Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 14
MAPK pathways influence the release of .NO and PGE2 in IL-1# stimulated
chondrocyte/agarose constructs subjected to dynamic compression
T T Chowdhury, K Elliot*, D M Salter*, D L Bader and D A Lee
Queen Mary, University of London, Mile End Road, London. E1 4NS. UK
*Edinburgh University Medical School, Edinburgh. EH16 4TJ
E-mail : t.t.chowdhury@qmul.ac.uk
Introduction: Nitric oxide (.NO) and prostaglandin E2 (PGE2) are catabolic mediators
induced by IL-1# and are therefore potentially important pharmacological targets in
osteoarthritis (OA). In chondrocytes, the application of mechanical load has been shown to
counteract the IL-1# induced .NO and PGE2 release. Furthermore, the cytokine can activate
all three members of the MAPK family. However, the role of the MAPK subtypes in the
release of .NO and PGE2 in chondrocytes is complex and may primarily involve
phosphorylation of p38 or JNK and activation of NF!B and AP-1. The current study
examines whether inhibition of these pathways will abolish the IL-1# induced .NO and PGE2
release in mechanically stimulated chondrocyte/agarose constructs.
Methods: Bovine chondrocytes seeded in agarose gel were cultured under free-swelling
conditions, for 48 hrs in medium containing 0 or 10 ng.ml-1 IL-1#, supplemented with 0 to
1000 ng.ml-1 SB203580 (inhibitor of p38 MAPK) or 0 to 50 nM JNK II (inhibitor of JNK-1,
2, 3), or 0 to 1000 ng.ml-1 PDTC (inhibits NFkB activation) or 0 to 1000 ng.ml-1 curcumin
(inhibitor of AP-1). For the mechanical loading experiments, all constructs were subjected to
15 % dynamic compression (1 Hz) for 48 hrs under the following conditions: 0 or 10 ng.ml-1
IL-1#, +/- 10 ng.ml-1 SB203580 or 5 nM JNK II or 10 ng.ml-1 PDTC or 10 ng.ml-1
curcumin.
Results: For constructs cultured under free-swelling conditions, SB203580 and JNK II
resulted in a dose-dependent inhibition of nitrite and PGE2 release in IL-1# stimulated
constructs. PDTC and curcumin reversed the IL-1# induced nitrite release whereas PGE2
levels were only partially inhibited. For the mechanical loading experiments, dynamic
compression could counteract the IL-1# induced nitrite release. Compression-induced
inhibition of nitrite release was abolished by SB203580, PDTC and curcumin and partially
reduced by JNK II. The application of dynamic compression resulted in the strain-induced
inhibition of PGE2 release in IL-1# stimulated constructs. This effect was abolished by
SB203580 and JNK II and partially reduced by PDTC and curcumin.
Discussion: These results suggest the involvement of MAPKs and the transcription factors,
AP-1 and NFkB in the IL-1# induced release of .NO and PGE2. Further work will determine
the sequential activation of the signalling cascades associated with mechanical load and IL-
1#. Moreover, the temporal organization of the multiple pathways will provide important
information for the pharmacological and biophysical treatment of degenerative joint disease
as observed in OA.
Acknowledgements:
This work was supported by a project grant from the Wellcome Trust, project number,
073972
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