Implication of prostaglandin E(2) in TNF-alpha-induced release of m-calpain from HCS-2/8 chondrocytes. Inhibition of m-calpain release by NSAIDs.
OBJECTIVE
Calpains are known as Ca(2+)-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-alpha (TNF-alpha). 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-alpha 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-alpha (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 E(2) (PGE(2)) induced by 10 ng/ml TNF-alpha. Exogenously added PGE(2) accelerated the release of m-calpain in response to a lower concentration of TNF-alpha (1 ng/ml). AH6809, an EP1/2 antagonist, but not SC19220 (an EP1 antagonist), effectively inhibited TNF-alpha-induced m-calpain release. In contrast, butaprost, an EP2 agonist, accelerated release of m-calpain by 1 ng/ml TNF-alpha.


CONCLUSIONS
These results suggest that TNF-alpha stimulates upregulation and release of m-calpain in chondrocytic HCS-2/8 cells, and that stimulation of EP2-PGE(2) receptor by produced PGE(2) is deeply involved in this process.
Fushimi
K
K
Department of Orthopaedic Surgery, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan.
Nakashima
S
S
Banno
Y
Y
Akaike
A
A
Takigawa
M
M
Shimizu
K
K
D000527:Alprostadil / Q000031:analogs & derivatives* / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D017209:Apoptosis / Q000502:physiology; D001241:Aspirin / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D012531:Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide / Q000494:pharmacology; D004008:Diclofenac / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis / Q000494:pharmacology; D006801:Humans; D009578:Nitrobenzenes / Q000494:pharmacology; D010666:Phenylpropionates / Q000494:pharmacology; D011448:Prostaglandin Antagonists / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation; D044004:Xanthones / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D009578:Nitrobenzenes; D010666:Phenylpropionates; D011448:Prostaglandin Antagonists; D013449:Sulfonamides; D014409:Tumor Necrosis Factor-alpha; D044004:Xanthones; C080955:N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; D004008:Diclofenac; D012531:Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; C053876:6-isopropoxy-9-oxoxanthene-2-carboxylic acid; C040656:loxoprofen; D002154:Calpain; C449000:m-calpain; D000527:Alprostadil; C048491:butaprost; D015232:Dinoprostone; D001241:Aspirin