Modulation of TGF-beta signaling by proinflammatory cytokines in articular chondrocytes

Osteoarthritis Cartilage. 2007 Dec;15(12):1367-77. doi: 10.1016/j.joca.2007.04.011. Epub 2007 Jun 29.


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-beta) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) exert a strong catabolic effect. Recent evidence has shown that IL-1beta, and TNF-alpha, and the TGF-beta 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-beta by IL-1beta or TNF-alpha 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 6h before treatment with IL-1beta alone, TNF-alpha alone or IL-1beta followed by TGF-beta. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-kappaB) 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-alpha (46%) or IL-1beta treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1beta or TNF-alpha. TGF-beta-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1beta in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1beta partially reversed the TGF-beta stimulation of Smad7 mRNA and protein levels in TGF-beta-treated human OA cells. In contrast, TGF-beta-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1beta.

Conclusions: IL-1beta or TNF-alpha exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-beta-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1beta partially reversed the increase in TGF-beta-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-beta signaling induced by IL-1beta or TNF-alpha in articular chondrocytes. The balance between the IL-1beta or TNF-alpha and the TGF-beta signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aged
  • Animals
  • Blotting, Western
  • Cartilage, Articular / cytology*
  • Cattle
  • Cells, Cultured
  • Chondrocytes / drug effects
  • Chondrocytes / metabolism*
  • Collagen Type II / metabolism
  • Cytokines / pharmacology*
  • DNA-Binding Proteins / metabolism
  • Electrophoretic Mobility Shift Assay / methods
  • Female
  • Gene Expression Regulation
  • High Mobility Group Proteins / genetics
  • Humans
  • In Vitro Techniques
  • Interleukin-1beta
  • Knee Joint
  • Male
  • Middle Aged
  • NF-kappa B / metabolism
  • Osteoarthritis, Knee / pathology*
  • Polymerase Chain Reaction
  • Proteoglycans / metabolism
  • RNA, Messenger / metabolism
  • SOX9 Transcription Factor
  • Signal Transduction / drug effects
  • Smad Proteins / metabolism
  • Transcription Factors / genetics
  • Transforming Growth Factor beta / antagonists & inhibitors*
  • Tumor Necrosis Factor-alpha / pharmacology


  • Collagen Type II
  • Cytokines
  • DNA-Binding Proteins
  • High Mobility Group Proteins
  • Interleukin-1beta
  • NF-kappa B
  • Proteoglycans
  • RNA, Messenger
  • SOX9 Transcription Factor
  • SOX9 protein, human
  • Smad Proteins
  • Transcription Factors
  • Transforming Growth Factor beta
  • Tumor Necrosis Factor-alpha