Does osteoprotegerin have a role to play in inflammatory bowel diseases?

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Transcript of Does osteoprotegerin have a role to play in inflammatory bowel diseases?

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Osteoprotegerin: diagnostic and/or prognostic marker in inflammatory bowel disease?Raghunath Ramanarasimhaiah

Wnt/-catenin pathway is involved in cell survival, proliferation and differentiation1.

Crypt-villus architecture is dependent on this pathway2.

OPG expression is regulated by Wnt/-catenin pathway3.Batlle E, et al. Cell.2002 Oct 18;111(2):251-63. Pinto D,Gregorieff A,Begthel H andClevers H. Genes Dev.2003 Jul 15;17(14):1709-13.De Toni EN, et al. Clin Cancer Res. 2008 Aug 1;14(15):4713-8. Osteoprotegerin in intestines

Title. Bullets. OPG, apoptosis and immune system. DCs. De Toni EN, et al. Clin Cancer Res. 2008 Aug 1;14(15):4713-8. siRNA against -catenin resulted in strong reduction in OPG mRNA and protein expression in intestinal epithelial cell lines.Batlle E, et al. Cell.2002 Oct 18;111(2):251-63. Nuclear -catenin is hallmark of Wnt signaling. Proliferating cells in the crypts show nuclear -catenin. Pinto D,Gregorieff A,Begthel H andClevers H. Genes Dev.2003 Jul 15;17(14):1709-13. Inhibiting Wnt/-catenin pathway (by ectopic expression of Dickkopf1) causes deleterious effects on crypt-villus architecture.

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Osteoprotegerin and Inflammatory Bowel DiseaseSylvester FA, et al. InflammBowelDis. 2011 Aug;17(8):1726-30. Fecal OPG concentration correlates with disease severity of ulcerative colitis.

Franchimont N, et al. Clin Exp Immunol.2004 December;138(3): 491498. Serum OPG levels increased in CD (active and inactive CD). OPG in colon biopsy culture of inflamed regions higher.

Miheller P, et al. Inflamm Bowel Dis.2007 Nov;13(11):1379-84. Serum OPG levels increased in CD.

Moschen AR, et al. Gut.2005 Apr;54(4):479-87. OPG plasma levels increased in IBD. Explant cultures of inflamed colon showed high levels of OPG. Intestinal epithelial cells, colonic lymphoid aggregates and lamina muscularis had increased expression of OPG. Macrophages and DCs were major sources.

Ashcroft AJ, et al. Immunity. 2003 Dec;19(6):849-61. Fc-OPG had beneficial effects against colitis in IL2-/- mice.

Segregate prognostic and diagnostic. Cut down on words. 3

Proliferating intestinal epithelial cells will express OPG.ObjectivesTo determinereactivity and specificity of anti-OPG antibody (IMG 103A)if T84, Caco2, HCT116 and MG63 cell lines express OPGappropriate concentration of anti-OPG and anti--catenin antibodies (C7207) distribution of OPG and -catenin with in cells of above mentioned cell linesGreenspan EJ, Madigan JP, Boardman LA and Rosenberg DW. Cancer Prev Res (Phila). 2011 Jan;4(1):161-71. -catenin staining, with C7207, in SW480 cell line.

Tile: hypothesis. 4

Rationale behind using T84, Caco2, HCT116 and MG63 cell linesCells of intestinal epithelial origin (T84, Caco2 and HCT116) and MG63 a positive control for OPG expression1.Single cell type.Validation of techniques and reagents.Precious human and animal samples.Caco2 cell monolayers can be used as an in vitro model of intestinal epithelial cell differentiation and dedifferentiation:Caco2 cells are known to differentiate into absorptive intestinal epithelium2-4.Conducive environment allows differentiated Caco2 cells to return back to undifferentiated state4, 5.Brndstrm H, et al. Biochem Biophys Res Commun.1998 Jul 30;248(3):454-7.Chantret I, et al. Cancer Res.1988 Apr 1;48(7):1936-42.Halbleib JM, et al. Mol Biol Cell.2007 Nov;18(11):4261-78. Leoni BD, et al. Differentiation.2012 Mar;83(3):116-27.Wang L, et al. Biomaterials.2010 Oct;31(29):7586-98.

Brief about cell lines utilizedCell lineMutation3-5-catenin expression6-9OPG expressionTissue of origin13T84APCMembrane boundExpressed10Lung metastasis of colon carcinomaCaco-2APC and -cateninMembrane and nuclearExpressed11ColonHCT116-cateninMembrane and nuclearmRNA12ColonMG631, 2(Osteosarcoma)p53 null Membrane boundExpressedBone

Ariyasu T, et al. In VitroCellDev Biol Anim.2002 Jan;38(1):30-4.Brndstrm H, et al. Biochem Biophys Res Commun.1998 Jul 30;248(3):454-7.Rowan AJ, et al Proc Natl Acad Sci U S A.2000 Mar 28;97(7):3352-7.Forsyth NR, et al. Neoplasia.2004 May-Jun;6(3):258-65.Hu X,J Exp Clin Cancer Res.2010 May 7;29:43.Hopkins AM, J Cell Sci.2003 Feb 15;116(Pt 4):725-42.Jones LJ andVeale RB. CancerCellInt.2003 Aug 15;3(1):13.Conacci-Sorrell M, et al. J Cell Biol.2003 Nov 24;163(4):847-57.Cai Y, et al. J Pathol.2010 Jan;220(1):24-33.Mohamed JA, et al. J Infect Dis.2009 Feb 15;199(4):477-85.Vidal K, et al. Am J Physiol Gastrointest Liver Physiol.2004 Oct;287(4):G836-44.De Toni EN, et al. ClinCancerRes.2008 Aug 1;14(15):4713-8. http://www.atcc.org/

0.2pg0.1pg0.05pgT84 (100g)Caco 2 HCT116MG63

anti-OPGIsotype

(rOPG)0.2pg0.1pg0.05pgT84 (100g)Caco 2 HCT116MG63

(rOPG)Goat anti-mouse conjugated with HRPWestern Blot Results2502007550372015MWMMouse anti-human OPG (IMG 103A, Imgenex) or isotype at 0.002mg/ml (ON at 40C)Secondary Goat anti-mouse antibody conjugated with HRP (SC-2969) at 0.0004mg/ml (2hrs at RT)

Western Blot - DiscussionSimonet WS, et al. Cell. 1997 Apr 18;89(2):309-19. Recombinant murine OPG produced in CHO cells: monomer in lysate 55kDa, dimer in supernatant 110kDaTsuda E, et al. Biochem Biophys Res Commun. 1997 May 8;234(1):137-42.Native Osteoclastogenesis inhibitory factor (OCIF) from media supporting human embryonic lung fibroblasts, monomer ~60kDa, dimer ~120kDaYasuda H, et al. Endocrinology. 1998 Mar;139(3):1329-37. OPG and OCIF have identical amino acid sequence.

46.026kDahttp://www.uniprot.org/uniprot/O00300

ObjectivesTo determinereactivity and specificity of anti-OPG antibody (IMG 103A)if T84, Caco2, HCT116 and MG63 cell lines express OPGappropriate concentration of anti-OPG and anti--catenin antibodies (C7207) distribution of OPG and -catenin with in cells of above mentioned cell lines

Tile: hypothesis. 10

Primary antibodySecondary antibody1:501:501:1001:501:2001:501:4001:501:501:1001:1001:1001:2001:1001:4001:1001:501:2001:1001:2001:2001:2001:4001:2001:501:4001:1001:4001:2001:4001:4001:400

Grid to calculate the appropriate concentrations of anti-OPG and anti--catenin antibody for ICCPrimary antibody Imgenex, IMG 103A: Stock 0.5mg/mlorPrimary antibody Sigma Aldrich C7207: Stock 3mg/mlandSecondary antibody Invitrogen, A-11001: Stock 2mg/ml

No absNo Primary,Sec onlySpecific primary and Sec. abs.Isotype and Sec. abs.

Nucleus/DAPIGreen/Alexa Fluor 488/OPGDAPI+Alexa Fluor 488/OPGExperiment layoutCell line: MG63OPG

Green/Alexa Fluor 488/OPGDAPI+Alexa Fluor 488/OPGCell line MG63OPG

To determineappropriate concentration of anti-OPG antibodyPrimary antibodySecondary antibody1:501:501:1001:501:2001:501:4001:501:501:1001:1001:1001:2001:1001:4001:1001:501:2001:1001:2001:2001:2001:4001:2001:501:4001:1001:4001:2001:4001:4001:400

anti-OPG 0.01mg/mlSecondary 0.01mg/mlPrimary antibody Imgenex, IMG 103A: Stock 0.5mg/ml

Secondary antibody Invitrogen, A-11001: Stock 2mg/ml

No absNo Primary,Sec onlySpecific primary and Sec. abs.Isotype and Sec. abs.Experiment layoutCell line MG63-catenin

Nucleus/DAPIGreen/Alexa Fluor 488DAPI+Alexa Fluor 488

Green/Alexa Fluor 488DAPI+Alexa Fluor 488Cell line MG63-catenin

To determineappropriate concentration of anti--catenin antibodyPrimary antibodySecondary antibody1:501:501:1001:501:2001:501:4001:501:501:1001:1001:1001:2001:1001:4001:1001:501:2001:1001:2001:2001:2001:4001:2001:501:4001:1001:4001:2001:4001:4001:400

anti--catenin 0.015mg/mlSecondary 0.01mg/mlPrimary antibody Sigma Aldrich C7207: Stock 3mg/ml

Secondary antibody Invitrogen, A-11001: Stock 2mg/ml

Proliferating intestinal epithelial cells will express OPG.ObjectivesTo determinereactivity and specificity of anti-OPG antibody (IMG 103A)if T84, Caco2, HCT116 and MG63 cell lines express OPGappropriate concentration of anti-OPG and anti--catenin antibodies (C7207) distribution of OPG and -catenin with in cells of above mentioned cell linesOPG, a secretory biomolecule, will be cytosolic before secreted out of the cell.

Tile: hypothesis. 18

Iso2ndry only-OPGDAPI/-OPGMG63 Overnight anti-OPG

T84 Overnight anti-OPGIso2ndry only-OPGDAPI/ -OPG

Iso2ndry onlyCaco2 7 days anti-OPG-OPGDAPI/ -OPG

Iso2ndry only-OPGDAPI/ -OPGHCT116 Overnight - -OPG

OPG expression

OPGDAPI/OPG

DAPI/ OPG Isotype

T84Caco2HCT116MG63

Iso2ndry only--cateninDAPI/--cateninMG63 Overnight anti--catenin

T84 Overnight anti--cateninIso2ndry only--cateninDAPI/-catenin

Caco2 7 days anti--cateninIso2ndry only--cateninDAPI/-catenin

HCT116 Overnight anti--cateninIso2ndry only--cateninDAPI/-catenin

T84Caco2HCT116MG63-catenin-catenin expressionDAPI/-catenin

DAPI/ Isotype

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T84Caco2HCT116MG63OPG

Comparison of -catenin and OPG expression-catenin

-cateninOPGConfocal microscopy of Caco-2 cells

-catenin and OPG expression in Caco-2 cells-catenin: 1, 2, 3, 4, 5OPG: 6, 7, 8, 9, 1012367845109

Immunocytochemistry discussion-catenin was expressed by all the cell lines investigated and was membrane bound in all. Nuclear -catenin staining was visible only in Caco-2 cells.Hopkins AM, J Cell Sci.2003 Feb 15;116(Pt 4):725-42.Jones LJ andVeale RB. CancerCellInt.2003 Aug 15;3(1):13.Conacci-Sorrell M, et al. J Cell Biol.2003 Nov 24;163(4):847-57.Cai Y, et al. J Pathol.2010 Jan;220(1):24-33.Ilyas M, et al. Proc Natl Acad Sci U S A.1997 Sep 16;94(19):10330-4.OPG was expressed by all the cell lines and was cytosolic in all. OPG staining in the nuclear region was visible in Caco-2 cells. Nuclear distribution of OPG in Caco-2 cells was investigated by confocal microscopy, which suggested to be so.Mohamed JA, et al. J Infect Dis.2009 Feb 15;199(4):477-85.Vidal K, et al. Am J Physiol Gastroint