HIF-1α regulates hypoxia-induced EP1 expression in osteoblastic cells

Changes in regional oxygen tension that occur during skeletal development and fracture stimulate local bone cell activity to regulate bone formation, maintenance, and repair. The adaptive responses of bone cells to hypoxia are only beginning to be understood. The transcription factor hypoxia-inducible factor-1α (HIF-1α) is activated under hypoxia and promotes expression of genes required for adaptation and cell survival, and also regulates both bone development and fracture repair. We have previously demonstrated that hypoxic osteoblasts increase PGE₂ release and expression of the PGE₂ receptor EP1. In the present studies, we investigated the impact of altered HIF-1α activity and expression on EP1 expression in osteoblasts. HIF-1α stabilization was induced in cells cultured in 21% oxygen by treatment with dimethyloxaloglycine (DMOG) or siRNA targeted against PHD2. To implicate HIF-1α in hypoxia-induced EP1 expression, osteoblastic cells were treated with siRNA targeted against HIF-1α prior to exposure to hypoxia. EP1 expression was significantly increased in cells cultured in 21% oxygen with DMOG or PHD2 siRNA treatment compared to controls. Hypoxia responsive element (HRE) activation in hypoxia was attenuated in cells treated with HIF-1α siRNA compared to controls, indicating HIF-1α as the functional HIF-α isoform in this system. Furthermore, hypoxic cells treated with HIF-1α siRNA demonstrated reduced EP1 expression in hypoxia compared to controls. Inhibition of SAPK/JNK activity significantly reduced hypoxia-induced EP1 expression but had no impact on HIF-1α expression or activity. These data strongly implicate a role for HIF-1α in hypoxia-induced EP1 expression and may provide important insight into the mechanisms by which HIF-1α regulates bone development and fracture repair.