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(Circulation Research. 2004;94:1351.)
© 2004 American Heart Association, Inc.

Cellular Biology

Store-Operated Ca²⁺ Entry Activates the CREB Transcription Factor in Vascular Smooth Muscle

Renee A. Pulver, Patricia Rose-Curtis, Michael W. Roe, George C. Wellman, Karen M. Lounsbury

From the Department of Pharmacology (R.A.P., P.R.-C., G.C.W., K.M.L.), University of Vermont, Burlington, Vt; and the Department of Medicine (M.W.R.), University of Chicago, Chicago, Ill.

Correspondence to Karen M. Lounsbury, Department of Pharmacology, University of Vermont, Burlington, VT 05405. E-mail Karen.Lounsbury{at}uvm.edu

	Abstract

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Ca²⁺-regulated gene transcription is a critical component of arterial responses to injury, hypertension, and tumor-stimulated angiogenesis. The Ca²⁺/cAMP response element binding protein (CREB), a transcription factor that regulates expression of many genes, is activated by Ca²⁺-induced phosphorylation. Multiple Ca²⁺ entry pathways may contribute to CREB activation in vascular smooth muscle including voltage-dependent Ca²⁺ channels and store-operated Ca²⁺ entry (SOCE). To investigate a role for SOCE in CREB activation, we measured CREB phosphorylation using immunofluorescence, intracellular Ca²⁺ levels using a fluorescence resonance energy transfer (FRET)–based Cameleon indicator, and c-fos transcription using RT-PCR. In this study, we report that SOCE activates CREB in both cultured smooth muscle cells and intact arteries. Depletion of intracellular Ca²⁺ stores with thapsigargin increased nuclear phospho-CREB levels, intracellular Ca²⁺ concentration, and transcription of c-fos. These effects were abolished by inhibiting SOCE through lowering extracellular Ca²⁺ concentration or by application of 2-aminoethoxydiphenylborate and Ni²⁺. Inhibition of Ca²⁺ influx through voltage-dependent Ca²⁺ channels using nimodipine partially blocked intact artery responses, but was without effect in cultured smooth muscle cells. Our findings indicate that Ca²⁺ entry through store-operated Ca²⁺ channels leads to CREB activation, suggesting that SOCE contributes to the regulation of gene expression in vascular smooth muscle.

Key Words: SERCA • gene transcription • calcium channels • arteries

	Introduction

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Vascular smooth muscle cells (VSMCs) possess an ability to transition between differentiated and proliferative phenotypes in response to environmental cues.¹ Although the proliferative phenotype is essential for vasculogenesis, uncontrolled proliferation and migration caused by changes in VSMC gene transcription are associated with the development of vascular pathologies such as atherosclerosis, hypertension, postangioplasty restenosis, and tumor-stimulated angiogenesis.^2,3 Disease-related variations in VSMC phenotype correlate with atypical Ca²⁺ signaling, elevated intracellular Ca²⁺, and gene transcription.^4–6 As yet, the interrelationships between Ca²⁺ signaling and transcriptional control of gene expression in VSMCs remain unresolved.

Regulation of gene expression by Ca²⁺ can be mediated by Ca²⁺-dependent phosphorylation of the transcription factor CREB (Ca²⁺/cAMP-response element binding protein). Regulation of c-fos and other immediate early genes is in part Ca²⁺-dependent and requires CREB.^7,8 CREB activation requires phosphorylation at ¹³³Serine to facilitate formation of an active transcriptional complex including recruitment of CREB binding protein (CBP300) and other cofactors to the Ca²⁺/cAMP-response element (CRE) in the promoter of many genes.^9–11 CREB phosphorylation can be mediated by multiple kinases including cAMP-dependent protein kinase, ribosomal S6 kinase, mitogen- and stress-activated protein kinases, and calmodulin-dependent protein kinase (CaMK).⁹ We have previously determined that membrane depolarization increases phosphorylated CREB (P-CREB) levels and c-fos transcription in VSMCs.¹² This effect is dependent on Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels (VDCCs) and CaMK activation.^8,12 In addition, cerebral arteries from hypertensive rats exhibit elevated intracellular Ca²⁺ and an increased level of basal P-CREB and c-fos transcription.⁶

Multiple sources of Ca²⁺ may participate in regulation of gene expression in VSMCs. Elevation of Ca²⁺ in smooth muscle cells can result from entry of extracellular Ca²⁺ as well as release from Ca²⁺ sequestered within organelles such as the sarcoplasmic reticulum (SR).^13–15 Ca²⁺ influx across the plasma membrane is mediated by voltage-dependent Ca²⁺ channels, and voltage-independent cation channels including store-operated Ca²⁺ channels. Store-operated calcium entry (SOCE), also known as capacitative Ca²⁺ entry, has been detected in VSMCs^16,17 and is thought to play an essential role in the regulation of contraction, cell proliferation, and apoptosis.^18,19 Activation of Ca²⁺ influx through store-operated Ca²⁺ channels is triggered by a reduction in SR Ca²⁺ concentration.^17,18 Transient discharge of SR Ca²⁺ occurs during the course of signaling events that activate inositol 1,4,5-trisphosphate receptors (IP₃R) or ryanodine receptors in the SR membrane.^15,20 SR Ca²⁺ stores also can be depleted by inhibiting sarcoendoplasmic reticulum Ca²⁺ ATPases (SERCA) with thapsigargin or cyclopiazonic acid.^21,22

A role for SOCE in the regulation of gene expression in VSMCs is unclear. In the present study, we examined the signaling pathway linking SR Ca²⁺ store depletion to CREB phosphorylation in cultured VSMCs and intact arterial myocytes. Our findings indicate that Ca²⁺ entry through SOCE contributes to Ca²⁺ homeostasis and induces CREB activation, suggesting a novel mechanism for the regulation of gene expression by Ca²⁺ in VSMCs.

	Materials and Methods

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Cell Culture, Animals, and Reagents
Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, 1985) following protocols approved by the University of Vermont IACUC. Female Sprague-Dawley rats (Harlan, Indianapolis, Ind) (12 weeks, 200 g) were euthanized (pentobarbital 150 mg/kg IP), and the aorta, middle, and posterior cerebral arteries were dissected in cold HBS (HEPES buffered saline). Rat VSMCs were cultured from aorta explants, maintained in DMEM containing 10% fetal bovine serum as detailed previously.¹² hcVSMCs were obtained from human cerebral artery explants (IRB No. CHRMS 01-195; informed consent) and cultured in SMGM2 media (Cambrex). VSMCs were used between passages 2 and 4.

Thapsigargin (TG), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), nimodipine (Nim), and ionomycin were purchased from Calbiochem, and 2-aminoethoxydiphenylborate (2-APB) was from Tocris Cookson, Inc. Cell culture reagents were obtained from Gibco. All other chemicals were purchased from Sigma.

P-CREB Immunofluorescence
Immunofluorescence was performed using anti-P-CREB antibodies (Cell Signaling Technology) [1:250] and Cy3-anti-rabbit IgG secondary antibody (Jackson ImmunoResearch Labs) [1:500] as described,¹² with the following exceptions. VSMCs were fixed with 4% formaldehyde, and 0.1% Triton X-100 was added to blocking and antibody dilution solutions. YOYO-1 (Molecular Probes) [1:10,000] containing 250 µg/mL RNase was added for 30 minutes at 37°C to counterstain the cell nuclei. For immunolabeling of intact arteries, Triton X-100 concentration was 0.2%, and Cy5 goat anti-rabbit IgG (Jackson ImmunoResearch Labs) [1:500] was used as the secondary antibody. Images were captured using a Bio-Rad 1000 laser scanning confocal microscope with a 40x objective. Fluorescence intensities from 30 to 90 nuclei were determined per condition from at least three independent experiments as described.²³

RNA Isolation, RT-PCR, and Quantitative RT-PCR
Total RNA was extracted from treated hcVSMCs using TriZol reagent and quantified using the NanoDrop spectrophotometer. For c-fos detection by RT-PCR, RNA (25 ng) was reverse-transcribed using Sensiscript RT kit (Qiagen) according to the manufacturer’s instructions, and cDNA was amplified using c-fos and ß-actin primers. PCR products were separated by agarose gel electrophoresis and quantified using Quantity One software. ß-Actin was used as an internal standard. For c-fos detection by quantitative RT-PCR, RNA (2 µg) was reverse-transcribed using Omniscript RT kit (Qiagen) according to the manufacturer’s instructions. The resulting cDNA was amplified using Assays-on-Demand gene expression products kits (Applied Biosystems) and analyzed with a 7900HT Sequence Detection System (TaqMan, Applied Biosystems). Assays were run in duplicate for each independent experiment according to manufacturer’s recommendations. HPRT was used as the internal standard. Data analysis was fully automated and performed using Sequence Detection 2.1 software (Applied Biosystems).

Determination of Intracellular Ca²⁺
VSMCs were grown on coverslips and growth media was replaced with DMEM containing 0.1% fetal bovine serum. Cells were transfected with 1 µg/mL of yellow cameleon2.1 (YC2.1), a fluorescence resonance energy transfer (FRET)–based calcium biosensor²⁴ using Lipofectamine 2000 (Invitrogen). After 2 hours, the media was replaced and cells were maintained in a humidified incubator 24 hours before use. Coverslips were placed into a SA-NIK chamber (Warner Instruments) mounted on a Nikon Diaphot 200 inverted microscope equipped for epifluorescence. Cells were superfused with HBS at room temperature. YC2.1 excitation was 440 nm and emission recorded with an ORCA-ER charge-coupled device (Hamamatsu) at 480 nm (FRET donor, enhanced cyan fluorescent protein, ECFP) and 535 nm (FRET acceptor, enhanced yellow fluorescent protein, EYFP). Data were analyzed using Metafluor 3.0 Imaging Software (Universal Imaging Corporation). FRET emission ratio (ratio 535/480) was used as a measure of intracellular Ca²⁺ concentration. Normalized ratio was obtained by dividing the Ratio 535/480 by the starting baseline ratio value. To calculate the percent maximal FRET ratio change, data were converted to a range of 100% using maximum, Ionomycin (10 µmol/L), and minimum, EGTA (2 mmol/L), values obtained in each experiment. Area under the curve (AUC)±SEM was calculated using Sigma Plot.

Statistical Analysis
Student t test and Student-Newman-Keuls multiple comparisons test were used to determine statistical significance between treatment groups.

An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

	Results

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SERCA Inhibition Promotes CREB Phosphorylation and c-fos Transcription Independent of L-Type Ca²⁺ Channel Activity in Cultured VSMCs
Ca²⁺ entry through L-type VDCCs has been shown to increase phosphorylation and activation of the transcription factor CREB.^8,12 To determine whether elevations in Ca²⁺ triggered by depletion of SR Ca²⁺ stores can also initiate CREB phosphorylation, SERCA was irreversibly inhibited with thapsigargin. CREB phosphorylation at ¹³³Ser was detected by immunofluorescence using P-CREB antibodies and quantified using nuclear pixel intensity. P-CREB nuclear fluorescence increased in response to thapsigargin in a concentration-dependent manner (Figure 1A). The EC₅₀ (28.2±10.7 nmol/L, n=3) was similar to the EC₅₀ for thapsigargin-induced arterial contraction (Wellman and Phillips, unpublished observation, 2004) and is consistent with SERCA inhibition.²¹ Cyclopiazonic acid (CPA), a reversible inhibitor of SERCA, also stimulated CREB phosphorylation (not shown). Elevation of P-CREB was evident 1 minute after thapsigargin addition, peaked at 5 minutes, and returned to baseline within 30 minutes of treatment (Figure 1B). These results suggest that SERCA inhibition can induce transient CREB activation in VSMCs.

View larger version (18K): [in this window] [in a new window]   Figure 1. Thapsigargin induces a dose-dependent transient increase in CREB phosphorylation in cultured VSMCs. A, Cells were incubated with increasing concentrations of thapsigargin (TG) for 5 minutes, and P-CREB was detected by immunofluorescence. Top, Representative images of P-CREB immunofluorescence in nuclei of VSMCs. Bottom, Graph represents quantification of nuclear P-CREB immunofluorescence intensities (±SEM, n=3). Bar=100 µm. B, Cells were incubated with 100 nmol/L TG over a time period of 1 minute to 24 hours followed by detection of P-CREB by immunofluorescence. Time points for TG addition were staggered such that all cells were simultaneously fixed at 24 hours. Top, Representative images of P-CREB for untreated control and 5-minute and 120-minute time points. Bottom, Graph represents quantification of nuclear P-CREB immunofluorescence intensities normalized by the untreated control P-CREB intensity (±SEM, n=3).

As SERCA inhibition depletes SR Ca²⁺ stores and consequently stimulates SOCE,¹⁴ we explored a role for Ca²⁺ influx through store-operated Ca²⁺ channels in the observed thapsigargin-induced CREB phosphorylation. Reducing extracellular Ca²⁺ or chelating extracellular Ca²⁺ with BAPTA significantly decreased thapsigargin-induced P-CREB levels (Figure 2A). Nimodipine, an L-type VDCC blocker, was without effect, whereas nickel (Ni²⁺), a divalent blocker of cation channels,²⁵ and 2-aminoethoxydiphenylborate (2-APB), a blocker of SOCE,²⁶ significantly reduced thapsigargin-mediated CREB phosphorylation (Figure 2B).

View larger version (19K): [in this window] [in a new window]   Figure 2. Thapsigargin-mediated CREB phosphorylation requires Ca2+ influx and is reduced by blockers of SOCE in cultured VSMCs. A, VSMCs were incubated in HBS with normal Ca2+ (2 mmol/L), 100 nmol/L Ca2+, or preincubated with 5 mmol/L BAPTA for 30 minutes then treated with 100 nmol/L thapsigargin (TG) for 5 minutes. P-CREB was detected by immunofluorescence, and shown are histograms of nuclear P-CREB intensities normalized by the untreated control P-CREB intensity (±SEM, n=3). ***P<0.001 compared with 2 mmol/L Ca2+. B, Where indicated, VSMCs were preincubated with 100 nmol/L nimodipine (Nim) for 15 minutes (n=3), 100 µmol/L 2-APB for 10 minutes (n=4), or 500 µmol/L nickel for 10 minutes (n=4) then treated with 100 nmol/L TG for 5 minutes. Detection and quantification of P-CREB were performed as in A. ***P<0.001 compared with TG alone.

To confirm that the observed effects on P-CREB correlated with changes in its transcriptional activity, we measured the effect of thapsigargin on transcription of the CRE-regulated gene, c-fos, using both standard and quantitative RT-PCR. Transcription of c-fos was induced by thapsigargin, and this effect was insensitive to nimodipine, but blocked by 2-APB, closely paralleling the data profile for CREB phosphorylation (Figure 3). Together, these results provide evidence that depletion of SR Ca²⁺ using thapsigargin leads to CREB activation through a mechanism requiring Ca²⁺ influx and functional store-operated Ca²⁺ channels.

View larger version (24K): [in this window] [in a new window]   Figure 3. Thapsigargin promotes transcription of c-fos that is insensitive to nimodipine and inhibited by the SOCE blocker 2-APB in cultured VSMCs. A and B, hcVSMCs were incubated in HBS containing 100 nmol/L thapsigargin (TG) or with isotonic elevation of K+ to 120 mmol/L for 30 minutes followed by extraction of RNA and RT-PCR using primers recognizing c-fos. Shown are representative agarose gel (A) of the resulting PCR products and histogram (B) of c-fos band intensities relative to ß-actin (±SEM, n=5). **P<0.01, ***P<0.001. C, hcVSMCs were treated as described, and where indicated, cells were preincubated with 100 nmol/L nimodipine (Nim) for 15 minutes or 100 µmol/L 2-APB for 10 minutes. RNA was isolated and c-fos transcript levels were determined using quantitative RT-PCR. Histograms represent data normalized to the TG response (average±SEM) of duplicate determinations from 2 independent experiments. *P<0.05.

Thapsigargin-Induced Ca²⁺ Signaling Involves Store-Operated Ca²⁺ Channels
The mechanism of CREB activation by thapsigargin was consistent with Ca²⁺ entry through store-operated Ca²⁺ channels. To support these data using single cell measurements of Ca²⁺, we expressed the Cameleon YC2.1, a calmodulin-based FRET biosensor in VSMCs.²⁴ Because this indicator has not been previously characterized in VSMCs, we first examined FRET ratio changes over a range of Ca²⁺ concentrations. Cells were incubated with buffers of known Ca²⁺ concentration²⁷ in the presence of the Ca²⁺ ionophore, ionomycin. A 20% FRET ratio change occurred at 200 nmol/L Ca²⁺ and the indicator appeared saturated at concentrations above 10 µmol/L (Figure 4A).

View larger version (24K): [in this window] [in a new window]   Figure 4. Thapsigargin elicits a transient rise in cytoplasmic Ca2+ levels and depletes SR Ca2+ stores as measured using a Cameleon FRET Ca2+ indicator. A, Cultured VSMCs were transfected with the YC2.1 Cameleon then incubated with buffers of known Ca2+ concentration containing 10 µmol/L ionomycin, and images of 535 nm (YFP) and 480 nm (CFP) emissions were recorded and plotted as change in ratio using a log-linear scale (±SEM, n=3). B, Pseudocolor images of the FRET emission ratio of a single VSMC transfected with the Ca2+ indicator YC2.1. Color range represents ratio of YFP/CFP from 1.0 to 1.5. Time 0 represents the addition of the reagent, either 1% serum or 100 nmol/L TG. C, Normalized FRET ratio (535/480 nm) plot of fluorescence emission from a representative single VSMC transfected with YC2.1. Treatments were added as a bolus to the bath as indicated by the horizontal lines on the plot. Maximum and minimum values were determined by addition of 10 µmol/L ionomycin and 2 mmol/L EGTA. D, Area under the curve [integration of normalized FRET ratioxtime (min)] was calculated for serum-induced FRET responses before and after the addition of 100 nmol/L TG (±SEM, n=6). **P<0.01.

Administration of serum, known to stimulate IP3-mediated Ca²⁺ release from thapsigargin-sensitive stores,^28–30 resulted in a transient rise in Ca²⁺. Thapsigargin caused a rapid and more sustained increase in Ca²⁺ and its administration inhibited subsequent serum responses, indicating that it effectively diminished SR Ca²⁺ (Figure 4B through 4D). Similar to our results related to CREB phosphorylation, the increase in Ca²⁺ initiated by thapsigargin was significantly attenuated by BAPTA or 2-APB (Figure 5). These findings indicate that the source of Ca²⁺ leading to CREB activation by thapsigargin is likely through SOCE.

View larger version (18K): [in this window] [in a new window]   Figure 5. Blockers of store-operated Ca2+ channels significantly reduce thapsigargin-induced Ca2+ signals. Cultured VSMCs expressing YC2.1 were treated with 100 nmol/L TG, and where indicated, cells were preincubated with 5 mmol/L BAPTA for 30 minutes or 100 µmol/L 2-APB for 10 minutes. A, Shown is a representative plot of percent maximal FRET ratio change of emissions at 535/480 nm based on 10 µmol/L ionomycin (maximum) and 2 mmol/L EGTA (minimum). B, Area under the curve [integration of percent maximal FRET ratio changextime (min)] was calculated for the responses shown in A (±SEM, n=5). **P<0.01 compared with TG alone.

Store-Operated Ca²⁺ Entry Leads to CREB Phosphorylation in Intact Arteries
VSMCs maintained in culture undergo multiple phenotypic changes.⁵ It is therefore possible that SOCE and Ca²⁺ signaling responses may be different in smooth muscle cells present in intact arteries. To measure the effect of SR Ca²⁺ store depletion on CREB phosphorylation in arterial myocytes, rat cerebral arteries were isolated and treated in vitro with thapsigargin, followed by detection of P-CREB using immunofluorescence. Thapsigargin induced an increase in P-CREB fluorescence that colocalized with nuclei (Figure 6A). In agreement with our previous findings,⁸ induction of CREB phosphorylation following membrane depolarization by elevated K⁺ was prevented by nimodipine or reducing extracellular Ca²⁺. The thapsigargin-induced CREB phosphorylation was partially inhibited by nimodipine, but was ablated by reducing extracellular Ca²⁺ (Figure 6A and 6B). Furthermore, the nimodipine-insensitive CREB phosphorylation was eliminated by treatment with 2-APB or Ni²⁺ (Figure 6C), suggesting that thapsigargin-mediated CREB activation is accomplished by Ca²⁺ signaling through voltage-dependent Ca²⁺ channels and SOCE in intact arteries.

View larger version (31K): [in this window] [in a new window]   Figure 6. SOCE plays a role in CREB phosphorylation in intact arteries. Rat cerebral arteries were isolated and incubated in HBS with normal Ca2+ (2 mmol/L), 100 nmol/L Ca2+, or 100 nmol/L nimodipine (Nim) for 15 minutes. Arteries were then exposed to 100 nmol/L TG for 15 minutes or 60 mmol/L K+ for 10 minutes. CREB phosphorylation was detected by anti–P-CREB immunofluorescence. A, Confocal images representing P-CREB (red), YOYO nuclear stain (green), and overlap of P-CREB and YOYO (white). Bar=100 µm. B, Histograms of nuclear P-CREB immunofluorescence intensities normalized to untreated control (±SEM, n=3). *P<0.05, ***P<0.001 compared with the TG-induced response; #P<0.05, ##P<0.01 compared with the 60 K+-induced response. C, TG-induced P-CREB is sensitive to 2-APB and Ni2+. Arteries were treated with TG after preincubation with 100 nmol/L Nim, and where indicated, 100 µmol/L 2-APB or 500 µmol/L Ni2+ was included for 15 minutes (±SEM, n=3). ***P<0.001.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gene expression profile of arterial smooth muscle cells is a critical determinant of the differentiated versus proliferative phenotype. CREB is implicated in both promoting VSMC proliferation and conversely in the protection of arteries from smooth muscle cell dedifferentiation. P-CREB levels and c-fos transcription are increased in smooth muscle cells of hypertensive arteries, and inhibition of CREB activity through expression of dominant negative CREB prevents apoptosis and augments mitogenesis of VSMCs.^8,31 However, CREB content of vascular tissues inversely correlates with VSMC proliferation and migration.^32–34 In light of its regulation by multiple pathways, CREB likely has pleiotropic effects on smooth muscle cell functions that may explain its regulation of opposing events, depending on the signal source and duration.

The underlying Ca²⁺-dependent signaling mechanisms involved in CREB activation and VSMC gene transcription are not completely understood. In this study, we used pharmacological tools and measurements of intracellular Ca²⁺ to establish a role for SOCE in the activation of CREB in VSMCs. We report that influx of Ca²⁺, caused by thapsigargin-induced depletion of SR Ca²⁺, results in transient phosphorylation of CREB and transcription of c-fos. Ca²⁺ influx through VDCCs did not affect thapsigargin-induced CREB phosphorylation or c-fos transcription in cultured VSMCs derived from vascular explants, but did contribute to P-CREB formation in intact arteries. The effect of SOCE on CREB activation suggests that SR Ca²⁺ store homeostasis is important in regulating gene expression in vivo and supports the hypothesis that store-operated Ca²⁺ influx pathways are involved in CREB-mediated transcriptional events in both physiological arterial signaling and in pathological growth changes associated with the development of hypertension and atherosclerosis.^8,35

Although Ca²⁺ is a ubiquitous signaling ion affecting many aspects of VSMC physiology, the relative contribution of different modes of Ca²⁺ entry or intracellular Ca²⁺ release in the induction of gene transcription is uncertain. Coupling of Ca²⁺ influx and intracellular Ca²⁺ mobilization pathways to CREB activation has been observed in neurons.³⁶ Our work suggests that similar mechanisms are present in VSMCs. Results in intact arteries indicate that influx of Ca²⁺ through either VDCCs or store-operated Ca²⁺ channels can contribute to regulation of CREB, and suggest that P-CREB formation occurs after global increases in Ca²⁺. The simplest explanation for the discrepancy between VSMCs from aortic explants and intact arterial myocytes is the reduction in L-type VDCC expression in the cultured cells and indirect effects of thapsigargin on membrane potential.^3,5 The VDCC-independent component of CREB phosphorylation was sensitive to inhibition of SOCE, supporting the hypothesis that SR Ca²⁺ and SOCE regulate Ca²⁺-dependent gene expression in intact arterial myocytes. Consistent with SOCE playing a role in the change between the differentiated and proliferative VSMC phenotypes, previous studies have demonstrated upregulation of store-operated channels in vascular smooth muscle during proliferation³⁷ and growth arrest of smooth muscle cells after loss of SERCA expression.³⁸

The kinases activated downstream of SOCE were not identified in this study. In neurons, CaM kinases have been implicated in the immediate phase of Ca²⁺-activated CREB phosphorylation, whereas the Ras/MAP kinase pathway has been linked to sustained CREB phosphorylation.³⁹ CaM kinase activity has also been shown to play an important role in CREB phosphorylation after membrane depolarization in vascular smooth muscle.⁸ The transient nature of CREB phosphorylation after SERCA inhibition that we observed in the present study suggests that SOCE activates the immediate pathway involving CaM kinases.

CREB phosphorylation has been established as an important molecular switch to control gene transcription driven by CREs. In this study, we have identified changes in c-fos transcription that correlate with SOCE-induced CREB phosphorylation. It is likely that the interplay between SR Ca²⁺ homeostasis and SOCE contributes to transcriptional regulation of multiple genes through CREB phosphorylation and interactions with other proteins in transcriptional complexes.^40–42 Moreover, different spatial and temporal patterns of Ca²⁺ gradients in VSMCs may add another level of transcriptional regulation.

In summary, we have established that SOCE stimulates phosphorylation of CREB, an essential step in the activation of this transcription factor. Future studies that determine the relative contributions of Ca²⁺ signals arising from multiple sources to the diverse patterns of CRE-mediated gene expression will contribute greater understanding of Ca²⁺ regulation of VSMC phenotype and development of vascular pathologies.

	Acknowledgments

 
This work was supported by grants from the NIH (R01HL67351) and the Totman Center for Cerebrovascular Research. P.R.-C. was supported by a minority supplement to HL67351. The authors would like to thank Dr Roger Y. Tsien for generously providing YC2.1 and Dr Deborah Damon for supplying arteries for this study. The authors also acknowledge the Vermont Cancer Center Imaging and DNA Analysis Facilities for their assistance in cell imaging and quantitative RT-PCR.

	Footnotes

 
Original received December 29, 2003; resubmission received February 23, 2004; revised resubmission received March 26, 2004; accepted March 29, 2004.

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