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Am J Physiol Heart Circ Physiol 291: H97-H105, 2006. First published February 3, 2006; doi:10.1152/ajpheart.00753.2005
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Ca2+ source-dependent transcription of CRE-containing genes in vascular smooth muscle

Renee A. Pulver-Kaste,1,2 Christy A. Barlow,1 Jeffery Bond,3 Anjanette Watson,4 Paul L. Penar,1,2,5 Bruce Tranmer,2,5 and Karen M. Lounsbury1,2

1Department of Pharmacology, 2Totman Center for Cerebrovascular Research, 3Department of Microbiology and Molecular Genetics, 4Department of Biology, and 5Department of Surgery, Division of Neurological Surgery, University of Vermont, Burlington, Vermont

Submitted 15 July 2005 ; accepted in final form 1 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Altered Ca2+ handling has immediate physiological and long-term genomic effects on vascular smooth muscle function. Previously we showed that Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs) or store-operated Ca2+ channels (SOCCs) results in phosphorylation of the Ca2+/cAMP response element (CRE)-binding protein in cerebral arteries. Here, oligonucleotide array analysis was used to determine gene transcription profiles resulting from these two Ca2+ entry pathways in human cerebrovascular smooth muscle cell cultures. Results were confirmed and expanded using quantitative RT-PCR, Western blot, and immunofluorescence. A distinct, yet overlapping, set of CRE-regulated genes was induced by VDCC activation using K+ membrane depolarization vs. SOCC activation by thapsigargin (TG). Membrane depolarization selectively induced a sustained increase in early growth response-1 (Egr-1) mRNA and protein, which were inhibited by the VDCC blocker nimodipine and the SOCC inhibitor 2-aminoethoxydiphenylborate (2-APB). TG selectively induced a sustained increase in MAPK phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB, but not by nimodipine. The physiological agonist ANG II also stimulated expression of Egr-1 and MKP-1. Coadministration of 2-APB prevented expression of Egr-1 and MKP-1, whereas nimodipine blocked only Egr-1 expression. TG and ANG II induced phosphorylation of ERK, which was sensitive to 2-APB and was selectively required for CRE-binding protein phosphorylation. Our findings thus indicate that Ca2+ entry through VDCCs and store-operated Ca2+ entry can differentially regulate CRE-containing genes in vascular smooth muscle and also imply that agonist-induced signals involved in modulation of gene transcription can be controlled by multiple sources of Ca2+.

microarray; calcium signaling; arterial remodeling; vascular smooth muscle cell proliferation

VASCULAR SMOOTH MUSCLE CELLS (VSMCs) transition between differentiated and proliferative phenotypes in response to environmental cues, enabling a balance that preserves a healthy, contractile response (34). Although essential for vasculogenesis, unchecked VSMC proliferation and migration lead to the development of vascular pathologies, such as atherosclerosis, hypertension, postangioplasty restenosis, and tumor angiogenesis (10). Disease-related changes in VSMC phenotype correlate with atypical Ca2+ signaling, elevated intracellular Ca2+, and altered gene transcription (28, 37). Although the connection between specific Ca2+ signals and transcriptional control of gene expression in VSMCs is probably important, it has not been well characterized.

Multiple sources of Ca2+ may participate in regulation of gene expression in VSMCs. Elevation of Ca2+ in smooth muscle can result from entry of extracellular Ca2+ as well as release of Ca2+ from intracellular stores (24, 33). Ca2+ entry is mediated by voltage-dependent Ca2+ channels (VDCCs) and voltage-independent cation channels, including store-operated Ca2+ channels (SOCCs). Store-operated Ca2+ entry (SOCE), also known as capacitative Ca2+ entry, has been detected in VSMCs and is thought to play an essential role in the regulation of contraction, cell proliferation, and apoptosis (41, 42).

Ca2+ entry into VSMC serves as a trigger for transcription through Ca2+-regulated signal transduction. However, not all routes of Ca2+ entry are equivalent in their ability to induce specific transcriptional events (1). Different signaling cascades can also be initiated by increases in intracellular Ca2+ concentrations, including cAMP-dependent protein kinase, MAPKs, and calmodulin-dependent protein kinase (35).

Ca2+-induced gene expression can be mediated by Ca2+-dependent phosphorylation of the transcription factor Ca2+/cAMP response element (CRE)-binding protein (CREB). CREB activation requires phosphorylation at Ser133 to facilitate formation of an active transcriptional complex, including recruitment of CREB-binding protein (CBP300) and other cofactors to the CRE found as a functional promoter in >100 characterized genes and with conserved sites in >4,000 genes (23, 35, 50). Modulation of c-fos and other immediate early genes is, in part, Ca2+ dependent and requires CREB (8, 21), signifying the impact of altered Ca2+ handling on expression of genes that contain a CRE.

We previously showed that Ca2+ entry through VDCCs and SOCCs increases levels of phosphorylated CREB (p-CREB) in cultured VSMCs and intact cerebral arteries (30, 37), yet the differential effects of these two Ca2+ signaling pathways on gene transcription remains unknown. In the present study, we explored the role of SOCE and Ca2+ entry through VDCCs in the regulation of gene expression in cultured human cerebral VSMCs. The results revealed that Ca2+ signals trigger different patterns of gene expression depending on the source of Ca2+. These findings also indicate that SOCE and Ca2+ entry through L-type Ca2+ channels predominantly regulate genes containing a CRE, suggesting an important role for CREB-dependent gene regulation by these Ca2+ signals in VSMCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture, reagents, and solutions. This project was approved by the University of Vermont’s Institutional Review Board (CHRMS 01-195), and informed consent was obtained from all subjects. Human cerebral VSMCs (hcVSMCs) and rat cerebral VSMCs at passages 2–4 were obtained from cerebral artery explants and maintained in smooth muscle growth medium 2 (Clontech, Palo Alto, CA) as previously described (46). Cells were grown to ~60% confluence and serum starved in DMEM containing 0.1% FBS 24–48 h before treatment.

Thapsigargin (TG, 100 nM), nimodipine (100 nM pretreatment for 15 min), PD-9805 (50 µM pretreatment for 3 h), and ANG II were purchased from Calbiochem (San Diego, CA); 2-aminoethoxydiphenylborate (2-APB, 100 µM pretreatment for 10 min) from Tocris Cookson (Ellisville, MO); and cell culture reagents from GIBCO (Grand Island, NY). All other chemicals were obtained from Sigma (St. Louis, MO).

The composition of HEPES-buffered saline (HBS) was (in mM) 10 HEPES, pH 7.4, 6 KCl, 140 NaCl, 2 CaCl2, 1 MgCl2, and 10 glucose. The composition of 100 nM Ca2+ HBS was (in mM) 10 HEPES, pH 7.4, 6 KCl, 140 NaCl, 0.1 CaCl2 (effectively 100 nM due to EGTA), 1 MgCl2, 10 glucose, and 1.1 EGTA. For 120 mM K+ HBS, NaCl was replaced isotonically by KCl (120 mM KCl and 26 mM NaCl final concentrations). For 120 mM K+ HBS with 100 nM Ca2+, 100 nM Ca2+ was added to HBS and NaCl was isotonically replaced by KCl (120 mM KCl and 26 mM NaCl final concentrations, with effectively 100 nM Ca2+ due to EGTA).

RNA isolation. Total RNA was extracted from treated hcVSMCs using TRIzol reagent and chloroform. RNA was precipitated using isopropanol, washed with 75% ethanol, dissolved in RNase-free water, and quantified using the NanoDrop spectrophotometer (36).

Microarray gene expression analysis. Oligonucleotide microarray analysis of RNA expression levels was performed using the Affymetrix GeneChip Human Genome U133 set (HG-U133A) according to manufacturer's recommendations. For each of three independent experiments, hcVSMCs were exposed to no treatment, TG, or 120 mM K+ for 30 min. Total RNA was then isolated using TRIzol reagent and quantified as described above. Double-stranded cDNA was then synthesized (Omniscript RT kit, Qiagen, Valencia, CA) and subjected to an in vitro transcription reaction and a fragmentation reaction to produce biotin-labeled cRNA fragments that were hybridized to the probe arrays at 45°C for 16 h. The probe arrays were washed, bound biotin-labeled cRNA fragments were detected with a streptavidin-phycoerythrin conjugate, and the signal was amplified by staining with a biotinylated anti-streptavidin antibody. The probe arrays were scanned (Hewlett-Packard GeneArray Scanner, Agilent Technologies) to quantify the fluorescence intensity associated with each oligonucleotide probe.

Probe-level intensities were calculated from scanned images using GCOS software (Affymetrix, Santa Clara, CA). Probe-level expression data were normalized using the Qspline method (47), a cubic spline/quantile normalization. The expression index was calculated using the robust multichip average (RMA) method (5, 18). For each gene and for each of the two treatments, we estimated the average change in the RMA expression index, the probability (not corrected for multiple comparisons) from the one-sample t-test on the three evaluations, and the average expression index. Data were placed in the MIAME GEO database (accession GEO series record GSE2883). CRE site-containing genes were identified through their assignment in the "CRE_TATA" class by use of a searchable database created by Zhang et al. (http://natural.salk.edu/CREB) (50).

Quantitative RT-PCR. For validation, RNA (2 µg) was isolated as described above and reverse transcribed using an Omniscript RT kit (Qiagen). Gene expression product kits (Assays-on-Demand, Applied Biosystems, Foster City, CA) were used to detect c-fos, MAPK phosphatase-1 (mkp-1), and early growth response-1 (egr-1). PCR products were detected by TaqMan real-time RT-PCR, as previously described (30), using the hypoxanthine guanine phosphoribosyltransferase gene hprt as the internal standard. Standard curves were produced to validate comparison of the threshold cycle between the gene of interest and hprt for data analysis. Each determination was recorded from duplicate samples in at least two independent experiments.

Western blots. After treatment, hcVSMCs were washed with PBS (in mM: 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.6 CaCl2, 1.2 MgCl2, 0.023 EDTA, 11 glucose, and 24 NaHCO3, pH 7.4), collected in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF), and centrifuged at 10,000 g for 15 min at 4°C. Protein concentrations were determined by Bradford protein assay (Bio-Rad, Hercules, CA), and 20 µg from each sample were separated by 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membranes and analyzed by Western blot using rabbit anti-MKP-1 (or anti-Egr-1) antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:1,000 dilution), as previously described (30). Band density was quantified using Quantity One (Bio-Rad) and normalized to a nonspecific band not altered by treatments. All experiments were performed in triplicate.

Immunofluorescence. Cells were fixed with paraformaldehyde, and immunofluorescence was performed as described previously (30). Primary antibodies included rabbit anti-MKP-1, rabbit anti-Egr-1 (Santa Cruz Biotechnology; 1:400 dilution), rabbit anti-p-CREB, and mouse anti-p-ERK (Cell Signaling, Danvers, MA; 1:250 dilution). Secondary antibodies were Alexa Fluor 568 goat anti-rabbit IgG for MKP-1 and Egr-1, Cy3 goat anti-rabbit IgG for p-CREB, and Cy5 goat anti-mouse IgG for p-ERK (Molecular Probes, Eugene, OR; 1:500 dilution). All incubations were performed in PBS containing 2% BSA and 0.1% Triton X-100. Images were captured using a laser scanning confocal microscope (model 1000, Bio-Rad) with a x40 objective. Nuclear fluorescence intensity of ≥30 cells was quantified from ≥3 independent experiments (37).

Data analysis. With the exception of the microarray analysis described above, a Student-Newman-Keuls test for multiple comparisons was used to determine statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ca2+ elevation via depolarization and sarco/endoplasmic reticulum Ca2+-ATPase inhibition differentially regulates gene expression in cultured hcVSMCs. To explore patterns of gene transcription elicited by different Ca2+ signals, VDCCs were activated by K+ depolarization and SOCCs were activated by TG, an irreversible inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Changes in gene transcription were detected by oligonucleotide array analysis using the Affymetrix GeneChip Human Genome U133A set, which contains >1 x 106 oligonucleotides to probe for 22,283 genes. A significant (≥1.5-fold) change in transcript level compared with the untreated control (n = 3) was detected for 12 genes in response to TG (Table 1) and for 32 genes in response to elevated K+ (Table 2). There was an overlap of four responsive genes, including fos, between the two treatment groups.


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  		Table 1. Genes regulated by thapsigargin according to microarray analysis in human VSMCs

 

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  		Table 2. Genes regulated by 120 mM K+ according to microarray analysis in human VSMCs

 
Because we previously showed that Ca2+ entry through L-type VDCCs and SOCCs increases phosphorylation and activation of the transcription factor CREB (8, 30, 37), focus was placed on genes that contain a CRE. The work of Zhang et al. (50) allowed us to determine those genes that contain a CRE consensus sequence in their promoter region. Membrane depolarization and SERCA inhibition induced significant upregulation, over untreated control, of 8 and 14 CRE-containing genes, respectively (Tables 1 and 2). These results suggest that although many genes are regulated by Ca2+ independently of its source, an important subset of CRE-containing genes involved in cell growth are differentially regulated by VDCCs compared with SOCCs.

Fisher exact test was used to examine the prospective hypothesis that genes regulated by TG or K+ depolarization have a CRE consensus sequence. The resulting volcano plots show that CRE site-containing genes were highly overrepresented among all upregulated genes after TG (Fig. 1A; P < 3 x 10–8) or K+ depolarization (Fig. 1B; P < 4 x 10–7), strongly supporting an association between the presence of a CRE and the probability of increased expression. Differentially regulated CRE-containing genes were distributed among three of the four quartiles of expression index (Fig. 1, C and D), suggesting that the expression level did not predict the probability of response.


Figure 1
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  		Fig. 1. Ca2+/cAMP response element (CRE) consensus site-containing genes are preferentially represented among upregulated genes in vascular smooth muscle cells (VSMCs) treated with thapsigargin (TG) and K+. A and B: volcano plots representing correlation between robust multichip average expression index [Response: TG (A) and K+ (B)] and P value (–log10 based on Student's t-test) for each of the 22,283 gene probe sets. mkp-1, MAPK phosphatase-1 gene. C and D: graphical representations [modified plot of log intensity ratio vs. mean log intensity (MvA plot)] of response [TG (C) and K+ (D)] vs. average expression index for each gene probe set. {circ}, CRE-containing genes; bullet, non-CRE-containing genes; vertical dashed lines, 1.5-fold thresholds; horizontal dashed lines, expression index quartiles.

 
To validate and expand on the changes in gene transcription observed using array analysis, the effect of TG and elevated K+ on transcription of the CRE-containing genes was measured using quantitative RT-PCR. According to microarray analysis, c-fos transcript levels were upregulated 3.175-fold by elevated K+ (Table 1) and 1.520-fold by TG treatment. Quantitative RT-PCR experiments confirmed the K+ and TG results, but a time course revealed a more sustained induction of c-fos by TG, demonstrating that regulation of c-fos expression by different Ca2+ sources varies in intensity and duration (Fig. 2A).


Figure 2
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  		Fig. 2. TG and K+ depolarization differentially regulate c-fos transcription in human cerebral VSMCs (hcVSMCs). A: hcVSMCs were incubated with TG or in HEPES-buffered saline (HBS) with isotonic elevation of K+ to 120 mM (K+) over 10 min to 2 h. Addition of TG and K+ was staggered, such that all cells were simultaneously fixed at 2 h. Levels of c-fos transcription were measured using quantitative RT-PCR. Data were normalized to TG or K+ response at 30 min. Values are means ± SE (n = 3). *P < 0.05 vs. K+ at 120 min. B: hcVSMCs were preincubated with nimodipine (Nim) or 2-aminoethoxydiphenylborate (2-APB) and then treated with TG or K+ for 30 min. Levels of c-fos transcription were determined by quantitative RT-PCR. Data were normalized to TG or K+ alone for each of the data series. Values are means ± SE (n = 2). Significantly different from K+ alone: ***P < 0.001; significantly different from TG alone: ###P < 0.001.

 
Ca2+ channel blockers were used to explore a role for different sources of Ca2+ influx in the induction of c-fos transcription. Nimodipine, an L-type VDCC blocker, significantly reduced induction of c-fos transcription by high K+ but had no effect on the TG response (Fig. 2B). Blockage of SOCE by 2-APB (6) significantly reduced TG- and K+-mediated c-fos transcription, closely paralleling the data profile for CREB phosphorylation (30). 2-APB did not reduce the TG-induced response at 2 h (not shown), suggesting that, after extended periods of time, c-fos may be activated through Ca2+-independent pathways. These data indicate that TG-induced c-fos transcription requires Ca2+ signaling through SOCCs, whereas depolarization-mediated c-fos transcription additionally requires functional L-type Ca2+ channels.

SOCE leads to MKP-1 expression independent of L-type Ca2+ channel activity. According to microarray analysis, mkp-1 transcript levels were selectively upregulated by TG (Table 2). Quantitative RT-PCR confirmed that transcription of mkp-1 was significantly and selectively induced by TG at 30 min (Fig. 3A). Similar to the TG-mediated c-fos response, this effect was insensitive to nimodipine and partially sensitive to 2-APB. Reduction of extracellular Ca2+ (100 nM) had a partial inhibitory effect at 30 min but dramatically blocked TG-induced mkp-1 transcription at 2 h (Fig. 3B).


Figure 3
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  		Fig. 3. TG promotes transcription of mkp-1, which is prevented by 2-APB in hcVSMCs. Levels of mkp-1 transcription were determined by quantitative RT-PCR. A: cells were incubated with TG or in HBS with isotonic elevation of K+ to 120 mM (K+) over 10 min to 2 h. Addition of TG and K+ was staggered, such that all cells were simultaneously fixed at 2 h. Data were normalized to TG or K+ at 0 min. Values are means ± SE (n = 3). *P < 0.05 vs. K+ at 30 min. B: hcVSMCs were incubated in HBS with normal (2 mM) Ca2+ or 100 nM Ca2+. Cells were preincubated with Nim or 2-APB and then treated with TG for 30 min or 2 h. Data were normalized to untreated control. Values are means ± SE (n = 2). ***P < 0.001 vs. TG alone. Significantly different from untreated control: #P < 0.05; ##P < 0.01.

 
To establish that the observed trends in mkp-1 transcription correlated with ultimate changes in its protein expression, MKP-1 levels were measured by Western blot and immunofluorescence using anti-MKP-1 antibodies. MKP-1 protein expression was significantly induced by TG, with peak expression at 1 h (Fig. 4). The induction of expression was not sensitive to nimodipine but was significantly blocked by 2-APB, which parallels the trend of the mkp-1 transcription data. Together these findings indicate that TG-mediated reduction of intracellular store Ca2+ induces transcription of mkp-1 and MKP-1 protein expression through a pathway that requires SOCE.


Figure 4
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  		Fig. 4. TG-induced mkp-1 expression is blocked by 2-APB in hcVSMCs. A: determination of MKP-1 protein expression levels using Western blot analysis. Cells were incubated with TG over 30 min to 2 h. Addition of TG and K+ was staggered, such that all cells were simultaneously fixed at 2 h. Data were normalized to TG at 0 min. Values are means ± SE (n = 3). Significantly different from 0 min TG: *P < 0.05; **P < 0.01. Inset: representative Western blot of resulting protein products. B: detection of nuclear MKP-1 by immunofluorescence. hcVSMCs were incubated in HBS with normal (2 mM) Ca2+ or 100 nM Ca2+ and pretreated with Nim or 2-APB and then exposed to TG for 1 h. Data were normalized to untreated control MKP-1 intensity. Values are means ± SE (n = 3). ***P < 0.001 vs. TG alone. ###P < 0.001 vs. untreated control. Inset: representative images of MKP-1 immunofluorescence in nuclei of hcVSMCs.

 
Elevated K+ selectively triggers transcription of egr-1 mRNA and expression of Egr-1 protein. According to microarray analysis, egr-1 transcript levels were upregulated 1.800-fold by K+ depolarization (Table 2). Quantitative RT-PCR confirmed that transcription of egr-1 was significantly induced by elevated K+ and unaffected by TG treatment (Fig. 5). The induction was partially inhibited by nimodipine (P = 0.053) and effectively blocked by 2-APB, suggesting a role for L-type VDCCs and SOCE. Reducing extracellular Ca2+ (100 nM) unexpectedly increased egr-1 transcription, suggesting that its transcription is also stimulated by disruption of the extracellular Ca2+ pool.


Figure 5
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  		Fig. 5. K+ promotes transcription of early growth response-1 gene (egr-1), which is decreased by Nim and inhibited by 2-APB in hcVSMCs. Levels of egr-1 transcription were determined using quantitative RT-PCR from ≥2 independent experiments in duplicate after extraction of RNA. A: hcVSMCs were incubated in HBS with isotonic elevation of K+ to 120 mM or with TG over 10 min to 2 h. Addition of TG and K+ was staggered, such that all cells were simultaneously fixed at 2 h. Data were normalized to K+ or TG at 0 min. Values are means ± SE (n = 3). *P < 0.05 vs. TG at 120 min. B: hcVSMCs were incubated in HBS with normal (2 mM) Ca2+ or 100 nM Ca2+ and preincubated with Nim or 2-APB and then treated with HBS containing 120 mM K+ for 30 min. Data were normalized to untreated control. Values are means ± SE (n = 2). Significantly different from 120 K+ alone: *P < 0.05; ***P < 0.001. ###P < 0.001 vs. untreated control.

 
To confirm that the observed trends of egr-1 transcription correlated with changes in its protein expression, we measured Egr-1 levels by Western blot and immunofluorescence using anti-Egr-1 antibodies. Similar to the egr-1 transcription results, Egr-1 protein expression was significantly induced by K+ depolarization, peaking at 1 h, and this effect was completely blocked by nimodipine and 2-APB (Fig. 6). In contrast to the transcription data, reducing extracellular Ca2+ blocked Egr-1 expression, suggesting that the egr-1 mRNA induced by loss of extracellular Ca2+ is not translated into protein. These data provide evidence that depolarization of hcVSMC membranes by elevation of K+ leads to Egr-1 expression through a mechanism that requires functional L-type Ca2+ channels and SOCE.


Figure 6
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  		Fig. 6. Depolarization-induced Egr-1 expression requires Ca2+ influx and is reduced by 2-APB and Nim in hcVSMCs. A: determination of Egr-1 protein expression levels by Western blot analysis. Cells were incubated in HBS containing 120 mM K+ over 30 min to 2 h with addition of K+, such that all cells were simultaneously fixed at 2 h. Data were normalized to K+ at 0 min. Values are means ± SE (n = 3). **P < 0.01 vs. 0 min K+. Inset: representative Western blot of resulting protein products. B: detection of Egr-1 by immunofluorescence. hcVSMCs were incubated in HBS with normal (2 mM) Ca2+ or 100 nM Ca2+ and preincubated with nimodipine or 2-APB and then treated with HBS containing 120 mM K+ for 1 h. Data were normalized to untreated control. Values are means ± SE (n = 3). ***P < 0.001 vs. 120 mM K+. ###P < 0.001 vs. untreated control. Inset: representative images of Egr-1 immunofluorescence in nuclei of hcVSMCs.

 
ANG II promotes an increase in expression of Ca2+- and CRE-regulated genes. ANG II, a physiological inducer of sarcoplasmic reticulum-mediated and voltage-gated Ca2+ signaling, significantly induced transcription of c-fos, mkp-1, and egr-1 (Fig. 7A), in agreement with previous findings (4, 22, 32). Immunofluorescence was used to verify that the ANG II-induced transcriptional responses could be correlated to changes in protein levels and to expand on the roles of SOCE and Ca2+ entry through L-type Ca2+ channels (Fig. 7, B and C). Protein expression of MKP-1 and Egr-1 was stimulated by ANG II, and this effect was significantly reduced by reducing extracellular Ca2+ (100 nM). 2-APB reduced the expression of MKP-1 (P = 0.072) and Egr-1 (P < 0.01), whereas nimodipine significantly reduced only Egr-1 expression. These ANG II data serve to strengthen the biological relevance by identifying a physiological trigger for expression of CRE-containing genes through two independent Ca2+ signaling pathways.


Figure 7
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  		Fig. 7. ANG II promotes an increase in expression of genes linked to proliferation in hcVSMCs. A: quantitative RT-PCR determination of egr-1, c-fos, and mkp-1 transcript levels. Cells were incubated with 100 nM ANG II for 30 min. Data were normalized to untreated control. Values are means ± SE. Significantly different from untreated control: *P < 0.05; **P < 0.01. B and C: hcVSMCs were incubated in HBS with normal (2 mM) Ca2+ or 100 nM Ca2+ and preincubated with Nim or 2-APB and then treated with ANG II for 2 h. B: representative confocal images of MKP-1 and Egr-1 immunofluorescence in nuclei of hcVSMCs. C: nuclear intensity of MKP-1 and Egr-1. Data were normalized to untreated control. Values are means ± SE (n = 3). Significantly different from ANG II: *P < 0.05; **P < 0.01. Significantly different from untreated control: #P < 0.05; ###P < 0.001.

 
TG and ANG II, but not K+, stimulate CREB phosphorylation in a SOCE- and ERK-dependent manner. To determine a role for the ERK MAPK as a specific intermediary between signaling from SOCE to CREB, cells were treated with TG or elevated K+ in the presence of the MEK inhibitor PD-9805. Inhibition of ERK signaling blocked only TG-mediated CREB phosphorylation, suggesting a specific role for ERK in SOCE-mediated signaling (Fig. 8A). Furthermore, ERK phosphorylation was increased by TG in a time course that paralleled CREB phosphorylation and was sensitive to SOCE inhibition by 2-APB (Fig. 8, B and C). Unexpectedly, 2-APB alone induced a small, but significant, increase in the level of p-ERK. ANG II also transiently stimulated ERK and CREB phosphorylation, which was inhibited by 2-APB, supporting a dominant role of SOCE in the resulting ERK and CREB phosphorylation (Fig. 9). Together these results indicate that ERK plays an important role in SOCE-mediated signaling to CREB and that SOCE has a role in ANG II signaling to ERK and CREB.


Figure 8
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  		Fig. 8. Store-operated Ca2+ entry is required for TG-induced CRE-binding protein (CREB) phosphorylation through ERK in rat cerebral vascular smooth muscle cells (rcVSMCs). A: immunofluorescence detection of phosphorylated CREB (p-CREB) in rcVSMCs stimulated with 120 mM K+ (K+) or 100 nM TG for 10 min. Cells were preincubated with 0.1% DMSO (vehicle) or 50 µM PD-9805. B: immunofluorescence detection of p-CREB (blue) and phosphorylated ERK (p-ERK, red) in rcVSMCs stimulated with TG as described in A in the presence or absence of 2-APB. Nuclei (green) were identified using YOYO-1 nuclear stain (30). C: quantification of nuclear p-ERK fluorescence in B. Values are means ± SE (n = 3). *P < 0.05 vs. control. #P < 0.05 vs. TG.

 

Figure 9
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  		Fig. 9. ANG II stimulates ERK and CREB phosphorylation in a store-operated Ca2+ channels-dependent manner in rcVSMCs. A: immunofluorescence detection of p-CREB (blue) and p-ERK (red) in rcVSMCs treated with 100 nM ANG II for 5 min after pretreatment with 0.1% DMSO (vehicle) or 100 µM 2-APB. Nuclei (green) were identified using YOYO-1 nuclear stain (see Fig. 8). B: quantification of p-ERK and p-CREB nuclear fluorescence in A. C: p-ERK nuclear fluorescence at different times of ANG II treatment. Values are means ± SE (n = 3). Significantly different from control (Con): *P < 0.05; **P < 0.01. Significantly different from ANG II: #P < 0.05; ##P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Data presented here reveal that Ca2+ signals lead to distinct patterns of gene expression in VSMCs depending on the source of Ca2+. These findings also show that Ca2+ entry predominantly regulates CRE-containing genes, suggesting an important role for CREB in Ca2+-dependent gene regulation. The Ca2+ hypothesis proposes that diverse Ca2+ signals serve to differentially control gene expression profiles (3). Variations in gene expression may be caused by the entry of Ca2+ through different channels with physically associated signaling molecules that translocate to the nucleus, ultimately inducing CREB activation. Alternatively, the capacity of a Ca2+ channel to increase the concentration of nuclear Ca2+ leading to nuclear kinase-induced CREB activation has been implicated in determining transcriptional differences (16). Furthermore, the duration of Ca2+-mediated CREB activation leads to variations in transcription that are associated with distinct pathways, such as involvement of calmodulin-dependent protein kinases in transient CREB activation and involvement of Ras/MAPK pathways in sustained CREB activation (48).

A role for CREB in the regulation of VSMC proliferation is apparent but controversial. Cerebral arteries from hypertensive animals exhibit tonic membrane depolarization, elevated arterial wall Ca2+, increased CREB phosphorylation, and increased c-fos expression (37, 38). Additionally, SOCCs are upregulated during vascular proliferation (15). Uncontrolled VSMC proliferation positively correlates with increased transcription of immediate early genes and inhibition of apoptosis, both of which are linked to upregulation of MAPK signaling through ERK and activation of CREB (12, 25, 39). Conversely, the presence of CREB has been shown to be inversely proportional to proliferation and migration, implicating a role for CREB in the protection of VSMCs from dedifferentiation (19, 31, 44). These seemingly contradictory functions of CREB may serve to provide a balance between proliferation and apoptosis that becomes altered in the development of vascular pathologies.

The mechanisms by which Ca2+-dependent signaling pathways differentially regulate VSMC gene transcription are not entirely understood, but recent evidence suggests mechanisms that involve activation of multiple transcription factors and regulation of specific cofactors such as myocardin (2, 43). In this study, we used microarray analysis and pharmacological tools to establish a differential role for SOCE vs. Ca2+ entry through VDCCs in the gene expression profile of VSMCs. We report that stimulation of different Ca2+ influx pathways creates divergent patterns of gene transcription overall, as well as among genes that contain a CRE. The differential regulation of genes that contain a CRE by SOCE supports the hypothesis that sarcoplasmic reticulum Ca2+ homeostasis influences transcription in arteries under normal physiological conditions and suggests that deregulation of Ca2+ signals may trigger the pathological transition of VSMCs from the differentiated to the proliferative phenotype observed in hypertension and atherosclerosis (30, 45).

In results presented here, Ca2+ influx caused by depletion of sarcoplasmic reticulum Ca2+ induced transcription and protein expression of MKP-1, which was not affected by Ca2+ entry through VDCCs, suggesting that SOCE may play a role in limiting MAPK activity and VSMC proliferation. A role for MAPK signaling in vascular pathologies is supported by other analyses of VSMC transcription patterns. Using microarray analysis of VSMCs stimulated with ANG II, Campos et al. (7) established a role for MAPK pathways in the expression of matricellular proteins involved in vascular lesion formation. Increased MAPK activation has also been linked to stroke risk by a study that compared gene expression patterns of stroke-prone spontaneously hypertensive rats (SHRs) with that of stroke-resistant SHRs (13). Inhibition of the ERK MAPK pathway has also been shown to normalize ANG II-mediated signaling and attenuate contraction in SHRs (40).

MKP-1 is induced by and inactivates the three primary MAPK pathways (ERK, p38, and Jun kinase) and thus functions as an important negative regulator of the mitogenic response in VSMCs (4). Inhibition of MKP-1 has been shown to prolong ANG II-induced MAPK signaling (11). Overexpression of MKP-1 also results in growth arrest of VSMCs in the G1 phase, suggesting that reduced MKP-1 expression may contribute to proliferation after vascular injury (20). Our results demonstrate induction of MKP-1 by a mechanism consistent with SOCE and, thus, indicate that Ca2+ signaling through SOCE may play an important role in VSMC proliferation by indirectly modulating MAPK signaling.

In contrast to MKP-1, Egr-1 was induced selectively by Ca2+ entry through VDCCs. Egr-1 is an early growth response transcription factor that shares induction kinetics similar to those of c-Fos and is overexpressed in endothelial cells after vascular injury (29). Previous studies have demonstrated that sustained ERK activation phosphorylates and stabilizes c-Fos and is necessary for Egr-1 expression and nuclear translocation (27, 49). Egr-1 expression and subsequent translocation are stimulated by mechanical strain in VSMCs, whereas c-Fos is not (26). Over 300 Egr-1 target genes, including upregulated growth factors and cytokines, and suppression of genes previously linked to VSMC apoptosis were identified by microarray analysis of human endothelial cells overexpressing Egr-1, suggesting the importance of Egr-1 in promoting vascular remodeling (14). Furthermore, Egr-1 and nuclear factor of activated T cells are known to act synergistically in the activation of cytokine expression (9). Thus it is likely that the known activation of nuclear factor of activated T cells by these Ca2+ signals in VSMCs will be further enhanced by transcription of the CRE-dependent genes identified (17).

Additional findings presented here expand the understanding of the underlying mechanism by identifying SOCE as an important mediator of ERK and CREB phosphorylation in response to ANG II and by implicating SOCE in the repression of basal ERK phosphorylation. These findings are also consistent with MKP-1 induction through SOCE limiting the time course of ERK activation after agonist stimulation.

Together these data support the overall hypothesis that Ca2+-dependent signaling pathways regulate cerebral VSMC proliferation through CRE-mediated control of gene expression. In this study, we have determined the relative contribution of two separate Ca2+ signals to the patterns of CRE-mediated gene expression. It is likely that differential gene regulation allows SOCE and Ca2+ signaling through VDCCs to provide a balance that maintains Ca2+ homeostasis and normal arterial function. In addition, our findings suggest a novel role for SOCE in the differential regulation of signal transduction by various Ca2+ sources stimulated by ANG II in vascular smooth muscle. Future studies that explore the molecular mechanisms by which diverse Ca2+ signals determine transcriptional differences and activation of MAPK pathways will further the understanding of the Ca2+-mediated control of the VSMC phenotype.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-67351 and the Totman Center for Cerebrovascular Research. Array analysis was supported by the Vermont Genetics Network through National Institutes of Health National Center for Research Resources Biomedical Research Infrastructure Program Grant 1-P20-RR-16462.


   ACKNOWLEDGMENTS
 
The authors thank Radostina Petrova and Stacia Rymarchyk for experimental contributions and Scott Tighe and Timothy Hunter (Vermont Cancer Center DNA Analysis Facility) for expert assistance in performing array analysis and quantitative RT-PCR.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Lounsbury, Dept. of Pharmacology, Univ. of Vermont, 89 Beaumont Ave., Burlington, VT 05405 (e-mail: Karen.Lounsbury{at}uvm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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P. Rose, J. Bond, S. Tighe, M. J. Toth, T. L. Wellman, E. M. B. de Montiano, M. M. Lewinter, and K. M. Lounsbury
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