Objective: The normal structure and function of articular cartilage are the result of a precisely balanced interaction between anabolic and catabolic processes. The transforming growth factor-beta (TGF-β) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) exert a strong catabolic effect. Recent evidence has shown that IL-1β, and TNF-α, and the TGF-β signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-β by IL-1β or TNF-α signaling pathways occurs through regulation of activity and availability of mothers against DPP (Drosophila) human homologue (Smad) proteins. Methods: Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in poly-(2-hydroxyethyl methacrylate)-coated dishes with either 10% fetal bovine serum media or serum-deprived media 6 h before treatment with IL-1β alone, TNF-α alone or IL-1β followed by TGF-β. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-κB) and Smad3/4 deoxyribonucleic acid (DNA) binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, type II collagen alpha 1 (COL2A1), aggrecan and SRY-related high mobility group-Box gene 9 (SOX-9) mRNA expression were measured by real-time polymerase chain reaction (PCR). Results: The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was downregulated by TNF-α (46%) or IL-1β treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1β or TNF-α. TGF-β-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1β in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1β partially reversed the TGF-β stimulation of Smad7 mRNA and protein levels in TGF-β-treated human OA cells. In contrast, TGF-β-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1β. Conclusions: IL-1β or TNF-α exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-β-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1β partially reversed the increase in TGF-β-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-β signaling induced by IL-1β or TNF-α in articular chondrocytes. The balance between the IL-1β or TNF-α and the TGF-β signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA. © 2007 Osteoarthritis Research Society International.
Roman-Blas, J. A., Stokes, D. G., & Jimenez, S. A. (2007). Modulation of TGF-β signaling by proinflammatory cytokines in articular chondrocytes. Osteoarthritis and Cartilage, 15(12), 1367–1377. https://doi.org/10.1016/j.joca.2007.04.011