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Investigating the PKR‐signalling pathway in the articular joint
Author(s) -
Gilbert S.J.,
Duance V.C.,
Mason D.J.
Publication year - 2004
Publication title -
international journal of experimental pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.671
H-Index - 72
eISSN - 1365-2613
pISSN - 0959-9673
DOI - 10.1111/j.0959-9673.2004.369ay.x
Subject(s) - ceramide , protein kinase r , signal transduction , chemistry , microbiology and biotechnology , kinase , tumor necrosis factor alpha , protein kinase a , activator (genetics) , apoptosis , biology , biochemistry , endocrinology , mitogen activated protein kinase kinase , receptor
Our previous studies have shown that the protein kinase, PKR and its activator, PACT are involved in TNF‐α signalling in articular cartilage (Gilbert et al . 2002). The sphingolipid ceramide is known to play an important role in signal transduction of TNF‐α and is a potent apoptotic agent. The PKR pathway is also known to be activated by ceramide (Ruvolo et al . 2001). Recent studies have shown that ceramide stimulates proteoglycan degradation and mRNA expression of MMP‐1, MMP‐3 and MMP‐13 in rabbit cartilage suggesting a role for this second messenger in cartilage degradation (Sabatini et al . 2000). In the current study, we investigated the role of ceramide, TNF‐α and PKR in bovine articular cartilage degradation. In addition, we treated human primary synoviocytes with TNF and investigated whether PKR is activated and if NFkB, a transcription factor downstream of PKR, is translocated to the nucleus. Materials and methods Bovine articular cartilage explants were stimulated with C 2 ‐ceramide (50 µm) or TNF‐α (100 ng/ml) for 24 h. To inhibit the activation of PKR, 2‐aminopurine (10 mm) was added to duplicate cultures 1 h prior to the addition of treatments. Media was collected and MMPs analysed by gelatin zymography. Proteoglycan release was measured by the DMMB assay and cell viability determined by the Cytotox ® assay. Human primary synoviocytes were stimulated with TNF‐α (10 ng/ml) for 30 min, and phosphorylated PKR and the Rel A p65 subunit of NFκB were detected by immunofluorescence. Results C 2 ‐ceramide treatment resulted in a significant release of both pro and active MMP‐2. Explants incubated with the PKR inhibitor, 2‐aminopurine, prior to TNF‐α or C 2 ‐ceramide treatment resulted in a marked reduction in both MMP‐2 and MMP‐9 activation. A significant increase in proteoglycan release was observed following treatment with TNF‐α and C 2 ‐ceramide, which was significantly inhibited by 2‐aminopurine. A significant loss of cell viability was observed when explants were treated with C 2 ‐ceramide, which was also found to be regulated by a PKR‐dependant pathway. Studies using antisense oligonucleotides to PACT and PKR are currently being undertaken to confirm these results. An increase in phosphorylation of PKR and NFkB translocation was observed in synoviocytes treated with TNF. Discussion Our data has shown that PKR potentially is an important mediator of degradative and death pathways in chondrocytes. Collectively, these results suggest a novel role for the PKR pathway in the turnover of articular cartilage and support our hypothesis that PKR and PACT are implicated in the cartilage loss that occurs in arthritic disease.