Published online before print November 9, 2006, doi: 10.1161/01.RES.0000252289.79841.d3
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© 2006 American Heart Association, Inc.
Molecular Medicine |
Wilms’ Tumor 1–Associating Protein Regulates the Proliferation of Vascular Smooth Muscle Cells
From the Robarts Research Institute (T.W.S., Z.B., C.B., C.O., Z.N., C.K., J.S., J.M., J.G.P.), London Health Sciences Centre (J.M., J.G.P.), Departments of Medicine (Cardiology) (J.G.P.), Biochemistry (J.G.P.), Medical Biophysics (J.G.P.), Anatomy and Cell Biology (W.R., N.R.), and Microbiology and Immunology (J.M.), University of Western Ontario, London, Canada.
Correspondence to J. Geoffrey Pickering, MD PhD, London Health Sciences Centre, 339 Windermere Rd., London, Ontario N6A 5A5. E-mail gpickering{at}robarts.ca
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Abstract |
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Smooth muscle cells (SMCs) are called on to proliferate during vascular restructuring but must return to a nonproliferative state if remodeling is to appropriately terminate. To identify mediators of the reacquisition of replicative quiescence, we undertook gene expression screening in a uniquely plastic human SMC line. As proliferating SMCs shifted to a contractile and nonproliferative state, expression of TIMP-3, Axl, and KIAA0098 decreased whereas expression of complement C1s, cathepsin B, cellular repressor of E1A-activated genes increased. Wilms’ tumor 1-associating protein (WTAP), a nuclear constituent of unknown function, was also upregulated as SMCs became nonproliferative. Furthermore, WTAP in the intima of injured arteries was substantially upregulated in the late stages of repair. Introduction of WTAP complementary DNA into human SMCs inhibited their proliferation, with a corresponding decrease in DNA synthesis and an increase in apoptosis. Knocking down endogenous WTAP increased SMC proliferation, because of increased DNA synthesis and G1/S phase transition, together with reduced apoptosis. WTAP was found to associate with the Wilms’ tumor-1 protein in human SMCs and WTAP overexpression inhibited the binding of WT1 to an oligonucleotide containing a consensus WT1 binding site, whereas WTAP knockdown accentuated this interaction. Expression of the WT1 target genes, amphiregulin and Bcl-2, was suppressed in WTAP-overexpressing SMCs and increased in WTAP-deficient SMCs. Moreover, exogenous amphiregulin rescued the antiproliferative effect of WTAP. These findings identify WTAP as a novel regulator of the cell cycle and cell survival and implicate a WTAP-WT1 axis as a novel pathway for controlling vascular SMC phenotype.
Key Words: amphiregulin • smooth muscle cells • Wilms’ tumor 1-associating protein • vascular smooth muscle cell proliferation
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Introduction |
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Phenotype plasticity is a feature of adult vascular smooth muscle cells (SMCs). A widely studied example of this is the dedifferentiation of mature, nonproliferative SMCs into proliferative SMCs, a process central to vascular remodeling.1,2
Although less well studied, an equally important manifestation of SMC plasticity is the reverse shift, whereby proliferative adult SMCs convert back to a nonproliferative state. This particular phenotype switch is essential for limiting SMC accumulation and for terminating vascular remodeling. As such, the regulatory factors that drive proliferative SMCs into a nonproliferative state, and hold them in that state, are critical for effective vascular remodeling and for limiting vascular disease.
We have generated unique lines of nonimmortalized human SMCs that are capable of converting between proliferative and nonproliferative states.2,3 In the presence of serum, these SMCs proliferate, migrate, and elaborate extracellular matrix similar to primary SMCs. On withdrawal of serum however they undergo a reproducible program of cellular maturation whereby they exit the cell cycle, migrate into multilayered aggregates, and acquire the ability to contract briskly. Because the SMCs are clonal, this shift to a nonproliferative and mature phenotype can be attributed to cellular plasticity, rather than selective expansion (or loss) of distinct cell subpopulations. The lines are thus well suited for studying the molecular basis of SMC phenotype switching.
We have sought out genes that are differentially expressed as human SMCs shift from a proliferative to a nonproliferative state and we report herein the identification of seven of these. We present detailed analysis on the product of one of these genes, Wilms’ tumor 1-associating protein (WTAP), a protein of unknown function not identified in the vasculature before. We demonstrate that WTAP is substantially upregulated as SMCs attain replicative quiescence, both in vitro and in vivo. We further show that WTAP associates with the Wilms’ tumor-1 (WT1) protein in SMCs, prevents this transcription factor from binding to target DNA, and thereby negatively regulates SMC growth. The findings identify a novel molecular control system for SMC proliferation.
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Materials and Methods |
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For an expanded Materials and Methods, please see the online data supplement available at http://circres.ahajournals.org.
Cell Culture
Experiments were performed using HITB5 and HITC6 SMCs, human clonal lines derived from the media of distal internal thoracic artery, as described previously.2,3 Cultures of rat vascular SMCs were generated by explant outgrowth from the thoracic aortas of Wistar Kyoto rats (Harlan, Indianapolis, Ind).
Transcript Analysis
Differentially expressed SMC transcripts were screened using a polymerase chain reaction (PCR) display approach, described in the online data supplement available at http://circres.ahajournals.org. Northern blot analysis was performed4 using subcloned PCR products as probes. Real-time quantitative RT-PCR was performed as described previously,5 with optimized primer-probe sets (Applied Biosystems, Foster City, Calif). Human GAPDH or rat ß2 microglobulin transcript abundance was used as an endogenous RNA control.
Laser Capture Microdissection of Rat Carotid Artery Intima
The left carotid artery of adult male Sprague-Dawley rats was balloon-injured, as described.6,7 The intima of harvested arteries was microdissected by laser capture (Veritas LCM System, Arcturus Engineering, Inc., Mountain View, Calif) from 8 µm-thick frozen sections stained with the HistoGene Staining Kit. RNA was extracted using TRIzol (Life Technologies) with addition of linearized polyacrylamide (2 mg/mL, Ambion, Austin, Tex) after phase separation.
Western Blot and Coimmunoprecipitation Analyses
Expression of WTAP and WT1 protein were evaluated by Western blot analysis.4 WTAP was detected using an affinity-purified rabbit polyclonal antibody raised against the peptide sequence N-CHVQNGLDSSVNV-C (Genemed Synthesis Inc.) and WT1 detected using a monoclonal antibody (Santa Cruz, Calif). For coimmunoprecipitation analysis, SMC protein extracts precleared with protein G PLUS agarose beads were mixed with either antibody or respective control antibody (Santa Cruz, Calif). SDS-PAGE-separated proteins were then immunoblotted for WTAP or WT1. Interference from IgG heavy chain signal was avoided by covalently linking all antibodies used for immunoprecipitation to G PLUS-Agarose.8
Overexpression of WTAP in Human SMCs
Full-length complementary (cDNA) encoding WTAP was amplified from HITB5 SMC mRNA by RT-PCR and subcloned into the pIRES-PURO3 vector (Clontech). The WTAP-IRES-PURO bicistronic fragment was then subcloned into the pQCXIP retroviral vector (Clontech). Retrovirus containing WTAP cDNA was produced and stable SMC transductants were generated, as described.9
Knockdown of WTAP Expression by RNA Interference
WTAP knockdown was accomplished by infecting human SMCs with retrovirus containing sequences encoding small hairpin RNA (shRNA) fragments.5 Three different 19-nucleotide sense and reverse complement WTAP-targeting sequences, starting at nucleotides 330, 638 and 1039 from the open reading frame, were designed (shRNA330 5'-CAATGGTAGACCCAGCGAT-3'; shRNA638 5'-GAAGTAGAGGGTATGCAGA-3'; shRNA1039 5'-CTCTCTCACACACCAATCA-3'). Control inserts (nonsilencing nsRNA) contained the gene-specific 19-nucleotide sense sequence but not the antisense sequence.
Cell Proliferation and DNA Synthesis
SMC proliferation and DNA synthesis were determined as previously described.2 Thymidine incorporation was normalized to DNA content, assessed by incubating an aliquot with Hoechst 33258 (500 µg/mL) and quantifying the signal by spectrophotometry (Hitachi F-4010).
Flow Cytometric Analysis of DNA Content
SMCs were dissociated with trypsin-EDTA and incubated in a detergent-trypsin solution.10 DNA was stained with 175 µg/mL propidium iodide and quantified on FACScalibur flow cytometer (BD Biosciences). DNA histogram analysis was performed using Cell Quest software (BD Biosciences).
Assessment of Apoptosis by Annexin V Staining
SMCs were suspended with trypsin-EDTA and incubated with FITC-Annexin V or R-phycoerythrin-Annexin V conjugates (Molecular Probes) together with 7-amino-actinomycin D (7-AAD, 1 µg/mL, Sigma). Apoptosis and necrosis were analyzed by flow cytometry (FACScalibur) using Cell Quest software.
DNA Binding Activity of WT1
Binding of WT1 to an oligonucleotide containing a consensus WT1-binding site (5'-TCGACCCTCGCCCCCGCGCCGGGC-3')11 was assessed using an ELISA-based DNA-binding assay (NoShift Transcription Factor Assay Kit, Novagen, Inc). Nuclear protein (NucBuster, Novagen, Inc, San Diego, Calif) was incubated with target, biotinylated DNA duplex in streptavidin-coated wells and bound WT1 protein was detected using an anti-WT1 monoclonal antibody.
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Results |
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Differential Gene Expression as Proliferative Human SMCs Convert to a Nonproliferative Phenotype
To identify genes involved in the switch from a proliferative to a nonproliferative adult human SMC, we compared transcripts expressed in replicating HITB5 SMCs with those expressed 72 hours after serum withdrawal. At this time, HITB5 SMCs have exited the cell cycle and acquired a stable, contractile phenotype.2 A total of 2100 PCR products were compared and 28 differentially expressed products were identified. The sequence of each product was determined, and the respective expression profiles were reevaluated by Northern blot analysis. This process identified 9 genes of interest. Two of these, Pre-B-cell colony-enhancing factor (nicotinamide phosphoribosyltransferase) and a novel subunit of prolyl 4-hydroxylase (
[III]) have been reported by us elsewhere.5,12 In addition to these genes, TIMP-3, Axl, and KIAA0098 were found to be downregulated as SMCs shifted to quiescence. Complement C1s, Cathepsin B, cellular repressor of E1A-activated genes (CREG), and Wilms’ tumor 1-associating protein (WTAP) were upregulated. Northern blots depicting the expression profiles of each of these transcripts are depicted in Figure 1 and 2
A.
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P<0.01 vs day 14.
WTAP is Upregulated in Mature, Nonproliferative SMCs In Vitro and In Vivo
Of these differentially expressed genes, WTAP was chosen for further study because: 1) it has not been detected in SMCs before; 2) it was the only gene known to encode a protein localized exclusively in the nucleus; and 3) it is a poorly understood protein. WTAP was first identified in a kidney cell line, based on an association with the Wilms’ tumor-1 (WT1) protein,13 but its function remains unknown.
To assess the consistency with which WTAP was dynamically expressed in SMCs, we studied a second human SMC line, HITC6, that also enters a stable, nonproliferative state after serum withdrawal.3 As shown in Figure 2A, WTAP transcript abundance in HITC6 SMCs tripled (3.0±0.2 P<0.01) 8 days after serum withdrawal, as assessed by quantitative RT-PCR.
To determine whether there was a corresponding increase in WTAP protein, a polyclonal anti-WTAP antibody was raised in rabbits. This antibody identified a single band at 47 kDa that was no longer apparent when cell lysates were mixed with the peptide from which the antibody was raised, confirming antibody specificity. WTAP protein abundance increased 4.8±0.4-fold (P<0.01) as HITC6 SMCs matured and became nonproliferative (Figure 2C).
To determine whether WTAP was upregulated during SMC maturation in vivo, we undertook balloon-injury of the rat carotid artery and harvested the intima 7, 14, and 28 days after injury, by laser capture microdissection. In this model the early intima contains activated, proliferative SMCs that cease proliferating over the subsequent weeks.6 As shown in Figure 2D, intimal WTAP expression was relatively low 7 days after injury but by 28 days had increased by 6.3-fold (P<0.001). Thus, WTAP is dynamically expressed in the remodeling artery wall and, as in culture, is strikingly upregulated as SMC proliferation terminates.
Overexpression of WTAP Inhibits SMC Proliferation
To investigate the function of WTAP, we cloned full-length WTAP cDNA and introduced this into HITC6 SMCs using retrovirus. This yielded an approximate 3-fold (range 2.4 to 3.6) increase in WTAP protein in stably transduced SMCs. Overexpression of WTAP had no apparent effect on SMC morphology. However, WTAP-overexpressing SMCs proliferated slowly and after 9 days had accumulated to only 25±4% that of control, vector-infected SMCs (P<0.01, Figure 3A). A similar decline in proliferation was observed with primary cultures of SMCs overexpressing WTAP (data not shown).
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To determine whether the slower proliferation of WTAP-overexpressing SMCs was because of a decline in DNA synthesis rate, thymidine incorporation was quantified. As shown in Figure 3B, [3H]thymidine incorporation in WTAP-overexpressing SMCs was reduced to 0.59±.04 that of vector-infected SMCs (P<0.01). To further delineate the effect of WTAP on the cell cycle, flow cytometry of propidium iodide-labeled SMCs was undertaken. This indicated that the proportion of WTAP-overexpressing SMCs in S-phase was 0.37±0.02 that of vector-infected SMCs (P<0.01), with a corresponding increase on the proportion of SMCs in G0/G1 phase and no effect on the proportion of cells in G2/M phase, confirming an inhibitory effect of WTAP on the G1-S-phase transition (Figure 3C).
Knock-Down of Endogenous WTAP Increases SMC Proliferation
To elucidate the role of endogenous WTAP, HITC6 SMCs were infected with retrovirus containing cDNA encoding shRNA. To ensure specificity of the shRNA approach for WTAP knockdown, 3 different targeting fragments were used and both WTAP messenger RNA and WTAP protein were quantified. As shown in Figure 4A, SMCs transduced with any 1 of the 3 WTAP RNA targeting fragments displayed significantly suppressed expression of WTAP messenger RNA and protein, the latter to 49±9% (P<0.01) of cells expressing the corresponding nonsilencing RNA (nsRNA) fragment.
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Interestingly, each of the WTAP-knockdown SMCs was smaller and less elongated than control SMCs (Figure 4, A and B). In addition, all 3 lines proliferated substantially faster than VSMCs expressing the respective control nsRNA fragment (Figure 5A), with a 54±3% shortening of the doubling time (P<0.01). WTAP-knockdown SMCs also showed a 2.0±0.3-fold increase in incorporation of [3H]thymidine (Figure 5B) and a 1.8±0.3-fold increase in the proportion of SMCs in S-phase (Figure 5C). These findings establish that WTAP serves to retain SMCs in an elongated and relatively slowly proliferating state, the latter by inhibiting cell cycle progression.
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WTAP Regulates SMC Apoptosis
To determine whether WTAP also regulates cell survival, SMC apoptosis was assessed by surface expression of phosphatidylserine using fluorescence-labeled Annexin V. As shown in Figure 6A, the proportion of apoptotic SMCs was significantly higher in the WTAP-overexpressing population than in SMCs expressing the corresponding control vector (P<0.05). In keeping with this finding, SMCs with suppressed WTAP expression had a significantly lower prevalence of apoptosis than the respective control SMCs (P<0.05, Figure 6B). Thus, WTAP retards the rate of accumulation of SMCs not only by slowing cell cycle kinetics but also by increasing the rate of apoptosis.
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WTAP and WT1 are reciprocally regulated in SMCs in vitro and in vivo
To determine whether there was a relationship between WTAP and WT1 in SMCs, we assessed expression of both proteins in vivo and in vitro. As depicted in the Western blot of Figure 7A, WTAP was detected within the medial layer of the adult rat aorta and its expression was notably higher (3.0±0.3-fold, P<0.01) than in SMCs isolated from this vessel and grown in culture (2nd subculture). Interestingly, WT1 was also expressed in the media of the aorta. In contrast to WTAP however, WT1 expression was higher in cultured aortic SMCs than in aortic SMCs in vivo (2.9±0.4-fold, P<0.01).
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WTAP Interacts with WT1 in SMCs
Having established that both WTAP and WT1 were expressed in arterial SMCs, we determined if these proteins interacted in this cell type. As shown in Figure 7B, WT1-immunoprecipitates of human SMCs contained WTAP protein, as assessed by immunoblot analysis. Likewise, WT1 was readily detectable in WTAP immunoprecipitates (Figure 7B). Control immunoprecipitations using nonspecific rabbit or mouse IgG showed no signal for WTAP or WT1. All immunoprecipitations were undertaken using antibody covalently linked to agarose beads,8 to avoid confounding signal from the IgG heavy chain.
WTAP Impairs WT1-DNA Binding
Because WT1 is a zinc-finger transcription factor, we next assessed the impact of WTAP on the ability of WT1 to bind target DNA, using an ELISA-based DNA-protein interaction assay. As shown in Figure 8A, WT1 in nuclear extracts harvested from WTAP-overexpressing SMCs displayed a 51.8±5.8% (P<0.05) decrease in the capacity to bind to an oligonucleotide containing a WT1 consensus site compared with WT1 in extracts from vector-infected SMCs (Figure 8A). In contrast, WT1 within nuclear extracts from SMCs containing WTAP shRNA (shRNA1039) displayed 32.8±6.4% increase (P<0.05) in DNA binding, relative to that from control SMCs expressing nsRNA. WT1 protein levels were found to be unaffected by the altered WTAP gene dosage. Therefore, the efficiency with which WT1 binds to target DNA is dependent on the level of WTAP in the nucleus.
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WTAP Inhibits Expression of WT1 Transcriptional Target Genes in SMCs
We next determined if expression of transcriptional target genes for WT1 was impacted by the level of WTAP. For this, expression of amphiregulin and Bcl-2 were measured by quantitative RT-PCR. Amphiregulin was chosen because it is a strong mitogen for SMCs14 and its transcription is potently activated by WT1, which binds directly to its promoter.15 The proto-oncogene Bcl-2 is also transcriptionally activated by WT1 leading to suppressed apoptosis.16 As depicted in Figure 8B, transcripts for both amphiregulin and Bcl-2 were significantly reduced in SMCs overexpressing WTAP. In contrast, in SMCs with shRNA-mediated WTAP knockdown (shRNA1039) there was a significant increase in both amphiregulin and Bcl-2 transcripts, compared with control HITC6-nsRNA VSMCs (nsRNA1039). Thus, WTAP exerts a concentration-dependent inhibitory effect on the expression of WT1-sensitive genes that regulate both mitogenic and survival pathways.
WTAP-Induced Inhibition of SMC Proliferation Can be Rescued by Amphiregulin
To determine whether the antiproliferative effect of WTAP was a consequence of reduced expression of WT1-sensitive genes, we tested whether addition of exogenous amphiregulin could rescue WTAP-induced inhibition of SMC DNA synthesis. As shown in Figure 8C, addition of amphiregulin (50 ng/mL) to WTAP-overexpressing SMCs restored the DNA synthesis rate close to that of control, vector-expressing SMCs. Furthermore, both the relative and absolute increases in DNA synthesis afforded by amphiregulin were significantly higher for WTAP-overexpressing SMCs than that for control SMCs (59±2 versus 31±6%, P<0.01; 1269±41 versus 799±23 cpm/µg DNA, P<0.05). Thus, the antiproliferative effect of WTAP was functionally linked to the decreased expression of amphiregulin which, in turn, was a result of disrupted WT1-mediated gene expression.
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Discussion |
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Replication of adult vascular SMCs is an essential feature of vascular repair. However, to avoid excess SMC accumulation and effectively terminate remodeling, there must be a process by which proliferating SMCs efficiently convert to a nonproliferative state. To seek out molecular regulators of this process, we studied a human SMC line with the unique capacity to shift to a nonreplicating phenotype that is stable for an extended period. In so doing we established that as SMCs attain replicative quiescence, TIMP-3, Axl, and KIAA0098 are downregulated and complement C1s, cathepsin B, CREG, and WTAP are upregulated. This panel of genes is noteworthy because it includes poorly understood genes as well as genes with little to no previously known relationship with SMC phenotype.
From these SMC phenotype-associated genes, we selected WTAP for detailed analysis. WTAP was particularly attractive in this regard not only because of its unequivocal upregulation in nonproliferating SMCs but also because it is a nuclear protein of unknown function that had not been identified in SMCs before. Upregulation of WTAP in nonproliferating and contractile SMCs proved to be a robust phenomenon, observed in 2 different contraction-competent human SMC lines and when SMCs of the intact adult arterial media were compared with those growing in culture. Furthermore, WTAP was expressed in the intima of balloon-injured arteries where it underwent striking upregulation as the intima organized and matured to a stage where SMC proliferation is known to be low.6
WTAP expression not only fluctuated with changes in SMC phenotype but it played an important role in determining the phenotype. Introducing a constitutively expressed WTAP gene into human SMCs suppressed their proliferation, an effect seen with levels of WTAP 
3-fold higher than baseline. This inhibition of SMC accumulation was partly because of effects on the cell cycle, with decreased DNA synthesis and a concomitant reduction in the proportion of SMCs undergoing the transition from G1 to S-phase. In addition, overexpression of WTAP impacted SMC survival, with the specific effect of activating apoptosis. Thus, WTAP coordinately affected 2 pathways that can functionally converge to suppress the accumulation of SMCs.
Knock-down of endogenous WTAP in SMCs produced effects that were generally opposite to those of WTAP overexpression. WTAP-deficient SMCs were characterized by small, rapidly proliferating cells that assumed a cobblestone appearance at confluence. DNA synthesis was substantially increased in these cells, with a corresponding increase in the G1-S phase transition. In addition, reduced WTAP expression inhibited apoptosis. These finding, verified using 3 different WTAP shRNA-expressing SMC lines, establish that the acquisition or maintenance of a nonproliferative SMC phenotype depends on WTAP expression. They also raise the possibility, given the morphological changes, that WTAP may be a determinant of epithelioid versus spindle-shaped SMCs, subsets of SMCs that may differentially contribute to vascular remodeling3,17
We propose that the mechanism by which WTAP inhibits progress through the cell cycle and stimulates apoptosis in SMCs is through partnering with WT1. WT1 is a zinc finger protein implicated in the development of several organs, including the heart.18,19 WT1 has been found to be expressed in proliferating vascular SMCs in response to ischemia20 and has recently been shown to be required for coronary vessel development.21 Thus, although traditionally viewed as a factor in kidney development and tumors, a role for WT1 in vascular remodeling is emerging.
The basic cellular actions of WT1 are diverse, because of, in part, different WT1 isoforms. It is well established however that WT1 can act as a transcriptional regulator. It is also emerging that the transcriptional activity of WT1 can be modulated by interaction with various protein partners.22 Our findings establish that WTAP is one such partner. Reciprocal coimmunoprecipitation analysis revealed that WTAP interacted with WT1 in human SMCs. Furthermore, several lines of evidence indicated that WTAP not only binds to WT1 but interferes with its transcriptional activity. First, the capacity with which WT1 could bind to an oligonucleotide containing a consensus WT1 binding sequence was inversely related to WTAP gene dosage. Second, expression of WT1 transcriptional target genes was suppressed in SMCs that had increased levels of WTAP and amplified in WTAP-knockdown SMCs.
One such WT1 target gene was amphiregulin, one of few genes known to be direct transcriptional targets of WT1 and a sensitive indicator of WT1 transcriptional activity.15 Amphiregulin is a member of the epidermal growth factor gene family and is a potent SMC mitogen.14 Not only was amphiregulin expression suppressed by WTAP but exogenous amphiregulin rescued the antiproliferative effect of WTAP, implicating this WT1 target in the functional actions of WTAP. Like amphiregulin, Bcl-2 has a WT1 binding site on its promoter through which its transcription is activated.16 Bcl-2 has been shown to suppress apoptosis in SMCs which, again, is concordant with the functional responses observed when WTAP gene dosage was modified.23,24 Based on these findings, we propose a model whereby WTAP buffers the ability of WT1 to stimulate proliferation and suppress apoptosis, by inhibiting WT1 transcriptional activity.
This model is consistent with previous data regarding binding of WTAP to different WT1 isoforms. Two major isoforms of WT1 are defined by the presence or absence of a lysine-threonine-serine (KTS) insert between the third and fourth zinc fingers. The transcriptional properties of WT1 are afforded primarily by the -KTS isoform, whereas the +KTS isoform binds DNA poorly and may be more involved in RNA processing.22 WTAP has been found to have a greater binding affinity for the -KTS isoform of WT1 than the +KTS isoform,13 a preference consistent with our model of WTAP as a regulator WT1 transcriptome dynamics. It is conceivable that WTAP prevents the zinger fingers of the –KTS WT1 from accessing promoter targets. This interaction might also shift the actions of the –KTS isoform of WT1 to a nontranscriptional role, although this possibility remains to be tested.
It is also plausible that the actions of WTAP in VSMCs are mediated by an effect on mRNA splicing, either dependent or independent of WT1. This possibility exists because WTAP has been identified as a component of the spliceosome and because WTAP is homologous to the Drosophila sex-determining splicing protein FL(2)D.25–27 We have found that the abundance of heavy-caldesmon and meta-vinculin is reduced, relative to their corresponding splicing alternative, in WTAP-knockdown SMCs (data not shown) and a role of WTAP in regulating splicing thus merits investigation.
Although we have focused our analysis on WTAP, the other 6 differentially expressed genes are also intriguing candidates for regulating SMC phenotype. Two of these genes, the receptor tyrosine kinase, Axl, and the matrix metalloproteinase inhibitor, TIMP-3, are emerging as proteins that can activate SMC proliferation28,29 and the observed downregulation in contractile SMCs strengthens this relationship. Complement C1s, a serine proteinase, cathepsin B, a cysteine proteinase, and CREG, a secreted glycoprotein that binds the mannose 6-phosphate/insulin-like growth factor II receptor,30 have not previously been linked to SMC behavior. Their upregulation during SMC maturation suggests novel pathways for regulating SMC phenotype. Finally, KIAA0098 has yet to have a function ascribed to it but its downregulation implicates it as candidate mediator of SMC behavior.
In summary, we have identified a panel of novel and poorly understood SMC phenotype-associated genes. Included in this panel was WTAP, a nuclear protein that we have determined to be an inhibitor of the cell cycle and an activator of apoptosis. These actions of WTAP are because of, at least in part, repressing the transcriptional activity of WT1. We conclude that interplay between WTAP and WT1 constitutes a novel molecular control system for SMC phenotype, and possibly that of other cells.
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Acknowledgments |
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We gratefully acknowledge the assistance of J. Rylett and T. Dobransky, Robarts Research Institute, in generating the WTAP antibody.
Sources of Funding
This work was supported by the Canadian Institutes of Health Research (MOP-11715), the Heart and Stroke Foundation of Canada (T5675, PRG4854), the Lawson Research Foundation, and The Krembil Foundation. J.G.P. holds a Career Investigator Award from the Heart and Stroke Foundation of Ontario and J.M. holds a Canada Research Chair in Transplantation and Immunobiology.
Disclosures
None.
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Footnotes |
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Original received July 14, 2006; revision received October 16, 2006; accepted November 1, 2006.
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References |
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- 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] - Li S, Sims S, Jiao Y, Chow LH, Pickering JG. Evidence from a novel human cell clone that adult vascular smooth muscle cells can convert reversibly between noncontractile and contractile phenotypes. Circ Res. 1999; 85: 338–348.
[Abstract/Free Full Text] - Li S, Fan YS, Chow LH, Van Den Diepstraten C, van Der Veer E, Sims SM, Pickering JG. Innate diversity of adult human arterial smooth muscle cells: cloning of distinct subtypes from the internal thoracic artery. Circ Res. 2001; 89: 517–525.
[Abstract/Free Full Text] - Rocnik EF, Van Der Veer E, Cao H, Hegele RA, Pickering JG. Functional linkage between the endoplasmic reticulum protein Hsp47 and procollagen expression in human vascular smooth muscle cells. J Biol Chem. 2002; 277: 38571–28578.
[Abstract/Free Full Text] - van der Veer E, Nong Z, O’Neil C, Urquhart B, Freeman D, Pickering JG. Pre-B-cell colony-enhancing factor regulates NAD+-dependent protein deacetylase activity and promotes vascular smooth muscle cell maturation. Circ Res. 2005; 97: 25–34.
[Abstract/Free Full Text] - Clowes A, Reidy M, Clowes M. Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium. Lab Invest. 1983; 49: 327–333.[Medline] [Order article via Infotrieve]
- Rocnik E, Saward L, Pickering JG. HSP47 expression by smooth muscle cells is increased during arterial development and lesion formation and is inhibited by fibrillar collagen. Arterioscler Thromb Vasc Biol. 2001; 21: 40–46.
[Abstract/Free Full Text] - Dobransky T, Davis WL, Rylett RJ. Functional characterization of phosphorylation of 69-kDa human choline acetyltransferase at serine 440 by protein kinase C. J Biol Chem. 2001; 276: 22244–22250.
[Abstract/Free Full Text] - Fera E, O’Neil C, Lee W, Li S, Pickering JG. Fibroblast growth factor-2 and remodeled type I collagen control membrane protrusion in human vascular smooth muscle cells: biphasic activation of Rac1. J Biol Chem. 2004; 279: 35573–35582.
[Abstract/Free Full Text] - Vindelov LL, Christensen IJ, Nissen NI. A detergent-trypsin method for the preparation of nuclei for flow cytometric DNA analysis. Cytometry. 1983; 3: 323–327.[CrossRef][Medline] [Order article via Infotrieve]
- Johnstone RW, Wang J, Tommerup N, Vissing H, Roberts T, Shi Y. Ciao 1 is a novel WD40 protein that interacts with the tumor suppressor protein WT1. J Biol Chem. 1998; 273: 10880–10887.
[Abstract/Free Full Text] - Van Den Diepstraten C, Papay K, Bolender Z, Brown A, Pickering JG. Cloning of a novel prolyl 4-hydroxylase subunit expressed in the fibrous cap of human atherosclerotic plaque. Circulation. 2003; 108: 508–511.
[Abstract/Free Full Text] - Little NA, Hastie ND, Davies RC. Identification of WTAP, a novel Wilms’ tumour 1-associating protein. Hum Mol Genet. 2000; 9: 2231–2239.
[Abstract/Free Full Text] - Shin HS, Lee HJ, Nishida M, Lee MS, Tamura R, Yamashita S, Matsuzawa Y, Lee IK, Koh GY. Betacellulin and amphiregulin induce upregulation of cyclin D1 and DNA synthesis activity through differential signaling pathways in vascular smooth muscle cells. Circ Res. 2003; 93: 302–310.
[Abstract/Free Full Text] - Lee SB, Huang K, Palmer R, Truong VB, Herzlinger D, Kolquist KA, Wong J, Paulding C, Yoon SK, Gerald W, Oliner JD, Haber DA. The Wilms tumor suppressor WT1 encodes a transcriptional activator of amphiregulin. Cell. 1999; 98: 663–673.[CrossRef][Medline] [Order article via Infotrieve]
- Mayo MW, Wang CY, Drouin SS, Madrid LV, Marshall AF, Reed JC, Weissman BE, Baldwin AS. WT1 modulates apoptosis by transcriptionally upregulating the bcl-2 proto-oncogene. Embo J. 1999; 18: 3990–4003.[CrossRef][Medline] [Order article via Infotrieve]
- Hao H, Gabbiani G, Bochaton-Piallat ML. Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development. Arterioscler Thromb Vasc Biol. 2003; 23: 1510–1520.
[Abstract/Free Full Text] - Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R. WT-1 is required for early kidney development. Cell. 1993; 74: 679–691.[CrossRef][Medline] [Order article via Infotrieve]
- Moore AW, McInnes L, Kreidberg J, Hastie ND, Schedl A. YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis. Development. 1999; 126: 1845–1857.[Abstract]
- Wagner KD, Wagner N, Bondke A, Nafz B, Flemming B, Theres H, Scholz H. The Wilms’ tumor suppressor Wt1 is expressed in the coronary vasculature after myocardial infarction. Faseb J. 2002; 16: 1117–1119.
[Abstract/Free Full Text] - Wagner N, Wagner KD, Theres H, Englert C, Schedl A, Scholz H. Coronary vessel development requires activation of the TrkB neurotrophin receptor by the Wilms’ tumor transcription factor Wt1. Genes Dev. 2005; 19: 2631–2642.
[Abstract/Free Full Text] - Roberts SG. Transcriptional regulation by WT1 in development. Curr Opin Genet Dev. 2005; 15: 542–547.[CrossRef][Medline] [Order article via Infotrieve]
- Perlman H, Sata M, Krasinski K, Dorai T, Buttyan R, Walsh K. Adenovirus-encoded hammerhead ribozyme to Bcl-2 inhibits neointimal hyperplasia and induces vascular smooth muscle cell apoptosis. Cardiovasc Res. 2000; 45: 570–578.
[Abstract/Free Full Text] - Ekhterae D, Platoshyn O, Krick S, Yu Y, McDaniel SS, Yuan JX. Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol. 2001; 281: C157–65.
[Abstract/Free Full Text] - Ortega A, Niksic M, Bachi A, Wilm M, Sanchez L, Hastie N, Valcarcel J. Biochemical function of female-lethal (2)D/Wilms’ tumor suppressor-1-associated proteins in alternative pre-mRNA splicing. J Biol Chem. 2003; 278: 3040–3047.
[Abstract/Free Full Text] - Zhou Z, Licklider LJ, Gygi SP, Reed R. Comprehensive proteomic analysis of the human spliceosome. Nature. 2002; 419: 182–185.[CrossRef][Medline] [Order article via Infotrieve]
- Penalva LO, Ruiz MF, Ortega A, Granadino B, Vicente L, Segarra C, Valcarcel J, Sanchez L. The Drosophila fl(2)d gene, required for female-specific splicing of Sxl and tra pre-mRNAs, encodes a novel nuclear protein with a HQ-rich domain. Genetics. 2000; 155: 129–139.
[Abstract/Free Full Text] - Melaragno MG, Wuthrich DA, Poppa V, Gill D, Lindner V, Berk BC, Corson MA. Increased expression of Axl tyrosine kinase after vascular injury and regulation by G protein-coupled receptor agonists in rats. Circ Res. 1998; 83: 697–704.
[Abstract/Free Full Text] - Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase-1, -2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis. J Clin Invest. 1998; 101: 1478–1487.[Medline] [Order article via Infotrieve]
- Di Bacco A, Gill G. The secreted glycoprotein CREG inhibits cell growth dependent on the mannose-6-phosphate/insulin-like growth factor II receptor. Oncogene. 2003; 22: 5436–5445.[CrossRef][Medline] [Order article via Infotrieve]
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