Regulation of the expression of 5-lipoxygenase–activating protein/5-lipoxygenase and the synthesis of leukotriene B4 in osteoarthritic chondrocytes: Role of transforming growth factor β and eicosanoids
Abstract
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 B4 (LTB4) production, the effect of different factors on this system, and the influence of increased LTB4 production on the synthesis of catabolic factors of cartilage.
Methods
FLAP and 5-LOX expression and LTB4 production were monitored following treatment with transforming growth factor β1 (TGFβ1; 5 ng/ml) and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3; 50 nM) alone or in combination with selective or nonselective cyclooxygenase (COX) inhibitors, naproxen (90 μg/ml), NS-398 (10 μM), or FR122047 (5 μM), or a dual inhibitor of COX/5-LOX activity, licofelone (2.6 μM). LTB4, prostaglandin E2 (PGE2), 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. TGFβ1 and/or 1,25(OH)2D3 induced a rapid and marked enhancement (∼4–13-fold) in FLAP messenger RNA (mRNA) levels, which was associated with a subsequent and late increase in LTB4 production and PGE2 synthesis. Treatment with COX inhibitors in the absence or presence of TGFβ1 and 1,25(OH)2D3 induced a rapid increase in LTB4 production; this response was mediated by the sustained and significant (P < 0.01) up-regulation (∼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 TGFβ1 plus 1,25(OH)2D3 did not affect FLAP or 5-LOX levels. In the presence of TGFβ1 plus 1,25(OH)2D3, naproxen, but not licofelone, induced MMP-1 production and both drugs decreased nitric oxide levels.
Conclusion
Both the eicosanoids PGE2 and LTB4 are important cofactors in regulating FLAP/5-LOX expression; the inhibition of PGE2 up-regulates 5-LOX while down-regulating FLAP gene expression, and LTB4 appears to be an up-regulating factor on the 5-LOX gene. Importantly, nonsteroidal antiinflammatory drugs up-regulate the synthesis of LTB4, supporting the shunt hypothesis from COX to 5-LOX. We also demonstrated that LTB4 likely contributes to the up-regulation of important catabolic factors involved in the pathophysiology of OA, such as MMP.
