Pulver Renee A, Rose-Curtis Patricia, Roe Michael W, Wellman George C, Lounsbury Karen M
Department of Pharmacology, University of Vermont, Burlington, VT 05405, USA.
Circ Res. 2004 May 28;94(10):1351-8. doi: 10.1161/01.RES.0000127618.34500.FD. Epub 2004 Apr 8.
Ca2+-regulated gene transcription is a critical component of arterial responses to injury, hypertension, and tumor-stimulated angiogenesis. The Ca2+/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, is activated by Ca2+-induced phosphorylation. Multiple Ca2+ entry pathways may contribute to CREB activation in vascular smooth muscle including voltage-dependent Ca2+ channels and store-operated Ca2+ entry (SOCE). To investigate a role for SOCE in CREB activation, we measured CREB phosphorylation using immunofluorescence, intracellular Ca2+ levels using a fluorescence resonance energy transfer (FRET)-based Cameleon indicator, and c-fos transcription using RT-PCR. In this study, we report that SOCE activates CREB in both cultured smooth muscle cells and intact arteries. Depletion of intracellular Ca2+ stores with thapsigargin increased nuclear phospho-CREB levels, intracellular Ca2+ concentration, and transcription of c-fos. These effects were abolished by inhibiting SOCE through lowering extracellular Ca2+ concentration or by application of 2-aminoethoxydiphenylborate and Ni2+. Inhibition of Ca2+ influx through voltage-dependent Ca2+ channels using nimodipine partially blocked intact artery responses, but was without effect in cultured smooth muscle cells. Our findings indicate that Ca2+ entry through store-operated Ca2+ channels leads to CREB activation, suggesting that SOCE contributes to the regulation of gene expression in vascular smooth muscle.
钙离子调节的基因转录是动脉对损伤、高血压和肿瘤刺激的血管生成反应的关键组成部分。钙离子/环磷酸腺苷反应元件结合蛋白（CREB）是一种调节许多基因表达的转录因子，通过钙离子诱导的磷酸化作用被激活。多种钙离子内流途径可能参与血管平滑肌中CREB的激活，包括电压依赖性钙离子通道和储存-操作性钙离子内流（SOCE）。为了研究SOCE在CREB激活中的作用，我们使用免疫荧光法测量CREB磷酸化，使用基于荧光共振能量转移（FRET）的钙荧光指示剂测量细胞内钙离子水平，并使用逆转录-聚合酶链反应（RT-PCR）测量c-fos转录。在本研究中，我们报告SOCE在培养的平滑肌细胞和完整动脉中均能激活CREB。用毒胡萝卜素耗尽细胞内钙离子储存会增加细胞核磷酸化CREB水平、细胞内钙离子浓度以及c-fos的转录。通过降低细胞外钙离子浓度或应用2-氨基乙氧基二苯硼酸和镍离子抑制SOCE可消除这些效应。使用尼莫地平抑制通过电压依赖性钙离子通道的钙离子内流可部分阻断完整动脉的反应，但对培养的平滑肌细胞无影响。我们的研究结果表明，通过储存-操作性钙离子通道的钙离子内流导致CREB激活，提示SOCE参与血管平滑肌中基因表达的调控。
