Identification of the p53-Responsive Element in the Promoter Region of the Human Decorin Gene

We have obtained some evidence that shows that the decorin gene is the target of p53 transactivation. Luciferase reporter plasmid, which contained the promoter region between positions -252 and -205, was activated by p53 dosedependently up to 170-fold. The promoter region involved a sequence, 5’-AGGCAAGTAG-3’, similar to p53-binding consensus sequence, 5’-PuPuPuC(A/T)(A/T)GPyPyPy-3’. Chromatin immunoprecipitation assay using p53 antibodies revealed that the region between -413 and -232 of the promoter of the decorin gene was co-precipitated with p53. p53binding to this region was further demonstrated by electrophoretic mobility shift assay, in which the complex between decorin promoter DNA and proteins decreased by pretreatment with anti-p53 antibodies. The mRNA expression levels of decorin increased after treatment with p53-activating nutlin-3 greatly and with genotoxic reagent, adriamycin, to some extent. Consequently, decorin promoter is useful to evaluate the p53 transactivation ability. *Corresponding author: Takaki Hiwasa, Department of Biochemistry and Genetics, Graduate School of Medicine, Chiba University, Inohana 1-8-1, Chuoku, Chiba 260-8670, Japan; Tel: +81-43-226-2541; Fax: +81-43-26-2037; E-mail: hiwasa_takaki@faculty.chiba-u.jp Received April 23, 2015; Accepted May 21, 2015; Published May 29, 2015 Citation: Hiwasa T, Iwase K, Suichi T, Hino Y, Kimura R, et al. (2015) Identification of the p53-Responsive Element in the Promoter Region of the Human Decorin Gene. Mol Biol 4: 124. doi:10.4172/2168-9547.1000124 Copyright: © 2015 Hiwasa T, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Mutations in the p53 gene are observed in many tumor cells most frequently [1]. p53 plays a main role in suppression of tumorigenesis because the deletion or mutation results in frequent oncogenesis [2,3]. p53 consists of N-terminal transactivation domain, DNA-binding domain and tetramerization domain. Wild-type p53 causes effects mainly as a transcription factor and regulates cell cycle, DNA repair, and apoptosis. For example, p53 induces cell cycle arrest by transactivating p21, GADD45, 14-3-3σ, and so on [4], and induces apoptosis by direct binding to Bcl-2 and/or transactivating Bax, Noxa, Puma, and so on. [5][6][7]. A comprehensive search for target genes of p53 transcription factor has been attempted using p53-binding consensus sequence [8], ChIP-on-Chip [9,10] and microarray [11]. However, despite such identification of many p53-target genes, the key molecule, which can explain the tumor suppression by p53, remains to be obscure.
In our previous study, the enforced expression of COUP-TFI in activated ras-transformed NIH3T3 mouse fibroblasts (ras-NIH3T3) resulted in growth inhibition as well as phenotypic reversion [12]. Microarray analysis suggested that the expression levels of decorin, cyclin G, lipocortin and LAMP-2 were elevated in COUP-TFIexpressing cells. Similarly, increased expression of decorin, biglycan, amyloid precursor protein and selenoprotein P was observed by enforced expression of C/EBPα and C/EBPβ, which caused growth inhibition as well as phenotypic and morphological reversion [13]. Thus, decorin may play a key role not only in growth inhibition but also in tumor suppression. Similar growth-suppressive and tumorsuppressive roles were also reported for p53 [1][2][3][4]. Therefore, we examined whether p53 can transactivate the decorin gene, and found that the decorin promoter was activated by p53. Plasmids pCMV-p53WT [14] was provided by Dr. Bert Vogelstein (Howard Hughes Medical Institute). pGL3-basic and SV40-Rluc reporter plasmids were purchased from Promega (Madison, WI). The promoter region from -1338 to -205 or from -204 to +42 of human decorin gene was amplified by PCR using genomic DNA isolated from human esophageal carcinoma cell line, T.Tn [15], as a template. After subcloning by TA cloning, the promoter regions were inserted into Xho I/Hind III site of pGL3-Basic vector (Promega) as described previously [12]. Deletion mutants such as Deco (-1237:-

Cell lines and culture
ras-NIH3T3 [16] were cultured in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 5% bovine serum and 100 μg/ml of kanamycin. Human glioblastoma U-87 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 μg/ml of kanamycin [17]. with p53 expression plasmids together with firefly luciferase and control Renilla luciferase reporter plasmid, SV40-Rluc, by using LipofectAMINE-Plus (Thermo Fisher Scientific, Waltham, MA). Two days after the transfection, firefly and Renilla luciferase activities were determined with a Dual Luciferase Assay System (Promega) and a luminescencer (Atto, Tokyo, Japan) as reported previously [17,18]. Firefly luciferase activities were normalized with the Renilla luciferase control activities.

Chromatin immunoprecipitation (ChIP) assay
U-87 glioblastoma cells were treated with adriamycin at a concentration of 10 μM for 24 h, and the chromatin fraction was prepared. ChIP assay was carried out using ChIP Assay Kit (Active Motif) according to the manufacturer's instruction. Briefly, chromatin preparation was treated with DNase I, and immunoprecipitated with anti-p53 antibodies such as DO-1 and Bp53-12 (Santa Cruz Biotechnology). Using the precipitated DNA as a template, the decorin promoter region between positions -413 and -232 was amplified by PCR using primers of 5'-AACTGGTGGACAGGGAGAAAG-3' and 5'-TCGGATTCCTACTTGCCTTGG-3' . The PCR products were separated by agarose gel electrophoresis.

Reverse transcription-quantitative real-time PCR (RT-qPCR)
U-87 human glioblastoma cells were treated with nutlin-3 (10 μM), 5-FU (1 μg/ml), VP-16 (500 ng/ml), CPT (10 nM), ADM (1 μg/ml) or solvent DMSO (0.1%) for 2 h, and total cellular RNA was isolated from using the AquaPure RNA Isolation kit (Bio-Rad, Hercules, CA). Reverse transcription was performed with an oligo(dT) 20 primer using the ThermoScript RT-PCR System (Thermo Fisher Scientific). The expression levels of decorin transcripts were quantitated by real-time RT-PCR with the standard of GAPDH using the following primers and probes (Universal Probe Library; Roche, Basel, Switzerland). PCR was performed using Fast Start Taq Man probe Master (Roche) and an ABI PRISM 7000 Sequence Detection System (Applied Biosystems LLC) as follows: an initial denaturation step at 95°C for 2 min, followed by 55 cycles of denaturation at 95°C for 15 sec and annealing/ extension at 60°C for 30 sec. The mean and SD for the -ΔΔCT were calculated and normalized to the GAPDH value as described [19].

Activation of decorin promoter by p53
We examined the effects of a typical tumor suppressor p53 on the expression of the decorin gene. The reporter plasmid, decorin-Luc, was constructed by insertion of the PCR product of decorin promoter from -1338 to -205 or from -204 to +42 into the XhoI/HindIII site of pGL3-basic (Promega). These reporter plasmids as well as the deletion constructs were co-transfected into ras-NIH3T3 cells with the control Renilla luciferase reporter, SV40-Rluc, and the p53 expression plasmid. The luciferase activity was increased by co-transfection with the p53 expression plasmid as compared with that of the control empty expression plasmid (Figure 1a). Most prominent activation was observed for Deco (-252:-205)-Luc, which was activated by p53 dosedependently up to 170-fold (Figure 1b). Similar results were observed by transfection into U-87 glioblastoma cells ( Figure 2). These suggest that the inhibitory sequences against p53 responsiveness exist in the region between -1338 and -252 of the promoter region of the decorin gene. In

ChIP assay verified p53 binding to the decorin promoter
U-87 cells were treated with adriamycin, and the chromatin was digested with DNase I and immunoprecipitated with anti-p53 antibody (DO-1, Santa Cruz Biotechnology). PCR amplification using coprecipitated DNA as a template revealed the DNA band of the size similar to the expected 182 bp (Figure 4). A similar result was observed using another p53 antibody (Bp53-12, Santa Cruz Biotechnology), but not without an antibody. This suggests that p53 binds to the region between -413 and -232 of the promoter of the decorin gene.

EMSA showed the complex between p53 and the decorin promoter
The synthetic probe of decorin promoter containing -256 to -232 was labeled with digoxigenin and incubated with nuclear extract of ADM-treated U87 cells. The mixture was electrophoresed through polyacrylamide gel, and blotted onto the PVDF membrane [18], which was then treated with anti-digoxigenin antibody followed by staining with CSPD. The bands of low-mobility represent DNA-protein complexes ( Figure 5, lane 2). The density decreased by pretreatment with anti-p53 antibodies, N-19 or M-19, but not with control anti-   NFκB p65 antibody ( Figure 5, lane [3][4][5]. Similar results were obtained by using different p53 antibodies such as Pab240, Bp53-12 and DO-1 (Supplementary Figure S1). This band disappeared after the addition of the excess amount of unlabelled probes ( Figure 5, lane 6).

Decorin expression level was elevated by p53-activating nutlin-3
It has been well-known that p53 is activated after treatment with genotoxic anticancer drugs such as 5-FU, VP-16, CPT and ADM. p53activating drug named Nutlin-3 has been developed as an inhibitor of MDM2 [21]. The mRNA expression levels of decorin were elevated by treatment with Nutlin-3 almost 15-fold ( Figure 6). ADM also increased the level significantly but only 2.5-fold. None of 5-FU, VP-6 or CPT enhanced the decorin expression levels. Thus, certain signaling pathways leading to p53 but not by all of the p53-activating signals may induce the expression of decorin.

Activation of decorin promoter by COUP-TFI, C/EBPα, C/ EBPβ and C/EBPδ
Our previous microarray analysis revealed that the expression of decorin was elevated by enforced expression of COUP-TFI and C/EBPα [12,13]. Thus, we examined the effects of COUP-TFI and C/EBP family on the decorin promoter. The regions between -287 to -252 and between -204 and +42 of the promoter of the decorin gene were most activated by COUP-TFI (Figure 7a), whereas the regions between -414 and -287 and between -204 and +42 were responsible to C/EBPβ (Figure 7c). C/ EBPα and C/EBPδ activated mainly the region between -204 and +42 of the decorin promoter (Figure 7b and d). These regions were different from the region between -252 and -205 which was responsible to p53.

Discussion
We have demonstrated that the decorin gene is the target of p53 transactivation. Decorin is a member of leucine-rich proteoglycan family, and causes effects on proliferation, apoptosis, carcinogenesis and metastasis by binding to growth factor receptors such as EGFR, ErbB4 and IGF-IR [22,23]. Despite the comprehensive screening of p53 target genes [8][9][10][11], decorin has not yet been identified. The sequence, 5'-AGGCAAGTAG-3' , similar to the consensus sequence, 5'-PuPuPuC(A/T)(A/T)GPyPyPy-3' [20] in the promoter region of the decorin gene may be responsible for p53 transactivation (Supplementary Figure S1) because the p53 transactivation ability was much reduced in the mutant with core CAAG replaced by AAAT. On the other hand, recent reports have shown that two repeated sequence of 5'-PuPuPuC(A/T)(A/T)GPyPyPy-3' was responsible to p53 [8]. Similar consensus sequence was also observed in the decorin promoter at positions between -275 and -266 (Supplementary Figure S1). However, Deco (-414:-205)-Luc containing this region was less responsible to p53 than Deco (-252:-205)-Luc (Figure 1a). The promoter region between positions -414 and -253 may involve binding sites for other regulatory molecules. Both basal and p53-activated expression levels of Deco (-204:+42)-Luc was very low (Figure 1a), suggesting that the region between positions -204 and +42 may not be involved in the regulation of gene expression.
Pleiotropic effects of p53 can be explained by the target molecules transactivated by p53; for example, growth-inhibition induced by p21 [4] and 14-3-3σ [24], apoptosis induced by Bax [5], Noxa [6] and Puma [7], senescence induced by PAI-1 [25], DNA repair induced by p53R2 [26] and Ku70 [27]. We have carried out a microarray analysis of ras-NIH3T3 cells transfected with various transcription factors, and found that decorin expression was commonly elevated by enforced expression ether of COUP-TFI, C/EBPα or C/EBPβ [12,13]. Consistently, Deco (-1237:-205)-Luc was also activated by COUP-TFI, C/EBPα, C/ EBPβ and C/EBPδ, yet the different regions were responsible to each molecule ( Figure 7). In addition to COUP-TFI [12], growth-inhibitory activity of C/EBPα and C/EBPβ has been reported [28,29]. C/EBPα acts as a tumor suppressor in multiple tissues [30]. Thus, it is possible that decorin plays a key role not only in inhibition of proliferation but also in suppression of tumorigenesis. Interestingly, in the p53-binding site, AGGCAAGTAG, T and last G are SNP sites, rs13312819 and rs13312820, respectively. Further genome-wide analysis study in this region may clarify the tumor-suppressive role of decorin.