Published online before print October 14, 2004, doi: 10.1161/01.RES.0000147961.09840.fb
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© 2004 American Heart Association, Inc.
Molecular Medicine |
A G/C Element Mediates Repression of the SM22
Promoter Within Phenotypically Modulated Smooth Muscle Cells in Experimental Atherosclerosis
From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville.
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
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Abstract |
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A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22
is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22 promoter required for this process. Transgenic mice carrying the SM22 promoter–ß-galactosidase (ß-gal) reporter transgene were crossed to apolipoprotein E (ApoE)–/– mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of ß-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22 promoter prevented the decrease in SM22 promoter–ß-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22 in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB–induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22 promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22. Together, these results support a model whereby a G/C repressor element within the SM22 promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.
Key Words: atherosclerosis • proliferation • smooth muscle differentiation • transcriptional regulation • vascular smooth muscle cell proliferation
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Introduction |
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Atherosclerosis, the principal cause of heart attack and stroke in Western nations, is a complex inflammatory-fibroproliferative response to various forms of insult to the endothelium and vessel wall resulting in phenotypic modulation of medial smooth muscle cells (SMCs) and ultimately luminal narrowing.1 There is unequivocal evidence demonstrating that SMCs within human atherosclerotic lesions or experimental atherosclerosis show an altered phenotype compared with normal medial SMCs.2 This process is known as phenotypic switching or modulation and is a feature of lesion SMCs irrespective of their origins from pre-existing SMCs or circulating stem cells.2,3 In addition to a profoundly altered morphological appearance compared with normal medial SMCs, phenotypically modified SMCs express much reduced levels of a variety of proteins characteristic of the differentiated SMC, including smooth muscle
-actin (SM -actin), smooth muscle myosin heavy chain (SMMHC), SM22, caldesmon, viniculin, and desmin. In contrast, phenotypically modulated SMCs show concomitant increases in expression of genes associated with lesion progression, including collagen, proteoglycans, growth regulatory genes, and matrix metalloproteinases.2
However, a major challenge in understanding phenotypic switching of SMCs is that this cell type has remarkable plasticity and can exhibit a broad range of phenotypic states and associated gene expression patterns as a function of changing environmental cues associated with normal development and vascular disease. Of interest, in the case of atherosclerosis, the nature of these phenotypic changes appears to occur at different stages of lesion development. For example, during the development of the fibrous cap, SMC proliferation and migration are required to stabilize the plaque, although in end-stage disease, these cells can alter gene expression patterns that contribute to destabilization of the fibrous plaque and plaque rupture. Whereas regulation of these processes is extremely dynamic and complex, and undoubtedly involves many different regulatory mechanisms, a common feature of SMC phenotypic switching in each of these cases is a reduction in expression of SMC-specific/SMC-selective proteins such as SM -actin, SMMHC, and SM22. As such, identification of mechanisms that mediate suppression of SMC marker genes in atherosclerosis are likely to provide key insights toward understanding the overall process of SMC phenotypic switching.
Although it may seem intuitive that suppression of SMC differentiation marker genes associated with phenotypic modulation in atherosclerosis would be transcriptionally mediated, there is no direct evidence that this is the case. Indeed, there is extensive evidence in an in vitro model of SMC phenotypic switching in response to platelet-derived growth factor (PDGF)-BB demonstrating that repression of SMC gene expression is mediated post-transcriptionally through selective changes in stability of mRNAs encoding for SMC contractile proteins such as SM -actin.4 However, we showed recently that repression of a number of SMC marker genes, including SM22, SM -actin and SMMHC, in response to severe mechanical trauma to the vessel wall in vivo, was mediated at least in part by transcriptional repression and in the case of the SM22 gene, this was dependent on a G/C repressor contained within this promoter.5 Importantly, however, the environmental cues that evoke SMC phenotypic switching associated with mechanical injury of a normal vessel and those that mediate this process within multicellular spontaneously occurring atherosclerotic lesions are undoubtedly very different, and at present, virtually nothing is known regarding the molecular mechanisms and environmental factors that control SMC phenotypic switching in association with atherosclerotic disease.
A major limitation in determining molecular mechanisms and environmental factors associated with SMC phenotypic modulation is that it is very difficult to directly test causality in models of experimental atherosclerosis. Moreover, the fact that SMCs can take on many phenotypic states throughout lesion development greatly limits identification of these cells by traditional immunohistological techniques for SMC differentiation markers. Thus, as an alternative, our laboratory has used a strategy focused on transcriptional regulation of SMC differentiation marker genes. That is, one can use SMC differentiation marker gene promoter–reporter transgenes to determine common cis elements and trans factors that are required for gene repression and then subsequently determine factors and mechanisms that regulate the activity of these elements in vivo and in vitro. Using this "inside-out" approach, studies presented here provide evidence that the decreased expression of the SMC differentiation marker gene SM22 associated with phenotypic switching in advanced experimental atherosclerotic lesions is attributable to transcriptional repression. Moreover, we have identified a cis"repressor" element in the SM22 promoter that, when mutated, prevents repression of SM22 promoter activity in phenotypically modified SMCs of experimental atherosclerotic lesions, and we show that PDGF-BB and the trans factor Sp1 suppress SM22 via this same repressor element.
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Materials and Methods |
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Assessment of Transgene Expression in the Apolipoprotein E–Deficient Mouse Model of Atherosclerosis
Animal protocol models were approved by the University of Virginia Animal Care and Use Committee. Transgenic founder lines described in the Table were described previously by Regan et al.5 Mice carrying the SM22
promoter–ß-galactosidase (ß-gal) reporter transgene or the SM22GC (-256/-249 deletion 5'-ggccgccc) promoter–ß-gal reporter transgene (B6.CBA backcrossed to B6) were crossed to apolipoprotein E (ApoE)–deficient mice (B6.129P2-Apoetm1Unc; The Jackson Laboratory) and placed on a Western-type diet (by weight 21% fat, 0.15% cholesterol, 19.5% casein; Harlan/Teklad) for 18 to 20 weeks. Mice were euthanized in a carbon dioxide chamber. Approximately 0.5 cc of blood was drawn from the left ventricle for total cholesterol assays (Infinity Cholesterol Reagent; Sigma). Mice were perfusion fixed with 2% formaldehyde/0.2% glutaraldehyde, the aortic tree harvested, and stained for ß-gal activity using X-gal (5-bromo-4-chloro-3-indolylb-D-galactoside; Sigma) as described previously.5 Care was taken to ensure that all samples were stained under the same conditions in parallel on the same day. The aortic arch was processed for routine histology and sections were counterstained with eosin.
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Cell Culture, PDGF-BB Stimulation, Transient Transfections
Cultured rat aortic SMCs were transiently transfected and stimulated with PDGF-BB (Upstate Biotechnology) or vehicle (fatty acid-free BSA [2 µg/mL] and acetic acid [10 mmol/L]) for the noted time period as described previously.6 The promoter luciferase constructs included: SMMHC-luc (–420/
11 600 bp), SM22-luc (–447/89 bp), and SM22GC-luc (-256/-249 G/C box deletion ggccgccc). CMV-Sp1 was a gift from Robert Tijian (Howard Hughes Medical Institute, University of California).
Short Interfering RNA Plasmids
As described previously, a plasmid-based system for the production of short interfering RNA (siRNA) using the minimal mouse H1 promoted was used (pMighty).7,8 To generate the siRNA specific for Sp1 (si-Sp1), an oligonucleotide (ttgagtcacccaatgagaa; specific 19-bp target sequence to rat Sp1) was inserted downstream of the H1 promoter of pMighty. As a control for reported nonspecific/"off-target" si-RNA effects, an empty H1 vector (si-Empty) and a random 19-bp oligonucleotide sequence (si-Scramble; gtcgactgtggattggcat) were generated to achieve no sequence identity for reported human, mouse, or rat mRNA sequences.
Quantitative Chromatin Immunoprecipitation Assay
Quantitative chromatin immunoprecipitation (ChIP) was performed as described previously.8 Antibodies included rabbit polyclonal anti-serum response factor (SRF; Santa Cruz Biotechnology), rabbit polyclonal anti-Sp1 (Santa Cruz Biotechnology and Upstate Biotechnologies). Recovered DNA was quantified by fluorescence with picogreen reagent (Molecular Probes) according to manufacturer recommendations. Real-time polymerase chain reaction (PCR) was performed on 1 ng genomic DNA from ChIP experiments. A total of 20 pmol of each primer with Sybergreen reagent (SG) or dual-fluorescence labeled probes were used in each reaction. Real-time PCR primers were designed to flank the 5' CArG–GC–CArG promoter motifs of SM22 and SMMHC and the c-fos 5' CArG as a control. Primer/probe sequences were as follows: SM22 (rat) FOR 5'-tttcgtggtcctgcccataaaag-3', REV 5'-tcactccacacaggctccatatt-3' PROBE 5'-tcccgccaccctcagcaccgc-3'; SMMHC (rat, SG) FOR 5'-ctgcgcgggaccatatttagtcagggggag-3', REV 5'-ctgggcgggagacaacc caaaaaggccagg-3'; c-fos (rat, SG) FOR 5'-cggttccccccctgcgctgcaccctcagag-3', REV 5'-agaacaacagggaccggccgtggaaacctg-3'. Quantification of protein:DNA interaction/enrichment was determined by the following equation: 2Ct(Ref)–Ct(IP)–2Ct(Ref)–Ct(No antibody control).
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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A Conserved G/C Repressor Element Within the SM22
Promoter Was Required for Transcriptional Repression of the ß-Gal Transgene In Vivo in Advanced Experimental Atherosclerotic LesionsTo determine the role of the SM22
promoter G/C repressor element in transcriptional regulation of the SM22 promoter–ß-gal transgene in advanced experimental atherosclerotic lesions, SM22–ß-gal and SM22GC–ß-gal reporter transgenic mice were bred to ApoE–/– mice to generate mice carrying either the wild-type or G/C mutant SM22 promoter–ß-gal reporter transgene construct and homozygous for the ApoE gene knockout. At 6 weeks of age, SM22–ß-gal /ApoE–/– and SM22GC–ß-gal/ApoE–/– mice were placed on a Western diet for 18 to 20 weeks. Both crosses showed a significant increase in total blood cholesterol when compared with SM22–ß-gal/ApoE+/– or SM22GC–ß-gal/ApoE+/– and ApoE+/– without either promoter transgene construct (Table). To determine whether mutation of the G/C repressor element altered the SM22–ß-gal reporter transgene expression, the aortic tree and heart were dissected intact and stained for ß-gal activity. Mutation of the G/C repressor element resulted in minimal loss of transgene expression (ß-gal staining) in regions of the aortic tree that are highly susceptible to atherosclerotic lesion development at 18 to 20 weeks9 (Figure 1a versus 1f). On more detailed histological analysis of lesions of the aortic arch, it was apparent that the G/C mutation in the SM22 promoter prevented ß-gal downregulation in cells located in the fibrotic cap of the lesion, cells at the shoulder of the fibrotic lesion, and cells residing in the medial layer below the lesion and proximal to the internal elastic lamina (Figure 1h through 1j) compared with wild-type SM22 promoter–ß-gal expression patterns (Figure 1c through 1e). Of major importance, in nonatherosclerotic regions of the thoracic aorta, ß-gal staining was not significantly different between SM22–ß-gal and SM22GC–ß-gal (Figure 1b and 1g), thus providing an internal control for ß-gal staining between mice and clearly showing that attenuated repression of the G/C mutant SM22 promoter transgene compared with the wild-type SM22 promoter transgene was highly restricted to regions of lesion formation. These results indicate that the SM22 G/C repressor element plays a key role in mediating transcriptional repression of this gene in cells that compose the advanced atherosclerotic lesion, the fibrous cap, and even SMCs of the media. Moreover, results provide novel evidence that the G/C mutant SM22 promoter transgene, unlike the wild-type promoter transgene, retains its activity within phenotypically modulated SMCs within lesions, which may have major implications for lineage tracing studies in the field as well as for efforts aimed at targeting expression of therapeutic genes to the phenotypically modulated SMC. It is important to note that the wild-type and G/C mutant SM22 promoter transgenic lines used in these studies contain a normal endogenous SM22 gene such that one would not expect alterations in lesion formation as was observed. Indeed, a major advantage of this transgenic model is that it allows one to ascertain mechanisms that contribute to SMC gene expression in the context of normal experimental lesion formation.
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PDGF-BB Suppressed SM22 Promoter Activity Via the G/C Repressor
Results shown in Figure 1 clearly show that mutation of the SM22 G/C repressor was sufficient to greatly attenuate loss of SM22 promoter transgene expression within SMCs in advanced atherosclerotic lesions. There is a wide plethora of cytokines and signaling pathways that are implicated in spontaneous intimal lesion development of the ApoE–/– atherosclerotic mouse that could potentially contribute to SMC phenotypic modulation and specifically to G/C repressor dependent downregulation of the SM22 transgene, including PDGF-BB.10 PDGF-BB is unique in its ability to potently and selectively repress expression of virtually all known SMC differentiation marker genes, including SM22, SM -actin, and SMMHC at the transcriptional and post-transcriptional levels.4,6,11 Finally, there is extensive evidence showing that expression of PDGF A and B chains and PDGF receptors are elevated within the intima of human and experimental atherosclerotic lesions.12,13 Of particular relevance to the present studies, we have demonstrated previously that the G/C repressor located within the SM22 and SMMHC promoters binds to Sp1 and Sp3 within SMC nuclear extracts.5,14,15 However, as yet, no studies have been done to test whether Sp1 mediates transcriptional repression or whether it plays a role in PDGF-BB–induced phenotypic switching in cultured SMCs. Results of transient transfection assays in cultured rat aortic SMCs showed that PDGF-BB treatment decreased SM22 and SMMHC promoter activity in a dose-dependent manner (Figure 2). Of major interest, PDGF-BB–induced suppression of the SM22 promoter was greatly reduced by the same mutation of the G/C repressor shown to abrogate suppression of the SM22 promoter reporter transgene in vivo within atherosclerotic lesions (Figure 1). These effects were concentration dependent in that the G/C-rich repressor was required for PDGF-BB–induced suppression at PDGF-BB concentrations of 1 and 10 ng/mL but not at 30 ng/mL (Figure 2).
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PDGF-BB Suppresses SM22 in Part Via the Transcription Factor Sp1
Sp1 is one member of a family of four zinc finger transcription factors (Sp1–Sp4). Previous studies by our laboratory showed Sp1 protein expression was increased in the neointima after balloon injury of the rat carotid artery.14 Similarly, McCaffery et al16 showed a relative increase in Sp1 message levels in the fibrous cap of human atherosclerotic lesions, consistent with the hypothesis that Sp1 may be relevant to SMC phenotypic switching in humans. However, it is unknown whether Sp1 serves as a repressor of SMC differentiation marker promoter activity or an activator and whether PDGF-BB–mediated regulation of these genes involves Sp1. Results of the present studies show that overexpression of plasmid DNA containing Sp1 resulted in a dose-dependent decrease in SM22 and SMMHC promoter activity (Figure 3). Mutation of the SM22 G/C repressor prevented Sp1-induced suppression of SM22 at lower plasmid DNA concentrations and attenuated the response at higher concentrations. PDGF-BB treatment of cultured rat aortic vascular SMCs resulted in an increase in expression of Sp1 as well as nuclear localization (Figure 4a and 4b). Results showed near-maximal induction of Sp1 mRNA (Figure 4c) and protein (Figure 4d) at 2 to 4 hours.
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To determine whether endogenous Sp1 and PDGF-BB–induced expression of Sp1 regulate SMC differentiation marker gene expression, the effect of an siRNA specific for Sp1 was examined by a plasmid-based siRNA production system. Rat aortic SMCs were transfected with an siRNA construct to Sp1 (si-Sp1) and Sp1 protein expression measured at 24, 48, and 72 hours. As depicted in Figure 5a, relative Sp1 protein expression was decreased by 50% at 48 hours and by 70% at 72 hours. Cotransfection of the SM22 or SMMHC promoter–reporter construct with si-Sp1 resulted in an 3.5-fold and 7.0-fold increase in promoter activity, respectively, when compared with cells cotransfected with an empty siRNA plasmid (si-Empty) or a scrambled siRNA plasmid (si-Scramble) as an "off-target" control (Figure 5b and 5c). si-Sp1 overexpression increased basal and partially blocked the PDGF-BB–induced decrease in SM22 and SMMHC promoter activity compared with controls (Figure 5b and 5c). These results provide direct evidence that Sp1 acts as a negative regulator of basal SM22 and SMMHC promoter activity and appears to contribute to PDGF-BB–induced suppression of SM22 and SMMHC. To rule out a generalized effect of the si-Sp1 construct as an activator of promoter reporter constructs, we tested the effect of si-Sp1 on the aortic carboxy-like peptidase (ACLP) promoter–reporter construct. Previous studies by Layne et al17 showed that ACLP promoter–ß-gal activity was increased in injured carotids of transgenic mice and that overexpression of Sp1 increased ACLP promoter–reporter activity in cultured cells. Of interest, in contrast to effects of the si-Sp1 on SM22 and SMMHC, results showed that although PDGF-BB had no effect on ACLP promoter activity, cotransfection with si-Sp1 decreased ACLP promoter activity by 50%, consistent with endogenous Sp1 acting as a positive regulator of ACLP (Figure 5d).
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PDGF-BB Decreased SRF Enrichment of the CArG1 and CArG2 SM22 Promoter Region Within Intact Chromatin
Sequence comparisons of the SM22 and SMMHC promoters have identified a common motif of cis regulatory elements with the G/C-rich repressor element situated between 2 positive cis regulatory elements, SRF response elements (SRE or CArG; ie, the CArG2–G/C–CArG1 motif; Figure 6a). Previous studies in our laboratory showed that the SM22 and SMMHC G/C repressor bound Sp1 by electrophoretic mobility shift assays.5,14,15 However, we further demonstrated that Sp1 binding to the SMMHC G/C repressor did not prevent SRF binding to the adjacent CArG2, which is separated from the G/C repressor by 6 nucleotides14 (Figure 6a). Inhibition of SRF binding to CArG1 by Sp1 is also unlikely given that CArG1 is located 100 nucleotides from the G/C repressor (Figure 6a). Although these data argue that Sp1 does not alter SRF binding to CArG boxes, there is lack of direct evidence for this mechanism and the involvement of Sp1 regulation of SRF binding to the endogenous SMC marker genes within the context of intact chromatin. Quantitative ChIP assays8 were thus used to determine the effect of PDGF-BB treatment on SRF and Sp1 enrichment of the CArG2–G/C–CArG1 motif in the SM22 or SMMHC promoters in intact chromatin. We did not detect PDGF-BB–induced Sp1 enrichment of the SM22 or SMMHC CArG2–G/C–CArG1 promoter regions after PDGF-BB treatment. However, PDGF-BB treatment resulted in a large decrease in SRF enrichment of the SM22 and SMMHC CArG2–G/C–CArG1 promoter regions by 54% and 74%, respectively (Figure 6b). In contrast, PDGF-BB treatment resulted in increased SRF enrichment of the c-fos CArG region by 79% (Figure 6b), results that are consistent with the PDGF-BB–induced activation of the c-fos promoter.6 These results suggest that Sp1-induced repression of SMC genes may involve more complex mechanisms than simple binding of Sp1 to the G/C repressor and subsequent inhibition of cooperative interactions between CArG elements (see Discussion for a detailed consideration of this issue). However, the inability to detect PDGF-BB–induced enrichment in the ChIP assay may simply be because of epitope masking by chromatin or a higher-order complex of transcription factors.
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Discussion |
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The major focus of the studies presented in this report was to identify molecular mechanisms that regulate SMC differentiation marker gene expression associated with SMC phenotypic switching in experimental atherosclerosis. A number of novel results are presented. First, we provide convincing evidence that suppression of SM22
gene expression within spontaneously occurring atherosclerotic lesions, at least in an animal model, are mediated in part by transcriptional repression. Second, we show that mutation of the G/C cis element in SM22 promoter–ß-gal transgene resulted in nearly complete loss in downregulation of this gene in SMCs within intimal atherosclerotic lesions in the aortic arch of ApoE–/– mice, as well as cells in the fibrous cap and shoulder of the plaque. These regions of the lesion, in particular, have been proposed to be composed of vascular SMCs, the function of which is to stabilize the complex plaque by forming a fibrotic cap. As shown here, these cells fail to express the wild-type SM22 promoter–ß-gal transgene, similar to findings by Feil et al18 that showed decreased SM22 protein expression in advanced ApoE–/– atherosclerotic lesions. Only when the SM22 G/C repressor was mutated were we able to detect SM22 promoter–ß-gal transgene expression in these regions of the lesion containing phenotypically altered SMCs. As such, the SM22 G/C mutant promoter–reporter transgene permits identification of phenotypically modulated SMCs even within advanced experimental atherosclerotic lesions, a property that may have considerable practical utility for studying SMC lineage, including in cells derived from bone marrow stem cells, as well as for purposes of targeting gene therapies to these cells. Moreover, of key importance, these results provide novel insights regarding the molecular mechanisms that contribute to SMC phenotypic switching in vivo, at least with respect to the SM22 gene, as well as other marker genes such as SMMHC, which has been shown to exhibit dependence on the G/C repressor.14,15
Of interest we found that PDGF-BB and Sp1 suppressed SM22 in part via the G/C cis repressor element. Moreover, an siRNA to Sp1 increased SM22 and SMMHC promoter activity and attenuated PDGF-BB–induced suppression, suggesting that Sp1 is a downstream repressor factor regulated by PDGF-BB. Although ChIP assays clearly showed that PDGF-BB greatly decreased SRF enrichment of the CArG2–G/C–CArG1 motif of both the SM22 and SMMHC promoters, we were not able to detect Sp1 enrichment of this promoter region associated with PDGF-BB treatment, motifs that were shown previously to bind Sp1 by in vitro gel shift assays.5 These results suggest that Sp1-induced repression of SMC genes may not be a simple function of binding of Sp1 to the G/C repressor and subsequent inhibition of cooperative interactions between CArG elements, as suggested previously by our laboratory.14 There are several alternative possibilities. First, it is possible that Sp1 interacts with other transcription factors required for SRF binding to CArG boxes, such as myocardin,7,19 and thereby inhibits SRF binding to SMC differentiation marker promoter CArG boxes. Indeed, a similar model has been proposed for the zinc finger-containing factor YY1, which is induced in the media of injured vessels after balloon injury20 and proposed to act as an endogenous inhibitor of SRF-dependent gene expression by displacing SRF from CArG boxes of SMC differentiation marker genes.21 Such a model would be applicable to Sp1, YY1, GKLF, ets-1, egr-1, and other transcription factors that are increased with vascular injury6,14,20,22 and atherosclerosis16 and would allow for SRF to selectively bind to CarG-related growth regulatory genes, such as c-fos, as we observed in our ChIP assays (Figure 6). Consistent with this model, recent studies by Wang et al23 showed that PDFG–BB-induced suppression of SM22 was partially mediated by elk-1 interaction with myocardin. Second, decreased SRF binding to CArGs may simply be a function of post-translational modification24 or decreased SRF expression. Regarding the latter, SRF expression in response to PDGF-BB either does not change23 or increases.25 Third, Sp1 may recruit histone deacetylases to SMC differentiation marker gene promoter regions thus silencing the gene, as has been shown to occur in silencing of the human telomerase reverse transcriptase promoter by Sp1.26 Recent studies by our laboratory support this model in that PDGF-BB decreases H4 acetylation in SMC differentiation marker CArG promoter regions but increases H4 acetylation in the c-fos CArG region (O.G.M., B.R.W., and G.K.O., 2004, unpublished data), the latter being consistent with gene activation.
The finding that Sp1 suppressed SM22 and SMMHC expression is interesting because Sp1 is generally considered to be an activator of gene expression. However, there are a number of previous studies by others that have also implicated Sp1 in transcriptional repression. For example, Silverman et al27 demonstrated that egr-1 displaced Sp1 from the PDGF-A chain promoter to activate transcription, suggesting that Sp1 can function as a repressor in this system. An alternative possibility is that the effects of Sp1 in suppressing SMC differentiation marker gene expression may be indirect and occur by activating other repressor pathways. Studies by Rafty et al28 partially support this model whereby Sp1 activation regulates PDGF-B chain expression in newborn rat aortic SMCs, potentially activating the PDGF-BB–mediated repressor pathway for suppression of SMC differentiation marker genes. Sp1 has also been shown to activate the ACLP promoter via G/C cis regulatory elements,17 a result confirmed in the present studies (Figure 5). Interestingly, Layne et al17 also showed that ACLP promoter–ß-gal transgene activity was increased in acute vascular injury. Although the function of ACLP as a regulator of SMC phenotypic modulation or a distinct marker of the phenotypically altered SMC remains uncertain, these results are quite interesting in that they suggest that common transcription factor pathways may be in involved in differential regulation of genes that are either activated or repressed during SMC phenotypic switching.
The preceding culture studies suggest that there may be several redundant pathways by which Sp1 and other transcription factors suppress SMC differentiation marker gene expression. However, the results presented in the present studies suggest that this may not be the case in vivo. That is, abrogation of a single repressor pathway alone (ie, the SM22 G/C repressor) was sufficient to nearly block SM22 promoter–ß-gal transgene suppression within complex experimental atherosclerotic lesions (Figure 1). Thus, if redundant suppressor mechanisms exist, they failed to overcome the loss of this pathway, suggesting that it may play a dominant role in suppression of SM22. Extensive further studies will be required to determine the broad significance of this finding for additional SMC differentiation marker genes and to identify environmental factors present within lesions that activate these repressor mechanisms. Whereas we have presented no direct evidence that PDGF-BB–dependent signaling contributes to SMC phenotypic switching or repression of SM22 gene expression in vivo, we have presented extensive evidence in support of the hypothesis that it may play a key role in mediating this response. However, because PDGF-B chain or ß-receptor knockout mice die during embryonic development,29,30 direct proof of this hypothesis will require generation of SMC-targeted conditional knockout mice. Until such studies become feasible, we feel that further use of transgenic and ApoE knockout mouse model systems as used in the present studies have tremendous utility for elucidating candidate factors and regulatory mechanisms that contribute to the plethora of changes in gene expression characteristic of SMCs within spontaneously occurring atherosclerotic lesions.
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Acknowledgments |
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This work was supported by National Institutes of Health grants R37 HL57353, RO1 HL38854, and P01 HL19242 to G.K.O.; MSTP 2T32-GM07267-22 to O.G.M. and M.H.H.; American Heart Association Fellowship to S.S.; and American Physiological Society Physiological Genomics Fellowship to B.R.W. We thank Rupa Tripathi and Diane Raines for their excellent technical assistance and Sheri L. Vanhoose of the Histology Core.
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Footnotes |
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Original received August 16, 2004; revision received September 27, 2004; accepted October 6, 2004.
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References |
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