Mechanisms of HIF‐1α Stabilization by Intermittent Hypoxia: Role of Ca ²⁺ ‐mTOR signaling

We recently reported that intermittent hypoxia (IH) activates HIF‐1 mediated transcription via CaMKII‐dependent phosphorylation of the co‐activator p300 (Yuan et al. J. Biol. Chem.2005). In the present study we examined whether Ca2+ also plays a role in HIF‐1α stabilization by IH. Experiments were performed on PC12 cells exposed to either 20% O2 (normoxic control) or 60 cycles of IH (30 sec at 1.5% O2 followed by 4 min at 20% O2). BAPTA‐AM, a calcium chelator, or 2‐APB, a membrane‐permeable inhibitor of the inositol 1,4,5‐trisphosphate (IP3) receptor prevented HIF‐1 α stabilization by IH. Because activation of PLCγ is required for IP‐3 receptor activation, we examined the involvement of PLCγ in IH‐induced HIF‐1α accumulation. IH increased PLCγ phosphorylation, and genistein an inhibitor of tyrosine kinase prevented this effect. U73122, inhibitor of PLC abolished IH‐induced HIF‐1α accumulation. Since it is known that PLCγ –mediated Ca 2+ can potentially regulate mTOR signaling, the involvement of mTOR was examined. IH increased phosphorylation of mTOR and downstream S6Kinase1 and these effects were blocked by either genistein or by U73122. The involvement of mTOR was further confirmed using rapamycin, an inhibitor of mTOR, which effectively inhibited IH‐induced HIF‐1α accumulation. These observations demonstrate that Ca 2+ activation by PLCγ and mTOR pathways contribute in part to the stabilization of HIF‐1α protein by IH. Supported by NIH‐HL‐25830, HL‐55338.