Sasaki M, Gonzalez-Zulueta M, Huang H, Herring W J, Ahn S, Ginty D D, Dawson V L, Dawson T M
Departments of Neurology, Neuroscience, and Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8617-22. doi: 10.1073/pnas.97.15.8617.
Neuronal nitric oxide (NO) synthase (nNOS) is dynamically regulated in response to a variety of physiologic and pathologic stimuli. Although the dynamic regulation of nNOS is well established, the molecular mechanisms by which such diverse stimuli regulate nNOS expression have not yet been identified. We describe experiments demonstrating that Ca(2+) entry through voltage-sensitive Ca(2+) channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca(2+). Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca(2+) response elements (CREs) that are immediately upstream of the transcription start site. CREB binds to the CREs within the nNOS gene. Mutation of the nNOS CREs as well as blockade of CREB function results in a dramatic loss of nNOS transcription. These findings suggest that nNOS is a Ca(2+)-regulated gene through the interactions of CREB on the CREs within the nNOS exon 2 promoter and that these interactions are likely to be centrally involved in the regulation of nNOS in response to neuronal injury and activity-dependent plasticity.
神经元型一氧化氮（NO）合酶（nNOS）会根据各种生理和病理刺激进行动态调节。尽管nNOS的动态调节已得到充分证实，但尚未确定这些不同刺激调节nNOS表达的分子机制。我们描述的实验表明，通过电压敏感钙通道进入的Ca(2+)通过在皮质神经元中使用交替启动子来调节nNOS表达，并且nNOS外显子2包含对Ca(2+)作出反应的调控序列。对nNOS外显子2启动子的缺失和突变分析揭示了位于转录起始位点上游紧邻的两个关键的cAMP/Ca(2+)反应元件（CREs）。CREB与nNOS基因内的CREs结合。nNOS CREs的突变以及CREB功能的阻断导致nNOS转录显著丧失。这些发现表明，nNOS是一个通过CREB与nNOS外显子2启动子内的CREs相互作用而受Ca(2+)调节的基因，并且这些相互作用可能在响应神经元损伤和活动依赖性可塑性时对nNOS的调节中起核心作用。
