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

凝血酶诱导内皮细胞表面ICAM-1的表达在肺血管损伤的发病机制中起重要作用。凝血酶诱导内皮细胞ICAM-1的稳定表达需要通过转录因子NF-kappaB的信号传导。在项目3中，我们将探讨凝血酶诱导的钙内流在核因子-kB活化机制中的作用。 ICAM-1表达与中性粒细胞(PMN)黏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钙浓度的增加既依赖于钙库的耗尽，也依赖于钙库耗尽所介导的钙内流。此外，我们还发现凝血酶诱导的钙内流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TRPC4基因缺失(TRPC4-/-)可阻止凝血酶诱导的钙内流。在支持数据中，我们使用药理学和基因敲除方法表明，凝血酶诱导的钙内流在肺内皮细胞中信号转导NF-kappaB激活和ICAM-1表达中起关键作用。此外，抑制钙依赖的PKC-α显著降低凝血酶诱导的ICAM-1的表达。然而，钙调神经磷酸酶(钙/钙调蛋白依赖的磷酸酶)的抑制增加了凝血酶诱导的肺内皮细胞ICAM-1的表达。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内流调节核因子-kappaB的激活、ICAM-1的表达以及PMN与肺血管内皮细胞的黏附。在目标2中，我们将验证一种假设，即通过激活核因子-kappaB，ICAM-1基因转录需要PKC-α的钙激活。在目标3中，我们将测试凝血酶诱导的钙调神经磷酸酶激活在负反馈中起作用的假设。 通过阻止核因子-kappaB的激活来抑制ICAM-1基因的表达。在目的4研究中，我们将验证一种假说，即核因子-kappaB激活TRPC1的表达增加了钙内流，并由此产生的ICAM-1的表达和PMN与肺血管内皮细胞的黏附。研究将使用内皮细胞培养、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内钙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。

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.