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-1alpha 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 ERK1, ERK2, JNK, PKC-alpha, and PKC-gamma, 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 Ca2+ 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-1alpha, 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.
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-1alpha 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 ERK1 , ERK2 , JNK , PKC - alpha , and PKC - gamma , 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 Ca2 + 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-1alpha , 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 .
Which kinase is inhibited by the small molecule KN-93?
Which kinase is inhibited by the small molecule KN-93 ?
CaM kinase II
The calcium/calmodulin-dependent protein kinase-II CaM kinase II CAMK2