Tài liệu Báo cáo khoa học: ¨ Induction of Kruppel-like factor 4 by high-density lipoproteins promotes the expression of scavenger receptor class B type I pptx

Induction of Kru
¨
ppel-like factor 4 by high-density
lipoproteins promotes the expression of scavenger
receptor class B type I
Tao Yang
1,2,3,
*, Caihong Chen
4,
*, Bin Zhang
1
, He Huang
1
, Ganqiu Wu
1
, Jianguo Wen
1
and
Junwen Liu
1
1 Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, Changsha, Hunan, China
2 College of Chemistry and Bioengineering, Changsha University of Science and Technology, Hunan, China
3 College of Food Science and Engineering, Central South University of Forestry and Technology, Changsha, Hunan, China
4 School of Science, Central South University of Forestry and Technology, Changsha, Hunan, China
Introduction
Atherosclerosis is a chronic inflammatory response in
the walls of arteries, in large part due to the accumula-
tion of macrophages and white blood cells, and pro-
moted by low-density lipoproteins (LDL) without
adequate removal of fats and cholesterol from the
macrophages by functional high-density lipoproteins

(HDL). Vascular smooth muscle cells (VSMCs), endo-
thelial cells and macrophages are the three predomi-
nant cell types involved in atherosclerosis, and the
proliferation, migration, differentiation and activation
of cells are always highlights for researchers.
Kru
¨
ppel-like factor 4 (KLF4) was first identified in
the epithelial lining of the gut and skin, and subse-
quent studies have shown it to play a role in the regu-
lation of cellular growth and differentiation in these
tissues [1]. Recently, it has been shown that KLF4
Keywords
atherosclerosis; gene regulation; high-
density lipoproteins; Kru
¨
ppel-like factor 4;
scavenger receptor class B type I
Correspondence
J. Liu, Department of Histology and
Embryology, School of Basic Medical
Sciences, Central South University,
Changsha, Hunan 410013, China
Fax: 86 731 82650400
Tel: 86 731 82650436
E-mail:
*These authors contributed equally to this
work
(Received 27 April 2010, revised 12 June
2010, accepted 14 July 2010)

doi:10.1111/j.1742-4658.2010.07779.x
Kru
¨
ppel-like factor 4 (KLF4) is an evolutionarily conserved zinc finger-
containing transcription factor. In the present study, peripheral blood
mononuclear cells and phorbol 12-myristate 13-acetate-differentiated THP-
1 cells were treated with oxidized low-density lipoproteins and high-density
lipoproteins to determine the expression of KLF4 and scavenger receptor
class B type I (SR-BI). A full-length cDNA of KLF4 or short interference
RNA against KLF4 was transfected into THP-1 cells, and the subsequent
expressions of SR-BI were analysed by real-time PCR and western blot.
The binding and transcriptional activities of KLF4 to the SR-BI promoter
were detected by electrophoretic mobility shift assay, chromatin immuno-
precipitation assay and luciferase reporter assay. The results showed that
induction of KLF4 by high-density lipoproteins could promote the expres-
sion of SR-BI, resulting from the binding to putative KLF4 binding
element on the promoter of SR-BI. All results indicate a potential function
of KLF4 in the pathogenesis of atherosclerosis through the regulation
effect on atherosclerotic-related genes.
Abbreviations
ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; HDL, high-density lipoproteins; hSR-BI, human scavenger
receptor class B type I; IFN, interferon; KLF4, Kru
¨
ppel-like factor 4; LDL, low-density lipoproteins; LPS, lipopolysaccharide; oxLDL, oxidized
low-density lipoprotein; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate;
siRNA, short interference RNA; SR-BI, scavenger receptor class B type I; TESS, transcription element search system; VSMC, vascular
smooth muscle cell.
3780 FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS
plays an important role in the activation of endothelial
cells and macrophages, as well as the differentiation

and proliferation of VSMCs. Overexpression of KLF4
induced expression of multiple anti-inflammatory and
antithrombotic factors, whereas knockdown of KLF4
led to the enhancement of tumour necrosis factor
a-induced vascular cell adhesion molecule-1 and tissue
factor expression, resulting in markedly decreased
inflammatory cell adhesion to the endothelial surface
and prolongation of clotting time following the induc-
tion of KLF4 under inflammatory states, and implicat-
ing KLF4 as a regulator of endothelial activation in
response to proinflammatory stimuli [2]. Overexpres-
sion of KLF4 in J774a macrophages induced the mac-
rophage activation marker inducible nitric oxide
synthase and inhibited the transforming growth factor-
b1 and Smad3 target gene plasminogen activator
inhibitor-1. Conversely, KLF4 knockdown markedly
attenuated the ability of interferon-c (IFN-c), lipopoly-
saccharide (LPS) or IFN-c plus LPS to induce the
inducible nitric oxide synthase promoter, whereas it
augmented macrophage responsiveness to transforming
growth factor-b1 and Smad3 signalling, implicating
KLF4 as a regulator of key signalling pathways that
control macrophage activation [3]. Furthermore, it has
also been demonstrated that KLF4 is required for the
expression of VSMC differentiation marker genes
induced by all-trans retinoic acid [4]; KLF4 could
induce inhibition of proliferation of VSMC, which is
mechanistically linked to a KLF4-induced enhance-
ment of the expression of the tumour suppressor gene
p53 [5]. Because of the important roles of KLF4 on

the above three cell types, we postulated the novel
effect of KLF4 in atherogenesis.
Scavenger receptors are a group of receptors that
recognize modified LDL by oxidation or acetylation.
In atherosclerotic lesions, macrophages that express
scavenger receptors on their plasma membrane aggres-
sively uptake the oxidized LDL (oxLDL) deposited in
the blood vessel wall inside and become foam cells,
and they secrete various inflammatory cytokines and
accelerate the development of atherosclerosis [6]. Scav-
enger receptor class B type I (SR-BI) was first identi-
fied as an oxLDL receptor and classified into class B.
It can interact not only with oxLDL, but also with
normal LDL and HDL. It is best known for its role in
facilitating the uptake of cholesteryl esters from HDLs
in the liver. This process drives the movement of cho-
lesterol from peripheral tissues towards the liver for
excretion, which is known as reverse cholesterol trans-
port and is a protective mechanism against the devel-
opment of atherosclerosis. By using the matinspector
Professional program () and
the Transcription Element Search System (TESS;
), we found that the
promoter of SR-BI contained multiple putative KLF4
binding sites. However, the direct effect of KLF4 on
the expression of SR-BI remains unknown.
Here, the expression of KLF4 in response to oxLDL
or HDL was investigated in both human peripheral
blood mononuclear cells (PBMCs) and human THP-1
monocytes. In addition, the effects of KLF4 on the

expression of SR-BI and the primary mechanism were
also investigated.
Results
HDL induces the expression of KLF4 and SR-BI in
PBMC and phorbol 12-myristate 13-acetate
(PMA)-differentiated THP-1 macrophages
We first determined KLF4 expression in PBMC and
PMA-differentiated THP-1 macrophages treated with
oxLDL (80 lgÆmL
)1
), HDL
2
(80 lgÆmL
)1
) or HDL
3
(80 lgÆmL
)1
) for 24 h in serum-free medium for the
effective dose and time of the treatment [7]. As shown
in Fig. 1A,B, oxLDL treatment did not influence the
expression of KLF4; although both HDL
2
and HDL
3
led to an induction of KLF4 in mRNA and protein
levels in PBMC and PMA-differentiated THP-1
macrophages, the increment level induced by HDL
3
was much higher than that by HDL

2
. Therefore,
HDL
3
was chosen as the stimulus in the subsequent
experiments.
The expression of SR-BI was also investigated
in PBMC and THP-1 cells. As shown in Fig. 1C,D,
oxLDL decreased the expression levels of SR-BI, and
HDL
3
increased the levels of SR-BI.
KLF4 influences the expression of SR-BI in
PMA-differentiated THP-1 macrophages
We overexpressed KLF4 in PMA-differentiated THP-1
macrophages using a pcDNA3.1-hKLF4 construct.
The transfection did not affect cell viability signifi-
cantly, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide MTT (data not shown).
As demonstrated in Fig. 2A,B, overexpression of
KLF4 did not influence the expression of SR-BI in
control and oxLDL-stimulated cells, but further
increased the expression of SR-BI in response to
HDL
3
stimulation compared with the vector control
group.
In order to observe the effect of KLF4 inhibition on
the expression of SR-BI, we transfected short interfer-
ence (si)RNAs against human KLF4 into PMA-differ-

T. Yang et al. SR-BI induction by KLF4
FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS 3781
entiated THP-1 macrophages. As shown in Fig. 2C,D,
following the basal inhibition of KLF4, the expression
of SR-BI was not influenced substantially in the con-
trol or oxLDL-stimulated cells. Consequently, HDL
3
treatment failed to induce expression of SR-BI further
compared with the control group.
KLF4 regulates SR-BI promoter in
PMA-differentiated THP-1 macrophages
To determine whether there are potential KLF4 bind-
ing sites on the SR-BI promoter, we performed electro-
phoretic mobility shift assay (EMSA). Figure 3A
shows that the KLF4-specific binding activity (at posi-
tion )342 to )329 bp) was promoted in the nuclear
extract of PMA-differentiated THP-1 macrophages
stimulated by HDL
3
. The specificity of the assay was
verified by using mutant oligonucleotides, which failed
to bind to KLF4, and by antibody competition. Mean-
while, the site at )320 to )307 bp had no obvious
binding activity with KLF4 protein (data not shown).
Furthermore, a chromatin immunoprecipitation
(ChIP) assay was used to determine whether KLF4
can bind to the SR-BI promoter. Figure 3B shows the
PCR product after the immunoprecipitation of the
cross-linked chromatin with the KLF4 antibody. As a
specific control, purified rabbit IgG in parallel did not

yield a detectable PCR product. Collectively, these
data support that KLF4 binds to the SR-BI promoter,
which spans the sequence from )359 to )200 in the
SR-BI promoter sequence.
In order to understand how KLF4 can induce SR-BI,
we assessed its effect on SR-BI promoter activity. A
strong transactivation effect of KLF4 on the SR-BI pro-
moter in response to HDL
3
is shown in Fig. 3C. Further-
more, this transactivation was almost abolished upon
further point mutations of the corresponding KLF4
binding site. The specificity of transcriptional activity of
KLF4 on SR-BI promoter was further confirmed by
another transcription factor, KLF2, as a control.
Discussion
KLF4 is a gut-enriched, zinc finger-containing tran-
scription factor that has been widely investigated in
both normal development and carcinogenesis. In nor-
mal conditions, the expression of KLF4 mRNA is
most abundant in the colon and skin in mice, whereas
expression of KLF4 is decreased in intestinal adeno-
mas of multiple intestinal neoplasia mice and in colo-
nic adenomas of familial adenomatous polyposis
patients. In this investigation, we first determined the
A
0
20
40
60

80
100
120
PBMC THP-1
Ratio of KLF4 mRNA
/GAPDH
Ctrl
oxLDL
HDL2
HDL3
B
KLF4
GAPDH
0
0.2
0.4
0.6
0.8
1
PBMC THP-1
Ratio of KLF4 protein
/GAPDH
Ctrl
oxLDL
HDL2
HDL3
* *
PMBC THP-1
Ctrl oxLDL HDL
2

HDL
3
Ctrl oxLDL HDL
2
HDL
3
* *
C
0
100
200
300
400
500
PBMC THP-1
Ratio of hSR-BI mRNA
/GAPDH
Ctrl
oxLDL
HDL3
D
SR-BI
GAPDH
0
0.5
1
1.5
2
2.5
3

PBMC THP-1
Ratio of hSR-BI protein
/GAPDH
Ctrl
oxLDL
HDL3
* *
*
*
Ctrl oxLDL HDL
3
Ctrl oxLDL HDL
3
PMBC THP-1
*
*
* *
Fig. 1. Expressions of KLF4 and SR-BI in oxLDL- and HDL-stimu-
lated PBMC and THP-1. PBMC and PMA-differentiated THP-1
macrophages were stimulated with oxLDL (80 lgÆmL
)1
), HDL
2
(80 lgÆmL
)1
) or HDL
3
(80 lgÆmL
)1
) for 24 h. (A) mRNA levels of

KLF4 were determined by real-time PCR. (B) Protein levels of KLF4
were determined by western blot. (C) mRNA levels of hSR-BI were
determined by real-time PCR. (D) Protein levels of hSR-BI
were determined by western blot. The relative values of all results
were determined and expressed as mean ± standard error of the
mean of three experiments in duplicate. *P < 0.05.
SR-BI induction by KLF4 T. Yang et al.
3782 FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS
expression of KLF4 in PBMC and PMA-differentiated
THP-1 macrophages induced by oxLDL and HDL.
PBMCs are monocytes and the PMA-differentiated
THP-1 cells are macrophages. The results showed that
KLF4 levels were increased in response to HDL
3
, but
were not changed significantly following oxLDL stimu-
lation. The induction level of KLF4 by HDL
3
was
much higher than that by HDL
2
. It has been shown
that HDL
3
exerts more powerful antioxidative and
protective effects against atherosclerosis than HDL
2
[8]. We then used HDL
3
as the stimulation in further

experiments. Recently, KLF4 has been shown to be
induced by IFN-c, LPS and tumour necrosis factor-a
in macrophages, and by a kind of oxidized phospho-
lipid, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phos-
phocholine, in VSMCs [3,9]. As a transcriptional
factor, the induction of KLF4 plays a role in the
corresponding pathogenesis. Galbois et al. [10] demon-
strated that reconstituted HDL abolishes the LPS-
induced overproduction of proinflammatory cytokines
in whole blood from patients with severe cirrhosis, as
well as in isolated monocytes from these patients. Our
laboratory also found that KLF4 could increase inter-
leukin-10 expression in LPS-induced RAW264.7 mac-
rophages [11]. We postulated that HDL abolishing the
overproduction of proinflammatory cytokines induced
by LPS might potentially and partially result from the
KLF4 anti-inflammatory effect. Certainly, it should be
confirmed by further investigations. As for no obvious
influence of oxLDL on the expression of KLF4, the
potential reason may be the deficiency of a corre-
sponding ligand–receptor interaction.
Here, the changes in the SR-BI response to oxLDL
were consistent with previous results [12], which also
indicated the effectiveness of stimulus and normal cell
status. Interestingly, we found that induction of KLF4
by HDL
3
could further induce the expression of SR-BI.
A variety of stimuli have been demonstrated to regulate
0

100
200
300
400
500
600
Neo KLF4
Ratio of hSR-BI mRNA
/GAPDH
Normal
oxLDL
HDL3
SR-BI
GAPDH
0
0.2
0.4
0.6
0.8
Neo KLF4
Ratio of hSR-BI protein
/GAPDH
Normal
oxLDL
HDL3
*
*
*
*
*

Norm oxLDL HDL
3
Norm oxLDL HDL
3
Neo KLF4
*
*
*
* *
KLF4
GAPDH
0
50
100
150
200
250
300
350
Ctrl Mock siRNA
Ratio of hSR-BI mRNA
/GAPDH
Normal
oxLDL
HDL3
SR-BI
GAPDH
0
0.2
0.4

0.6
0.8
1
Ctrl Mock siRNA
Ratio of hSR-BI protein
/GAPDH
Normal
oxLDL
HDL3
*
*
* * *
*
Ctrl Mock siRNA Ctrl Mock siRNA Ctrl Mock siRNA
Norm oxLDL HDL
3
* * *
*
*
*
Ctrl Mock siRNA
A
B
C
D
E
Fig. 2. Effect of KLF4 on expression of hSR-BI in PMA-differenti-
ated THP-1 macrophages. (A,B) PMA-differentiated THP-1 macro-
phages were transiently transfected with pcDNA3.1-hKLF4 and
were then treated with oxLDL or HDL

3
as indicated for 24 h.
mRNA levels of hSR-BI were determined by real-time PCR (A) and
protein levels of hSR-BI were determined by western blot (B).
Neo, the vector control group; KLF4, the KLF4 overexpression
group. (C–E) PMA-differentiated THP-1 macrophages were tran-
siently transfected with siRNA of KLF4, and were then treated with
oxLDL or HDL
3
as indicated for 24 h. KLF4 inhibition was detected
by western blot (C). mRNA levels of hSR-BI were determined by
real-time PCR (D) and protein levels of hSR-BI were determined by
western blot (E). Ctrl, PMA-differentiated THP-1 macrophages were
treated only with lipofectamine; Mock, PMA-differentiated THP-1
macrophages were transiently transfected with control siRNA; siR-
NA, PMA-differentiated THP-1 macrophages were transiently trans-
fected with siRNA of KLF4. The relative values of all results were
determined and expressed as mean ± standard error of the mean
of three experiments in duplicate. *P < 0.05.
T. Yang et al. SR-BI induction by KLF4
FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS 3783
SR-BI expression [13]. Oestrogen and adrenocorticotro-
pic hormone have been observed to alter SR-BI expres-
sion. In addition, modified LDL has been shown to
increase SR-BI in human monocyte-derived macro-
phages, whereas a high cholesterol diet lowered SR-BI
expression in rat liver parenchymal cells. Despite a
number of studies demonstrating regulation of SR-BI,
relatively little is known about the basic mechanisms
involved. Recent promoter studies have shown that

members of the Sp1 transcription factor family are
essential for transcription of the rat SR-BI gene in
mouse Lydig tumour cells. It has also been shown that
the sterol response element binding protein activates
transcription of the rat SR-BI promoter in a variety of
cell lines [14] and that steroidogenic factor 1 binds to
and activates the human SR-BI promoter in mouse
adrenocortical cells [15]. Moreover, it was shown that
ligand activated peroxisome proliferator activated
receptor increases SR-BI expression in human mono-
cytes and macrophages [16]. As a transcriptional factor,
many target genes of KLF4 have been identified,
including CYP1A1, human keratin 4, intestinal alkaline
phosphatase, ornithine decarboxylase, histidine decar-
boxylase and cyclin D1 [17]. KLF4 regulates the target
genes by binding to the potential KLF4 binding ele-
ments in the promoters. By using matinspector and
TESS, we found the promoter of human scavenger
receptor class B type I (hSR-BI) containing multiple
putative KLF4 binding sites. Among them, the KLF
binding site at position )342 to )329 bp had the high-
est predicting value from both matinspector and
TESS. We also demonstrated that KLF4 could bind to
the corresponding KLF4 binding site (position )342 to
)329 bp) in vivo and in vitro, and transactivate the pro-
moter activity of hSR-BI in response to HDL
3
stimula-
tion. Sp1 and Sp3 have been shown to be essential
transcriptional factors for transcription of the rat SR-

BI gene [18]; as one of Sp1-like ⁄ KLF family members,
the regulation effect of KLF4 on the hSR-BI gene shall
reveal a novel function for investigations on atheroscle-
rotic-related genes. Moreover, it has been shown that a
hemizygous deficiency of KLF2 increased diet-induced
Fig. 3. DNA binding activity and transcription activity of KLF4 to
the KLF binding element of hSR-BI promoter in PMA-differentiated
THP-1 macrophages. (A) Binding activity of KLF4 to the correspond-
ing probes containing KLF4 binding element on the promoter of the
hSR-BI gene. oxLDL, cells stimulated by oxLDL (80 lgÆmL
)1
) for
24 h; HDL
3
, cells stimulated by HDL
3
(80 lgÆmL
)1
) for 24 h; Cold
probe, competition with cold probe (200-fold excess concentration);
Mutant probe, competition with mutant cold probe (200-fold excess
concentration); KLF4 Ab, supershift group by KLF4 antibody. (B)
Recruitment of KLF4 to the binding element of the SR-BI promoter
region. The ChIP assay was used to detect the binding of KLF4 to
the SR-BI promoter. The cross-linked protein-DNA complexes were
immunoprecipitated with the KLF4 antibody (lane 6) or with a puri-
fied rabbit IgG as a negative control (lane 3), or with the KLF2 anti-
body as a specific control (lane 4). PCR of the input (a sample
representing PCR amplification from a 1 : 25 dilution of total input
chromatin from the ChIP experiment) is shown in lane 5. The PCR

control represents the PCR amplification in the absence of DNA
(lane 2). M, marker; Water control, negative control; IgG control,
negative control for KLF4 antibody; KLF2 ab, KLF2 antibody; Input,
positive control; KLF4 ab, KLF4 antibody. (C) PMA-differentiated
THP-1 macrophages were cotransfected transiently with an expres-
sion plasmid of full-length KLF4 (500 ng) or null (500 ng) and a
reporter driven by hSR-BI promoter (500 ng) or mutant hSR-BI pro-
moter (500 ng). Luciferase activities were detected using the Dual
Luciferase Reporter System. All transfections were performed at
least three times in triplicate. Neo, the vector control group; KLF4,
KLF4 overexpression group; Mut, the cell group transfected with
pGL3-mutSR-BI plus HDL
3
treatment (80 lgÆmL
)1
for 24 h).
*P < 0.05.
SR-BI induction by KLF4 T. Yang et al.
3784 FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS
atherosclerosis in apolipoprotein E-deficient mice, and
KLF2 played an important role in primary macrophage
foam cell formation via the potential regulation of the
key lipid binding protein adipocyte protein 2 ⁄ fatty acid
binding protein 4 [19]. All indicate that KLF4 may play
an antiatherosclerotic role, which needs further investi-
gation.
In summary, our study demonstrated the increasing
expression of KLF4 in PBMC and THP-1 cells, and
identified that induction of KLF4 by HDL
3

promoted
the expression of hSR-BI. It has been shown that dis-
ruption of SR-BI in mice impairs HDL-cholesterol
delivery to the liver and induces susceptibility to
atherosclerosis. The regulatory effect of KLF4 on SR-
BI reveals a novel pathway to elucidate the mechanism
of SR-BI in the development of atherosclerosis. Of
course, other KLF members may have the potential
regulation effect on hSR-BI under certain circum-
stances. Further research will provide us with a more
complete picture on corresponding signalling pathways
to learn the mechanism taking effect in atherogenesis.
Materials and methods
HDL isolation and LDL oxidization
HDL
2
(density = 1.063–1.125 gÆmL
)1
), HDL
3
(density =
1.125–1.210 gÆmL
)1
) and LDL (density = 1.019–1.063
gÆmL
)1
) were isolated from human plasma of normolipidae-
mic healthy volunteers by sequential ultracentrifugation
and stored in phosphate-buffered saline (PBS) containing
200 lm EDTA [20,21]. The EDTA was removed from

HDL and LDL by passing the lipoprotein through a PD 10
column (GE healthcare, Piscataway, NJ, USA). LDL was
oxidized in Ham’s F-10 medium by exposure to 10 lm
CuSO
4
at 37 °C for 24 h [20]. The HDL
3
, HDL
2
, native
LDL and oxLDL were then filtered (filter membrane aper-
ture: 0.22 lm) and stored at 4 °C.
Cell culture
Human THP-1 monocytes were purchased from the Shang-
hai Type Culture Collection and cultured in RPMI-1640
(Invitrogen, Carlsbad, CA, USA) supplemented with 10%
heat-inactivated fetal bovine serum, 2 mm glutamine and
an antibiotic–antimycotic mix in a humidified incubator
with 5% CO
2
and 95% air. Differentiation into macro-
phages was achieved in supplemented RPMI-1640 medium
containing 160 nm PMA (Promega, Madison, WI, USA)
for 24 h. Human PBMCs were isolated from healthy donor
blood (n = 5) by Ficoll density gradient centrifugation and
cultured in RPMI-1640 medium with 10% heat-inactivated
human serum and 2 mm glutamine overnight. Nonadherent
cells were subsequently removed, and adherent monocytes
were cultured continually for 2 days and then stimulated
with oxLDL or HDL

3
at various concentrations. Informed
consent was obtained from donors.
Generation of constructs
Oligonucleotide primers were designed to amplify the cod-
ing sequence of homo KLF4 cDNA. The oligonucleotide
primers were as follows: 5¢-CCC GGA TCC ATG GCT
GTC AGC GAC GCG C-3 ¢ (forward) and 5¢-CCC GAA
TTC TTA AAA TGC CTC TTC ATG TGT A-3¢ (reverse)
[22]. The PCR product was electrophoresed on to 0.9%
agarose, the fragment was purified with the Gel Extraction
kit (Qiagen, Hilden, Germany), then inserted into the
pcDNA3.1 vector (Strategene, Cedar Creek, TX, USA) and
sequenced commercially (Invitrogen). Meanwhile, full-
length homo KLF2 cDNA was also generated by PCR and
inserted into the pcDNA3.1 vector for plasmid construc-
tion, as described previously [23,24].
Lipofectamine-mediated gene transfection
Transfection of cells was carried out according the manu-
facturer’s instructions (LIPOFECTAMINE 2000Ô, Invitro-
gen) [11]. Briefly,  5 · 10
5
cells per bottle containing
5 mL appropriate complete growth medium were seeded,
and incubated at 37 °C with 5% CO
2
until the cells were
70–80% confluence (24 h). After being rinsed with serum-
free and antibiotic-free medium, the cells were transfected
separately with pcDNA3.1-KLF4 10 lg ⁄ lipofectamine

20 lL (experimental group), pcDNA3.1 10 lg ⁄ lipofecta-
mine 20 lL (vector control), followed by incubation at
37 °CinaCO
2
incubator for 6 h. The medium was then
replaced with RPMI-1640 culture medium containing 10%
fetal bovine serum.
RNA interference
The siRNAs against human KLF4 and its control were
purchased from Santa Cruz Biotechnology (Santa Cruz,
CA, USA). Transfection of KLF4siRNA was performed
using siPORT Amine (Ambion, Austin, TX, USA). To
ensure the knockdown of KLF4 protein production, a wes-
tern blot was performed with KLF4 antibody.
RNA extraction and real-time PCR
Total RNA was isolated using Trizol
Ò
reagent (Invitrogen)
in accordance with the manufacturer’s protocol. After
extraction, 5 lg total RNA was then used as a template to
synthesize the complimentary cDNA using the First Strand
Synthesis Kit (Invitrogen). The cDNA from this synthesis
was then used in quantitative real-time PCR analysis with
the TaqMan system (ABI-Prism 7700 Sequence Detection
T. Yang et al. SR-BI induction by KLF4
FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS 3785
System, Applied Biosystems, Foster City, CA, USA) using
SYBR Green dye. The following primer pairs of human
origin were used [25,26]: KLF4, 5¢-CAA GTC CCG CCG
CTC CAT TAC CAA-3¢ (forward) and 5¢-CCA CAG CCG

TCC CAG TCA CAG TGG-3¢ (reverse); SR-BI, 5¢-CCT
TCA ATG ACA ACG ACA CCG-3¢ (forward) and 5¢-CCA
TGC GAC TTG TCA GGC T-3 ¢ (reverse); glyceraldehyde-
3-phosphate dehydrogenase, 5¢-GAC ATC AAG AAG
GTG GTG AAG C-3¢ (forward) and 5¢-GTC CAC CAC
CCT GTT GCT GTA G-3¢ (reverse).
Western blot analysis
After various treatments, proteins in the whole cell lysate
were resolved on 10% SDS ⁄ PAGE and then transferred on
to poly(vinylidene difluoride) membranes (Schleicher &
Schuell, Dassel, Germany). The membranes were blocked
overnight in PBS containing 10% nonfat dry milk and
0.5% Tween-20, and incubated with the primary antibodies
for 2 h and the secondary antibodies for 1 h, successively.
The immunoreactive bands were visualized using diamino-
benzidine (DAB) (Boster Biological Technology, Wuhan,
Hubei, China). The following antibodies were used: rabbit
SRBI polyclonal antibody (1 : 1000, Abcam, Cambridge,
MA, USA); rabbit KLF4 polyclonal antibody (1 : 1000,
Santa Cruz Biotechnology); mouse glyceraldehyde-3-phos-
phate dehydrogenase monoclonal antibody (1 : 1000,
Sigma, St Louis, MO, USA); horseradish peroxidase-conju-
gated anti-mouse and anti-rabbit IgG (1 : 1000, Boster Bio-
logical Technology).
Nuclear extract preparation and EMSA
For nuclear extract preparation, cells were harvested and
washed twice with cold PBS. The nuclear extract was pre-
pared as described previously [11]. EMSA was carried out
using the Lightshift Chemiluminescent EMSA kit (Thermo
Scientific, Rockford, IL, USA). Supershift antibody for

KLF4 was incubated with nuclear extracts of KLF4 overex-
pressing cells for 1 h at 4 °C prior to the addition of biotin-
labelled oligonucleotide. The concentration of cold probe
was 100 times higher than that of the biotin-labelled probe.
DNA probes were also generated to the KLF binding site
at position )342 to )329 bp of the hSR-BI promoter as
double-stranded, biotin-labelled oligonucleotides corre-
sponding to the wild-type sequences (5¢-AGA AAG GG-
G AAG GG-3¢) and mutant sequences [27] (5¢ -AGA AAG
TGC AAG CG-3¢).
ChIP assay
ChIP assays were performed according to the provider’s
protocol (Cell Signaling Technology, Danvers, MA, USA).
In brief, cells were grown to 80–90% confluence. After
cross-linking for 10 min with 1% formaldehyde in serum-
free medium, phosphate-glycine buffer was added to a final
concentration of 0.125 m, and cells were washed twice with
ice-cold PBS. The chromatin lysate was sonicated on ice to
an average DNA length of 600 bp. Chromatin was precle-
ared with blocked Sepharose A, and ChIP assays were per-
formed with either the KLF4 antibody or the KLF2
antibody (Santa Cruz Biotechnology) as the specific con-
trol, and control IgG as the negative control. The final
PCR step was performed to amplify the fragment spanning
the nucleotides from )359 to )200 of the promoter
sequence using the primers (forward: 5¢-GTG GGG GAA
GGG GTA GGA GA-3¢; reverse: 5¢-CCA AGA CAA
GCC CCG CCA TG-3¢). Reaction products were analysed
on a 1.5% agarose ⁄ Tris-borate ⁄ EDTA gel stained with
ethidium bromide and visualized under UV light.

Luciferase reporter gene assay
The assay was performed according to the instructions of
the Dual Luciferase Reporter System (Promega). Genera-
tion of hSR-BI promoter construct ()500 to +10) was
carried out by PCR using human genomic DNA as the
template and cloned into pGL3-Basic, and authenticity was
verified by sequencing (data not shown). Moreover, the
mutant promoter construct with the point mutations (G–T
at position )336; G–C at position )330) was also per-
formed using the PGL3-hSR-BI construct as the template
for overlap extension PCR. For the luciferase reporter
assay, cells were seeded in 24-well culture dishes. Transfec-
tions were carried out as described above. All transfections
were performed in triplicate from at least three independent
experiments. Each transfection experiment contained
500 ng pGL3-hSR-BI promoter reporter construct or
pGL3-mutSR-BI promoter construct with 500 ng
pcDNA3.1-KLF4 vector or 500 ng pcDNA3.1 vector and
with 20 ng pRL-null vector (Promega) as an internal trans-
fection control.
Statistical analysis
Each experiment was performed at least three times, and
the data were expressed as mean ± standard error of the
mean, or representative data were shown. The statistical
analysis was performed using a two-tailed Student’s t-test.
P < 0.05 was considered significant.
Acknowledgements
The work was supported by research funding from the
Postdoctoral Science Foundation of Central South
University of Forestry and Technology, the Science

and Technology Program of Hunan Province
(2009FJ3169), the National Natural Science Founda-
SR-BI induction by KLF4 T. Yang et al.
3786 FEBS Journal 277 (2010) 3780–3788 ª 2010 The Authors Journal compilation ª 2010 FEBS
tion of China (30900623), and the Doctoral Fund
of Ministry of Education of China (Fund for New
Teacher, 20090162120020).
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