Basic calcium phosphate crystal-induced prostaglandin E2 production in human fibroblasts: role of cyclooxygenase 1, cyclooxygenase 2, and interleukin-1beta.
OBJECTIVE
To elucidate the mechanism of basic calcium phosphate (BCP) crystal-induced prostaglandin E(2) (PGE(2)) production in human foreskin fibroblasts (HFFs), to identify the signaling pathway involved in the induction of cyclooxygenase 2 (COX-2) messenger RNA (mRNA) by BCP crystals, to examine the effect of BCP crystals on interleukin-1beta (IL-1beta) mRNA expression, and to investigate the potential of phosphocitrate to abrogate the BCP crystal-induced effects.


METHODS
PGE(2) levels were quantified using a commercial enzyme immunoassay kit. COX-2 and COX-1 transcript levels were quantified using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Induction of IL-1beta and COX-2 mRNA was examined by end-point RT-PCR. COX-2 protein expression was assessed by Western blotting.


RESULTS
PGE(2) production measured 4 and 30 hours after BCP crystal treatment was higher in BCP crystal-treated (mean +/- SEM 1,891 +/- 273 pg/microg and 1,792 +/- 233 pg/microg, respectively) than in untreated (88 +/- 5 pg/microg and 205 +/- 93 pg/microg, respectively) HFFs. The PGE(2) produced after 4 hours was sensitive to inhibition with NS398, a selective COX-2 inhibitor, implying that it was COX-2 mediated, whereas the PGE(2) produced at 30 hours could not be completely inhibited by NS398. Real-time RT-PCR demonstrated a 23-fold increase in COX-2 mRNA that was maximal at 4 hours, whereas analysis of mRNA for COX-1 showed up-regulation of transcript peaking at 24 hours poststimulation (1.75-fold increase). The protein kinase C and phosphatidylinositol 3-kinase signal-transduction inhibitors bisindolylmaleimide I and LY294002, respectively, blocked BCP crystal-induced COX-2 mRNA in HFFs. In addition, BCP crystals were found to up-regulate the proinflammatory cytokine IL-1beta (maximal at 8 hours). The induction of both COX-2 and IL-1beta by BCP crystals was attenuated when the cells were treated with phosphocitrate.


CONCLUSIONS
These findings indicate that BCP crystals may be an important amplifier of PGE(2) production through induction of the COX enzymes and the proinflammatory cytokine IL-1beta.
Morgan
Maria P
MP
Department of Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St Stephens Green, Dublin 2, Ireland. mmorgan@rcsi.ie
Whelan
Linda C
LC
Sallis
John D
JD
McCarthy
Conor J
CJ
Fitzgerald
Desmond J
DJ
McCarthy
Geraldine M
GM
D002130:Calcium Phosphates / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D003460:Crystallization; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis*; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins; D010003:Osteoarthritis / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology; D013583:Synovial Membrane / Q000166:cytology / Q000201:enzymology; D015854:Up-Regulation
D002130:Calcium Phosphates; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007375:Interleukin-1; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; C020243:calcium phosphate; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone