Hypoxia inducible factor-1α directly induces the expression of receptor activator of nuclear factor-κB ligand in periodontal ligament fibroblasts

Abstract

During orthodontic tooth movement, local hypoxia and enhanced osteoclastogenesis are observed in the compression side of periodontal tissues. The receptor activator of nuclear factor-kappaB ligand (RANKL) is an osteoblast/stromal cell-derived factor that is essential for osteoclastogenesis. In this study, we examined the effect of hypoxia on RANKL expression in human periodontal ligament fibroblasts (PDLFs) to investigate the relationship between local hypoxia and enhanced osteoclastogenesis in the compression side of periodontal tissues. Hypoxia significantly enhanced the levels of RANKL mRNA and protein as well as hypoxia inducible factor-1alpha (HIF-1alpha) protein in PDLFs. Constitutively active HIF-1alpha alone significantly increased the levels of RANKL expression in PDLFs under normoxic conditions, whereas dominant negative HIF-1alpha blocked hypoxia-induced RANKL expression. To investigate further whether HIF-1alpha directly regulates RANKL transcription, a luciferase reporter assay was performed using the reporter vector containing the RANKL promoter sequence. Exposure to hypoxia or overexpression of constitutively active HIF-1alpha significantly increased RANKL promoter activity, whereas dominant negative HIF-1alpha blocked hypoxia-induced RANKL promoter activity. Furthermore, mutations of putative HIF-1alpha binding elements in RANKL promoter prevented hypoxia-induced RANKL promoter activity. The results of chromatin immunoprecipitation showed that hypoxia or constitutively active HIF-1alpha increased the DNA binding of HIF-1alpha to RANKL promoter. These results suggest that HIF-1alpha mediates hypoxia-induced up-regulation of RANKL expression and that in compression side periodontal ligament, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in PDLFs. ⓒ 2011 The Korean Society for Molecular and Cellular Biology and Springer Netherlands.