Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids.
OBJECTIVE
To explore the modulation of 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX) expression in human osteoarthritic (OA) chondrocytes, their relative implications in leukotriene B(4) (LTB(4)) production, the effect of different factors on this system, and the influence of increased LTB(4) production on the synthesis of catabolic factors of cartilage.


METHODS
FLAP and 5-LOX expression and LTB(4) production were monitored following treatment with transforming growth factor beta1 (TGFbeta1; 5 ng/ml) and 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3); 50 nM) alone or in combination with selective or nonselective cyclooxygenase (COX) inhibitors, naproxen (90 mug/ml), NS-398 (10 muM), or FR122047 (5 muM), or a dual inhibitor of COX/5-LOX activity, licofelone (2.6 muM). LTB(4), prostaglandin E(2) (PGE(2)), and matrix metalloprotease 1 (MMP-1) production were measured by specific enzyme-linked immunosorbent assays, nitric oxide by the Griess reaction, and FLAP and 5-LOX expression by quantitative polymerase chain reaction.


RESULTS
Human OA chondrocytes expressed both FLAP and 5-LOX. TGFbeta1 and/or 1,25(OH)(2)D(3) induced a rapid and marked enhancement ( approximately 4-13-fold) in FLAP messenger RNA (mRNA) levels, which was associated with a subsequent and late increase in LTB(4) production and PGE(2) synthesis. Treatment with COX inhibitors in the absence or presence of TGFbeta1 and 1,25(OH)(2)D(3) induced a rapid increase in LTB(4) production; this response was mediated by the sustained and significant (P < 0.01) up-regulation ( approximately 1.5-fold) of 5-LOX mRNA levels. Conversely, treatment with licofelone showed no effect on 5-LOX but significantly reduced FLAP expression levels. Coincubation of licofelone with TGFbeta1 plus 1,25(OH)(2)D(3) did not affect FLAP or 5-LOX levels. In the presence of TGFbeta1 plus 1,25(OH)(2)D(3), naproxen, but not licofelone, induced MMP-1 production and both drugs decreased nitric oxide levels.


CONCLUSIONS
Both the eicosanoids PGE(2) and LTB(4) are important cofactors in regulating FLAP/5-LOX expression; the inhibition of PGE(2) up-regulates 5-LOX while down-regulating FLAP gene expression, and LTB(4) appears to be an up-regulating factor on the 5-LOX gene. Importantly, nonsteroidal antiinflammatory drugs up-regulate the synthesis of LTB(4), supporting the shunt hypothesis from COX to 5-LOX. We also demonstrated that LTB(4) likely contributes to the up-regulation of important catabolic factors involved in the pathophysiology of OA, such as MMP.
Martel-Pelletier
Johanne
J
Osteoarthritis Research Unit, Centre Hospitalier de l'Université de Montréal, Hôpital Notre-Dame, Montreal, Quebec, Canada. jm@martelpelletier.ca
Mineau
François
F
Fahmi
Hassan
H
Laufer
Stefan
S
Reboul
Pascal
P
Boileau
Christelle
C
Lavigne
Martin
M
Pelletier
Jean-Pierre
JP
D058927:5-Lipoxygenase-Activating Proteins; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D002117:Calcitriol / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002358:Cartilage, Articular / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000378:metabolism; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D007975:Leukotriene B4 / Q000096:biosynthesis*; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D015854:Up-Regulation
D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; D002352:Carrier Proteins; D016861:Cyclooxygenase Inhibitors; D004338:Drug Combinations; D008565:Membrane Proteins; D012333:RNA, Messenger; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D007975:Leukotriene B4; D009569:Nitric Oxide; D001094:Arachidonate 5-Lipoxygenase; D020781:Matrix Metalloproteinase 1; D002117:Calcitriol; D015232:Dinoprostone