Hypoxia Inducible Factor-1α Directly Induces the Expression of Receptor Activator of Nuclear Factor-κB Ligand in MLO-Y4 Osteocytes

Hypoxia Inducible Factor-1α Directly Induces the Expression of Receptor Activator of Nuclear Factor-κB Ligand in MLO-Y4 Osteocytes

Osteocytes may function as mechanotransducers byregulating local osteoclastogenesis. Reduced availability ofoxygen, i.e. hypoxia, could occur during disuse, bonedevelopment, and fracture. Receptor activator of nuclearfactor-κB ligand (RANKL) is an osteoblast/stromal cellderived essential factor for osteoclastogenesis. The hypoxiainduced osteoclastogenesis via increased RANKL expressionin osteoblasts was demonstrated. Hypoxic regulation of geneexpression generally involves activation of thehypoxia-inducible factor (HIF) transcription pathway. In thepresent study, we investigated whether hypoxia regulatesRANKL expression in murine osteocytes and HIF-1αmediates hypoxia-induced RANKL expression bytransactivating RANKL promoter, to elucidate the role ofosteocyte in osteoclastogenesis in the context of hypoxiccondition. The expression levels of RANKL mRNA andprotein, as well as hypoxia inducible factor-1α (HIF-1α)protein, were significantly increased in hypoxic condition inMLO-Y4s. Constitutively active HIF-1α alone significantlyincreased the levels of RANKL expression in MLO-Y4sunder normoxic conditions, whereas dominant negativeHIF-1α blocked hypoxia-induced RANKL expression. Tofurther explore to find if HIF-1α directly regulates RANKLtranscription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity,whereas mutations of putative HIF-1α binding elements inRANKL promoter prevented this hypoxia-induced RANKLpromoter activity in MLO-Y4s. These results suggest thatHIF-1α mediates hypoxia-induced up-regulation of RANKLexpression, and that in osteocytes of mechanically unloadedbone, hypoxia enhances osteoclastogenesis, at least in part, viaan increased RANKL expression in osteocytes.

Osteocytes may function as mechanotransducers byregulating local osteoclastogenesis. Reduced availability ofoxygen, i.e. hypoxia, could occur during disuse, bonedevelopment, and fracture. Receptor activator of nuclearfactor-κB ligand (RANKL) is an osteoblast/stromal cellderived essential factor for osteoclastogenesis. The hypoxiainduced osteoclastogenesis via increased RANKL expressionin osteoblasts was demonstrated. Hypoxic regulation of geneexpression generally involves activation of thehypoxia-inducible factor (HIF) transcription pathway. In thepresent study, we investigated whether hypoxia regulatesRANKL expression in murine osteocytes and HIF-1αmediates hypoxia-induced RANKL expression bytransactivating RANKL promoter, to elucidate the role ofosteocyte in osteoclastogenesis in the context of hypoxiccondition. The expression levels of RANKL mRNA andprotein, as well as hypoxia inducible factor-1α (HIF-1α)protein, were significantly increased in hypoxic condition inMLO-Y4s. Constitutively active HIF-1α alone significantlyincreased the levels of RANKL expression in MLO-Y4sunder normoxic conditions, whereas dominant negativeHIF-1α blocked hypoxia-induced RANKL expression. Tofurther explore to find if HIF-1α directly regulates RANKLtranscription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity,whereas mutations of putative HIF-1α binding elements inRANKL promoter prevented this hypoxia-induced RANKLpromoter activity in MLO-Y4s. These results suggest thatHIF-1α mediates hypoxia-induced up-regulation of RANKLexpression, and that in osteocytes of mechanically unloadedbone, hypoxia enhances osteoclastogenesis, at least in part, viaan increased RANKL expression in osteocytes.