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Effect of interleukin-1beta on osteogenic protein 1-induced signaling in adult human articular chondrocytes.

ABSTRACT: Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1beta (IL-1beta)-induced cartilage degeneration to investigate the effect of IL-1beta on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes.Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1beta on BMP receptors was studied by reverse transcription-polymerase chain reaction and flow cytometry. Phosphorylation of R-Smads was tested in cells treated with IL-1beta (10 ng/ml), OP-1 (100 ng/ml), or the combination of IL-1beta and OP-1. Cell lysates were analyzed by Western blotting with polyclonal antibodies against 2 R-Smad phosphorylation sites (BMP- and MAPK-mediated) or with total, nonphosphorylated R-Smad as a control. To identify which MAPKs play a role in IL-1beta activation of the linker region, chondrocytes were preincubated with specific MAPK inhibitors (PD98059 for MAP/ERK, SP600125 for JNK, and SB203580 for p38).IL-1beta reduced the number of activin receptor-like kinase 2 (ALK-2) and ALK-3 receptors, inhibited expression of Smad1 and Smad6, delayed and prematurely terminated the onset of OP-1-mediated R-Smad phosphorylation, and affected nuclear translocation of R-Smad/Smad4 complexes. The alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) was observed to be a possible mechanism through which IL-1beta offsets OP-1 signaling and the response to OP-1. Conversely, OP-1 was found to directly inhibit phosphorylation of p38.These findings describe new mechanisms of the crosstalk between OP-1 and IL-1beta in chondrocytes. The study also identifies potential targets for therapeutic interventions in the treatment of cartilage-degenerative processes.

SUBMITTER: Elshaier AM

PROVIDER: S-EPMC2626196 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Effect of interleukin-1beta on osteogenic protein 1-induced signaling in adult human articular chondrocytes.

Elshaier Amel M AM Hakimiyan Arnavaz A AA Rappoport Lev L Rueger David C DC Chubinskaya Susan S

Arthritis and rheumatism 20090101 1

<h4>Objective</h4>Two major receptor-activated Smad (R-Smad) signaling pathways, bone morphogenetic protein (BMP) and MAPK, were examined in a model of interleukin-1beta (IL-1beta)-induced cartilage degeneration to investigate the effect of IL-1beta on osteogenic protein 1 (OP-1) signaling in adult human articular chondrocytes.<h4>Methods</h4>Chondrocytes from the ankles of 26 normal human donors were cultured in high-density monolayers in serum-free medium. The effect of IL-1beta on BMP recepto ...[more]

PMID: 19116903

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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:Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPARgamma activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPARgamma expression in cartilage are poorly defined. This study was undertaken to investigate the quantitative expression and distribution of PPARgamma in normal and OA cartilage and to evaluate the effect of IL-1beta, a prominent cytokine in OA, on PPARgamma expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPARgamma protein expression were significantly lower in OA cartilage than in normal cartilage. Using real-time RT-PCR, we demonstrated that PPARgamma1 mRNA levels were about 10-fold higher than PPARgamma2 mRNA levels, and that only PPARgamma1 was differentially expressed: its levels in OA cartilage was 2.4-fold lower than in normal cartilage (p < 0.001). IL-1 treatment of OA chondrocytes downregulated PPARgamma1 expression in a dose- and time-dependent manner. This effect probably occurred at the transcriptional level, because IL-1 decreases both PPARgamma1 mRNA expression and PPARgamma1 promoter activity. TNF-alpha, IL-17, and prostaglandin E2 (PGE2), which are involved in the pathogenesis of OA, also downregulated PPARgamma1 expression. Specific inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580) and c-Jun N-terminal kinase (SP600125), but not of extracellular signal-regulated kinase (PD98059), prevented IL-1-induced downregulation of PPARgamma1 expression. Similarly, inhibitors of NF-kappaB signaling (pyrrolidine dithiocarbamate, MG-132, and SN-50) abolished the suppressive effect of IL-1. Thus, our study demonstrated that PPARgamma1 is downregulated in OA cartilage. The pro-inflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-kappaB signaling pathways. The IL-1-induced downregulation of PPARgamma expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation.

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

Effect of interleukin-1beta on osteogenic protein 1-induced signaling in adult human articular chondrocytes.

Publications

Effect of interleukin-1beta on osteogenic protein 1-induced signaling in adult human articular chondrocytes.

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