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J. Biol. Chem., Vol. 281, Issue 26, 18246-18256, June 30, 2006
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**1
From the
Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales 2031, Australia, the Department of Medicine, University of Sydney, New South Wales 2006, Australia, ¶Bioanalytical Mass Spectrometry Facility, University of New South Wales, New South Wales 2031, Australia, the ||Department of Cancer, Berlex Biosciences, Richmond, California 94804, the **Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Randwick, New South Wales 2031, Australia
Received for publication, January 30, 2006 , and in revised form, April 3, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(RAR) gene transcription. The RA response element (RARE) is the essential DNA sequence required for retinoid-induced RAR transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a RARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RAR transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RAR expression. EBBP increased RARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, , and 
subtypes). The ligand-receptor complex initiates the retinoid signal by changes that occur at consensus DNA sequences or RA response elements (RAREs) in the regulatory 5'-untranslated sequence of so-called "target" genes.
RAR is a retinoid target with a well defined RARE, which is necessary but not sufficient for the RA anti-cancer signal (4). Repression of basal or RA-induced RAR transcription is a common event in a wide range of human tumor types, and numerous studies indicate that in specific circumstances RAR acts as a tumor suppressor gene (2, 5-8). A frequent mechanism of RAR repression is DNA methylation-induced silencing (9). Additionally, histone deacetylation is also associated with retinoid resistance, in the presence and absence of RAR2 hypermethylation, indicating that multiple mechanisms appear to contribute to RAR repression (10).
Intrinsic and acquired retinoid resistance has limited the clinical activity of retinoid-based therapy and chemoprevention. RA treatment in acute promyelocytic leukemia has been particularly valuable in illuminating mechanisms of retinoid resistance. Most acute promyelocytic leukemia cases present with the reciprocal t(15,17) translocation, resulting in the fusion product PML-RAR (11). Dominant negative effects of the PML-RAR fusion protein have been associated with increased affinity for co-repressor proteins in acute promyelocytic leukemia cells. The signaling is constitutively repressed by co-repressor-associated histone deacetylase activity at physiologic RA levels. Co-repressor association with PML-RAR is disrupted, following recruitment of co-activator complexes and differentiation induced at pharmacologic RA dosages (12-14). PML protein localizes to discrete nuclear domains termed PML bodies, which represent one of many subnuclear domains in the eukaryotic cell nucleus (15, 16). Treatment of acute promyelocytic leukemia cells in culture with RA correlates with degradation of the PML-RAR fusion protein, allowing wild-type PML protein to dimerize or multimerize in PML bodies (17).
In common with other hormone receptor proteins, RARs can act as either transcriptional repressors or activators (18). This dual mode of regulation is dependent on the ability of RARs to associate with either co-activator or co-repressor proteins. Retinoid ligand binding to nuclear retinoid receptors induces a conformational change in these proteins, already bound at target gene promoter sequences, leading to new protein-protein interactions. One of the key factors determining the transcriptional and cellular response to RA is the dynamic change in co-regulator protein binding. These interactions are determined by the nuclear location, the post-translational modification, and the expression level of co-regulator proteins (19-21).
Acetylation, methylation, or phosphorylations of specific residues in histone tails, mediated by multiprotein complexes, control the activity of nuclear receptors. Most nuclear hormone receptors are phosphoproteins, and phosphorylation is a potent regulator of nuclear receptor function. Depending on the receptor, phosphorylation can regulate receptor DNA binding, dimerization, coactivator recruitment, and transactivation (22-26). These findings led us to hypothesize that the retinoid anti-cancer effect may be determined by other proteins that undergo post-translational modification following retinoid treatment of cancer cells, and, moreover, that such changes in co-regulator proteins may represent mechanisms for overcoming retinoid resistance in cancer cells.
To identify novel regulators of retinoid signaling, we developed a technique for directly isolating RARE-binding proteins, using electromobility shift assays, two-dimensional polyacrylamide gel electrophoresis (2-DE), direct isolation of differentially displayed protein spots, and peptide microsequencing to better understand the dynamic changes in protein-protein and protein-DNA complexes at this focal point of the RA anti-cancer signal. Using this approach, we have identified the estrogen-responsive B box protein (EBBP) as a RARE-binding protein, which undergoes post-translational modification, affecting protein stability and expression level at PML bodies, following RA treatment of retinoid-sensitive cancer cells. Importantly, EBBP derepressed RA-responsive RAR transcription in retinoid-resistant cancer cells, suggesting a novel mechanism for RA-regulated gene transcription.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Decoy Experiments—RARE decoy (GAT CGG GTA GGG TTC ACC GAA AGT TCA CTC G) or scrambled (AGC TCT CTG TTC TCC TGG TGT TCT GAA GCT) oligonucleotides were annealed to their respective complementary sequence by heating to 80 °C for 5 min and then cooling down to room temperature. BE cells cultured in T25 flasks were transfected with either RARE decoy (5 µM) or scrambled (5 µM) oligonucleotides using 192 µg/ml of SuperFect (Qiagen) for 2 h. The medium was then replaced and supplemented with 10 µM aRA for 24 h. To test the efficacy of the oligonucleotide decoy at blocking responses through the RARE, BE neuroblastoma cells with low level constitutive RAR expression, as measured by a competitive reverse transcription (RT)-PCR (27), were used.
Plasmid Construction—pGL3-3xRARE was constructed by ligating a double-stranded RARE oligonucleotide into the plasmid pGL3.basic vector (Promega) at the SmaI multicloning site. The EBBP full-length cDNA (gift from Dr. Zajchowski) was subcloned into the pcDNA3.1(-) vector (Invitrogen) at the EcoRI multicloning site. The EBBP full-length and deleted EBBP cDNA mutant constructs were created using PCR fragments cloned into plasmid pcDNA3.1(-)/Myc.His (Invitrogen) at EcoRI and KpnI sites. The oligonucleotides used for the PCR assay were as follows: 5'-TTGGGGGTGACTGCTCCCTATGGTACCAATG-3' (
B1B2BoxF1) forward primer for mutant 1; 5'-AATGGAATTCATGAACACTGAAGACATCACCTTC-3' (B1B2BoxCoilF1) forward primer for mutant 2; 5'-AACTTAAGCTTGGTACCGAG-3' (EBBPF1) forward primer for the full-length and mutants 3 and 4; 5'-CATTGGTACCATAGGGAGCAGTCACCCCCAA-3' (EBBPm/h R1) reverse primer for full-length, mutant 1, and mutant 2; 5'-CATTGGTACCATACGCATATTGGAGGAACTG-3' (RFPm/hR1) reverse primer for mutant 3; and 5'-CATTGGTACCATACCGCTCCAAGTCTAACTG-3' (CoilRFPm/hR2) reverse primer for mutant 4.
Isolation of Candidate RARE-associated Proteins—Nuclear proteins were isolated from BE cells untreated or treated with 10 µM aRA for 24 or 48 h. Proteins were then combined with a radiolabeled RARE, and an electrophoretic mobility shift assay was performed as described previously (6). Following an electrophoretic mobility shift assay, the polyacrylamide gels were transferred onto 3 MM blotting paper (Whatman) and dried on a slab gel drier prior to autoradiography. Protein-RARE complexes were excised directly from the gels and then passively eluted from the DNA-protein complexes (passive elution buffer: 100 mM sodium acetate, 0.1% SDS, and 10 mM dithiothreitol). The proteins were then separated using 2-DE, which was performed according to the standard protocols (28). All gels were run in triplicate, and then comparisons were made between aRA-treated and untreated gels. Differentially bound protein spots were excised and sent for mass spectroscopy and amino acid analysis (Australian Proteome Analysis Facility, Macquarie University, Sydney). The samples underwent an in-gel 16-h tryptic digest at 37 °C, and the resulting peptides were extracted from the gel, placed in microtiter plates, and subjected to matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) (29).
Transient Transfection and Reporter Gene Analysis—Cells (2.3 x 105) were seeded in a 6-well culture plates. Transient transfection assays were carried out using the SuperFect Transfection Reagent, according to the manufacturer's instructions. The cells were co-transfected with 2 µg of the pGL3-3xRARE luciferase (firefly luciferase) reporter plasmid, 2 µg of EBBP expression vector, and 0.75 µg of the pRL-TK vector (Renilla luciferase) as an internal control for the transfection efficiency. After 48 h, the treated cells were washed with phosphate-buffered saline (PBS), harvested by scraping in 250 µl of passive lysis buffer (Promega), and lysed by freezing/thawing two times. Lysate samples were cleared by centrifugation for 30 s at 4 °C. The supernatants were used for measuring both firefly and Renilla luciferase activities with the Dual-Luciferase Reporter Assay System (Promega) using a luminometer (TD-20/20; Turner Designs, CA). Luciferase reagent substrate (100 µl) was mixed with cell lysates (20 µl), and then initiated the Renilla luciferase reaction. Stop & GloR solution (100 µl) was added to the mixture, and Renilla luciferase activity was measured. The ratio between firefly and Renilla luciferase activities was referred to as relative luciferase activity.
Analysis of Gene Expression by RT-PCR—RAR and EBBP mRNA expression levels in neuroblastoma cells were assayed using a competitive semiquantitative RT-PCR technique, which we have previously described (30). The RT-PCR involved determining a ratio between the level of expression of the RAR and EBBP genes and that of the control 2-microglobulin (2M) gene in total RNA samples. The RT-PCR primer sequences for 2M have been described (31). The gene-specific RAR2 forward PCR primer was 5'-CTACACTGCGAGTCCGTCTT-3', and the reverse PCR primer was 5'-CAGAGCTGGTGCTCTGTGTT-3'. This primer pair generated a 131-bp product extending from bp 375 to 506 of the published RAR2 sequence (4). The gene-specific EBBP forward PCR primer was 5'-CAGGCTCCAGGCTAACCAAAAG-3', and reverse PCR primer was 5'-TCCTCTAAGAAGGGCATCACATTG-3'. This primer pair generated a 131-bp PCR product extending from bp 812 to 943 of the published EBBP coding sequence (32). In order to maximize RT-PCR efficiency in RA-resistant SK-MES-1 cells, the FailSafe PCR System (Epicenter Biotechnologies) was used. This involved using the FailSafe PCR enzyme mix and a specific PCR C buffer.
Quantitative Real Time PCR—RAR mRNA expression levels in lung and breast cancer cells were assayed using a quantitative real time PCR technique. Total RNA was isolated from cell lines using RNeasy Mini Kit (Qiagen). For each RT-PCR, 2 µg of total RNA was converted into cDNA using Moloney murine leukemia virus reverse transcriptase (Promega). Two microliters of reverse transcription reaction was then quantified by quantitative real time PCR using PRISM 7700 Sequence Detection System and ABI TaqMan® PCR Master Mix (Applied Biosystems). The quantitative results of RAR mRNA were normalized to 2M mRNA from the same sample. Primers were synthesized at Invitrogen, and probe was purchased from Applied Biosystems. Primers used for RAR were as follows: forward primer, 5'-tgaaaatcacagatctccgtagca-3' and reverse primer, 5'-ccaggaatttccattttcaaggt-3'. The TaqMan® probe used for RAR was as follows: 5'-6-carboxyfluorescein-ctctgcacctttagcactg-3' molecular groove-binding non-fluorescence quencher. Primers used for 2M were as follows: forward primer, 5'-actggtctttctatctcttgtactacactga-3'; reverse primer, 5'-tgatgctgcttacatgtctcgat-3'. The TaqMan® probe used for 2M was 5'-VIC-tgcctgccgtgtgaaccatgtgac-3' 6-carboxy-tetramethyl-rhodamin.
Immunofluorescence Staining for EBBP and Myc Tag Antibodies—Immunohistochemical staining for EBBP expression was carried out as follows. All procedures were performed at room temperature. BE cell cultures adherent to the substratum on the glass slide were rinsed in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) in wells. Cells were fixed and permeabilized by incubation in 500 µl of cold 4% paraformaldehyde in TBS for 20 min prior to incubation in 500 µl of 0.2% Triton X in TBS in wells for 10 min. After washing in TBS, cultures were incubated in 100 µl of blocking buffer 1 (1% fetal bovine serum, 1% bovine serum albumin in TBS) and then incubated in 100 µl of polyclonal rabbit anti-human EBBP antibody diluted 1:30 in TBS or monoclonal mouse anti-Myc tag antibody diluted 1:500 in 5% fetal calf serum, PBS for 1 h. After washes in TBS, cells were incubated in 100 µl of secondary antiserum, goat anti-rabbit fluorescein isothiocyanate diluted 1:150 in TBS for 1 h or Cy3 anti-mouse secondary antibody diluted 1:1000 in 5% fetal calf serum, PBS buffer for 40 min. After washing, slides were mounted in fluorescence anti-fade solution and sealed with clear nail polish.
Confocal Scanning Fluorescence Microscopy Images of EBBP and PML Body Immunostaining—Cells were fixed in 10% formalin for 20 min, followed by permeabilization with 0.2% Triton X for 10 min. Cells were then blocked in 1% fetal bovine serum and 1% bovine serum albumin in PBS for 1 h and then incubated in rabbit anti-EBBP (1:30) for 1 h, followed by anti-rabbit fluorescein isothiocyanate (1:100) for 1 h. Cells were then reblocked for 10 min in 10% bovine serum albumin in PBS before incubation in mouse anti-PML (1:10) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h and then incubated in biotinylated anti-mouse IgG (1:100) for 1 h and finally labeled with streptavidin Alexa 594. Coverslips were then mounted on slides in Vectashield antifade solution and sealed with nail polish ready for fluorescence microscopy.
Confocal scanning fluorescence microscopy was performed on a TCS SP2 System (Leica) fitted with a HCxPL APO x63/1.20 W CORR objective. Fluorochromes fluorescein isothiocyanate and Alexa 594 were excited with the 488-nM argon laser and the 530-nm helium/neon laser, respectively. Emission fluorescence was collected in red (>580 nM) and green (500-560 nM) channels sequentially. Images were collected and processed using the imaging software provided by the Leica TCS NT system. Images were randomly taken from at least three different fields of view, and the immunofluorescence was measured from 6-8 cells/treatment. Nuclear areas were considered EBBP-positive if they displayed a fluorescence of over 1501 (Arbitrary Fluorescence Unit).
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To determine the EBBP protein stability, BE cells were treated with or without cycloheximide (3.35 µM) and aRA (10 µM). At the indicated time points, cell cultures were harvested, and nuclear extracts were prepared using an NE-PER nuclear and cytoplasmic extraction reagents kit (Pierce). Equal amounts of protein (50 µg) from each sample were analyzed by Western blotting using an anti-EBBP or anti-RAR antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:400 dilution. The relative amounts of EBBP and RAR proteins were quantified using a Bio-Rad Quantity One program. Identical blots were analyzed by Western analysis using anti-actin antibodies as the loading control.
Bio-Plex Phosphoprotein Assays—For analysis of phosphoproteins, neuroblastoma cells were treated with 10 µM aRA for 0, 3, 6, 9, and 24 h on 24-well plates. The cell lysates were prepared using the Bio-Plex cell lysis kit (Bio-Rad) according to the manufacturer's instructions. Protein recovery was determined with a bicinchoninic acid protein assay (Pierce). Protein extracts were aliquoted and stored at -80 °C prior to analysis. Bio-Plex CooH (carboxylated) beads 45 and the Bio-PlexTM amine coupling kit were purchased from Bio-Rad. Covalent coupling of the captured EBBP antibody to polystyrene beads was performed using the manufacturer's instructions. The beads were counted with a hemacytometer and stored at a final concentration of 4 x 106 beads/ml in the dark at 4 °C. Coupling efficiency of EBBP polyclonal antibody was tested by staining 10,000 beads with a phycoerythrin-conjugated goat anti-rabbit immunoglobulin G (Southern Biotechnology Associates, Birmingham, AL). Functionality of the coupling was determined by incubating 2,000 beads with 10 µg of protein in assay buffer 2 (Upstate Biotechnology) and shaking overnight at 4 °C and then incubating protein samples with anti-phospho-Ser/Thr or anti-phosphotyrosine mouse monoclonal IgGs (Upstate Biotechnology) for 1 h, followed by Beadlyte biotinylated goat anti-mouse IgG antibody (Upstate Biotechnology) for 30 min. After incubation with BeadlyteTM streptavidin-phycoerythrin (Upstate Biotechnology) and washing with Upstate Biotechnology washing buffer, the beads were measured and analyzed using the Bio-Plex system (Bio-Rad) in combination with the Bio-Plex Manager software (33).
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Small Interfering RNA (siRNA)—siRNA for EBBP (ID 3573) and negative control siRNA (catalogue number) were purchased directly from Ambion as double-stranded EBBP predesigned siRNA. The sequences of the siRNA were targeted EBBP exon 6; the sequences were patented and cannot be published according to Ambion. BE neuroblastoma cells at 1 x 10 ml were simultaneously plated and transfected with 2 µl/ml Lipofectamine (Invitrogen), which mixed with 10 nM siRNA in serum-free Dulbecco's modified Eagle's medium. At 8 h post-transfection, the medium volume was doubled with 20% fetal calf serum, Dulbecco's modified Eagle's medium. The cells were then treated with 10 µM aRA or ethanol solvent control at 24 h and were harvested for RNA or protein at 72 h. Transfection efficiency and its effect on potential target gene expression was assessed by RT-PCR and/or immunoblot.
Chromatin Immunoprecipitation Assay (ChIP Assay)—ChIP analysis was performed with the ChIP kit (Upstate Biotechnology) according to the manufacturer's instructions with minor modifications. EBBP-specific antibody and control rabbit IgG antibody (Santa Cruz Biotechnology) were used in the experiments. Chromatin was immunoprecipitated from 2 x 106 BE cells transiently transfected with empty vector or pcDNA3.1(-)/EBBP construct. For PCR, the primers included the RAR P2 sense primer 5'-GCCGAGAACGCGAGCGATCC-3' and the RAR P2 antisense primer 5'-GGCCAATCCAGCCGGGGC-3' (6).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RARE-binding Proteins during RA-induced RAR Transcription—To provide evidence that the specific RARE in the RAR2 gene promoter was required for RA-induced RAR induction, we transiently transfected RA-sensitive human neuroblastoma BE cells with a double-stranded decoy oligonucleotide that corresponded to the RARE (4) to compete for and block RA-activated receptor complex binding at the RARE. Transfection of the RARE decoy oligonucleotide, but not the scrambled oligonucleotide, significantly inhibited RA-induced RAR mRNA expression (Fig. 1). We next performed an electrophoretic mobility shift assay with the RARE oligonucleotide and nuclear protein lysates from aRA-treated and -untreated BE cells at different time points following aRA treatment. Protein-DNA complexes were directly isolated from the polyacrylamide gel and then electrophoretically separated on 2-DE. Protein gels were compared for differential protein display over the 48 h of aRA treatment. Protein spots were excised directly from the gel, and microsequencing of tryptic peptides was determined by MALDI-MS analysis. Using this technique, we identified EBBP as a candidate RARE-associated protein of 67 kDa, which exhibited an alteration in pH from pH 5.4 to 6.0 during aRA treatment, suggesting post-translational modification (Fig. 2A). The peptide masses were searched against Swiss-Prot using PeptIdent (available on the World Wide Web at www.expasy.ch/tools/peptident.html) (Fig. 2C). To confirm these findings, we transiently transfected BE cells with an expression vector containing the full-length human EBBP cDNA, treated the cells with aRA, and performed a 2-DE immunoblot of nuclear protein lysates hybridized with an EBBP-specific antibody (Fig. 2B). A marked increase in EBBP protein expression levels and altered pH at 24 and 48 h after aRA treatment was observed.
EBBP was first identified in a screen for transcripts induced by estrogen in human mammary epithelial cells (32) and is a member of the RBCC (34) or tripartite motif (TRIM) (35) protein family, sharing the B box and coiled-coil domains with PML and transcriptional intermediary factor (TIF1) (Fig. 2D). When compared with PML and TIF1, EBBP is missing the NH2-terminal RING finger domain but has an additional COOH-terminal RFP or B30.2 domain of unknown function. This additional domain is shared with several other TRIM proteins, such as MID1 and PYRIN, which have been shown to be mutated in the human disorders Opitz syndrome and familial Mediterranean fever, respectively (36). Next we used the ChIP assay with EBBP and acetylated histone H4 antibodies to analyze in vivo the composition of proteins binding to the RARE of the RAR2 promoter in EBBP transfectants (Fig. 2E). Overexpression of EBBP caused EBBP binding and acetylation of histone H4 at the RARE of the RAR promoter when compared with empty vector transfectants.
RA Alters EBBP Protein Stability—In order to examine the mechanism of the aRA-induced increase in EBBP expression, we measured the level of EBBP mRNA expression in RA-responsive BE and SY neuroblastoma cells, treated with 10 µM aRA for 0 h, 1 h, 24 h, 3 days, and 7 days, using gene-specific PCR primers and control 2M primers in a semiquantitative RT-PCR. These cell lines undergo significant growth arrest, cell death, and neuritic differentiation following aRA treatment (37). RT-PCR analysis showed no change in EBBP mRNA level during aRA treatment (data not shown). However, EBBP protein levels increased in aRA-treated cells by more than 2-fold in immunoblotting experiments using an EBBP-specific antibody in both cell lines (Fig. 3A). To determine if aRA treatment affected EBBP protein stability, BE cells were treated with the protein synthesis inhibitor, cycloheximide, in conjunction with retinoid (Fig. 3B). The aRA-induced levels of EBBP protein expression increased in a similar manner in both the cycloheximide-treated and -untreated cells over 24 h, in contrast to aRA-induced expression of RAR, which required new protein synthesis (data not shown). Nuclear extracts from cells treated with cycloheximide only (absence of RA) were subjected to Western analysis, which showed no changes in EBBP protein level during the time course (Fig. 3C).
Post-translation Modification of EBBP—To investigate the nature of the RA-induced post-translational modification of EBBP, we examined the phosphorylation status of EBBP protein during 24 h of RA treatment. Protein lysates from BE cells treated with aRA were incubated with polystyrene beads that had been coupled to an EBBP-specific antibody. Samples were then incubated with either an anti-phospho-Ser/Thr or anti-phosphotyrosine anti-mouse antibody. Streptavidin-phycoerythrin was then added to bind to the biotinylated anti-mouse IgG antibody on the bead surface before being analyzed with a dual laser, flow-based microplate reader system (Bio-Plex suspension array system). This system identifies both antibodies and shows the level of phospho-EBBP protein in the well as fluorescence intensity using Bio-Plex Manager software (Fig. 3D). In contrast to untreated cells, EBBP protein phospho-Ser/Thr levels increased 5-fold at 6 and 9 h following aRA treatment, whereas EBBP phosphotyrosine levels were relatively unchanged.
Retinoid-induced EBBP Protein Co-localizes with PML to Nuclear Bodies—We next determined the pattern of subcellular localization of EBBP following RA treatment, using confocal scanning fluorescence microscopy in BE cells (Fig. 4). In untreated BE cells, EBBP staining was scattered throughout the nucleus. Following 10 µM aRA treatment, however, the nuclei of aRA-treated cells displayed an increase in the intensity of EBBP staining at 24 h, which had coalesced into a single, strongly stained region by 48 h (Fig. 4A). Quantitative analysis of EBBP-positive regions within the nuclei of untreated or aRA-treated cells demonstrated that the number of EBBP-positive regions increased within 2 h of RA treatment and peaked at 24 h before declining at 48 h (Fig. 4B). The strongly staining nuclear EBBP region seen at 48 h after aRA treatment resembled the large multiprotein complexes, known as PML nuclear bodies (15, 16). Accordingly, we examined whether EBBP and PML proteins co-localized to PML bodies. PML, like EBBP, had a scattered, nuclear distribution and was expressed in every cell. PML expression did not discernibly change in level or localization with aRA treatment (data not shown). Under confocal fluorescent microscopy, PML co-localized with EBBP in 50% of untreated or aRA-treated cells. However, at 48 h of aRA treatment, EBBP appeared to aggregate into large nuclear bodies and was co-localized with PML (Fig. 4C).
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Transcription and Transactivation through the RARE in Retinoid-sensitive Neuroblastoma Cells—To further define the role of the EBBP protein in determining retinoid sensitivity of neuroblastoma cells, we transiently transfected human EBBP cDNA into the cells. Transient overexpression of EBBP led to a significant decrease in viable BE cells numbers when compared with empty vector control (p < 0.05), as measured by trypan blue exclusion at 72 h after transfection. In the cell proliferation study, BrdUrd incorporation by BE cells was decreased by 50% in cells transfected with EBBP plasmid DNA, after 48 h of aRA treatment or without treatment, compared with empty vector control (p < 0.01) (Fig. 5A). The functional consequences of RA-induced EBBP expression were assessed by examining the effect of increased EBBP expression on endogenous RAR transcription and transactivation from the RARE. We transiently transfected full-length human EBBP cDNA and empty vector into retinoid-sensitive BE and SY neuroblastoma cells. Transfection of EBBP increased endogenous RAR mRNA expression following 10 µM aRA treatment in both neuroblastoma cell lines at 24 and 48 h of aRA treatment compared with empty vector control (Fig. 5B). We next evaluated whether an excess of EBBP would enhance the transactivating ability of a transiently transfected RARE-luciferase construct in the presence of aRA. Following 24 and 48 h of aRA treatment, both cell lines demonstrated a 1.5-2.0-fold increase in RARE-luciferase activity, when EBBP was co-transfected with RARE-luciferase, in comparison with the empty vector alone (Fig. 5C), suggesting that RA-induced EBBP expression has functional consequences for endogenous RAR gene expression.
Overexpressed EBBP Enhances RAR Transcription and Reduces Cell Proliferation in Retinoid-resistant Lung and Breast Cancer Cells—The effects of an excess of EBBP were examined in retinoid-resistant SK-MES-1, MDA-MB-231, and MDA-MB-468 cancer cells, which are known to be phenotypically resistant to aRA and do not induce RAR transcription following aRA treatment (38). To determine whether the repression of RAR expression in the aRA-resistant cancer cells was due to DNA methylation, these cancer cells were treated with the demethylation agent, 5-aza-2-deoxycytidine. Treatment with 0.1, 1.0, and 10 µM 5-aza-2-deoxycytidine in combination with 10 µM aRA for 3 days did not restore RAR expression in the aRA-resistant cells when analyzed by RT-PCR (data not shown). Since the level of histone acetylation can influence gene transcription, the effects of the histone deacetylase inhibitor, trichostatin A, on RAR expression in SK-MES-1, MDA-MB-231, and MDA-MB-468 were investigated. Cells were treated with 0.3 µM trichostatin A (39) in combination with 1 µM aRA for 24 h and 3 days and analyzed for RAR expression. RAR transcription was not detected in these cancer cells by RT-PCR (data not shown). However, when SKMES-1 MDA-MB-231 and MDA-MB-468 cells were transfected with an EBBP expression vector, endogenous RAR mRNA expression at 24 and 48 h of aRA treatment was restored (Fig. 6A). Furthermore, proliferation of these cancer cells was also significantly reduced by transient overexpression of the EBBP expression vector with aRA treatment for 48 h compared with empty vector control measured by BrdUrd incorporation (Fig. 6B). These results suggested that overexpression of EBBP in retinoid-resistant cancer cells may overcome repression of retinoid-induced transcription, which was resistant to both demethylation and histone acetylation.
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Gene Expression with Specific EBBP siRNA—Next we examined the hypothesis that knockdown of EBBP expression will affect RA-responsive RAR transcription. The efficiency of EBBP siRNA in reducing RAR was evaluated after 72 h of transfection (Fig. 7). The siRNA concentration was kept as low as 10 nM in order to eliminate the possibility of nonspecific effects. A very efficient decrease in EBBP mRNA expression was obtained, with only 40% remaining expression in untreated BE cells and 18% remaining in 48-h 10 µM aRA-treated BE cells (Fig. 7A). RAR mRNA expression was significantly decreased after transfection of 10 nM siRNA EBBP for 72 h in the presence of 10 µM aRA for 48 h and compared with control siRNA (p < 0.05) (Fig. 7B). As shown in Fig. 7C, the level of RAR protein was decreased by 40% in the absence or presence of aRA treatment in comparison with control siRNA.
The EBBP Coiled-coil Domain Is Necessary for the Effect of EBBP on RARE Activity—To determine which protein domain of EBBP was required for its effect on RARE activity, we created four EBBP deletion mutant constructs, where the mutant protein was expressed with both a Myc epitope and a His6 tag (Fig. 8). We used both immunofluorescent staining and immunoblot with Myc epitope antibodies to confirm that the mutant proteins were expressed in BE cells following transient transfection (Fig. 8, A and B). Without aRA treatment, the RARE-luciferase activity was increased in EBBP full-length, mutant 1, and mutant 3 constructs compared with empty vector control (p < 0.05). Following 48-h treatment with 10 µM aRA, only mutants 1 and 3, which each share the coiled-coil domain, retained the ability to enhance RARE activity to the same degree as full-length EBBP (Fig. 8C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and, may play a significant role in the retinoid signaling pathway. Our data indicate that RA treatment of retinoid-sensitive cells results in EBBP phosphorylation and elevated EBBP nuclear expression in aggregates with PML at PML nuclear bodies. Overexpression of EBBP increases the level of exogenous and endogenous RARE transactivation. Transfection of EBBP siRNA significantly decreased basal and RA-induced RAR gene expression in neuroblastoma cells. A ChIP assay showed that EBBP protein directly interacted with the RARE. Furthermore, overexpression of EBBP induced growth inhibition in RA-sensitive neuroblastoma cells as well as RA-resistant lung and breast cancer cells. Most importantly, EBBP restores RA-responsive RAR transcription in RA-resistant lung cancer cells, which were resistant to both a histone deacetylase inhibitor and demethylating agent. In addition, deletion mutant analysis localized the RARE transactivating function to the coiled-coil domain of EBBP, a region thought to be responsible for protein-protein interactions.
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Our screen for changes in RARE-associated proteins detected a 67-kDa protein whose isoelectric point (pI) changed from pH 5.4 to pH 6 during aRA treatment of RA-sensitive neuroblastoma cells. Since the calculated molecular mass is only 64 kDa, it seems likely that the protein is post-translationally modified and that this may vary dependent on the cellular context. Our results are consistent with previous data obtained with HepG2, COS-1, and HaCaTt cells (32, 40). Phosphorylation is one form of post-translational modification that can produce a more acidic protein isoform on 2-DE SDS-polyacrylamide gel and so could not explain the shift to a more alkaline pH, which we observed in the initial 2-DE EBBP blot, following RA treatment. Our data indicate that, following RA treatment, EBBP undergoes serine-threonine phosphorylation and, in addition, an as yet unrecognized post-translational modification, which results in cathodal shift.
Cathodal shifts during 2-DE or isoelectric focusing of proteins are typically observed when there is loss of sialic acid as a consequence of abnormal glycosylation (41, 42), loss of phosphate groups (43), or loss of negatively charged amino acids, such as aspartic or glutamic acid (44, 45). Loss of negatively charged residues with resultant heterogeneity in pI could result from alternative splicing (46) or post-translational proteolytic processing (47). EBBP has glutamic acid residues within close proximity to both the N and C termini, so it is conceivable that some proteolytic processing might alter its net charge and cause a cathodic shift. However, anomalous behavior during isoelectric focusing of proteins has also been reported in the case of hemoglobin E7A and E6V/E7A mutants (48). This kind of anomaly may also be due to the local effects of surrounding amino acids altering the theoretical pI or changes in protein-protein noncovalent interactions (49). Determination of the exact reason for the apparently anomalous behavior of EBBP in our case will require further experimental work.
Many transcription factors and their associated cofactors have been shown to oligomerize, and this property is often essential for their in vivo biological functions. Among the RBCC/TRIM family proteins, the PML1 and MID1 proteins have been shown to participate in both homo- and hetero-oligomeric complexes (36, 50). Furthermore, another member of the RBCC/TRIM family, RFP, can hetero-oligomerize and co-localize with PML, and this interaction is necessary for the location of RFP nuclear bodies, which play an important role in regulation of cellular growth and differentiation (51). The apparent sequence and functional similarities between EBBP and PML suggested that EBBP might also undergo nuclear translocation, and indeed, this was the case. This suggests that EBBP may play an important role in the induction of the differentiation pathway in neuroblastoma cells.
In RA-sensitive neuroblastoma cells, we showed that EBBP enhanced RA-induced RAR transcription, and this effect can be blocked by a specific EBBP siRNA that demonstrated that EBBP is an endogenous control point for RAR gene expression. Most importantly, a similar result was also observed in RA-resistant lung (SK-MES-1) and breast (MDA-MB-231 and MDA-MB-468) cancer cell lines. We had previously been unable to restore RA-responsive RAR transcription in this cell line by combining RA with either a demethylating agent (5-azacytidine) or a histone deacetylase inhibitor (trichostatin A). These findings therefore suggest that EBBP may represent a novel mechanism for relieving repressed RA-responsive transcription, critical to the RA anti-cancer signal.
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-helices that form a coiled-coil domain. Two motifs within these -helices, FXXLL and LXXLL, have been identified as nuclear receptor interaction domains in transcriptional co-factors such as TIF1 and cAMP-response element-binding protein (52, 53). Mutations in the B-box and deletion of the distal two coils of RFP eliminate the hetero-oligomerization between RFP and PML and interfere with their co-localization in nuclear bodies (51). Our finding that the coiled-coil domain was necessary for transactivation of the RARE in RA-sensitive cells is consistent with previous data that suggested that the coiled-coil region of TIF1 is necessary for interaction with other proteins (54). In particular, the coiled-coil domain is responsible for interaction with nuclear receptors, such as the RARs (52, 53). In summary, we have identified the EBBP protein as a novel co-regulator of the retinoid signaling pathway. Our results indicate that, during RA treatment, EBBP may physically associate with PML or other proteins in PML bodies and that this binding may occur via the coiled-coil domain. Our results also suggest the possibility that phosphorylation of EBBP may be required for the RA anti-cancer signal and, moreover, that an excess of EBBP can overcome transcriptional repression in cells that are resistant to demethylation or histone acetylation, by mechanisms yet to be determined. Most importantly, overexpression of EBBP protein increased cell growth inhibition in several human cancer cells. Understanding the mechanism by which EBBP regulates cell cycle progression and defining an effective method to increase EBBP protein expression in human cancer cells may provide a novel strategy in cancer therapy.
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FOOTNOTES |
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1 To whom correspondence should be addressed: Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Randwick, NSW, 2031, Australia. Tel.: 61-2-9382-1721; Fax: 61-2-9382-1789; E-mail: g.marshall{at}unsw.edu.au.
2 The abbreviations used are: RA, retinoic acid; RAR, retinoic acid receptor; RARE, RA response element; 2-DE, two-dimensional polyacrylamide gel electrophoresis; BE, BE(2)-C; SY, SH-SY5Y; aRA, all-trans-retinoic acid; RT, reverse transcription; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; PBS, phosphate-buffered saline; EBBP, estrogen-responsive B box protein; 2M, 2-microglobulin; BrdUrd, bromodeoxyuridine; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; TRIM, tripartite motif; PML, promyelocytic leukemia.
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ACKNOWLEDGMENTS |
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REFERENCES |
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