L-type Ca2+ channel activation regulates induction of c-fos transcription by hypoxia

In the present study we examined the intracellular pathways that link hypox ia to activation of c-fos gene expression. Experiments were performed on ra t pheocromocytoma-12 (PC-12) cells. c-fos mRNA and promoter activities were analyzed by RT-PCR and reporter gene assays, respectively. BAPTA, a Ca2+ c helator, inhibited c-fos mRNA and promoter activation by hypoxia. Nitrendip ine, an L-type Ca2+-channel blocker, abolished, whereas BAY K 8644, an L-ty pe channel agonist, enhanced c-fos activation by hypoxia. Ca2+ currents wer e augmented reversibly by hypoxia, suggesting that Ca2+ influx mediated by L-type Ca2+ channels is essential for c-fos activation by hypoxia. We next determined downstream pathways activated by intracellular Ca2+ concentratio n. Immunoblot analysis revealed Ca2+/calmodulin-dependent kinase II (CaMKII ) protein in PC-12 cells and revealed that hypoxia increased the enzyme act ivity. KN-93, a CaMK inhibitor, blocked CaMKII activation and c-fos promote r stimulation by hypoxia. Ectopic expression of an active mutant of CaMKII (pCaNMII290) stimulated c-fos promoter activity under normoxia. Hypoxia inc reased phosphorylation of CREB at the serine residue 133 (Ser-133), and KN- 93 attenuated this effect. Point mutations at the Ca2+/cAMP-responsive cis- element (Ca/CRE) attenuated, whereas point mutations in the serum-responsiv e cis-element (SRE) abolished transcriptional activation of c-fos by hypoxi a. These results demonstrate that c-fos activation by hypoxia involves CaMK activation and CREB phosphorylation at Ser-133 and requires Ca/CRE and SRE . These observations demonstrate that Ca2+-dependent signaling pathways pla y a crucial role in induction of c-fos gene expression, which may underlie long-term adaptive responses to hypoxia.