Evaluation of coexpression of microsomal prostaglandin E synthase-1 and cyclooxygenase-2 in interleukin-1-stimulated equine articular chondrocytes

Abstract

Objective - To characterize expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and regulation of prostaglandin E2 (PGE2) production by equine articular chondrocytes. Sample population - Articular cartilage from the metacarpophalangeal joints of 7 adult horses. Procedure - Equine chondrocyte monolayer cultures were stimulated with different concentrations (2.5, 5, 10, and 20 ng/mL) of recombinant human interleukin-1β (rhIL-1β) for 24 hours and then with rhIL-1β (5 ng/mL) for 3, 6, 9, 12, and 24 hours. Concentration of PGE2 in the media was measured via radioimmunoassay. Total RNA was extracted from harvested chondrocytes, and regulation of COX-2 and mPGES-1 mRNA was studied via reverse transcriptase-polymerase chain reaction assay and Southern blot analysis with equine-specific probes. Western blot analyses were performed on cellular extracts to characterize expression of COX-2 and mPGES-1 protein. Results - Stimulation with 5, 10, and 20 ng of rhIL-1β/mL caused a significant increase in PGE2 concentrations in the culture media, and incubation of cells with rhIL-1β (5 ng/mL) for 6 to 24 hours increased PGE2 production significantly. The increase in prostaglandin production was associated with an induction of COX-2 and mPGES-1 transcripts. There also was an rhIL-1β-dependent induction in COX-2 and mPGES-1 protein expression. Conclusions and clinical relevance - Collectively, results indicated that the rhIL-1β-dependent increase in PGE2 production in equine chondrocytes in monolayer culture was associated with coordinated upregulation of COX-2 and mPGES-1 expression. The pathophysiologic consequences of upregulated COX-2 and mPGES-1 expression and of PGE2 synthesis in rhIL-1β-stimulated equine chondrocytes remain to be elucidated.