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Blood, 15 December 2004, Vol. 104, No. 13, pp. 4226-4235. Prepublished online as a Blood First Edition Paper on August 19, 2004; DOI 10.1182/blood-2003-10-3583.
NEOPLASIA
Expression of SMRT
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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fusion oncoprotein, whose abnormal transcriptional function is successfully targeted by pharmacologic levels of all-trans-retinoic acid (ATRA). Mutations in the ligand-binding domain of PML/RAR that confer resistance to ATRA have been studied by expression in nonhematopoietic cells, such as Cos-1. Here, we show that ATRA binding and transcriptional activation by the same PML/RAR mutant differ markedly between nonhematopoietic and leukemic cell lines. Differential expression of the corepressor isoform silencing mediator for retinoid and thyroid receptors 
(SMRT) correlates with increased ligand binding and transcription by the mutant PML/RAR. Transient and stable overexpression of SMRT in hematopoietic cells that only express SMRT increased ATRA binding, ligand-induced transcription, and ATRA-induced cell differentiation. This effect may not be limited to abnormal nuclear receptors, because overexpression of SMRT increased ATRA-induced binding and transcriptional activation of wild-type receptors PML/RAR and RAR. Our results suggest a novel role for the SMRT isoform whereby its cell-specific expression may influence the binding and transcriptional capacities of nuclear receptors, thus providing new evidence of distinct functions of corepressor isoforms and adding complexity to transcriptional regulation. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Many nuclear hormone receptors can either repress or activate expression of a given target gene depending on the presence of hormones, nature of the promoter, and the cell context. These regulatory properties are closely linked to the ability of these receptors to recruit corepressors and coactivators, which help mediate the transcriptional response.2 Thus, in absence of all-trans-retinoic acid (ATRA), retinoic acid receptor- (RAR) binds to corepressors silencing mediator for retinoid and thyroid receptors (SMRT) and nuclear receptor corepressor (NCoR), which in turn recruit a larger protein complex containing histone deacetylase (HDAC). The best characterized is the NCoR/SMRT-HDAC3-TBL1 complex first identified in HeLa cells that contains HDAC3 and TBL1 (transducin –like protein 1). Subsequently, GPS2 (G protein pathway suppression 2) was also shown to be a component of this TBL1 or TBLR1 (TBL1-related protein 1) complex.3-6 This complex hypoacetylates chromatin and restricts accessibility of the DNA template to the transcriptional machinery.7-9 Conversely, binding of ATRA leads to a conformational change in the receptor that releases the corepressor complex and recruits instead coactivator complexes, many of which possess histone acetyltransferase activity. Histone acetylation is believed to create a more accessible and therefore more readily transcribed chromatin structure.2,10,11
SMRT and NCoR are modular proteins that contain N-terminal repression domains (RDs) and C-terminal nuclear receptor interaction domains (RIDs). Two variant cDNA clones have been identified in the screening for the N-terminal region of the m-SMRT, which potentially represent products of an alternative splicing event. The longer 10-kb isoform, designated SMRT, refers to full-length SMRT. The shorter 8.5-kb isoform is designated SMRT and harbors a natural deletion of amino acids 36 to 254.12 Based on sequence similarity to NCoR, this deletion removes most of the repressor domain 1 (RD1), including a portion of the TBL1/GPS2-binding region. The region of SMRT corresponding to NCoR RD1 has the most potent repressor activity, which is presumably caused by the presence of the TBL1/GPS2 interaction domain. However, no cell-specific differential function of these isoforms has been reported to date.
A model for the interaction of nuclear receptors and coregulators in cancer cells has been provided by acute promyelocytic leukemia (APL), in which a translocation invariably involving the retinoic acid receptor- (RAR) leads to the uncontrolled growth and failure of normal differentiation of promyelocytes. In most cases of APL, a translocation of PML and RAR genes produces a fusion protein that acts as a dominant negative inhibitor of retinoid-mediated transcription.13,14 PML/RAR is found exclusively in APL patients15,16 and causes APL in transgenic mice.17,18
The PML/RAR protein retains most functional domains of RAR and behaves as an abnormal receptor with altered transactivation functions. The physiological importance of an active transcriptional repression is demonstrated by the role of corepressors in APL. In APL, the fusion protein can bind ATRA, but higher concentrations are required to activate transcription.19,20 This may be due to a more tightly associated PML/RAR corepressor complex, where the corepressor is not released at physiological ATRA concentrations.21-23 The release of corepressor induced by higher ATRA concentrations allows recruitment of coactivators and may underlie the cytodifferentiation of APL cells.21-24
Similar concentrations of ATRA can be achieved in APL patients, leading to dramatic remissions associated with granulocytic differentiation of leukemic cells.15,25 Nevertheless, APL cells develop resistance to ATRA in vitro and in vivo. Although combined cytotoxic chemotherapy with ATRA cures a high percentage of patients with APL, relapse with ATRA-resistant cells still occurs.26-31
Although altered pharmacokinetics may play a role, we and others have reported NB4 subclones that are rendered highly resistant to retinoid-induced cytodifferentiation by expression of dominant negative mutations of the PML/RAR fusion protein.32-37 Similar mutations of PML/RAR have been reported in APL cells from a growing number of patients who developed ATRA resistance, demonstrating the clinical relevance of these genetic alterations as a mechanism of resistance.31,38-41
PML/RAR mutations found in ATRA-resistant APL patients have been studied by expression of the cloned mutated receptor in established cell lines, such as Cos-1 or CV-1 cells, which do not express the PML/RAR protein. We and others have found that these amino acid changes cause a variety of abnormalities in ligand binding, transactivation of retinoic acid response elements (RAREs), and ligand-dependent interactions with the transcriptional coregulators SMRT and activator of thyroid and retinoic acid receptors (ACTR).42,43 Interestingly, a recent report found no correlation between in vitro response in Cos-1 cells and the clinical response to ATRA combined with HDAC inhibitors in 5 patients with resistant APL involving different PML/RAR mutations.40 However, these in vitro analyses did not examine whether the cell line in which PML/RAR mutations are studied affects the results obtained. In this report, we evaluate the use of Cos-1 cells as a model for the characterization of patient-derived PML/RAR mutations and find that ligand binding and transcriptional activation by a PML/RAR mutant overexpressed in Cos-1 cells differ markedly from those observed in APL cells. We report a difference in the expression of transcriptional coregulators in APL cells and Cos-1 cells that may be responsible for the differences we observed. Specifically, we show that expression of the isoform of the transcriptional corepressor SMRT positively modulates the ability of PML/RAR mutant I410T to bind ligand, activate transcription, and induce cell differentiation. Our results therefore demonstrate an isoform-specific ability of transcriptional corepressor SMRT to coactivate a response to ligand, suggesting a novel mechanism for transcriptional regulation of nuclear receptors.
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Materials and methods |
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The establishment and conditions for cell culture of the ATRA-resistant APL cell line NB4-MRA1 have been published previously.43 NB4, NB4-MRA1, Jurkat, U937, and Cos-1 cells are routinely cultured in ATRA-free RPMI 1640 medium (Life Technologies [GIBCO], Burlington, ON, Canada) supplemented with 10% fetal calf serum (FCS) (Wisent, St-Bruno, QC, Canada) at 37°C with 5% CO2. ATRA was obtained from Sigma (St Louis, MO), dissolved in ethanol, and kept at -80°C in the dark.
Stable transfection
The SMRT cDNA was subcloned in the PINCO retroviral vector. Virus production, cell infection, and purification of green fluorescent protein (GFP)–positive cells were performed as described previously.44
Plasmid constructs
PML/RAR with the I410T mutation was cloned into the pSG5 mammalian expression vector harboring wild-type PML/RAR (Long) cDNA using a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The construct was verified by sequencing analysis using the dsDNA Cycle Sequencing System (Life Technologies).
Northern blot analysis
Total RNA was extracted from different cells with guanidine thiocyanate, and Northern blotting was performed as previously described.45 Blots were probed to an 885-bp pGEX2T-NCoR-IDN/BamHI fragment46 and a 285-bp pGEX-KG-SMRT-IDII/BamHI-HindIII fragment.21 The filters subsequently were stripped and rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe for RNA loading normalization.
Western blot analysis
Nuclear extracts were diluted 1:1 with 2 x sodium dodecyl sulfate (SDS) sample buffer. Proteins were then fractionated by electrophoresis on 4% to 12% precast SDS polyacrylamide gels (Invitrogen, Carlsbad, CA), electroblotted on nitrocellulose membrane (Protran; Schleicher & Schuell, Dassel, Germany), and detected by the enhanced chemiluminescence (ECL) Western blotting detection kit (Amersham, Piscataway, NJ). Proteins that reacted with either the anti-RAR RP (Ab directed against F region of RAR) antibody47 or the anti-RAR (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) antibody were used at a 1:1000 dilution. Proteins that reacted with either the anti-SMRT (S93920; Transduction Laboratories, Lexington, KY) antibody or anti-SMRT (PA1-842; Affinity BioReagents, Golden, CO) antibody were used at a 1:200 dilution and a 1:250 dilution, respectively.
Assay for ligand-binding activity
Nuclear extracts were prepared from 1 to 5 x 107 cells and incubated for 18 hours at 4°C with 10 nmol/L [3H]-ATRA (50.7 Ci/mmol [1.88 TBq/mmol]; DuPont-NEN, Boston, MA) or with [3H]-ATRA in the presence of 200-fold excess of unlabeled ATRA. The extracts were subsequently fractionated at 4°C by high-performance liquid chromatography (HPLC) using a Superose 6 HR 10/30 size exclusion column (Pharmacia, Uppsala, Sweden) as previously described.19
Transient transfection experiments for transcriptional activity
Suspension cells (NB4, NB4-MRA1, A1-GFP, A1-GFP-SMRT, Jurkat, U937) were grown in RPMI 1640 with 10% FCS, and 5 x 106 cells per transfection were used. Cells were rinsed with serum-free OPTI-MEM (Life Technologies) and transfected by electroporation with 5 µg per transfection of the reporter plasmids DR5-tk-CAT,48 1 to 5 µg receptor (pSG5-RAR, pSG5-PML/RAR, or pSG5-PML/RAR-I410T) plasmid DNA, and 1 to 5 µg pCMX-SMRT or pCMX-SMRT plasmid DNA.12 In all transfection experiments, the amount of transfected DNA was equalized with empty vector DNA (pSG5, pCMX). A1-GFP and A1-GFP-SMRT were transfected with 10 µg per transfection of the reporter plasmid DR5-tk-CAT. Cells were electroporated and replenished with 5 mL RPMI 1640 with 10% FCS and grown for 48 hours in the absence or presence of different concentrations of ATRA. The chloramphenicol acetyltransferase (CAT) activity was measured using a modified protocol of the organic diffusion method as described previously.42 After reporter gene activity had been measured, the values were normalized with respect to protein content of the cell lysates to obtain the relative CAT activity. Each transfection was performed in triplicate and repeated 3 times.
Adherent cells (Cos-1) were grown in RPMI 1640 with 10% FCS and were seeded at 120 000 cells per well in 6-well plates 1 day before transfection. Cells were rinsed with serum-free OPTI-MEM (Life Technologies) and transfected by the lipofectamine method (Life Technologies) with 0.7 µg wild-type or mutant fusion protein plasmid, 1 µg reporter plasmid DR5-tk-CAT, and 0.3 µg pCMV-galactosidase (gal) as an internal control for the transfection efficiency. Cells were transfected for 5 hours, replenished with 2 mL RPMI 1640 with 10% FCS, and grown for 24 hours in the absence or presence of different concentrations of ATRA. CAT activity was measured using a modified protocol of the organic diffusion method as described previously.42 After reporter gene activity had been measured, the values were normalized with respect to gal to obtain the relative CAT activity. Each transfection was performed in triplicate and repeated 3 times. The Student t test was used to analyze the significance of the results obtained in transfection experiments for transcriptional activity.
Cell differentiation
Cell differentiation was evaluated by direct immunofluorescence staining of CD11b and CD11c cell-surface myeloid-specific antigens (nos. 555388 and 555392, PharMingen, Mississauga, ON, Canada) (FACSCalibur flow cytometer, BD BioSciences, Mississauga, ON, Canada) and by nitrobluetetrazolium (NBT) dye reduction assays as previously described.49 The cells were also analyzed for the isotype control phycoerythrin (PE)–conjugated mouse immunoglobulin G1
(IgG1) (no. 555749; PharMingen). In each sample viable cells were gated, and expression of CD11b and CD11c surface markers of 5 x 103 cells was evaluated.
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Results |
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mutant I410T differs in ATRA binding and transcriptional activity when endogenously expressed in the ATRA-resistant NB4-MRA1 APL cells than when expressed in Cos-1 cells
The resistant subclone NB4-MRA1 harbors a missense mutation in the ligand-binding domain (LBD) of PML/RAR in the ATRA-resistant NB4-MRA1 APL cells.43 This mutation results in amino acid substitution of isoleucine (Ile, I) (ATC) for threonine (Thr, T) (ACC) in the long form (L, Bcr1) of PML/RAR protein, which corresponds to codon 410 (I410T) in wild-type RAR. The mutation is centrally located in the highly conserved core of the activator function-2 (AF-2) activation domain within the helix 12 (H12).
To evaluate how this point mutation of PML/RAR alters the function of the LBD, we examined the binding of [3H]-ATRA to nuclear extracts from the ATRA-sensitive NB4 and the ATRA-resistant NB4-MRA1 cells. The size exclusion HPLC profile of extracts from NB4 cells expressing the wild-type form of PML/RAR is characterized by the 50-kDa peak representing the binding of ATRA to endogenous RARs and the approximately 670-kDa peak representing macromolecular complexes formed by the interaction of PML/RAR with itself and/or other nuclear proteins.19,50 Nuclear extracts from NB4-MRA1 cells expressing the PML/RAR mutation I410T showed an HPLC profile consistent with ATRA binding to the mutated chimeric protein but at a much decreased level (Figure 1A). PML/RAR mutations found in ATRA-resistant APL patients have been studied by expression of the cloned mutated receptor in established cells, such as Cos-1, Cos-7, or CV-1, which do not express the PML/RAR protein. To validate the use of Cos-1 cells as a model for the evaluation of these patient-derived PML/RAR mutations, we compared ATRA-binding data of PML/RAR mutant I410T expressed in Cos-1 cells with binding of the same mutation in its APL cells of origin, NB4-MRA1. In contrast to the PML/RAR mutant I410T expressed in its cells of origin, the I410T mutant transfected into Cos-1 cells presents a specific ATRA-binding profile similar to that of the wild-type PML/RAR (Figure 1B).
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We then compared transcriptional activation by the mutant PML/RAR protein in its cell of origin, NB4-MRA1, with transcription when overexpressed in Cos-1 cells. As shown in Figure 1C, significant ligand-dependent transcriptional activity of the wild-type PML/RAR chimeric protein was observed in ATRA-sensitive NB4 cells and in Cos-1 cells at concentrations of ATRA from 0.01 to 1 µM. Induction of transcriptional activity was substantially impaired in the ATRA-resistant NB4-MRA1 cells at all concentrations of ATRA. However, when transfected into Cos-1 cells, consistent with the differences in binding, the PML/RAR mutant I410T showed a significant increase in transcriptional activity in the presence of 0.1 to 1.0 µM ATRA (Figure 1C). Thus, ATRA binding and transcriptional activation by the same PML/RAR mutant are different in these 2 cell lines. Right panels of Figure 1A-B show that the NB4 and NB4-MRA1 cell lines, as well as Cos-1 cells transfected with wild-type or mutated PML/RAR, expressed similar levels of the PML/RAR fusion proteins. Thus, the cell-specific differences we observed in ATRA-binding capacity and transcriptional activation of PML/RAR were not due to a variation in protein expression.
U937 leukemic cells expressing PML/RAR I410T show ATRA binding and transcriptional activity similar to that of NB4 APL cells
We sought to determine whether the differences in binding and transcriptional activity of mutant PML/RAR we observed in NB4-MRA1 and Cos-1 cells are specific to those cells or reflect more general differences between cell types. We analyzed the HPLC ATRA-binding capacity and the transcriptional activity of the mutant I410T expressed in a non-APL leukemic cell line, U937. In these cells, as in the NB4 subclone, the mutant I410T has a minimal ATRA-binding capacity (Figure 2A) and transcriptional activity (Figure 2B). When expressed in additional nonhematopoietic adherent cell lines (HeLa and CV-1), ATRA binding and transcriptional activity of PML/RAR I410T were similar to those observed when expressed in Cos-1 cells (data not shown).
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The isoform of the corepressor SMRT is expressed in nonhematopoietic cells but is not expressed in hematopoietic cells
Previous work emphasizing the importance of coactivators and corepressors of transcription in the response to altered nuclear receptors led us to hypothesize that differential expression of transcriptional coregulators may be responsible for the discrepancy we observed between cell lines in HPLC ATRA binding and transcriptional analyses. To test this hypothesis, we compared the RNA expression levels of the transcriptional corepressors NCoR and SMRT in our APL clones with those of Cos-1, U937, and a variety of other cell lines by Northern blot analysis. Figure 3A shows that the expression level of the corepressor NCoR is similar in all the cell lines tested. However, analysis of the expression of the corepressor SMRT shows marked differences among the cell lines. ATRA-sensitive and ATRA-resistant APL cells as well as other hematopoietic cells, U937 and Jurkat, express only the SMRT 10-kb transcript, whereas the nonhematopoietic cells, Cos-1, Cos-7, CV-1, and HeLa, express both SMRT and SMRT isoforms as transcripts of 10 kb and 8.5 kb, respectively. Figure 3B also shows marked differences among those cell lines at the level of protein expression as analyzed by Western blotting for SMRT. ATRA-sensitive NB4, as well as another hematopoietic cell line, Jurkat, express only the SMRT isoform (Figure 3B, top band), whereas the nonhematopoietic cells, Cos-1, HeLa, and T47-D, express both SMRT and SMRT isoforms (Figure 3B, bottom band). These results suggest that the presence of the SMRT isoform in nonhematopoietic cell lines may modulate the capacity of the PML/RAR mutant I410T to bind ligand and subsequently activate transcription.
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Overexpressed SMRT isoform increases ATRA-binding capacity of the PML/RAR mutant I410T in ATRA-resistant NB4-MRA1 APL cells
To directly test whether SMRT isoform modulates the ability of the PML/RAR mutant I410T to bind ligand, we transfected the SMRT or SMRT isoforms into ATRA-resistant NB4-MRA1 APL cells and performed an HPLC ATRA-binding analysis (Figure 3C). In the absence of the transfected corepressor SMRT isoforms (Figure 3C, "mock"), the mutant I410T fusion protein exhibited minimal binding capacity to ATRA as represented by the absence of the 670-kDa peak. The presence of the 670-kDa peak in NB4-MRA1 transfected with SMRT indicates that expression of the SMRT isoform increased the ATRA-binding capacity of the PML/RAR mutant I410T, while increased expression of SMRT isoform in those cells has no significant effect on ligand binding. The right panel of Figure 3C shows a similar PML/RAR protein level in NB4-MRA1 compared with those cells transfected with SMRT or SMRT, confirming that expression of SMRT isoforms does not alter the levels of PML/RAR protein. These results are in agreement and support our hypothesis that difference in expression of SMRT isoforms might be responsible for cell-specific responsiveness to hormone.
Overexpressed SMRT isoform promotes ligand-induced transactivation of PML/RAR mutant I410T in ATRA-resistant NB4-MRA1 APL cells and other cell lines
The activity of SMRT ATRA-binding analysis prompted us to address the role of SMRT in regulating the transcriptional activity of the PML/RAR mutant I410T in cell lines that varied in expression of this isoform. We conducted transient cotransfection assays to determine the effects of SMRT and SMRT overexpression on I410T activity using a DR5-tk-CAT reporter gene in 4 cell lines (Figure 4). We found that expression of SMRT resulted in an activation of transcription by PML/RAR I410T in ATRA-resistant NB4-MRA1 APL cells in response to 1 µM ATRA treatment, producing a 24.1-fold induction compared with a 10.6-fold induction with the empty vector pCMX (Figure 4A, "control"). We next analyzed the effect of overexpression of SMRT isoforms in the hematopoietic Jurkat cells. Jurkat cells were cotransfected either with an empty expression vector or with expression vectors for PML/RAR I410T and specific vectors for SMRT or SMRT. As shown in Figure 4B, the mutant PML/RAR activated DR5-tk-CAT expression 26.7-fold in the presence of ATRA. Interestingly, expression of SMRT in Jurkat increases this activation to 72.6-fold, while additional expression of SMRT had no significant effect. We also analyzed the effect of overexpression of SMRT isoforms in the hematopoietic U937 cells. As shown in Figure 4C, the mutant PML/RAR activated DR5-tk-CAT expression 16.2-fold in the presence of ATRA. Expression of SMRT in U937 cells increases this activation to 32.1-fold, while SMRT isoform decreases the activation to 9.9-fold. Finally, we analyzed the effect of overexpression of SMRT isoforms in the nonhematopoietic Cos-1 cells. As shown in Figure 4D, the mutant PML/RAR activated DR5-tk-CAT expression 16.9-fold in the presence of ATRA. Interestingly, overexpression of SMRT decreases the induction to 6.1-fold. Expression of SMRT isoforms without PML/RAR I410T had no significant effect on transcriptional activity (data not shown). In general, expression of either SMRT isoform was associated with decreased basal transcription, consistent with its ability to repress transcription, but this repression was relieved by ligand with the expression of SMRT. Taken together these results indicate that SMRT induces a ligand-dependent transcriptional activation by PML/RAR I410T in all the cell lines tested. This suggests an antirepression role for the SMRT isoform in the presence of ligand and that its cell-specific presence may influence the binding and ligand-dependent transcriptional capacities of nuclear receptors.
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Expression of SMRT activates transcription by wild-type nuclear receptors PML/RAR and RAR
We next sought to determine whether the ligand-induced activation by SMRT we observed with the PML/RAR mutant I410T is limited to this mutated receptor or reflects a more general action of SMRT on activation of retinoid receptors. We tested the effects of coexpression of SMRT with wild-type retinoid receptors PML/RAR and RAR in Jurkat cells, which do not endogenously express SMRT and show low levels of endogenous RAR activity.51 pCMX-SMRT was cotransfected with a DR5-tk-CAT reporter plasmid and a pSG5-PML/RAR into Jurkat cells and tested for ligand response. As shown in Figure 5A, in the absence of cotransfected corepressor, PML/RAR exhibited a 12.6-fold ligand-mediated induction, while coexpression of SMRT increased induction to about 28.9-fold. Expression of empty vectors or SMRT alone had no significant effect on the ATRA-induced transcription. To investigate the effects of overexpressing SMRT on RAR-mediated reporter gene expression, increasing amounts of pCMX-SMRT were cotransfected with a DR5-tk-CAT reporter plasmid and a pSG5-RAR into Jurkat cells. As shown in Figure 5B, increasing coexpression of SMRT resulted in an increased transcriptional activation. Expression of empty vectors or SMRT has no significant effect on the ATRA-induced transcription. In addition, coexpression of SMRT did not enhance transcription at any concentration of ligand tested (data not shown). The right panels in Figure 5 show PML/RAR and RAR protein levels in mock and transfected Jurkat. In Figure 5B, note the very low level of endogenous expression of RAR in mock Jurkat as compared with the positive control HeLa, confirming the results reported by Ballow et al.51
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Stable expression of SMRT in the ATRA-resistant NB4-MRA1 cells promotes ligand binding, ligand-induced transcriptional activation, and ATRA-induced cell differentiation
The activity of transiently expressed SMRT observed in ATRA binding and transcriptional activity analyses in various cell lines prompted us to stably express the SMRT isoform in ATRA-resistant NB4-MRA1 APL cells (A1-GFP-SMRT). The right panel of Figure 6A confirms that the ATRA-resistant NB4-MRA1 cells stably transfected with the empty GFP vector (A1-GFP) expressed only SMRT isoform (Figure 6A, top band), whereas NB4-MRA1 stably transfected with GFP-SMRT (A1-GFP-SMRT) expressed SMRT and SMRT (Figure 6A, bottom band), as analyzed by Western blotting.
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In an HPLC binding analysis of NB4-MRA1 stably expressing the GFP empty vector (A1-GFP), the mutant I410T fusion protein exhibited minimal binding capacity to ATRA, as represented by a decreased 670-kDa peak (Figure 6A). The increased 670-kDa peak in A1-GFP-SMRT cells indicates increased ATRA-binding capacity of the PML/RAR mutant I410T, confirming the results we obtained in transient transfections. Interestingly, there is also an increased 50-kDa peak, suggesting that expression of SMRT promotes ATRA binding to the wild-type RAR receptor as well. This is consistent with the results we obtained in Figure 5 showing a more general action of SMRT on transcriptional activation of wild-type retinoid receptors PML/RAR and RAR.
We determined the effects of stable SMRT overexpression on I410T transcriptional activity using a transiently transfected DR5-tk-CAT reporter gene (Figure 6B). We found increased transcription by PML/RAR I410T in A1-GFP-SMRT cells in response to 0.01 and 1 µM ATRA treatment, compared with A1-GFP stably transfected with a GFP empty vector. The right panel of Figure 6B demonstrates similar levels of PML/RAR protein in A1-GFP and A1-GFP-SMRT, showing that stable expression of SMRT into NB4-MRA1 cells does not affect the expression or stability of the PML/RAR protein. These results again confirm those we obtained in transient transfections of SMRT.
Finally, we evaluated the biologic response to ATRA of A1-GFP-SMRT cells stably expressing the SMRT corepressor. We tested whether the presence of SMRT increases sensitivity of A1-GFP-SMRT cells to ATRA-induced differentiation by evaluating the expression of 2 cell-surface markers of myeloid differentiation by flow cytometry. NB4-MRA1 cells stably expressing a GFP empty vector or a GFP-SMRT vector were treated for 5 days with 0.01 µM and 1 µM ATRA. We observed no evidence of differentiation in NB4-MRA1 expressing the GFP empty vector, whereas stable expression of SMRT in A1-GFP-SMRT cells led to increases in expression of CD11b (Figure 6C) and CD11c (Figure 6D) in response to ATRA treatment. We obtained similar results using the same ATRA concentrations for a 3-day treatment (data not shown). To confirm these data with an additional assay of myeloid differentiation, a nitrobluetetrazolium (NBT) assay was performed on cells treated for 9 days with 10 µM ATRA. In control A1-GFP cells, ATRA treatment yielded 11% NBT reduction, while in SMRT-expressing A1-GFP-SMRT cells, NBT increased to 41% with ATRA treatment.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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, have been studied by expression of the cloned oncoprotein in established cell lines, such as Cos-1, Cos-7, or CV-1 cells. In this report, we utilized an ATRA-resistant NB4 subclone, NB4-MRA1, to evaluate the use of Cos-1 cells as a model for the characterization of patient-derived PML/RAR mutations.
In NB4-MRA1 cells, the replacement of isoleucine at position 410 by threonine abolished almost completely the capacity of the fusion protein PML/RAR to bind ATRA and significantly impaired ligand-dependent transcriptional activity on a RARE. In contrast, when transiently expressed in Cos-1 cells, the same I410T mutant presents an ATRA-binding profile and an increased transcriptional activity similar to that of the wild-type PML/RAR. Expression in additional nonleukemic cell lines gave results similar to those seen in Cos-1, while ATRA binding and transcriptional activation by PML/RAR I410T expressed in other leukemic cell lines resemble that of NB4.
Our evaluation of the levels of expression of the transcriptional corepressors NCoR and SMRT suggested a potential mechanism for these differences. All the cell lines tested express a similar RNA level of NCoR and the longer 10 kb transcript of SMRT. Interestingly, only the nonhematopoietic adherent cell lines express the shorter SMRT transcript of 8.5 kb. The tested hematopoietic cell lines, including ATRA-sensitive and ATRA-resistant cells, express only the longer SMRT transcript and do not express the shorter transcript. This tissue-specific difference in expression was confirmed by Western blotting.
Figure 7 shows a schematic representation of NCoR and SMRT. The full-length form of either corepressor harbors 3 adjacent domains, denoted repressor domain (RD1-RD3), which play important roles in corepressor-mediated transcriptional repression. The SMRT isoform harbors a natural deletion that removes most of the RD1 domain. The most extensively characterized interaction is the ability of RD1 to bind to GPS2, TBL1, and related proteins, which in turn facilitate recruitment of histone deacetylase and transcriptional repression of unliganded nuclear receptors.3-6,52 A recent report has shown that this NCoR/SMRT-TBLR1-HDAC corepressor complex is recruited by unliganded RAR, PML/RAR, and PLZF/RAR, leading to histone deacetylation and transcriptional repression in vivo of ATRA-responsive promoters.53 The ability to assemble this SMRT/TBL1/HDAC complex is closely linked to the ability of SMRT to repress, so that the absence of RD1 can impair or prevent transcriptional repression. The effect of this deletion on SMRT function has been confirmed by cotransfection experiments comparing repression by SMRT and SMRT.12
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We find cell-specific differences in ligand binding and transcriptional activity of a mutated nuclear receptor that reflect differences in SMRT isoform expression. Expression of the SMRT into hematopoietic cells that express only SMRT confirmed that SMRT modulates ligand binding and transcription. Expression of SMRT, but not SMRT, increased ATRA binding and transcriptional activation by the mutant PML/RAR, while increased expression of SMRT in Cos-1 cells decreased transcription. The results from transient transfection of SMRT isoforms have been confirmed in cells stably transfected with SMRT. Furthermore, the SMRT-induced increase of ligand binding and transcriptional activity of PML/RAR I410T is associated with evidence of ATRA-induced differentiation of NB4-MRA1 cells stably expressing SMRT. Taken together, these data suggest that specific corepressors can play a novel role as mediators of cell-specific responsiveness to ATRA.
Indeed, we have evidence of a more general role in activation of retinoid receptors by SMRT. Expression of SMRT in Jurkat cells enhanced the transcriptional activity of nonmutated PML/RAR as well as wild-type nuclear receptor RAR (Figure 5), consistent with the increased binding of ligand to wild-type RAR seen in the stably transfected A1-GFP-SMRT cells (Figure 6). These results suggest that the role of coactivator that we identified for SMRT is not restricted to a specific PML/RAR mutation
promotes ligand-induced activation of mutated and wild-type retinoid receptors
) with the PML/RAR
) cells. Nuclear extracts of NB4 and NB4-MRA1 cells were subjected to Western blot analysis. The left-pointing arrow indicates expression of the 110-kDa PML/RAR
) and subjected to HPLC analysis using a 6 HR 10/30 size exclusion column. Down-pointing arrows indicate approximate retention times of void volume, PML/RAR
) or SMRT
