoter prevented this hypoxia-induced RANKL promoter activity in ATDC5s. Our results suggest that RANKL is a target gene of HIF-1α and that hypoxia plays a role in osteoclastogenesis during bone development, at least in part, through the induction of RANKL expression in chondrocytes.