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Differential effects of p38MAP kinase inhibitors on the expression of inflammation-associated genes in primary, interleukin-1beta-stimulated human chondrocytes.

ABSTRACT: BACKGROUND AND PURPOSE: A main challenge in the therapy of osteoarthritis (OA) is the development of drugs that will modify the disease. Reliable test systems are necessary to enable an efficient screening of therapeutic substances. We therefore established a chondrocyte-based in vitro cell culture model in order to characterize different p38MAPK inhibitors. EXPERIMENTAL APPROACH: Interleukin-1beta (IL-1beta)-stimulated human OA chondrocytes were treated with the p38MAPK inhibitors Birb 796, pamapimod, SB203580 and the new substance CBS-3868. Birb 796- and SB203580-treated cells were analysed in a genome-wide microarray analysis. The efficacy of all inhibitors was characterized by quantitative gene expression analysis and the quantification of PGE(2) and NO release. KEY RESULTS: Microarray analysis revealed inhibitor-specific differences in gene expression. Whereas SB203580 had a broad effect on chondrocytes, Birb 796 counteracted the IL-1beta effect more specifically. All p38MAPK inhibitors significantly inhibited the IL-1beta-induced gene expression of COX-2, mPGES1, iNOS, matrix metalloproteinase 13 (MMP13) and TNFRSF11B, as well as PGE(2) release. Birb 796 and CBS-3868 showed a higher efficacy than SB203580 and pamapimod at inhibiting the expression of COX-2 and MMP13 genes, as well as PGE(2) release. In the case of mPGES1 and TNFRSF11B gene expression, CBS-3868 exceeded the efficacy of Birb 796. CONCLUSIONS AND IMPLICATIONS: Our test system could differentially characterize inhibitors of the same primary pharmaceutical target. It reflects processes relevant in OA and is based on chondrocytes that are mainly responsible for cartilage degradation. It therefore represents a valuable tool for drug screening in between functional in vitro testing and in vivo models.

SUBMITTER: Joos H

PROVIDER: S-EPMC2936033 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Differential effects of p38MAP kinase inhibitors on the expression of inflammation-associated genes in primary, interleukin-1beta-stimulated human chondrocytes.

Joos H H Albrecht W W Laufer S S Brenner R E RE

British journal of pharmacology 20100701 5

<h4>Background and purpose</h4>A main challenge in the therapy of osteoarthritis (OA) is the development of drugs that will modify the disease. Reliable test systems are necessary to enable an efficient screening of therapeutic substances. We therefore established a chondrocyte-based in vitro cell culture model in order to characterize different p38MAPK inhibitors.<h4>Experimental approach</h4>Interleukin-1beta (IL-1beta)-stimulated human OA chondrocytes were treated with the p38MAPK inhibitors ...[more]

PMID: 20590617

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Project description:ObjectivesTo investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression.MethodsThe presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry.ResultsERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2.ConclusionsOur data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

Project description:Epigallocatechin-3-gallate (EGCG) is a bioactive polyphenol of green tea and exerts potent anti-inflammatory effects by inhibiting signaling events and gene expression. Interleukin-1beta (IL-1?) is the principal cytokine linked to cartilage degradation in osteoarthritis (OA). The objective of this study was to evaluate the global effect of EGCG on IL-1?-induced expression of proteins associated with OA pathogenesis in human chondrocytes.Primary OA chondrocytes were pretreated with EGCG (10 to 100 uM) and then stimulated with IL-1? (5 ng/ml) for 24 hours. Culture supernatants were incubated with cytokine antibody arrays and immunoreactive proteins (80 proteins) were visualized by enhanced chemiluminiscence. Effect of EGCG on IL-1?-induced expression of 18 selected genes was verified by Real time-PCR and effect on IL-6, IL-8 and tumor necrosis factor-alpha (TNF-?) production was determined using specific ELISAs. Western immunoblotting was used to analyze the effect of EGCG on the interleukin-1 receptor-associated kinase 1 (IRAK-1) and TNF receptor-associated factor 6 (TRAF-6) proteins in IL-1?-stimulated chondrocytes. The role of nuclear factor kappa-B (NF-?B) and mitogen activated protein kinases (MAPKs) in the regulation of selected genes and the mechanism involved in EGCG mediated modulation of these genes was determined by using specific inhibitors for NF- ?B (MG132) and MAPKs (p38-MAPK, SB202190; JNK-MAPK, SP600125, ERK-MAPK, PD98059).Out of 80 proteins present on the array, constitutive expression of 14% proteins was altered by EGCG treatment. No significant stimulatory effect was observed on the proteins associated with cartilage anabolic response. Stimulation with IL-1? enhanced the expression of 29 proteins. Expression of all 29 proteins up-regulated by IL-1? was found to be suppressed by EGCG. EGCG also inhibited the expression of the signaling intermediate TRAF-6 at 50 and 100 uM concentrations (P < 0.05). Our results identified several new targets of EGCG, including epithelial neutrophil activating peptide-78 (ENA-78), granulocyte macrophage colony stimulation factor (GM-CSF), growth- related oncogene (GRO), GRO-?, IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), MCP-3, macrophage inflammatory protein-1beta (MIP-1?), granulocyte chemotactic protein-2 (GCP-2), MIP-3alpha, interferon-gamma-inducible protein-10 (IP-10), nucleosome assembly protein-2 (NAP-2) and leukemia inhibitory factor (LIF). The inhibitory effects of EGCG were mainly mediated by inhibiting the activation of NF-?B and c-Jun N-terminal Kinase (JNK)-MAPK in human chondrocytes.Our results suggest that the potential of EGCG in OA treatment/prevention may be related to its ability to globally suppress the inflammatory response in human chondrocytes. These results identify additional new targets of EGCG and advocate that EGCG may be a potent chondroprotective agent in OA.

Dataset Information

Differential effects of p38MAP kinase inhibitors on the expression of inflammation-associated genes in primary, interleukin-1beta-stimulated human chondrocytes.

Publications

Differential effects of p38MAP kinase inhibitors on the expression of inflammation-associated genes in primary, interleukin-1beta-stimulated human chondrocytes.

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