does osteoprotegerin have a role to play in inflammatory bowel diseases?
TRANSCRIPT
Osteoprotegerin: diagnostic and/or prognostic marker in inflammatory
bowel disease?
Raghunath Ramanarasimhaiah
Wnt/β-catenin pathway is involved in cell survival, proliferation and differentiation1.
Crypt-villus architecture is dependent on this pathway2.
OPG expression is regulated by Wnt/β-catenin pathway3.
1) Batlle E, et al. Cell. 2002 Oct 18;111(2):251-63. 2) Pinto D, Gregorieff A, Begthel H and Clevers H. Genes Dev. 2003 Jul 15;17(14):1709-13.3) De Toni EN, et al. Clin Cancer Res. 2008 Aug 1;14(15):4713-8.
Osteoprotegerin in intestines
Osteoprotegerin and Inflammatory Bowel Disease
1. Sylvester FA, et al. Inflamm Bowel Dis. 2011 Aug;17(8):1726-30. Fecal OPG concentration correlates with disease severity of ulcerative colitis.
2. Franchimont N, et al. Clin Exp Immunol. 2004 December; 138(3): 491–498. Serum OPG levels increased in CD (active and inactive CD). OPG in colon biopsy culture of inflamed regions higher.
3. Miheller P, et al. Inflamm Bowel Dis. 2007 Nov;13(11):1379-84. Serum OPG levels increased in CD.
4. 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.
5. Ashcroft AJ, et al. Immunity. 2003 Dec;19(6):849-61. Fc-OPG had beneficial effects against colitis in IL2-/- mice.
Proliferating intestinal epithelial cells will express OPG.
ObjectivesTo determine1) reactivity and specificity of anti-OPG antibody (IMG 103A)
2) if T84, Caco2, HCT116 and MG63 cell lines express OPG
3) appropriate concentration of anti-OPG and anti-β-catenin
antibodies (C7207)
4) distribution of OPG and β-catenin with in cells of above mentioned
cell lines
Greenspan EJ, Madigan JP, Boardman LA and Rosenberg DW. Cancer Prev Res (Phila). 2011 Jan;4(1):161-71. β-catenin staining, with C7207, in SW480 cell line.
Rationale behind using T84, Caco2, HCT116 and MG63 cell lines
1. Cells of intestinal epithelial origin (T84, Caco2 and HCT116) and MG63 a positive control for OPG expression1.
2. Single cell type.3. Validation of techniques and reagents.4. Precious human and animal samples.5. Caco2 cell monolayers can be used as an in vitro model of intestinal
epithelial cell differentiation and dedifferentiation:a. Caco2 cells are known to differentiate into absorptive intestinal
epithelium2-4.b. Conducive environment allows differentiated Caco2 cells to return
back to undifferentiated state4, 5.1) Brändström H, et al. Biochem Biophys Res Commun. 1998 Jul
30;248(3):454-7.2) Chantret I, et al. Cancer Res. 1988 Apr 1;48(7):1936-42.3) Halbleib JM, et al. Mol Biol Cell. 2007 Nov;18(11):4261-78. 4) Leoni BD, et al. Differentiation. 2012 Mar;83(3):116-27.5) Wang L, et al. Biomaterials. 2010 Oct;31(29):7586-98.
Brief about cell lines utilized
Cell line Mutation3-5 β-catenin expression6-9
OPG expression
Tissue of origin13
T84 APC Membrane bound Expressed10
Lung metastasis
of colon carcinoma
Caco-2 APC and β-catenin
Membrane and nuclear Expressed11 Colon
HCT116 β-catenin Membrane and nuclear mRNA12 Colon
MG631, 2
(Osteosarcoma) p53 null Membrane bound Expressed Bone
1. Ariyasu T, et al. In Vitro Cell Dev Biol Anim. 2002 Jan;38(1):30-4.2. Brändström H, et al. Biochem Biophys Res Commun. 1998 Jul 30;248(3):454-7.3. Rowan AJ, et al Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3352-7.4. Forsyth NR, et al. Neoplasia. 2004 May-Jun;6(3):258-65.5. Hu X, J Exp Clin Cancer Res. 2010 May 7;29:43.6. Hopkins AM, J Cell Sci. 2003 Feb 15;116(Pt 4):725-42.7. Jones LJ and Veale RB. Cancer Cell Int. 2003 Aug 15;3(1):13.8. Conacci-Sorrell M, et al. J Cell Biol. 2003 Nov 24;163(4):847-57.9. Cai Y, et al. J Pathol. 2010 Jan;220(1):24-33.10.Mohamed JA, et al. J Infect Dis. 2009 Feb 15;199(4):477-85.11.Vidal K, et al. Am J Physiol Gastrointest Liver Physiol. 2004 Oct;287(4):G836-
44.12.De Toni EN, et al. Clin Cancer Res. 2008 Aug 1;14(15):4713-8. 13.http://www.atcc.org/
0.2pg
0.1pg
0.05p
g
T84
(100μg)
Caco
2 HCT
116
MG63
anti-OPG Isotype
(rOPG)
0.2pg
0.1pg
0.05p
g
T84
(100μg)
Caco
2 HCT
116
MG63
(rOPG)
Goat anti-mouse conjugated with HRP
Western Blot Results
250200
7550372015
MWM
1) Mouse anti-human OPG (IMG 103A, Imgenex) or isotype at 0.002mg/ml (ON at 40C)2) 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.
46.026kDa
http://www.uniprot.org/uniprot/O00300
ObjectivesTo determine1) reactivity and specificity of anti-OPG antibody (IMG 103A)
2) if T84, Caco2, HCT116 and MG63 cell lines express OPG
3) appropriate concentration of anti-OPG and anti-β-catenin
antibodies (C7207)
4) distribution of OPG and β-catenin with in cells of above mentioned
cell lines
Primary antibody
Secondar
y antibody
1:501:50
1:1001:50
1:2001:50
1:4001:50
1:501:100
1:1001:100
1:2001:100
1:4001:100
1:501:200
1:1001:200
1:2001:200
1:4001:200
1:501:400
1:1001:400
1:2001:400
1:4001:400
Grid to calculate the appropriate concentrations of anti-OPG and anti-β-catenin antibody for ICC
Primary antibody Imgenex, IMG 103A: Stock 0.5mg/mlor
Primary antibody Sigma Aldrich C7207: Stock 3mg/mland
Secondary antibody Invitrogen, A-11001: Stock 2mg/ml
No absNo
Primary,Sec only
Specific primary and Sec.
abs.
Isotype and Sec.
abs.
Nucleus/DAPI
Green/Alexa Fluor
488/OPG
DAPI+Alexa Fluor
488/OPG
Experiment layout
Cell line: MG63OPG
Green/Alexa Fluor
488/OPG
DAPI+Alexa Fluor
488/OPG
Cell line MG63OPG
To determineappropriate concentration of anti-OPG antibody
Primary antibody
Secondar
y antibody
1:501:50
1:1001:50
1:2001:50
1:4001:50
1:501:100
1:1001:100
1:2001:100
1:4001:100
1:501:200
1:1001:200
1:2001:200
1:4001:200
1:501:400
1:1001:400
1:2001:400
1:4001:400
anti-OPG 0.01mg/mlSecondary 0.01mg/ml
Primary antibody Imgenex, IMG 103A: Stock 0.5mg/ml
Secondary antibody Invitrogen, A-11001: Stock 2mg/ml
No absNo
Primary,Sec only
Specific primary and Sec.
abs.
Isotype and Sec.
abs.
Experiment layoutCell line MG63
β-catenin
Nucleus/DAPI
Green/Alexa Fluor 488
DAPI+Alexa Fluor 488
Green/Alexa Fluor 488
DAPI+Alexa Fluor 488
Cell line MG63
β-catenin
To determineappropriate concentration of anti-β-catenin antibody
Primary antibody
Secondar
y antibody
1:501:50
1:1001:50
1:2001:50
1:4001:50
1:501:100
1:1001:100
1:2001:100
1:4001:100
1:501:200
1:1001:200
1:2001:200
1:4001:200
1:501:400
1:1001:400
1:2001:400
1:4001:400
anti-β-catenin 0.015mg/mlSecondary 0.01mg/ml
Primary antibody Sigma Aldrich C7207: Stock 3mg/ml
Secondary antibody Invitrogen, A-11001: Stock 2mg/ml
Proliferating intestinal epithelial cells will express OPG.
ObjectivesTo determine1) reactivity and specificity of anti-OPG antibody (IMG 103A)
2) if T84, Caco2, HCT116 and MG63 cell lines express OPG
3) appropriate concentration of anti-OPG and anti-β-catenin
antibodies (C7207)
4) distribution of OPG and β-catenin with in cells of above mentioned
cell lines
OPG, a secretory biomolecule, will be cytosolic before secreted out of the cell.
Iso 2ndry only
α-OPG DAPI/α-OPG
MG63 – Overnight – anti-OPG
T84 – Overnight – anti-OPG
Iso 2ndry only
α-OPG DAPI/ α-OPG
Iso 2ndry only
Caco2 – 7 days – anti-OPG
α-OPG DAPI/ α-OPG
Iso 2ndry only
α-OPG DAPI/ α-OPG
HCT116 – Overnight - α-OPG
OPG expression
OPG
DAPI/OPG
DAPI/ OPG Isotype
T84 Caco2 HCT116 MG63
Iso 2ndry only
α-β-catenin DAPI/α-β-catenin
MG63 – Overnight – anti-β-catenin
T84 – Overnight – anti-β-catenin
Iso 2ndry only
α-β-catenin DAPI/β-catenin
Caco2 – 7 days – anti-β-catenin
Iso 2ndry only
α-β-catenin DAPI/β-catenin
HCT116 – Overnight – anti-β-catenin
Iso 2ndry only
α-β-catenin DAPI/β-catenin
T84 Caco2 HCT116 MG63
β-catenin
β-catenin expression
DAPI/β-catenin
DAPI/ Isotype
T84 Caco2 HCT116 MG63
OPG
Comparison of β-catenin and OPG expression
β-catenin
β-catenin OPG
Confocal microscopy of Caco-2 cells
β-catenin and OPG expression in Caco-2 cells
β-catenin: 1, 2, 3, 4, 5 OPG: 6, 7, 8, 9, 10
1
2
3
6
7
8
4
5 10
9
Immunocytochemistry discussionβ-catenin was expressed by all the cell lines investigated and was membrane bound in all. Nuclear β-catenin staining was visible only in Caco-2 cells.
1. Hopkins AM, J Cell Sci. 2003 Feb 15;116(Pt 4):725-42.2. Jones LJ and Veale RB. Cancer Cell Int. 2003 Aug 15;3(1):13.3. Conacci-Sorrell M, et al. J Cell Biol. 2003 Nov 24;163(4):847-57.4. Cai Y, et al. J Pathol. 2010 Jan;220(1):24-33.5. 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. 1. Mohamed JA, et al. J Infect Dis. 2009 Feb 15;199(4):477-85.
2. Vidal K, et al. Am J Physiol Gastrointest Liver Physiol. 2004 Oct;287(4):G836-44.
3. De Toni EN, et al. Clin Cancer Res. 2008 Aug 1;14(15):4713-8.
Ongoing:Western blot and Immunohistochemical staining
for OPG in mouse (WT and OPG-/-) intestine
Imminent Future:Immunohistochemical staining for OPG in human
(? Inflammatory Bowel Disease) colon biopsies
Future:Devise a protocol to utilize OPG expression in
diagnosing IBD in human patients
RANKL-RANK DC survival and maturation
T-lymphocyte survival and maturation
TRAIL-TRAILR Apoptosis of TRAILR expressing cells
Immune and non-immune cells
OPG
OPG and its ligands
Salient Histopathological features of Inflammatory Bowel Disease
Distortion of intestinal architecture: Disrupted mucosal surface, short and decreased crypt numbers, increased crypt branching, continuous attempt towards intestinal epithelial cell renewal (attenuated and flattened epithelial cells).
Signs of inflammation: increased cellularity of lamina propria (including basal plasmacytocis, increased neutrophils), crypt abscesses (ulcerative colitis), granulomas (Crohn’s disease).
1) Jenkins D, et al. J Clin Pathol. 1997 Feb;50(2):93-105.2) Geboes K and Dalle I. Gut. 2002 May;50 Suppl 3:III37-42.
Immunofluorescence staining of cell lines for OPG
1) Primary antibody (Imgenex, IMG 103A, Mouse mAb, 0.01mg/ml, overnight, 40C)
2) Secondary antibody (Invitrogen, A-11001, Goat anti-mouse Alexa Fluor 488, 0.01mg/ml, 2hrs, RT)
To determineappropriate concentration of anti-OPG antibody
Immuno-cytochemistry protocol1) Cells seeded at 10,000 cells/chamber (4 chamber slide)2) Incubated at 370C, 5% CO2 for 48 hrs or Caco 2 (7 days)3) Media decanted, cells fixed (cold 4% Paraformaldehyde, 30mins at 40C)4) Wash (1xPBS, thrice, 5mins each, RT)5) Permiabilization (0.1% Triton X100 in 1XPBS, 20mins, RT)6) Wash7) Block (Power block, 30mins, RT)8) Primary antibody (C7027 0.015mg/ml or IMG 103A 0.01mg/ml in antibody-
dilution buffer, overnight, 40C)9) Wash10) Secondary antibody (Invitrogen, Goat anti-mouse Alexa Fluor 488, 0.01mg/ml,
2hrs, RT)11) Wash12) Vectashield-DAPI, cover slip and seal13) Image
Immunofluorescence staining of cell linesβ-catenin
1) Primary antibody (Sigma Aldrich, C7207, 0.015mg/ml, Mouse mAb, 0.015mg/ml, overnight, 40C)
2) Secondary antibody (Invitrogen, A-11001, Goat anti-mouse Alexa Fluor 488, 0.01mg/ml, 2hrs, RT)
To determineappropriate concentration of anti-β-catenin antibody
Primary antibody
Secondar
y antibody
1:501:50
1:1001:50
1:2001:50
1:4001:50
1:501:100
1:1001:100
1:2001:100
1:4001:100
1:501:200
1:1001:200
1:2001:200
1:4001:200
1:501:400
1:1001:400
1:2001:400
1:4001:400
Concentration grid for anti-β-catenin
Primary antibody Sigma Aldrich C7207: Stock 3mg/ml
Secondary antibody Invitrogen, A-11001: Stock 2mg/ml