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-densi
Trang 1lipoproteins promotes the expression of scavenger
receptor class B type I
Tao Yang1,2,3,*, Caihong Chen4,*, Bin Zhang1, He Huang1, Ganqiu Wu1, Jianguo Wen1and
Junwen Liu1
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: liujunwenying@126.com
*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.
Trang 2plays 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 classiidenti-fied 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 (http://www.genomatix.de) and
the Transcription Element Search System (TESS; http://www.cbil.upenn.edu), 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), HDL2 (80 lgÆmL)1) or HDL3 (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 HDL2 and HDL3 led to an induction of KLF4 in mRNA and protein levels in PBMC and PMA-differentiated THP-1 macrophages, the increment level induced by HDL3 was much higher than that by HDL2 Therefore, HDL3 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 HDL3increased 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 HDL3 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
Trang 3PMA-differ-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, HDL3 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 HDL3 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 HDL3is 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
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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), HDL2
(80 lgÆmL)1) or HDL3 (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.
Trang 4expression 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 HDL3, but were not changed significantly following oxLDL stimu-lation The induction level of KLF4 by HDL3 was much higher than that by HDL2 It has been shown that HDL3 exerts more powerful antioxidative and protective effects against atherosclerosis than HDL2 [8] We then used HDL3 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 HDL3could further induce the expression of SR-BI
A variety of stimuli have been demonstrated to regulate
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A
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D
E
Fig 2 Effect of KLF4 on expression of hSR-BI in PMA-differenti-ated THP-1 macrophages (A,B) PMA-differentiPMA-differenti-ated THP-1 macro-phages were transiently transfected with pcDNA3.1-hKLF4 and were then treated with oxLDL or HDL3 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 HDL3as 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.
Trang 5SR-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 HDL3 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 containcorrespond-ing KLF4 bindcorrespond-ing element on the promoter of the hSR-BI gene oxLDL, cells stimulated by oxLDL (80 lgÆmL)1) for
24 h; HDL3, cells stimulated by HDL3(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 HDL3 treatment (80 lgÆmL)1 for 24 h).
*P < 0.05.
Trang 6atherosclerosis 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 HDL3 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
HDL2 (density = 1.063–1.125 gÆmL)1), HDL3 (density =
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
CuSO4 at 37C for 24 h [20] The HDL3, HDL2, native
LDL and oxLDL were then filtered (filter membrane
aper-ture: 0.22 lm) and stored at 4C
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
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
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
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 2000, Invitro-gen) [11] Briefly, 5 · 105
cells per bottle containing
5 mL appropriate complete growth medium were seeded, and incubated at 37C with 5% CO2 until the cells were 70–80% confluence (24 h) After being rinsed with serum-free and antibiotic-serum-free medium, the cells were transfected
lipofecta-mine 20 lL (vector control), followed by incubation at
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 Trizolreagent (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
Trang 7System, 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);
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
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
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 4C 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
double-stranded, biotin-labelled oligonucleotides
corre-sponding to the wild-type sequences (5¢-AGA AAG
GG-G AAGG-G GG-GGG-G-3¢) and mutant sequences [27] (5¢-AGG-GA AAGG-G
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
sequence using the primers (forward: 5¢-GTG GGG GAA
GCC CCG CCA TG-3¢) Reaction products were analysed
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)
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
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 transfecTransfec-tions were performed in triplicate from at least three independent
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
Trang 8Founda-tion of China (30900623), and the Doctoral Fund
of Ministry of Education of China (Fund for New
Teacher, 20090162120020)
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