Constitutively Elevated Nuclear Export Activity Opposes Ca -dependent NFATc3 Nuclear Accumulation in Vascular Smooth Muscle

The transcription factor NFAT (nuclear factor of activated T-cells) is a cytosolic phosphoprotein that accumulates in the nucleus following dephosphorylation by the calcium (Ca )/calmodulin-dependent phosphatase, calcineurin. A defining feature of stimuli that induce NFAT nuclear accumulation/activation is a sustained increase in global intracellular Ca . Contrary to expectations, we have found that a sustained elevation of intracellular Ca , induced by membrane potential depolarization and mediated by voltage-dependent Ca 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 Ca 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 Ca -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. The NFAT (nuclear factor of activated T-cells) transcription factor family is comprised of four well characterized members, designated NFATc1 (NFAT2/c), NFATc2 (NFAT1/p), NFATc3 (NFAT4/x), and NFATc4 (NFAT3). First identified as the transcription factor responsible for mediating Ca -dependent transcription of genes involved in T-cell activation (3, 4), NFAT has since been shown to play a role in mediating Ca -dependent gene transcription in diverse cell types outside of the immune system, including neurons and cardiac, skeletal, and smooth muscle cells (5–7). NFAT activation is regulated primarily through control of its subcellular localization (8) and reflects the dynamic interplay between influences that promote and oppose nuclear accumulation (reviewed in Refs. 5 and 6). In the absence of a Ca elevating stimulus, NFAT is a transcriptionally inactive cytosolic phosphoprotein. Elevation of intracellular Ca , produced by a variety of mechanisms, activates the Ca /calmodulin-dependent protein phosphatase, calcineurin, which dephosphorylates NFAT on multiple N-terminal serine residues. Dephosphorylation results in a conformational change in the NFAT molecule that exposes nuclear localization signals (9), allowing NFAT nuclear import to take place, presumably through nuclear pores. Calcineurin also plays a role in promoting nuclear retention of NFAT by masking nuclear export signals (NES) recognized by the exportin protein, Crm-1, which has been shown to mediate NES-dependent NFAT nuclear export (10– 13). Calcineurin-dependent NFAT nuclear accumulation is subject to further regulation by the activity of serine/threonine kinases (14), which may act in the nucleus to promote the export of nuclear NFAT, or in the cytosol, to inhibit NFAT import. Some of these regulatory mechanisms rely on sites that are conserved in all NFAT isoforms and are likely to apply universally, whereas other kinases exhibit apparent subtypeselective regulation (reviewed in Refs. 5 and 6). Among the serine/threonine kinases that have been shown to regulate NFAT nuclear accumulation are members of the c-Jun terminal kinase (JNK) family, comprised of JNK1–3 isoforms. JNK1 has been shown to target the NFATc1 isoform, phosphorylating the PxIxIT docking site required for NFAT interaction with calcineurin, resulting in a disruption of calcineurin binding and inhibition of NFAT nuclear import (15). This site is conserved in the NFATc2 isoform but not in the NFATc3 or NFATc4 isoforms. A second more distal target of JNK1, which may also contribute to the inhibitory effect of JNK1 on NFAT * This work was supported by National Institutes of Health Grants HL63722, DDK53832, HL44455, HL07647-12, T32 HL/AR 07944, and HL07944; American Heart Association Grant 022559713; the Swedish Heart & Lung Foundation; the Crafoord and Bergvall Trusts; the Swedish Physiographical Society; and the Totman Trust for Medical Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Present address: Dept. of Physiological Sciences, University of Lund, BMC F12, S-22184 Lund, Sweden. § Present address: Dept. of Molecular Physiology and Biological Physics, University of Virginia Health System, Charlottesville, VA 22908. ¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of Vermont, 89 Beaumont Ave., Burlington, VT 05405. E-mail: [email protected]. 1 The abbreviations used are: NFAT, nuclear factor of activation T-cells; Ca , calcium; K , potassium; HK, high potassium; JNK, c-Jun terminal kinase; P-JNK, phosphorylated JNK; NES, nuclear export signal; PBS, phosphate-buffered saline. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 47, Issue of November 21, pp. 46847–46853, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.