NItric oxide and CREB
This paper goes with the first
Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism
1. Masayuki Sasaki*,
2. Mirella Gonzalez-Zulueta*,
3. Hui Huang*,
4. William J. Herring*,
5. Sohyun Ahn,
6. David D. Ginty,
7. Valina L. Dawson*,,, and
8. Ted M. Dawson*,,
+ Author Affiliations
Departments of *Neurology, Neuroscience, and Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287
Edited by Louis J. Ignarro, University of California, Los Angeles, CA, and approved May 15, 2000 (received for review January 28, 2000)
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 Ca2+ entry through voltage-sensitive Ca2+ channels regulates nNOS expression through alternate promoter usage in cortical neurons and that nNOS exon 2 contains the regulatory sequences that respond to Ca2+. Deletion and mutational analysis of the nNOS exon 2 promoter reveals two critical cAMP/Ca2+ 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 Ca2+-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.
Nitric oxide (NO) is an important biological messenger that plays a prominent role in the physiology of the central nervous system. Three isoforms account for NO production and include neuronal NO synthase (nNOS; type I), inducible NO synthase (iNOS; type II), and endothelial NO synthase (eNOS; type III). In the nervous system, nNOS accounts for the majority of the physiologic actions of NO (1, 2). As a diffusible messenger molecule, NO is ideally suited to modulate and regulate synaptic function by acting as a spatial signal (3). Many investigations have shown that nNOS expression is dynamically regulated by both physiological and pathophysiological stimuli; however, the molecular mechanisms controlling the expression of nNOS in response to these stimuli are not known (1, 47).
The structure of the nNOS gene is extremely complicated. Its genomic structure in humans spans more than 240 kilobases, and its expression is potentially regulated by more than nine separate alternative first exons, which splice to a common exon 2 that contains a large 5′ untranslated region (UTR) before the start methionine (8). nNOS expression may be regulated at multiple levels, which could be relevant to a variety of physiologic functions of NO, ranging from a modulator of neuronal plasticity and behavior to a mediator of neuronal cell death (4, 9). To begin to understand how diverse stimuli regulate nNOS expression, we sought to identify the signaling pathways that mediate nNOS expression in neurons. In this study, using primary embryonic cortical neurons, we show that neuronal activity controls nNOS expression through influx of Ca2+ into neurons through L-type voltage-sensitive Ca2+ channels (VSCCs). Furthermore, we find that Ca2+ influx through L-type VSCCs stimulates transcription from the nNOS promoter contained within exon 2 by means of a CREB family transcription factor-dependent mechanism.