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(Circulation Research. 2004;95:406.)
© 2004 American Heart Association, Inc.

Cellular Biology

L-type Voltage-Gated Ca²⁺ Channels Modulate Expression of Smooth Muscle Differentiation Marker Genes via a Rho Kinase/Myocardin/SRF–Dependent Mechanism

B.R. Wamhoff, D.K. Bowles, O.G. McDonald, S. Sinha, A.P. Somlyo, A.V. Somlyo, G.K. Owens

From the Department of Molecular Physiology and Biological Physics (B.R.W., O.G.M., S.S., A.P.S., A.V.S., G.K.O.), University of Virginia, Charlottesville; and the Department of Biomedical Sciences (D.K.B.), University of Missouri, Columbia.

Correspondence to Gary K. Owens, PhD, Department of Molecular Physiology and Biological Physics, University of Virginia, PO Box 800736, Charlottesville, VA 22908-0736. E-mail gko{at}virginia.edu

	Abstract

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Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca²⁺ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca²⁺ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM -actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM -actin but had no effect on c-fos expression. Conversely, the Ca²⁺/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM -actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM -actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM -actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM -actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM -actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca²⁺ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.

Key Words: smooth muscle cells • calcium channel • Rho kinase • transcription • myocardin

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
L-type voltage-gated Ca²⁺ channels (VGCC) and intracellular Ca²⁺ ([Ca²⁺]_i) play a critical role in regulating many cellular functions in vascular smooth muscle cells (SMC), including contraction and relaxation. Although there is extensive evidence showing a link between changes in [Ca²⁺]_i levels and cell-specific gene expression in skeletal muscle, cardiac muscle, and neurons, very little is known regarding this interaction in SMCs. This may be of importance because alterations in the expression of VGCCs and [Ca²⁺]_i levels have been linked to several pathological processes that involve vascular SMCs, including hypertension,¹ coronary vasospasm,² and atherosclerosis,^3,4 as well as cardioprotective adaptations in response to chronic exercise training.^4,5 However, the signaling mechanisms linking physiological and/or pathophysiological changes in [Ca²⁺]_i regulation to changes in cell-selective gene expression in SMCs are not well-understood.

Seminal studies by Cartin et al showed that activation of VGCCs in intact cerebral arteries was coupled to phosphorylation of the cAMP-responsive element-binding protein (CREB) and subsequent increases in expression of the immediate-early gene, c-fos.⁶ This process was mediated in part by Ca²⁺/calmodulin-dependent protein kinase (CaMK). Although these studies defined a fundamental physiological pathway linking Ca²⁺ to c-fos gene expression, VGCC [Ca²⁺]_i CaMK P-CREB c-fos, very little is known about the role of intercellular Ca²⁺ in mediating changes in the expression of a subset of genes that typify the differentiated cell type. This distinction is important in that unlike markers defining the differentiated SMC phenotype, eg, SM -actin, smooth muscle myosin heavy chain (SMMHC), or SM22, c-fos is expressed in all cell types and thus is not a marker for any terminally differentiated cell. Moreover, the expression patterns of markers that typify the differentiated SMC can be highly variable because the SMC is prone to rapid phenotypic switching in response to vascular injury or pathological stimuli.⁷ Finally, it is unknown whether there is convergence of pathways that regulate smooth muscle cell contraction and expression of SMC differentiation marker genes.⁷

One such pathway that appears to have a dual role in SMC contraction and gene expression is the RhoA/Rho kinase pathway.⁸ RhoA, a Rho GTPase, and its downstream effector, Rho kinase (ROK), play a critical role in modulating SMC contraction and also influence transcription of SMC differentiation marker genes.^8,9 There is evidence demonstrating that both membrane depolarization- and receptor-induced contraction are regulated in part by Ca²⁺-dependent activation of RhoA and ROK.^10,11 For example, Sakurda et al showed that activation of VGCCs by depolarization resulted in a sustained increase in GTP-bound RhoA (active RhoA) that correlated to a sustained increase in contraction, a response that was blocked by a dihydropyridine Ca²⁺ channel antagonist or the selective ROK inhibitor Y-27632.¹¹ Studies by our laboratory demonstrated that overexpression of constitutively active RhoA stimulated transcription of multiple CArG-dependent SMC genes, including SM -actin, SMMHC, and SM22.⁹ In contrast, administration of either C3 transferase, which ADP ribosylates and irreversibly inactivates RhoA, or the ROK inhibitor, Y-27632, decreased expression of these genes. Importantly, these effects were selective in that no effects were seen on the c-fos gene promoter that contains only one CArG/serum response element, whereas SMC differentiation marker gene promoters typically contain pairs of CArGs. CArG boxes [CC(A/T)₆GG], also known as promoter serum response elements, bind the transcription factor serum response factor (SRF).

Recently, our laboratory and others have shown that myocardin, a potent coactivator of SRF, plays a key role in regulating the expression of CArG-dependent SMC marker genes in SMCs.^12–15 Moreover, we showed that the contractile agonist angiotensin-II increased SMC marker gene expression in part via myocardin.¹⁶ However, as yet, there have been no reports investigating whether physiological signals that couple excitation to contraction, ie, VGCC-mediated activation of the RhoA/ROK cascade, could also contribute to regulation of SMC differentiation marker gene expression through a similar mechanism.

The goals of this study were to determine the potential role of VGCCs in regulating SMC differentiation marker gene expression and to define the role of RhoA/ROK, myocardin, and SRF in this process. Our results provide novel evidence showing that activation of VGCCs by depolarization increased SMC differentiation marker gene expression through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements, VGCC [Ca²⁺]_i RhoA/ROK myocardin/SRF SMMHC/SM-actin.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Rat aortic SMCs were isolated and cultured (passage 11 to 15) as previously described. These cells are used routinely by our laboratory and stably express all known SMC differentiation markers identified to date.⁷

Embryoid Body Model of SMC Differentiation
Generation of embryoid bodies (EB) and SMC differentiation was based on the protocol described by Drab et al,¹⁷ with minor modifications.

Whole Cell Voltage Clamp Recordings
Whole cell currents were determined with a standard whole cell voltage clamp technique as previously described.^3,4

Transient Transfection and Luciferase Assays
Rat aortic SMCs were seeded at 1.5x10⁴ cells/cm² onto 12-well plates and transfected with plasmids using FuGENE6 (Roche) when the cells were 80% confluent. Cells were grown to confluence in DF10 and then changed to SFM for 2 days, at which time cells were treated with various drugs as described in the Results section. The total amount of DNA per well was kept constant (500 ng). Luciferase activity (Promega) was measured and normalized to cellular protein concentrations (Pierce). Each sample was examined in duplicate and it was repeated in 3 (minimum) different experiments.

Quantitative Polymerase Chain Reaction
Total RNA was prepared from cultured rat aortic SMCs using TRIzol reagent (Invitrogen) and cDNA synthesized using the iScript cDNA Synthesis Kit (BioRad). To quantify the expression of mRNA in all experiments, real-time polymerase chain reaction (PCR) analysis (iCycler, BioRad) was performed on cDNA using dual-fluorescence labeled probes (IDT). All results were normalized to 18S rRNA and expressed as a percent of control.

RhoA Translocation
Cytosolic and particulate fractions of RhoA were determined by Western blot as previously described.¹⁸ Cytosolic (CY) and membrane fractions (MB; detergent-soluble particulate fraction) were normalized to the whole cell extract fraction (WE) for a given sample by densitometry.

Quantitative Chromatin Immunoprecipitation Assay
Chromatin immunoprecipitation (ChIP) was performed as previously described with modifications allowing for a quantitative analysis of protein:DNA interactions.¹⁹ Recovered DNA was quantified by fluorescence with picogreen reagent (Molecular Probes) according to manufacturer’s recommendations. Real-time PCR was performed on 1 ng genomic DNA from ChIP experiments as described by Litt et al,²⁰ with minor modifications. Real-time PCR primers were designed to flank the 5'-CArG elements of SM--actin and SMMHC, the c-fos CArG, and the aortic carboxy-like peptidase (ACLP) promoter region containing 3 GC boxes and no CArGs. Quantification of protein:DNA interaction/enrichment was determined by the following equation:

Statistical Analysis
Data are expressed as mean±SE. Statistical significance among treatment groups was confirmed with a 1-way ANOVA when appropriate. Statistical significance between specific groups was determined by a post-hoc multiple comparison Student-Newman-Keuls test (P<0.05).

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

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cultured Rat Aortic SMCs Express Functional L-type Voltage-Gated Ca²⁺ Channels
Our initial aim was to determine whether the cultured rat aortic SMCs that are used routinely by our laboratory to study mechanisms of SMC differentiation marker expression express functional VGCCs. Rat aortic SMCs were grown to confluence in DF10 and growth-arrested for 3 days in SFM. Figure 1A and 1B represent typical current traces and current-voltage (I-V) relationships of VGCC current using 10 mmol/L Ba²⁺ as the charge carrier. The L-type VGCC activator BayK 8644 (5.0 µmol/L) augmented peak inward Ba²⁺ current (I_Ba) 2-fold and shifted the I-V relationship negatively. The L-type VGCC blocker nifedipine (1.0 µmol/L) attenuated inward current with 10 mmol/L Ba²⁺ as the external charge carrier. Taken together, the pharmacology and voltage-dependence described here indicate that L-type VGCC current is the dominant current, not T-type. Please see online data supplement.

View larger version (23K): [in this window] [in a new window]   Figure 1. Expression of L-type VGCC current in rat aortic SMCs. A, Representative VGCC current traces from confluent 3-day growth-arrested cultured rat aortic SMCs (10 mmol/L Ba2+, external) with and without 1 µmol/L nifedipine and 5 µmol/L BayK 8644. B, Representative typical whole cell current-voltage (I-V) relationships.

VGCC Activation Promotes SMC Differentiation Marker Gene Expression
To determine the effects of VGCC activation on endogenous SMC differentiation marker gene expression, cultured rat aortic SMCs were exposed to elevated levels of extracellular KCl to depolarize the cells. Membrane depolarization with 60 mmol/L KCl resulted in a nifedipine-sensitive increase in the endogenous expression of SM -actin, SMMHC, and myocardin mRNA as measured by real-time reverse-transcription (RT) PCR (Figure 2A). The effects of depolarization on the activity of a series of promoter-luciferase constructs were tested: SM -actin (–2.6/+2.8 kb), SMMHC (–4.2/+11.6 kb), SM22 (–447/+89 bp), c-fos (–356/+109 bp), and ACLP (–2502/+176 bp). Depolarization with 60 mmol/L KCl resulted in a 2-fold increase in SM -actin, SMMHC, SM22, and c-fos promoter activity that was completely blocked by 1.0 µmol/L nifedipine (Figure 2B). Moreover, the non-CArG-mediated ACLP promoter construct showed no changes in promoter activity in response to depolarization, suggesting that CArG cis-regulatory elements, which bind SRF, may play a critical role in the response to depolarization.

View larger version (38K): [in this window] [in a new window]   Figure 2. VGCC activation induces SMC differentiation marker gene expression. A, Rat aortic SMCs were treated with 60 mmol/L KCl (60K) and/or nifedipine (NIF) (1 µmol/L) for 24 hours and expression of SM -actin, SMMHC, myocardin, and 18S rRNA mRNA levels were quantified by real-time PCR. Ratios of SM -actin, SMMHC, and myocardin to 18S rRNA expression were calculated. B, Transcriptional activation of various promoter-reporter constructs in cultured SMCs by membrane depolarization. Transiently transfected SMCs were treated with 60K and/or NIF for 24 hours and assayed for luciferase activity. For A and B, data are expressed as percent of control (vehicle treated cells) ± SEM, *P<0.05.

The SM -actin promoter contains 3 CArG boxes (CArGB, CArGA, and an intronic CArG) that are required for SM -actin promoter activity in vivo and in vitro.^15,21 Mutation of single CArG elements within the –2.6/+2.8-kb SM -actin promoter resulted in a significantly reduced response to depolarization (Figure 3). Combination mutations of each of the 3 CArG elements (A+B, A+int, B+int) and CArG B+A+int mutant prevented depolarization-induced increase in SM -actin transcription. These results demonstrate that CArG elements are necessary for VGCC-dependent activation of SM -actin transcription.

View larger version (26K): [in this window] [in a new window]   Figure 3. VGCC activation increased SM -actin transcription in a CArG-dependent manner. SMCs were transfected with the –2.6/+2.8-kb SM -actin promoter or its mutants, treated with 60K for 24 hours and assayed for luciferase activity. Data are expressed as a percent of control (vehicle treated cells) ± SEM.

To further test the role of VGCC activation in regulation of SMC gene expression, a series of studies were performed in an in vitro embryoid body model of SMC differentiation from which highly differentiated, contractile SMCs that express functional VGCCs have been derived.¹⁷ Indeed, the heterotypic nature of these cultures and the fact that SMCs evolve spontaneously within them suggest that they may have at least some properties distinct from conventional SMC cultures derived from adult blood vessels whereby cells undergo extensive phenotypic switching in response to changing environmental cues in vitro.⁷ The 28-day embryoid bodies were transiently transfected with either SM -actin or SMMHC promoter-luciferase constructs. At 28 days, embryoid bodies maximally express endogenous SM -actin and SMMHC, and regions of nifedipine-sensitive spontaneous phasic smooth muscle contraction can be readily observed (Figure 4A to H). These regions immunostain for SMMHC (data not shown). Depolarization with 60 mmol/L KCl resulted in a nifedipine-sensitive increase in SM -actin and SMMHC promoter activity (Figure 4I). Furthermore, unlike the studies in cultured rat aortic SMCs (Figure 2), nifedipine alone and nifedipine plus 60 mmol/L KCl reduced SM -actin and SMMHC promoter activity below basal or vehicle-treated levels, implying a critical role for VGCCs in regulating basal SMC differentiation marker gene expression. It is important to note that although the EB system is a heterogeneous cell population, the SMMHC (–4.2/+11.6 kb) promoter construct used herein has been shown to be exclusively expressed in SMC in transgenic mice.^19,21 As such, expression of this gene is likely restricted to SMC within the EB system.

View larger version (73K): [in this window] [in a new window]   Figure 4. Effect of VGCC activation on SMC differentiation marker gene expression in embryoid bodies. A to G, Images at 0.5-second intervals of a spontaneously phasic contracting region of a 28-day embryoid body. This contraction was blocked by 1.0 µmol/L NIF (H). Scale denotes 1 mm. I, Embryonic stem cells were cultivated in cell aggregates for 26 days to form embryoid bodies, transfected with promoter-reporter constructs on day 27, treated with 60 mmol/L KCl (60K) and/or NIF (1 µmol/L) on day 28 for 48 hours and assayed for luciferase activity. Activity was normalized to protein content and expressed as mean±SEM.

siRNA Specific for Myocardin Attenuated Depolarization-Induced Transcription of SM -Actin and SMMHC
As shown in Figure 2A, depolarization increased endogenous myocardin expression in SMCs. To directly test whether endogenous myocardin contributed to depolarization-induced activation of SMMHC/SM -actin gene expression, effects of an siRNA specific for myocardin were examined by cotransfection studies. Consistent with previous studies by our laboratory,^15,16 pMighty-siMyocardin, an siRNA expression plasmid for myocardin, reduced basal SMMHC/SM -actin promoter activity by 50% and had no effect on c-fos promoter activity (Figure 5), validating the selectivity of myocardin for promoters containing 2 CArGs. Moreover, siRNA to myocardin significantly attenuated depolarization-induced SM -actin/SMMHC promoter activity to near basal levels. There was no difference between si-Myo vehicle and si-Myo 60K.

View larger version (28K): [in this window] [in a new window]   Figure 5. siRNA to myocardin attenuated VGCC-induced activation of SMC differentiation marker transcription. SMCs were cotransfected with 250 ng of pMighty-siScramble (siScr) or pMighty-siMyocardin (siMyo) and 250 ng of a promoter luciferase construct, treated with 60 mmol/L KCl (60K) for 24 hours and assayed for luciferase activity. Data are expressed as a percent of control (siScr vehicle-treated cells) ± SEM. *Significant difference compared with si-Scr 60K, P<0.05.

Transcriptional Regulation by VGCC Activation Is Mediated Partially by Rho Kinase
Activation of the RhoA pathway in smooth muscle by agonists leads to RhoA dissociation from Rho-GDI, nucleotide exchange of GTP for GDP, and translocation of RhoA and ROK from the cytosol to the membrane fraction to initiate signaling cascades.⁸ Figure 6A depicts a typical Western blot showing nifedipine-sensitive depolarization-induced translocation of RhoA from the cytosolic fraction (CY) to the MB fraction (detergent-soluble particulate fraction) compared with the vehicle-treated MB (P<0.05). Note the increase of RhoA in the MB fraction (*) and decrease in the CY fraction with 60K. To determine whether depolarization and VGCC activation mediate SMC-specific transcription through ROK, cultured SMCs were treated with the highly specific ROK inhibitors, Y-27632²² (gift from Mitsubishi Pharma Corp, Koyata, Japan) or H11562²³ (Calbiochem) in the presence or absence of depolarization. Treatment with either 10 µmol/L Y-27632 or 10 µmol/L H1152 decreased SM -actin and SMMHC promoter activity by 50% (Figure 6B). Importantly, Y-27632 and H1152 completely abrogated the depolarization-induced activation of SM -actin and SMMHC promoter activity (Figure 6B), and Y-27632 inhibited depolarization-induced expression of endogenous SM -actin, SMMHC, and myocardin mRNA (Figure 6C). However, as previously shown by Mack et al,⁹ neither Y-27632 nor H1152 decreased c-fos basal promoter activity or depolarization-induced increase in c-fos promoter activity (Figure 6B). In intact cerebral blood vessels, Cartin et al showed that the Ca²⁺/calmodulin-dependent protein kinase (CaMK) inhibitor KN93 selectively inhibited depolarization-induced expression of endogenous c-fos gene expression.⁶ Consistent with these results, 30 µmol/L KN93 (Calbiochem) decreased basal and abrogated the depolarization-induced increase in c-fos promoter activity but had only a minimal effect on SM -actin and SMMHC promoter activity (Figure 6D). Of interest, neither Y-27632 nor H1152 had an effect on the non-CArG-containing ACLP construct, whereas KN93 decreased activity by 55%. These data support a signaling mechanism whereby increased ROK activity caused by depolarization and activation of VGCCs results in transcription of SMC differentiation marker genes but not the immediate-early response gene, c-fos.

View larger version (34K): [in this window] [in a new window]   Figure 6. Rho kinase (ROK) partially mediates VGCC-induced activation of SMC differentiation marker gene expression. A, Rat aortic SMCs were grown to confluence, growth-arrested for 3 days, and treated with 60 mmol/L KCl (60K) ± nifedipine (NIF, 1 µmol/L) for 30 minutes. Western blot for RhoA was performed on whole cell extract (WE, loading control), cytosolic (CF), and membrane detergent-soluble particulate fractions (MB). *Significant difference in RhoA translocation to MB compared with vehicle and 60K+NIF, P<0.05. B, Transiently transfected rat aortic SMCs were treated with 60K and/or the ROK inhibitors 10 µmol/L Y-27632 or 10 µmol/L H1152 for 24 hours and assayed for luciferase activity. *Significant difference from vehicle, P<0.05. C, SMCs were treated with 60K and/or Y-27632 for 24 hours and expression of SM -actin, SMMHC, myocardin, and 18S rRNA mRNA levels were quantified by real-time PCR. Ratios of SM -actin, SMMHC, and myocardin to 18S rRNA expression were calculated. * P<0.05. D, SMCs were treated with 60K and/or the Ca2+/calmodulin-dependent protein kinase (CaMK) inhibitor KN93 (30 µmol/L) for 24 hours and assayed for luciferase activity. Data are expressed as percent of control (vehicle treated cells) ± SEM. *Significant difference compared with vehicle, P<0.05.

VGCC Activation and ROK Mediate SRF Binding to CArG Promoter Regions Within Intact Chromatin
The RhoA/ROK signaling pathway has been shown to regulate SRF expression and mediate SRF translocation to the nucleus in SMCs.²⁴ Moreover, it has been shown in neurons that membrane-induced depolarization can confer SRF binding to the c-fos CArG as measured by electrophoretic mobility shift assay.²⁵ However, there is lack of direct evidence linking depolarization and RhoA/ROK to alterations in the interaction of SRF with CArG regions in SMC differentiation marker gene promoters in intact chromatin. Therefore, quantitative ChIP assays were used to directly test whether VGCC activation or ROK altered SRF enrichment of CArG-containing regions in the endogenous SM -actin, SMMHC, or c-fos promoters and the non-CArG-containing ACLP promoter. Depolarization-induced activation of VGCCs resulted in a 2- to 3-fold enrichment of SRF to the CArG-containing regions in the SM -actin, SMMHC, and c-fos endogenous promoters (Figure 7A), not the ACLP promoter (data not shown). Depolarization-induced SRF enrichment was completely blocked by nifedipine. Subsequent studies tested whether the ROK inhibitor Y-27632 effected SRF enrichment of these promoters in the presence and absence of depolarization. Indeed, 10 µmol/L Y-27632 alone resulted in 90% decrease in SRF enrichment of the SM -actin CArG-containing region, 50% decrease in SMMHC but had no effect on SRF enrichment of the c-fos CArG region, further validating the selectivity of ROK for smooth muscle-selective CArG-containing gene promoters compared with c-fos (Figure 7B). Moreover, Y-27632 prevented the depolarization-induced enrichment of SRF in SM -actin and SMMHC (Figure 7B).

View larger version (28K): [in this window] [in a new window]   Figure 7. VGCC activation and ROK mediate SRF binding to CArG promoter regions within intact chromatin. Quantitative chromatin immunoprecipitation (ChIP) assays were performed to determine the effects of 60K ±1.0 µmol/L nifedipine (A) and 10 µmol/L Y-27632 alone and 60K ± Y-27632 (B) on SRF enrichment of endogenous promoter regions containing CArG cis-regulatory elements. SRF enrichment of CArG regions is expressed as percent of control (vehicle-treated cells) ± SEM. A, *P<0.05. B, *Significant difference from vehicle, P<0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vascular SMC contraction is mediated in part by VGCC-induced activation of the RhoA/ROK signaling cascade.^10,11 Moreover, RhoA/ROK signaling has been shown to selectively regulate SMC differentiation marker gene expression and not c-fos in SMCs.⁹ Thus, the goal of this study was to examine the interplay between VGCC activation and RhoA/ROK signaling in the regulation of genes that typify the differentiated contractile SMC phenotype, specifically SMMHC and SM -actin. To this end, our results provide evidence for a novel mechanism whereby L-type VGCC activation in SMCs stimulates CArG-dependent increased expression of the SMC differentiation marker genes, ie, SM -actin/SMMHC. Furthermore, regulation of these genes by VGCCs is dependent on RhoA/ROK signaling and myocardin, resulting in increased binding of SRF to CArG cis regulatory elements in the endogenous SM -actin and SMMHC promoters (Figure 8).

View larger version (30K): [in this window] [in a new window]   Figure 8. Proposed mechanism of L-type VGCC-dependent activation of SMC differentiation marker gene expression. Membrane depolarization increases [Ca2+]i through activation of nifedipine-sensitive L-type VGCCs. This results in increased SRF enrichment of 2 distinct subsets of genes that contain CArG cis-regulatory elements: SMC differentiation marker genes (2 CArGs: SM -actin, SMMHC) and the proliferation gene c-fos (1 CArG). Consistent with previous studies by Cartin et al,6 VGCC-dependent activation of c-fos is mediated predominantly through CaM kinase (CaMK): VGCC [Ca2+]i CaMK c-fos transcription. Here we propose a model whereby depolarization-induced SRF enrichment of endogenous SM -actin and SMMHC CArG promoter regions, not c-fos, are regulated by the RhoA/ROK signaling pathway: VGCC [Ca2+]i RhoA/ROK myocardin/SRF SM -actin/SMMHC. The mechanisms by which agonist-induced changes alter [Ca2+]i, either by activation of VGCCs and/or intracellular Ca2+ store release, are coupled to RhoA/ROK activation and concomitant SMC differentiation marker gene expression remain to be determined. Black thick lines indicate mechanisms addressed herein; black thin lines, previously described mechanisms; dashed thin lines, undetermined mechanisms; GPCR, G protein-coupled receptors; AII, angiotensin II; ET-1, endothelin-1.

Of major significance is the novel finding that activation of VGCCs was associated with enrichment of SRF binding to CArG-containing regions of SMC differentiation marker gene promoters within intact chromatin through a RhoA/ROK-dependent mechanism. Of interest, this effect exhibited gene selectivity in that SRF enrichment was not observed within the CArG region of the c-fos gene promoter or the ACLP gene promoter, which contains no CArG elements.²⁶ RhoA/ROK activation is required for growth factor-induced expression of c-fos, and evidence suggests that the growth response of SMCs to angiotensin II is also mediated by RhoA.²⁷ Indeed, in non-SMCs, RhoA potentiates CArG-SRF-dependent activation of the c-fos promoter.²⁸ However, herein and in previous studies by our laboratory, it has been demonstrated that c-fos promoter activity is not substantially upregulated by overexpression of constitutively active RhoA in SMCs or by toxin-induced reorganization of the actin cytoskeleton.⁹ Interestingly, the c-fos response to Ca²⁺ in neurons is partially regulated by SRF phosphorylation by CaMK and increased SRF binding to the c-fos CArG.²⁵ Therefore, it is plausible that VGCC activation stimulates multiple distinct signaling pathways in SMCs that regulate distinct subsets of genes, such as genes that define the "contractile" SMC, eg, SM -actin and SMMHC, versus genes involved in mediating cell growth, eg, c-fos.

Consistent with this hypothesis, Carten et al showed that phosphorylation of CREB by CaMK coupled VGCC activation in intact cerebral arteries to increased expression of c-fos.⁶ However, the RhoA/ROK signaling pathway was not tested. Similarly, results of the present studies showed that the CaMK inhibitor KN-93, but not the ROK inhibitors Y-27632 or H1152, prevented VGCC-induced activation of c-fos promoter, whereas the RhoA/ROK pathway, not CaMK, regulates SM -actin or SMMHC promoter activity, providing evidence that VGCC activation stimulates separate signaling pathways in SMCs (Figure 8).

The finding that VGCC activation results in increased SMC differentiation marker gene expression and c-fos expression in SMCs presents a unique dichotomy. How can 2 disparate gene subsets (differentiation genes versus growth-response/proliferation genes) share a common pathway of activation, VGCC-mediated activation? There are several potential explanations for this apparent dichotomy. First, it has been shown that SMC differentiation and proliferation/growth are not mutually exclusive.⁷ For example, during development, SMC differentiation coincides with rapid SMC proliferation.²⁹ Similarly, in cultured SMCs, angiotensin II induces SMC hypertrophy and activates both SMC differentiation marker gene expression¹⁶ and c-fos expression,²⁷ mechanisms that are potentially recapitulated in hypertension where medial SMC hypertrophy, not hyperplasia, dominates.³⁰ Indeed, L-type VGCC expression is also increased in SMCs from hypertensive vessels,¹ thus providing possible physiological relevance to the increased SMC differentiation marker expression observed with depolarization in embryoid bodies and cultured SMCs. Second, it is well-recognized that the nature of the [Ca²⁺]_i signal will ultimately dictate the integration of Ca²⁺ and transcriptional activation, ie, steady-state and oscillatory Ca²⁺ signals can preferentially recruit Ca²⁺-activated transcription factors.³¹ Thus, in an in vivo setting where VGCC activation and [Ca²⁺]_i levels are subjected to regulation by multiple signaling molecules (such as endothelin-1, angiotensin II, nitric oxide) and mechanical forces that selectively regulate various second messenger signaling pathways, eg, RhoA/ROK, the precise nature of the Ca²⁺-generated signal could potentially differentiate between 2 separate subsets of genes based on selective transcription factor activation. In support of this paradigm, depolarization increased expression of myocardin, a potent coactivator of the transcription factor SRF that mediates CArG-dependent transcription of multiple SMC marker genes that contain pairs of CArG cis regulatory elements (SMMHC/SM -actin), but not c-fos (contains 1 CArG).^12–15 Moreover, we show here that siRNA specific to myocardin attenuated depolarization-induced SM -actin/SMMHC transcription. Thus, we postulate that one potential mechanism for differential regulation of SMC differentiation marker genes by VGCCs compared with c-fos is caused by activation of myocardin and increased enrichment of SRF to SMC differentiation marker promoter CArG regions, possibly through a RhoA/ROK-mediated pathway (Figure 8). Indeed, in non-SMCs, it has been shown that CREB-binding protein cooperates with SRF to transactivate the c-fos promoter.³² Thus, we can only speculate in SMCs that other cofactors, possibly NFAT and CREB,³³ interact with SRF or independently to regulate c-fos.

The coupling of VGCC activation and RhoA/ROK signaling to contraction and SMC differentiation marker gene expression also suggests that short-term regulation of SMC contractile force may be coupled to long-term regulation of SMC differentiation marker gene expression. In support of this, results of the present studies showed that the VGCC is critical for basal and depolarization-induced activation of SMC differentiation marker gene expression in embryoid bodies (Figure 4). This is perhaps not surprising given that the VGCC is also required for myoblast differentiation into myotubes and skeletal -actin gene expression.³⁴ In vivo, one would thus predict that the VGCC is critical for SMC differentiation and vessel maturation. Although mice globally lacking the VGCC (Ca_v1.2) die in utero before day 15 postcoitum,³⁵ it is unknown whether this is because of SMC differentiation and vascular development. Although we did not test the role of the RhoA/ROK pathway in differentiation of SMCs from embryonic stem cells, Lu et al showed that coronary SMC differentiation from proepicardial cells is mediated by RhoA, specifically RhoA-mediated actin reorganization.³⁶ Taken together, we speculate that VGCCs and RhoA/ROK signaling may be important in the early stages of control of SMC differentiation.

In summary, data presented here support a model whereby VGCC-dependent increases in [Ca²⁺]_i are associated with activation of expression of SMC differentiation marker genes through mechanisms that are dependent on ROK and increased binding of SRF to CArG cis regulatory elements (Figure 8). These findings represent the first mechanistic data linking SMC differentiation marker gene expression to common signaling pathways, VGCC and RhoA/ROK, intimately involved in controlling SMC contraction. Key challenges are to determine the mechanisms by which agonist-induced changes in [Ca²⁺]_i, either by activation of VGCCs and/or intracellular Ca²⁺ store release, are coupled to RhoA/ROK activation and concomitant SMC differentiation marker gene expression (Figure 8). Moreover, it is unknown if these mechanisms regulate SMC differentiation marker gene expression in intact blood vessels in vivo, and whether alterations in these control pathways contribute to development of cardiovascular disease.

	Acknowledgments

 
This work was supported by National Institutes of Health grants PO1 HL19242 to G.K.O., A.V.S., and A.P.S.; R37 HL57353, RO1 HL3884 to G.K.O.; HL52490 to D.K.B., PO1 HL48807 to A.P.S.; MSTP 2T32-GM07267-22 to O.G.M.; and American Heart Association Fellowship to S.S.; American Physiological Society Fellowship to B.R.W. We thank Rupa Tripathi, Mary McCanna, Shizhen Luo, Tadashi Yoshida (University of Virginia), and Darla Tharp (University of Missouri, Columbia) for technical assistance.

	Footnotes

 
Original received March 15, 2004; revision received June 25, 2004; accepted June 30, 2004.

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