Induction of HIF-1 alpha Expression by Intermittent Hypoxia: Involvement of NADPH Oxidase, Ca2+ Signaling, Prolyl Hydroxylases, and mTOR

Sleep-disordered breathing with recurrent apnea (periodic cessation of breathing) results in chronic intermittent hypoxia (IH), which leads to cardiovascular and respiratory pathology. Molecular mechanisms underlying IH-evoked cardio-respiratory co-morbidities have not been delineated. Mice with heterozygous deficiency of hypoxia-inducible factor I alpha (HIF-I alpha) do not develop cardio-respiratory responses to chronic IH. HIF-I alpha protein expression and HIF-I transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH-evoked HIF-I alpha accumulation. PC12 cells were exposed to aerobic conditions (20% O-2) or 60 cycles of IH (30 sec at 1.5% O-2 followed by 5 min at 20% O-2). Our results show that IH-incluced HIF-I alpha accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS-dependent Ca2+ signaling pathways involving phospholipase C-gamma (PLC gamma) and protein kinase C activation are required for IH-evoked HIF-I alpha accumulation. IH leads to activation of mTOR and S6 kinase (S6K) and rapamycin partially inhibited IH-incluced HIF-I alpha accumulation. IH also decreased hydroxylation of HIF-I alpha protein and anti-oxidants as well as inhibitors of Ca+2 signaling prevented this response. Thus, both increased mTOR-dependent HIF-I alpha synthesis and decreased hydroxylase-dependent HIF-I alpha degradation contribute to IH-evoked HIF-I alpha accumulation. Following IH, HIF-I alpha, and phosphorylated mTOR levels remained elevated during 90 min of re-oxygenation despite re-activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF-I alpha levels during re-oxygenation indicating that mTOR-dependent protein synthesis is required for the persistent elevation of HIF-I alpha levels during re-oxygenation.