Published online before print February 21, 2002, doi: 10.1161/01.RES.0000013289.97650.C8
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© 2002 American Heart Association, Inc.
Molecular Medicine |
Characterization of the Mouse Aortic Carboxypeptidase-Like Protein Promoter Reveals Activity in Differentiated and Dedifferentiated Vascular Smooth Muscle Cells
From the Pulmonary and Critical Care (M.D.L., S.-F.Y., X.L., B.I., M.A.P.) and Cardiovascular (K.M., C.-M.H., M.-E.L.) Divisions and the Department of Medicine (M.D.L, S.-F.Y., C.-M.H., M.-E.L., M.A.P.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Matthew D. Layne, PhD, Brigham and Women’s Hospital, Pulmonary Division TH1333, 75 Francis St, Boston, MA 02115. E-mail mlayne{at}rics.bwh.harvard.edu
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Abstract |
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The dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) contribute to the formation of vascular lesions. In this study, the regulation of aortic carboxypeptidase-like protein (ACLP) expression in VSMCs was investigated. After mouse carotid injury, the expression of ACLP increases in the dedifferentiated VSMCs of the neointima in a pattern that differs from that of smooth muscle
-actin. To better understand the regulation of ACLP in VSMCs, we characterized the 21-exon mouse ACLP gene and 5'-flanking region and examined its promoter activity. In transient transfection assays, 2.5 kb of the ACLP 5'-flanking sequence directed high levels of luciferase reporter activity in primary cultured rat aortic smooth muscle cells, and this activity was not dependent on serum response factor. We identified a positive element between base pairs -156 and -122 by analysis of 5' deletion and mutant constructs. By use of electrophoretic mobility shift assays with rat aortic smooth muscle cell nuclear extracts, Sp1 and Sp3 transcription factors bound to this region, and transfection assays in D.Mel.2 cells revealed that both Sp1 and Sp3 transactivated the ACLP promoter. Transgenic mice harboring the -2.5-kb ACLP promoter upstream from a nuclear-targeted LacZ gene were generated, and expression was detected in the VSMCs of large blood vessels, arterioles, and veins. Interestingly, ACLP promoter–LacZ reporter activity increased within the neointimal VSMCs of injured carotid vessels, consistent with the expression of the endogenous ACLP protein. The ACLP promoter may provide a novel tool to target gene expression to dedifferentiated VSMCs.
Key Words: vascular biology • carotid injury • aortic carboxypeptidase-like protein • transgene
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Introduction |
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Vascular smooth muscle cells (VSMCs) are derived from both neural crest and local mesenchymal cell precursors during embryonic development.1–3 In the adult, these highly specialized cells express a unique set of proteins that are necessary for proper vessel structure and contractile function.3 In response to injury, VSMCs undergo a phenotypic change, adopting a proliferative and synthetic phenotype, reminiscent of embryonic smooth muscle cells (SMCs).4–7 Concomitant with the change from the contractile to the synthetic phenotype, the expression of numerous gene products is altered. These changes are elicited and modified by extracellular stimuli, including growth factors, cytokines, and extracellular matrix molecules.3 Alterations of the VSMC phenotypic state contribute to the progression of vascular disease, including atherosclerosis and restenosis after vessel injury.7,8
The transcriptional regulation of genes in vascular smooth muscle during development and in pathogenic states is beginning to be elucidated. In particular, serum response factor (SRF), a transcription factor that binds to the CArG box, is involved in the regulation of several smooth muscle–specific genes, including SM22, smooth muscle myosin heavy chain (SM-MHC),9 smooth muscle -actin (SM -actin),10,11 and calponin.12 In addition, other transcription factors contribute to the regulation of VSMC gene expression. For example, myocyte-specific enhancer factor-2 is important for SM-MHC expression13 and vascular development,14 upstream stimulatory factor (USF) contributes to SM -actin gene expression, 15 and Sp1 activates SM-MHC gene expression.16 The regulatory sequences of many VSMC-expressed genes have been studied by using transgenic animals, which have yielded important information on the transcriptional regulation of VSMC genes in vivo.9,10,17–21 However, these promoters are often downregulated in response to vessel injury; thus, they cannot be used to target therapeutic gene products to potentially prevent VSMC proliferation.20
Previously, we identified aortic carboxypeptidase-like protein (ACLP), a protein that is highly expressed in the VSMCs of blood vessels.22 ACLP is an extracellular protein that contains a domain with similarity to the slime mold protein discoidin I and blood coagulation factors V and VIII.23 In addition, ACLP has a catalytically inactive metallocarboxypeptidase domain at its carboxyl terminus.24 ACLP expression is induced during the differentiation of neural crest precursors to the SMC lineage.22 During embryonic development, ACLP is expressed highly in the VSMCs of the blood vessels, in the dermal layer of the skin, and in the developing skeleton.25 Initial characterization of ACLP-null mice revealed impaired abdominal wall development and deficient dermal wound healing.25 Although we know that ACLP mRNA expression is regulated in cultured and differentiating VSMCs,22 nothing is presently known about the mechanism of its regulation in blood vessels in vivo.
The focus of the present study was to characterize the expression of ACLP in normal and injured blood vessels and to investigate the regulation of the mouse ACLP promoter in VSMCs. We designed experiments to identify potential cis-acting elements within the ACLP promoter and the transcription factors that bind to these sites. In addition, we wanted to investigate the regulation of the ACLP promoter in vivo in transgenic mice.
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Materials and Methods |
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Cloning and Characterization of the Mouse ACLP Promoter
Genomic clones were isolated from a 129 SvJ mouse
DASH II genomic library (Stratagene) as described.25 Southern blot analysis was performed as described.21 The ACLP 5'-flanking sequence, including exon 1, was isolated as an 
3-kb HindIII fragment from phage DNA and sequenced (GenBank accession No. AF332596).
Plasmid Constructs
ACLP promoter deletion fragments and site-directed mutations were generated by polymerase chain reaction with the use of Pfu polymerase (Stratagene) and cloned into the luciferase reporter pGL2-Basic (Promega). The plasmid pCMVß was obtained from Clontech. Sp1 and Sp3 pPAC expression constructs were provided by Dr Jonathan M. Horowitz (Raleigh, NC).26 The expression vector pCGN-SRF was provided by Dr Michael Gilman (Cambridge, Mass), and the dominant-negative construct pCGN-DN-SRF (amino acids 1 to 269) was generated by polymerase chain reaction and cloned into pCGN. The Drosophila expression plasmids pPAC and phsp82LacZ were provided by Dr Tom Maniatis (Cambridge, Mass).27
Cell Culture and Transient Transfection Assays
Rat aortic smooth muscle cells (RASMCs) were isolated and cultured as described.22,28 RASMCs (2x105 cells per 35-mm well) were transiently transfected by using FuGENE 6 reagent (Roche Molecular Biochemicals), and luciferase and ß-galactosidase activity were measured after 2 days.21 Schneider’s Drosophila line 2 (D.Mel.2) cells (Invitrogen) were transfected with luciferase reporter, expression plasmids, and phsp82LacZ to normalize for transfection efficiency with the use of CellFECTIN (Invitrogen).
Electrophoretic Mobility Shift Assays
Nuclear proteins were isolated,29 and protein concentrations were determined by using the Bio-Rad protein assay kit. Complementary oligonucleotides were synthesized by Life Technologies, annealed, and end-labeled with the use of T4 polynucleotide kinase (New England Biolabs) and [
-32P]ATP (NEN Life Science Products). DNA-binding assays using 5-µg nuclear extract were performed essentially as described.30 For supershift assays, 2 µg of Sp1, Sp3, or USF2 (Santa Cruz) antibody was added to the reaction mixture. For competition assays, unlabeled competitors were added at 100-fold molar excess.
Transgenic Mouse Production
The -2502 to 176-bp ACLP promoter fragment was ligated upstream from the nuclear localized LacZ reporter vector (pPD46.21, provided by Dr Andrew Fire, Baltimore, Md). Transgenic mice were generated, and tissues were stained as described.21 Animal use conformed to federal guidelines and institutional policies.
Mouse Arterial Remodeling Model and Histological Analysis
The left common carotid artery of male FVB mice (6 to 12 weeks) was ligated with a 5-0 suture just below the bifurcation, as described.31 Tied and control vessels were analyzed histologically, as described,25 by using antibodies against ACLP, 22 SM -actin (Sigma Chemical Co), and platelet and endothelial cell adhesion molecule (PECAM, Pharmingen) or for ß-galactosidase activity.21
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Results |
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ACLP Expression in Injured Carotid Arteries
Previously, we cloned the human ACLP cDNA from a human aorta cDNA library and its mouse homologue and determined that ACLP is highly expressed in VSMCs in vitro and in vivo.22 To examine the expression of ACLP in the vasculature after injury, we used a mouse model of carotid ligation that induces VSMC proliferation and neointimal formation after the cessation of blood flow.31 Immunohistochemical staining revealed strong staining for ACLP in the medial layer of the uninjured control right carotid artery (Figure 1A) in a pattern similar to that of SM
-actin (Figure 1C). In addition, ACLP expression was not expressed in endothelial cells delineated by the marker PECAM (CD31) (Figure 1E). The left carotid artery was ligated for 14 days (Figures 1B, 1D, and 1F). ACLP expression was detected within the medial layer as well as in the neointima (Figure 1B). Although there are similarities, this pattern differs somewhat from that of SM -actin, which is not present throughout the neointima and is more prominent in the media and in the VSMCs closest to the lumen (Figure 1D). PECAM staining indicated that the endothelium of the injured blood vessel remained intact (Figure 1F). The endothelial cells did not express ACLP (Figure 1B). These results indicate that ACLP expression persists in the neointimal SMCs of injured carotid arteries.
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Characterization of the Mouse ACLP Gene and Promoter
To better understand the regulation of ACLP expression in VSMCs, we cloned the mouse ACLP gene and promoter. The ACLP gene is composed of 21 exons spanning 12 kb in the mouse genome (Figure 2A and Layne et al25). Many of the exons of the ACLP gene are small (<200 bp). The first exon encodes the 5'-untranslated region and the initiating methionine (ATG). Exon 21 contains the termination codon (TGA) and the 3'-untranslated region. The discoidin-like domain is encoded by exons 11 through 14, whereas the carboxypeptidase-like domain of ACLP is generated by exons 15 through most of exon 21. Mouse ACLP and human ACLP are single-copy genes, which is indicated by the simple hybridization pattern on genomic Southern blotting (Figure 2B). Radiation hybrid mapping has indicated that human ACLP localizes near marker D7S478 on chromosome 7 (data not shown), which is homologous with mouse chromosome 11 (The Jackson Laboratory, Mouse Genome Informatics). Exon-intron junctions and the splice sites are consistent with the 5'-GT and 3'-AG consensus (Figure 2C). We have not detected any splice variants of mouse ACLP encoded by additional exons (data not shown).
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Sequence Analysis of the Mouse ACLP 5'-Flanking Region
To study the transcriptional regulation of ACLP and to evaluate regulatory elements that contribute to ACLP expression in VSMCs, we cloned and sequenced 2.5 kb of the 5'-flanking region of the mouse ACLP gene (Figure 3). The transcription start site, a CA pair, which is the most frequent transcription start site, 32 was determined by 5' rapid amplification of cDNA ends (RACE)22 and confirmed by RNase protection assays (data not shown). Analysis of the upstream and exon 1 sequence searching for RNA polymerase II recognition sites (BCM Search Launcher, Baylor College of Medicine Human Genome Sequencing Center) determined that this region is the predicted transcription start site. The ACLP gene does not contain a canonical TATA box; however, the promoter does contain multiple GC-rich elements consistent with non-TATA–initiated transcription (Figure 3).33 The mouse ACLP promoter contains a B1-type repetitive element (underlined).34 We did not detect any CArG boxes [CC(A/T)6GG] within the -2.5-kb promoter; this site has been shown to regulate the expression of many VSMC genes.9–12,35
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Characterization of ACLP Promoter Activity in VSMCs
We cloned the region from base pairs -2502 to 176 of the mouse ACLP promoter 5'-flanking sequence into the luciferase reporter vector pGL2-Basic. In transient transfection experiments in RASMCs, the largest construct (-2502) exhibited high luciferase activity (60% of pGL2-control). To identify the elements responsible for this transcriptional activity, we generated several 5' deletion constructs. Deletion constructs -354, -257, and -156 retained the majority of ACLP promoter activity compared with the longest construct (Figure 4). However, promoter activity of the -140 ACLP construct decreased substantially, and further decreases in activity were observed in the -122 construct. These results indicate that a strong positive element(s) exists within this small region. Deletion to -100 did not further alter the promoter activity, whereas the smallest (-75 and -58) constructs retained minimal basal promoter activity (Figure 4). Similar results were obtained in transfected mouse aortic SMCs (data not shown).
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ACLP Promoter Activity Is SRF Independent
Many SMC-expressed genes rely on SRF–CArG box interactions for their promoter and enhancer activities. To test whether SRF is involved in ACLP promoter regulation, we cotransfected a dominant-negative SRF (DN-SRF) expression construct with the -2502 ACLP promoter construct into RASMCs. Our experiments indicated that ACLP promoter activity was not altered by DN-SRF and not dependent on SRF in SMCs, whereas the CArG box containing -441 SM22 promoter was inhibited significantly by DN-SRF (Figure 4B).
Sp1 and Sp3 Transcription Factors Bind to ACLP Promoter Elements
To characterize the transcription factors that bind to the -156 to -122 region of the promoter, we performed electrophoretic mobility shift assays (EMSAs) with nuclear extracts prepared from RASMCs. Comparison of the sequence of this region with transcription factor databases (BCM Search Launcher, TRANSFAC, MatInspector) showed similarity with Sp1 and zinc-finger protein-binding sites. Oligonucleotide sequences used in the EMSA experiments are indicated (Figure 5A). With the use of the largest (-157 to -119) region as an oligonucleotide probe, two major complexes were identified in RASMC nuclear extracts (Figure 5B, I and II). To identify the composition of these complexes, antibodies specific for Sp1, Sp3, or the unrelated USF2 antibody were added to the binding reactions (Figure 5B). Both Sp1 and Sp3 antibodies supershifted the I and II complexes; this was indicated by the reduction in intensity of the lower complex and the appearance of a larger complex (Figure 5B, top two arrows). To determine the specificity of these complexes and to further localize the position of their binding within the -157 to -119 region, unlabeled oligonucleotides were included as competitors. The identical oligonucleotide (157/119) as well as consensus Sp1 oligonucleotides abolished complexes I and II (Figure 5B). However, the 157/138 oligonucleotide did not compete away these Sp1 and Sp3 complexes. The 140/119 cold competitor at 100-fold molar excess partially competed away the Sp1 and Sp3 complexes. The incomplete competition with either the 5' (157/138) or 3' (140/119) portions of the 155/122 oligonucleotide could indicate that the Sp1 and Sp3 binding sites are located near the center of this region. To test this possibility, we generated a new oligonucleotide (147/128) derived from the center of this region. EMSAs using RASMC nuclear extracts revealed similar complexes, I and II (Figure 5C). These complexes were supershifted with both Sp1 and Sp3 antibodies and competed with identical and Sp1 consensus oligonucleotides but not with the unrelated E-box oligonucleotide (Figure 5C). These results indicate that the binding site for the Sp1 and Sp3 transcription factors is within the -147 to -128 region. This result is consistent with the ACLP reporter activity in RASMCs (Figure 4).
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To further localize the binding site for the Sp1 transcription factors, we generated a series of mutant oligonucleotides and converted the GC-rich sites to A or T (Figure 5A). Gel shifts were performed with 100-fold molar excess of unlabeled competitors (Figure 5D). Mutants 1 and 3 failed to compete away the binding complexes I and II, whereas mutant 2 was effective in competing away these complexes (Figure 5D). These results indicate that binding sites I and II are contained within the region covered by these mutations. When the mutated oligonucleotides were used as probes, only the 157/119 mutation 2 retained complexes I and II, similar to the 157/119 wild type (Figure 5E). Binding was nearly abolished when the 157/119 mutation 1 was used as a probe and the 157/119 mutation 3 had minimal binding of complexes I and II relative to the wild-type oligonucleotide (Figure 5E).
Analysis of the ACLP Promoter Mutations
To analyze the functional importance of these sites, we generated mutations in the context of the -2502 ACLP promoter construct (Figure 5A). Transfection into RASMCs revealed that a single mutation (mutation 1 and, to a lesser extent, mutations 2 and 3) significantly inhibited the promoter activity compared with the wild-type construct (Figure 6A). Mutation of these sites in combination was more effective in inhibiting promoter activity, indicating the importance of these sites in the regulation of ACLP in SMCs (Figure 6A).
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Sp1 and Sp3 Transactivate the ACLP Promoter
To test the functional effects of Sp1 and Sp3 on the ACLP promoter, we transfected the Drosophila cell line D.Mel.2 because it lacks endogenous Sp1.36 These studies could not be performed in RASMCs because they express high levels of Sp1 and Sp3 (Figure 5), and exogenous Sp1 regulates the viral promoters used for transfection normalization (data not shown). Sp1 and Sp3 expression vectors driven by the actin 5C promoter (pPAC) were transfected into D.Mel.2 cells together with either the -156 or -100 ACLP luciferase reporter constructs and phsp82LacZ to normalize for transfection efficiency. Both Sp1 and Sp3 dose-dependently activated the -156 ACLP construct, with Sp3 being more potent in these assays (Figure 6). Combining Sp1 and Sp3 expression vectors (250 ng each) resulted in an additive effect on the -156 ACLP promoter activity (45-fold induction). Analysis of the -100 construct revealed that Sp1 and Sp3 also activated this construct but to a lesser extent than the -156 ACLP (Figure 6). These results indicate the presence of multiple positive regulatory sites (GC-rich Sp1/Sp3 sites) within the proximal promoter (Figure 3). These results are consistent with those obtained in RASMCs, in which the -100 ACLP construct retained activity (Figure 4).
Transgenic Mice Harboring ACLP Promoter Express Reporter Activity in VSMCs In Vivo
To test whether the ACLP promoter was active in vivo, we generated transgenic mice by using a construct with the -2.5-kb ACLP promoter cloned upstream from the nuclear-targeted LacZ gene. Three independent lines were analyzed (Figure 7 and data not shown). Tissues from the adult transgenic mice were removed, fixed, permeabilized with detergent, and stained for ß-galactosidase activity.21 We observed transgene expression in the SMCs of large arterial blood vessels, such as the descending aorta, but not in endothelial cells (Figure 7A). Strong expression was detected in the branch points of the descending aorta, which connect to the intersegmental arteries (Figure 7B). The expression of the transgene was not limited to arterial SMCs; ß-galactosidase activity was present in venous SMCs but not in endothelial cells (Figure 7C). In addition, ß-galactosidase activity was detected in the blood vessels supplying fatty tissue but not in the adipocytes (Figure 7D). Whole-mount views of the subcutaneous region revealed intense staining in the blood vessels (Figure 7E). Staining was observed in SMCs in the vascular cutaneous plexus (Figure 7F). Histological analysis of the blood vessels revealed that ß-galactosidase activity was present in the VSMCs but not in the endothelial cells of medium-sized blood vessels (Figure 7G) and arterioles (Figures 7H and 7I). Additional sites of expression, which were consistent with ACLP expression in vivo,25 included skeletal structures, such as the ribs, and the dermal layer of the skin (data not shown). We did not detect transgene expression in other muscle types, including skeletal, cardiac, or visceral smooth muscle (data not shown). These results indicate that the -2.5-kb ACLP promoter is sufficient to direct expression in VSMCs in adult mice and that it recapitulates the endogenous expression pattern of ACLP in vivo.
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ACLP Promoter Activity in Injured Carotid Arteries
The promoters of SM -actin, SM22, and SM-MHC are downregulated in response to vascular injury.20 Different from these genes, ACLP is expressed in the neointima of vascular lesions (Figure 1). To test whether the ACLP promoter driving ß-galactosidase was active in these dedifferentiated VSMCs, we ligated the left carotid artery and collected vessels after 7 and 14 days; the right carotid artery from the same transgenic mouse served as an uninjured control. The uninjured carotid artery contained positively stained VSMCs (Figures 8A and 8C). After 7 days, the ligated vessel contained many positively stained nuclei, indicating that the ACLP promoter was active (Figure 8B). Interestingly, after 14 days, transgene expression was very intense in the neointimal VSMCs (Figure 8D). These results are consistent with our immunostaining results (Figure 1) and indicate that the ACLP promoter is active in both differentiated and dedifferentiated VSMCs in vivo.
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Discussion |
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To investigate the transcriptional regulation of the ACLP gene in VSMCs, we cloned and analyzed its promoter first in vitro and then in vivo. The single-copy mouse ACLP gene is composed of many small, closely spaced exons (Figure 1). The exon structure of the discoidin domain of ACLP is generally conserved with other discoidin domains containing proteins such as coagulation factor VIII37 and the discoidin domain tyrosine kinase receptors.38 In addition, the carboxypeptidase-like domain of mouse ACLP is similar in structure to the rat carboxypeptidase E gene.39 It is possible that ACLP could have been generated during evolution through the process of exon shuffling.40
The ACLP promoter is regulated in RASMCs via a strong positive element (-156 to -122) (Figure 4), which is bound and transactivated by Sp1 and Sp3 transcription factors (Figures 5 through 7). Sp1 and Sp3 are ubiquitously expressed proteins that regulate numerous genes.41 It is difficult to explain the regulation of VSMC transcription through these factors alone. However, Sp1 interacts with other transcription factors, such as MyoD and SRF, to generate a muscle-specific multiprotein complex in cardiac cells.42 It remains to be determined whether additional proteins are interacting with Sp1 and Sp3 to activate the ACLP promoter through this site in VSMCs or whether additional smooth muscle–expressed coactivators are required.43
The expression of ACLP is similar in vivo to elastin, which is required for proper blood vessel morphogenesis and structure.44 Analogous to ACLP, Sp1 regulates elastin transcription.45,46 However, different from ACLP, the elastin promoter is not activated by Sp3 in Drosophila cells, whereas either Sp1 or Sp3 activated the ACLP promoter (Figure 6). Sp1 also regulates SM-MHC expression through an element16 that is similar in sequence to the -156 to -122 region of the ACLP promoter (Figures 3 and 5). Taken together, this information provides a potential regulatory mechanism for both ACLP and SM-MHC in VSMCs. Consistent with this hypothesis is our observation that ACLP and SM-MHC are both induced late in the differentiation of neural crest precursors to the smooth muscle lineage.22,47
The -2.5-kb ACLP promoter drove expression of the reporter gene activity in the VSMC cells of transgenic mice, including both venous and arterial SMCs. Moreover, reporter gene activity was present in both large and small vessels (arterioles) (Figure 7) in a pattern consistent with endogenous ACLP expression (Layne et al25 and data not shown). This expression pattern differs from other VSMC-specific transgenes, such as SM22, which is expressed preferentially in large arteries,17,18 or the SmLIM/CRP2 promoter, which is expressed in arterial but not venous VSMCs in vivo.21
The ACLP promoter was active in dedifferentiated VSMCs in injured carotid arteries (Figure 8). Although other transgenic animals with VSMC expression exhibit a downregulation of expression in the injured vessels, Regan et al20 identified a construct with a mutated G/C-rich repressor element that attenuated the injury-induced downregulation. This repressor element is found in both the SM22 and SM-MHC promoters and is bound by Sp1 and Sp3 factors.20,48,49 It is conceivable that the transcriptional complex that is repressing the SM-MHC and SM22 promoters in states of VSMC dedifferentiation is also responsible for activating the ACLP promoter or that these promoters are competing for limited coactivators.
The present study has identified an important element within the ACLP promoter that regulates its expression in cultured VSMCs and a larger (-2.5-kb) ACLP promoter that is sufficient to direct expression to VSMCs in vivo. Of particular interest is the expression of ACLP promoter activity in the neointimal SMCs (Figure 8) and in the arteriole-sized VSMCs (Figure 7). The continued expression of the ACLP promoter in the neointima may facilitate the targeting of proliferation-inhibitory gene products to prevent restenosis after arterial injury. In addition, targeted expression of specific products to the smaller resistance vessels may also be beneficial in the regulation of blood pressure. Future studies will examine the role of the identified Sp1 and Sp3 binding sites in the regulation of ACLP expression in vivo and potentially identify regions of the ACLP promoter that regulate expression in different VSMC subtypes.
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Acknowledgments |
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This study was supported by NIH Grants HL-10113 and AR-47861 (to M.D.L), HL-57977 (to M.-E.L and S.-F.Y), and HL-65639 (to M.A.P) and a grant from the March of Dimes Birth Defects Foundation (to M.-E. L and M.A.P).
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Footnotes |
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Deceased. 
Received October 24, 2001; revision received January 31, 2002; accepted February 7, 2002.
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