Recent developments in understanding the role of osteoprotegerin in colonic intestinal epithelium at Sylvester lab

Raghunath Ramanarasimhaiah

Which evolved first? Gut or bone!

http://answers.mheducation.com/sciences/life-science/biology/animal-kingdom

Expression of OPG in different cell types

van Agtmaal EL, et al. The shear stress-induced transcription factor KLF2 affects dynamics and angiopoietin-2 content of Weibel-Palade bodies. PLoS One. 2012;7(6):e38399.

Chollet ME, et al. Evidence of a colocalisation of osteoprotegerin (OPG) with von Willebrand factor (VWF) in platelets and megakaryocytes alpha granules. Studies from normal and grey platelets. Br J Haematol. 2010 Mar;148(5):805-7.

Sylvester lab

MG63 osteoblastic cells

OPG DAPI Merge

Caco 2 Intestinal epithelial cells

Expression of OPG in different tissues

Carda C, et al. Osteoprotegerin (OPG) and RANKL expression and distribution in developing human craniomandibular joint. Tissue Cell. 2005 Jun;37(3):247-55.

Ueland T, et al. Dysregulated osteoprotegerin/RANK ligand/RANK axis in clinical and experimental heart failure. Circulation. 2005 May 17;111(19):2461-8.

Sylvester lab

Questions

• Does treatment with a detergent increase detectable concentration of osteoprotegerin (OPG) in spent Caco 2 media?

• Does exposure to commensal bacteria alter OPG expression in Caco 2 cells?

• Does OPG bind to bacteria? If yes, can it be released from the bacteria?

• Expression of OPG in appendix?

Question

• Does treatment with a detergent increase detectable concentration of OPG in spent Caco 2 media?

Experimental procedureSpin down

Collect supernatant

ELISAIL1β

Media only

Add 1% Tween 20

Incubate on ice for 30 M

12 well culture plate supporting Caco 2

Over night370C

Spin down

Collect supernatant

Add 1% Tween 20

Incubate on ice for 30 M

ELISA

Detergent treatment of Caco 2 supernatant does not increase the detectable OPG by ELISA

3000

6000

9000

12000

15000

1% Tween 20

IL1β

- - ++

+ -

Cell culture inserts

• Seed and grow Caco 2 cells as a monolayer• After achieving confluence, allow 21 days for

polarization• Check TEER (transepithelial electrical

resistance)

Apical

Cell monolayer

Baso-lateral

Questions

• Does exposure to commensal bacteria (ATCC 25922) alter OPG expression in Caco 2 cells?

Experimental procedure

Live E.coli

Spindown Collect supernatant

ELISAApical

Baso lateral

Over night370C

OPG expression appears suppressed on exposure to commensal bacteria

A B A B A B A B A B A B106 105 104 102 EC Sup Media

2000

4000

6000

8000

10000

No. of EC/insert A: ApicalB: BasolateralEC: Escherichia coliEC Sup: Filtered spent medium of EC

pg/m

L O

PG

Questions

• Does OPG bind to bacteria? If yes, can it be released from the bacteria?

Experimental procedure

Spindown Collect supernatant

ELISAOn ice 30 M

0.05% Tween 20

Spindown Collect supernatant

ELISAOn ice 30 MELISA

Spindown

Live E.coli

Killed E.coli

Over night370C

TEER

Live Escherichia coli

+

Live E.coli 106 104 102 EC sup EC med Con med

2000

4000

6000

- +- +- +- +- +-

EC sup: E.coli spent mediaEC med: Fresh LB brothCon med: Caco 2 media

Tween 20 (0.05%)

Killed Escherichia coli

Tween 20 (0.05%)

Killed E.coli 106 104 102 EC sup EC med Con med

2000

4000

6000

+- +- +- +- +- +-

EC sup: E.coli spent mediaEC med: Fresh LB brothCon med: Caco 2 media

Baso-lateral compartment

L K + - + - + -E.coli

106 104 102 EC sup EC med Con med

2000

4000

6000

L K L K

L: LiveK: KilledEC sup: E.coli spent mediaEC med: Fresh LB brothCon med: Caco 2 media

Conclusions

• ELISA detects the true concentration of OPG in Caco 2 supernatants and is not altered by detergent (0.05% Tween 20)

• Osteoprotegerin (OPG) binds to commensal bacteria and the bound OPG can be released by detergent treatment