Ca2+ Signaling, ICAM-1 Expression, and Lung Vascular Injury

Ca2 信号传导、ICAM-1 表达和肺血管损伤

• 批准号: 7457949
• 负责人: CHINNASWAMY TIRUPPATHI
• 金额: $ 28.28万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7457949
• 关键词: Address; Adhesions; Calcineurin; Calmodulin; Cell surface; Data; Endothelial Cells; Feedback; Gene Deletion; Gene Expression; Genetic Transcription; ICAM1 gene; Inflammation; Intercellular adhesion molecule 1; Lung; Mediating; Mus; NF-kappa B; Pathogenesis; Phosphoric Monoester Hydrolases; Play; Protein Kinase C Alpha; Role; Signal Transduction; Testing; Thrombin; Tissues; Vascular Endothelial Cell; base; insight; knockout gene; lung vascular injury; neutrophil; novel; prevent; receptor; transcription factor

Thrombin-induced endothelial cell surface ICAM-1 expression plays a critical role in the pathogenesis of lung vascular injury. Signaling via the transcription factor NF-kappaB is required for the stable thrombin-induced ICAM-1 expression in endothelial cells. In Project 3, we will address the role of thrombin-induced Ca2" entry in the mechanism of NF-KB activation and signaling ICAM-1 expression and neutrophil (PMN) adhesion-mediated lung vascular injury. Our studies indicate that the thrombin-induced increase in intracellular Ca2+ concentration is dependent on both Ca2+ store depletion and the Ca2+ store depletion-mediated Ca2+ influx. Further, we have shown that thrombin-induced Ca2+ influx occurs via transient receptor potential channels (TRPCs) expressed in endothelial cells. TRPC4 gene deletion (TRPC4-/-) in mice prevented thrombin-induced Ca2+ influx. In Supporting Data, we show using pharmacological and gene knockout approaches that the thrombin-induced Ca2+ influx is critically involved in signaling NF-kappaB activation and ICAM-1 expression in lung endothelial cells. Moreover, inhibition of Ca2+-dependent PKC-alpha markedly reduced the thrombin-induced ICAM-1 expression. However, the inhibition of calcineurin (Ca2+/ calmodulin-dependent phosphatase) increased thrombin-induced ICAM-1 expression in pulmonary endothelial cells. Based on these preliminary results, in Aim 1, we will test the hypothesis that TRPC-mediated Ca2+ influx regulates NF-kappaB activation, ICAM-1 expression, and PMN adhesion to pulmonary vascular endothelial cells. In Aim 2, we will test the hypothesis that the Ca2+ activation of PKC-alpha is required for ICAM-1 gene transcription via NF-kappaB activation. In Aim 3, we will test the hypothesis that thrombin-induced calcineurin activation acts in a negative feedback manner to inhibit ICAM-1 gene expression by preventing NF-kappaB activation. In Aim 4 studies, we will test the hypothesis that NF-kappaB activated TRPC1 expression augments Ca2+ influx, and the resultant ICAM-1 expression and PMN adhesion to pulmonary vascular endothelial cells. Studies will be carried out using endothelial cell culture, genetically modified mice (e.g., TRPC4-/- mice), and intact lungs. With these studies, we will be able to provide novel insights into the role of Ca2+ influx in endothelial cells in the mechanism of lung vascular injury and tissue inflammation.

凝血酶诱导的内皮细胞表面ICAM-1表达在肺血管损伤的发病机制中发挥着关键作用。内皮细胞中凝血酶诱导的 ICAM-1 稳定表达需要通过转录因子 NF-kappaB 进行信号传导。在项目 3 中，我们将探讨凝血酶诱导的 Ca2+ 进入在 NF-KB 激活机制中的作用和 信号传导 ICAM-1 表达和中性粒细胞 (PMN) 粘附介导的肺血管损伤。我们的研究表明，凝血酶诱导的细胞内 Ca2+ 浓度增加取决于 Ca2+ 储存消耗和 Ca2+ 储存消耗介导的 Ca2+ 流入。此外，我们还发现凝血酶诱导的 Ca2+ 流入是通过瞬时受体发生的 在内皮细胞中表达的潜在通道（TRPC）。小鼠中 TRPC4 基因缺失 (TRPC4-/-) 可阻止凝血酶诱导的 Ca2+ 内流。在支持数据中，我们使用药理学和基因敲除方法表明，凝血酶诱导的 Ca2+ 内流与肺内皮细胞中 NF-kappaB 激活和 ICAM-1 表达信号传导至关重要。此外，抑制 Ca2+ 依赖性 PKC-α 显着降低了凝血酶诱导的 ICAM-1 表达。然而，抑制钙调神经磷酸酶（Ca2+/钙调蛋白依赖性磷酸酶）会增加肺内皮细胞中凝血酶诱导的 ICAM-1 表达。基于这些初步结果，在目标 1 中，我们将检验 TRPC 介导的假设 Ca2+ 流入调节 NF-kappaB 激活、ICAM-1 表达以及 PMN 与肺血管内皮细胞的粘附。在目标 2 中，我们将测试以下假设：通过 NF-kappaB 激活，ICAM-1 基因转录需要 Ca2+ 激活 PKC-alpha。在目标 3 中，我们将检验凝血酶诱导的钙调磷酸酶激活以负反馈方式起作用的假设 通过阻止 NF-κB 激活来抑制 ICAM-1 基因表达。在 Aim 4 研究中，我们将检验以下假设：NF-kappaB 激活的 TRPC1 表达会增加 Ca2+ 内流，以及由此产生的 ICAM-1 表达和 PMN 对肺血管内皮细胞的粘附。研究将使用内皮细胞培养物、转基因小鼠（例如 TRPC4-/- 小鼠）和完整的肺进行。通过这些研究，我们将能够对内皮细胞中 Ca2+ 内流在肺血管损伤和组织炎症机制中的作用提供新的见解。