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Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs.

ABSTRACT: 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.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.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.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.

SUBMITTER: Chowdhury TT

PROVIDER: S-EPMC2453754 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs.

Chowdhury T T TT Arghandawi S S Brand J J Akanji O O OO Bader D L DL Salter D M DM Lee D A DA

Arthritis research & therapy 20080318 2

<h4>Background</h4>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 ...[more]

PMID: 18348730

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Project description:Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.

Project description:We investigated the mechanism of suppression of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) by ergolide, sesquiterpene lactone from Inula britannica. iNOS activity in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages was markedly attenuated by the treatment with ergolide. Its inhibitory effect on iNOS was paralleled by decrease in nitrite accumulation in culture medium of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner. However, its inhibitory effect does not result from direct inhibition of the catalytic activity of NOS. Ergolide markedly decreased the production of prostaglandin E(2) (PGE(2)) in cell-free extract of LPS/IFN-gamma-stimulated RAW 264.7 macrophages in a concentration-dependent manner, without alteration of the catalytic activity of COX-2 itself. Ergolide decreased the level of iNOS and COX-2 protein, and iNOS mRNA caused by stimulation of LPS/IFN-gamma in a concentration-dependent manner, as measured by Western blot and Northern blot analysis, respectively. Ergolide inhibited nuclear factor-kappaB (NF-kappaB) activation, a transcription factor necessary for iNOS and COX-2 expression in response to LPS/IFN-gamma. This effect was accompanied by the parallel reduction of nuclear translocation of subunit p65 of NF-kappaB as well as IkappaB-alpha degradation. In addition, these effects were completely blocked by treatment of cysteine, indicating that this inhibitory effect of ergolide could be mediated by alkylation of NF-kappaB itself or an upstream molecule of NF-kappaB. Ergolide also directly inhibited the DNA-binding activity of active NF-kappaB in LPS/IFN-gamma-pretreated RAW 264.7 macrophages. These results demonstrate that the suppression of NF-kappaB activation by ergolide might be attributed to the inhibition of nuclear translocation of NF-kappaB resulted from blockade of the degradation of IkappaB and the direct modification of active NF-kappaB, leading to the suppression of the expression of iNOS and COX-2, which play important roles in inflammatory signalling pathway.

Dataset Information

Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs.

Publications

Dynamic compression counteracts IL-1beta induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs.

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