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

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

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

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-

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

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

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

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
*
*
*
*
*

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

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-

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

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

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

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.

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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