Research article

Regulation of microsomal prostaglandin E₂ synthase-1 and 5-lipoxygenase-activating protein/5-lipoxygenase by 4-hydroxynonenal in human osteoarthritic chondrocytes

Shu-Huang Chen , Hassan Fahmi , Qin Shi and Mohamed Benderdour

Arthritis Research & Therapy 2010, 12:R21doi:10.1186/ar2926

Published:	9 February 2010

Abstract (provisional)

Introduction

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-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase (5-LOX).

Methods

For COX-2 and mPGES-1 studies, human osteoarthritic (OA) chondrocytes were stimulated at different incubation times (up to 24 hours) with a single or repetitive addition of 10 microM 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 microM 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-beta1. The protein levels of COX-2, mPGES-1 and early growth response factor-1 (Egr-1) transcription factor were evaluated by Western blot and that of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-beta1were 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.

Results

Single addition of 10 microM 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, LTB4 level rose through 5-LOX and FLAP up-regulation. 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-beta1 production. The addition of anti-TGF-beta1 antibody reduced HNE-induced 5-LOX and FLAP expression by 40%, indicating the partial involvement of a TGF-beta1-dependent mechanism.

Conclusions

Our data demonstrate that the shunt to the FLAP and 5-LOX pathway in HNE-induced human OA chondrocytes is attributed to COX-2 and mPGES-1 inhibition, probably due to HNE depletion. PGE2 and TGF-beta1 are suggested to be involved in this regulation.