title: L-type Ca(2+) channel activation regulates induction of c-fos transcription by hypoxia.
abstract: In the present study we examined the intracellular pathways that link hypoxia to activation of c-fos gene expression. Experiments were performed on rat pheocromocytoma-12 (PC-12) cells. c-fos mRNA and promoter activities were analyzed by RT-PCR and reporter gene assays, respectively. BAPTA, a Ca(2+) chelator, inhibited c-fos mRNA and promoter activation by hypoxia. Nitrendipine, an L-type Ca(2+)-channel blocker, abolished, whereas BAY K 8644, an L-type channel agonist, enhanced c-fos activation by hypoxia. Ca(2+) currents were augmented reversibly by hypoxia, suggesting that Ca(2+) influx mediated by L-type Ca(2+) channels is essential for c-fos activation by hypoxia. We next determined downstream pathways activated by intracellular Ca(2+) concentration. Immunoblot analysis revealed Ca(2+)/calmodulin-dependent kinase II (CaMKII) protein in PC-12 cells and revealed that hypoxia increased the enzyme activity. KN-93, a CaMK inhibitor, blocked CaMKII activation and c-fos promoter stimulation by hypoxia. Ectopic expression of an active mutant of CaMKII (pCaMKII290) stimulated c-fos promoter activity under normoxia. Hypoxia increased phosphorylation of CREB at the serine residue 133 (Ser-133), and KN-93 attenuated this effect. Point mutations at the Ca(2+)/cAMP-responsive cis-element (Ca/CRE) attenuated, whereas point mutations in the serum-responsive cis-element (SRE) abolished transcriptional activation of c-fos by hypoxia. 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 Ca(2+)-dependent signaling pathways play a crucial role in induction of c-fos gene expression, which may underlie long-term adaptive responses to hypoxia.
authors.affiliation: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Premkumar
authors.forename: R R
authors.initials: RR
authors.lastname: Mishra
authors.forename: J L
authors.initials: JL
authors.lastname: Overholt
authors.forename: M S
authors.initials: MS
authors.lastname: Simonson
authors.forename: N S
authors.initials: NS
authors.lastname: Cherniack
authors.forename: N R
authors.initials: NR
authors.lastname: Prabhakar
mesh_terms: D000818:Animals; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002614:Chelating Agents / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D000860:Hypoxia / Q000235:genetics / Q000503:physiopathology*; D016716:PC12 Cells; D010766:Phosphorylation; D017354:Point Mutation / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002614:Chelating Agents; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D004533:Egtazic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid