Chinese Journal of Cancer Research 18(4): 251-256, 2006
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ESTROGEN REGULATION OF LRP16 GENE EXPRESSION INVOLVES SP1 TRANSCRIPTION FACTOR SI Yi-ling (司艺玲)1, HAN Wei-dong (韩为东)1*, ZhAO Ya-li(赵亚力)1, LI Qi (李琦)1, 1 1 2 HAO Hao-jie (郝好杰) , SONG Hai-jing (宋海静) , MU Yi-ming(母义明) , YU Li (于力)3 1 3
Department of Molecular Biology, Institute of Basic Medicine; 2Department of Endocrinology; Department of Hematology, PLA General Hospital, Beijing 100853
CLC number: Q78
Document code: A
Article ID: 1000-9604(2006)04-0251-06
ABSTRACT Objective: To investigate the role of Sp1 as transcription factor required for transactivation of LRP16 gene by estrogen. Methods: Specific antibodies of ERα and Sp1 were used to precipitate the target DNA/protein complexes of MCF-7 cells at different time points after estrogen treatment (Chromatin immunoprecipitation assay), the promoter region of LRP16 gene was amplified by semi-nested polymerase chain reaction (snPCR). Small interfering RNA (siRNA) against Sp1 was transiently cotransfected with LRP16-Luc (containing the region from -213bp to -126bp of LRP16 gene promoter) in MCF-7 cells. The luciferase activities were measured by dual-luciferase assay. Results: The results of chromatin immunoprecipitation assay showed that Sp1 protein directly bound to the -213bp to -126bp region of LRP16 gene, and ERα could enhance the affinity of Sp1 to DNA. Sp1-siRNA specifically decreased the transactivation of LRP16-Luc by 17β-estradiol to 70-80%. Conclusion: The estrogen-induced transactivation of the human LRP16 gene was mediated by Sp1 protein. Moreover, the interactions of ERα/Sp1 functional complex with LRP16 promoter DNA were required for enhanced LRP16 gene transactivation. Key words: Sp1; LRP16 gene; Small interference RNA; Estrogen; Gene expression
Estrogens have been shown to play a central role in breast cancer development. An important pathway for the action of estrogen on the target cells is mediated by estrogen receptor (ER). In human, two subtypes of ER exist, ERα and ERβ, which display different expressions in specific tissues and organs. ERs can modulate the expression of target genes not only through specific binding to the estrogen response elements (EREs), but also without coming directly into contact with the DNA, by means of functional ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯
Received date: Jun 23, 2006; Accepted date: Sep 28, 2006 Foundation item: This work was supported by the National Natural Science Foundation of China (No.30572096) and Beijing Natural Science Foundation (No.5052024). * Author to whom Correspondence should be addressed. E-mail:
[email protected] Biography: SI Yi-ling(1972-), female, doctor of medicine, assistant professor, PLA General Hospital, majors in molecular biology of tumor. E-mail:
[email protected]
interactions with other transcription factors, such as Sp1 and the fos/jun complex, that bind to their cognate DNA elements within the promoter of the genes[1-3] In particular, Sp1 has proven an important transcription factor to regulate target genes expression[4-6]. LRP16 is a novel gene that was cloned from human lymphocyte cells by our group in 1999 using restriction length genomic scanning (RLGS), and then the cDNA was isolated using the rapid amplification of cDNA end (RACE) technique (GenBank Accession No. AF202922)[7,8]. LRP16 gene locates in 11q11.1 and contains an open reading frame for a protein of 325 amino acids[9]. The encoding product mainly distributes in the nucleus. Based on the computeraided SAGE pattern analysis of LRP16 gene in MCF-7 cells and previous studies[10,11], we have demonstrated that 17β-estradiol (17β-E2) activated LRP16 mRNA levels in MCF-7 human breast cancer cell via ERα/Sp1 binding to DNA. In recent studies, our group identified an E2-responsive region from -213bp to -126bp at the 5′-flanking promoter of the
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LRP16 gene, which was essential for the maximal responsiveness for estrogen[12]. Sequence analysis showed that the classical ERE (estrogen response element) was absent within this region, but six GC-rich sites exist, which were potential sites for binding of Sp1 transcription factor. The aim of the current study was to further investigate the mechanism associated with the role of Sp1 on the E2-induced transactivation of LRP16 gene expression. Chromatin immunoprecipitation assay (ChIP) and Sp1-siRNA experiments were used to illustrate the interaction of Sp1 with the region from -213bp to -126bp of LRP16 gene promoter.
MATERIALS AND METHODS Chemicals, Biochemicals, and Oligonucleotides The primers of PCR were synthesized by ShengGong Biotechnology, Inc. (Shanghai, China). Specific antibodies of Sp1 and ERα were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chromatin immunoprecipitation (ChIPs) assay kits were purchased from Upstate Co. (NY, USA). LA Taq DNA polymerase and various restriction enzymes were purchased from TaKaRa (Otsu, Shiga, Japan). 17β-estrdiol (17β-E2) was purchased from Sigma, Corp (St. Louis, MO). Fetal calf serum (FCS) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from GibcoBRL (Grand Island, N.Y, USA). Transfection reagent Lipofectamine 2000 was purchased from Invitrogen Corp (USA). Dual-Luciferase® Reporter Assay System and TD-20/20n Luminometry System were purchased from Promega Corp. (USA). Chromatin Immunoprecipitation (ChIP) Assays MCF-7 cells (1×106) were grown in 10-cm tissue culture plates and treated with 100 nmol/L E2 for various times, then cross-linked with formaldehyde. Subsequently, cells were washed twice using ice-cold PBS containing protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin A), and then were scraped, collected and sonicated for 45 sec to shear DNA to lengths between 200 and 1000 base pairs. Specific antibodies of Sp1 and ERα or nonspecific IgG were added to precipitate the DNA/protein complex. Samples were then briefly centrifuged and the supernatant that contained unbound, non-specific DNA was carefully removed. The protein A agarose/antibody/protein complex was collected according to manufacturer’s
instructions provided by Upstate Co. (ChIP Assay Kit). Subsequently, semi-nested polymerase chain reaction (snPCR) was used to detect the presence of promoter regions immunoprecipitated by ERα or Sp1 antibodies (Santa Cruz Biotechnology, Inc.). The outer primers and the inner primers allow amplification of a sequence within -213 to -24bp upstream of the LRP16 gene translation starting site. In the first amplification round, the outer primers -213, 5′-GAGCTC(Sac I) ACGAGTGCGTGGGCCCATCCGG-3′ and -24, 5′-AAGCTT(Hind III) CCGCCCACTTGGACTCTATTT-3′ were used to amplify a fragment of 202-bp, followed by a second amplification round, using primer -213 and inner primer -126, 5′-AAGCTTAGCGGGAGGCGCGGCCAGA-3′ to obtain a fragment of 184-bp. The parameter of PCR was 95℃ for 10 min (pre-denaturing) and 33 cycles of 94℃ for 1min (denaturing), 62℃ for 30s (annealing) and 72℃ for 1min (extension) and then 1 cycle of 72℃ for 5 min to extend the reaction using a GeneAmp PCR System 9700. To avoid false positive or negative results, all experiments were carried out under stringent conditions and in parallel with negative and positive controls. The 1% input DNA was used as template in the positive control PCR. The primers 5′-GGGGTGGCTGTGAAGGTGGA-3′ and 5′-CGTTGACGATGGCGTCCACC-3′ was used to allow amplification of a 115-bp sequence within the third exon in LRP16 gene in the negative control PCR. Sp1-siRNA Sp1-siRNAs were designed as previously described[13] and prepared by Shanghai GeneChem Co., Ltd (Shanghai, China), which targeted the coding region 1811–1833 relative to the start codon of Sp1 genes. The negative control was purchased from Shanghai GeneChem Co., Ltd (Shanghai, China). The iRNA duplexes used in this study are indicated as follows: Sp1 5′-AUCACUCCAUGGAUGAAAUGATT-3′ 3′-TTUAGUGAGGUACCUACUUUACU-5′ Non-silencing 5′-UUCUCCGAACGUGUCACGUTT-3′ 3′-TTAAGAGGCUUGCACAGUGCA-5′ The 20μmol/L siRNA was prepared 24h before transfection experiment by adding 125μl 1×universal buffer to 2.5nmol siRNA duplexes. Single stranded iRNAs were annealed by incubating 20 μmol/L of each strand in annealing buffer (100 mmol/L
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potassium acetate, 30 mmol/L HEPES buffer at pH 7.4, 2 mmol/L magnesium acetate) for 2 min at 90°C followed by 1h at 37 ℃. Cell Culture, Transient Transfection and DualLuciferase Assay MCF-7 cell line was purchased from the American Type Culture Collection (ATCC, Rockvile, MD) and maintained in DMEM supplemented with 2 mmol/L L-glutamine, 10 mg/ml bovine insulin. Media for these cells were supplemented with 10% fetal calf serum (FCS) plus 100 U/ml penicillin-streptomycin and 100 U/ml penicillin-streptomycin. Cells were grown in humid atmosphere with 5% CO2 at 37℃. For transfection experiments, MCF-7 cells were plated in 3.5cm culture dishes and grown until 80%
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confluent. iRNA duplexes and/or reporter gene constructs pS10 (contain the promoter region from -213bp to -126bp)[12] were transfected using Lipofectamine 2000 Reagent (Invitrogen). For each dish (Table 1), 100μl Opti-MEM supplemented with pS10 (2μg) and/or pGL3-Basic (750ng), ERα expression vector (500ng), and 0.75 or 1.5μg iSp1 at a final concentration of 50 or 100 nmol/L, were applied and incubated for 4-6h, then the mixture were removed and replaced by the fresh medium. The effects of iSp1 on hormone-induced transaction was investigated in MCF-7 cells treated with 10 mmol/L E2 and harvested 36h after transfection by manual scraping in 1×lysis buffer (Promega). The cells were lysed for 15min according to the manufacturer’s instructions of DualLuciferase Assay Kit and measured by the TD-20/20 n Luminometry System. The experiments for each treatment group were carried out at least in triplicate.
Table 1. Cotransfection assay of pS10 and Sp1-SiRNA Cell line MCF-7
group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Construct pRL-SV40 pS10( μ g) (μ g) 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001 2 0.001
pSG5-ER (μg) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
pGL3-Basic (μ g) 1.5 1.5 0.75 0.75 0.75 0.75 0.75 0.75 1.5 1.5 -
Sp1-siRNA (μ g) 0.75 0.75 0.75 1.5 1.5 1.5 -
Negative control 17β-E2 (10-7M) (μ g) + + + + + 0.75 + 0.75 + 0.75 + + + + + + 1.5 + 1.5 + 1.5 +
Statistical Analysis
-213bp to -126bp of LRP16 Gene Promoter
Experiments were repeated two or more times, and data were expressed as ⎯x±s at least three replicates for each treatment group. Statistical differences between treatment groups were determined by Statview and Student’s t-test. Treatments were considered significantly different from controls if P<0.05, and the levels of probability were noted for each experiment.
Sp1 antibody immunoprecipitated the region from -213bp to -126bp of LRP16 gene, the predicted 184-bp DNA fragment was obtained in untreated MCF-7 cells and in cells treated with 100nmol/L E2 for 30, 60 and 90min (Figure 1A). ERα antibody did not immunoprecipitated this region of the LRP16 promoter at 0 min; however, after 30 min, the predicted band was observed obviously and indicated a hormone-induced increase up to 90 min (Figure 1B). These data showed that the interactions of ERα and Sp1 with the human LRP16 gene promoter occurred at 30-, 60- and 90-min time points after treatment with
RESULTS Interactions of ER and Sp1 with the Region from
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E2. As a positive control, the fragment from -213bp to -126bp was detected using 1% input DNA as template negative control experiment, the above mentioned fragment was not detected in the nonspecific IgG antibody group. Moreover, ERαand Sp1 antibodies did not immunoprecipitate the third exon region of the LRP16 gene (another negative control) (Figure 1C and 1D).
Chinese Journal of Cancer Research 18(4): 251-256, 2006 (A-C): The -213bp to -126bp region of the LRP16 gene promopter. After immunoprecipitation of cross-linked complexes by Sp1 (A), ERα (B) antibody or non-specific IgG (C), respectively; the chromatin was analyzed by PCR, as described in Materials and methods. (D): The third exon region of LRP16 gene. After immunoprecipitation by Sp1 or ERα antibody, the third exon region of the LRP16 gene was detected by PCR, as described in Materials and methods.
iSp1 Inhibits MCF-7 Cells
E2-induced
Transactivation
in
The results in Figure 2 summarize the effects of iSp1 on luciferase activity in MCF-7 cells cotransfected with pS10 and iSp1. In this study, there was >40% decrease in basal activity in cells transfected with iSp1 (data not shown). Moreover, E2 induced luciferase activity in MCF-7 cells cotransfected with pS10 as previously described, and in cells transfected with 50nmol/L iSp1 there was 22.2% decrease in hormone-induced transactivation, and there was 29% decrease in cells transfected with 100 nmol/L iSp1(Figure 2). Thus, iSp1 inhibited both basal and E2 induced luciferase activity in MCF-7 cells cotransfected with pS10, and there was no significant difference between the groups treated with 50nmol/L or 100nmol/L iSp1.
Fig.2. Sp1-siRNA-mediated inhibition of transactivation in MCF-7 cells cotransfected with pS10 and treated with 17 β-E2. Significant decrease of reporter gene were observed mediated by Sp1-siRNA (*P<0.05). There was not significant difference in the groups of 50 nmol/L and 100 nmol/L (#P>0.05).
Fig. 1. Specific antibodies of ERα and Sp1 immunoprecipitated the region from -213bp to -126bp of LRP16 gene promoter.
DISCUSSION E2-dependent transactivation via ERα/Sp1 is
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mediated by at least two pathways, namely (1) interactions of the ERα/Sp1 complex with GC-rich sites in which only Sp1 protein binds DNA and (2) interaction of ERα/Sp1 proteins with half-site (1/2) ERE(N)xSp1 or Sp1(N)xERE (1/2) motif DNA elements where both ERα and Sp1 bind DNA elements. Previous studies have shown that E2 induced LRP16 gene expression and the effect of E2-induced transactivation are caused by direct interaction of ERα with the promoter region of LRP16 gene. A cis-element within the 5′-flanking region of LRP16 gene has been identified, which confers the E2-action via ERα/Sp1 protein complex binding to DNA[14-16]. Recently, we identified a fragment from -213bp to -126bp that was essential for the E2responsiveness[12]. By computer-aided analysis, we demonstrated that this region does not contain any perfect palindrome ERE and any cis-element that can bind to other transcriptional factors, such as AP1, USF1/2 et al (data not shown), it only contains three GC-rich sites that may bind to Sp1 protein. As far as our information goes, ERα/Sp1-mediated activation of GC-rich sites has been characterized in several E2-responsive gene promoters, including bcl-2, cathepsin D, heat shock protein 27, et al [2,17,18]. In this study, we attempted to investigate the interactions of ERα/Sp1 with the LRP16 gene promoter using a chromatin immunoprecipitation (ChIP) assay in MCF-7 cells at different time point after E2 treatment. The results showed that ERα and Sp1 antibodies immunoprecipitated the E2-responsive region (-213bp to -126bp) of the LRP16 gene after treatment with E2 at 30, 60, 90 min. The above mentioned fragment was also immunoprecipitated at 0 min when Sp1 antibody was used, but not when ERα antibody was used. In addition, the fragment immunoprecipitated by Sp1 was faint at 0 min, suggesting that Sp1 weakly interact with the promoter region of LRP16 gene without E2 treatment; however, enhanced interactions were observed after treatment with E2 for up to 90 min. These results indicate that Sp1 association with the LRP16 gene promoter is ligand-independent, whereas interaction of ERα and LRP16 gene promoter is ligand-dependent, this is in good correlation with the findings of Han WD, et al[14,19,20]. Take together, these results provided direct evidence for Sp1 binding to the proximal promoter region from -213bp to -126bp of the LRP16 gene, and demonstrated that the E2-induced interaction of ERα and Sp1 enhanced the Sp1/DNA binding affinity. The results of cotransfection assays combining with Sp1-SiRNA indicated 20-30% decrease in E2-induced transactivity in MCF-7 cells mediated by
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iSp1, lower than the results reported by Abdelrahim M[13]. The molecular mechanism may involve the doze-dependent character of DNA fragment or target gene on Sp1 protein. These demonstrated that Sp1 protein is required for the responsiveness of region B to estrogen, but the expression of LRP16 gene is independent on the quantity of Sp1 protein. In summary, we have localized elements required for E2-transactivation that bind to Sp1 trans-acting factor, and have demonstrated that activation of LRP16 gene expression in MCF-7 cells by E2 is dependent on Sp1/ERα interaction with the promoter region mentioned above. These observations strongly suggest an important role for these two proteins on expression of the LRP16 gene. We anticipate that the results of this study will be a basis to further investigate the mechanism involved in the regulation of LRP16 gene expression.
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