Differential but complementary roles of HIF-1a and HIF-2a in the regulation of bone homeostasis

Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1a and HIF-2a in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1a and HIF-2a. We found that increased HIF-1a expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1a via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2a upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1a in regulating RANKL expression, verified through an experiment of HIF-2a knockdown after HIF-1a overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1a plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2a modulation. Overall design: RNA-seq analysis was performed to determine the gene expression profile caused by overexpression of HIF-1a and HIF-2a during primary culture calvarial preosteoblast differentiation.