title: In vitro studies on the antimicrobial peptide human beta-defensin 9 (HBD9): signalling pathways and pathogen-related response (an American Ophthalmological Society thesis).
abstract: OBJECTIVE
  Human β-defensins (HBDs) are an important part of the innate immune host defense at the ocular surface. Unlike other defensins, expression of HBD9 at the ocular surface is reduced during microbial infection, but activation of toll-like receptor 2 (TLR2) in corneal epithelial cells has been shown to up-regulate HBD9. Our purpose was to test the hypothesis that TLR2 has a key role in the signalling pathway(s) involved in the overexpression or underexpression of HBD9, and accordingly, different pathogens would induce a different expression pattern of HBD9.
  
  
  METHODS
  The in vitro RNAi silencing method and response to dexamethasone were used to determine key molecules involved in signalling pathways of HBD9 in immortalized human corneal epithelial cells. The techniques included cell culture with exposure to specific transcription factor inhibitors and bacteria, RNA extraction and cDNA synthesis, quantitative real-time polymerase chain reaction, and immunohistology.
  
  
  RESULTS
  This study demonstrates that TLR2 induces HBD9 mRNA and protein expression in a time- and dose-dependent manner. Transforming growth factor-β-activated kinase 1 (TAK1) plays a central role in HBD9 induction by TLR2, and transcription factors c-JUN and activating transcription factor 2 are also involved. Dexamethasone reduces TLR2-mediated up-regulation of HBD9 mRNA and protein levels in mitogen-activated protein kinase phosphatase 1 (MKP1)-dependent and c-JUN-independent manner. HBD9 expression differs with gram-negative and gram-positive bacteria.
  
  
  CONCLUSIONS
  TLR2-mediated MKPs and nuclear factor-κB signalling pathways are involved in HBD9 expression. TAK-1 is a key molecule. These molecules can be potentially targeted to modulate HBD9 expression. Differential expression of HBD9 with different bacteria could be related to differences in pathogen-associated molecular patterns of these organisms.
authors: Academic Department of Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, England.
authors: Harminder S
authors: HS
authors: Dua
authors: Academic Department of Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, England.
authors: Ahmad Muneer
authors: AM
authors: Otri
authors: Academic Department of Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, England.
authors: Andrew
authors: A
authors: Hopkinson
authors: Academic Department of Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, England.
authors: Imran
authors: I
authors: Mohammed
mesh_terms: D051698:Activating Transcription Factor 2 / Q000502:physiology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism; D015818:Eye Infections, Bacterial / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000502:physiology*; D006094:Gram-Positive Bacteria / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D020930:MAP Kinase Kinase Kinases / Q000502:physiology; D012313:RNA / Q000378:metabolism; D000077278:RNA, Mitochondrial; D015398:Signal Transduction / Q000502:physiology*; D051195:Toll-Like Receptor 2 / Q000502:physiology*; D023083:beta-Defensins / Q000378:metabolism*; D000099151:MAP Kinase Kinase Kinase 7
chemical_list: D051698:Activating Transcription Factor 2; D000893:Anti-Inflammatory Agents; D003907:Dexamethasone; D048031:JNK Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D012313:RNA; D000077278:RNA, Mitochondrial; D051195:Toll-Like Receptor 2; D023083:beta-Defensins; D000099151:MAP Kinase Kinase Kinase 7; C526629:DEFB-109 peptide, human; C495342:TLR2 protein, human
references: Infect Immun. 1999 Apr;67(4):2005-9
references: 10085049
references: FASEB J. 2003 Sep;17(12):1727-9
references: 12958190
references: Antimicrob Agents Chemother. 1997 Aug;41(8):1738-42
references: 9257752
references: Ann Anat. 2011 Dec 20;193(6):479-85
references: 22056339
references: J Med Microbiol. 1998 Sep;47(9):837-42
references: 9736166
references: Peptides. 2003 Nov;24(11):1647-54
references: 15019196
references: Infect Immun. 2002 Dec;70(12):6524-33
references: 12438321
references: Infect Immun. 2005 Apr;73(4):2147-56
references: 15784557
references: Adv Exp Med Biol. 2002;506(Pt A):601-5
references: 12613966
references: J Clin Invest. 1985 Oct;76(4):1427-35
references: 2997278
references: Exp Eye Res. 1999 Nov;69(5):483-90
references: 10548468
references: J Clin Invest. 1988 Aug;82(2):552-6
references: 2841356
references: Nature. 1997 Jun 26;387(6636):861
references: 9202117
references: J Biol Chem. 2008 Jan 4;283(1):137-44
references: 17965022
references: Exp Eye Res. 2004 Aug;79(2):203-8
references: 15325567
references: Exp Eye Res. 2003 Jul;77(1):17-26
references: 12823984
references: Lancet. 1998 Feb 7;351(9100):387-8
references: 9482289
references: Electrophoresis. 2010 Jun;31(11):1853-61
references: 20506419
references: Trans Am Ophthalmol Soc. 2002;100:243-71
references: 12545697
references: Cell Mol Life Sci. 2011 Jul;68(13):2201-13
references: 21584809
references: Cornea. 1992 Sep;11(5):370-5
references: 1424662
references: Graefes Arch Clin Exp Ophthalmol. 2006 Feb;244(2):143-8
references: 16044323
references: Curr Opin Pharmacol. 2006 Oct;6(5):468-72
references: 16890021
references: J Biol Chem. 2008 May 23;283(21):14277-85
references: 18356163
references: Hum Gene Ther. 2002 Nov 20;13(17):2017-25
references: 12489997
references: Biotechnol Lett. 2005 Sep;27(18):1337-47
references: 16215847
references: J Leukoc Biol. 2005 Apr;77(4):476-86
references: 15629886
references: J Leukoc Biol. 2004 Feb;75(2):190-7
references: 14576362
references: J Biol Chem. 1996 Jun 28;271(26):15428-35
references: 8663196
references: Invest Ophthalmol Vis Sci. 2003 Sep;44(9):3795-801
references: 12939294
references: Am J Pathol. 1998 May;152(5):1247-58
references: 9588893
references: Invest Ophthalmol Vis Sci. 2013 Sep;54(9):6373-81
references: 23989188
references: Antimicrob Agents Chemother. 2006 Feb;50(2):806-9
references: 16436752
references: Nat Immunol. 2001 Dec;2(12):1133-7
references: 11694882
references: Infect Immun. 2011 Feb;79(2):595-605
references: 21115716
references: Lancet. 1998 Aug 8;352(9126):451-2
references: 9708756
references: J Exp Med. 2006 Aug 7;203(8):1883-9
references: 16880258
references: J Biol Chem. 2001 Feb 23;276(8):5707-13
references: 11085990
references: Curr Eye Res. 1998 Apr;17(4):348-62
references: 9561826
references: J Immunol. 2005 Nov 15;175(10):6676-85
references: 16272323
references: Cont Lens Anterior Eye. 2013 Jun;36(3):140-5
references: 23290099
references: FASEB J. 2008 Jun;22(6):1807-16
references: 18230685
references: Am J Physiol Lung Cell Mol Physiol. 2004 Mar;286(3):L514-20
references: 14594730
references: Am J Respir Cell Mol Biol. 2004 Feb;30(2):193-201
references: 12871849
references: J Biol Chem. 2000 Sep 22;275(38):29731-6
references: 10882713
references: Blood. 2009 May 7;113(19):4566-74
references: 19196865
references: Acta Paediatr. 2001 Jul;90(7):757-64
references: 11519978
references: J Invest Dermatol. 2006 Jan;126(1):121-7
references: 16417227
references: Mediators Inflamm. 2003 Feb;12(1):37-46
references: 12745547
references: Invest Ophthalmol Vis Sci. 2007 Sep;48(9):3945-53
references: 17724171
references: Oral Microbiol Immunol. 2004 Apr;19(2):111-7
references: 14871351
references: Invest Ophthalmol Vis Sci. 2012 Feb;53(2):757-61
references: 22232436
references: Respir Res. 2009;10:96
references: 19835594
references: Exp Eye Res. 2004 Feb;78(2):173-85
references: 14729350
references: Lancet. 2002 Oct 12;360(9340):1144-9
references: 12387964
references: J Biol Chem. 2004 Jun 11;279(24):25876-82
references: 15075345
references: Science. 1999 Oct 1;286(5437):113-7
references: 10506557
references: J Immunol. 2003 Dec 15;171(12):6820-6
references: 14662888
references: Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8686-90
references: 9238038
references: Annu Rev Physiol. 2002;64:709-48
references: 11826286
references: Infect Immun. 2011 Jul;79(7):2709-16
references: 21555403
references: J Antimicrob Chemother. 1999 Sep;44(3):403-6
references: 10511411
references: Br J Dermatol. 2004 Sep;151(3):534-9
references: 15377337
references: Blood. 1993 Jul 15;82(2):641-50
references: 8329717
references: Virulence. 2010 Sep-Oct;1(5):440-64
references: 21178486
references: Infect Immun. 2000 Jan;68(1):113-9
references: 10603376
references: J Immunol. 2004 Nov 1;173(9):5398-405
references: 15494486
references: Int J Biochem Cell Biol. 2001 Mar;33(3):205-20
references: 11311852
references: Nature. 2002 Jan 24;415(6870):389-95
references: 11807545
references: J Biol Chem. 1998 Feb 6;273(6):3718-24
references: 9452503
references: Antimicrob Agents Chemother. 2003 Feb;47(2):494-500
references: 12543649
references: Biosci Rep. 2001 Aug;21(4):463-80
references: 11900322
references: Br J Ophthalmol. 2006 Mar;90(3):378-82
references: 16488966
references: J Clin Invest. 1998 Oct 15;102(8):1583-90
references: 9788972
references: Curr Eye Res. 2005 Jul;30(7):505-15
references: 16020284
references: J Proteome Res. 2005 Nov-Dec;4(6):2052-61
references: 16335950
references: Exp Eye Res. 2008 Mar;86(3):457-8
references: 17412322
references: Respir Res. 2000;1(3):141-50
references: 11667978
references: Am J Ophthalmol. 2013 Jun;155(6):961-970.e2
references: 23601656
references: J Biol Chem. 1996 Mar 15;271(11):6497-501
references: 8626452
references: Ocul Surf. 2004 Oct;2(4):229-47
references: 17216098
references: J Infect Dis. 1995 Aug;172(2):380-8
references: 7622881
references: J Biol Chem. 2004 Oct 22;279(43):44731-9
references: 15284227
references: Ocul Surf. 2013 Apr;11(2):75-92
references: 23583043
references: Cell Mol Life Sci. 2011 Jun;68(11):1941-52
references: 20967562
references: J Biol Chem. 2009 Sep 25;284(39):26803-15
references: 19648110
references: Mol Endocrinol. 1993 Apr;7(4):570-84
references: 8388998
references: Nature. 1999 Mar 18;398(6724):252-6
references: 10094049
references: J Histochem Cytochem. 2002 May;50(5):651-60
references: 11967276
references: Curr Eye Res. 2001 Jan;22(1):64-7
references: 11402380
references: Br J Ophthalmol. 1999 Jun;83(6):737-41
references: 10340986
references: Antimicrob Agents Chemother. 1999 Jun;43(6):1317-23
references: 10348745
references: Microbes Infect. 2006 May;8(6):1513-21
references: 16697678
references: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14083-8
references: 12384573
references: J Immunol. 2006 Feb 1;176(3):1899-907
references: 16424221
references: Microbiology. 2007 May;153(Pt 5):1297-306
references: 17464044
references: Korean J Ophthalmol. 2013 Jun;27(3):199-203
references: 23730113
references: J Immunol. 2006 Dec 1;177(11):7497-504
references: 17114416
references: Inflamm Res. 2000 Feb;49(2):73-9
references: 10738945
references: Curr Eye Res. 1998 Nov;17(11):1082-6
references: 9846627
references: Biochem Soc Trans. 2011 Aug;39(4):1045-50
references: 21787345
references: Nat Immunol. 2005 Nov;6(11):1087-95
references: 16186825
references: Exp Mol Med. 2004 Jun 30;36(3):204-10
references: 15272231
references: Invest Ophthalmol Vis Sci. 2003 May;44(5):1859-65
references: 12714616
references: Clin Chim Acta. 2006 Jul 15;369(1):17-28
references: 16516878
references: Am J Respir Cell Mol Biol. 2010 Jun;42(6):635-43
references: 20395631
references: J Immunol. 1995 Nov 1;155(9):4476-84
references: 7594610
references: J Mol Endocrinol. 2000 Oct;25(2):169-93
references: 11013345
references: Invest Ophthalmol Vis Sci. 1995 Mar;36(3):614-21
references: 7534282
references: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14961-6
references: 9843998
references: J Proteome Res. 2004 May-Jun;3(3):410-6
references: 15253421
references: Curr Eye Res. 2004 May;28(5):297-306
references: 15287366
references: J Invest Dermatol. 2003 Jul;121(1):28-36
references: 12839560
references: Invest Ophthalmol Vis Sci. 2012 May;53(6):2845-55
references: 22467583
references: Cell Mol Life Sci. 2006 Jun;63(11):1294-313
references: 16710608
references: Hum Gene Ther. 1999 Apr 10;10(6):957-64
references: 10223729
references: J Leukoc Biol. 2002 Jan;71(1):154-62
references: 11781391
references: Invest Ophthalmol Vis Sci. 2001 Sep;42(10):2157-63
references: 11527925
references: Gene. 1998 Nov 19;222(2):237-44
references: 9831658
references: Mol Endocrinol. 2009 Jan;23(1):86-99
references: 18945810
references: Science. 1995 Oct 13;270(5234):286-90
references: 7569976
references: Exp Eye Res. 2001 May;72(5):605-8
references: 11311052
references: Ophthalmic Res. 2009;41(2):60-75
references: 19122467
references: Infect Immun. 1999 Nov;67(11):6084-9
references: 10531270
references: Nature. 2004 Aug 12;430(7001):793-7
references: 15306813
references: FEMS Immunol Med Microbiol. 2007 Oct;51(1):171-84
references: 17645739
references: Mol Divers. 2006 Nov;10(4):585-97
references: 16969722
references: Invest Ophthalmol Vis Sci. 2000 Sep;41(10):3026-31
references: 10967060
references: Int J Antimicrob Agents. 2013 Jan;41(1):5-10
references: 23121833
references: CLAO J. 1996 Apr;22(2):114-7
references: 8728617
references: Exp Eye Res. 1998 Feb;66(2):129-37
references: 9533839
references: Adv Exp Med Biol. 2002;506(Pt A):547-53
references: 12613959
references: Gut. 2001 Feb;48(2):176-85
references: 11156637
references: J Exp Med. 2006 Jan 23;203(1):131-40
references: 16380513
references: J Biol Chem. 2009 Sep 4;284(36):24006-16
references: 19592489
references: Am J Respir Cell Mol Biol. 1995 Mar;12(3):351-7
references: 7873202
references: Clin Microbiol Rev. 2009 Apr;22(2):240-73, Table of Contents
references: 19366914
references: Invest Ophthalmol Vis Sci. 2011 Jun;52(7):4914-22
references: 21551409
references: Cell. 1996 Sep 20;86(6):973-83
references: 8808632
references: Infect Immun. 2000 May;68(5):2748-55
references: 10768969
references: J Biol Chem. 1997 Jun 13;272(24):15258-63
references: 9182550
references: PLoS Pathog. 2013;9(7):e1003457
references: 23874197
references: Exp Eye Res. 2002 Feb;74(2):293-9
references: 11950239
references: Exp Eye Res. 2013 Dec;117:53-61
references: 23880529
references: Br J Ophthalmol. 2006 May;90(5):609-11
references: 16622092
references: Invest Ophthalmol Vis Sci. 2008 Jan;49(1):28-33
references: 18172071
references: J Invest Dermatol. 2005 May;124(5):931-8
references: 15854033
references: Immunology. 2002 May;106(1):20-6
references: 11972628
references: Invest Ophthalmol Vis Sci. 2005 Apr;46(4):1379-85
references: 15790905
references: Proc Natl Acad Sci U S A. 2006 Feb 14;103(7):2274-9
references: 16461893
references: Trends Pharmacol Sci. 2002 Nov;23(11):494-6
references: 12413797
references: J Immunol. 2003 Sep 15;171(6):3262-9
references: 12960356
references: Invest Ophthalmol Vis Sci. 2002 May;43(5):1414-21
references: 11980855
references: Br J Ophthalmol. 2002 Dec;86(12):1412-6
references: 12446377
references: Mol Vis. 2011;17:113-26
references: 21245952
references: J Mol Endocrinol. 2008 Oct;41(4):239-49
references: 18682532
references: Lancet Infect Dis. 2001 Oct;1(3):156-64
references: 11871492
references: Invest Ophthalmol Vis Sci. 2010 Sep;51(9):4677-82
references: 20375350
references: Invest Ophthalmol Vis Sci. 2007 May;48(5):2050-61
references: 17460260
references: Plast Reconstr Surg. 2002 Apr 1;109(4):1338-43
references: 11964988
references: PLoS One. 2011;6(1):e16400
references: 21298015
references: Cell. 1990 Sep 21;62(6):1217-26
references: 2169353
references: FEBS Lett. 1995 Jul 17;368(2):331-5
references: 7628632
references: Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L799-805
references: 11053013
references: J Biol Chem. 1992 Nov 15;267(32):23216-25
references: 1429669
references: Science. 1999 Oct 15;286(5439):498-502
references: 10521339
references: J Leukoc Biol. 2009 Mar;85(3):344-51
references: 18955543
references: Infect Immun. 1995 Apr;63(4):1291-7
references: 7890387
references: Regul Pept. 2001 Sep 15;101(1-3):157-61
references: 11495691
references: Invest Ophthalmol Vis Sci. 2006 Jun;47(6):2369-80
references: 16723446
references: Cornea. 2011 Aug;30(8):899-904
references: 21499082
references: Proc Natl Acad Sci U S A. 2006 Jan 31;103(5):1516-21
references: 16432216
references: Histochem Cell Biol. 2010 Jul;134(1):59-73
references: 20526610
references: Mol Cell Endocrinol. 2001 Apr 25;175(1-2):67-79
references: 11325517
references: J Biol Chem. 2000 Oct 27;275(43):33969-73
references: 10942762
references: Curr Eye Res. 2007 Jul-Aug;32(7-8):595-609
references: 17852183
references: Antimicrob Agents Chemother. 2001 May;45(5):1558-60
references: 11302828
references: Cell Tissue Res. 2011 Jan;343(1):175-88
references: 20730446
references: Exp Eye Res. 2004 Aug;79(2):175-80
references: 15325564
references: Curr Opin Pharmacol. 2003 Aug;3(4):404-11
references: 12901950
references: Pharmacol Rev. 2003 Mar;55(1):27-55
references: 12615953
references: Br J Ophthalmol. 2000 May;84(5):523-6
references: 10781518
references: Biochim Biophys Acta. 2009 Mar;1793(3):439-46
references: 19110010
references: Br J Ophthalmol. 2010 Nov;94(11):1523-7
references: 20679078
references: Curr Opin Immunol. 1994 Aug;6(4):584-9
references: 7946046
references: Infect Immun. 2003 Jul;71(7):3730-9
references: 12819054
references: Microbes Infect. 2006 Feb;8(2):380-9
references: 16242370
references: J Histochem Cytochem. 1989 Apr;37(4):493-8
references: 2926127
references: J Clin Invest. 1990 Mar;85(3):904-15
references: 2312733
references: J Clin Invest. 1991 Oct;88(4):1080-91
references: 1918365
references: Cell. 1997 Feb 21;88(4):553-60
references: 9038346