Open AccessBasic calcium phosphate crystal–induced prostaglandin E 2 production in human fibroblasts: Role of cyclooxygenase 1, cyclooxygenase 2, and interleukin‐1β
Author(s) -
Morgan Maria P.,
Whelan Linda C.,
Sallis John D.,
McCarthy Conor J.,
Fitzgerald Desmond J.,
McCarthy Geraldine M.
Publication year - 2004
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.20223
Subject(s) - cyclooxygenase , messenger rna , blot , prostaglandin e2 , prostaglandin , prostaglandin e , microbiology and biotechnology , chemistry , reverse transcription polymerase chain reaction , real time polymerase chain reaction , enzyme , medicine , endocrinology , biology , biochemistry , gene
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‐1β (IL‐1β) 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‐1β 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/μg and 1,792 ± 233 pg/μg, respectively) than in untreated (88 ± 5 pg/μg and 205 ± 93 pg/μg, 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‐1β (maximal at 8 hours). The induction of both COX‐2 and IL‐1β by BCP crystals was attenuated when the cells were treated with phosphocitrate. Conclusion 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‐1β.