Orthopaedic Research Laboratory, Hôpital du Sacré-Caeur de Montréal, Department of Surgery, University of Montreal, 5400 Gouin Blvd West, Montreal, QC H4J 1C5, Canada.
Arthritis Res Ther. 2010;12(1):R21. doi: 10.1186/ar2926. Epub 2010 Feb 9.
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).
For 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 muM 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 muM 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 those of prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and TGF-beta1 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.
Single addition of 10 muM 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-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.
Our 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-beta1 are suggested to be involved in this regulation.
本研究旨在探讨羟壬烯醛（HNE）耗竭是否导致环氧化酶-2（COX-2）和微粒体前列腺素 E2 合酶-1（mPGES-1）向 5-脂氧合酶激活蛋白（FLAP）和 5-脂氧合酶（5-LOX）的转变。
在 COX-2 和 mPGES-1 研究中，人类骨关节炎软骨细胞在不同的孵育时间（长达 24 小时）内受到单一或重复添加 10 μM HNE 的刺激，每隔 2 小时添加一次，最长可达 14 小时。在 5-LOX 和 FLAP 研究中，细胞用 10 μM HNE 处理 24 小时、48 小时和 72 小时，同时存在或不存在萘普生（一种非特异性 COX-2 抑制剂）或抗转化生长因子-β1（TGF-β1）抗体。通过 Western blot 评估 COX-2、mPGES-1 和早期生长反应因子-1（Egr-1）转录因子的蛋白水平，并用商业试剂盒测定前列腺素 E2（PGE2）、白三烯 B4（LTB4）和 TGF-β1 的水平。通过实时 RT-PCR 测量 mPGES-1、FLAP 和 5-LOX mRNA 的水平。进行瞬时转染以确定 mPGES-1 和 5-LOX 启动子活性。
向培养的软骨细胞中单次添加 10 μM HNE 诱导 PGE2 释放以及 COX-2 和 mPGES-1 蛋白和 mRNA 水平的表达，分别在孵育 8 小时和 16 小时达到平台期，随后随后下降。然而，HNE 的重复处理阻止了单次添加醛时 COX-2 和 mPGES-1 表达的下降。HNE 诱导 mPGES-1 启动子活性，可能通过转录因子 Egr-1 的激活。48 小时后，当 COX-2 表达下降时，LTB4 水平通过 5-LOX 和 FLAP 的上调而升高。向培养的软骨细胞中添加萘普生表明，HNE 对 FLAP 和 5-LOX 的调节需要 PGE2 的产生。此外，我们的数据表明 HNE 显著诱导 TGF-β1 的产生。添加抗 TGF-β1 抗体可使 HNE 诱导的 5-LOX 和 FLAP 表达减少 40%，表明部分涉及 TGF-β1 依赖性机制。
我们的数据表明，HNE 诱导的人骨关节炎软骨细胞中向 FLAP 和 5-LOX 途径的分流归因于 COX-2 和 mPGES-1 的抑制，可能是由于 HNE 的耗竭。提示 PGE2 和 TGF-β1 参与了这种调节。
