Abstract
The transcription factor NFAT (nuclear factor of activated T-cells) plays a central role in mediating Ca(2+)-dependent gene transcription in a variety of cell types. Sustained increases in intracellular calcium concentration ([Ca(2+)]i) are presumed to be required for NFAT dephosphorylation by the Ca(2+)/calmodulin-dependent protein calcineurin and its subsequent nuclear translocation. Here, we provide the first identification and characterization of NFAT in native smooth muscle, showing that NFAT4 is the predominant isoform detected by reverse transcriptase-polymerase chain reaction and Western blot analysis. PDGF induces NFAT4 translocation in smooth muscle, leading to an increase in NFAT transcriptional activity. NFAT4 activation by PDGF depends on Ca(2+) entry through voltage-dependent Ca(2+) channels, because its nuclear accumulation is prevented by the Ca(2+) channel blocker nisoldipine and the K(+) channel opener pinacidil. Interestingly, elevation of [Ca(2+)]i by membrane depolarization or ionomycin treatment are not effective stimuli for NFAT4 nuclear accumulation, indicating that Ca(2+) influx is necessary but not sufficient for NFAT4 activation. In contrast, membrane depolarization readily activates the Ca(2+)-dependent transcription factor CREB (cAMP-responsive element-binding protein). The calcineurin blockers CsA and FK506 also prevented the PDGF-induced NFAT4 nuclear localization. These results indicate that both the nature of the calcium signal and PDGF-induced modulation of nuclear import-export of NFAT are critical for NFAT4 activation in this tissue.
Highlights
Calcium ions (Ca2ϩ) play a central role in the physiology of all cells
We have recently demonstrated that activation of the cAMP-responsive element-binding protein (CREB) and subsequent c-fos expression can be induced by depolarization in native arterial smooth muscle, a process that is mediated by calmodulin-dependent kinase (CaMK) and is dependent on Ca2ϩ influx through voltage-dependent Ca2ϩ channels (VDCC) [6]
Previous results from experiments employing the rat A7r5 aortic smooth muscle cell line suggested that the NFAT1 and NFAT2 isoforms are expressed in smooth muscle [7], expression of additional isoforms could not be ruled out in this study
Summary
The subcellular localization of NFAT is dynamically dependent on a balance between the cytosolic phosphatase activity of calcineurin and the activity of incompletely characterized nuclear kinases, which promote export of NFAT cyclosporin A; RT-PCR, reverse transcriptase-polymerase chain reaction; CREB, cAMP-responsive element-binding protein; CaMK, calmodulin-dependent kinase; GSK-3, glycogen synthase kinase-3; JNK, c-Jun N-terminal kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nc, neurotransmitter inhibitor mixture; PBS, phosphatebuffered saline; P-CREB, phosphorylated CREB. We further show that Ca2ϩ influx alone is not sufficient to induce nuclear accumulation of NFAT4 and demonstrate differential effects of distinct Ca2ϩ signals on the activation of the transcription factors NFAT4 and CREB
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