Eligini Sonia, Barbieri Silvia Stella, Cavalca Viviana, Camera Marina, Brambilla Marta, De Franceschi Michela, Tremoli Elena, Colli Susanna
E. Grossi Paoletti Center, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.
Cardiovasc Res. 2005 Feb 15;65(3):683-93. doi: 10.1016/j.cardiores.2004.10.024.
This study examines whether cyclooxygenase 2 (Cox-2) synthesis in human endothelial cells involves different signaling pathways when induced by the proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha) or by the tumor and angiogenic promoter phorbol ester (PMA). Moreover, the hypothesis that reactive oxygen species (ROS) and an altered redox status within the cell are fundamental steps for Cox-2 synthesis is verified.
Human endothelial cells isolated from umbilical vein (HUVEC) were exposed to PMA and TNF alpha and Cox-2 protein and mRNA levels were evaluated by Western blot and Real-Time Quantitative Reverse Transcription-PCR analysis. Prostaglandin E(2) (PGE(2)) and 6-keto prostaglandin F(1 alpha) (6-keto-PGF(1 alpha)) levels were measured in cell medium as an index of Cox-2 activity. Intracellular ROS formation was detected by flow cytometry in HUVEC loaded with the oxidant-sensitive 2',7'-dichlorofluorescein diacetate (DCFH-DA) and by nitroblue tetrazolium (NBT) reduction. Reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC.
Data show that TNF alpha and PMA signal for early Cox-2 induction through distinct pathways. PMA-induced Cox-2 expression involves a small GTPase-dependent pathway acting via tyrosine kinase, activation of protein kinase C (PKC) and of the mitogen-activated protein kinase (MAPK) ERK1/2. Conversely, MAPK p38 is critical for Cox-2 induction by TNF alpha. Of interest, intracellular ROS generation and consequent GSH/GSSG ratio reduction represents a common step through which PMA and TNF alpha signal for early Cox-2 induction. In addition, we provide evidence that phosphatidylinositol 3 (PI3)-kinase activation plays a regulatory role for Cox-2 synthesis in HUVEC.
Cox-2 represents a critical link among vascular homeostasis, inflammatory response, angiogenesis and tumor growth. The finding that two independent pathways and an overlapping upstream event signal for Cox-2 induction in HUVEC may be of relevance to develop strategies aimed at selectively interfering with Cox-2 regulating pathways.
本研究旨在探讨在人内皮细胞中，促炎细胞因子肿瘤坏死因子-α（TNFα）或肿瘤及血管生成促进剂佛波酯（PMA）诱导环氧合酶2（Cox-2）合成时是否涉及不同的信号通路。此外，还验证了活性氧（ROS）及细胞内氧化还原状态改变是Cox-2合成的基本步骤这一假说。
从人脐静脉分离的内皮细胞（HUVEC）暴露于PMA和TNFα，通过蛋白质印迹法和实时定量逆转录-PCR分析评估Cox-2蛋白和mRNA水平。在细胞培养基中测量前列腺素E2（PGE2）和6-酮前列腺素F1α（6-酮-PGF1α）水平作为Cox-2活性指标。通过流式细胞术检测负载氧化敏感型2',7'-二氯荧光素二乙酸酯（DCFH-DA）的HUVEC中的细胞内ROS形成，并通过硝基蓝四氮唑（NBT）还原法检测。通过高效液相色谱法测量还原型和氧化型谷胱甘肽（GSH和GSSG）。
数据表明，TNFα和PMA通过不同途径早期诱导Cox-2。PMA诱导的Cox-2表达涉及一条通过酪氨酸激酶作用的小GTP酶依赖性途径，激活蛋白激酶C（PKC）和丝裂原活化蛋白激酶（MAPK）ERK1/2。相反，MAPK p38对TNFα诱导Cox-2至关重要。有趣的是，细胞内ROS生成及随后的GSH/GSSG比值降低是PMA和TNFα早期诱导Cox-2信号传导的共同步骤。此外，我们提供证据表明磷脂酰肌醇3（PI3）-激酶激活在HUVEC中Cox-2合成中起调节作用。
Cox-2是血管稳态、炎症反应、血管生成和肿瘤生长之间的关键联系。在HUVEC中，两条独立途径和一个重叠的上游事件信号诱导Cox-2这一发现可能与制定旨在选择性干扰Cox-2调节途径的策略相关。
