Induction of HIF-1alpha expression by intermittent hypoxia: involvement of NADPH oxidase, Ca2+ signaling, prolyl hydroxylases, and mTOR

J Cell Physiol. 2008 Dec;217(3):674-85. doi: 10.1002/jcp.21537.


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 1alpha (HIF-1alpha) do not develop cardio-respiratory responses to chronic IH. HIF-1alpha protein expression and HIF-1 transcriptional activity are induced by IH in PC12 cells. In the present study, we investigated the signaling pathways associated with IH-evoked HIF-1alpha 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-induced HIF-1alpha accumulation is due to increased generation of ROS by NADPH oxidase. We further demonstrate that ROS-dependent Ca(2+) signaling pathways involving phospholipase Cgamma (PLCgamma) and protein kinase C activation are required for IH-evoked HIF-1alpha accumulation. IH leads to activation of mTOR and S6 kinase (S6K) and rapamycin partially inhibited IH-induced HIF-1alpha accumulation. IH also decreased hydroxylation of HIF-1alpha protein and anti-oxidants as well as inhibitors of Ca(+2) signaling prevented this response. Thus, both increased mTOR-dependent HIF-1alpha synthesis and decreased hydroxylase-dependent HIF-1alpha degradation contribute to IH-evoked HIF-1alpha accumulation. Following IH, HIF-1alpha, and phosphorylated mTOR levels remained elevated during 90 min of re-oxygenation despite re-activation of prolyl hydroxylase. Rapamycin or cycloheximide, blocked increased HIF-1alpha levels during re-oxygenation indicating that mTOR-dependent protein synthesis is required for the persistent elevation of HIF-1alpha levels during re-oxygenation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Calcium / metabolism
  • Calcium Signaling* / drug effects
  • Cell Hypoxia / drug effects
  • Enzyme Activation / drug effects
  • Hydroxylation / drug effects
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • Mice
  • Models, Biological
  • NADPH Oxidases / metabolism*
  • Oxygen / pharmacology
  • PC12 Cells
  • Phospholipase C gamma / metabolism
  • Procollagen-Proline Dioxygenase / metabolism*
  • Protein Biosynthesis / drug effects
  • Protein Kinase C / metabolism
  • Protein Kinases / metabolism*
  • Rats
  • Reactive Oxygen Species / metabolism
  • Ribosomal Protein S6 Kinases / metabolism
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases
  • Thermodynamics


  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Reactive Oxygen Species
  • Procollagen-Proline Dioxygenase
  • NADPH Oxidases
  • Protein Kinases
  • mTOR protein, mouse
  • Ribosomal Protein S6 Kinases
  • TOR Serine-Threonine Kinases
  • Protein Kinase C
  • Phospholipase C gamma
  • Oxygen
  • Calcium
  • Sirolimus