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