HIF-1alpha 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-1alpha (HIF-1alpha) 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 PGE(2) release and expression of the PGE(2) receptor EP1. In the present studies, we investigated the impact of altered HIF-1alpha activity and expression on EP1 expression in osteoblasts. HIF-1alpha stabilization was induced in cells cultured in 21% oxygen by treatment with dimethyloxaloglycine (DMOG) or siRNA targeted against PHD2. To implicate HIF-1alpha in hypoxia-induced EP1 expression, osteoblastic cells were treated with siRNA targeted against HIF-1alpha 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-1alpha siRNA compared to controls, indicating HIF-1alpha as the functional HIF-alpha isoform in this system. Furthermore, hypoxic cells treated with HIF-1alpha 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-1alpha expression or activity. These data strongly implicate a role for HIF-1alpha in hypoxia-induced EP1 expression and may provide important insight into the mechanisms by which HIF-1alpha regulates bone development and fracture repair. 2009 Wiley-Liss, Inc.