Mechanism of basic calcium phosphate crystal-stimulated matrix metalloproteinase-13 expression by osteoarthritic synovial fibroblasts: inhibition by prostaglandin E2.
OBJECTIVE
To determine the mechanism of matrix metalloproteinase (MMP)-13 upregulation in osteoarthritic synovial fibroblasts (OASF) in response to stimulation with basic calcium phosphate (BCP) crystals and to investigate the effect of prostaglandin (PG)E2 on BCP crystal-stimulated MMP expression.


METHODS
Primary OASF were stimulated with BCP crystals; mRNA expression was measured by real-time reverse transcription-polymerase chain reaction and protein levels were assessed by Western blotting.


RESULTS
BCP crystals upregulated MMP-13 mRNA expression over 20-fold and increased MMP-13 protein production in OASF. BCP crystal-stimulated MMP-13 mRNA expression was blocked by inhibition of the extracellular regulated kinase (ERK1/2) and p38 mitogen activated protein kinase (MAPK) pathways and inhibition of the activation of nuclear factor kappaB. Addition of exogenous PGE2 downregulated BCP crystal-stimulated MMP-13 expression. In contrast, PGE2 upregulated, and had no effect, on BCP crystal stimulated MMP-3 and MMP-1 mRNA expression, respectively. These effects of PGE2 were diminished by L-161,982, a selective EP4 receptor antagonist, and mimicked by CAY10399, a selective EP2 receptor agonist, and forskolin, an adenylate cyclase activator.


CONCLUSIONS
These data suggest that BCP crystal induction of MMP-13 expression may involve the ERK1/2 and p38 MAPK pathways and activation of nuclear factor kappaB; this upregulation of MMP-13 may contribute to the accelerated cartilage breakdown in BCP crystal-associated osteoarthritis. PGE2 had contrasting effects on BCP crystal-stimulated MMP-3 and MMP-13 mRNA expression, mediated in an EP2/EP4/cAMP-dependent manner, suggesting that PGE2 may have beneficial as well as deleterious effects in BCP crystal-associated osteoarthritis.
Molloy
E S
ES
Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, National Orthopaedic Hospital, Dublin, Ireland. eamonn.molloy@ireland.com
Morgan
M P
MP
Doherty
G A
GA
McDonnell
B
B
O'Byrne
J
J
Fitzgerald
D J
DJ
McCarthy
G M
GM
D002130:Calcium Phosphates / Q000494:pharmacology*; D002478:Cells, Cultured; D003460:Crystallization; D003513:Cycloheximide / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000502:physiology; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D018078:Receptors, Prostaglandin E / Q000502:physiology; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015398:Signal Transduction; D013583:Synovial Membrane / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D002130:Calcium Phosphates; D004791:Enzyme Inhibitors; D018942:Macrolides; D016328:NF-kappa B; C545644:PTGER2 protein, human; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058307:Receptors, Prostaglandin E, EP2 Subtype; D058309:Receptors, Prostaglandin E, EP4 Subtype; C040929:bafilomycin A1; C020243:calcium phosphate; D003513:Cycloheximide; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053509:Matrix Metalloproteinase 13; D015232:Dinoprostone