title: Functional KCa3.1 channels regulate steroid insensitivity in bronchial smooth muscle cells.
abstract: Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma we see in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles that can be modeled in vitro by exposing cultured ASM cells to TNF-α/IFN-γ. This action drives GC insensitivity via protein phosphatase 5-dependent impairment of GC receptor phosphorylation. In this study, we investigated whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and immunofluorescence staining confirmed the nuclear localization of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNF-α/IFN-γ was assessed using complementary inhibitory strategies, including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific small hairpin RNA delivered by adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5, and CCL11 gene and protein expression. KCa3.1 channel blockade also restored fluticasone-induced GC receptor-α phosphorylation at Ser(211) and transactivation properties via the suppression of cytokine-induced protein phosphatase 5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary, KCa3.1 channels contribute to the regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in severe asthma.
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
authors.forename: Latifa
authors.initials: L
authors.lastname: Chachi
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
authors.forename: Aarti
authors.initials: A
authors.lastname: Shikotra
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
authors.forename: S Mark
authors.initials: SM
authors.lastname: Duffy
authors.affiliation: Department of Pharmaceutical Sciences, Thomas Jefferson University, Jefferson School of Pharmacy, Philadelphia, PA, USA.
authors.forename: Omar
authors.initials: O
authors.lastname: Tliba
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
authors.forename: Christopher
authors.initials: C
authors.lastname: Brightling
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
authors.forename: Peter
authors.initials: P
authors.lastname: Bradding
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, University Road, Leicester, LE1 7RH, U.K.
authors.forename: Yassine
authors.initials: Y
authors.lastname: Amrani
mesh_terms: D000328:Adult; D001249:Asthma / Q000188:drug therapy / Q000378:metabolism*; D015153:Blotting, Western; D001980:Bronchi / Q000378:metabolism*; D002478:Cells, Cultured; D004351:Drug Resistance / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005938:Glucocorticoids; D006801:Humans; D007150:Immunohistochemistry; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D008297:Male; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D005938:Glucocorticoids; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human
references.citation: Arterioscler Thromb Vasc Biol. 2008 Jun;28(6):1084-9
references.pmid: 18309114
references.citation: Eur J Immunol. 2005 Apr;35(4):1027-36
references.pmid: 15770697
references.citation: Proc Am Thorac Soc. 2008 Jan 1;5(1):106-12
references.pmid: 18094092
references.citation: Am J Respir Cell Mol Biol. 2010 Jan;42(1):9-15
references.pmid: 19805480
references.citation: Blood. 2003 Mar 15;101(6):2412-8
references.pmid: 12433690
references.citation: J Immunol. 1997 Feb 15;158(4):1841-7
references.pmid: 9029124
references.citation: Am J Respir Crit Care Med. 2006 Apr 1;173(7):729-35
references.pmid: 16387804
references.citation: Pharmacol Ther. 2012 Apr;134(1):54-67
references.pmid: 22212616
references.citation: Am J Respir Cell Mol Biol. 2008 Apr;38(4):463-72
references.pmid: 17947510
references.citation: Eur J Immunol. 2007 Jun;37(6):1653-62
references.pmid: 17474152
references.citation: Thorax. 2008 May;63 Suppl 4:iv1-121
references.pmid: 18463203
references.citation: Am J Respir Cell Mol Biol. 2006 Aug;35(2):243-51
references.pmid: 16574942
references.citation: Curr Pharm Des. 2011;17(7):667-73
references.pmid: 21406060
references.citation: J Leukoc Biol. 2001 Aug;70(2):233-40
references.pmid: 11493615
references.citation: N Engl J Med. 2007 Mar 29;356(13):1327-37
references.pmid: 17392302
references.citation: Expert Rev Clin Pharmacol. 2010 May;3(3):385-96
references.pmid: 22111618
references.citation: Am J Respir Cell Mol Biol. 2013 Feb;48(2):212-9
references.pmid: 23204391
references.citation: J Neurosci. 2007 Jan 3;27(1):234-44
references.pmid: 17202491
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 23;105(38):14442-6
references.pmid: 18796614
references.citation: Am J Respir Crit Care Med. 2007 Dec 15;176(12):1185-91
references.pmid: 17901415
references.citation: Am J Respir Crit Care Med. 2005 May 15;171(10):1103-8
references.pmid: 15879427
references.citation: FASEB J. 2003 Mar;17(3):452-4
references.pmid: 12514117
references.citation: Thorax. 2001 Mar;56 Suppl 1:i1-21
references.pmid: 11158709
references.citation: Am J Physiol Lung Cell Mol Physiol. 2002 Dec;283(6):L1280-90
references.pmid: 12388374
references.citation: J Immunol. 2001 Jan 15;166(2):936-44
references.pmid: 11145670
references.citation: Am J Respir Cell Mol Biol. 1996 Jul;15(1):55-63
references.pmid: 8679222
references.citation: Thorax. 2008 Sep;63(9):784-90
references.pmid: 18492738
references.citation: FASEB J. 2005 Jun;19(8):1006-8
references.pmid: 15817638
references.citation: J Allergy Clin Immunol. 2004 Jun;113(6):1100-8
references.pmid: 15208591
references.citation: Clin Exp Allergy. 2012 Jan;42(1):85-94
references.pmid: 22092970
references.citation: Am J Respir Cell Mol Biol. 2007 Nov;37(5):525-31
references.pmid: 17585114
references.citation: Am J Respir Crit Care Med. 2006 Dec 1;174(11):1179-88
references.pmid: 16959919
references.citation: Biochim Biophys Acta. 2009 May;1787(5):351-63
references.pmid: 19111672
references.citation: Nat Protoc. 2008;3(6):1101-8
references.pmid: 18546601
references.citation: J Biol Chem. 2009 Sep 4;284(36):24542-52
references.pmid: 19586900
references.citation: Proc Natl Acad Sci U S A. 2000 Jul 5;97(14):8151-6
references.pmid: 10884437
references.citation: Am J Respir Cell Mol Biol. 1994 May;10(5):471-80
references.pmid: 8179909
references.citation: Am J Respir Cell Mol Biol. 2012 Oct;47(4):464-73
references.pmid: 22592921
references.citation: Ann N Y Acad Sci. 2004 Jun;1024:86-101
references.pmid: 15265775
references.citation: Curr Pharm Des. 2010;16(32):3554-73
references.pmid: 20977420
references.citation: J Exp Med. 1997 Nov 3;186(9):1567-74
references.pmid: 9348314
references.citation: J Immunol. 1999 Aug 15;163(4):2128-34
references.pmid: 10438953
references.citation: Am J Respir Crit Care Med. 1999 Jun;159(6):1933-42
references.pmid: 10351942
references.citation: J Appl Physiol (1985). 2004 Nov;97(5):1946-53
references.pmid: 15258133
references.citation: Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1541-6
references.pmid: 20080610
references.citation: Mol Pharmacol. 2006 Feb;69(2):588-96
references.pmid: 16291871
references.citation: Am J Respir Cell Mol Biol. 2011 Nov;45(5):962-8
references.pmid: 21493782
references.citation: J Pharmacol Exp Ther. 2011 Aug;338(2):528-36
references.pmid: 21602424
references.citation: Am J Respir Crit Care Med. 2000 Dec;162(6):2341-51
references.pmid: 11112161
references.citation: Mol Cell. 2006 Dec 8;24(5):665-675
references.pmid: 17157250
references.citation: PLoS One. 2013;8(1):e53936
references.pmid: 23349769
references.citation: J Allergy Clin Immunol. 2008 Mar;121(3):580-4
references.pmid: 18234319
references.citation: J Immunol. 2006 Feb 1;176(3):1860-8
references.pmid: 16424217
references.citation: Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13942-7
references.pmid: 11717451
references.citation: Am J Physiol Cell Physiol. 2009 Feb;296(2):C285-95
references.pmid: 19052260
references.citation: J Allergy Clin Immunol. 2007 Apr;119(4):863-71
references.pmid: 17339047
references.citation: Thorax. 2006 Oct;61(10):880-5
references.pmid: 16809411
references.citation: Curr Pharm Des. 2011;17(7):674-84
references.pmid: 21406059
references.citation: J Clin Invest. 2008 Sep;118(9):3025-37
references.pmid: 18688283
references.citation: Am J Respir Crit Care Med. 2006 May 1;173(9):965-9
references.pmid: 16456145
references.citation: J Allergy Clin Immunol. 2002 Apr;109(4):649-57
references.pmid: 11941315
references.citation: Am J Respir Cell Mol Biol. 2004 Jul;31(1):36-42
references.pmid: 14764428
references.citation: J Allergy Clin Immunol. 2011 Dec;128(6):1303-1309.e2
references.pmid: 21872912
references.citation: J Immunol. 1993 Oct 1;151(7):3460-6
references.pmid: 8376786
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 2004 Jun;369(6):602-15
references.pmid: 15127180
references.citation: J Allergy Clin Immunol. 2012 Jan;129(1):104-11.e1-9
references.pmid: 21975173