Abstract
Cytokine-mediated induction and overexpression of matrix metalloproteinases (MMPs) is recognized as an important factor in the pathogenesis of arthritis. Interleukin (IL)-1β is a proinflammatory cytokine that is known to superinduce the expression and production of MMP-13 in many cell types. Phenyl N-tert-butylnitrone (PBN), a spin trap agent, inhibited the IL-1β-induced expression of MMP-13 in human osteoarthritis (OA) chondrocytes. Down-regulation of MMP-13 expression correlated with the inhibition of mitogen-activated protein kinase (MAPK) subgroups c-Jun NH2-terminal kinase (JNK) and p38-MAPK activation, accumulation of phospho-c-jun, and the DNA binding activity of activating protein-1 (AP-1). Results of in vitro kinase assays showed that exogenously added PBN completely blocked the c-Jun phosphorylating activity of JNK. Interestingly, using in vitro kinase assay, we also found that chondrocyte p38-MAPK phosphorylate c-Jun and that PBN was not very effective in inhibiting c-Jun phosphorylating activity of p38-MAPK. In addition, PBN did not block the ATF-2 phosphorylating activity of p38-MAPK and Elk-1 phosphorylating activity of extracellular regulated kinase p44/p42 in vitro, indicating that PBN may act selectively to inhibit the phosphorylation of c-Jun in OA chondrocytes. Together, our results for the first time demonstrate that PBN suppresses the IL-1β-stimulated expression of MMP-13 in OA chondrocytes and that this was achieved by inhibiting the activation of JNK and AP-1. These results suggest that use of PBN or compounds derived from it may be of potential benefit in inhibiting signaling events associated with cartilage degradation in arthritis.
Footnotes
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↵ 1 Current address: Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202 002, India.
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This work was supported by National Institutes of Health Grants AR-44902, AR-48782, and AR-37726 and funds from the Department of Orthopedics.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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DOI: 10.1124/jpet.102.048611.
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ABBREVIATIONS: IL, interleukin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; AP-1, activating protein-1; MMP, matrix metalloproteinase; CII, type II collagen; OA, osteoarthritis; PBN, phenyl N-tert-butylnitrone; NAC, N-acetyl cysteine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; PAGE, polyacrylamide gel electrophoresis; RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; MKK, mitogen-activated kinase kinase.
- Received December 27, 2002.
- Accepted March 3, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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