Abstract
IntroductionThis study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) to 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX).MethodsFor COX-2 and mPGES-1 studies, human osteoarthritic chondrocytes were stimulated at different incubation times (up to 24 hours) with a single or repetitive addition of 10 μM HNE to the cultures at 2-hour intervals, up to 14 hours. For 5-LOX and FLAP studies, cells were treated with a single addition of 10 μM HNE for 24 hours, 48 hours, and 72 hours in the presence or absence of naproxen (a nonspecific COX-2 inhibitor) or antibody anti-transforming growth factor-beta 1 (TGF-β1). The protein levels of COX-2, mPGES-1 and early growth response factor-1 (Egr-1) transcription factor were evaluated by western blot, and those of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-β1 were determined with commercial kits. The levels of mPGES-1, FLAP and 5-LOX mRNA were measured by real-time RT-PCR. Transient transfection was performed to determine promoter activities of mPGES-1 and 5-LOX.ResultsSingle addition of 10 μM HNE to cultured chondrocytes induced PGE2 release as well as COX-2 and mPGES-1 expression at the protein and mRNA levels, with a plateau reached respectively at 8 and 16 hours of incubation, followed by a subsequent decline. However, repeated treatments with HNE prevented the decline of COX-2 and mPGES-1 expression that occurred with a single aldehyde addition. HNE induced mPGES-1 promoter activity, possibly through transcription factor Egr-1 activation. After 48 hours, when COX-2 expression decreased, the LTB4 level rose through 5-LOX and FLAP upregulation. The addition of naproxen to cultured chondrocytes revealed that FLAP and 5-LOX regulation by HNE required PGE2 production. Furthermore, our data showed that HNE significantly induced TGF-β1 production. The addition of anti-TGF-β1 antibody reduced HNE-induced 5-LOX and FLAP expression by 40%, indicating the partial involvement of a TGF-β1-dependent mechanism.ConclusionsOur data demonstrate that the shunt to the FLAP and 5-LOX pathway in HNE-induced human osteoarthritic chondrocytes is attributed to COX-2 and mPGES-1 inhibition, probably due to HNE depletion. PGE2 and TGF-β1 are suggested to be involved in this regulation.
Highlights
This study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 to 5-lipoxygenaseactivating protein (FLAP) and 5-lipoxygenase (5-LOX)
It has been shown that microsomal prostaglandin E2 synthase-1 (mPGES-1) expression is upregulated in animal models of rheumatoid arthritis and in patients suffering from OA [4,5] and is downregulated by antiinflammatory drugs [6,7]. mPGES-1 expression is regulated by several transcription factors, such as early growth response-1 (Egr-1) and NF-B, in different cell types [8,9]
The objectives of the present project were to investigate whether COX-2 and mPGES-1 downregulation, attributed to HNE depletion, is responsible for the switch from COX-2 and mPGES-1 to 5-LOX and Functional 5-LOX requires binding to 5-lipoxygenaseactivating protein (FLAP), and to elucidate the molecular mechanisms underlying their expression in HNE-treated human OA chondrocytes
Summary
This study aimed to investigate whether hydroxynonenal (HNE) depletion is responsible for the switch from cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) to 5-lipoxygenaseactivating protein (FLAP) and 5-lipoxygenase (5-LOX). IL-1b and TNFa stimulate the production of prostaglandins, such as prostaglandin E2 (PGE2), whose roles in the inflammatory process and proteoglycan degradation in human OA cartilage have been reported [2]. PGE2, a major prostaglandin produced via arachidonic acid (AA) metabolism, is involved in many physiological events, such as cell growth, immune regulation, inflammation and arthritis [3]. Cyclooxygenase (COX) and prostaglandin E2 synthase (PGES) are key enzymes for PGE2 biosynthesis under inflammatory conditions. MPGES-1 expression is regulated by several transcription factors, such as early growth response-1 (Egr-1) and NF-B, in different cell types [8,9] It has been shown that mPGES-1 expression is upregulated in animal models of rheumatoid arthritis and in patients suffering from OA [4,5] and is downregulated by antiinflammatory drugs [6,7]. mPGES-1 expression is regulated by several transcription factors, such as early growth response-1 (Egr-1) and NF-B, in different cell types [8,9]
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