McEvoy Alice N, Bresnihan Barry, FitzGerald Oliver, Murphy Evelyn P
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
Arthritis Rheum. 2004 Apr;50(4):1132-45. doi: 10.1002/art.20157.
To determine a mechanism by which corticotropin-releasing hormone (CRH) promotes human inflammatory joint disease progression.
An ex vivo synovial tissue culture system was established to investigate the functional properties of CRH at peripheral sites of inflammation. CRH- and interleukin-1 beta (IL-1 beta)-induced prostaglandin E(2) (PGE(2)) production from 10 fresh rheumatoid arthritis (RA) synovial tissue (ST) explants was quantified using a competitive enzyme-linked immunosorbent assay. Modulation of PGE(2) levels was further examined following selective and nonselective cyclooxygenase 2 (COX-2) inhibition. Nuclear extracts were analyzed by electrophoretic mobility shift assays to determine functional cAMP response element binding protein (CREB) activity in response to CRH and PGE(2) in isolated primary synovial cell populations. Western blot analysis measured levels of total and activated (phosphospecific) CREB/activating transcription factor (ATF) family members prior to and following stimulation.
CRH, in a time- and dose-dependent manner, significantly (P = 0.022) up-regulated PGE(2) production from 10 fresh RA ST explants. Costimulation of RA ST with CRH and IL-1 beta significantly augmented (P = 0.036) the effects on PGE(2) production additively over 24 hours. We demonstrated that selective COX-2 inhibitors prevent the induction of PGE(2) by both CRH and IL-1 beta. Further, we provided evidence that CRH and PGE(2) signal through the induction of CREB and phosphorylated CREB/ATF family members in RA ST and in isolated primary RA cell populations.
Our findings underscore the pathogenic role that CRH may play in modulating inflammatory joint disease and establish the CREB/ATF family of transcription factors as principal effector molecules of proinflammatory mediator action in RA.
确定促肾上腺皮质激素释放激素（CRH）促进人类炎性关节病进展的机制。
建立体外滑膜组织培养系统，以研究CRH在炎症外周部位的功能特性。使用竞争性酶联免疫吸附测定法定量10个新鲜类风湿关节炎（RA）滑膜组织（ST）外植体中CRH和白细胞介素-1β（IL-1β）诱导的前列腺素E2（PGE2）生成。在选择性和非选择性环氧化酶2（COX-2）抑制后，进一步检测PGE2水平的调节情况。通过电泳迁移率变动分析对核提取物进行分析，以确定在分离的原代滑膜细胞群体中，响应CRH和PGE2的功能性环磷酸腺苷反应元件结合蛋白（CREB）活性。蛋白质印迹分析测量刺激前后CREB/激活转录因子（ATF）家族总成员和激活（磷酸化特异性）成员的水平。
CRH以时间和剂量依赖性方式显著（P = 0.022）上调10个新鲜RA ST外植体中PGE2的生成。CRH和IL-1β对RA ST的共刺激在24小时内显著增强（P = 0.036）对PGE2生成的叠加效应。我们证明选择性COX-2抑制剂可阻止CRH和IL-1β诱导PGE2。此外，我们提供证据表明，CRH和PGE2通过在RA ST和分离的原代RA细胞群体中诱导CREB和磷酸化CREB/ATF家族成员来发出信号。
我们的研究结果强调了CRH在调节炎性关节病中可能发挥的致病作用，并确立了CREB/ATF转录因子家族作为RA中促炎介质作用的主要效应分子。
