Membrane Depolarization Mediates Phosphorylation and Nuclear Translocation of CREB in Vascular Smooth Muscle Cells

Experimental Cell Research 263, 118 –130 (2001) doi:10.1006/excr.2000.5107, available online at http://www.idealibrary.com on

Membrane Depolarization Mediates Phosphorylation and Nuclear Translocation of CREB in Vascular Smooth Muscle Cells Andra´ S. Stevenson, Laura Cartin, Theresa L. Wellman, Melissa H. Dick, Mark T. Nelson, and Karen M. Lounsbury 1 Department of Pharmacology, University of Vermont, Burlington, Vermont 05405

Diverse signals have the potential to modulate gene transcription through the Ca 2ⴙ and cAMP response element binding protein (CREB) in vascular smooth muscle cells (VSMCs). A key step in the transmission of these signals is import into the nucleus. Here, we provide evidence that the Ran GTPase, which regulates nuclear import, exerts different regulation over PDGF-BB, Ca 2ⴙ, and cAMP signaling to CREB in VSMCs. PDGF-BB, membrane depolarization, and forskolin increased levels of activated CREB (P-CREB) and c-fos in VSMCs and intact aorta. The calcium channel antagonist nimodipine reduced the level of P-CREB stimulated by membrane depolarization, but not by PDGF-BB or forskolin. Block of Ran-mediated nuclear import, by wheat germ agglutinin or an inactivating Ran mutant (T24N Ran), significantly reduced nuclear P-CREB in response to PDGF-BB or membrane depolarization, but enhanced levels of P-CREB in response to forskolin. Contrary to expectation, block of nuclear import led to the appearance of PCREB in the cytoplasm after depolarization. Furthermore, blocking nuclear export with leptomycin B reduced P-CREB stimulation by both depolarization and PDGF-BB. These results suggest that translocation of CREB between the nucleus and the cytoplasm provides an important role in CREB activating pathways in VSMCs. © 2001 Academic Press Key Words: CREB; Ran; nuclear transport; calcium; vascular smooth muscle; phosphorylation; gene transcription; nucleus; cAMP response element.

INTRODUCTION

Although contraction is a major function of vascular smooth muscle cells (VSMCs), proliferative responses of VSMCs are important for recovery from injury and the pathogenesis of hypertension, atherosclerosis, postangioplasty restenosis, and tumor-stimulated an1 To whom correspondence and reprint requests should be addressed at the Department of Pharmacology, University of Vermont, Burlington, VT 05405. Fax: (802) 656-8892. E-mail: Karen. [email protected].

0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

giogenesis [1, 2]. Because multiple signaling pathways generate both contraction and gene expression [3–5], many questions remain regarding the ability of VSMCs to interpret these signals into optimal physiological responses. Proliferation of VSMCs correlates well with induction of immediate early genes such as c-fos and activation of cell cycle regulators such as cyclins A and E [6 –9]. The promoter regions of both the c-fos gene and cyclins A and D include a Ca 2⫹ and cyclic AMP response element (CRE) [10, 11]. Activation of transcription through the CRE is mediated by the CRE binding protein (CREB). Phosphorylation of CREB at Ser-133 enables CREB to modulate transcription of genes containing upstream CREs [12]. Because Ser-133 of CREB is a potential substrate for several kinases, including Ca 2⫹/calmodulin-dependent kinases (CaMKs), protein kinase A (PKA), ribosomal S-6 kinase 2, and protein kinase C, the specific signaling pathways mediating significant changes in gene expression are not fully established [13–16]. A key checkpoint in the signaling to CREB is the transport of kinases or regulatory proteins (i.e., phosphatases, kinase activators) across the nuclear envelope through nuclear pores. Nuclear pores allow diffusion of small molecules (⬍40 kDa) and mediated transport of larger proteins containing a nuclear localization sequence (NLS) (reviewed by [17]). The small GTPase, Ran, and multiple proteins interacting at the nuclear pore regulate nuclear transport in both directions [18]. Ran-mediated nuclear import is inhibited by introducing a dominant negative mutant of Ran (T24N Ran) or by wheat germ agglutinin (WGA), which binds carbohydrate groups on nuclear pore proteins [19, 20]. The role of Ran-mediated nuclear transport in signals leading to CREB activation in VSMCs has not been studied and may be an important mechanism for VSMCs to distinguish cytoplasmic versus nuclear events. We have recently shown that Ca 2⫹ influx through voltage-gated Ca 2⫹ channels leads to CREB phosphorylation and c-fos transcription in intact arteries [21].

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The goal of this study was both to identify pathways leading to CREB activation in early passage cultures of VSMCs and to further explore regulation of these signals at the nuclear pore. The results demonstrate that voltage-dependent Ca 2⫹ signaling, activation of PKA, and activation through PDGF receptors all promote CREB phosphorylation. Our results also indicate that Ran-mediated nuclear transport has important and distinct roles in these diverse signaling pathways. Furthermore, these results suggest a novel pathway for Ca 2⫹-mediated CREB phosphorylation that includes nuclear export of CREB. METHODS Cell culture and reagents. Adult female rats were euthanized with pentobarbital and the aorta was removed and placed in ice-cold DMEM (low glucose). Cross-sectional rings of aorta were planted on scored culture dishes in DMEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1000 units/ml penicillin, and 1000 ␮g/ml streptomycin. After 3 to 5 days, rings were aspirated off and colonies of migrated VSMCs were trypsinized and passaged for experiments. Cells were used between passages 2 and 4. For K ⫹ treatments, control cells were incubated in Hepes buffer containing (in mM): 10 Hepes, pH 7.4, 6 KCl, 140 NaCl, 2 CaCl 2, 1 MgCl 2, and 10 glucose, and high K ⫹ cells were treated with Hepes buffer with isotonic replacement of NaCl by KCl (final: 120 mM KCl, 10 mM NaCl). Chemicals were obtained from Sigma (St. Louis, MO), unless otherwise noted. Cell culture reagents were obtained from Life Technologies (Gaithersburg, MD). Immunofluorescence. Cultured VSMCs were grown on glass coverslips in six-well culture dishes. The cells were serum-starved in medium containing 0.1% FBS for 36 – 42 h before experimental treatments were initiated. After treatment, the cells were washed with ice-cold phosphate-buffered saline (PBS), pH 7.4. Cells were fixed with ⫺20°C methanol for 15 min, washed in PBS, and blocked for ⱖ1 h with 2% milk in PBS. Primary antibody dilutions in 2% BSA/PBS were added: rabbit anti-P-CREB (New England Biolabs, Beverly, MA) (1:250 dilution) and monoclonal anti-smooth muscle ␣-actin (Sigma) (1:400 dilution) for 1 h at 37°C. Secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA), CY3–anti-rabbit IgG and FITC–anti-mouse IgG (1:500 dilution), were applied for 1 h at 37°C. Cells were then washed with PBS and mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence was detected using a Bio-Rad 1000 laser scanning confocal microscope with a 40⫻ objective. Note. Western analysis with anti-P-CREB antibodies after PDGF treatment resulted in a single band at 45 kDa that corresponded with anti-CREB; no other specific bands were detected (not shown). Laser scanning cytometry. Cells were fixed and treated with antiP-CREB antibodies as above. Oregon green anti-rabbit IgG (1:400) was used for secondary antibody, and cells were incubated with 5 ␮g/ml propidium iodide and 100 ␮g/ml RNase A for 30 min at 37°C to stain DNA. Cells were washed and mounted as above. Cells were scanned using red (propidium iodide) to set nuclear contours. The threshold setting for fluorescence detection (pixel intensity 2000) was determined by the background contour. Northern analysis. RNA was isolated from cultured vascular smooth muscle cells by lysis with Trizol LS Reagent (Life Technologies). Isolated RNA was run on a 1% high-gelling agarose (Type V) gel containing (3.7%) formaldehyde. The gel was transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) and then baked at 42°C for 10 min followed by vacuum oven baking at 70 – 80°C for 2 h to crosslink the RNA. The nitrocellulose membrane was prehybrid-

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ized overnight at 42°C with a deionized formamide solution containing 10⫻ SSC, 50⫻ Denhardt’s solution, 0.1% SDS, and 10 mg/ml salmon testes DNA. Probes to detect c-fos or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were generated by labeling 25 ng of cDNA encoding c-fos DNA or GAPDH (a gift from Dr. Brooke Mossman, University of Vermont) with [␣- 32P]dCTP using the Promega Labeling Kit (Promega Corp., Madison, WI). The probes were purified using NICK Spin Columns (Pharmacia Biotech, Piscataway, NJ). After purification, 1.0 ⫻ 10 6 cpm/ml of either probe was used for hybridization. The RNA transfers were incubated with probe at 42°C overnight then washed in 4⫻ SSC/0.1% SDS, 2⫻ SSC/0.1% SDS, and 1⫻ SSC/0.1% SDS solutions at room temperature for 30 min each and a final wash in 0.1⫻ SSC/0.1% SDS solution at 42°C for 30 min. Calcium measurements. Cultured VSMCs were grown on glass coverslips to about 75% confluency. After 48 h in low serum (0.1% FBS) medium, cells were rinsed in low-K ⫹ Hepes buffer and loaded with 1 ␮M Fura-2-AM (Molecular Probes, Eugene, OR) and a 1:1 ratio of 20% pluronic acid (Molecular Probes) for 45 min at room temperature in the dark [22]. Cells were rinsed in low-K ⫹ Hepes buffer and Fura-2-AM was allowed to de-esterify for 45 min. Coverslips were transferred to a RC-26G recording chamber (Warner Instruments Corporation), and intracellular calcium changes (340/380 nm excitation ratio) were measured for individual cells using a Nikon inverted microscope with Image 1 software. c-fos detection by RT-PCR in intact aorta. CD-1 mice were euthanized with pentobarbital and the aorta was removed. The aorta was placed in ice-cold physiological saline solution (PSS) containing (in mM): 119 NaCl, 4.7 KCl, 24 NaHCO 3, 1.2 K 2HPO 4, 1.6 CaCl 2, 1.2 MgSO 4, 0.23 EDTA, and 11 glucose (pH 7.4), continuously bubbled with 95% O 2/5% CO 2. The aorta was cut into two equal-sized pieces and incubated in various agents at 37°C for 15–30 min. Aorta segments were transferred to sterile microfuge tubes, flash-frozen in liquid nitrogen, and stored at ⫺80°C for future use. RNA extraction, cDNA synthesis, and amplification of c-fos and GAPDH were performed as described [21]. Microinjection. To prepare the synthetic import substrate, Bphycoerythrin (Molecular Probes) was conjugated to the SV40 NLS peptide using sulfo-SMCC (Pierce, Rockford, IL) as in Moore and Blobel [20]. The conjugate was dialyzed against microinjection buffer containing 10 mM sodium phosphate, pH 7.2, 70 mM KCl, and 1 mM MgCl 2 [19]. Ran cDNA constructs (a gift from Dr. Ian Macara, University of Virginia) were amplified and expressed in Escherichia coli as GST fusion proteins and purified by glutathione–Sepharose affinity chromatography as described previously [23]. Purified Ran proteins were cleaved from the GST by addition of thrombin and concentrated to 4 mg/ml. TRITC– dextran (250,000 MW) (2 mg/ml) was included as an injection marker when required. WGA (2 mg/ml), wild-type Ran (1 mg/ml), or T24N Ran (1 mg/ml) was added to the injection solution where indicated. VSMCs for microinjection were grown to approximately 75% confluency on CELLocate gridded coverslips (Eppendorf, Inc., Fremont, CA) and then serum-starved (0.1% FBS) for 48 h before injection. An Eppendorf 5242 system attached to a Nikon Diaphot inverted microscope was used for injection. Cells were injected over a 15-min period (approximately 50 –100 cells) and then incubated at 37°C for 30 min to allow for recovery from the injection. Cells were then exposed to various conditions and processed for immunofluorescence as described above. Cell transfection. VSMCs grown on coverslips in six-well plates were transfected with 1 ␮g/ml pKH 3-Ran or pKH 3-T24N Ran [23] using Trans-IT reagent (Mirus Corp.) according to the manufacturer’s instructions. Cell medium was replaced at 4 h, cells were serum starved at 28 h, and cells were analyzed 48 h after transfection. Expression of HA-tagged Ran proteins was detected using 12CA5 antibodies as previously described [23].

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STEVENSON ET AL. Data analysis. For all experiments the “n” value represents the number of independent experiments. For laser scanning data at least 2000 cells were counted per condition and the integral green fluorescence was determined to quantify phosphorylated CREB (PCREB) immunostaining within the nuclei. These results were expressed as a histogram, and the mean integral nuclear fluorescence was used to compare different conditions. The percentage of VSMC nuclei containing activated CREB (percentage P-CREB positive) was determined by qualitative scoring (⫹ or ⫺) of random fields for at least 100 cells per condition. Levels of mRNA from Northern blots were analyzed by autoradiography and quantified by Phosphoimager (Bio-Rad, Hercules, CA). The c-fos DNA intensity was then normalized to GAPDH expression and expressed as percentage increase over control. In microinjection and transfection experiments, nuclear and cytoplasmic P-CREB intensities were quantified using the histogram intensity tool of Corel Photopaint with background subtracted. At least 10 cells were scored per condition in three or more independent experiments.

RESULTS

Phosphorylation/Activation of CREB and c-fos Transcription in VSMCs through Diverse Signaling Pathways To explore potential pathways leading to CREB activation/phosphorylation in VSMCs, P-CREB was measured by immunostaining with anti-P-CREB antibodies after treatment with PDGF-BB, high-K ⫹ depolarization buffer, or forskolin (Fig. 1A). Cells also were stained with anti-␣-smooth muscle actin, a marker for smooth muscle phenotype. Control cells exhibited ␣-actin staining, but few displayed P-CREB staining (5.5 ⫾ 1.4%). Activation with PDGF-BB, 120 mM K ⫹, or forskolin resulted in a marked increase in the percentage of cells with P-CREB staining (91.3 ⫾ 2.3, 72.0 ⫾ 5.5, and 62.8 ⫾ 10.6%, respectively). To determine the extent of CREB phosphorylation, in response to PDGF-BB, 120 mM K ⫹, or forskolin, the mean nuclear P-CREB fluorescence was also determined using laser scanning cytometry [24]. All three treatments showed a significant increase in the P-CREB intensity over control (Figs. 1B and 1C). To establish that CREB phosphorylation correlates with an increase in transcription of c-fos, a Northern analysis was performed on RNA extracted from VSMCs. As shown in Fig. 2, treatment with PDGF-BB, 120 mM K ⫹, or forskolin increased c-fos mRNA over vehicle control (40 ⫾ 20, 46 ⫾ 16, and 53 ⫾ 21% increase over control, respectively). Together these re-

FIG. 1. Phosphorylation/activation of CREB in response to PDGF-BB, depolarization, and forskolin in VSMCs. (A) CREB phosphorylation detected by anti-P-CREB immunofluorescence. Cells were incubated with (a) control, (b) 5 ng/ml PDGF-BB for 1 h, (c) 120

mM K ⫹ for 10 min, or (d) 10 ␮M forskolin for 30 min. Cells were then fixed and immunostained for smooth muscle ␣-actin (left) and PCREB (right). Bar, 25 ␮m. (B) Representative histogram of nuclear P-CREB immunofluorescence intensity distributions using laser scanning cytometry. Arrows denote treatment conditions as in A. (C) Graphical representation of mean integral nuclear fluorescence from compiled laser scanning data ⫾SEM; *P ⬍ 0.005 (control, n ⫽ 13; K ⫹, n ⫽ 13; PDGF, n ⫽ 11; forskolin, n ⫽ 11).

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no effect on the observed results (not shown). Thus, Ca 2⫹ influx through L-type Ca 2⫹ channels is necessary for depolarization-induced CREB phosphorylation but is not required for CREB phosphorylation through PDGF or PKA pathways. Dose and Time Dependence of CREB Phosphorylation in Response to PDGF, Depolarization, and Forskolin The time course of CREB phosphorylation was determined for treatment with PDGF-BB, K⫹ depolarization, and forskolin and analyzed using laser scanning cytometry (Fig. 4A). CREB phosphorylation in response to PDGF-BB peaked at 15 min and remained elevated for 60 min. Cells treated with 120 mM K⫹ exhibited a transient increase in P-CREB-positive cells that returned to baseline after 60 min (Fig. 4A). The increase in P-CREBpositive cells stimulated by forskolin peaked at 30 min FIG. 2. c-fos transcription induced by PDGF-BB, depolarization, and forskolin. VSMCs were treated with agents as in Fig. 1A. RNA was analyzed by Northern blot using probes recognizing c-fos (top) or GAPDH (bottom). (A) Response to 5 ng/ml PDGF-BB and depolarization with 120 mM K ⫹; n ⫽ 4. (B) Response to 10 ␮M forskolin with DMSO vehicle control; n ⫽ 4. Shown are representative autoradiographs.

sults suggest that VSMC responses resulting in CREB activation also result in an increase in c-fos transcription. Depolarization-Stimulated Ca 2⫹ Influx and CREB Phosphorylation Require Voltage-Dependent Ca 2⫹ Channel Function To determine the role of voltage-dependent Ca 2⫹ channels in depolarization-mediated CREB phosphorylation, the effects of the Ca 2⫹ channel antagonist, nimodipine, on [Ca 2⫹] i and CREB phosphorylation were determined. [Ca 2⫹] i in single VSMCs was measured using Fura-2-AM ratiometric imaging. Membrane depolarization with 120 mM K ⫹ resulted in a reversible rise in [Ca 2⫹] i (Fig. 3A). Maximal [Ca 2⫹] i was observed by the addition of the Ca 2⫹ ionophore ionomycin. The response to depolarization was sensitive to the dihydropyridine, nimodipine, suggesting that the observed rise in [Ca 2⫹] i was through L-type Ca 2⫹ channels and that the VSMC model elicits responses similar to freshly isolated or in situ vascular smooth muscle cells [25]. Blocking L-type Ca 2⫹ channels with nimodipine also significantly reduced the number of P-CREB-positive cells by 63% following depolarization with 120 mM K ⫹ (P ⬍ 0.05); however, responses to PDGF-BB or forskolin were not affected (Fig. 3B). Altering the times of nimodipine treatment before or during stimulation had

FIG. 3. Depolarization-stimulated Ca 2⫹ influx and CREB phosphorylation require L-type Ca 2⫹ channel function. (A) L-type Ca 2⫹ channel signaling in VSMCs. Ratiometric (F 340/F 380) measurements of [Ca 2⫹] i were made in VSMCs loaded with Fura-2-AM. Arrows indicate exposure to 120 mM K ⫹ for depolarization, 100 nM nimodipine to block L-type Ca 2⫹ channels, and 5 ␮g/ml ionomycin to indicate maximal [Ca 2⫹] i. (B) Nimodipine blocks depolarization-stimulated CREB phosphorylation. VSMCs were treated with 0.1% DMSO (Vehicle) or 100 nmol/L nimodipine for 15 min and then exposed to PDGF, K ⫹, or forskolin and processed for immunofluorescence as in Fig. 1. Numbers represent % cells positive for nuclear P-CREB ⫾SEM (*P ⬍ 0.05).

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FIG. 4. Time and dose dependence of nuclear P-CREB fluorescence intensity. (A) VSMCs were treated with 5 ng/ml PDGF-BB, 120 mM K ⫹, or 10 ␮M forskolin for the indicated times and processed for immunofluorescence and laser scanning cytometry as in Fig. 1C (n ⫽ 3). (B–D) Dose responses for CREB phosphorylation (n ⫽ 3). (B) K ⫹ 10 min (n ⫽ 4); (C) PDGF 1 h (n ⫽ 3); (D) forskolin 30 min (n ⫽ 3). Values represent increases in mean nuclear fluorescence over control ⫾SEM.

and remained elevated for 60 min. Exposure of cells to forskolin produced dramatic changes in the actin polymerization, and treatment for periods longer than 30 min resulted in dramatic cytoskeletal changes and cell shrinkage. The observed differences in time course of CREB activation suggest activation through pathways with distinct temporal regulation. P-CREB immunoreactivity increased in a dose-dependent manner in response to PDGF-BB, K ⫹ depolarization, and forskolin, analyzed by laser scanning cytometry (Figs. 4B– 4D). The relationship between PDGF-BB concentration and CREB phosphorylation was similar to profiles for PDGF-BB stimulation of DNA synthesis, migration, and MAP kinase activation in VSMCs [26]. c-fos Transcription Is Stimulated by PDGF-BB, Depolarization, and Forskolin in Intact Aorta We have recently shown that depolarization mediates c-fos transcription in intact cerebral arteries [21].

To confirm and expand these results in intact aorta, RT-PCR experiments measuring c-fos transcription were performed on freshly isolated mouse aorta. Aorta segments were treated with PDGF-BB, 60 mM K ⫹, or forskolin at 37°C, followed by RNA isolation, reverse transcription, and cDNA amplification to quantify RNA transcripts. Consistent with the results using cultured VSMCs, treatment with PDGF-BB, 60 mM K ⫹, or forskolin promoted an increase in c-fos mRNA levels (Fig. 5). The depolarization response was sensitive to nimodipine, suggesting that influx through Ltype Ca 2⫹ channels is necessary for the observed increase in c-fos transcription. Ran-Mediated Nuclear Transport Has Opposite Regulatory Roles in PDGF- vs Forskolin-Mediated CREB Activation in VSMCs Although diverse stimuli have the ability to increase the phosphorylation of CREB in the nuclei of VSMCs, the signaling pathways leading to CREB phosphoryla-

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FIG. 5. Transcription of c-fos in intact aorta. Freshly isolated mouse aorta in PSS were stimulated with 5 ng/ml PDGF (30 min), 60 mM K ⫹ (15 min), or 10 ␮m forskolin (30 min) at 37°C. RNA was analyzed by RT-PCR using probes recognizing c-fos or GAPDH. Quantification of c-fos transcription relative to GAPDH was determined by pixel intensity of scanned gels (top). Errors represent SEM; **P ⬍ 0.001, n ⫽ 3. A representative gel of RT-PCR products stained with ethidium bromide is shown at the bottom.

tion are likely to be distinct. We examined nuclear transport regulation as a potential key step in the differential processing of these signals. Entry of signals into the nucleus occurs either through passive diffusion, for small molecules, or by signal-mediated nuclear import, regulated by the ubiquitous GTPase Ran. A role for Ran-mediated nuclear import in pathways leading to CREB activation would indicate that transport of a protein containing an NLS is required for signal communication to the nucleus. To examine Ran-mediated nuclear transport in VSMCs, WGA or a dominant negative mutant of the Ran GTPase (T24N Ran) was microinjected into cells to block Ran-mediated nuclear import. The effectiveness of this technique in blocking nuclear import was confirmed using a conjugate of B-phycoerythrin and a peptide containing the SV40 nuclear localization sequence [20]. After microinjection into the cytoplasm of VSMCs, the NLS substrate accumulated efficiently in nuclei after 30 min at 37°C (Fig. 6A). Co-injection with wild-type Ran had no effect on import. Co-injection with either WGA or T24N Ran diminished nuclear localization of the NLS substrate, suggesting that this method efficiently blocked active nuclear import. To determine the requirement for active nuclear

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transport in CREB activation through PDGF, cells were injected with WGA or T24N Ran and then stimulated with PDGF-BB. Cells were co-injected with TRITC– dextran (250,000 MW) to identify injected cells. Injection of TRITC– dextran alone or wild-type Ran had no significant effect on the intensity of PCREB immunofluorescence in the nuclei after stimulation (Figs. 6B and 6C). Injection with WGA or T24N Ran reduced the intensity of nuclear P-CREB fluorescence by 87 and 74%, respectively, suggesting an important role for active nuclear transport in signaling from PDGF receptors to CREB (Figs. 6B and 6C). Unlike responses to PDGF, treatment with submaximal concentrations of forskolin (1 ␮M), following injection of WGA or T24N, enhanced the forskolin response over control injected cells (Figs. 6B and 6C). Because the catalytic subunit of PKA enters the nucleus by diffusion [27], these results suggest that T24N Ran does not inhibit diffusion of activated PKA into the nucleus. Furthermore, the increase in forskolin-stimulated P-CREB in the presence of T24N Ran indicates that Ran is necessary for the normal termination of PKA signaling to CREB. Ran-Mediated Nuclear Import Regulates Ca 2⫹ Signaling to CREB in VSMCs Ca 2⫹ or Ca 2⫹/CaM should readily diffuse through nuclear pores, thus a requirement for Ran-mediated nuclear transport would implicate a cytoplasmic Ca 2⫹activated kinase in the Ca 2⫹–CREB pathway [28]. To explore a possible role for Ran in depolarization-induced CREB activation, cells were injected with WGA or T24N Ran to block nuclear import. As shown in Fig. 7, blocking Ran reduced depolarization-induced phosphorylation of CREB (68 and 51% reduction, respectively). Furthermore, an unexpected observation was the detection of significant P-CREB immunofluorescence in the cytoplasm of injected cells, suggesting that CREB may be phosphorylated in the cytoplasm of VSMCs. Two plausible mechanisms could explain the detection of P-CREB in the cytoplasm after depolarization: (1) CREB shuttles constitutively between the nucleus and the cytoplasm, and blocking nuclear import disrupts the reentry of CREB into the nucleus; (2) CREB exports from the nucleus in response to a rise in [Ca 2⫹] i, and blocking nuclear import prevents its reentry into the nucleus. To determine if CREB shuttles constitutively between the nucleus and the cytoplasm, total CREB was detected by immunofluorescence in cells injected with T24N Ran. Blocking nuclear import with cytoplasmic injection of T24N Ran did not affect the distribution of CREB until the cells were depolarized (Fig. 8). Blocking nuclear export by injection of T24N Ran into the nucleus, however, did not affect the

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FIG. 6. Role of Ran-mediated nuclear import in CREB phosphorylation mediated by PDGF and PKA. (A) Nuclear import of NLS substrate requires active nuclear transport. VSMCs were chilled for 10 min and then injected with B-phycoerythrin conjugated to the SV40 NLS. Where indicated, cells were co-injected with buffer containing 2 mg/ml wheat germ agglutinin (WGA) or 1 mg/ml inactivating Ran mutant (T24N Ran). Following injection, cells were fixed and imaged using confocal microscopy; n ⫽ 3. (B) Activation of CREB through

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FIG. 7. Blocking Ran-mediated nuclear import leads to cytoplasmic P-CREB after depolarization. (A) Cells were injected with 2 mg/ml TRITC– dextran alone (Control) or mixed with 2 mg/ml WGA or 1 mg/ml T24N Ran. Cells were then exposed to K ⫹ depolarization buffer (120 mM K ⫹) for 10 min and processed for anti-P-CREB immunofluorescence. Red, TRITC– dextran injection marker. Green, P-CREB immunostaining. (B) Graphical representation of cytoplasmic vs nuclear P-CREB fluorescence measured by pixel intensity for results in A. At least 10 injected cells were quantified for each condition in three separate experiments. Errors represent SEM; *P ⬍ 0.05.

distribution of CREB after depolarization (Fig. 8, bottom). Because CREB was not detected in the cytoplasm of unstimulated cells with nuclear import blocked, these data do not support the constitutive shuttling model for CREB nuclear transport, but instead support a model including depolarization- and Ran-mediated export of CREB into the cytoplasm. Ran-Mediated Nuclear Export Is Important for Signaling to CREB by both PDGF and Ca 2⫹ in VSMCs Microinjection of T24N Ran into the cytoplasm inhibits nuclear import, but does not effectively block nuclear export. However, expression of T24N Ran by transient transfection effectively blocks both nuclear import and nuclear export [19, 29]. Thus, to explore a role for Ran-mediated export on CREB function, the effect of T24N expression on the phosphorylation and subcellular localization of CREB was monitored before and after stimulation with PDGF or 120 mM K ⫹. Expression of wild-type Ran had no effect on CREB phosphorylation or subcellular localization after treatment with either PDGF or 120 mM K ⫹ (Fig. 9A, top). Cells expressing T24N Ran exhibited a reduction in CREB phosphorylation stimulated by either PDGF or 120 mM K ⫹ (Fig. 9A, bottom). Approximately 20% of the T24N-expressing cells exposed to 120 mM K ⫹ exhibited intense cytoplasmic P-CREB immunostaining, similar to that seen after

cytoplasmic injection of T24N Ran (Fig. 9A). These results are possibly due to the higher sensitivity of nuclear import vs export to the effects of T24N Ran. Cytoplasmic P-CREB was not observed in any T24Nexpressing cells treated with PDGF. Thus, disruption of Ran-mediated nuclear import and export by expression of T24N Ran prevents signaling from both PDGF and Ca 2⫹ to the CREB transcription factor, and blocking Ran-mediated nuclear export prevents accumulation of P-CREB in the cytoplasm after membrane depolarization. A specific role for Ran-mediated nuclear export in CREB signaling was examined using the specific nuclear export inhibitor leptomycin B [30]. Cells pretreated with leptomycin B exhibited a threefold increase in P-CREB immunofluorescence compared with control. However, leptomycin B significantly inhibited the increase in P-CREB stimulated by 120 mM K ⫹ or PDGF to a level similar to that seen in control cells (Fig. 9B). Thus, nuclear export has important functions in both activation and inhibition of CREB phosphorylation in VSMCs. DISCUSSION

A central issue in vascular smooth muscle physiology is how a cell can distinguish mitogenic, contractile, and relaxant stimuli and how these stimuli effect specific changes in gene transcription in the nucleus. Our re-

PDGF, but not PKA, requires active nuclear transport. Cells were injected with 2 mg/ml TRITC– dextran alone (injection marker) or mixed with 2 mg/ml WGA or 1 mg/ml T24N Ran. Cells were then exposed to 5 ng/ml PDGF or 1 ␮M forskolin as indicated. TRITC– dextran (red) served as an injection marker and P-CREB (green) was detected by immunofluorescence. Bar, 25 ␮m. (C) Graphical representation of P-CREB nuclear fluorescence measured by pixel intensity for results in B. At least 10 injected cells were quantified for each condition in three separate experiments. Errors represent SEM; **P ⬍ 0.005.

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tion in the signaling of vascular smooth muscle cells through PDGF-BB, membrane depolarization, and cAMP. A correlation between CREB activation and c-fos transcription further demonstrates the importance of CREB in transcriptional regulation by diverse external stimuli. The results from intact aorta support and expand our observations in cultured VSMCs and intact cerebral arteries [21]. These data suggest that up-regulation of c-fos transcription can occur not only in proliferating cells, but also in differentiated smooth muscle cells. These responses are thus likely to be an important component of basic smooth muscle physiology and potentially important in pathological responses in proliferative disorders such as hypertension and atherosclerosis. Regulation of PDGF-BB Nuclear Signaling to CREB by the Ran GTPase

FIG. 8. Microinjection of T24N Ran affects localization of total CREB after membrane depolarization. Cells were injected with 1 mg/ml T24N Ran and 2 mg/ml TRITC– dextran (Injection Marker) and then exposed to buffer containing 6 mM K ⫹ (Con) or 120 mM K ⫹ (K ⫹) for 10 min. Note. Bottom images depict injection into the nucleus with T24N Ran. Cells were then fixed and processed for immunofluorescence with anti-CREB antibodies to visualize total CREB.

sults indicate that mitogenic (PDGF-BB), contractile (membrane depolarization), and relaxant (forskolin) stimuli all lead to a common functional outcome, activation of the transcription factor CREB and elevation of transcription of the immediate early gene, c-fos. However, inhibition of nuclear import revealed differences in the mechanisms by which these signals reach the nucleus to activate CREB. Our results thus provide the first evidence that the Ran GTPase has a key role in differentiating these signals by regulating signal entry into the nucleus of vascular smooth muscle cells. Pathways Leading to CREB Activation and c-fos Transcription in Cultured and Intact Vascular Smooth Muscle The transcription factor CREB is an important mediator of signals originating from Ca 2⫹ influx and elevated cAMP levels. In neurons, membrane depolarization, leading to Ca 2⫹ influx and subsequent CREB activation, is necessary for long-term potentiation and synaptic plasticity [31]. Results presented here provide the first evidence supporting a role for CREB activa-

The results obtained by blocking Ran-mediated nuclear transport suggest differential requirements for signaling to CREB by PDGF, depolarization, and cAMP. Blocking active nuclear import abolished activation of CREB by PDGF-BB, securing a role for Ranmediated nuclear transport in PDGF signaling to the nucleus (Fig. 7A). Signaling through PDGF receptors includes activation of MAP kinase pathways, the phosphoinositide-3 kinase pathway, and phospholipase C pathways [32]. Blocking nuclear import does not distinguish which pathway is required for CREB activation; however, results confirm that links to nuclear signaling through PDGF signaling involve Ran-mediated nuclear transport of downstream kinases. Deactivation of cAMP Signaling to CREB Regulated by the Ran GTPase The finding that blocking nuclear import increases the intensity of P-CREB in cells stimulated with forskolin suggests different requirements for activation and deactivation of the PKA–CREB pathway. Our results suggest that nuclear import of the PKA catalytic subunit does not require Ran-mediated nuclear import (Fig. 7B). The catalytic subunit of PKA is 38 kDa in size and it diffuses into the nucleus [27]. Thus, these data confirm that injection with WGA or T24N Ran selectively inhibits mediated nuclear import and not diffusion. These data also suggest that activation of CREB through PKA is independent of pathways involving PDGF or Ca 2⫹ signaling to the nucleus. Increased intensity of P-CREB after blocking mediated import supports a mechanism in which either a phosphatase (possibly protein phosphatase 1 [13]) or the PKA inhibitory protein (PKI) requires Ran-mediated nuclear entry to reverse activation by PKA. Nuclear export of the PKA–PKI complex requires Ran-medi-

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FIG. 9. Inhibiting nuclear export reduces CREB activation by PDGF and membrane depolarization. (A) Cells were transfected with pKH 3-T24N Ran and then exposed to buffer containing 6 mM K ⫹ (Control), 5 ng/ml PDGF-BB for 30 min (PDGF), or 120 mM K ⫹ for 10 min (120 K ⫹). Cells were then fixed and processed for immunofluorescence with anti-HA antibodies to visualize T24N Ran (HA-tag) and anti-P-CREB antibodies to visualize P-CREB (P-CREB). (%) indicates frequency of phenotype. (B) Where indicated, cells were treated for 1 h with 200 nM leptomycin B (LMB), followed by stimulation with 120 mM K ⫹ for 10 min or 5 ng/ml PDGF-BB for 30 min. Cells were then fixed and processed for immunofluorescence with anti-P-CREB antibodies. Nuclear P-CREB fluorescence intensity was quantified by averaging pixel intensities from at least 10 cells per condition as in Fig. 7B. Errors represent SEM from three independent experiments. **P ⬍ 0.001 relative to control, ##P ⬍ 0.001 relative to K ⫹ control, ⌽⌽P ⬍ 0.001 relative to PDGF control.

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FIG. 10. Proposed model for regulation of CREB by Ca 2⫹ and the Ran GTPase.

ated nuclear export [33], and mediated import of PKI has been implicated [34]. Regulation of Voltage-Gated Ca 2⫹ Signaling to CREB by Ca 2⫹ and the Ran GTPase Interpreting the Ca 2⫹ signaling cascades leading to CREB-mediated gene transcription is decidedly complex [35]. Influx of Ca 2⫹ through L-type Ca 2⫹ channels generates signaling cascades including small molecules (i.e., Ca 2⫹, CaM) and large proteins (i.e., CaMK, calcineurin), and there is evidence in neurons that nuclear entry of both diffusible and mediated signaling molecules may be important for Ca 2⫹-mediated transcription through the CRE [36 –38]. Because blocking active nuclear import with specific inhibitors is not likely to have an effect on the diffusion of Ca 2⫹ across the nuclear pore or release of Ca 2⫹ into the nuclear compartment [28], our results suggest that an NLS-containing protein is integral to communicating signals from a rise in Ca 2⫹ in the cytoplasm to the phosphorylation of CREB in the nucleus. These results are in good agreement with Chawla et al., who found that, although CRE-mediated transcription requires nuclear Ca 2⫹, CREB phosphorylation itself can occur in the absence of nuclear Ca 2⫹ [37, 39]. Our data further reveal that a complex mechanism regulates depolarization-mediated CREB signaling (Fig. 10). Whereas CREB is normally detected in the nucleus, the observation that CREB is located in the cytoplasm after inhibiting active nuclear transport raises the possibility that CREB transiently exports from the nucleus in response to a rise in [Ca 2⫹] i. CREB contains a nuclear localization signal within its DNA recognition sequence (aa 286 –295) [40]. A nuclear export signal has not been identified within the CREB sequence; however, two leucine-rich domains near the C-terminus have the potential to function as nuclear

export signals. The specificity of CREB export to depolarization-induced signals may indicate that phosphorylation of CREB in response to Ca 2⫹ influx occurs in the cytoplasm rather than in the nucleus as proposed previously [41]. Nuclear export of CREB mediated by membrane depolarization suggests a novel role for Ca 2⫹ signaling in pathways leading to CREB activation in the nucleus and raises new questions regarding the function of CREB export and the role of subcellular Ca 2⫹ signaling in the dynamics of CREB regulation. A role for nuclear Ca 2⫹ in transcription through the CRE has already been established [37], and further study of the role of nuclear versus cytoplasmic Ca 2⫹ in the translocation of CREB will establish mechanisms of CREB regulation at the level of nuclear localization. In addition, the function of transient CREB export remains to be determined. Because P-CREB was detected in the cytoplasm, it is plausible that CREB enters the cytoplasm in order to come in contact with cytoplasmic kinases; however, the experiments described here were not able to distinguish the form of CREB that exports from the nucleus. Overall, this study revealed requirements for CREB signaling in the nucleus of VSMCs. PDGF-BB, membrane depolarization (Ca 2⫹), and forskolin (cAMP) all increase levels of phosphorylated CREB and of c-fos mRNA in vascular smooth muscle. The Ran GTPase differentially processes these signals at the nuclear pore and may play a role in the dynamics of CREB subcellular localization. Selectivity of signaling through the Ran GTPase thus provides an important mechanism whereby smooth muscle cells interpret diverse vascular signals through regulation in both the cytoplasm and the nucleus. The authors thank Dr. Deborah Damon (University of Vermont) for supplying rat tissues, Dr. Brooke Mossman (University of Vermont) for supplying c-fos cDNA constructs, Dr. Ian Macara (University of Virginia) for supplying Ran cDNA constructs, and the Vermont Cancer Center Cell Imaging Facility for assistance in confocal and laser scanning cytometry. This study was supported primarily by grants from the American Heart Association, AHA9930218N (K.M.L.); the National Heart, Lung and Blood Institute, HL44455 (M.T.N.); and the Totman Center for Cerebrovascular Research. A.S.S. was supported by a Graduate Research Supplement to HL44455 (M.T.N.).

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