Regulation of microsomal prostaglandin E2synthase-1 and 5-lipoxygenase-activating protein-5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytesReportar como inadecuado

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Arthritis Research and Therapy

, 12:R21

First Online: 09 February 2010Received: 15 December 2009Revised: 25 January 2010Accepted: 09 February 2010


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.

AbbreviationsAAarachidonic acid


Ctthreshold cycle

DMEMDulbecco-s modified Eagle-s medium

Egr-1early growth response factor 1

ELISAenzyme-linked immunosorbent assay

FLAP5-lipoxygenase-activating protein




LTB4leukotriene B4


mPGES-1microsomal prostaglandin E2 synthase-1

NFnuclear factor


PCRpolymerase chain reaction

PGE2prostaglandin E2

PGESprostaglandin E2 synthase

RTreverse transcription

TGF-β1transforming growth factor-beta 1

TNFtumor necrosis factor.

Electronic supplementary materialThe online version of this article doi:10.1186-ar2926 contains supplementary material, which is available to authorized users.

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Autor: Shu-Huang Chen - Hassan Fahmi - Qin Shi - Mohamed Benderdour


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