Mechanism of basic calcium phosphate crystal-stimulated cyclo-oxygenase-1 up-regulation in osteoarthritic synovial fibroblasts.
OBJECTIVE
Basic calcium phosphate (BCP) crystals have been implicated in the pathogenesis of OA and stimulate cyclo-oxygenase (COX) expression and PGE(2) production. This study aimed to elucidate the mechanism of COX-1 up-regulation by BCP crystals and to characterize the PGs produced in OA synovial fibroblasts (OASFs) in response to BCP crystals.


METHODS
OASFs were stimulated with BCP crystals in vitro. mRNA expression was measured by real-time PCR, PG production by EIA and protein production by western blot.


RESULTS
Maximal (19-fold) up-regulation of COX-1 mRNA occurred 32 h after stimulation with BCP crystals; increased COX-1 protein production was also seen. At 32 h post-stimulation with BCP crystals, PGE(2) (and prostacyclin) production was COX-1 dependent. In contrast, maximal (17-fold) up-regulation of COX-2, with corresponding COX-2-dependent PG production, occurred 4 h after BCP crystal stimulation. There was no appreciable increased production of other PGs such as PGF(2alpha), thromboxane A(2) or cyclopentanone PGs including 15d-PGJ(2). Inhibition of protein kinase C (PKC) and extracellular regulated kinase 1/2 (ERK1/2) signal transduction pathways blocked BCP crystal-induced COX-1 mRNA expression. Bafilomycin A1, an inhibitor of intra-lysosomal BCP crystal dissolution, diminished BCP crystal-induced COX-1 mRNA expression.


CONCLUSIONS
These findings indicate that BCP crystals can augment PG production in OASF through induction of COX-1 and COX-2. Intra-lysosomal BCP crystal dissolution and activity of the PKC and ERK1/2 signal transduction pathways are required for BCP crystal-induced COX-1 up-regulation. These data add to the evidence suggesting that the constitutive COX-1/inducible COX-2 concept is an over-simplification and suggest that non-selective COX inhibition may be preferable to COX-2 selective inhibition in BCP crystal-associated OA.
Molloy
E S
ES
Department of Molecular and Cellular Therapeutics, Royal College of Surgeons of Ireland. eamonn.molloy@ireland.com
Morgan
M P
MP
Doherty
G A
GA
McDonnell
B
B
Hilliard
M
M
O'Byrne
J
J
Fitzgerald
D J
DJ
McCarthy
G M
GM
D002130:Calcium Phosphates / Q000494:pharmacology*; D002478:Cells, Cultured; D003460:Crystallization; D051545:Cyclooxygenase 1 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000096:biosynthesis / Q000235:genetics; D015232:Dinoprostone / Q000096:biosynthesis; D011464:Epoprostenol / Q000096:biosynthesis; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D053583:Interleukin-1beta / Q000502:physiology; D010003:Osteoarthritis / Q000201:enzymology* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013583:Synovial Membrane / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D015854:Up-Regulation / Q000187:drug effects*
D002130:Calcium Phosphates; D053583:Interleukin-1beta; D012333:RNA, Messenger; C020243:calcium phosphate; D011464:Epoprostenol; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D015232:Dinoprostone