TY - JOUR
T1 - Ca2+/calmodulin kinase-dependent activation of hypoxia inducible factor 1 transcriptional activity in cells subjected to intermittent hypoxia
AU - Yuan, Guoxiang
AU - Nanduri, Jayasri
AU - Bhasker, C. Raman
AU - Semenza, Gregg L.
AU - Prabhakar, Nanduri R.
PY - 2005/2/11
Y1 - 2005/2/11
N2 - Intermittent hypoxia (IH) occurs in many pathological conditions. However, very little is known about the molecular mechanisms associated with IH. Hypoxia-inducible factor 1 (HIF-1) mediates transcriptional responses to continuous hypoxia. In the present study, we investigated whether IH activates HIF-1 and, if so, which signaling pathways are involved. PC12 cells were exposed to either to 20% O2 (non-hypoxic control) or to 60 cycles consisting of 30 s at 1.5% O2, followed by 4 min at 20% O2 (IH). Western blot analysis revealed significant increases in HIF-1α protein in nuclear extracts of cells subjected to IH. Expression of a HIF-1-dependent reporter gene was increased 3-fold in cells subjected to IH. Although IH induced the activation of BRK1, ERK2, JNK, PKC-α, and PKC-γ, inhibitors of these kinases and of phosphatidylinositol 3-kinase did not block HIF-1-mediated reporter gene expression induced by IH, indicating that signaling via these kinases was not required. In contrast, addition of the intracellular Ca 2+ chelator BAPTA-AM or the Ca2+/calmodulin-dependent (CaM) kinase inhibitor KN93 blocked reporter gene activation in response to IH. CaM kinase activity was increased 5-fold in cells subjected to IH. KN 93 prevented IH-induced transactivation mediated by HIF-1α, and its coactivator p300, which was phosphorylated by CaM kinase II in vitro. Expression of the HIF-1-regulated gene encoding tyrosine hydroxylase was induced by IH and this effect was blocked by KN93. These observations suggest that IH induces HIF-1 transcriptional activity via a novel signaling pathway involving CaM kinase.
AB - Intermittent hypoxia (IH) occurs in many pathological conditions. However, very little is known about the molecular mechanisms associated with IH. Hypoxia-inducible factor 1 (HIF-1) mediates transcriptional responses to continuous hypoxia. In the present study, we investigated whether IH activates HIF-1 and, if so, which signaling pathways are involved. PC12 cells were exposed to either to 20% O2 (non-hypoxic control) or to 60 cycles consisting of 30 s at 1.5% O2, followed by 4 min at 20% O2 (IH). Western blot analysis revealed significant increases in HIF-1α protein in nuclear extracts of cells subjected to IH. Expression of a HIF-1-dependent reporter gene was increased 3-fold in cells subjected to IH. Although IH induced the activation of BRK1, ERK2, JNK, PKC-α, and PKC-γ, inhibitors of these kinases and of phosphatidylinositol 3-kinase did not block HIF-1-mediated reporter gene expression induced by IH, indicating that signaling via these kinases was not required. In contrast, addition of the intracellular Ca 2+ chelator BAPTA-AM or the Ca2+/calmodulin-dependent (CaM) kinase inhibitor KN93 blocked reporter gene activation in response to IH. CaM kinase activity was increased 5-fold in cells subjected to IH. KN 93 prevented IH-induced transactivation mediated by HIF-1α, and its coactivator p300, which was phosphorylated by CaM kinase II in vitro. Expression of the HIF-1-regulated gene encoding tyrosine hydroxylase was induced by IH and this effect was blocked by KN93. These observations suggest that IH induces HIF-1 transcriptional activity via a novel signaling pathway involving CaM kinase.
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U2 - 10.1074/jbc.M407706200
DO - 10.1074/jbc.M407706200
M3 - Article
C2 - 15569687
AN - SCOPUS:14244256421
SN - 0021-9258
VL - 280
SP - 4321
EP - 4328
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 6
ER -