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Dynamic regulation of neuronal NO synthase transcription by calcium influx through a CREB family transcription factor-dependent mechanism

  1. Ted M. Dawson*†§
  1. Departments of *Neurology, †Neuroscience, and ‡Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287

  1. Edited by Louis J. Ignarro, University of California, Los Angeles, CA, and approved May 15, 2000 (received for review January 28, 2000)

Abstract

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.

Footnotes

    • ↵§ To whom reprint requests should be addressed at: Department of Neurology and Neuroscience, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Carnegie 2–214, Baltimore, MD 21287. E-mail: tdawson{at}jhmi.edu.

    • This paper was submitted directly (Track II) to the PNAS office.

    • Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF135825–AF135827).

  • Abbreviations

    nNOS,
    neuronal NO synthase;
    UTR,
    untranslated region;
    VSCCs,
    voltage-sensitive Ca2+ channels;
    En,
    embryonic day n;
    DIV,
    days in vitro;
    β-gal,
    β-galactosidase;
    RT-PCR,
    reverse transcription–PCR;
    GAPDH,
    glyceraldehyde-3-phosphate dehydrogenase;
    GFP,
    green fluorescent protein;
    5′-RACE,
    rapid amplification of 5′-cDNA ends;
    CRE,
    cAMP/Ca2+ response element
    • Received January 28, 2000.

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