Title: 9-cis-Retinoic Acid Up-regulates Expression of Transcriptional Coregulator PELP1, a Novel Coactivator of the Retinoid X Receptor α Pathway
Abstract: Retinoid X receptor α (RXRα), functioning as either a homodimer or a heterodimer with peroxisome proliferator receptors, is known to be involved in manifesting antiproliferative effects in cells. Consequently, studies of RXRα functions and its coregulators have been in the focus for therapeutic approaches against cancer. Here we have discovered that 9-cis-retinoic acid (9-cis-RA), a RXRα-specific ligand, up-regulated the expression of transcriptional coregulatory protein PELP1 (proline-, glutamic acid-, and leucine-rich protein 1). PELP1 functioned as a coactivator of RXRα, increasing its transactivation function in response to 9-cis-RA as evident by the retinoid X receptor response element-luciferase assays. PELP1 was found to be a binding partner of RXRα, and the binding interactions were confirmed both in vitro and in vivo. An electrophoretic mobility shift assay showed greater formation and stability of RXRα homodimers on consensus oligonucleotides in PELP1-overexpressing clones in comparison to the pcDNA clones. The presence of PELP1 in these oligonucleotide-bound RXRα homodimers was proved by the supershift of the complex when incubated with PELP1-specific antibody. PELP1-overexpressing stable MCF-7 cells exhibited a significantly higher extent of 9-cis-RA-induced apoptosis than the control pcDNA clones. Silencing of PELP1 expression in parental MCF-7 cells and PELP1-overexpressing clones using PELP1-specific RNA-mediated interference compromised the susceptibility to 9-cis-RA-induced apoptosis. PELP1 could also function as a coactivator of the RXRα-peroxisome proliferator-activated receptor (PPARγ) heterodimer as evident by the peroxisome proliferator-activated receptor response element-luciferase assay in response to both 9-cis-RA and PPARγ-specific ligands. This was reinforced by the higher propensity of PELP1-overexpressing clones to undergo differentiation in response to PPARγ-specific ligands. This study has revealed a novel facet of PELP1 functions and identified it to be an important potentiator of the antiproliferative effects of 9-cis-RA and PPARγ-specific ligands. Retinoid X receptor α (RXRα), functioning as either a homodimer or a heterodimer with peroxisome proliferator receptors, is known to be involved in manifesting antiproliferative effects in cells. Consequently, studies of RXRα functions and its coregulators have been in the focus for therapeutic approaches against cancer. Here we have discovered that 9-cis-retinoic acid (9-cis-RA), a RXRα-specific ligand, up-regulated the expression of transcriptional coregulatory protein PELP1 (proline-, glutamic acid-, and leucine-rich protein 1). PELP1 functioned as a coactivator of RXRα, increasing its transactivation function in response to 9-cis-RA as evident by the retinoid X receptor response element-luciferase assays. PELP1 was found to be a binding partner of RXRα, and the binding interactions were confirmed both in vitro and in vivo. An electrophoretic mobility shift assay showed greater formation and stability of RXRα homodimers on consensus oligonucleotides in PELP1-overexpressing clones in comparison to the pcDNA clones. The presence of PELP1 in these oligonucleotide-bound RXRα homodimers was proved by the supershift of the complex when incubated with PELP1-specific antibody. PELP1-overexpressing stable MCF-7 cells exhibited a significantly higher extent of 9-cis-RA-induced apoptosis than the control pcDNA clones. Silencing of PELP1 expression in parental MCF-7 cells and PELP1-overexpressing clones using PELP1-specific RNA-mediated interference compromised the susceptibility to 9-cis-RA-induced apoptosis. PELP1 could also function as a coactivator of the RXRα-peroxisome proliferator-activated receptor (PPARγ) heterodimer as evident by the peroxisome proliferator-activated receptor response element-luciferase assay in response to both 9-cis-RA and PPARγ-specific ligands. This was reinforced by the higher propensity of PELP1-overexpressing clones to undergo differentiation in response to PPARγ-specific ligands. This study has revealed a novel facet of PELP1 functions and identified it to be an important potentiator of the antiproliferative effects of 9-cis-RA and PPARγ-specific ligands. Nuclear receptors (NR) 2The abbreviations used are: NR, nuclear receptor(s); PELP1, proline-, glutamic acid-, and leucine-rich protein 1; RXRα, retinoid X receptor α; PPARγ, peroxisome proliferator-activated receptor γ; 9-cis-RA, 9-cis-retinoic acid; RNAi, RNA-mediated interference; PIC, preinitiation complex; ER, endoplasmic reticulum; CBP, cAMP-response element-binding protein (CREB)-binding protein; GST, glutathione sulfur-transferase; RT, reverse transcription; CRBP II, cellular retinol-binding protein; EMSA, electrophoretic mobility shift assay; WT, wild type; RXRE, retinoid X receptor response element; PPRE, peroxisome proliferator-activated receptor response element. are ligand-activated transcription factors that on binding to small lipophilic signal molecules facilitate gene transcription and regulate diverse vital biological processes such as cell survival, proliferation, differentiation, and apoptosis (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6164) Google Scholar). On activation, the NR in their homodimeric or heterodimeric complexes with other NR bind to specific DNA sequences referred to as “response elements,” which are present in the regulatory elements of target genes. On DNA binding, the NR orchestrate gene transcription by interacting with the basal transcriptional machinery through bridging factors, referred to broadly as coactivator proteins. One of the potential functions of the coactivator proteins is to directly or indirectly remodel the local chromatin structure through covalent modification of histones (acetylation, phosphorylation, or methylation) resulting in opening of chromatin, greater accessibility of the target gene promoter to transcriptional machinery, and ultimately, gene transcription. Numerous families of coactivators of different NR have been identified. Their mode of interaction with the receptors and the molecular mechanism by which they function has been established. The most well studied coactivators include p300/CBP, p300/CBP-associated factor, and members of the p160 families, which include SRC-1, GRIP1/TIF2/SRC-2, and ACTR/AIB1/RAC3/SRC-3 (for review, see Ref. 2Lonard D.M. O'Malley B.W. Trends Biochem. Sci. 2005; 30: 126-132Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). These coactivators function by facilitating the initial penetration of the chromatin by histone acetylation. Coactivators, which utilize histone methylation for the same purpose like CARM1 and PRMT, have also been identified (3Stallcup M.R. Oncogene. 2001; 20: 3014-3020Crossref PubMed Scopus (156) Google Scholar). In addition to these families, several other coactivator proteins have been identified that function directly at the level of the preinitiation complex (PIC), enhancing its activity. This group includes TATA box-binding protein-associated factors (4Verrijzer C.P. Tjian R. Biochem. Sci. 1996; 21: 338-342Crossref PubMed Scopus (319) Google Scholar), positive cofactors PC1, PC2, PC3, PC5, and PC52 (5Roeder R.G. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 201-218Crossref PubMed Scopus (145) Google Scholar), and several multiprotein complexes that are related to thyroid hormone receptor-associated protein-mediator complex (6Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) was a recent addition to the family of transcriptional coregulatory proteins and was identified as a novel estrogen receptor (ER) coactivator (7Vadlamudi R.K. Wang R.-A. Mazumdar A. Kim Y. Shin J. Sahin A. Kumar R. J. Biol. Chem. 2001; 276: 38272-38279Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). It is a protein of 1273 amino acids that is unusually rich in prolines (13.2%), glutamic acid (12.4%), and leucine (12.9%) and, hence, was named as PELP1. It has nine LXXLL motifs (NR box motifs), seven toward the N-terminal region, and two in the central region. It interacts with and significantly enhances the transcriptional function of ERα, indicating that it functions as an ER coactivator. PELP1 did not have the same effect on progesterone receptor and glucocorticoid receptor, indicating that it may be an ER-specific coactivator. Even though it has a molecular mass of 160 kDa, sequence analysis showed it was distinct from the p160 family of coactivators. The conserved domains of p160 proteins such as bHLH, PER, ARNT, and SIM homology domains are absent in PELP1. It could interact with transcriptional activators like CBP and p300 in vivo, which suggest that PELP1 activates ER transcription by recruiting general coactivators such as CBP and p300. PELP1 also interacts with pRb (retinoblastoma protein), a cell cycle switch protein, inducing its hyperphosphorylation in an estrogen-dependent manner, increasing estrogen-stimulated cell proliferation (8Balasenthil S. Vadlamudi R.K. J. Biol. Chem. 2003; 276: 22119-22127Abstract Full Text Full Text PDF Scopus (77) Google Scholar). Upon stimulation of cells with estrogen, there was an enhanced recruitment of PELP1 to the estrogen-responsive promoters and colocalization with the acetylated histone H3. Increased levels of PELP1-associated acetyltransferase activity were observed on estrogen stimulation. PELP1 also interacts with histones H1 and H3 and increases transcription by chromatin modification involving the displacement of H1 (9Nair S.S. Mishra S.K. Yang Z. Balasenthil S. Kumar R. Vadlamudi R.K. Cancer Res. 2000; 64: 6416-6423Crossref Scopus (85) Google Scholar). In another study, PELP1 was confirmed to act as an ER coactivator but as a corepressor of glucocorticoid receptor and of non-NR sequence-specific transcription factors tested, including activating protein 1, nuclear factor κB (NF-κB), and ternary complex factor/serum response factor (10Choi Y.B. Ko J.K. Shin J. J. Biol. Chem. 2004; 279: 50930-50941Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The noted repressor activity of PELP1 was due to its ability to recruit HDAC2, which in turn might mask the acetylation of histones H3 and H4 and prevent them from acting as substrates for histone acetyltransferases. Ligand binding to the ER reverses the repressor role of PELP1, with a parallel increase in the status of histone hyper-acetylation. More recently it was also shown that PELP1 functions as a coactivator of signal transducers and activator 3 (STAT3), stimulating its transcriptional activity (11Manavathi B. Nair S.S. Wang R.-A. Kumar R. Vadlamudi R.K. Cancer Res. 2005; 65: 5571-5577Crossref PubMed Scopus (51) Google Scholar). This positive regulation is brought about by the ability of PELP1 to augment growth factor-induced phosphorylation of serine 727 of STAT3 via activation of the Src-mitogen-activated protein kinase pathway. Collectively, emerging evidence suggests that PELP1 can function as a positive and a negative regulator of transcription in a transcription factor-specific manner. Retinoids (natural retinoic acids and their synthetic derivatives) are derivatives of vitamin A. They are non-steroidal hormones that play a vital role in the development and homeostasis of almost every vertebrate tissue by regulating embryogenesis, cell differentiation, proliferation, and apoptosis (12Altucci L. Gronemeyer H. Trends Endocrinol. Metab. 2001; 12: 460-468Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 13Ross S.A. McCaffery P.J. Drager U.C. De Luca L.M. Physiol. Rev. 2000; 80: 1021-1054Crossref PubMed Scopus (756) Google Scholar). Retinoids transduce their signals through two well studied classes of receptors namely, retinoic acid receptor (RAR) and retinoid X receptor (RXR). Three isotypes in each class of the retinoid receptors have been identified and are referred to as α, β, and γ RARs (or RXRs), and each isotype is encoded by a separate gene (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6164) Google Scholar, 14Chambon P. FASEB J. 1996; 10: 940-954Crossref PubMed Scopus (2621) Google Scholar). RXRs represent a unique class of NR as they function as obligate members of majority of NR heterodimers reported. They heterodimerize not only with RARs but also with several other receptors such as thyroid hormone receptors, vitamin D receptors (15Kliewer S.A. Umesono K. Mangelsdorf D.J. Evans R.M. Nature. 1992; 355: 446-449Crossref PubMed Scopus (1268) Google Scholar), and peroxisome-proliferator-activated receptors (PPARs) (16Bardot O. Aldridge T.C. Latruffe N. Green S. Biochem. Biophys. Res. Commun. 1993; 192: 37-45Crossref PubMed Scopus (236) Google Scholar). The RXRs bind with stereo-selectivity to the retinoid 9-cis-retinoic acid (9-cis-RA). In addition to forming heterodimers, RXRs bind to 9-cis-RA and form functionally active homodimers that bind to the target DNA sequences (RXRE) and activate gene transcription (17Sarraf P Mueller E Jones D. King F.J. DeAngelo D.J. Partridge J.B. Holden S.A. Chen L.B. Singer S. Fletcher C. Spiegelman B.M. Nat. Med. 1998; 4: 1046-1052Crossref PubMed Scopus (936) Google Scholar). In the present investigation we discovered that 9-cis-RA transcriptionally up-regulates expression of the PELP1 gene. PELP1, in turn physically binds with RXRα and functions as a coactivator, activating the transcriptional functions of RXRα homodimer and also of its permissive heterodimer with PPARγ. Consequently, PELP1 potentiates the apoptotic effects of 9-cis-RA and the differentiation-inducing effects of PPARγ-activating ligands. Cell Culture and Reagents—MCF-7 cells were purchased from American Tissue Culture Collection (ATCC) and were maintained in Dulbecco's modified Eagle's medium and F-12 medium (1:1) (Mediatech, Herndon, VA) supplemented with 10% fetal calf serum. Antibodies for RXRα and PPARγ were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Her2 antibody was from Labvision Corp (Fremont, CA). Anti-T7 tag antibody was from Bethyl Laboratories (Montgomery, TX), and anti-vinculin antibody was from Sigma. Anti-PELP1antibody was used as reported earlier (7Vadlamudi R.K. Wang R.-A. Mazumdar A. Kim Y. Shin J. Sahin A. Kumar R. J. Biol. Chem. 2001; 276: 38272-38279Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Anti-mouse and anti-rabbit horseradish peroxidase or alkaline phosphatase-conjugated antibodies were from Amersham Biosciences. 9-cis-Retinoic acid, ciglitazone, and troglitazone were from Biomol (Plymouth Meeting, PA). The making and characterization of pcDNA, T7-PELP1, and T7-PELP1 H1-mutant protein-overexpressing cell lines were described earlier (9Nair S.S. Mishra S.K. Yang Z. Balasenthil S. Kumar R. Vadlamudi R.K. Cancer Res. 2000; 64: 6416-6423Crossref Scopus (85) Google Scholar). These cells were cultured in RPMI medium (Mediatech) with 5% fetal calf serum and G418 (1 mg/ml). Reporter Luciferase Assays—Cells were seeded in 6-well tissue culture plates 24 h before transfection. Subconfluent cells were transfected with PPRE-luciferase (17Sarraf P Mueller E Jones D. King F.J. DeAngelo D.J. Partridge J.B. Holden S.A. Chen L.B. Singer S. Fletcher C. Spiegelman B.M. Nat. Med. 1998; 4: 1046-1052Crossref PubMed Scopus (936) Google Scholar) or RXRE-tk-luciferase (18Tanaka T. Dancheck B.L. Trifiletti L.C. Birnkrant R.E. Taylor B.J. Garfield S.H. Thorgeirsson U. De Luca L.M. Mol. Cell. Biol. 2004; 24: 3972-3982Crossref PubMed Scopus (32) Google Scholar) using FuGENE 6 transfection reagent (Roche Applied Science). After 24 h the cells were treated with RXR- or PPAR-specific ligands as appropriate. Luciferase assay was done after 24 h using the luciferase assay kit (Promega). Each experiment was repeated a minimum of three times. The PELP1 promoter luciferase constructs were used as reported earlier (19Mishra S.K. Balasenthil S. Nguyen D. Vadlamudi R.K. Gene (Amst.). 2004; 330: 115-122Crossref PubMed Scopus (32) Google Scholar). Glutathione S-transferase (GST) Pulldown Assay—In vitro transcription and translation of RXRα was done using a T7-TNT kit (Promega), where 1 μg of cDNA in pcDNA 3.1 vector was translated in the presence of [35S]methionine in a reaction volume of 50 μl. The reaction mixture was diluted to 1 ml with Nonidet P-40 lysis buffer (25 mm Tris, 50 mm NaCl, and 1% Nonidet P-40). An equal aliquot was used for each GST pulldown assay. Translation and product size were verified by subjecting 2 μl of the reaction mixture to SDS-PAGE and autoradiography. The GST pulldown assays were performed by incubating equal amounts of GST and GST-tagged PELP1 deletion constructs, immobilized on glutathione-Sepharose beads (Amersham Biosciences) with in vitro translated 35S-labeled RXRα to which binding was being tested. Bound proteins were isolated by incubating the mixture for 3 h at 4 °C, washing 5 times with Nonidet P-40 lysis buffer, eluting the proteins with 2× SDS buffer, and separating them by SDS-PAGE. The bound proteins were then visualized by autoradiography. RT-PCR Analysis—Total RNA from the cells was extracted using TRIzol reagent (Invitrogen) and treated with DNase for 15 min after which DNase was inactivated by heating the samples at 65 °C for 10 min. The cellular retinol-binding protein (CRBP II) mRNA levels were analyzed by RT-PCR with specific primers 5′-cactagcacattccgcaact-3′ and 5′-aggtcagctccaggtacagc-3′. Primers used for acyl-CoA synthase were 5′-ttatcgaccggaaaaagcac-3′ and 5′-gtcagaaggccattgtcgat-3′. Immunofluorescence and Confocal Microscopy—Cells grown on glass coverslips were fixed in 4% phosphate-buffered paraformaldehyde for 15 min. Cells were permeabilized in methanol at –20 °C for 4 min. After permeabilization, cells were incubated with primary antibodies for 2 h at room temperature, washed 3 times in phosphate-buffered saline, and then incubated with secondary antibodies conjugated with 546-Alexa (red) or 488-Alexa (green) from Molecular Probes (Invitrogen). The DNA dye Topro-3 (Molecular Probes) was used for nuclear localization (blue). Confocal scanning analysis was performed using an Olympus FV300 laser scanning confocal microscope in accordance with established methods, utilizing sequential laser excitation to minimize the possibility of fluorescence emission bleed-through. Each image is a three-dimensional reconstructed stack of serial Z sections at the same cellular level and magnification. Co-localization of two proteins is shown yellow for red and green fluorescence. Lipid Droplet Staining with Oil Red O—Cells were seeded into 6-well plates containing a glass coverslip in each well and cultured for 5 days in the absence or presence of 5 μm troglitazone. Subsequently, cells were fixed in 3.7% formaldehyde and washed with phosphate-buffered saline for 10 min at room temperature and later stained with 1% oil red O for 10 min. Excess stain was washed with 60% isopropanol followed by 1 water wash. Cells were later stained with hematoxylene for 1–2 min followed by 1 wash with water. RNAi Transfection—PELP1 RNAi was a Smartpool mix purchased from Dharmacon (Lafayette, CO). RNAi transfection was performed using Oligofectamine (Invitrogen) according to the manufacturer's protocol. Cell Death and Cell Cycle Progression Assay—For the cell proliferation assay, 10,000 cells were plated/well in a 6-well culture plate. After 24 h cells were treated with 9-cis-RA (2.5 μm). 5 days later cell numbers were measured by using Beckman Coulter Counter. A comparison of the cell numbers of untreated cells to those treated with 9-cis-RA provided a measure of the extent of cell death. For cell cycle progression assay, cells were treated with 9-cis-RA (2.5 μm) and after 3 days stained with propidium iodide and analyzed by fluorescence-assisted cell sorting (BD Biosciences). Early apoptosis was detected by using an annexin V-apoptosis detection kit (Sigma) after cells were treated with 9-cis-RA (2.5 μm) for 16 h. Electrophoretic Mobility Shift Assay (EMSA)—EMSA analysis with radiolabeled probe was done with commercial double-stranded RXRE consensus sequence probes purchased from Santa Cruz Biotechnology. The probes were end-labeled using T4-polynucleotide kinase (Invitrogen) and 32P-labeled ATP (PerkinElmer Life Sciences). 9-cis-Retinoic Acid Transcriptionally Up-regulates PELP1 Expression—The promoter region of PELP1 has been cloned and characterized with regard to the estrogen-induced transcriptional up-regulation (19Mishra S.K. Balasenthil S. Nguyen D. Vadlamudi R.K. Gene (Amst.). 2004; 330: 115-122Crossref PubMed Scopus (32) Google Scholar). Analysis of the 2-kilobase PELP1 promoter sequence for putative transcription factor binding elements employing the program Matinspector Professional Version 7.2 (20Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2432) Google Scholar) showed at least 15 possible RXRα heterodimer binding sites. This prompted us to check whether 9-cis-RA, an RXR-specific ligand, would transcriptionally up-regulate PELP1 expression. Treatment of MCF-7 cells with 9-cis-RA (2.5 μm) led to enhancement of the PELP1 mRNA expression with in 6 h of treatment as evident by the RT-PCR analysis (Fig. 1A). Treatment of MCF-7 cells with 9-cis-RA for 24 h and subsequent Western blotting analysis of the total cell proteins with PELP1-specific antibody showed a clear increase in the level of PELP1 protein when compared with the control cells treated with Me2SO in a dose-dependent manner (Fig. 1B, upper panel). A similar up-regulation of PELP1 was observed in HeLa cells on treatment with 9-cis-RA (Fig. 1B, lower panel). Simultaneous treatment of cells with 9-cis-RA and actinomycin D, an inhibitor of transcription, blocked this up-regulation of PELP1 expression (Fig. 1C). On the other hand, similar treatment of cells with cyclohexamide, a protein synthesis inhibitor, did not affect the 9-cis-RA-induced up-regulation of PELP1 expression (Fig. 1C). This suggests that the increase in the level of PELP1 induced by 9-cis-RA was at the transcriptional level. To further validate the transcription stimulating effect of 9-cis-RA on the PELP1 promoter and identify the possible 9-cis-RA-responsive region, we next carried out luc-reporter assays with PELP1 promoter and its deletion luciferase constructs (Fig. 1D). In response to 9-cis-RA treatment, a 2-fold increase in the luciferase activity with construct A, which has the complete 2-kilobase PELP1 promoter, was observed. The activity of PELP1 promoter construct C, encompassing the 700-bp region upstream of the putative start site, exhibited more than a 3-fold induction of luciferase activity. This region has four putative RXRα binding sites including one RXR/LXR, one PXR/RXR half-site, and two CAR/RXR binding consensus sequence, suggesting that this region might be responsible for the responsiveness of 9-cis-RA. Together, these results presented in Fig. 1 established that the PELP1 gene is a target of 9-cis-RA signaling. PELP1 Functions as a Coactivator of RXRα—In addition to serving as a coactivator of ERα, PELP1 is a broad-range coregulator of several transcription factors (7Vadlamudi R.K. Wang R.-A. Mazumdar A. Kim Y. Shin J. Sahin A. Kumar R. J. Biol. Chem. 2001; 276: 38272-38279Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 9Nair S.S. Mishra S.K. Yang Z. Balasenthil S. Kumar R. Vadlamudi R.K. Cancer Res. 2000; 64: 6416-6423Crossref Scopus (85) Google Scholar, 10Choi Y.B. Ko J.K. Shin J. J. Biol. Chem. 2004; 279: 50930-50941Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Manavathi B. Nair S.S. Wang R.-A. Kumar R. Vadlamudi R.K. Cancer Res. 2005; 65: 5571-5577Crossref PubMed Scopus (51) Google Scholar). Because 9-cis-RA up-regulates PELP1 expression (Fig. 1), we next examined the possibility of PELP1 in regulating retinoid signaling. To investigate this we examined the effect of PELP1 on the RXRE-tk-luc-reporter assay in MCF-7 cells with transient overexpression of PELP1. Stimulation of cells with 9-cis-RA resulted in about a 2.5-fold induction of reporter activity by PELP1 as compared with about a 1.3-fold stimulation by control pcDNA, suggesting that PELP1 acts as a coactivator of RXRα (Fig. 2A). To gain further insights into PELP1 regulation of the RXRα pathway, we next used MCF-7 cells that stably overexpress PELP1. Results showed that 9-cis-RA was a potent stimulator of RXRα transactivation activity in cells with overexpression of PELP1 (Fig. 2B). In addition to recruiting chromatin modifiers such as CBP/p300, the coactivator function of PELP1 also requires binding and displacement of histone H1, which binds to DNA flanking the nucleosome core and represses the basal transcriptional activity of a variety of DNA binding factors including NRs. To show the significance of the histone H1 binding region of PELP1, we also conducted the RXRE-luc assay in MCF-7 cells stably expressing PELP1 histone H1-binding mutant protein lacking the C-terminal 253 amino acid stretch and incapable of binding to histone H1 (reported in Nair et al. Ref. 9Nair S.S. Mishra S.K. Yang Z. Balasenthil S. Kumar R. Vadlamudi R.K. Cancer Res. 2000; 64: 6416-6423Crossref Scopus (85) Google Scholar). We found a significant reduction in the ability of PELP1-H1 mutant to transactivate RXRα, as there was only a 2-fold induction of RXRE-luc-reporter by 9-cis-RA in MCF-7/PELP1-H1 mutant cells as compared with a 4-fold induction in the case of MCF-7/PELP1 cells (Fig. 2B). These results confirmed that PELP1 could function as coactivator of RXRα and, thus, potentiate the effect of 9-cis-RA signaling in cells. The expression of T7-tagged PELP1-WT and PELP1 H1-mutant form in the respective stable clones has been shown (Fig. 2C, upper panel). The expression of similar levels of RXRα and PPARγ proteins in MCF-7/pcDNA, MCF-7/PELP1, and MCF-7/PELP-H1 mutant cells was demonstrated by the Western blot analysis (Fig. 2C, lower panel). This ensured that the differential up-regulation of RXRE-luc observed in cells was due primarily to up-regulation of a fully functional PELP1 and not to alteration of the levels of the RXRα itself. To show the physiological relevance of the role of PELP1 as RXRα coactivator, RT-PCR analysis was done in the stable clones for the expression of CRBP II, an RXRα target gene. In response to 9-cis-RA stimulation, the expression of CRBP II in PELP WT clones was severalfold higher when compared with the pcDNA clones (Fig. 2D), which provides important proof of the potentiation of the RXRα transactivation function by PELP1. PELP1 Physically Interacts with RXRα—Because PELP1 is a coactivator of RXRα, we next investigated whether PELP1 interacts with RXRα. Results from the GST pulldown assays, utilizing GST-tagged PELP1 deletion constructs and 35S-labeled RXRα, indicated that the N-terminal 1–400 amino acid region of PELP1, which has seven of the nine NR boxes in PELP1, has the highest binding affinity to RXRα (Fig. 3A, construct A). A comparatively lower binding affinity was also shown by PELP1 construct B, which encompasses the region of amino acids 400–600 from the N-terminal region. No binding was observed by the deletion constructs C and D (Fig. 3A). The NR boxes are structural motifs that form an amphipathic helix employed by the coactivators to dock with the ligand activated NRs. Results from the above in vitro binding studies suggested that PELP1 may utilize its NR box-rich region to physically interact with RXRα. To validate the noted interaction of PELP1 with RXRα in the physiologic setting, we next performed co-immunoprecipitation followed by Western blot analysis from cellular extracts. Because the PELP1 antibody does not work well in immunoprecipitation assays, we used MCF-7/PELP1 cells, which allowed us to immunoprecipitate the T7-PELP1 by anti-T7 antibody. MCF-7/PELP1 and control cells were stimulated with 9-cis-RA or with the vehicle Me2SO, T7-PELP1 was immunoprecipitated from the cell lysates using an anti-T7-antibody, and immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with RXRα antibody. We found that RXRα indeed interacted with T7-PELP1, and such interaction was further enhanced by 9-cis-RA stimulation (Fig. 3B). Conversely, we immunoprecipitated the cell lysates with anti-RXRα antibody and showed co-precipitation of PELP1 (Fig. 3C). No PELP1 was detected in the lane where proteins were immunoprecipitated with control IgG. As an additional validation of in vivo PELP1-RXRα interaction, we used scanning confocal microscopy and co-localization of PELP1, and RXRα was visualized by immunofluorescence. Localization of PELP1 and RXRα was detected by red and green fluorescence, respectively. The majority of PELP1 was localized in the nucleus along with RXRα, and their co-localization was evident by yellow spots due to the overlapping of the two colors (Fig. 3D). To demonstrate the binding of PELP1 to RXRα homodimers bound to their consensus DNA sequences, EMSA was done with nuclear extract from pcDNA and PELP1 WT clones treated with 9-cis-RA using labeled synthetic double-stranded oligos with RXRE sequence (Fig. 3E). Incubation with the nuclear extracts resulted in the appearance of shifted bands, which represented the homodimer bound to the oligos. Increased binding of RXRα to the oligos was evident in both pcDNA and PELP WT clones on 9-cis-RA treatment (Fig. 3E, lanes 3 and 5, in comparison to lanes 2 and 4, respectively). Relatively, there was a higher leve