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: 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. 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 (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 ), 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. 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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. is required for the expression of VSMC differentiation marker genes induced by all-trans retinoic acid [4] ; KLF4 could induce inhibition of proliferation