Transforming Growth Factor-β/Smads Signaling Induces Transcription of the Cell Type–Restricted Ankyrin Repeat Protein CARP Gene Through CAGA Motif in Vascular Smooth Muscle Cells
Abstract—Transforming growth factor (TGF)-β plays a major role in the development of vascular diseases. Despite the pleiotropic effects of TGF-β on vascular smooth muscle cells (VSMCs), only a few genes have been characterized as direct targets of TGF-β in VSMCs. Cardiac ankyrin repeat protein (CARP) has been thought to be expressed exclusively in the heart. In the present study, we showed that CARP is expressed in the vasculature after balloon injury and in cultured VSMCs in response to TGF-β. Analysis of a half-life of the cytoplasmic CARP mRNA levels and the transient transfection of the CARP promoter/luciferase gene indicates that the regulation of CARP expression is increased by TGF-β at the transcriptional level. Transfection of expression vectors encoding Smads significantly activated the CARP promoter/luciferase activity. Deletion analysis and site-specific mutagenesis of the CARP promoter indicate that TGF-β response element is localized to CAGA motif at −108 bp relative to the transcription start site. Electrophoretic mobility shift assays showed that the binding activity to the CAGA motif was increased in nuclear extracts of cultured VSMCs by TGF-β. Cells transfected with adenovirus vector expressing CARP showed a significant decrease in DNA synthesis. Overexpression of CARP enhanced the TGF-β–mediated inhibition of the DNA synthesis. These data indicate that CARP is a downstream target of TGF-β/Smad signaling in VSMCs and suggest a role of CARP in mediation of the inhibitory effects of TGF-β on the proliferation of VSMCs.
Members of the transforming growth factor-β (TGF-β) superfamily play a critical role in the regulation of cellular growth and differentiation in a wide range of biologic systems, including vasculature.1 TGF-β has unique abilities to both positively and negatively regulate systems involved in cell proliferation, migration, extracellular matrix synthesis, and apoptosis in vascular smooth muscle cells (VSMCs). These pleiotropic effects of TGF-β are thought to result from its ability to induce or repress transcription of many genes that are critical in these processes.
TGF-β is often considered to have proatherosclerotic effects because (1) TGF-β causes an increase in the production of collagen2 and fibronectin,3 a decrease in the synthesis of proteases that degrade extracellular matrix, and an increase in the expression of protease inhibitors, such as plasminogen activator inhibitor type I (PAI-1)4 ; (2) TGF-β mRNA and immunoreactivity are increased in human restenotic lesions5 ; (3) the infusion of TGF-β or the transfection of TGF-β cDNA into injured arteries strongly accelerates lesion formation by increasing cellularity and markedly increasing extracellular matrix accumulation6 ; and (4) antibodies to TGF-β reduce the development of vascular lesions after balloon injury in rats.7 These data favored a model in which TGF-β promotes atherosclerosis and restenosis after vascular injury. However, considerable evidence implies that TGF-β exerts the antiatherosclerotic effects within the vascular wall. Besides the inhibitory effects of TGF-β on proliferation and migration of SMCs in vitro, alterations in active TGF-β level were associated with the progression of vascular disease in a manner consistent with an antiatherosclerotic effect of TGF-β.8
To date, only a few genes has been characterized as the primary targets of TGF-β signaling in VSMCs, including PAI-1,9 p21WAF1/CIP1,10 p15INK4B,11 α2(I) procollagen,12 and fibronectin.13 Cardiac ankyrin repeat protein (CARP) has been cloned as a nuclear factor that is expressed in a heart-specific manner,14 15 but CARP has also been identified in endothelial cells activated by inflammatory cytokines or in denervated skeletal muscle.16 17 Furthermore, the finding of the CARP expression in the cardiovascular tissue is supported by the recent report that CARP expression in transgenic mice harboring various CARP promoter/lacZ reporters was found not only in the heart but also in the conotruncal segments of the heart that form basal of pulmonary artery and ascending aorta.18 Although previous studies have implicated CARP as a modulator of transcription,16 the function of CARP has yet to be determined.
In the present study, we examined CARP expression in vascular injury and the regulatory mechanisms that underlie its inducible expression in VSMCs. The results show that CARP mRNA levels are increased in injured arteries and in cultured VSMCs in response to TGF-β. In addition, we provide evidences for transcriptional regulation of CARP gene expression by TGF-β through a direct binding of Smads to CAGA motif. Furthermore, results of the experiments with the adenovirus expressing CARP indicate that CARP overexpression inhibits the DNA synthesis probably through the induction of p21WAF1/CIP1 and resultant dephosphorylation of ppRb, the hyperphosphorylated form of pRb, the retinoblastoma protein. Together, the present results demonstrated the inducible expression of the CARP gene in VSMCs by TGF-β and suggested the role of CARP in inhibition of cell proliferation.
Materials and Methods
TGF-β1 and basic fibroblast growth factor (bFGF) were purchased from Boehringer Mannheim. Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) were obtained from Sigma Chemical Co. Endothelin-1 (ET-1) was purchased from Peptide Institute Inc.
C2/2 cells were previously described.19 PAE, COS-7, A549, CV-1, and NIH-3T3 cells were obtained from American Type Culture Collection. Neonatal rat cardiac myocytes were prepared as previously described.20
Balloon catheter denudation was accomplished on rats at 8 weeks of age as previously described.21 Rats were killed at various times after injury (0, 1, 2, and 4 weeks). Total RNA was reverse-transcribed with oligo(dT) primer with the use of avian myeloblastosis virus reverse transcriptase and amplified with Taq DNA polymerase (Takara). PCR consisted of 28 cycles at 94°C for 1 minute, at 60°C for 1 minute, and at 72°C for 1.5 minutes. This investigation conforms to the guide for the care and use of laboratory animals approved by Committee of Gunma University School of Medicine.
Northern Blot Analysis
Total RNA was extracted from the cultured cells and tissues with the use of ISOGEN (Nippon gene) according to the manufacturer’s instruction. Northern blot analysis was performed as previously described.21 The cDNA probes were radiolabeled with [α-32P]dCTP (Amersham) with a random primer DNA labeling kit (Boehringer Mannheim).
Construction of the CARP Promoter/Luciferase Gene
Human genomic clone encoding CARP was isolated by screening the human leukocyte genomic library (HL1006d; Clontech). For the generation of luciferase reporter genes, the following upstream primers with a KpnI site were used with PCR with a plasmid that contained a ≈5-kb DNA insert with the reverse primer (nucleotide +170) with an XhoI site, 5′-GCAGATCTCGAGGGGGGGCCCCTC-3′.
Sequences for PCR upstream primers were CARP−1828Luc, 5′-GGGGGGGTACCTGCAGCAAGTTACTTAATG-3′; CARP−206Luc, 5′-AGAAAGGTACCACTG GGGGTGTGA-3′; CARP−90Luc, 5′-TGTCCGGTACCTCCTGACAAATAG-3′; and CARP+1Luc, 5′-ATTCAGGTACCCAGCAGGGTTAGC-3′.
For the generation of mutants, CARP−120 MLuc was designed: 5′-CCCGGTACCCAATGTCAATGAGTGGCTGTC -3′.
Recombinant PCR with 2 rounds of amplification was performed. PCR products were subcloned into the promoterless luciferase reporter gene vector, pGL3 (Promega). The expression vectors of Flag-tagged Smad2, Smad3, Smad4, and Smad6 have been previously described.22 T204D and K232R vectors were kindly given by Joan Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY).23
Transient Transfection, Luciferase Assay, and Preparation of Cell Lysates
Cells were transfected with 1 μg of plasmid according to the calcium phosphate precipitation method as previously described.21 After 24 hours, cells were stimulated with 1 ng/mL TGF-β1 for 24 hours and harvested. Luciferase activity was measured with a luminometer.
Nuclear Extracts and Whole-Cell Extracts
Nuclear extracts were prepared from C2/2 cells treated with or without 1 ng/mL TGF-β1 for 30 minutes, as previously described.24 Whole cell extracts were prepared from COS-7 cells transfected with Smad3 or Smad4 expression vectors treated with TGF-β1 (1 ng/mL) for 30 minutes or C2/2 cells infected with AxCA/LacZ and AxCA/CARP as previously described.25
Western Blot Analysis
Western blot analyses were performed essentially as previously described.21 Anti-p21 antibody (C-19-G; Santa Cruz) and anti-Rb antibody (G3-245; PharMingen) was visualized with a horseradish peroxidase–linked anti-goat or anti-mouse IgG secondary antibody, respectively (Amersham). The complexes were detected using the ECL chemiluminescence detection system (Amersham).
Electrophoretic Mobility Shift Assays
Probes were labeled with T4 polynucleotide kinase and [γ-32P]dATP (Amersham) and electrophoresed with 5% polyacrylamide gels.
Recombinant Adenovirus Expression Constructs
The recombinant adenovirus vectors were generated as previously described.25 AxCA/LacZ and AxCA/CARP were prepared by inserting the β-galactosidase or CARP cDNAs into the Ad E1–deleted region under the control of the CAG promoter.
[3H]Thymidine Incorporation Assay
C2/2 cells stimulated with TGF-β1 or infected with AxCA/LacZ or AxCA/CARP were pulsed with 1 μCi/well [3H]thymidine (Amersham) and analyzed with a scintillation counter.
CARP Is Expressed in VSMCs
The expression of CARP was examined in the arterial wall and in VSMCs. RT-PCR analysis showed that the expression of CARP was detected in arterial tissue and that its expression was increased in injured artery (Figure 1A⇓). To determine whether CARP is expressed in VSMCs, total RNA was prepared from various cell lines. The expression of CARP mRNA was evident in C2/2 cells derived from rabbit aortic SMCs,18 and modest expression of CARP was detected in bovine pulmonary artery endothelial (PAE) cells. CARP expression was barely detectable in other cell lines, including NIH-3T3 (mouse fibroblast cells), CV-1 (monkey kidney cells), and A549 (human lung adenocarcinoma cells) (Figure 1B⇓). These results indicate that CARP is expressed in VSMCs as well as in cardiac myocytes and provided further evidence that CARP expression is cell type specific.
TGF-β Increases CARP mRNA Levels in VSMCs
To investigate the molecular mechanisms that underlie the regulated expression of CARP mRNAs, C2/2 cells were stimulated with Ang II, ET-1, TGF-β1, bFGF, and VEGF, all of which are known to play a major role in the development of vascular disease. Among these growth factors, TGF-β was most potent in inducing the expression of CARP mRNA levels (Figure 2⇓). Figures 2B⇓ and 2C⇓ show time course and concentration-dependent changes in CARP mRNA levels. An increase in CARP mRNA expression was evident at 1.0 ng/mL TGF-β. The time course of the change in CARP mRNA levels was modestly increased at 2 hours and reached maximal levels at 24 hours after stimulation.
TGF-β Increases CARP Expression at the Transcriptional Level
To determine whether TGF-β increases CARP expression at the transcriptional level, we performed a standard mRNA decay assay with actinomycin D, a potent inhibitor of RNA synthesis (Figure 3⇓). In this result, the CARP half-life was 11.3 and 8.5 hours in the absence and presence of TGF-β, respectively. Thus, the TGF-β–mediated increase in CARP mRNA levels in C2/2 cells was at least in part due to an increase in the stability of mRNA. To confirm the transcriptional regulation by TGF-β, CARP promoter/luciferase reporter gene was transiently transfected into C2/2 cells. As shown in Figure 4A⇓, luciferase activity derived from the CARP promoter spanning −1828 to +170 was increased by 3.2±0.5-fold in response to TGF-β. These results indicate that TGF-β increases CARP expression at the transcriptional level and that the promoter region downstream of −1828 contains TGF-β response element or elements.
Constitutively Active Mutant of Type I TGF-β Receptor Increases CARP Promoter Activity
Specific serine/threonine kinase type I receptors transduce intracellular signaling of TGF-β family members.26 Previous studies have demonstrated that mutation of Thr204 of the type I receptor of TGF-β (TβR-I) to aspartate residue (T204D) yields constitutively active receptor in the absence of TGF-β.23 Thus, we examined whether this mutant stimulates CARP promoter activity independent of TGF-β stimulation. As shown in Figure 4B⇑, cotransfection of T204D, but not the kinase-defective TβR-I mutant (K232R),23 increased luciferase activity of CARP promoter. These results suggest that CARP promoter is stimulated through a signaling pathway evoked by activation of the kinase domain of TβR-I.
Smads Increase CARP Promoter Activity
Signaling by TGF-β was recently shown to rely on Smad proteins. Activated TβR-I propagates the TGF-β signal by phosphorylating the pathway-restricted signal transducers Smad2 and Smad3. These Smads form a heteromer with the common signaling mediator Smad4 and then translocate to the nucleus and activate the gene transcription. Smad6 and Smad7, however, potently inhibit the function of the activated TβR-I.26
To examine whether Smad proteins were involved in the TGF-β–induced transcriptional activation of CARP promoter, we cotransfected into C2/2 cells with CARP(−1828/+170)Luc/reporter gene and an expression vector encoding the Smad6 protein. As shown in Figure 4C⇑, Smad6 inhibits the inducible expression of CARP promoter by TGF-β as well as by constitutively active TβR-I (T204D) in a dose-dependent manner. In our study, Smad6 inhibited the signaling pathway induced by T204D more strongly than that activated by TGF-β. These results indicate a possible involvement of Smad proteins in TGF-β–induced CARP expression. A previous study indicated that the overexpression of some Smad proteins activates transcription from TGF-β constructs, even in the absence of TGF-β.27 To determine whether Smads activate the expression of the CARP promoter, we cotransfected various Smad constructs with CARP(−1828/+170)Luc. Figure 4D⇑ shows that Smad3 markedly stimulated the CARP promoter, whereas either Smad2 or Smad4 had a modest effect on the CARP activity. The concomitant overexpression of Smad3 with Smad4 showed an induction of CARP promoter activity almost comparable to that seen with the overexpression of Smad3 alone. Taken together, these results indicate that TGF-β increases CARP promoter activity through mechanisms that involve Smad proteins.
Identification of TGF-β Response Element Within the CARP Promoter
To identify the TGF-β response element within the CARP promoter, a series of 5′-deletion constructs was generated and assayed for luciferase activity. Deletion to −206 bp from the transcriptional start site had relatively little effect on the inducible expression by TGF-β, but further deletion to −90 bp almost completely abolished the TGF-β inducibility (Figure 5A⇓). Thus, the positive regulatory region of CARP promoter activity by TGF-β seems to reside between −206 and −90 bp. Inspection of this region revealed the presence of one copy of 5′-CAGACAGC-3′ at −108 bp (Figure 5B⇓), matching very closely the consensus sequence of CAGA box [AG(C/A)CAGA] of the PAI-1 promoter identified as a TGF-β response element.28
To determine whether this CAGA motif could have a function as a TGF-β response element, mutation was introduced into this sequence. As shown in Figure 5C⇑, the mutation in the CAGA motif significantly reduced the promoter activity and, more important, almost completely eliminated the TGF-β–mediated induction of CARP promoter activity. To exclude the possibility that loss of responsiveness to TGF-β is due to the loss of basal promoter function, we examined whether mutation construct is responsive to ET-1, which significantly induces CARP expression in cardiac myocytes (Y. Aihara, H. Kanai, M. Kurabayashi, unpublished data, 2001). Although the responsiveness appears to be reduced compared with the wild type, constructs containing mutation within the CAGA motif were responsive to ET-1 (Figure 5C⇑). These results indicate that promoter-containing mutation within CAGA motif has an ability to respond to an irrelevant signal that does not work through Smads, thus allowing us to consider CAGA motif as a TGF-β response element.
Nuclear Factors Binding to the CARP −108-bp Element
To confirm the specific role of the CAGA motif within the CARP promoter, we performed electrophoretic mobility shift assays (EMSAs). Figure 6A⇓ shows 2 constitutive DNA-binding activities in unstimulated or TGF-β–treated cells. These complexes were formed in a sequence-specific manner because an excess of the unlabeled probe, but not of the mutated probe, displaced the corresponding bands. Furthermore, this complex was efficiently competed by a molar excess of bona fide CAGA box of PAI-1−280. These results suggest that the CAGA motif can serve as a binding site for Smads. To verify that Smads can bind to CAGA motif, whole cell extracts prepared from Smad3- or Smad4-overexpressed cells were subjected to EMSA. The incubation of nuclear extracts with either anti-Smad2/3 or anti-Smad4 antibody gave rise to a supershifted band (Figure 6B⇓).
Overexpression of CARP Inhibits DNA Synthesis in C2/2 Cells
TGF-β is known to have a dual effect on cellular growth depending on the cell types and culture conditions. To determine the effects of TGF-β on DNA synthesis of C2/2 cells, [3H]thymidine incorporation was measured. As shown in Figure 7A⇓, TGF-β–treated cells significantly reduced [3H]thymidine incorporation in a dose-dependent manner. C2/2 cells infected with adenovirus encoding CARP, AxCA/CARP, also exhibited an ≈40% decrease compared with AxCA/LacZ control adenovirus expressing β-galactosidase (Figure 7B⇓). The treatment of CARP-overexpressed cells with TGF-β markedly reduced the DNA synthesis compared with the untreated control. (Figure 7C⇓). These results are consistent with our supposition that CARP may play a role in the inhibition of cell proliferation and that the suppression of cellular growth by TGF-β might be in part mediated through the induction of CARP. To rule out that these inhibitory effects of AxCA/CARP are nonspecific toxic effects, we conducted some experiments. The lactose dehydrogenase released from infected cells was not changed in cells infected with AxCA/LacZ and AxCA/CARP at the multiplicity of infection used in this study (data not shown). Furthermore, housekeeping gene expression such as GAPDH was compared between AxCA/LacZ and AxCA/CARP; no significant differences have been seen in the AxCA/LacZ- and AxCA/CARP- infected cells (Figure 7D⇓). These results indicate that the effects of AxCA/CARP are not due to the nonspecific toxic effects.
To examine further the mechanisms of inhibitory effect of CARP on cellular growth, we performed Western blot analysis for cell cycle regulatory proteins. As shown in Figure 7E⇑, the expression of p21WAF1/CIP1 protein, a universal inhibitor of cyclin-dependent kinases,29 was increased modestly but reproducibly in the AxCA/CARP-infected cells. Consistent with the established role of p21WAF1/CIP1, which inhibits the phosphorylation of pRb in a cell cycle–dependent manner,30 relative levels of ppRb were decreased in the AxCA/CARP-infected cells compared with the control cells (AxCA/LacZ-infected cells). These results suggest that CARP might be one of the key regulators in VSMC proliferation that works through the p21WAF1/CIP1 protein expression.
Previous studies have shown that CARP expression is restricted to cardiac myocytes, endothelial cells, and conotruncal segments in cardiogenesis.14 15 16 17 18 The present study was designed to examine the expression of CARP in the vessel wall and to characterize the molecular mechanisms that underlie the regulated expression of the CARP gene in VSMCs. We show here that CARP mRNA is expressed in vascular tissue and that its expression levels were increased in injured aorta. The presence of CARP in VSMCs would provide a valuable clue for understanding the function of CARP in cardiovascular pathophysiology.
What are the molecular mechanisms that underlie the inducible expression of CARP in balloon-injured aorta? A number of lines of evidence indicate that neointimal SMCs differ from medial SMCs in many respects, including the proliferative capacity, the gene expression of contractile protein isoforms, and the ability to synthesize adhesion molecules, receptors for growth factors, and extracellular matrix proteins. Such phenotypic difference between neointimal and medial SMCs is considered to be governed by the transcription factors, which are preferentially or exclusively expressed in certain phenotypes. We found that point mutation in the CAGA motif reduced the basal promoter activity of the CARP gene in C2/2 cells. EMSAs demonstrate that CAGA motif-binding protein is constitutively present in the nuclear extracts from C2/2 cells. Thus, it is intriguing to speculate that abundant expression of CARP mRNA in untreated C2/2 cells may in part be ascribed to the binding of the nuclear factors to CAGA motif in the unstimulated cells.
A variety of growth factors have been inferred to play a role in the development of neointima; these include TGF-β, platelet-derived growth factor, bFGF, and Ang II.31 Among these peptides, TGF-β was most potent in inducing CARP expression in cultured VSMCs. This finding prompted us to investigate the molecular basis for the TGF-β–mediated CARP expression in VSMCs. Results of the present study clearly document that the CARP gene is a direct target of the TGF-β/Smads signaling. This conclusion has been drawn from the following 4 criteria. First, TGF-β stimulates the CARP expression in transient transfection assays of the CARP promoter/luciferase reporter gene. Second, overexpression of the plasmids encoding Smad3 and Smad4 as well as the constitutively active form of TβR-I increases CARP promoter activity, and this activity is downregulated by the cotransfection of inhibitory Smad, Smad6. Third, mutation of the CAGA motif almost completely eliminates the response of CARP promoter by TGF-β. Last, EMSAs indicate that TGF-β induces binding activity of nuclear factors to the CAGA motif.
Results of the site-specific mutation analysis indicate that CAGA motif is required for the TGF-β–mediated CARP promoter activation. This sequence matches the sequence termed “CAGA box,”28 which has been demonstrated to function as the binding sequences for Smad3/Smad4 of the PAI-1 promoter. The CAGA motif has been identified in the promoter region of the several other TGF-β–inducible genes, such as JunB,32 c-jun,33 and IgCα.34 Although several studies reported that AP-1 is involved in TGF-β–mediated gene expression,33 results of our study indicated that AP-1 does not play a major role in TGF-β–mediated CARP expression.
Several lines of experimental evidence in this study indicated that CARP acts as a negative regulator for cell cycle progression. First, overexpression of CARP reduced the DNA synthesis. Second, treatment with TGF-β of the CARP-overexpressed cells enhanced the inhibition of the DNA synthesis by TGF-β. Third, CARP increased the protein levels of p21WAF1/CIP1, which is known as cyclin-dependent kinase inhibitor.29 Fourth, CARP overexpression reduced ppRb compared with unphosphorylated pRb. Because ppRb has been assumed to play a role in growth arrest,30 it is likely that CARP-mediated inhibitory effects on cell proliferation occur in part via an induction of p21WAF1/CIP1 and subsequent dephosphorylation of ppRb. Further studies that focus on exploration of the possible interaction between CARP and p21WAF1/CIP1 should be warranted.
In conclusion, the present study demonstrates that cell-type–restricted CARP is expressed in injured aorta. We also showed that CARP expression is directly regulated by TGF-β signaling, which is mediated through the binding of Smad proteins to the CAGA motif within the CARP promoter. Consistent with the possible role of CARP in mediating the effects of TGF-β, results of our experiments with the adenovirus imply that CARP functions as an inhibitor of cell proliferation. Taken together, the identification of CARP as a direct target of TGF-β/Smads signaling and as a negative regulator of cell cycle progression will undoubtedly provide novel insight into the potential role of CARP in acting as an effector of the VSMC proliferation by TGF-β.
This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sport and Culture of Japan and a grant from the Japan Cardiovascular Foundation.
Original received February 9, 2000; resubmission received September 14, 2000; revised resubmission received October 30, 2000; accepted October 30, 2000.
- © 2001 American Heart Association, Inc.
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