A Role for the ␤-Catenin/T-Cell Factor Signaling Cascade in
Vascular Remodeling
Xiaohong Wang, Yan Xiao, Yongshan Mou, Ying Zhao, W. Matthijs Blankesteijn, Jennifer L. Hall
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Abstract—␤-Catenin and T cell factor (Tcf) are distal components of the highly conserved Wnt pathway that govern cell
fate and proliferation in lower organisms. Thus, we hypothesized that the regulation of ␤-catenin and Tcf played a
critical role in vascular remodeling. The first objective was to define ␤-catenin expression in vascular smooth muscle
cells (VSMCs) after balloon injury. Indeed, ␤-catenin mRNA and protein were significantly elevated 7 days after
balloon injury in the rat carotid artery. We hypothesized that ␤-catenin accumulation in response to vascular injury
inhibited VSMC apoptosis. In line with our hypothesis, transfection of a degradation-resistant ␤-catenin transgene into
rat VSMCs significantly inhibited apoptosis. Accumulation of ␤-catenin also resulted in a 10-fold increase in the
activation of Tcf. To test if Tcf was necessary to confer ␤-catenin–induced survival, loss of function studies were carried
out with a dominant negative Tcf-4 transgene lacking the ␤-catenin binding domain, Tcf4(N31). Indeed, loss of Tcf-4
activity abolished ␤-catenin–induced survival. We further postulated that ␤-catenin and Tcf promoted cell cycle
progression by activating cyclin D1, a target gene of Tcf-4. ␤-Catenin activated cyclin D1, and this activation was
partially blocked with loss of Tcf-4. In parallel, blockade of Tcf-4 resulted in inhibition of [3H]thymidine incorporation
and partial blockade of the G1-S phase transition. In conclusion, ␤-catenin and Tcf-4 play a dual role in vascular
remodeling by inhibiting VSMC apoptosis and promoting proliferation. (Circ Res. 2002;90:340-347.)
Key Words: vascular smooth muscle cells 䡲 apoptosis 䡲 proliferation 䡲 vascular injury
T
Blankesteijn et al10 demonstrated upregulated expression of
the Wnt receptor isoform Frizzled-2 following myocardial
infarction, suggesting a role for the Wnt pathway in the
spatial control of cardiac wound repair. Finally, Mao and
colleagues11 recently demonstrated altered expression of
the Frizzled receptor family in VSMCs in response to
vascular injury. Based on these collective findings in
VSMCs and other tissues, we hypothesized that the inactivation of GSK3␤ in VSMCs after vascular injury lead to
enhanced accumulation of ␤-catenin and activation of the
transcription factor, Tcf.
In addition to its role in cell fate, the GSK3␤/␤-catenin/Tcf
signaling pathway has been demonstrated to exert cell cycle
control via regulation of a cell cycle regulatory protein, cyclin
D1.14 –16 Cyclin D1 contains a Tcf response element within its
promoter region and is thought to be a rate-limiting mediator
of the G1 to S phase transition in the cell cycle.14,17 The
cyclin D1 gene encodes the regulatory subunit of the holoenzyme that phosphorylates the Rb protein resulting in its
inactivation. In addition, cyclin D1 has also been shown to act
as an oncogene.18 Thus, the second half of our working
hypothesis states that the induction of VSMC proliferation in
response to vascular injury is mediated in part through the
he dynamic process of vascular remodeling involves
numerous molecular signaling cascades governing
VSMC migration, differentiation, proliferation, and fate.1– 4
The highly conserved Wnt signaling cascade regulates many
of these same processes in lower organisms.5–7 Wnts are a
large family of secreted glycoproteins that bind to a class of
7 transmembrane receptors termed “frizzled.”5–7 Wnt binding
activates disheveled leading to inactivation of the serinethreonine kinase glycogen synthase kinase 3␤ (GSK3␤) and
stabilization and accumulation of ␤-catenin.5–7 ␤-Catenin
translocates to the nucleus and activates a family of transcription factors referred to as T-cell factors/lymphoid-enhancing
factors that collectively include Lef-1, Tcf-1, Tcf-3, and
Tcf-4.7 The roles of ␤-catenin and the Tcf family of HMG
box-containing DNA-binding proteins have not been defined
in VSMCs.
Recent work suggests a role for the Wnt/␤-catenin signaling pathway in the pathophysiological remodeling within the
cardiovascular system.8,10 –13 We recently demonstrated differential inhibition of GSK3␤ in the intimal tissue following
vascular injury that acted as a critical signal mediating VSMC
survival.8 This confirmed previous work in other cell systems
defining a role for GSK3␤ in regulating cell fate.9 Moreover,
Original received June 7, 2001; resubmission received November 27, 2001; revised resubmission received December 19, 2001; accepted December 19,
2001.
From the Cardiovascular Research Institute (X.W., Y.X., Y.M., Y.Z., J.L.H.), Morehouse School of Medicine, Atlanta, Ga; the Division of Cardiology,
Department of Medicine (X.W., J.L.H.), Lillehei Heart Institute, University of Minnesota, Minneapolis, Minn; and the Department of Pharmacology and
Toxicology (W.M.B.), Maastricht University, Maastricht, The Netherlands.
Correspondence to Jennifer L. Hall, PhD, Assistant Professor of Medicine, Cardiovascular Division, Lillehei Heart Institute, University of Minnesota,
420 Delaware St, Minneapolis, MN 55455. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/hh0302.104466
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Wang et al
regulation of a novel ␤-catenin/Tcf signaling pathway. Taken
together, we propose that the GSK3␤/␤-catenin/Tcf-4 signaling relay may play a dual role in vascular remodeling by
inhibiting apoptosis and promoting cell cycle progression.
The purpose of this study was 2-fold: (1) to define the
regulation of distal signaling elements mediated by the
posttranslational modifications of GSK3␤ in the vasculature
after injury and (2) to further characterize the role of this
pathway in mediating VSMC survival and cell cycle
regulation.
Materials and Methods
Materials
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Wild-type ␤-catenin construct (gift from S. Byers), ␤-catenin mutant
construct in which the conserved serine/threonine residues in the
amino terminus of ␤-catenin were mutated to alanines19 (gift D.
Kimelman), a dominant-negative form of Tcf-4, known as Tcf4⌬N31 (Tcf4(N31)) lacking the N-terminal 31 aa as well as the Tcf-4
control vector (pPGS-CMV-CITE-neo)20 (gifts from E. Fearon), the
reporter constructs Topflash and Fopflash, containing either 3 copies
of the optimal Tcf motif CCTTTGATC or 3 copies of the mutant
motif CCTTTGGCC upstream of a minimal c-Fos promoter driving
luciferase expression (gifts from B. Vogelstein),21 cyclin D1 luciferase reporter construct16 (gift from R. Pestell and C. Albanese).
Cell Culture
The clonal A7r5 rat aortic VSMCs were purchased from American
Type Culture Collection.
␤-Catenin and Tcf in Vascular Remodeling
341
Reporter Assays
Luciferase and EGFP activities in cell extracts were determined
according to the manufacturer’s directions. All data are expressed as
fold activation of luciferase activity/EGFP fluorescence over values
for control transfected cells under baseline conditions.
Quantitation of Apoptosis
Apoptosis was assessed by staining with H33342 and quantitating
the percentage of apoptotic nuclei (100 cells counted/sample) in the
transfected subset by identifying cells cotransfected with pDsRed1Mito as previously described.8,23 Our laboratory has extensively
cross-validated the use of H33342 staining with other apoptotic
indices.1,8,22,23
[3H]-Thymidine Incorporation
VSMCs stably expressing a control vector or Tcf4(N31) were placed
in DMEM plus 1% FBS to induce a quiescent state followed by
stimulation with 10% FBS for 18 hours. During the final 4 hours,
[3H]thymidine (1.0 ␮Ci/mL) was added.
Cell Cycle Analysis
Cell cycle analysis was assessed by FACS in VSMCs with constitutive expression of Tcf4(N31) or a control vector after a 24-hour
exposure to media containing 1% FBS followed by stimulation with
10% FBS for 18 hours.
Western Blotting
SDS-PAGE and Western blotting was carried out as previously
described.8 The membranes were re-probed with a mouse monoclonal vimentin antibody to verify equal loading.
Immunofluorescence
Rat Carotid Artery Balloon Injury Model
Balloon injury was performed as previously described with a 2f
balloon catheter in the carotid arteries of 10- to 14-week-old male
Sprague-Dawley rats (350 g, n⫽35; Harlan, Madison, Wis) in
accordance with protocols approved by the Standing Committee on
Animals at Morehouse School of Medicine.1,8,22
A7r5 VSMCs were incubated in serum free media in the presence
and absence of lithium chloride (50 mmol/L) for 6 hours. Cells were
fixed, blocked, and incubated in primary ␤-catenin antibody (1:100)
or an equal concentration of anti–mouse-IgG control. Fluoresceinconjugated secondary antibody (1:100) was then added and incubated for 1 hour.
RNA Isolation and cDNA Preparation
Immunohistochemistry
Total RNA was isolated with RNeasy columns with RNase-free
DNase treatment. Reverse transcription (RT) reactions were performed using oligo (dT)18 as a primer.
Quantitative Real-Time RT-PCR
Changes in mRNA levels under different experimental conditions
were compared by real-time quantitative RT–polymerase chain
reaction (PCR) analysis, using the Light Cycler thermocycler (Roche
Diagnostics Corp). Reactions were prepared in the presence of the
fluorescent dye SYBR green I for specific detection of doublestranded DNA. Quantification was performed at the log-linear phase
of the reaction and cycle numbers obtained at this point were plotted
against a standard curve prepared with serially diluted control
samples.
Transfection
Retrovirus-Mediated Transfection
AmphoPack 293 cells were transfected with the pPGS-CMV-CITEneo vector (control) or the pPGS-CMV-CITE-neo vector containing
the dominant negative Tcf4 mutant lacking the N terminal 31 aa
␤-catenin binding domain (Tcf4(N31))20 via calcium phosphate as
previously described.8 Virus produced from the 293 cells was
transferred to A7r5 VSMCs and stable lines were created.
Transient Transfection
A7r5 VSMCs were transiently transfected with Effectene with a total
of 0.3 ␮g DNA/well according to the manufacturer’s directions.
Paraffin sections (5 ␮m) were mounted on aminopropyltriethoxysilane-coated slides. Diluted (1:500) monoclonal anti–␤-catenin
antibody or equal concentrations of a control mouse IgG1 were
incubated with sections overnight at room temperature. The proliferating cell nuclear antigen (PCNA, 50 ␮g/mL) (DAKO) was stained
according to manufacturer’s directions.
Statistical Analysis
Comparisons between 2 groups were analyzed via a Student’s t test
(P⬍0.05), whereas comparisons between 3 groups were analyzed by
an analysis of variance (ANOVA) with a Student-Newman-Keuls
post hoc test (P⬍0.05). Data are presented as mean⫾SE.
An expanded Materials and Methods section can be found in the
online data supplement available at http://www.circresaha.org.
Results
Regulation of GSK3␤/␤-Catenin Signaling
Pathway After Balloon Injury in the Rat
We utilized real-time quantitative RT-PCR, Western blotting,
and immunohistochemistry to define the expression profile of
␤-catenin after vascular injury. ␤-Catenin mRNA was significantly increased 7 days after balloon injury in rat carotid
arteries (data are expressed as a normalized ratio of ␤-catenin
to GAPDH expression levels) (uninjured control vessel
1.2⫾0.1; injured vessel, 2.2⫾0.3; n⫽9, P⬍0.01) (Figure
1A). GAPDH mRNA was not significantly different between
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February 22, 2002
Figure 3. Representative Western blot demonstrating enhanced
phosphorylated (ser9) GSK3␤ protein expression in injured vessels 7 days after injury compared with uninjured control vessels.
Phosphorylated GSK3␤ protein expression assessed by Western blotting 7 days after balloon injury (Top); same blot
re-probed with vimentin to control for equal loading (Bottom). C
indicates control; I, injury.
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Figure 1. ␤-Catenin mRNA and protein expression in rat carotid
arteries 7 days after balloon injury. A, ␤-Catenin mRNA
assessed by real-time quantitative RT-PCR is expressed as a
normalized ratio of ␤-catenin to GAPDH expression (uninjured
control vessel 1.2⫾0.1; injured vessel, 2.2⫾0.3; n⫽9, P⬍0.01).
B, Representative Western blot of ␤-catenin protein 7 days after
balloon injury (Top); same blot re-probed with vimentin to control for equal loading (Bottom). C indicates control; I, injury.
injured and control (uninjured) vessels. Moreover, the accumulation of ␤-catenin protein was also increased at 7 days
after balloon injury as shown by both Western blotting and
immunohistochemistry (Figures 1B and 2). A defined time
course analysis demonstrated significant accumulation of
␤-catenin at 7 days after injury that was reduced at day 14 and
nearly absent at 28 days (Figure 2). No staining was seen with
the negative mouse IgG1 control antibody (Figure 2).
␤-Catenin is negatively regulated by GSK3␤. Phosphorylation of the regulatory serine 9 site of GSK3␤ inactivates the
kinase and promotes the accumulation of ␤-catenin in
cells.5–7 We utilized a phosphospecific GSK3␤ antibody
specific for the serine 9 site to determine phosphorylation
state of GSK3␤ after 7 days of balloon injury in the rat carotid
Figure 2. Time course of ␤-catenin accumulation assessed by
immunohistochemistry at 40⫻ magnification. A, Negative control
for ␤-catenin (a mouse monoclonal antibody IgG1) in the injured
vessel at 7 days. B through D, ␤-Catenin staining in the injured
rat carotid artery at 7 (B), 14 (C), and 28 (D) days after injury.
artery. In line with the accumulation of ␤-catenin at 7 days,
the phosphorylated and inactive form of GSK3␤ was differentially increased in the injured vessels compared with the
control uninjured vessels (Figure 3, top). The membrane was
re-probed with vimentin to verify equal loading (Figure 3,
bottom). Total GSK3␤ protein expression was unchanged
(data not shown).
␤-Catenin Stimulates an Antiapoptotic
Signaling Pathway
Earlier work from our laboratory demonstrated that the
activation state of GSK3␤ was an important control site
regulating VSMC fate.8 Activation of GSK3␤ was sufficient
to induce apoptosis, whereas inactivation of GSK3␤, as seen
in the intimal tissue, promoted survival.8 We hypothesized
that activation of GSK3␤ degraded ␤-catenin expression and
that ␤-catenin degradation was an important distal event in
the proapoptotic pathway. To test this we blocked ␤-catenin
degradation with a degradation-resistant ␤-catenin mutant
transgene in which the GSK3␤ serine phosphorylation sites
have been mutated to alanines19 and determined the apoptotic
rate in response to GSK3␤ activation (see Materials and
Methods). In line with our hypothesis, transfection of the
degradation-resistant ␤-catenin transgene significantly inhibited GSK3␤-induced apoptosis (control transfected, 13⫾2%
apoptotic nuclei; GSK3␤ transfected, 29⫾2%; GSK3␤⫹␤catenin mutant, 11⫾1%; n⫽6, P⬍0.001) (Figure 4A). This
indirectly suggested that GSK3␤-induced degradation of
␤-catenin was important in mediating the proapoptotic response. To directly test this hypothesis we examined the
ability of a wild-type ␤-catenin transgene (degradable by
GSK3␤) to block GSK3␤-induced apoptosis. In accord with
our hypothesis, upregulation of a wild-type ␤-catenin gene
was ineffective in blocking GSK3␤-induced death (27⫾3%,
n⫽6) (Figure 4A).
Previous work from our laboratory and others has demonstrated that growth factor withdrawal activates GSK3␤.8,9,24
Thus, as another means of testing our working hypothesis that
␤-catenin blocked GSK3␤-induced apoptosis, we subjected
VSMCs to serum withdrawal to activate GSK3␤ and assessed
the ability of ␤-catenin accumulation to inhibit apoptosis. In
line with our previous data, upregulation of the degradationresistant ␤-catenin mutant resulted in significant blockade of
serum withdrawal–induced apoptosis (control transfected,
26⫾3%; ␤-catenin transfected, 13⫾1%; n⫽7, P⬍0.001).
There was no significant change in the percentage of apoptotic nuclei in serum.
Wang et al
␤-Catenin and Tcf in Vascular Remodeling
343
Role of Tcf-4 in ␤-Catenin–Mediated
VSMC Survival
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Figure 4. A, Upregulation of ␤-catenin inhibits apoptosis
induced by GSK3␤. VSMCs were transiently cotransfected with
the reporter gene, pDsRed1-Mito, GSK3␤ or a control expression vector (pcDNA3.1), and a degradation-resistant ␤-catenin
mutant (␤-catmut), ␤-catenin wild type (␤-catwt), or pcDNA3.1.
A subset of cells were treated with LiCl (50 mmol/L). ⌻he percentage of apoptotic nuclei in the mito-tracker–positive subset
of transfected cells was quantitated by assessing nuclear chromatin morphology. (Control transfected, 13⫾2% apoptotic
nuclei; GSK3␤ transfected, 29⫾2% apoptotic nuclei; GSK3␤⫹␤catenin mutant, 11⫾1%; GSK3␤⫹wild-type ␤-catenin, 27⫾3%;
GSK3␤⫹␤-catenin wild type⫹LiCl, 12⫾2%; n⫽6, P⬍0.001).
Data are expressed as mean⫾SE. B through G, Immunofluorescence studies determining localization of ␤-catenin after treatment with vehicle (B through D) or LiCl (50 mmol/L) (E through
G). VSMCs stained with the nuclear stain propidium iodide (B
and E), VSMCs stained with ␤-catenin (C and F), overlay of propidium iodide and ␤-catenin staining (D and G) demonstrating
colocalization of ␤-catenin to the nucleus in cells treated with
LiCl (G) but not in cells treated with vehicle (D).
We hypothesized that ␤-catenin–induced activation of Tcf-4
was an important distal signaling event in the survival
pathway. Initial studies with real-time quantitative RT-PCR
demonstrated that the Tcf-4 isoform was the predominant Tcf
isoform in cultured rat and human VSMCs as well as the rat
carotid artery. To test the ability of ␤-catenin to stimulate
Tcf-4, we transiently transfected a Tcf-4 reporter gene construct (Topflash)21 along with a ␤-catenin degradation-resistant mutant or a control gene into rat VSMCs. Indeed,
upregulation of the ␤-catenin degradation-resistant mutant
resulted in significant activation of the Tcf-4 reporter construct (data expressed as fold activation of luciferase/EGFP)
(control⫹Topflash, 1.0⫾0.1; ␤-catenin mutant⫹Topflash,
22.3⫾1.1; n⫽6, P⬍0.001) (Figure 5A). ␤-catenin–induced
activation of Topflash was 10-fold greater than the mutated
Tcf reporter construct Fopflash (2.4⫾0.1, n⫽6), thereby
demonstrating specificity of the response.
To test whether activation of Tcf-4 was necessary to confer
the ␤-catenin–mediated survival pathway in VSMCs, we
utilized a retroviral strategy to establish a line of VSMCs with
stable upregulation of a FLAG-tagged mutant Tcf-4 gene
lacking the ␤-catenin binding domain (Tcf4(N31)). Characterization of these VSMCs by real-time quantitative RT-PCR
and Western blotting with an anti-FLAG antibody confirmed
that the mutant Tcf-4 transgene was constitutively upregulated (Figures 5B and 5C). In addition, functional assays
using the Tcf-4 reporter construct, Topflash, demonstrated
that ␤-catenin–induced Tcf-4 transactivation in Tcf4(N31)
VSMCs was significantly abolished (Figure 5D). In line with
our hypothesis, loss of ␤-catenin–induced Tcf-4 transactivation, significantly inhibited ␤-catenin–mediated survival in
response to serum withdrawal (stable control VSMC lines
transiently transfected with a control transgene (pcDNA3.1)
30⫾2%; or a ␤-catenin mutant, 15⫾2%; Tcf4(N31) stable
VSMC lines transfected with a control transgene, 30⫾1%; or
a ␤-catenin mutant, 26⫾2%; n⫽7, P⬍0.001) (Figure 5E).
Similar inhibition of Tcf activation and ␤-catenin–induced
survival was seen in transient cotransfection experiments in
which a degradation-resistant mutant ␤-catenin was cotransfected along with the Tcf-4 mutant (control, 36⫾3%;
␤-catenin mutant, 17⫾1%; Tcf4(N31)⫹control, 29⫾1%;
Tcf4(N31)⫹␤-catenin mutant, 31⫾2%; n⫽6, P⬍0.001).
Role of ␤-Catenin/Tcf-4 in Cell Cycle Regulation
As a final test of our hypothesis that ␤-catenin was a
critical distal mediator of VSMC fate, we utilized the wellcharacterized pharmacological inhibitor of GSK3␤, LiCl.
Immunofluorescence studies demonstrated that LiCl resulted
in a significant translocation of ␤-catenin into the nucleus of
VSMCs (Figures 4B through 4G). In line with our hypothesis,
LiCl induced nuclear translocation of endogenous ␤-catenin
coincided with a significant inhibition of GSK3␤-induced
apoptosis (12⫾2%, n⫽6) (Figures 4A through 4G). These
results suggest that accumulation and nuclear translocation of
␤-catenin block a proapoptotic signaling pathway induced by
growth factor withdrawal and GSK3␤ activation.
Previous work in other cell systems has demonstrated that the
␤-catenin/Tcf signaling pathway regulated cell cycle control.14 –16 Indeed, the cell cycle regulatory protein cyclin D1
contains a Tcf-responsive element in its promoter region.14
However, the role of ␤-catenin and Tcf-4 in governing
proliferative signaling pathways in VSMCs has not been
determined. To test the ability of ␤-catenin to stimulate cyclin
D1, we utilized a cyclin D1 promoter reporter construct.16 In
line with previous work in other cell systems, transfection of
a degradation-resistant ␤-catenin mutant resulted in an increase in cyclin D1 promoter activation (control, 1.0⫾0.0;
␤-catenin mutant, 2.3⫾0.2; n⫽6, P⬍0.001). Parallel experiments carried out in Tcf4(N31) VSMCs demonstrated a
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February 22, 2002
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significant blockade of ␤-catenin–induced cyclin D1 activation (control, 0.7⫾0.0; Tcf4(N31)⫹␤-catenin mutant,
1.5⫾0.4; n⫽6, P⬍0.001) (Figure 5F); suggesting that cyclin
D1 expression was regulated via a ␤-catenin/Tcf– dependent
signaling pathway.
To better understand the specific role of Tcf-4 in proliferative signaling pathways, we performed [3H]-thymidine experiments in VSMCs with stable upregulation of the
Tcf4(N31) mutant. Upregulation of Tcf4(N31) under conditions of 10% FBS resulted in a significant inhibition of
thymidine incorporation (Control, 32 671⫾1689 cpm/min;
Tcf4(N31), 24 549⫾272 cpm/min; n⫽12, P⬍0.001). This
suggested that the proliferative response of VSMCs after
vascular injury might be partially governed by the upregulation of ␤-catenin and activation of Tcf-4.
D-type cyclins have been suggested to be rate-limiting
mediators of the G1 to S phase transition in the cell cycle.17
To directly test this, we utilized FACS analysis to determine
the percentage of cells in the S phase of the cell cycle. In line
with our earlier data, we saw a 37% reduction in the
percentage of cells in the S phase of the cycle in the stable
Tcf4(N31) VSMC line compared with the stable control
Figure 5. A, Upregulation of ␤-catenin activates Tcf. VSMCs
were transiently cotransfected with a control vector or a
␤-catenin degradation-resistant mutant, the Tcf-luciferase
reporter construct Topflash, or the mutated reporter construct
Fopflash, and the reporter construct pEGFP-C1. Data are
expressed as fold activation of luciferase/EGFP over control
transfected cells (mean⫾SE) (control⫹Topflash, 1.0⫾0.1;
␤-catenin mutant⫹Topflash, 22.3⫾1.1; control⫹Fopflash,
1.3⫾0.1; ␤-catenin mutant⫹Fopflash, 2.4⫾0.1; n⫽6, P⬍0.001).
B, Tcf-4 mRNA expression assessed by real-time quantitative
RT-PCR and expressed as a ratio of Tcf-4 to GAPDH expression in control transfected rat VSMCs as well as VSMCs with
constitutive upregulation of the Tcf4(N31) transgene. Primers
were designed to the middle region of the gene thus recognizing both the native Tcf-4 as well as the constitutive overexpression of the Tcf4(N31) dominant negative gene. C, Representative Western blot for FLAG epitope expression in VSMCs with
constitutive upregulation of the FLAG-tagged Tcf4(N31) construct. D, ␤-Catenin–induced Tcf-4 transactivation as measured
by a luciferase promoter reporter construct is blocked in VSMCs
with constitutive upregulation of Tcf4(N31), providing functional
characterization (Tcf-4 control at baseline, 1.0⫾0.0; Tcf-4
control⫹␤-catenin mutant, 22.4⫾0.6; Tcf4(N31) at baseline,
1.0⫾0.1; Tcf4(N31)⫹␤-catenin mutant, 1.1⫾0.1; n⫽6, P⬍0.001).
Data are expressed as fold activation of luciferase/EGFP in control cells. E, ␤-Catenin–induced VSMC survival is mediated
through a Tcf-4 – dependent pathway. VSMCs with constitutive
upregulation of a control vector or Tcf4(N31) were transiently
transfected with ␤-catenin and the reporter construct mitotracker red. Apoptosis was assessed in response to serum withdrawal in the transfected subset (Control at baseline, 30⫾2%
apoptotic nuclei; Control⫹␤-catenin mutant, 15⫾2%; Tcf4(N31)
at baseline, 30⫾1%; Tcf4(N31)⫹␤-catenin mutant, 26⫾2%;
n⫽7, P⬍0.001). Data are expressed as mean⫾SE. F, Upregulation of ␤-catenin stimulates cyclin D1 transactivation through a
Tcf-4 – dependent signaling pathway. VSMCs with constitutive
upregulation of a control vector or Tcf4(N31) were transiently
transfected with ␤-catenin, a cyclin D1 luciferase promoter
reporter construct and the reporter construct pEGFP-C1. Data
are expressed as fold activation of luciferase/EGFP over control
cells. Expression was determined under conditions of 10%
serum (control at baseline, 1.0⫾0.0; control⫹␤-catenin mutant,
2.3⫾0.2; Tcf4(N31) at baseline, 0.7⫾0.0; Tcf4(N31)⫹␤-catenin
mutant, 1.5⫾0.4; n⫽6, P⬍0.001). Data are presented as
mean⫾SE.
Wang et al
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Figure 6. ␤-Catenin and PCNA immunohistochemical staining in
the rat carotid artery 7 days after injury shown at 60⫻ magnification. a, Negative control for ␤-catenin (mouse monoclonal
antibody IgG1) in the vessel 7days after injury. b, ␤-Catenin
staining in the injured rat carotid artery at 7 days. c, Negative
control for PCNA (mouse monoclonal antibody IgG2 in the vessel 7 days after injury. d, PCNA staining in injured vessel at 7
days after injury.
VSMC line (Control VSMC, 15.6⫾0.4% in S phase;
Tcf4(N31) VSMC, 9.9⫾0.5% in S phase; n⫽8, P⬍0.001).
Thus taken together, we have identified a novel signaling
pathway regulating cell survival and cell cycle control in
response to vascular injury.
To determine if ␤-catenin expression was colocalized in
proliferating VSMCs within the developing neointima, we
stained for proliferating cell nuclear antigen (PCNA), the
delta accessory protein of DNA polymerase synthesized in
late G1 and S phases of the cell cycle, and ␤-catenin 7 days
after balloon injury in the same vessel. As seen in Figure 6,
␤-catenin expression is partially colocalized in actively proliferating VSMCs. Taken together, our data suggests that
␤-catenin accumulation plays a critical role in VSMC proliferation in the context of vascular remodeling.
Discussion
The dynamic process of vascular remodeling involves numerous molecular signaling cascades governing VSMC migration, differentiation, proliferation, and fate. We hypothesized
that ␤-catenin played a critical regulatory role in remodeling
of the vessel wall in response to balloon injury. In accord with
our hypothesis, we demonstrated significant accumulation of
␤-catenin as well as inactivation of GSK3␤, an upstream
negative regulator of ␤-catenin, 7 days after balloon injury.
Transfection of a degradation-resistant ␤-catenin transgene
as well as translocation of endogenous ␤-catenin to the
nucleus resulted in a significant inhibition of apoptosis as
well as activation of the downstream transcription factor Tcf.
To test if Tcf-4 activation was necessary to confer the
antiapoptotic effect of ␤-catenin stabilization, we performed
loss of function studies in which VSMCs expressing a Tcf-4
dominant negative mutant transgene lacking the ␤-catenin
binding domain were transfected with the degradation-resis-
␤-Catenin and Tcf in Vascular Remodeling
345
tant ␤-catenin transgene. In accord with our hypothesis, loss
of Tcf-4 activation abolished ␤-catenin–induced survival.
Prior work from our laboratory and others has demonstrated that mitogens, nutrient signals, or receptor ligand
systems that induce an antiapoptotic effect in VSMCs also
have the capability to promote proliferation.2,8 Furthermore,
recent work has demonstrated that the GSK3␤/␤-catenin/Tcf
signaling promotes activation of cyclin D1.14 Based on these
findings, we further hypothesized that injury-induced activation of ␤-catenin and Tcf-4 played a dual role in the course of
vascular remodeling: stimulating cyclin D1 as well as inhibiting apoptosis. Indeed, upregulation of ␤-catenin resulted in
a significant activation of cyclin D1. The ␤-catenin–induced
activation of cyclin D1 was lost in VSMCs expressing the
mutant Tcf-4 gene lacking the ␤-catenin binding domain,
suggesting that Tcf-4 was mediating the ␤-catenin–induced
increase in cyclin D1. To our knowledge, this is the first study
to demonstrate a role for the ␤-catenin/Tcf-4 signaling
pathway in VSMCs.
We provide evidence from multiple experiments that accumulation of ␤-catenin in the neointima and activation of
Tcf-4 plays a critical role in VSMC proliferation. Brabletz et
al25 demonstrated that ␤-catenin expression in human tumors
did not correlate with proliferative indices but rather with
hypertrophy. However, regulation of Tcf-4 was not examined
in this study. We speculate that the ability of ␤-catenin to
transduce numerous signals to the cell including survival,
hypertrophy, and proliferation is coupled to the downstream
transcription factor(s) that it regulates. This likely depends on
the balance of outside signals, including growth factors, etc.
It is noteworthy that blockade of ␤-catenin binding to Tcf-4
at baseline does not significantly affect VSMC proliferation
as evident from the ability of our line of VSMCs with
constitutive expression of Tcf4(N31) to proliferate normally.
These findings suggest that Tcf-4 plays a significant role
under conditions of vascular remodeling when ␤-catenin is
elevated but likely plays a minor role at baseline. It is
noteworthy that mice lacking Tcf-4 die shortly after birth
with the single histopathological abnormality in these animals
being a lack of cell proliferation in the crypt regions within
the small intestine, with no denoted abnormalities in the
vasculature.26
The role of ␤-catenin in apoptotic regulation appears to be
cell-type specific.27–29 In line with our data, upregulation of
the ␤-catenin/Tcf-4 signaling pathway has been demonstrated
to inhibit apoptosis in fibroblasts.27 However, recent work has
reported that ␤-catenin promotes apoptosis in other cell
types.28,29 The disparity concerning the pro- or antiapoptotic
role of ␤-catenin in different cell types is not surprising to the
field of vascular biology given that numerous factors, including nitric oxide, glucose, and TGF␤, have opposing effects on
vascular endothelial cells compared with VSMCs.2 Our work
demonstrates that the ability of ␤-catenin to promote survival
and activate cyclin D1 was mediated through Tcf-4. Work in
other cell systems has established that upregulation of
␤-catenin activates several transcription factors, including
Tcf-4, lymphoid enhancing factor, TATA-binding protein,
Pontin, Teashirt, Sox13 and Sox17, and CREB.5–7,30 –34 It is
likely that the role of ␤-catenin in regulating cell fate in
346
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February 22, 2002
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different tissues is mediated through the complex regulation
of multiple transcription factors and cobinding factors.
It is also noteworthy that we see a discrepancy between the
time course of ␤-catenin accumulation and GSK3␤ phosphorylation state in the developing lesion. We previously demonstrated that 28 days after balloon injury GSK3␤ remains
inactivated8; however, ␤-catenin mRNA (data not shown) and
protein in the injured vessel had returned to baseline values.
This suggests that the regulation of the complex of GSK3␤,
␤-catenin, axin, glycogen binding protein, APC, and yet to be
defined factors is clearly dependent on multiple lines of
regulation in addition to the phosphorylation state of GSK3␤.
Moreover, parallel signaling elements including the cytoskeletal cadherin family are also likely to be regulating ␤-catenin
accumulation. Future studies will need to be completed to
more fully understand the multitude of factors regulating
␤-catenin expression.
The most widely studied areas of research involving the
Wnt cascade in mammals have been cancer biology,
development, and stem cell differentiation.5–7,35–37 Recent
work defines a role for ␤-catenin in stem cell differentiation into follicular keratinocytes.37 Furthermore, ␤-catenin
appears to be necessary in the development of skin and
hair.37 To our knowledge this is the first work to date to
define a role for ␤-catenin in the vasculature. Preliminary
data from our laboratory suggests an upregulation of the
upstream Wnt signaling mediator, disheveled 1, by in situ
hybridization in the developing lesion. Paired with work
by others demonstrating expression of Wnt and Frizzled
family members in the vasculature, we speculate that
␤-catenin stabilization and Tcf activation in the remodeled
vessel are regulated in part through the Wnt signaling
pathway. However, future studies will be needed to directly implicate a role of Wnts and the frizzled family of
receptors in vascular disease.
In conclusion, we have identified an integral role for
␤-catenin and Tcf-4 in the regulation of vascular remodeling.
To our knowledge, this is the first evidence of a role for these
well-conserved genes in the process of vascular remodeling.
Acknowledgments
This project was supported by a Scientific Development Grant from
the American Heart Association (J.L.H., 0030136N) as well as an
NIH Center of Excellence Award for Enhancement in Cardiovascular and related research areas and the Research Centers in Minority
Institutions program (G. Gibbons, Morehouse School of Medicine,
NIH/NHLBI 5 UH1 HL03676). The authors wish to thank D.
Kimmelman, A. Kowalcyzk, E. Fearon, B. Vogelstein, R. Pestell,
and C. Albanese for the constructs utilized in this project. In
addition, special thanks to G. Gibbons for his insightful suggestions,
advice, and support.
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A Role for the β-Catenin/T-Cell Factor Signaling Cascade in Vascular Remodeling
Xiaohong Wang, Yan Xiao, Yongshan Mou, Ying Zhao, W. Matthijs Blankesteijn and Jennifer
L. Hall
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Circ Res. 2002;90:340-347; originally published online January 3, 2002;
doi: 10.1161/hh0302.104466
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2002 American Heart Association, Inc. All rights reserved.
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