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

凝血酶诱导的内皮细胞表面ICAM-1的表达在肺血管损伤的发病机制中起关键作用。通过转录因子NF-κ B的信号传导是凝血酶诱导的内皮细胞中ICAM-1稳定表达所必需的。在项目3中，我们将讨论凝血酶诱导的Ca 2+内流在NF-κ B活化机制中的作用， 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Ca ~（2+）浓度增加依赖于Ca ~（2+）库耗竭和Ca ~（2+）库耗竭介导的Ca ~（2+）内流。此外，我们已经表明凝血酶诱导的Ca 2+内流通过瞬时受体发生， 潜在通道（TRPC）在内皮细胞中表达。TRPC 4基因缺失（TRPC 4-/-）可抑制凝血酶诱导的Ca 2+内流。在支持性数据中，我们使用药理学和基因敲除方法表明凝血酶诱导的Ca 2+内流在肺内皮细胞中的NF-κ B活化和ICAM-1表达的信号转导中起关键作用。此外，抑制Ca 2+依赖性PKC-α显著降低凝血酶诱导的ICAM-1表达。然而，钙调磷酸酶（钙/钙调素依赖性磷酸酶）的抑制增加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 Ca 2+内流调节NF-κ B活化、ICAM-1表达和PMN与肺血管内皮细胞的粘附在目的2中，我们将检验这样的假设，即PKC-α的Ca 2+激活是ICAM-1基因通过NF-κ B激活而转录所必需的。在目标3中，我们将检验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NF-κ B活化来抑制ICAM-1基因表达的方式。在目的4的研究中，我们将测试的假设，NF-κ B激活TRPC 1的表达增加Ca 2+内流，和由此产生的ICAM-1的表达和PMN粘附到肺血管内皮细胞。研究将使用内皮细胞培养物、遗传修饰的小鼠（例如，TRPC 4-/-小鼠）和完整肺。通过这些研究，我们将能够提供新的见解，在肺血管损伤和组织炎症的机制中，内皮细胞中的Ca 2+内流的作用。

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.