title: Transcriptional regulation of α1H T-type calcium channel under hypoxia.
abstract: The low-voltage-activated T-type Ca(2+) channels play an important role in mediating the cellular responses to altered oxygen tension. Among three T-type channel isoforms, α1G, α1H, and α1I, only α1H was found to be upregulated under hypoxia. However, mechanisms underlying such hypoxia-dependent isoform-specific gene regulation remain incompletely understood. We, therefore, studied the hypoxia-dependent transcriptional regulation of α1G and α1H gene promoters with the aim to identify the functional hypoxia-response elements (HREs). In rat pulmonary artery smooth muscle cells (PASMCs) and pheochromocytoma (PC12) cells after hypoxia (3% O2) exposure, we observed a prominent increase in α1H mRNA at 12 h along with a significant rise in α1H-mediated T-type current at 24 and 48 h. We then cloned two promoter fragments from the 5'-flanking regions of rat α1G and α1H gene, 2,000 and 3,076 bp, respectively, and inserted these fragments into a luciferase reporter vector. Transient transfection of PASMCs and PC12 cells with these recombinant constructs and subsequent luciferase assay revealed a significant increase in luciferase activity from the reporter containing the α1H, but not α1G, promoter fragment under hypoxia. Using serial deletion and point mutation analysis strategies, we identified a functional HRE at site -1,173cacgc-1,169 within the α1H promoter region. Furthermore, an electrophoretic mobility shift assay using this site as a DNA probe demonstrated an increased binding activity to nuclear protein extracts from the cells after hypoxia exposure. Taken together, these findings indicate that hypoxia-induced α1H upregulation involves binding of hypoxia-inducible factor to an HRE within the α1H promoter region.
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Georgia Regents University, Augusta, Georgia;
authors.forename: Hassan
authors.initials: H
authors.lastname: Sellak
authors.affiliation: Center for Lung Biology, University of South Alabama, Mobile, Alabama; Department of Pharmacology, University of South Alabama, Mobile, Alabama; and.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhou
authors.affiliation: Center for Lung Biology, University of South Alabama, Mobile, Alabama; Department of Pharmacology, University of South Alabama, Mobile, Alabama; and.
authors.forename: Bainan
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Georgia Regents University, Augusta, Georgia;
authors.forename: Hairu
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Physiology, University of South Alabama, Mobile, Alabama.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Lincoln
authors.affiliation: Department of Anesthesiology and Perioperative Medicine, Georgia Regents University, Augusta, Georgia; sowu@gru.edu.
authors.forename: Songwei
authors.initials: S
authors.lastname: Wu
mesh_terms: D000818:Animals; D001665:Binding Sites; D020747:Calcium Channels, T-Type / Q000235:genetics* / Q000378:metabolism; D015687:Cell Hypoxia; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D016716:PC12 Cells; D011651:Pulmonary Artery / Q000378:metabolism; D011667:Pulmonary Veins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020218:Response Elements; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
chemical_list: C500994:Cacna1g protein, rat; C509958:Cacna1h protein, rat; D020747:Calcium Channels, T-Type; D012333:RNA, Messenger
keywords: T-type calcium channel; gene expression; hypoxia; hypoxia-response element
references.citation: J Neurosci. 2010 Apr 7;30(14):4957-69
references.pmid: 20371816
references.citation: Am J Physiol Lung Cell Mol Physiol. 2013 Jul 15;305(2):L154-64
references.pmid: 23686856
references.citation: Proc Assoc Am Physicians. 1999 Sep-Oct;111(5):429-37
references.pmid: 10519164
references.citation: J Biol Chem. 2000 Jun 2;275(22):16530-5
references.pmid: 10749850
references.citation: Cell Calcium. 2008 Jan;43(1):49-58
references.pmid: 17532042
references.citation: Nat Cell Biol. 2007 Feb;9(2):210-7
references.pmid: 17220880
references.citation: Am J Physiol Lung Cell Mol Physiol. 2000 Mar;278(3):L610-7
references.pmid: 10710534
references.citation: Am J Physiol. 1995 Apr;268(4 Pt 1):C985-92
references.pmid: 7733247
references.citation: Circ Res. 2005 Apr 29;96(8):864-72
references.pmid: 15774856
references.citation: J Biol Chem. 2003 Jun 20;278(25):22316-24
references.pmid: 12679337
references.citation: Hypertension. 2010 Dec;56(6):1145-51
references.pmid: 21059997
references.citation: Circ Res. 1993 Oct;73(4):777-82
references.pmid: 8396509
references.citation: Arterioscler Thromb Vasc Biol. 1995 Aug;15(8):1161-5
references.pmid: 7627710
references.citation: Microvasc Res. 2004 Mar;67(2):139-51
references.pmid: 15020205
references.citation: Cell Calcium. 2006 Aug;40(2):135-46
references.pmid: 16797068
references.citation: Circ Res. 2003 Aug 22;93(4):346-53
references.pmid: 12869394
references.citation: Am J Physiol Lung Cell Mol Physiol. 2010 Jul;299(1):L86-97
references.pmid: 20435690
references.citation: Am J Physiol Heart Circ Physiol. 2009 Oct;297(4):H1304-13
references.pmid: 19666840
references.citation: Circ Res. 2006 Jun 9;98(11):1422-30
references.pmid: 16690884
references.citation: J Mol Cell Cardiol. 2010 Jan;48(1):65-70
references.pmid: 19729018
references.citation: Biophys J. 1999 Dec;77(6):3034-42
references.pmid: 10585925
references.citation: Cardiovasc Res. 2013 Nov 1;100(2):222-30
references.pmid: 23929524
references.citation: J Biol Chem. 2002 Mar 29;277(13):10824-33
references.pmid: 11799114
references.citation: Diabetes. 2000 Jan;49(1):59-64
references.pmid: 10615950
references.citation: Proteins. 2006 Aug 1;64(2):320-42
references.pmid: 16671074
references.citation: J Physiol. 2007 Oct 1;584(Pt 1):149-65
references.pmid: 17690152
references.citation: J Biol Chem. 2008 Apr 25;283(17):11841-9
references.pmid: 18303027
references.citation: J Biol Chem. 2012 May 4;287(19):15489-501
references.pmid: 22431737
references.citation: Cardiovasc Res. 2012 Dec 1;96(3):533-42
references.pmid: 22886848
references.citation: J Physiol. 2002 Apr 1;540(Pt 1):3-14
references.pmid: 11927664
references.citation: J Appl Physiol (1985). 2000 Jun;88(6):2303-9
references.pmid: 10846049
references.citation: J Neurosci. 2008 Dec 3;28(49):13341-53
references.pmid: 19052226
references.citation: J Mol Cell Cardiol. 2007 Jun;42(6):1045-53
references.pmid: 17498735
references.citation: J Physiol. 2009 May 1;587(Pt 9):1917-29
references.pmid: 19273573
references.citation: Glia. 2004 Nov 1;48(2):112-9
references.pmid: 15378657
references.citation: J Pharmacol Sci. 2005 Nov;99(3):211-3
references.pmid: 16272789
references.citation: Eur J Neurosci. 2003 May;17(9):1802-10
references.pmid: 12752779
references.citation: J Biol Chem. 2009 Sep 4;284(36):24025-34
references.pmid: 19567868
references.citation: Circ Res. 1998 Jul 13;83(1):103-9
references.pmid: 9670923
references.citation: Nature. 1998 Feb 26;391(6670):896-900
references.pmid: 9495342
references.citation: Cancer Res. 1999 Sep 15;59(18):4535-41
references.pmid: 10493502
references.citation: Physiol Rev. 2003 Jan;83(1):117-61
references.pmid: 12506128
references.citation: J Biol Chem. 2007 Apr 13;282(15):10873-80
references.pmid: 17303576
references.citation: J Biol Chem. 2001 Feb 9;276(6):3999-4011
references.pmid: 11073957
references.citation: Am J Physiol Lung Cell Mol Physiol. 2007 Apr;292(4):L833-44
references.pmid: 17172292