Abstract
The transcription factor NFAT (nuclear factor of activated T-cells) is a cytosolic phosphoprotein that accumulates in the nucleus following dephosphorylation by the calcium (Ca2+)/calmodulin-dependent phosphatase, calcineurin. A defining feature of stimuli that induce NFAT nuclear accumulation/activation is a sustained increase in global intracellular Ca2+. Contrary to expectations, we have found that a sustained elevation of intracellular Ca2+, induced by membrane potential depolarization and mediated by voltage-dependent Ca2+ channels, does not result in nuclear localization of the NFATc3 isoform in smooth muscle. However, vasoconstrictors (e.g. uridine triphosphate (UTP)) and growth factors, which elevate intracellular Ca2+ and engage multiple intracellular signaling pathways, induce a robust increase in smooth muscle nuclear NFATc3. Here we show that depolarizing stimuli that normally fail to induce NFATc3 nuclear accumulation in arterial smooth muscle effectively induce nuclear accumulation under conditions in which Crm-1-dependent or JNK2-mediated nuclear export processes are disrupted. Consistent with an important regulatory role for JNK, UTP exerts a suppressive effect on JNK activity in smooth muscle. Export of nuclear NFATc3 following UTP-induced nuclear accumulation is dramatically slowed in cerebral arteries from JNK2-/- animals. These data indicate that in smooth muscle, stimulation of Ca2+-dependent, calcineurin-mediated nuclear import and suppression of Crm-1/JNK-dependent nuclear export are both required for induction of NFATc3 nuclear accumulation. These results highlight the dynamic interplay between influences that promote and oppose NFAT nuclear accumulation and suggest that in arterial smooth muscle suppression of constitutive nuclear export activity is an important property of NFAT-activating stimuli.
Highlights
The transcription factor NFAT is a cytosolic phosphoprotein that accumulates in the nucleus following dephosphorylation by the calcium (Ca2؉)/calmodulin-dependent phosphatase, calcineurin
Depolarization-induced NFATc3 Nuclear Accumulation in Cerebral Arteries from JNK2-deficient Animals—We have previously shown that uridine triphosphate (UTP) and other Gq-coupled vasoconstrictors are effective stimuli for NFATc3 nuclear accumulation in cerebral artery smooth muscle [2], and platelet-derived growth factor is a potent stimulus for NFATc3 nuclear accumulation in ileal smooth muscle [1]
Depolarization with 60 mM Kϩ (HK), which increases intracellular Ca2ϩ levels in smooth muscle, fails to induce a corresponding increase in NFATc3 nuclear localization in either of these smooth muscle tissues [1, 2]. These results were somewhat unexpected given that sustained increases in intracellular Ca2ϩ are sufficient to induce NFAT nuclear accumulation in non-excitable cells, and elevations in intracellular Ca2ϩ induced by depolarizing pulses of HK have been shown to effectively induce NFAT nuclear accumulation in hippocampal neurons [20]
Summary
Tissue Samples—Adult JNK1Ϫ/Ϫ, JNK2Ϫ/Ϫ, C57BL/6, and CD1 mice (20 –25 g) were used. JNK1Ϫ/Ϫ and JNK2Ϫ/Ϫ mice were kindly provided by Dr Mercedes Rincon and have been described previously [21, 22]. Cy5-anti-rabbit IgG (Jackson ImmunoResearch Laboratories; 1:500 dilution), was applied for 1 h at room temperature. The fluorescent nucleic acid dyes YOYO-1 or SYTOX Green (Molecular Probes, 1:30,000 and 1:3000 dilution, and for NFATc3 and JNK experiments, respectively) were used. Specificity of immune staining was confirmed by the absence of fluorescence in arteries incubated with primary or secondary antibodies alone. For P-JNK quantification, grayscale images acquired in proprietary Bio-Rad format were imported into MetaMorph image analysis software (Universal Imaging Corp., Downingtown, PA). 40 ϫ 40 pixel dimension, were applied to five randomly selected areas within the smooth muscle layer of each artery. A threshold encompassing an intensity range of 50 –255 grayscale values was applied to the segmented regions of interest, and the integrated pixel values within the areas were measured using the software’s Integrated Morphometry Analysis module. Chemicals—All drugs and chemical reagents were purchased from Sigma unless otherwise specified
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