Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs.
BACKGROUND
Nitric oxide and prostaglandin E2 (PGE2play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1beta and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.


METHODS
Chondrocyte/agarose constructs were cultured under free-swelling conditions with or without IL-1beta and/or SB203580 (inhibitor of p38 MAPK) for up to 48 hours. Using a fully characterized bioreactor system, constructs were subjected to dynamic compression for 6, 12 and 48 hours under similar treatments. The activation or inhibition of p38 MAPK by IL-1beta and/or SB203580 was analyzed by western blotting. iNOS, COX-2, aggrecan and collagen type II signals were assessed utilizing real-time quantitative PCR coupled with molecular beacons. Release of nitrite and PGE2 was quantified using biochemical assays. Two-way analysis of variance and the post hoc Bonferroni-corrected t-test were used to examine data.


RESULTS
IL-1beta activated the phosphorylation of p38 MAPK and this effect was abolished by SB203580. IL-1beta induced a transient increase in iNOS expression and stimulated the production of nitrite release. Stimulation by either dynamic compression or SB203580 in isolation reduced the IL-1beta induced iNOS expression and nitrite production. However, co-stimulation with both dynamic compression and SB203580 inhibited the expression levels of iNOS and production of nitrite induced by the cytokine. IL-1beta induced a transient increase in COX-2 expression and stimulated the cumulative production of PGE2 release. These effects were inhibited by dynamic compression or SB203580. Co-stimulation with both dynamic compression and SB203580 restored cytokine-induced inhibition of aggrecan expression. This is in contrast to collagen type II, in which we observed no response with the cytokine and/or SB203580.


CONCLUSIONS
These data suggest that dynamic compression directly influences the expression levels of iNOS and COX-2. These molecules are current targets for pharmacological intervention, raising the possibility for integrated pharmacological and biophysical therapies for the treatment of cartilage joint disorders.
Chowdhury
T T
TT
School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London, E1 4NS, UK. t.t.chowdhury@qmul.ac.uk
Arghandawi
S
S
Brand
J
J
Akanji
O O
OO
Bader
D L
DL
Salter
D M
DM
Lee
D A
DA
D053674:Aggrecans / Q000096:biosynthesis; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D019902:Chondrocytes / Q000378:metabolism*; D024043:Collagen Type II / Q000096:biosynthesis; D051546:Cyclooxygenase 2 / Q000096:biosynthesis*; D015232:Dinoprostone / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053583:Interleukin-1beta / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D009573:Nitrites / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D048051:p38 Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*
D053674:Aggrecans; D024043:Collagen Type II; D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D009573:Nitrites; D012685:Sepharose; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone