Regulation of nuclear factor of activated T cells (NFAT) in vascular endothelial cells.
Proteins of the NFAT family (nuclear factor of activated T cells) are Ca(2+)-sensitive transcription factors, which are involved in hypertrophic cardiovascular remodeling. Activation and nuclear translocation is mediated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin (CaN). We identified Ca(2+) signals that induced nuclear translocation of NFAT in cultured calf pulmonary artery endothelial (CPAE) cells using confocal fluorescence microscopy to measure simultaneously [Ca(2+)](i) and subcellular localization of NFAT-GFP (isoforms NFATc1 and NFATc3). The vasoactive agonists ATP (5 microM) or bradykinin (20 microM) in the presence of 2 mM extracellular Ca(2+) induced Ca(2+) release from the endoplasmic reticulum (ER) and activated capacitative Ca(2+) entry (CCE), which caused robust translocation of NFAT to the nucleus. This effect was sensitive to the CaN-inhibitor cyclosporin A (1 microM). Influx of extracellular Ca(2+) via CCE, but not ER Ca(2+) release was identified as the activating Ca(2+) source. NFAT was also activated by Ca(2+) influx induced by cell swelling, reverse mode Na/Ca exchange or ionomycin treatment. NFAT regulation was isoform-specific. Whereas activation of NFATc1-GFP by ATP resulted in persistent nuclear localization, NFATc3-GFP was only transiently imported into the nucleus, followed by rapid export back to the cytoplasm. Inhibition of nuclear kinases, which mediate export of NFAT via phosphorylation, or direct block of nuclear export (Leptomycin B) resulted in stable nuclear localization of NFATc3. These data demonstrate that extracellular Ca(2+) entry mediates NFAT activation. Furthermore, the regulation of nuclear localization of NFAT is isoform-specific and dependent on nuclear export processes.
Rinne
Andreas
A
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA.
Banach
Kathrin
K
Blatter
Lothar A
LA
D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000378:metabolism*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008954:Models, Biological; D050778:NFATC Transcription Factors / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D011651:Pulmonary Artery / Q000166:cytology
D050778:NFATC Transcription Factors; D020033:Protein Isoforms; C445503:transcription factor NF-AT c3; D049452:Green Fluorescent Proteins; D002118:Calcium