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


METHODS
Articular cartilage from the metacarpophalangeal joints of 7 adult horses.


METHODS
Equine chondrocyte monolayer cultures were stimulated with different concentrations (2.5, 5, 10, and 20 ng/mL) of recombinant human interleukin-1beta (rhIL-1beta) for 24 hours and then with rhIL-1beta (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-1beta/mL caused a significant increase in PGE2 concentrations in the culture media, and incubation of cells with rhIL-1beta (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-1beta-dependent induction in COX-2 and mPGES-1 protein expression.


CONCLUSIONS
Collectively, results indicated that the rhIL-1beta-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-1beta-stimulated equine chondrocytes remain to be elucidated.
Farley
Judith
J
Département des Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC, Canada.
Sirois
Jean
J
MacFarlane
Patrick-Hubert
PH
Kombé
Aimé
A
Laverty
Sheila
S
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002358:Cartilage, Articular / Q000187:drug effects / Q000201:enzymology*; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology; D051546:Cyclooxygenase 2 / Q000096:biosynthesis* / Q000235:genetics; D015232:Dinoprostone / Q000096:biosynthesis; D004790:Enzyme Induction / Q000187:drug effects; D006736:Horses / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology*; D019746:Intramolecular Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D000071876:Prostaglandin-E Synthases; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary
D007375:Interleukin-1; D012333:RNA, Messenger; D051546:Cyclooxygenase 2; D019746:Intramolecular Oxidoreductases; C000605862:PTGES protein, human; D000071876:Prostaglandin-E Synthases; D015232:Dinoprostone