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(Circulation Research. 2005;97:427.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Depletion of Serum Response Factor by RNA Interference Mimics the Mitogenic Effects of Platelet Derived Growth Factor-BB in Vascular Smooth Muscle Cells

Nihal Kaplan-Albuquerque, Vicki Van Putten, Mary C. Weiser-Evans, Raphael A. Nemenoff

From the Department of Medicine, University of Colorado Health Sciences Center, Denver.

Correspondence to Dr Raphael A. Nemenoff, Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Box C-281, 4200 E 9th Ave, Denver, CO 80262. E-mail Raphael.Nemenoff{at}UCHSC.edu

	Abstract

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Promoters of many smooth muscle-specific genes (SM-genes) contain multiple CArG boxes, which represent a binding site for serum response factor (SRF). Transciptional control through these regions involves interactions with SRF and specific coactivators such as myocardin. We have previously reported that suppression of SM-gene expression by platelet derived growth factor (PDGF) is associated with redistribution of SRF, leading to lower intra-nuclear levels, and a reduction in SRF transactivation. To further assess the role of SRF depletion on VSMC phenotype, the current study used RNA interference (RNAi). Two SRF-specific sequences constructed as hairpins were stably expressed in rat VSMC. Clones expressing SRF RNAi had no detectable SRF expression by immunoblotting, and showed diminished levels of SM -actin protein and promoter activity. Unexpectedly, depletion of VSMC resulted in increased rates of proliferation and migration. Several genes whose expression is increased by PDGF stimulation, including c-Jun, were similarly induced in cells lacking SRF. Effects of SRF depletion were not attributable to altered PDGF receptor activity or alterations in activation of Akt. These data indicate that loss of SRF transactivation in VSMC, in this case through suppression via RNAi, induces biological responses similar to that seen with PDGF.

Key Words: vascular smooth muscle cells • serum response factor • RNA interference • platelet derived growth factor

	Introduction

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Phenotypic modulation of vascular smooth muscle cells (VSMC¹) is critical during development and in the onset of diseases such as atherosclerosis and hypertension. Atherosclerosis is a leading cause of heart disease and stroke and causes 50% of all deaths.^2,3 Therefore, understanding the physiology of large vessels will help to identify the cause of a primary cardiovascular disease. VSMC in mature animals express multiple contractile proteins to maintain vascular tone. These proteins are markers for the differentiated contractile phenotype. Unlike myocardial and skeletal muscle cells, VSMC are highly plastic and retain the ability to modulate their phenotype.^4–6 Environmental factors can alter the expression levels of these contractile proteins, leading to a switch from a differentiated phenotype, characterized by high expression, to a proliferative, dedifferentiated phenotype characterized by low expression. This occurs in pathophysiologic states of large arteries such as atherosclerosis, where migration and proliferation of neointimal VSMC lead to vascular remodeling.^7,8

In adult cultured VSMC, vasoconstrictors, such as angiotensin II or arginine vasopressin (AVP), increase expression of smooth muscle (SM) markers such as SMA and SM22, whereas growth factors such as platelet derived growth factor (PDGF) decrease protein and mRNA levels of these proteins.^1,9–11 We have shown that these effects are mediated through transcriptional regulation of SM specific promoter activity, and have defined specific signal transduction pathways that regulate SM specific gene expression by AVP or PDGF. AVP-induced increases in gene expression require c-jun amino terminal kinase and p38 MAP kinases, whereas PDGF-induced suppression is mediated by Ras, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways.^1,11–13 The promoters of these SM-genes contain common regulatory elements, specifically multiple CArG boxes, which have been shown to bind the MCM1, agamous, deficiens, serum response factor (MADS) box transcription factor, serum response factor (SRF).^14,15 Many SM-genes contain multiple CArG boxes in the proximal region of their promoter. Truncations, deletions, or point mutations in individual CArG elements of the SMA and SM22 promoters abolish both basal and AVP stimulated activation.^12,16 Single copies of these CArG elements are also found in the promoters of a number of immediate early genes (IEG) such as c-fos.^17,18 Selective regulation of SM-specific genes vis-à-vis IEG is mediated through specific associations of SRF with distinct families of coactivators. We have recently demonstrated that activation of the PI3K/Akt pathway by PDGF mediated down regulation of SM specific gene expression in part through relocalization of SRF to an extranuclear compartment.¹ This would effectively reduce the amount of SRF that is available to bind to the promoters of SRF-dependent genes. Changes in actin dynamics regulate the transcriptional activity of SRF-dependent genes in several muscle types,^19,20 and nonmuscle cells.²¹ Redistribution of SRF from the nucleus to the cytosol through changes in actin dynamics^22–25 and by reduced Rho activity²⁶ suggests that reduced SRF-dependent transcription may correlate with redistribution of the nucleus.^20,27 As an alternative way to inhibit SRF transactivation, and define the effects of blocking activation by this transcription factor, we have used an RNAi approach in cultured VSMC. The results from this study indicate that depletion of SRF not only suppresses expression of SM markers, but unexpectedly has broader effects in controlling the proliferation and migration of these cells. This study also places SRF as a potential regulatory node in events occurring during neointima formation.

	Materials and Methods

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Rat aortic VSMC were isolated and cultured as previously described.¹² Two sequences selected from different regions of the human SRF (Ac#J03161) coding sequence; Sequence A (human 648 to 669 cds): 5'-GACCTGCCTCAACTCGCCAGAC and Sequence B (human 1207 to 1228 cds): 5'-GTGGGTGGCCACATGATGTACC were used to construct corresponding hairpin SRF-RNAi sequences driven by U6 promoter (shRNAi; GenScript), which were then used in transient and stable transfection. For stable transfections, VSMC were transfected with 0.5 µg of SRF RNAi plasmids or control plasmid and grown in media containing 500 µg/mL G418 to select stable transfectants, and individual clones were picked. For transient transfections measuring promoter activity, a sequence encoding 765-bp of the rat SM--actin promoter coupled to luciferase together with cytomegalovirus-ß-galactosidase vector (CLONTECH) were used. The transfections also included 0.05 µg of SRF RNAi plasmids or control plasmid. Cell lysates were assayed for luciferase and ß-galactosidase activities as previously described.¹²

Proliferation assays were preformed by ³H-Thymidine incorporation. For add-back experiments, proliferation of cells transiently transfected with Green Fluorescent Protein (GFP) and SRF was determined by counting green cells. Migration of VSMC was measured using 5% serum in the bottom chamber of a Transwell chamber. The number of nuclei on the bottom of the Transwell membrane were stained with DAPI and counted. Change in mRNA levels was measured by Quantitative Real-Time PCR. Percent change in the arbitrary copy numbers normalized to Ribosomal RNA was used.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previously we demonstrated that PDGF-mediated activation of the PI3K/Akt pathway decreases nuclear levels of SRF as a result of accumulation in a cytosolic compartment. We proposed that this mechanism accounts, at least in part, for the ability of PDGF to decrease basal, and AVP-induced activation of SM-genes.¹ To determine whether ablation of SRF would have similar effects, 2 sequences specific for SRF were designed and inserted into a plasmid driven by U6 promoter (shRNAi). VSMC were transiently cotransfected with these SRF shRNAi constructs along with a promoter/reporter construct for SMA.¹⁶ Cells transfected with SRF shRNAi had reduced basal promoter activity, and AVP stimulation of the promoter was abolished, compared with cells cotransfected with an empty vector (Figure 1A) or a plasmid encoding an irrelevant shRNAi (data not shown). To confirm that the effects of the SRF RNAi sequences were attributable to depletion of SRF in rat VSMC, stable transfectants were selected by G418 resistance. Figure 1B shows western blotting with SRF and SMA antibodies of 6 independent clones transfected with Sequence B-SRF shRNAi compared with 2 independent clones transfected with control plasmid. Total actin levels were blotted as loading control. Expression levels of SRF were reduced in all 6 SRF RNAi clones selected. All of the clones also had reduced expression of SMA. Sequence B-Clone #3 had the highest SRF expression levels among the SRF shRNAi clones, and also had higher SMA levels. Sequence B-Clone #2 and Clone #6, which had the lowest SRF expression levels, were chosen for further analysis. To confirm that the decrease in SMA promoter activity was directly attributable to loss of SRF expression, cells expressing SRF RNAi were transiently transfected with an expression plasmid encoding SRF. As shown in Figure 1C, reexpression of SRF restored basal SMA promoter activity in a dose-dependent manner.

View larger version (23K): [in this window] [in a new window]   Figure 1. SRF depletion suppresses SMA promoter activity and protein levels. A, VSMC were transiently transfected with SMA promoter coupled to Luciferase reporter as described in the online supplement available at http://circres.ahajournals.org. Cells were stimulated with AVP (10–6 mol/L) or the vehicle (Basal) for 72 hours in the presence of plasmids containing either 2 different RNAi sequences (Seq A or B) or control plasmid. Promoter activity normalized to ß-galactosidase was determined. Results represent the mean and SEM of 3 experiments performed in duplicate, percent change over basal stimulation is shown. *P<0.05 vs Basal in pU6.1/Neo, #P<0.05 vs AVP in pU6.1/Neo. B, VSMC stably expressing Sequence B shRNAi were serum-restricted for 24 hours and extracts were immunoblotted with SRF, SMA, or total actin. C, VSMC stably expressing Sequence B shRNAi were transiently transfected with the indicated amounts of an expression plasmid encoding SRF, along with the SMA promoter construct, and luciferase activity normalized to ß-gal determined as indicated above. Results represent the mean and SEM of 3 experiments performed in duplicate, percent change over pU6.1/Neo#1 is shown. *P<0.05 vs pU6.1/Neo#1, #P<0.05 vs SRF shRNAi#6 in basal condition.

In addition to SMA, and SM22, depletion of SRF also reduced the expression of 2 SRF-dependent immediate early genes, c-Fos, and Egr-1 (Figure 2A). Levels of mRNA expression of the smooth and cardiac muscle-specific SRF coactivator, myocardin, a CArG containing gene¹⁴ were also reduced (Figure 2B). However, there was no significant change in the expression of Elk-1 (Figure 2A), which has been shown to be involved in induction of c-fos by growth factors.²⁸ On the other hand, the expression level of c-jun was unexpectedly increased to levels comparable to those achieved with PDGF stimulation of control cells (Figure 2A). These data suggested that ablation of SRF may effect expression of other genes, not directly containing SRF binding sites, and could mimic the biological responses of VSMC to PDGF. We therefore examined the effects of SRF ablation on 2 responses of VSMC to PDGF: proliferation and migration. Proliferation, of SRF depleted clones #2 and #6, was measured by ³H-Thymidine incorporation. Cells with depleted SRF levels (SRF shRNAi #2, and #6) had higher proliferative rates under low-serum conditions, and in response to PDGF-BB (20 ng/mL) stimulation compared with control cells (Figure 2C). However, proliferative rates in response to serum (10%) stimulation were not affected by depletion of SRF. This was confirmed by changes in cell number. Control cells doubled in number (from 20 000 cells to 48 000 cells) after 48 hours; however SRF depleted cells showed 4 to 5 fold increases (from 22 000 cells to 104 000 cells) in cell number. Examination of stable pools of Embryonic day 17 (E17) rat VSMC also showed similar increases in proliferation after SRF depletion (data not shown), despite their nonproliferative responsiveness to PDGF-BB.²⁹ To confirm that the increase in proliferation was directly attributable to loss of SRF expression, cells expressing SRF RNAi were transiently cotransfected with expression plasmids encoding SRF or empty vector and GFP. As shown in Figure 2D, numbers of GFP expressing cells were higher in SRF depleted cells, and reexpression of SRF reduced the number of GFP expressing cells to levels seen in control cells. To assess affects on migration, Transwell assays were used, with cells exposed to 5% serum in the bottom chamber. SRF depleted clones had dramatically higher number of migrating cells compared with the control cells (pU6.1/Neo #1 and #2) (Figure 2E).

View larger version (38K): [in this window] [in a new window]   Figure 2. SRF depletion increases VSMC proliferation and migration. VSMC stably expressing SRF RNAi plasmid with Sequence B (Clones 2 and 6) or control plasmid (Clones 1 and 2) were serum restricted in 0.2%-fetal calf serum (FCS)-EMEM (A) stimulated with AVP (10–6 mol/L) or PDGF for 6 hours. Western analysis was performed with antibodies against c-Fos, Egr-1, c-Jun, SRF, SMA, SM22, Elk-1, or total actin. B, Total RNA was isolated and real time quantitative PCR for Myocardin was performed as described in the online supplement Methods section. Ribosomal RNA is used for the normalization of cDNA from each sample. Results represent the mean and SEM of 2 independent samples performed in at least triplicate. C, Cells stimulated with PDGF or 10% serum for 24 hours, and processed for H3-Thymidine incorporation as described in the online supplement Methods section. The samples were counted and corrected with the protein amount in each well. *P<0.05 vs Basal in pU6.1/Neo#1, #P<0.05 within each stimulation. D, Cells were transiently cotransfected with an expression plasmid encoding for GFP and SRF or empty vector, then serum restricted in 0.2%FCS-EMEM for 48 hours, and GFP expressing cells were counted under a fluorescent microscope. *P<0.05 vs GFP+Empty vector in pU6.1/Neo#1, #: P<0.05 GFP+SRF vs GFP+Empty vector. E, VSMC stably expressing SRF RNAi plasmid with Sequence B (Clones 2 and 6) or control plasmid (Clone 1) were plated onto the upper chamber of the 8-µm pored transwell membranes. The chemotaxis was achieved in the presence of 5% serum in the bottom chamber as described in the online supplement Methods section. Percent change in cell migration is shown. *P<0.05 vs pU6.1/Neo#1.

To confirm that these effects were not a result of alterations in signaling through PDGF receptors, expression levels of PDGF receptor ß (PDGFRß) and its activation by PDGF were measured by Western blotting with antibodies against total and phospho-receptor. As shown in Figure 3A, there was no difference in expression of PDGFRß between SRF depleted and control cells, and stimulation for 5 minutes with PDGF resulted in equivalent levels of phospho-receptor. Stimulation of VSMC for 6 hours with PDGF-BB resulted in downregulation of PDGFRß expression, and this occurred to the same extent in SRF-depleted and control cells (Figure 3A). Finally, Akt, a downstream effector of PDGF receptor signaling, was activated to similar levels in SRF depleted clones compared with control cells (Figure 3B).

View larger version (54K): [in this window] [in a new window]   Figure 3. The effects of SRF depletion are not attributable to PDGF receptor activation. VSMC stably expressing SRF RNAi plasmid with Sequence B (Clones 2 and 6) or control plasmid (Clones 1 and 2) were serum restricted in 0.2%-FCS-EMEM and then stimulated with PDGF for 5 minutes or 6 hours. A, Western analysis was performed with antibodies against phosphorylated form of PDGFRß (upper) or unphosphorylated form of PDGFRß (lower). B, Western analysis was performed with antibodies against phosphorylated form of Akt (S473, upper) or unphosphorylated form of Akt (lower)

Based on the gene array analysis,³⁰ PDGF-BB increased the mRNA expression of a condroidin sulfate proteoglycan, Versican, and a cytokine, Gro, but decreased the expression of Collagen X 1. Quantitative Real-Time PCR (Q-RT-PCR) analysis showed that VSMC depleted of SRF (SRF shRNAi Clones #2, and #6) also had marked increases in mRNA levels for Versican when compared with control cells (U6.1/Neo clones #1 and #2). However, mRNA levels of Gro and ColX1 were not significantly changed (Figure 4A). We have also shown that PDGF-BB increases the protein expression of C/EBP, Ezrin, GADD45, and hemeoxygenase 1 (HmOx-1), and decreases the protein expression of Nexilin.³⁰ Western analysis of samples from VSMC depleted of SRF (SRF shRNAi #2 and #6) and control cells (pU6.1/Neo #1 and #2) showed that C/EBP, Ezrin, GADD45, and HmOx-1 expression levels were also increased in SRF depleted cells. Conversely, protein expression of Nexilin was dramatically decreased in SRF depleted clones (Figure 4B). Similarly, in SRF RNAi-treated E17 VSMC, a reduced SRF, and consequently a reduced SMA expression level was accompanied by increased C/EBP protein expression levels (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 4. VSMC stably expressing SRF RNAi plasmid with Sequence B (Clones 2 and 6) or control plasmid (Clones 1 and 2) were serum restricted in 0.2%-FCS-EMEM. A, The total RNA was isolated. Real-time quantitative PCR for Versican, Gro, and Collagen type X 1 was performed as described in the online supplement Methods section. Ribosomal RNA is used for the normalization of cDNA from each sample. Results represent the mean and SEM of 2 independent samples performed in at least triplicate. *P<0.05 vs pU6.1/Neo#1. B, Western analysis was performed with antibodies against C/EBP, Ezrin, GADD45, HmOx-1, and Nexilin. PDGF induced decreases are shown as (–) and increases are shown as (+).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
SRF is a MADS box transcription factor, which is critical in the regulation of muscle growth and development. It binds to specific cis-regulatory elements, namely CArG boxes, of muscle specific genes, and IEG. Mutations in the CArG boxes of SM-specific genes inhibit expression of SM-markers during development,^5,6,14 and regulation by hormonal factors in cultured VSMC.^12,13,31,32 Phenotypic remodeling in mature vessels is regulated by local environmental cues including humoral factors, cell-cell and cell-matrix interactions, mechanical stresses, and inflammatory stimuli. Our laboratory has observed the convergence of signaling from both differentiating and dedifferentiating signals on SRF transactivation. PDGF induces a variety of cellular responses in VSMC including cell proliferation, migration, dedifferentiation, and extracellular matrix deposition. Previously we have shown that PDGF-mediated suppression of SM-specific gene expression is mediated through an Akt-dependent pathway that leads to partial redistribution of SRF to a cytoplasmic location.¹ This, redistribution effectively decreases the concentration of SRF within the nucleus. Decreases in nuclear SRF levels will presumably lead to decreased binding to specific CArG boxes, and suppression of expression of SRF-dependent genes. In this study we have more directly examined the biological consequences of decreasing SRF expression through RNAi. Our data show that ablation of SRF results in inhibition of both SM-specific genes and IEG expression. However, to our surprise, SRF depletion mimicked the biological responses of these cells to PDGF stimulation in adult VSMC.

Previously, SRF has been shown to be dispensable for ES cell proliferation and cell cycle regulation,³³ and required for ES cell migration.³⁴ Surprisingly, in addition to inhibition of SM-specific gene expression, SRF depletion increased VSMC proliferation and migration (Figure 2), both effects seen with PDGF. Reexpression of SRF reversed the effects of SRF depletion on both muscle gene expression and proliferation, indicating that they are not a result of nonspecific effects of siRNA. VSMC proliferation and migration are the key elements in development of the blood vessels, as well as in the formation of neointima during vascular injury.^35,36 Induction of c-jun by PDGF-BB is important for VSMC proliferation during vascular injury,^37–39 as well as in the promotion of the migratory phenotype of VSMC.⁴⁰ In our cells, depletion of SRF by shRNAi resulted in increases in basal c-jun expression to levels achieved by PDGF stimulation of control cells (Figure 2A). This suggests that SRF acts to suppress c-jun expression, and that this suppression is relieved by PDGF stimulation. On the other hand, because PDGF was further able to increase c-jun expression in SRF depleted cells, likely that PDGF mediates induction of c-Jun through SRF-independent mechanisms as well. Sandbo et al⁴¹ reported that increased c-jun expression coincided with reduced SRF transactivation. However, these workers concluded the inhibition of SRF transactivation was mediated by increased c-jun expression,⁴¹ placing c-jun upstream of SRF. Our data suggest that in our cells SRF is proximal to regulation of c-jun expression.

In addition to c-jun expression, the expression levels of several, but not all PDGF-regulated genes were also changed in response to SRF depletion. Expression of growth arrest and DNA damage induced-45 (GADD45), which is associated with proliferation, was increased in cells expressing SRF RNAi. HmOx-1 is an antioxidant and antiinflammatory enzyme, which is increased during vascular injury as a feed-back response.⁴² Similar to PDGF stimulation,³⁰ SRF depletion also increased HmOx-1.

SRF depletion increased the expression of a PDGF inducible transcription factor CCAAT-enhanced binding protein delta (C/EBP).³⁰ C/EBP overexpression was correlated with increased PDGFR, and vascular remodeling,⁴³ and in our studies correlated with decreased SM-specific gene expression, and neointima formation.³⁰ In addition, the expression level of Versican (CSPG2), an extracellular matrix proteoglycan, was upregulated in SRF depleted cells. We have previously shown that versican is upregulated by PDGF stimulation.³⁰ Versican is secreted by VSMC in normal blood vessels, and is also a marker of atherosclerosis and vascular injury.^44–46 Nexilin, an F-actin binding protein localized to cell-matrix junctions⁴⁷ was downregulated, and Ezrin, which has metastatic properties^48,49 was upregulated in SRF depleted VSMC. Both of these effects were seen with PDGF. Expression of nexilin is increased by hypertrophic stimuli, and decreased expression is seen during neointima formation.³⁰ The regulatory regions of these genes are of interest; however the promoter regions have not been well defined for many of them. In fact, the promoter region of Versican has several C/EBP binding elements.³⁰

Depletion of SRF increases expression of several growth related genes, including GADD45, c-Jun, Versican, which in combination may have effects on the growth properties of these cells. SRF-mediated changes in expression of a transcription factor like C/EBP will lead to secondary changes in expression of genes that lack CArG boxes and SRF binding sites. Regulation of cell-matrix interactions might also play a major role in the migration, differentiation, and proliferation of VSMC, and SRF transcriptional activity might play a key role in keeping the balance between differentiated and dedifferentiated, migratory phenotype of VSMC.

The effects of SRF depletion were not attributable to altered signaling through the PDGFR-ß. Levels of PDGFRß expression, activation and desensitization by PDGF, and activation of the downstream effector Akt, were not altered in VSMC depleted of SRF. In addition, PDGF was still able to increase c-jun expression in SRF depleted cells, suggesting an intact signaling pathway.

Developmental studies of SRF depletion have shown that targeted deletion of SRF is embryo lethal at e11.5 to 13.5 attributable to impairment in the development of cardiovascular system^50,51 or attributable to skeletal hypoplasia.⁵² However, the role of SRF in modulating the phenotype of adult cells has not been clearly defined. Based on our data, we would propose that regulation of SRF transcriptional activity is central to the biological response of VSMC, beyond controlling expression of genes containing known CArG elements in their promoter. In fact, the pleiotropic response of VSMC to PDGF, ie, dedifferentiation, proliferation, extracellular matrix deposition, and migration, may require inactivation of SRF. These effects could occur through redistribution of the protein of the nucleus, as we have previously shown.¹ In fact, SRF transactivation is decreased in smooth muscle tumors.⁵³ Posttranlational modifications of SRF might be important in regulation of SRF transcriptional activity. Several studies have shown that SRF can be phosphorylated on several residues.^12,54–57 Interestingly, loss of SRF binding in aging fibroblasts has been associated with hyperphosphorylation of SRF.⁵⁸ Further studies on the relationship between SRF phosphorylation, subcellular distribution and transcriptional activity are required. Finally, because our data suggest that SRF dependent pathways mediate the effects of PDGF, including proliferation, migration, extracellular matrix deposition, and dedifferentiation, targeting the signaling pathways leading to SRF transcriptional inactivation may present a novel strategy in treatment of phenotypic modulation of VSMC associated with vessel injury and formation of atherosclerotic lesions.

	Acknowledgments

 
Supported by the National Institutes of Health grants DK 19928 and CA103618.

	Footnotes

 
Original received March 24, 2005; revision received July 19, 2005; accepted July 21, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Kaplan-Albuquerque N, Garat C, Desseva C, Jones PL, Nemenoff RA. Platelet-derived growth factor-BB-mediated activation of Akt suppresses smooth muscle-specific gene expression through Inhibition of mitogen-activated protein kinase and redistribution of serum response factor. J Biol Chem. 2003; 278: 39830–39838.[Abstract/Free Full Text]

2. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993; 362: 801–809.[CrossRef][Medline] [Order article via Infotrieve]

3. Lusis AJ. Atherosclerosis Nature. 2000; 407: 233–241.

4. Owens GK, Wise G. Regulation of differentiation/maturation in vascular smooth muscle cells by hormones and growth factors. Agents Actions Suppl. 1997; 48: 3–24.[Medline] [Order article via Infotrieve]

5. Owens GK. Molecular control of vascular smooth muscle cell differentiation. Acta Physiol Scand. 1998; 164: 623–635.[Medline] [Order article via Infotrieve]

6. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004; 84: 767–801.[Abstract/Free Full Text]

7. Ikeda U, Oguchi A, Okada K, Ishikawa S, Saito T, Ikeda M, Kano S, Takahashi M, Shiomi M, Shimada K. Involvement of LDL receptor in the proliferation of vascular smooth muscle cells. Atherosclerosis. 1994; 110: 87–94.[CrossRef][Medline] [Order article via Infotrieve]

8. Hayashi K, Takahashi M, Nishida W, Yoshida K, Ohkawa Y, Kitabatake A, Aoki J, Arai H, Sobue K. Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids. Circ Res. 2001; 89: 251–258.[Abstract/Free Full Text]

9. Geisterfer AA, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res. 1988; 62: 749–756.[Abstract/Free Full Text]

10. Hautmann MB, Thompson MM, Swartz EA, Olson EN, Owens GK. Angiotensin II-induced stimulation of smooth muscle alpha-actin expression by serum response factor and the homeodomain transcription factor MHox. Circ Res. 1997; 81: 600–610.[Abstract/Free Full Text]

11. Li X, Van Putten V, Zarinetchi F, Nicks ME, Thaler S, Heasley LE, Nemenoff RA. Suppression of smooth-muscle alpha-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2. Biochem J. 1997; 327: 709–716.[Medline] [Order article via Infotrieve]

12. Garat C, Van Putten V, Refaat ZA, Dessev C, Han SY, Nemenoff RA. Induction of smooth muscle alpha-actin in vascular smooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activated protein kinase. J Biol Chem. 2000; 275: 22537–22543.[Abstract/Free Full Text]

13. Kaplan-Albuquerque N, Garat C, Van Putten V, Nemenoff RA. Regulation of SM22{alpha} expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells. Am J Physiol Heart Circ Physiol. 2003; 285: H1444–H1452.[Abstract/Free Full Text]

14. Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol. 2003; 35: 577–593.[CrossRef][Medline] [Order article via Infotrieve]

15. Shore P, Sharrocks AD. The MADS-box family of transcription factors. Eur J Biochem. 1995; 229: 1–13.[Medline] [Order article via Infotrieve]

16. Van Putten V, Li X, Maselli J, Nemenoff RA. Regulation of smooth muscle alpha-actin promoter by vasopressin and platelet-derived growth factor in rat aortic vascular smooth muscle cells. Circ Res. 1994; 75: 1126–1130.[Abstract/Free Full Text]

17. Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986; 46: 567–574.[CrossRef][Medline] [Order article via Infotrieve]

18. Mohun T, Garrett N, Treisman R. Xenopus cytoskeletal actin and human c-fos gene promoters share a conserved protein-binding site. Embo J. 1987; 6: 667–673.[Medline] [Order article via Infotrieve]

19. Arai A, Spencer JA, Olson EN. STARS, a striated muscle activator of Rho signaling and serum response factor-dependent transcription. J Biol Chem. 2002; 277: 24453–24459.[Abstract/Free Full Text]

20. Liu HW, Halayko AJ, Fernandes DJ, Harmon GS, McCauley JA, Kocieniewski P, McConville J, Fu Y, Forsythe SM, Kogut P, Bellam S, Dowell M, Churchill J, Lesso H, Kassiri K, Mitchell RW, Hershenson MB, Camoretti-Mercado B, Solway J. The RhoA/Rho kinase pathway regulates nuclear localization of serum response factor. Am J Respir Cell Mol Biol. 2003; 29: 39–47.[Abstract/Free Full Text]

21. Psichari E, Balmain A, Plows D, Zoumpourlis V, Pintzas A. High activity of serum response factor in the mesenchymal transition of epithelial tumor cells is regulated by RhoA signaling. J Biol Chem. 2002; 277: 29490–29495.[Abstract/Free Full Text]

22. Geneste O, Copeland JW, Treisman R. LIM kinase and Diaphanous cooperate to regulate serum response factor and actin dynamics. J Cell Biol. 2002; 157: 831–838.[Abstract/Free Full Text]

23. Sotiropoulos A, Gineitis D, Copeland J, Treisman R. Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell. 1999; 98: 159–169.[CrossRef][Medline] [Order article via Infotrieve]

24. Miralles F, Posern G, Zaromytidou AI, Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell. 2003; 113: 329–342.[CrossRef][Medline] [Order article via Infotrieve]

25. Grosse R, Copeland JW, Newsome TP, Way M, Treisman R. A role for VASP in RhoA-Diaphanous signalling to actin dynamics and SRF activity. Embo J. 2003; 22: 3050–3061.[CrossRef][Medline] [Order article via Infotrieve]

26. Copeland JW, Treisman R. The Diaphanous-related formin mDia1 controls serum response factor activity through its effects on actin polymerization. Mol Biol Cell. 2002; 13: 4088–4099.[Abstract/Free Full Text]

27. Camoretti-Mercado B, Liu HW, Halayko AJ, Forsythe SM, Kyle JW, Li B, Fu Y, McConville J, Kogut P, Vieira JE, Patel NM, Hershenson MB, Fuchs E, Sinha S, Miano JM, Parmacek MS, Burkhardt JK, Solway J. Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor. J Biol Chem. 2000; 275: 30387–30393.[Abstract/Free Full Text]

28. Yang S-H, Yates PR, Whitmarsh AJ, Davis RJ, Sharrocks AD. The Elk-1 ETS-domain transcription factor contains a mitogen-activated protein kinase targeting motif. Mol Cell Biol. 1998; 18: 710–720.[Abstract/Free Full Text]

29. Cook CL, Weiser MC, Schwartz PE, Jones CL, Majack RA. Developmentally timed expression of an embryonic growth phenotype in vascular smooth muscle cells. Circ Res. 1994; 74: 189–196.[Abstract/Free Full Text]

30. Kaplan-Albuquerque N, Bogaert YE, Van Putten V, Weiser-Evans MC, Nemenoff RA. Patterns of gene expression differentially regulated by platelet-derived growth factor and hypertrophic stimuli in vascular smooth muscle cells: markers for phenotypic modulation and response to injury. J Biol Chem. 2005; 280: 19966–19976.[Abstract/Free Full Text]

31. Owens GK. Role of contractile agonists in growth regulation of vascular smooth muscle cells. Adv Exp Med Biol. 1991; 308: 71–79.[Medline] [Order article via Infotrieve]

32. Owens GK, Geisterfer AA, Yang YW, Komoriya A. Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells. J Cell Biol. 1988; 107: 771–780.[Abstract/Free Full Text]

33. Schratt G, Weinhold B, Lundberg AS, Schuck S, Berger J, Schwarz H, Weinberg RA, Ruther U, Nordheim A. Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells. Mol Cell Biol. 2001; 21: 2933–2943.[Abstract/Free Full Text]

34. Schratt G, Philippar U, Berger J, Schwarz H, Heidenreich O, Nordheim A. Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells. J Cell Biol. 2002; 156: 737–750.[Abstract/Free Full Text]

35. Ross R. Atherosclerosis–an inflammatory disease. N Engl J Med. 1999; 340: 115–126.[Free Full Text]

36. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999; 79: 1283–1316.[Abstract/Free Full Text]

37. Miano JM, Vlasic N, Tota RR, Stemerman MB. Localization of Fos and Jun proteins in rat aortic smooth muscle cells after vascular injury. Am J Pathol. 1993; 142: 715–724.[Abstract]

38. Suggs WD, Olson SC, Madnani D, Patel S, Veith FJ. Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model. Surgery. 1999; 126: 443–449.[Medline] [Order article via Infotrieve]

39. Kim S, Iwao H. Stress and vascular responses: mitogen-activated protein kinases and activator protein-1 as promising therapeutic targets of vascular remodeling. J Pharmacol Sci. 2003; 91: 177–181.[CrossRef][Medline] [Order article via Infotrieve]

40. Ioroi T, Yamamori M, Yagi K, Hirai M, Zhan Y, Kim S, Iwao H. Dominant negative c-Jun inhibits platelet-derived growth factor-directed migration by vascular smooth muscle cells. J Pharmacol Sci. 2003; 91: 145–148.[CrossRef][Medline] [Order article via Infotrieve]

41. Sandbo N, Qin Y, Taurin S, Hogarth DK, Kreutz B, Dulin NO. Regulation of serum response factor-dependent gene expression by proteasome inhibitors. Mol Pharmacol. 2005; 67: 789–797.[Abstract/Free Full Text]

42. Togane Y, Morita T, Suematsu M, Ishimura Y, Yamazaki JI, Katayama S. Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury. Am J Physiol Heart Circ Physiol. 2000; 278: H623–32.[Abstract/Free Full Text]

43. Yang ZH, Kitami Y, Takata Y, Okura T, Hiwada K. Targeted overexpression of CCAAT/enhancer-binding protein-delta evokes enhanced gene transcription of platelet-derived growth factor alpha-receptor in vascular smooth muscle cells. Circ Res. 2001; 89: 503–508.[Abstract/Free Full Text]

44. Geary RL, Wong JM, Rossini A, Schwartz SM, Adams LD. Expression profiling identifies 147 genes contributing to a unique primate neointimal smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol. 2002; 22: 2010–2016.[Abstract/Free Full Text]

45. Wight TN, Merrilees MJ. Proteoglycans in atherosclerosis and restenosis: key roles for versican. Circ Res. 2004; 94: 1158–1167.[Abstract/Free Full Text]

46. Shimizu-Hirota R, Sasamura H, Kuroda M, Kobayashi E, Hayashi M, Saruta T. Extracellular matrix glycoprotein biglycan enhances vascular smooth muscle cell proliferation and migration. Circ Res. 2004; 94: 1067–1074.[Abstract/Free Full Text]

47. Ohtsuka T, Nakanishi H, Ikeda W, Satoh A, Momose Y, Nishioka H, Takai Y. Nexilin: a novel actin filament-binding protein localized at cell-matrix adherens junction. J Cell Biol. 1998; 143: 1227–1238.[Abstract/Free Full Text]

48. Khanna C, Wan X, Bose S, Cassaday R, Olomu O, Mendoza A, Yeung C, Gorlick R, Hewitt SM, Helman LJ. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med. 2004; 10: 182–186.[CrossRef][Medline] [Order article via Infotrieve]

49. Yu Y, Khan J, Khanna C, Helman L, Meltzer PS, Merlino G. Expression profiling identifies the cytoskeletal organizer ezrin and the developmental homeoprotein Six-1 as key metastatic regulators. Nat Med. 2004; 10: 175–181.[CrossRef][Medline] [Order article via Infotrieve]

50. Parlakian A, Tuil D, Hamard G, Tavernier G, Hentzen D, Concordet JP, Paulin D, Li Z, Daegelen D. Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality. Mol Cell Biol. 2004; 24: 5281–5289.[Abstract/Free Full Text]

51. Miano JM, Ramanan N, Georger MA, de Mesy Bentley KL, Emerson RL, Balza RO Jr, Xiao Q, Weiler H, Ginty DD, Misra RP. Restricted inactivation of serum response factor to the cardiovascular system. Proc Natl Acad Sci U S A. 2004; 101: 17132–17137.[Abstract/Free Full Text]

52. Li S, Czubryt MP, McAnally J, Bassel-Duby R, Richardson JA, Wiebel FF, Nordheim A, Olson EN. Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice. Proc Natl Acad Sci U S A. 2005; 102: 1082–1087.[Abstract/Free Full Text]

53. Phiel CJ, Gabbeta V, Parsons LM, Rothblat D, Harvey RP, McHugh KM. Differential binding of an SRF/NK-2/MEF2 transcription factor complex in normal versus neoplastic smooth muscle tissues. J Biol Chem. 2001; 276: 34637–34650.[Abstract/Free Full Text]

54. Manak JR, Prywes R. Phosphorylation of serum response factor by casein kinase II: evidence against a role in growth factor regulation of fos expression. Oncogene. 1993; 8: 703–711.[Medline] [Order article via Infotrieve]

55. Marais RM, Hsuan JJ, McGuigan C, Wynne J, Treisman R. Casein kinase II phosphorylation increases the rate of serum response factor-binding site exchange. Embo J. 1992; 11: 97–105.[Medline] [Order article via Infotrieve]

56. Misra RP, Rivera VM, Wang JM, Fan PD, Greenberg ME. The serum response factor is extensively modified by phosphorylation following its synthesis in serum-stimulated fibroblasts. Mol Cell Biol. 1991; 11: 4545–4554.[Abstract/Free Full Text]

57. Papavassiliou AG. The role of regulated phosphorylation in the biological activity of transcription factors SRF and Elk-1/SAP-1. Anticancer Res. 1994; 14: 1923–1926.[Medline] [Order article via Infotrieve]

58. Atadja PW, Stringer KF, Riabowol KT. Loss of serum response element-binding activity and hyperphosphorylation of serum response factor during cellular aging. Mol Cell Biol. 1994; 14: 4991–4999.[Abstract/Free Full Text]

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