Krüppel-like factor 4 is
induced by rapamycin and
mediates the anti-
proliferative effect of
rapamycin in rat carotid
arteries after balloon injury
Ying Wang1,2, Beilei Zhao1, Yi Zhang2, Zhihui Tang1, Qiang Shen1,
Youyi Zhang1, Weizhen Zhang1, Jie Du2, Shu Chien3,4and
1Key Laboratory of Molecular Cardiovascular Science of Ministry of Education, Peking University
Health Science Center, Beijing, China,2Beijing Anzhen Hospital Affiliated to the Capital Medical
University, The Key Laboratory of Remodeling-Related Cardiovascular Diseases, Capital Medical
University, Ministry of Education, Beijing, China,3Department of Bioengineering, University of
California San Diego, La Jolla, CA, USA,4Department of Medicine, University of California San
Diego, La Jolla, CA, USA, and5Cardiovascular Research Center, Xi’an Jiaotong University School
of Medicine, Xi’an, China
Nanping Wang, Cardiovascular
Research Center, Xi’an Jiaotong
University School of Medicine,
Xi’an 710061, China. E-mail:
rapamycin; transcription; smooth
muscle cells; restenosis; gene
25 March 2011
30 August 2011
24 September 2011
BACKGROUND AND PURPOSE
The transcription factor, Krüppel-like factor 4 (KLF4), plays an important role in regulating the proliferation of vascular smooth
muscle cells. This study aimed to examine the effect of rapamycin on the expression of KLF4 and the role of KLF4 in arterial
Expression of KLF4 was monitored using real-time PCR and immunoblotting in cultured vascular smooth muscle cells. and in
rat carotid arteries in vivo after balloon injury. Adenovirus-mediated overexpression and siRNA-mediated knockdown of KLF4
were used to examine the role of KLF4 in mediating the anti-proliferative role of rapamycin . KLF4-regulated genes were
identified using cDNA microarray.
Rapamycin induced the expression of KLF4 in vitro and in vivo. Overexpression of KLF4 inhibited cell proliferation and the
activity of mammalian target of rapamycin (mTOR) and its downstream pathways, including 4EBP-1 and p70S6K in vascular
smooth muscle cells and prevented the neointimal formation in the balloon-injured arteries. KLF4 up-regulated the expression
of GADD45b, p57kip2and p27kip1. Furthermore, knockdown of KLF4 attenuated the anti-proliferative effect of rapamycin both
in vitro and in vivo.
CONCLUSIONS AND IMPLICATIONS
KLF4 plays an important role in mediating the anti-proliferative effect of rapamycin in VSMCs and balloon-injured arteries.
Thus, it is a potential target for the treatment of proliferative vascular disorders such as restenosis after angioplasty.
British Journal of
2378British Journal of Pharmacology (2012) 165 2378–2388
© 2011 The Authors
British Journal of Pharmacology © 2011 The British Pharmacological Society
Ad-KLF4, adenovirus expressing KLF4; ECs, endothelial cells; GADD45b, growth arrest and DNA damage inducible gene
b; IMT, intimal–medial thickness; KLF4, Krüppel-like factor 4; mTOR, mammalian target of rapamycin; mTOR-KD,
kinase-dead mTOR; p27kip1, cyclin-dependent kinase inhibitor 1b; p57kip2, cyclin-dependent kinase inhibitor 1c; p70S6K,
p70 S6 kinase; PDGF, platelet-derived growth factor; PTCA, percutaneous transluminal coronary angioplasty; tTA,
tetracycline-controlled transactivator; VSMCs, vascular smooth muscle cells; 4EBP1, eukaryotic translation initiation
factor 4E binding protein 1
Coronary artery disease is a leading cause of morbidity and
mortality in the Western countries as well as in Asia. Cur-
rently, percutaneous transluminal
(PTCA) is widely used to treat severe arterial stenosis.
However, restenosis has been a major drawback, occurring in
10–40% of the patients receiving angioplasty (Nobuyoshi
et al., 1988; Serruys et al., 1988). Although the pathophysi-
ological mechanisms remain to be fully elucidated, it is gen-
erally recognized that, in response to a range of cytokines
generated including platelet-derived growth factor (PDGF)
and fibroblast growth factor, the proliferation and migration
of vascular smooth muscle cells (VSMCs) play central roles in
the neointimal hyperplasia (Ferns et al., 1991; Jawien et al.,
1992; Agrotis et al., 2004). Rapamycin (sirolimus), a cytostatic
macrocyclic lactone with anti-proliferative properties, has
been used in drug-eluting stents to reduce the risk of resteno-
sis (Marx et al., 1995; Morris et al., 1995; Gallo et al., 1999;
Sousa et al., 2001). Rapamycin binds to cytosolic FKBP-12 and
inhibits the protein kinase mTOR (mammalian target of rapa-
mycin), which regulates the initiation of protein translation
by phosphorylating its downstream targets such as p70 S6
kinase (p70S6K) (Heitman et al., 1991; Marks, 1996; Inoki and
Guan, 2006). Recently, the therapeutic effects of rapamycin
have also been related to its ability to modulate the transcrip-
tional program (Zohlnhofer et al., 2004). However, the tran-
scription factor(s) that mediate the anti-proliferative effect of
rapamycin in VSMCs remain to be characterized.
Krüppel-like factors (KLF) are a family of evolutionarily
conserved zinc finger-containing transcription factors. KLF4,
or gut-enriched KLF, was initially considered to be an
epithelial-specific transcription factor functioning in the ter-
minal differentiation and regulation of gut and skin epithe-
lium (Garrett-Sinha et al., 1996; Shields et al., 1996; Segre
et al., 1999). However, KLF4 is also expressed in vascular
tissues including endothelial cells (ECs), VSMCs and in mac-
rophages (Yet et al., 1998; Adam et al., 2000; King et al., 2003;
Feinberg et al., 2005). In ECs, gene expression of KLF4 is
induced by laminar shear stress and was shown to negatively
inflammatory stimuli (Hamik et al., 2007). However, it
appears to have a pro-inflammatory effect in macrophages,
suggesting a cell type-specific role in inflammation (Feinberg
et al., 2005; Alder et al., 2008). In VSMCs, KLF4 is rapidly
induced by TGF-b and PDGF-BB (Adam et al., 2000; King
et al., 2003). In addition, overexpression of KLF4 suppressed
VSMC differentiation markers such as myocardin and con-
tributed to the phenotypic control of PDGF-BB (Liu et al.,
2005). However, deletion of KLF4 accelerated neointimal for-
in responseto pro-
mation following vascular injury in mice through reducing
cellular proliferation, indicating that KLF4 is an important
transcription factor in the vascular response to injury
(Yoshida et al., 2008). Nonetheless, the regulatory mecha-
nisms underlying KLF4 expression during vascular remodel-
ling are largely unknown. In the present study, we have
found that KLF4 expression was increased by rapamycin in
VSMCs and balloon-injured rat arteries and we have explored
the role of KLF4 in mediating the anti-proliferative effect of
rapamycin in the context of vascular injury.
Adenoviral vectors and plasmids
To generate the recombinant adenovirus expressing KLF4
(Ad-KLF4), a cDNA fragment containing the full-length
coding region of mouse KLF4 was subcloned from pMT3-
KLF4 (a generous gift from Dr Vincent Yang, Emory Univer-
sity) into pAdlox and recombined with an E1- and E3-deleted
adenovirus-5 genome DNA in a Cre-recombinase-expressing
293 cell line. The expression of the inserted gene was driven
by a 7¥ tet operon/minimal CMV promoter, which was
further under the control of tetracycline-controlled transac-
tivator (tTA). The adenoviruses were plaque-purified and
titrated in 293 cells (Wang et al., 2002). The expression plas-
mids encoding HA-TSC2, HA-TSC2 1-1080, mTOR and the
kinase-dead mTOR (mTOR-KD) were previously described
(Inoki et al., 2002; 2003).
All animal care and experimental procedures conformed to
the Guide for the Care and Use of Laboratory Animals pub-
lished by the US National Institutes of Health (NIH Publica-
tion no. 85-23, revised 1996) and were approved by the
Animal Research Committee of Peking University Health
Science Center. Sprague–Dawley rats were given standard rat
chow and water ad libitum and maintained on a 12 h light/
12 h dark schedule at 22–25°C with 45–65% humidity.
Animals were killed by overdose of sodium pentobarbital
(200 mg·kg-1, i.p.).
Balloon injury was performed as described previously
(Clowes et al., 1983; Tang et al., 2008). Briefly, male Sprague–
Dawley rats (total number = 42) weighing 350 to 400 g were
anaesthetized with sodium pentobarbital (60 mg·kg-1, i.p.).
To make sure the adequacy and depth of anaesthesia of
animals, respiratory pattern, mucous membrane colour and
reactions to manipulation were monitored and toe pinch
method was used. During the procedure, body temperature
was monitored by a rectal temperature probe and maintained
Rapamycin induces KLF4 in vascular smooth muscle cells
British Journal of Pharmacology (2012) 165 2378–23882379
at 37 ? 1°C by means of a heated operating table. The left
common carotid artery was injured with a 2F Fogarty balloon
catheter inserted from the external carotid artery. Rapamycin
was perivascularly delivered with 30% pluronic gel (100 mg
per artery). DMSO containing gel was used as control. Unin-
jured arteries were treated similarly.
At different time points following the arterial injury, vascular
croscopy system (Vevo770, Visualsonics, Toronto, Canada) as
previously described (Clowes et al., 1983; Razuvaev et al.,
2008). Briefly, rats were anaesthetized with pentobarbital
sodium (60 mg·kg-1, i.p.), and the hair on the jugular section
was removed. A 17.5 MHz scanning head (RMV-707B) with a
15 to 45 MHz frequency band was used. The real-time images
were captured. The same person did all examinations and
ments. Intimal–medial thickness (IMT) and the lumen size
were measured with the VEVO 770TM analysis software, IMT
was defined as the distance from the lumen–intima interface
to the media–adventitia interface. Lumen diameter was
the nearwall and the lumen–intima interface of the farwall.
Rat aortic VSMCs were isolated from thoracic aorta of male
200 mg·kg-1, i.p., n = 3) and grown in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and antibiotics.
Cell proliferation assays
VSMCs were synchronized by serum starvation (0.1% FBS) for
24 h and, then, stimulated with 10% FBS for 24 h. Harvested
cells were fixed with 70% ethanol and stained with pro-
pidium iodide (50 mg·mL-1) for 10 min. Cell cycle distribution
was analysed with a FACScan (Becton-Dickinson, Franklin
Lakes, NJ, USA). To detect the vascular cell replication rate in
vivo, rats were i.p. injected with BrdU (25 mg·kg-1) at 18 h and
2 h before killing (sodium pentobarbital, 200 mg·kg-1, i.p.).
The arteries were perfusion-fixed with 4% paraformaldehyde
in PBS under a pressure of 100 mmHg and the segments were
snap-frozen in OCT embedding compound. The cryosections
of arterial segments were denatured with 1.5 N HCl and sub-
jected to immunofluorescent staining.
Total protein lysates were prepared from VSMCs with the lysis
buffer (50 mM Tris–HCl, pH 7.5, 15 mM EGTA, 100 mM
NaCl, 0.1% Triton X-100 supplemented with protease inhibi-
tor cocktail) and resolved on SDS-PAGE. Cytoplasmic pro-
teins were extracted with the use of hypotonic lysis buffer
(10 mM Tris–HCl, pH 7.5, 1.5 mM MgCl2, 10 mM KCl, 0.5%
NP-40, supplemented with protease inhibitor cocktail), and
nuclear proteins were extracted with the use of a high-salt
buffer (20 mM Tris–HCl, pH 7.5, 1.5 mM MgCl2, 420 mM
NaCl, 10% glycerol, 0.2 mM EGTA, supplemented with pro-
tease inhibitor cocktail). Immunoblotting was performed
with appropriate primary antibodies and a horseradish per-
oxidase (HRP)-conjugated secondary antibody followed by
ECL detection (Amersham Biosciences, Piscataway, NJ, USA).
RNA samples were analysed with Affymetrix U230 2.0 arrays,
which contain over 31 000 probe sets representing 30 000
gene transcripts. RNA labelling, hybridization, washing and
scanning of the microarray were performed following the
manufacturer’s specifications (Affymetrix, Santa Clara, CA,
USA). The results were analysed with GeneChip Operating
Software 1.4. Primary analysis of raw cell data files was per-
formed using the dChip platform. Control RNA was consid-
Total RNA was isolated from VSMCs and arterial segments
with Trizol Reagent (Invitrogen, Grand Island, NY, USA).
cDNA was synthesized with the use of Superscript III reverse
transcriptase and oligo-(dT) primer. qPCR was performed
with the iQTMSYBR Green PCR Supermix in the DNA Engine
Opticon real-time system (Bio-Rad, Hercules, CA, USA), with
GAPDH used as an internal control. The primer sequences are
as follows: KLF4, 5′-CTGAACAGCAGGGACTGTCA-3′ (for-
ward), 5′-GTGTGGGT GGCTGTTCTTTT-3′ (reverse); GAPDH,
5′-ACCACAGTCCATGCCATCAC-3′ (forward), 5′-TCCACCA
CCCTGTTGCTGTA-3′ (reverse); p57kip2, 5′-TCTGAGCAGGT
CTCTGAGCA (forward), 5′-CAGGAGCCACGTTAGGAGAG
(reverse); GADD45b, 5′-GACAACGCGGTTCAGAAGAT (for-
ward), 5′-TGACAGTTCGTGACCAGGAG (reverse).
Small-interfering RNA (siRNA)-mediated gene
The siRNAs targeting rat KLF4 mRNA (NM_053713.1) were
5′-ACCUUGCCUUACACAUGAATT-3′; siKLF4-298, 5′-GGAC
CUAGACUUUAUCCUUTT-3′. The siRNA against an irrel-
evant sequence derived from the Thermotoga maritimia
genome was used as a control. The double-strand RNAs
(100 nM) were transfected into VSMCs with Lipofectamine
2000 (Invitrogen). The control siRNA was used at the same
dose. For in vivo use, 15 mg of the siRNA dissolved in 30%
pluronic gel solution was perivascularly delivered to the rat
carotid arteries immediately after injury as described previ-
ously (Simons et al., 1992).
All values are expressed as means ? SEM. Student’s t-test
(paired groups) or one-way ANOVA followed by Newman–
Keuls test (multi-group comparisons) were used to analyse the
statistical significance of differences. P < 0.05 was considered
significant. Non-quantitative results were representative of at
least three independent experiments.
Antibodies against KLF4 and BrdU were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA, USA), antibodies against
mTOR, phosphorylated mTOR (Ser2448), S6K, phosphorylated
Y Wang et al.
2380 British Journal of Pharmacology (2012) 165 2378–2388
S6K (Thr389), 4EBP1 (eukaryotic translation initiation factor
4E binding protein 1), phosphorylated 4EBP1 (Thr70),
(Danvers, MA, USA). Rapamycin, recombinant PDGF-BB and
antibody against GAPDH and p27kip1were from Sigma-
Aldrich, Grand Island, NY, USA.
Rapamycin induced KLF4 expression in vitro
and in vivo
To examine the effect of rapamycin on gene expression of
KLF4, VSMCs were treated with rapamycin (100 ng·mL-1) or
vehicle (DMSO) for 12 and 24 h. qRT-PCR showed that mRNA
level of KLF4 was significantly increased by rapamycin
(Figure 1A). Westernblotting
Rapamycin exerts its anti-proliferative effect via the sup-
pression of mTOR. To investigate the role of mTOR in the
regulation of KLF4 expression, VSMCs were transfected with
plasmids expressing wild-type mTOR or the kinase-dead
mutant (mTOR-KD). The results showed that expression of
mTOR, but not mTOR-KD, decreased gene expression of KLF4
in VSMCs (Figure 1C). Conversely, overexpression of the
tumour suppressor tuberous sclerosis complex (TSC)-2, which
functions as a complex with TSC1 to negatively regulate the
KLF4 was up-regulated by rapamycin in vitro and in vivo. (A) VSMCs were treated with rapamycin (100 ng·mL-1) or control (DMSO) for 12 and
24 h. KLF4 mRNA level was assessed with qRT-PCR and expressed as fold induction compared with the controls after normalizing to GAPDH. (B)
After treatment with a range of concentrations of rapamycin (2–200 ng·mL-1) for 24 h, nuclear extracts were immunoblotted with antibodies
against KLF4 and histone. Data are representative of five independent experiments. (C) VSMCs were transfected with plasmids expressing mTOR,
mTOR-KD or control plasmid. Total RNA was extracted 36 h later and subjected to qRT-PCR for KLF4. (D) KLF4 mRNA levels were detected in
VSMCs transfected with TSC2, TSC2-1080 or control plasmid. Total RNA was extracted 36 h later and subjected to qRT-PCR for KLF4. (E) Rat
carotid arteries were balloon-injured. Total RNA was extracted 72 h later and subjected to qRT-PCR for KLF4 (n = 6 for each group). Balloon-injured
arteries were treated perivascularly with pluronic gel containing rapamycin (100 mg per artery) or DMSO, for 72 h before RNA extraction (n = 3
for each group). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from control.
Rapamycin induces KLF4 in vascular smooth muscle cells
British Journal of Pharmacology (2012) 165 2378–2388 2381
activity of mTOR, increased the gene expression of KLF4.
However, TSC-2-1-1080, a truncated mutant lacking the
GTPase-activating domain, had little effect (Figure 1D). The
results suggested that KLF4 induction by rapamycin might
involve inhibition of the mTOR pathway.
To further examine the effect of rapamycin on KLF4 in
vivo, rat carotid arteries were injured and treated perivascu-
larly with pluronic gel containing rapamycin or vehicle
decreased in injured arteries (n = 6 for each group, P < 0.01),
and rapamycin significantly up-regulated KLF4 (n = 3 for each
group, P < 0.05) (Figure 1E). Similarly, rapamycin also
increased KLF4 in uninjured arteries (Figure S1) (n = 3 for each
group, P < 0.05).
that KLF4 expressionwas
Overexpression of KLF4 inhibited mTOR
activation and induced the expression
To explore the functional role of KLF4 in mediating the effect
of rapamycin on VSMCs, we constructed a tetracycline-
regulated adenovirus expressing KLF4 (AdKLF4). As shown in
Figure 2A, overexpression of KLF4 was observed in VSMCs
co-infected with AdKLF4 and AdtTA, a virus expressing the
tetracycline-controlled expression of KLF4. Furthermore,
immunofluorescence confirmed the nuclear localization of
the overexpressed KLF4 (Figure 2B).
As KLF4 was up-regulated by rapamycin and down-
regulated by overexpression of mTOR in VSMCs, we exam-
ined whether induced expression of KLF4 could also affect
mTOR activation. VSMCs were infected with AdKLF4 and
adtTA for 48 h and stimulated with PDGF-BB. PDGF-BB was
chosen because of its high affinity to three types of PDGF
receptors and its pathophysiological role in restenosis
(Raines, 2004). Western blotting showed that KLF4 overex-
pression markedly attenuated the PDGF-induced phosphory-
lation of mTOR and its downstream targets 4E-BP1 and
p70S6K (Figures 2C and S2).
As previous studies showed that p27kip1was essential for
the anti-proliferation effect of rapamycin, we also examined
whether overexpression of KLF4 could affect the expression
of p27kip1. VSMCs were infected with AdKLF4 and AdtTA for
48 h. Western blotting showed that KLF4 overexpression
induction of KLF4 might enhance the role of rapamycin
through inhibiting activation of mTOR and up-regulating
transactivator, whichdrives the
Overexpression of KLF4 inhibited the activation of mTOR. VSMCs were co-infected with AdKLF4 and AdtTA (20 MOI) and maintained in the
medium with or without tetracycline (Tc; 0.1 mg·mL-1). (A) Nuclear protein lysates were immunoblotted with antibodies against KLF4 or histone
H3 as an internal control. (B) Immunofluorescent staining was performed using a primary antibody against KLF4 and detected with a
rhodamine-conjugated secondary antibody. Nuclei were counterstained with Hoechst 33258. (C) VSMCs were treated with PDGF-BB (20 ng·mL-1)
for the indicated time, and total proteins were immunoblotted with antibodies against mTOR, p70S6K, 4EBP1 or their phosphorylated forms. Data
shown are representative of three independent experiments. (D) Nuclear protein lysates were immunoblotted with antibody against p27kip1and
histone H3. Data are representative of three independent experiments. **P < 0.01, *P < 0.05, significantly different from VSMCs infected with
AdtTA and AdKLF4 in the presence of Tc.
Y Wang et al.
2382British Journal of Pharmacology (2012) 165 2378–2388
KLF4 inhibited the proliferation and intimal
hyperplasia in injured arteries
To explore the functional role of KLF4 in neointimal hyper-
plasia, rat carotid arteries were balloon-injured and adenovi-
rally transduced to express KLF4 or LacZ as a control. KLF4
transgene expression in the vessel wall was confirmed with
immunohistochemical staining (Figure 3A). Intimal cell pro-
liferation was evaluated with BrdU incorporation 7 days after
balloon angioplasty, when the intimal proliferation peaked.
The results showed that BrdU-incorporated intimal cells were
decreased by approximately 50% in the KLF4-transduced
arteries comparedwith the
(Figure 3A), indicating that KLF4 inhibited intimal SMC pro-
liferation in vivo.
IMT and vascular lumen sizes were examined by ultra-
sound biomicroscopy on days 7, 14 and 21 after gene deliv-
ery. Overexpression of KLF4 effectively inhibited the increase
of IMT and preserved the lumen size, compared with the
vector control (Table S1). The animals were killed on day 21
after gene delivery, for morphometric analysis of the cross-
sections of the injured carotid arteries. The results (Figure 3B)
revealed that the carotid arteries infected with the control
adenovirus exhibited pronounced neointimal formation,
while those infected with AdKLF4 had a significantly reduced
neointimal hyperplasia (intimal areas: 184.4 ? 9.3 versus
72.1 ? 12.9 mm2¥ 10-3, P < 0.01, I/M ratio: 1.60 ? 0.62 versus
0.69 ? 0.11, P < 0.01, n = 11 for control, n = 10 for KLF4). No
difference in medial area was observed between the two
groups (data not shown). These results demonstrated that
KLF4 suppressed neointimal hyperplasia in injured arteries.
KLF4 up-regulated an anti-proliferative gene
profile in SMCs
To identify the potential targets of KLF4 that mediated the
inhibition of vascular remodelling, gene profiles were com-
pared between the AdKLF4- or mock-infected SMCs with
cDNA array by using Affymetrix U230 2.0 gene chips (Fig-
ure S3). Among the KLF4-regulated genes, proliferation-
related genes including two cell cycle regulators, cyclin-
dependent kinase inhibitor 1c (p57kip2) and growth arrest and
DNA damage inducible gene b (GADD45b), were validated in
vitro and in vivo. As shown in Figure 4A and B, overexpression
of KLF4 significantly induced the expression of p57kip2and
GADD45b in cultured SMCs and in balloon-injured arteries. It
is thus suggested that induction of these anti-proliferative
genes may contribute to the suppressive effects of KLF4 on
Overexpression of KLF4 inhibited neointimal formation. (A) Rat carotid arteries were balloon injured and infected with AdtTA and AdKLF4 or
AdLacZ. Vessel segments were harvested 7 days later for immunohistochemical staining of KLF4 (a rabbit IgG was used as negative control) and
BrdU. The bar graph indicates the percentage of BrdU-positive cells in the neointima (n = 4 for each group). (B) Rat carotid arteries were
balloon-injured and co-infected with AdtTA and AdKLF4 or with AdLacZ. Vessel segments were harvested 21 days later and cross-sections were
stained with haematoxylin–eosin to evaluate neointimal formation. Intimal and medial areas were measured and expressed as mean ? SEM of
neointimal area (mm2) and intima-to-media ratio (I/M) (n = 11 for Lac Z, n = 10 for KLF4) (Scale bar =100 mm, **P < 0.01, *P < 0.05, significantly
different from values with AdLacZ).
Rapamycin induces KLF4 in vascular smooth muscle cells
British Journal of Pharmacology (2012) 165 2378–23882383
RNA interference of KLF4 enhanced the
proliferation of VSMCs
To examine whether endogenous KLF4 plays a role in VSMC
proliferation, we transfected VSMCs with KLF4 siRNAs (two
sets of siRNA targeting different regions of KLF4 mRNA were
used to ensure efficient gene silencing) or control siRNA.
KLF4 siRNA efficiently decreased KLF4 at both mRNA and
protein levels (Figure 5A,B). Knockdown of endogenous KLF4
significantly increased the serum-induced DNA synthesis in
VSMCs (Figure 5C).
To elucidate the effect of KLF4 on the activation of mTOR,
VSMCs were transfected with KLF4 siRNA or control siRNA
for 48 h and stimulated with PDGF-BB. Knockdown of KLF4
potentiated the PDGF-BB induced phosphorylation of mTOR
and its downstream p70S6K and 4EBP1 (Figure 5D). These
results suggested that endogenous KLF4 played an important
role in the proliferation of VSMCs, by inhibiting their prolif-
eration and suppressing activation of the mTOR pathway,
induced by PDGF-BB.
Knockdown of KLF4 attenuated the
anti-proliferative role of rapamycin in vitro
and in vivo
To explore the possibility that KLF4 mediated the anti-
proliferative role of rapamycin, we examined the effect of
rapamycin upon KLF4 siRNA transfected-VSMCs and injured
arteries. We found that knockdown of KLF4 attenuated the
suppressive effect of rapamycin on VSMC proliferation
(Figure 6A) and the expression of proliferation cell nuclear
antigen (PCNA), a well-established marker for cell prolifera-
tion (Figure 6B). These results suggested that knockdown of
KLF4 reduced the sensitivity of VSMCs to the anti-
proliferative effect of rapamycin. We further examined the
role of endogenous KLF4 in mediating the anti-proliferative
effect of rapamycin in vivo. Perivascular delivery of KLF4
siRNA effectively decreased the KLF4 mRNA level in injured
carotid arteries (Figure S4) and resulted in increased BrdU
incorporation in injured arteries and neointimal formation
(BrdU incorporation ratio: 43.8% ? 1.8% vs. 55.8% ? 4.7%,
P < 0.05, n = 3 for each group. intimal/medial ratio: 0.27 ?
0.03 vs. 0.46 ? 0.04, P < 0.001, n = 3 for each group). KLF4
siRNA attenuated the inhibitory effects of rapamycin on
intimal proliferation in injured arteries (BrdU incorporation
ratio: 8.0% ? 1.5% vs. 32.0% ? 4.1%, P < 0.01, n = 3 for each
group. intimal/medial ratio: 0.13 ? 0.01 vs. 0.22 ? 0.01,
P < 0.05, n = 3 for each group) (Figure 6C,D). Taken together,
these results suggested that endogenous KLF4 functioned as a
negative regulator of VSMC proliferation and the induction
of KLF4 contributed to the anti-proliferative action of rapa-
mycin in vitro and in vivo.
In this study, we describe the novel finding that rapamycin
induced expression of KLF4 in vitro and in vivo. We also
demonstrated a critical role of KLF4 in mediating the anti-
proliferative effects of rapamycin on VSMC proliferation.
Rapamycin is an anti-proliferative agent, widely used to
inhibit post-angioplasty restenosis. In addition to a well-
recognized action on protein translational machinery via the
suppression of mTOR-mediated signal pathways, recent
studies indicated that the anti-proliferative effect of rapamy-
cin may also involve the regulation of transcriptional mecha-
nisms controlling cellular proliferation (Zohlnhofer et al.,
2004). However, the transcription factor(s) that mediate the
anti-proliferative effects of rapamycin remain less character-
ized. Here, our results show that rapamycin induced KLF4 in
VSMCs and arteries (Figures 1B,E and S1). However, because
of the low abundance of endogenous KLF4 protein in arteries,
the effect of rapamycin on KLF4 protein level in arteries still
needs further confirmation. Our results indicated that induc-
tion of KLF4 may represent a novel, transcriptional regulation
step, in the mediation of the pharmacological actions of
Previous groups have found that certain types of cells
exhibited a decreased sensitivity to rapamycin (Dumont
et al., 1995; Sugiyama et al., 1996). Here, we report, for the
first time, that KLF4 may affect the sensitivity to rapamycin
in VSMCs and injured arteries (Figure 6A,D). This effect may
be explained by a reciprocal inhibition between mTOR and
KLF4. This interaction is supported by our observation that
expression of KLF4 was suppressed by mTOR but not by its
kinase-dead mutant (Figure 1C), and was increased by TSC2,
a suppressor of mTOR (Figure 1D). Moreover, KLF4 inhibited
the phosphorylation of mTOR and its downstream targets
p70S6K and 4E-BP1 (Figures 2C and S2). As mTOR is activated
in PDGF-stimulated VSMCs and in restenosis, suppression of
mTOR may be involved in the anti-proliferative effect of KLF4
in VSMCs (Tang et al., 2008).
KLF4 regulated cell cycle-related genes in SMCs. (A) RNA was iso-
lated from SMCs infected with AdKLF4 or mock for 36 h. Gene
expression of p57kip2and GADD45b were validated with qRT-PCR. (B)
Rat carotid arteries were balloon-injured and infected with AdKLF4 or
AdLacZ with Ad tTA. RNA was extracted from the vessels 72 h after
infection and subjected to qRT-PCR. *P < 0.05, **P < 0.01, signifi-
cantly different from control group. n = 3 for each group.
Y Wang et al.
2384British Journal of Pharmacology (2012) 165 2378–2388
We also found that overexpression of KLF4 increased
p27kip1(Figure 2D), which is consistent with the previously
reported effect of rapamycin (Marx and Marks, 2001; Moss
et al., 2010). Conversely, siRNA-mediated knockdown of
KLF4 increased the activity of mTOR as well as its down-
stream targets (Figure 5D). Taken together with the in vivo
finding that knockdown of KLF4 attenuated the anti-
proliferative effect of rapamycin (Figure 6D), it appears that
induction of KLF4 contributes to the anti-proliferative effect
and that the mechanisms, among others, may involve a sup-
pression of mTOR by KLF4.
Furthermore, we asked whether KLF4 was essential for the
inhibition of mTOR downstream pathways by rapamycin. We
compared the effects of rapamycin on 4EBP1 phosphoryla-
tion stimulated by PDGF-BB in VSMCs transfected with KLF4
siRNA or scrambled control (Figure S5). The results show that
phosphorylation of 4EBP1 was increased in the KLF4-silenced
cells in the presence of rapamycin, whereas the effect of
rapamycin was only partially diminished. Thus, KLF4 may
have a contributing rather than a pivotal role in blocking the
pathways downstream of mTOR activation. In this regard,
our microarray study demonstrated that GADD45 and p57kip2,
among others, may also be involved in mediating the anti-
proliferative action of rapamycin.
Pro-inflammatory processes including the activation of
macrophages and VSMCs also play an important role in the
Knockdown of KLF4 increased the proliferation of VSMCs. VSMCs were transfected with KLF4 siRNAs (siKLF4-1416 or siKLF4-298) or control siRNA
(100 nM each) for 48 h. KLF4 expression level was assessed with qRT-PCR (A) and Western blotting (B). (C) Synchronized VSMCs were transfected
with two independent siRNA, siKLF4-1416 or siKLF4-298, and harvested for cell cycle analysis 24 h after serum stimulation with flow cytometry.
(D) SMCs were transfected with KLF4 siRNA (siKLF4-1416) or control siRNA for 48 h and then were stimulated with PDGF-BB with indicated times.
Total protein lysate was immunoblotted with antibodies against mTOR, p70S6K, 4EBP1 and their phosphorylated forms. *P < 0.05, **P < 0.01,
***P < 0.001, significantly different from siRNA control group.
Rapamycin induces KLF4 in vascular smooth muscle cells
British Journal of Pharmacology (2012) 165 2378–23882385
Genis et al., 2002; Shagdarsuren et al., 2009). The anti-
inflammatory effect of rapamycin also contributes to its
pharmacological actions (Zohlnhofer et al., 2004) but KLF4
may promote the activation of monocytes/macrophages and
prevent the differentiation of VSMCs (Liu et al., 2005; Fein-
berg et al., 2007; Yoshida et al., 2008). In addition, the effect
of KLF4 on VSMC migration, also a key step in the intimal
hyperplasia, remains to be studied.
In conclusion, the present study demonstrated that the
transcription factor KLF4 was induced by rapamycin and
played an important role in mediating the anti-proliferative
effect of rapamycin on VSMCs. It is suggested that KLF4 is a
ofrestenosis afterangioplasty (Bayes-
potential target for the development of novel approaches to
the treatment of proliferative vascular disorders, such as res-
tenosis after angioplasty.
This study was supported by the grants from the National
Natural Science Foundation of China (30670848, 30890041
and 30910280), the Ministry of Science and Techno-
logy (2010CB912500) and the National Health Institute
Knockdown of KLF4 suppressed the effect of rapamycin in vitro and in vivo. (A) After transfection with siRNAs, VSMCs were stimulated with 10%
FBS and exposed to different concentrations of rapamycin or DMSO for 48 h. Cell numbers were counted and expressed as percentage of the
DMSO-treated and control siRNA-transfected group. Comparison was made between siKLF4 and control siRNA groups at various concentrations
of rapamycin. (B) After transfection with siRNAs, VSMCs were stimulated with 10% FBS in the presence or absence of rapamycin. Nuclear extracts
were immunoblotted with antibodies against PCNA and histone 24 h later. (C) Rat carotid arteries were balloon-injured and treated with
perivascular pluronic gel containing siKLF4 or control siRNA (15 mg per artery) and rapamycin (100 mg per artery) or DMSO. The injured arteries
were harvested 7 days later and stained with haematoxylin–eosin. (D) BrdU incorporation was assessed 7 days later using anti-BrdU antibody
(TRITC). Nuclei were stained with Hoechst 33258. The bar graph indicates the percentage of BrdU-positive cells in the neointima (n = 3 for each
group). *P < 0.05, **P < 0.01, ***P < 0.001, significantly different from siRNA control group.
Y Wang et al.
2386 British Journal of Pharmacology (2012) 165 2378–2388
Conflicts of interest
Adam PJ, Regan CP, Hautmann MB, Owens GK (2000). Positive-
and negative-acting Kruppel-like transcription factors bind a
transforming growth factor beta control element required for
expression of the smooth muscle cell differentiation marker
SM22alpha in vivo. J Biol Chem 275: 37798–37806.
Agrotis A, Kanellakis P, Kostolias G, Di Vitto G, Wei C, Hannan R
et al. (2004). Proliferation of neointimal smooth muscle cells after
arterial injury. Dependence on interactions between fibroblast
growth factor receptor-2 and fibroblast growth factor-9. J Biol
Chem 279: 42221–42229.
Alder JK, Georgantas RW 3rd, Hildreth RL, Kaplan IM, Morisot S,
Yu X et al. (2008). Kruppel-like factor 4 is essential for
inflammatory monocyte differentiation in vivo. J Immunol 180:
Bayes-Genis A, Campbell JH, Carlson PJ, Holmes DR Jr, Schwartz RS
(2002). Macrophages, myofibroblasts and neointimal hyperplasia
after coronary artery injury and repair. Atherosclerosis 163: 89–98.
Clowes AW, Reidy MA, Clowes MM (1983). Mechanisms of stenosis
after arterial injury. Lab Invest 49: 208–215.
Dumont FJ, Staruch MJ, Grammer T, Blenis J, Kastner CA,
Rupprecht KM (1995). Dominant mutations confer resistance to the
immunosuppressant, rapamycin, in variants of a T cell lymphoma.
Cell Immunol 163: 70–79.
Feinberg MW, Cao Z, Wara AK, Lebedeva MA, Senbanerjee S,
Jain MK (2005). Kruppel-like factor 4 is a mediator of
proinflammatory signaling in macrophages. J Biol Chem 280:
Feinberg MW, Wara AK, Cao Z, Lebedeva MA, Rosenbauer F,
Iwasaki H et al. (2007). The Kruppel-like factor KLF4 is a critical
regulator of monocyte differentiation. EMBO J 26: 4138–4148.
Ferns GA, Raines EW, Sprugel KH, Motani AS, Reidy MA, Ross R
(1991). Inhibition of neointimal smooth muscle accumulation after
angioplasty by an antibody to PDGF. Science 253: 1129–1132.
Gallo R, Padurean A, Jayaraman T, Marx S, Roque M, Adelman S
et al. (1999). Inhibition of intimal thickening after balloon
angioplasty in porcine coronary arteries by targeting regulators of
the cell cycle. Circulation 99: 2164–2170.
Garrett-Sinha LA, Eberspaecher H, Seldin MF, de Crombrugghe B
(1996). A gene for a novel zinc-finger protein expressed in
differentiated epithelial cells and transiently in certain
mesenchymal cells. J Biol Chem 271: 31384–31390.
Hamik A, Lin Z, Kumar A, Balcells M, Sinha S, Katz J et al. (2007).
Kruppel-like factor 4 regulates endothelial inflammation. J Biol
Chem 282: 13769–13779.
Heitman J, Movva NR, Hall MN (1991). Targets for cell cycle arrest
by the immunosuppressant rapamycin in yeast. Science 253:
Inoki K, Guan KL (2006). Complexity of the TOR signaling
network. Trends Cell Biol 16: 206–212.
Inoki K, Li Y, Zhu T, Wu J, Guan KL (2002). TSC2 is
phosphorylated and inhibited by Akt and suppresses mTOR
signalling. Nat Cell Biol 4: 648–657.
Inoki K, Li Y, Xu T, Guan KL (2003). Rheb GTPase is a direct target
of TSC2 GAP activity and regulates mTOR signaling. Genes Dev 17:
Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, Clowes AW
(1992). Platelet-derived growth factor promotes smooth muscle
migration and intimal thickening in a rat model of balloon
angioplasty. J Clin Invest 89: 507–511.
King KE, Iyemere VP, Weissberg PL, Shanahan CM (2003).
Kruppel-like factor 4 (KLF4/GKLF) is a target of bone
morphogenetic proteins and transforming growth factor beta 1 in
the regulation of vascular smooth muscle cell phenotype. J Biol
Chem 278: 11661–11669.
Liu Y, Sinha S, McDonald OG, Shang Y, Hoofnagle MH, Owens GK
(2005). Kruppel-like factor 4 abrogates myocardin-induced
activation of smooth muscle gene expression. J Biol Chem 280:
Marks AR (1996). Cellular functions of immunophilins. Physiol Rev
Marx SO, Marks AR (2001). Bench to bedside: the development of
rapamycin and its application to stent restenosis. Circulation 104:
Marx SO, Jayaraman T, Go LO, Marks AR (1995). Rapamycin-FKBP
inhibits cell cycle regulators of proliferation in vascular smooth
muscle cells. Circ Res 76: 412–417.
Morris RE, Cao W, Huang X, Gregory CR, Billingham ME, Rowan R
et al. (1995). Rapamycin (Sirolimus) inhibits vascular smooth
muscle DNA synthesis in vitro and suppresses narrowing in arterial
allografts and in balloon-injured carotid arteries: evidence that
rapamycin antagonizes growth factor action on immune and
nonimmune cells. Transplant Proc 27: 430–431.
Moss SC, Lightell DJ Jr, Marx SO, Marks AR, Woods TC (2010).
Rapamycin regulates endothelial cell migration through regulation
of the cyclin-dependent kinase inhibitor p27Kip1. J Biol Chem 285:
Nobuyoshi M, Kimura T, Nosaka H, Mioka S, Ueno K, Yokoi H et al.
(1988). Restenosis after successful percutaneous transluminal
coronary angioplasty: serial angiographic follow-up of 229 patients.
J Am Coll Cardiol 12: 616–623.
Raines EW (2004). PDGF and cardiovascular disease. Cytokine
Growth Factor Rev 15: 237–254.
Razuvaev A, Lund K, Roy J, Hedin U, Caidahl K (2008).
Noninvasive real-time imaging of intima thickness after rat carotid
artery balloon injury using ultrasound biomicroscopy.
Atherosclerosis 199: 310–316.
Segre JA, Bauer C, Fuchs E (1999). Klf4 is a transcription factor
required for establishing the barrier function of the skin. Nat Genet
Serruys PW, Luijten HE, Beatt KJ, Geuskens R, de Feyter PJ,
van den Brand M et al. (1988). Incidence of restenosis after
successful coronary angioplasty: a time-related phenomenon. A
quantitative angiographic study in 342 consecutive patients at 1, 2,
3, and 4 months. Circulation 77: 361–371.
Shagdarsuren E, Djalali-Talab Y, Aurrand-Lions M, Bidzhekov K,
Liehn EA, Imhof BA et al. (2009). Importance of junctional
adhesion molecule-C for neointimal hyperplasia and monocyte
Rapamycin induces KLF4 in vascular smooth muscle cells
British Journal of Pharmacology (2012) 165 2378–23882387
recruitment in atherosclerosis-prone mice-brief report. Arterioscler
Thromb Vasc Biol 29: 1161–1163.
Shields JM, Christy RJ, Yang VW (1996). Identification and
characterization of a gene encoding a gut-enriched Kruppel-like
factor expressed during growth arrest. J Biol Chem 271:
Simons M, Edelman ER, DeKeyser JL, Langer R, Rosenberg RD
(1992). Antisense c-myb oligonucleotides inhibit intimal
arterial smooth muscle cell accumulation in vivo. Nature 359:
Sousa JE, Costa MA, Abizaid A, Abizaid AS, Feres F, Pinto IM et al.
(2001). Lack of neointimal proliferation after implantation of
sirolimus-coated stents in human coronary arteries: a quantitative
coronary angiography and three-dimensional intravascular
ultrasound study. Circulation 103: 192–195.
Sugiyama H, Papst P, Gelfand EW, Terada N (1996). p70 S6 kinase
sensitivity to rapamycin is eliminated by amino acid substitution of
Thr229. J Immunol 157: 656–660.
Tang Z, Wang Y, Fan Y, Zhu Y, Chien S, Wang N (2008).
Suppression of c-Cbl tyrosine phosphorylation inhibits neointimal
formation in balloon-injured rat arteries. Circulation 118:
Wang N, Verna L, Chen NG, Chen J, Li H, Forman BM et al. (2002).
Constitutive activation of peroxisome proliferator-activated
receptor-gamma suppresses pro-inflammatory adhesion molecules
in human vascular endothelial cells. J Biol Chem 277:
Yet SF, McA’Nulty MM, Folta SC, Yen HW, Yoshizumi M, Hsieh CM
et al. (1998). Human EZF, a Kruppel-like zinc finger protein, is
expressed in vascular endothelial cells and contains transcriptional
activation and repression domains. J Biol Chem 273:
Yoshida T, Kaestner KH, Owens GK (2008). Conditional deletion of
Kruppel-like factor 4 delays downregulation of smooth muscle cell
differentiation markers but accelerates neointimal formation
following vascular injury. Circ Res 102: 1548–1557.
Zohlnhofer D, Nuhrenberg TG, Neumann FJ, Richter T, May AE,
Schmidt R et al. (2004). Rapamycin effects transcriptional programs
in smooth muscle cells controlling proliferative and inflammatory
properties. Mol Pharmacol 65: 880–889.
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Effect of rapamycin on KLF4 expression of normal
arteries. Rat carotid arteries were isolated and pluronic gel,
containing DMSO or rapamycin (100 mg per artery), applied
perivascularly. Total RNA was isolated and subjected to qRT-
PCR to validate the expression of KLF4 and GAPDH 24 h later
(n = 3 for each group, *P < 0.05).
Figure S2 Quantification of Western blotting in Figure 2C.
For the Western blotting in Figure 2C, band intensities were
scanned, quantified with NIH Image J and expressed as fold of
control. Data are mean ? SEM from three to four indepen-
dent experiments. *P < 0.05, ***P < 0.001, compared with
control SMCs stimulated with PDGF-BB for 30 min.#P < 0.05,
###P < 0.001, compared with control SMCs stimulated with
PDGF-BB for 60 min. NS, not significant.
Figure S3 Microarray analysis of KLF4-overexpressed SMCs.
Scatter plot shows the differential gene expression in SMCs
with or without KLF4 overexpression.
Figure S4 SiRNA-mediated KLF4 knockdown in rat carotid
arteries. Rat carotid arteries were balloon injured and perivas-
cularly treated with pluronic gel containing siKLF4 or control
siRNA (15 mg per artery). Total RNA was extracted 72 h later
and subjected to qRT-PCR for KLF4. *P < 0.05, vs. control
siRNA, n = 3.
Figure S5 Effect of rapamycin on the downstream targets of
mTOR. SMCs were transfected with KLF4 siRNA or control
siRNA for 24 h and pretreated with rapamycin or DMSO for
30 min. Cells were then stimulated with PDGF-BB. Levels of
4EBP1 and its phosphorylated forms were detected with
Western blotting using GADPH as internal control. *P < 0.05,
compared with control SMCs stimulated with PDGF-BB,
#P < 0.05, compared with control SMCs treated with rapamy-
cin and PGDF-BB.
Table S1 Ultrasonic evaluation of carotid arteries
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
Y Wang et al.
2388 British Journal of Pharmacology (2012) 165 2378–2388