Comparative effects of IL-1β and hydrogen peroxide (H2O2)on catabolic and anabolic gene expression in juvenile bovine chondrocytes

Objective: To compare the effects of hydrogen peroxide (H2O2) to those of interleukin-1 beta (IL-1 beta) on gene expression in juvenile bovine articular chondrocytes (BAC). The study analyses the activation of nuclear factor-kappa B (NF-B-K) and activator protein-1 (AP-1) transcription factors, and the mRNA steady-state levels of the type II collagen, aggrecan core protein matrix, metalloproteinases (MMP-1, -3), and transforming growth factor-beta 1 (TGF-beta 1) genes. Methods: Confluent BAC cultures were treated for 3 and 24 h with IL-1 beta and/or different concentrations of H2O2 (Protocol 1). Following initial treatment, a part of the cells was further subjected to another 24 h with medium, in the presence of IL-1 beta, to determine the effect of the cytokine on H2O2 pre-treated cells (Protocol 2). Total RNA and nuclear protein extractions were performed to study mRNA steady-state levels (real-time polymerase chain reaction) and AP-1/NF-B-K DNA binding (Electrophoretic Mobility Shift Assays), respectively. Results: IL-1 beta enhanced both AP-1 and NF-B-K binding, whereas H2O2 only activated AP-1. H2O2 pre-treatment decreased the IL-1 beta activation of NF-B-K. Both H2O2 and IL-1 beta down-regulated type II collagen and aggrecan expression and increased that of MMP-1 and -3. When cells were pre-treated with H2O2, followed by IL-1 beta, the effects were the same as those observed with H2O2 alone. However, although H2O2 and IL-1 beta were capable of increasing TGF-beta 1 expression separately, subsequent incubation with both factors led to a partial or total abolition of TGF-beta 1 up-regulation. Conclusion: The different regulation of NF-B-K and AP-1 by H2O2 and IL-1 beta underlines the distinct roles played by the two transcription factors in the regulation of gene expression. H2O2 and IL-1 beta exert similar effects on matrix, MMPs and TGF-beta 1 gene expression. However, the association of H2O2 and IL-1 beta does not cause synergic effect, and rather leads, in some cases, to an opposite effect. These data provide further insights into the respective roles of reactive oxygen species and cytokine in the pathophysiology of joint diseases. (c) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.