Volume 10 Supplement 3
Computational identification and experimental validation of PPRE motifs in NHE1 and MnSOD genes of Human
© Venkatachalam et al; licensee BioMed Central Ltd. 2009
Published: 3 December 2009
Activation of PPARs has been reported to inhibit the proliferation of malignant cells from different lineages. They are involved in transcription regulation of genes upon activation by a ligand. The binding of PPARs to the promoter sequence either represses or activates the gene. Hence, PPARs represent promising targets for cancer treatment because of their anti-proliferative and pro-apoptotic activities. Here we computationally identified PPAR binding regions in NHE1 and MnSOD. We further validated the predictions in vitro.
Our results computationally predicted the presence of 2 PPRE motifs in NHE1 and 3 PPRE motifs in MnSOD. We experimentally confirmed the true motifs and their regulation by PPAR.
Our results suggest that both NHE1 and MnSOD have PPRE binding motif in their upstream/promoter region and hence are regulated by PPAR upon ligand binding.
PPARs (Peroxisome proliferators activated Receptor) belong to the nuclear receptor super family and are ligand activated transcription factors, regulating a wide variety of genes . Phylogenetic studies have shown that PPARs form a subfamily of the nuclear receptor superfamily, along with the receptors for thyroid hormone, retinoic acid (RA), vitamin D, ecdysone, and the orphan receptors Rev-ErbAa (5ear1;NR1D1) and E75 (NR1D3, from Drosophila), the two latter being the closest relatives of the PPARs . PPARs play important role in lipid metabolism, cell differentiation and development, inflammation, cancer and demyelination .
Three isoforms (α, β and γ) for PPAR have been identified so far in xenopus, mouse, human, rats and hamsters . The isoforms exhibit a tissue specific expression pattern. The first isoform PPAR α is expressed in tissues that play a role in fatty acid catabolism such as liver, kidney, heart, and intestine . This isoform is the first described receptor that is activated by peroxisome proliferators and hence the name . The main function of this isoform is to regulate the genes encoding lipid metabolizing enzymes and proteins participating in lipid metabolism . The second isoform is PPAR β. This receptor is also called as FAAR (fatty acid activated receptor). This isoform is expressed mainly in the brain, skeletal muscle, skin, adipose tissue, gut, placenta and has a very broad expression pattern. Based on the cell proliferation and differentiation, the expression profile of this isoform varies. Both isoforms PPAR α and β are involved in lipid utilization activities such as fatty acid oxidation, energy uncoupling reactions and response to fasting . PPAR γ has three alternatively spliced isoforms. PPAR γ1 is virtually expressed in almost all tissues . PPAR γ2 is primarily expressed in adipose tissue while PPAR γ3 is expressed in white adipose tissue, large intestine and macrophages. PPAR γ regulates the lipid storage and insulin sensitivity reactions such as lipogenesis, adipocyte differentiation, adipocyte survival and adipokine secretion . Activation of the peroxisome proliferator-activated receptor-γ (PPARγ) has been identified as an approach to induce differentiation and inhibit proliferation in a variety of cancer cells. Breast tissue, in particular, was found to express PPARγ in amounts greater than those found in normal breast epithelium . Moreover, activation of PPARγ through cells' exposure to synthetic PPARγ ligands such as proglitazone or rosiglitazone is shown to exert antitumor activity through cell growth inhibition and cellular differentiation. Activation of PPARs has been reported to inhibit the proliferation of malignant cells from different lineages such as liposarcoma, breast adenocarcinoma, prostate carcinoma, colorectal carcinoma, non-small cell lung carcinoma, pancreatic carcinoma bladder carcinoma, and gastric carcinoma both in vitro and in vivo.
Collection of PPRE motifs from literature
This collection contains 250 reported PPRE motifs from literature. Keywords "PPRE", "PPAR consensus sequences" "PPAR binding sequences" were used to search for literatures in pubmed and Google Scholar. The sequences reported only with experimental validation were added to this collection. For each PPRE data on its reported consensus, isoform specificity, in vivo and in vitro binding efficiencies and Pubmed IDs were collected (additional file 1).
Promoter sequence extraction and motif identification
The promoter sequences (5 kb) upstream of the transcription start site from transcripts of NHE1 and MnSOD were downloaded from NCBI http://www.ncbi.nlm.nih.gov using keywords NHE1 and MnSOD. The motif collection was searched/matched (string comparison) by a cgi-script against the extracted sequence of NHE1 and MnSOD, to check if there is presence of any of the motifs (string) in the upstream regions of the above gene sequences. Search of human NHE1 and MnSOD promoter sequences against this collection predicted/identified two putative DR2 PPRE motifs in NHE1 (Figure 1) and one DR1 PPRE and two DR2 PPRE motifs in MnSOD (Figure 2).
The general DR1 and DR2 consensus are AGGTCA N AGGTCA (6-N-6) and AGGTCA NN AGGTCA (6-NN-6), respectively. Both the above patterns were matched against the collection, first by looking for the surrounding hexamers and then looking for the spacers i.e. one nucleotide or 2 nucleotides separation. Thus, the motifs for DR1 and DR2 are identified based on the number of 'N's that separate the two hexamer consensus pattern.
These predicted motifs were experimentally validated as explained in the subsequent sections.
Validation of identified patterns in NHE1 and MnSOD
Reagents and antibodies
Roswell Park Memorial Institute (RPMI) 1640 medium, phosphate buffered saline (PBS), fetal bovine serum (FBS), L-glutamine and trypsin was purchased from Hyclone UT, USA. Pepstatin A, phenylmethanesulfonyl fluoride (PMSF), leupeptin, bovine serum albumin (BSA), mouse anti-β-actin monoclonal antibody were supplied by Sigma-Aldrich LO, USA. Aprotinin was purchased from Applichem Darmstadt, Germany. Mouse anti-HuNHE1 monoclonal antibody was purchased from Chemicon International MA, USA. Rabbit anti-HuMnSOD monoclonal antibody was purchased from Upstate, NY, USA. Stabilized goat anti-mouse horseradish peroxidase (HRP) was obtained from Pierce IL, USA. The PPARγ agonist, 15 d-PGJ2 was purchased from Alexis Biochemical CA, USA. 5'-biotinylated oligonucleotides for NoShift assay were synthesized by Proligo, Singapore.
Cell lines and culture conditions
Human MCF-7, MDA-MB-231, and MDA-MB-468 breast carcinoma cells were obtained from the American Type Culture Collection (ATCC MD, USA). MCF-7, MDA-MB-231, and MDA-MB-468 cells were propagated and maintained in RPMI medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1 mM gentamicin sulfate at 37°C and 5% CO2. Cultures were replenished with fresh medium every 2 to 3 days and passage 1:3 when they reached 80% confluence.
Western blot analysis
Whole cell lysates were prepared with RIPA lysis buffer containing 10 mM Tris-HCL pH7.4, 30 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, supplemented with 1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin and 1 mM PMSF before use. Protein concentration was determined for each sample and equal amounts of protein were warmed at 37°C in the water bath for NHE1 protein, boiled for 5 min for MnSOD, with 1 × SDS sample buffer and resolved by 8% (NHE1) or 12% (MnSOD) SDS-PAGE. Thereafter, proteins were transferred onto nitrocellulose membrane, blocked for 1 h at RT with 5% non-fat milk, and incubated overnight at 4°C with the primary antibody. After probing with secondary antibody for 1 h at 25°C, protein bands were detected by using the Supersignal West Pico Chemiluminescence (Pierce IL, USA). β-actin antibody was used as a loading control.
Reporter plasmid constructs
Chloramphenicol acetyltransferase (CAT) pUCSS-CAT reporter plasmid constructs: -1374/+16, -850/+16, and empty vector pUCSS-CAT were kindly provided by Dr. Alexey Kolyada, Dept of Medicine, Tufts University School of Medicine, Boston, USA (Kolyada et al., 1994). Full length and 5' deletion constructs of the human MnSOD promoter tagged to luciferase reporter gene were a gift from Dr. Daret K. St Clair, Graduate Center for Toxicology, University of Kentucky, USA (Xu et al., 2002).
Chloramphenicol acetyltransferase (CAT) ELISA
The levels of CAT protein were quantified using a CAT antigen capture enzyme-linked immunosorbent assay (ELISA) (Roche Molecular Biochemicals Mannheim Germany). All CAT quantitations were normalized to the protein concentration of the cell extract, as determined using the Coomasie Plus Protein Assay Reagent Kit (Pierce IL, USA).
Luciferase reporter assays
3 × PPRE promoter activity was assessed with a dual-luciferase assay kit (Promega WI, USA). Briefly, feeding medium was removed from the wells, washed once with 1× PBS, and lysed with ice-cold 100 μl of reporter lysis buffer. Ten microlitres of cell lysate was then added to 50 μl of luciferase substrate solution, following which 50 μl of stop & glow buffer was added for Renilla reading. Bioluminescence generated was measured using a Sirius luminometer (Berthold Technologies, Deutschland, Germany). The luminescence readings obtained were normalized to the protein content of the corresponding cell lysate. Activity was calculated by dividing luminescence readings expressed in relative light units (RLU)/renilla/μg total protein in 10 μl of cell extract used in the assay.
NoShift transcription factor assay
Nuclear and cytosol fractions of MDA-MB-231 cells treated with 15 d-PGJ2 were prepared using the NE-PER Nuclear and cytoplasmic Extraction kit (Pierce IL, USA). The binding affinities of PPARγ protein in nuclear extracts to three target PPREs on MnSOD promoter were determined with the NoShift transcriptional factor assay kit (Novagen Inc. NJ, USA). The oligonucleotide sequences (listed below) used for the three putative PPREs on MnSOD were first annealed in a PCR machine with 1× annealing buffer (1 mM TrisHCl pH 8, 50 mM NaCl, 10 mM MgCl2) and the annealed oligonucleotides then diluted to a final concentration of 100 ng/μl.
HuMnSODPPRE-1 Sense 5'-Biotin-GTAACTTGGGACACAGGTCAATCGACTG-3'
HuMnSODPPRE-1 Antisense 5'-Biotin-CAGTCGATTGACCTGTGTCCCAAGTTAC-3'
HuMnSODPPRE-2 Sense 5'-Biotin-CACTTGAGGTCAGGCGTTCGAGACCA-3'
HuMnSODPPRE-2 Antisense 5'-Biotin-TGGTCTCGAACGCCTGACCTCAAGTG-3'
HuMnSODPPRE-3 Sense 5'-Biotin-TGGAATAGGTCCCAAGGTCGGCTTAC-3'
HuMnSODPPRE-3 Antisense 5'-Biotin-GTAAGCCGACCTTGGGACCTATTCCA-3'
For measurement of PPARγ binding, each reaction mixture contained 5 μl of 4× NoShift Bind buffer, 1 μl of poly(dI-dC)·poly(dI-dC) (0.01 U/μl in 100 mM KCl, 20 mM HEPES, pH 8.0), 1 μl of salmon sperm DNA (500 ng/μl in nuclease-free water), 1 μl of biotinylated 10-pmol/μl target DNA duplex, and 20 μg of nuclear lysate in a total reaction volume of 20 μl. After 30 min incubation on ice, 80 μl of 1× NoShift Bind buffer was added to each reaction mixture, and the resulting 100 μl was dispensed into one well of a freshly washed streptavidin plate and incubated for 1 h at 37°C. After this incubation period, plates were washed with 1× NoShift Bind buffer, the binding of PPARγ was detected by incubation for 1 h at 37°C with 100 μl of anti-PPARγ diluted 1:250 in NoShift antibody dilution buffer. After repeated washings, horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) was added (1:1000 dilution in NoShift antibody dilution buffer). After 30 min of incubation at 37°C, wells were washed thoroughly. Finally, 100 μl of room temperature TMB (tetramethylbenzidine) substrate was added and the wells were incubated for 10-30 min at room temperature in the dark until a blue color developed. The reaction was stopped by adding 100 μl of 1 M HCl to each well, and A450 was measured using Spectrofluoro Plus spectroflurometer (TECAN, Grodig, Austria).
Cells were transfected using CalPhost Mammalian transfection kit (BD Bioscience, CA, USA) for 15 h. Cells were then washed twice with PBS and culture media added for another 24 h. Co-transfection with the Renilla plasmid (Clontech, CA, USA) was used to assess transfection efficiency in dual-luciferase reporter assay (Promega, WI, USA).
RNA isolation and mRNA determination by real-time PCR
Total RNA was isolated from cells by TRIZOL reagent (Invitrogen, CA, USA) as described by manufacturer's instructions with a DNAse treatment step incorporated into the protocol. Each RT reaction contains 2.5 μg of total RNA, 1× RT buffer, 5 mM MgCl2, 425 μM each of dNTPs, 2 μM random hexamers, 0.35 U/μl RNase inhibitor, 1.1 U/μl MultiScribe™ reverse transcriptase and made up to 10 μl with sterile water. RT reaction was carried out at 37°C for 1 h. 5 μl of the 10 μl cDNA reaction volume was used in realtime quantitative PCR using ABI PRISM 7500 (Applied Biosystems, CA, USA). Normalization was to either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or 18S RNA. Fluorescence was measured with the Sequence Detection Systems 2.0 software. PCR was performed in multiplex (both target and endogenous control co-amplified in the same reaction with distinct fluorescent dyes. Primers and probe for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S RNA, human NHE1 and human MnSOD were purchased as kits from Applied Biosystems (Assays on Demand).
Statistical analysis was performed using paired Student's t-test. A p-value of less than 0.05 was considered significant.
PPRE motif collection
It is known that the general DR1 and DR2 consensus are AGGTCA N AGGTCA (6-N-6) and AGGTCA NN AGGTCA (6-NN-6), respectively. The determining factor for PPAR binding to the PPRE are the surrounding hexamers 1 and 2 (AGGTCA). The DR1 and DR2 motifs share the same hexamers and PPARs have also been reported to bind hexamers with DR2 spacers. Thus, for PPARs optimal spacer nucleotide is either 1 or 2. Out of the 413 PPRE DR1 motifs collected from literature the binding strengths were reported for 236 motifs (Additional file 1). These 236 motifs were used to match for both DR1 and DR2 sequences. Based on sequence match, we identified two putative PPREs in NHE1 and three putative PPREs in MnSOD promoter sequences.
Identification of P eroxisome P roliferator R esponse E lements (PPREs) within the human NHE1 and MnSOD promoters
We identified two putative PPREs in the human NHE1 promoter, (Figure 1). The first PPRE site (PPRE1) is within a primate-specific Alu element (Sq) , within which there is a cluster of four hexamers half-sites recognized by various nuclear receptors [22–25], termed as Alu receptor response element (AluRRE). Kumar et al. have previously shown in the context of the human myeloperoxidase gene, a DR2 element is also recognized by PPARγ . DR2 element, in both PPRE1 and PPRE2 are seen here within the human NHE1 promoter. We identified three putative PPREs in the human MnSOD promoter region (Figure 2C). In the human MnSOD promoter, PPRE1 is a DR1 element, while PPRE2 and PPRE3 are of the DR2 elements. Here, PPRE2 is within a primate-specific Alu element (Sx) .
PPARγ activation by 15 d-PGJ2 represses NHE1 mRNA and protein levels
PPARγ activation by 15 d-PGJ2 represses MnSOD mRNA and protein levels
Experimental evidence for PPRESearch-derived prediction for PPARγ binding site in NHE1 promoter
Experimental evidence for PPRESearch-derived prediction for PPARγ binding site in human MnSOD promoter
Breast cancer is the third most common cancer globally and accounts for the highest morbidity and mortality. It is the second highest occurring cancer in women and one of the leading causes of death . Although anti-estrogens have provided an effective endocrine therapy, a large number of patients acquiring resistance to these drugs pose a significant problem. Recently, a new approach using activators of p eroxisome p roliferator-a ctivated r eceptor g amma (PPARγ) to inhibit proliferation and increase cell death in breast cancer cell lines has given hope to the development of a new class of anticancer drugs . Interestingly, tumor breast tissue expresses PPARγ in amounts greater than normal breast epithelium, suggesting for a novel therapeutic approach for ligand-activated PPARγ as an anti-tumor agent in differentiation-based breast cancer therapy.
Normal functioning of cell metabolism occurs within a restricted intracellular pH (pHi) range. Regulation of pHi is accomplished via active extrusion of H+ by the Na+/H+ Exchangers (NHEs), a membrane antiporter expressed in a variety of cell types. The NHE family consists of ten isoforms, NHE1 to NHE10. Interestingly, apart from its role as a principal regulator of pHi and cell volume, the ubiquitously expressed NHE1 has been implicated in cell proliferation and transformation. Conversely, tumor cells deficient in NHE1 either fail to grow or show severely retarded growth when implanted in immuno-deficient mice . More recently, we showed that down regulation of NHE1 expression by direct silencing of the gene expression or H2O2 treatment leads to cells' growth arrest and sensitization to etoposide or staurosporine [16, 17].
Development of breast cancer has also been correlated to oxidative stress, brought about by alterations to the delicate balance between reactive oxygen species (ROS) and oxidative defenses [28, 29] ROS stress seems to render cancer cells more dependent on MnSOD to protect them by maintaining cellular ROS balance. The importance of MnSOD became clearer when genetic knockout studies in mice indicate that MnSOD, but not other SODs, is essential for cell survival. Several recent studies reported that forced suppression of MnSOD expression by siRNA leads to decrease in breast cancer cells invasive property and to sensitization of ovarian cancer cells to anti-cancer drugs . Although the mouse MnSOD gene has been shown to be a PPARγ target gene , a direct association between PPARγ and human MnSOD from tumor cells' perspective has not been shown.
Taken together, our results suggest that NHE1 and MnSOD have PPRE binding motifs of high affinity. This confirms that these genes can be downregulated by PPARγ in presence of a ligand. Since, downregulation of NHE1 inhibits proliferation and increases cell death in breast cancer cell lines, the presented data provides new direction for development of a new class of anticancer drugs. Concurrently, downregulaion of MnSOD expression is reported to decrease cancer cells invasive property  and sensitizes cancer cells to anti-cancer drugs. Thus, the presented data suggests that PPARγ can regulate MnSOD in human cell linesPPARγ has been found to regulate MnSOD transcription by binding to a PPRE on the mouse MnSOD promoter region. This report for the first time suggests PPARγ activation and its involvement in downregulating MnSOD gene expression in human.
Other papers from the meeting have been published as part of BMC Bioinformatics Volume 10 Supplement 15, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Bioinformatics, available online at http://www.biomedcentral.com/1471-2105/10?issue=S15.
This work was supported by grant R-183-000-204-213 from the National Medical Research Council, Singapore to MVC, SP and APK. The authors also thank Dr. Alexey Kolyada, Dept of Medicine, Tufts University School of Medicine, Boston, USA for providing the pUCSS-CAT NHE1 reporter plasmid constructs: -1374/+16, -850/+16, and empty vector pUCSS-CAT and Dr. Daret K. St Clair, Graduate Center for Toxicology, University of Kentucky, USA for providing the full length human MnSOD promoter construct and the various 5' deletions derivatives tagged to a luciferase reporter (pGL3 vector).
This article has been published as part of BMC Genomics Volume 10 Supplement 3, 2009: Eighth International Conference on Bioinformatics (InCoB2009): Computational Biology. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2164/10?issue=S3.
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