Implication of prostaglandin E₂ in TNF-α-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs

Objectives: Calpains are known as Ca²⁺-dependent intracellular neutral cysteine proteases. However, m-calpain is detected in synovial fluid of arthritic joints and is shown to possess the proteoglycanase activity in vitro. The mechanism of m-calpain release into the extracellular spaces during arthritis has not yet been well characterized. In the present study, we have analyzed m-calpain release from cultured chondrocytes stimulated by a proinflammatory cytokine, tumor necrosis factor-α (TNF-α). The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on m-calpain release were also examined. Methods: Human chondrocytic HCS-2/8 cells were stimulated by TNF-α in the presence or absence of an NSAID. m-Calpain in the cells and culture medium was quantified by Western blot analysis using an anti-m-calpain antibody. Western blots were subjected to densitometric analysis and band intensities were determined. Results: TNF-α (10 ng/ml) stimulated m-calpain release with transient increase in cellular m-calpain in HCS-2/8 cells. NSAIDs examined (aspirin, loxoprofen-SRS, diclofenac sodium, indomethacin and NS398) inhibited m-calpain release and production of prostaglandin E₂ (PGE₂) induced by 10 ng/ml TNF-α. Exogenously added PGE₂ accelerated the release of m-calpain in response to a lower concentration of TNF-α (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-α-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-α. Conclusions: These results suggest that TNF-α stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE₂ receptor by produced PGE₂ is deeply involved in this process.