INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Nuclear hormone receptors are hormone-regulated transcription factors that bind to cognate hormone, bind to specific DNA sequences, and regulate the expression of adjacent target genes (3, 38, 39, 61). A wide variety of nuclear hormone receptors have been identified that mediate cellular responses to an assortment of different hormone ligands, including thyroid hormone, retinoids, steroids, vitamin D3, and a number of lipid metabolites. As a consequence, nuclear hormone receptors play key roles in many aspects of metazoan development, differentiation, and homeostasis (3, 38, 39, 61).

Many nuclear receptors are bipolar in function and are able to either repress or activate expression of target genes. Repression is conferred through the ability of nuclear receptors to recruit a complex of auxiliary proteins, designated corepressors, that mediate the molecular events necessary for transcriptional silencing (7, 10, 22, 54, 60, 66). The corepressor protein SMRT and its paralog, N-CoR, play a particularly important role in this process by serving as the principal point of contact of the corepressor complex with the nuclear receptors (6, 12, 21, 31, 50, 51, 67). Conversely, transcriptional activation is associated with release of SMRT/N-CoR from the nuclear receptor, followed by acquisition of a novel set of coactivator proteins (7, 10, 22, 25, 49, 54, 60, 66). Corepressors and coactivators regulate transcription through multiple mechanisms, including modifications of the chromatin template and interactions with the general transcriptional machinery (1, 24, 27, 32, 46, 65).

Thyroid hormone receptors (T3Rs) and retinoic acid receptors (RARs) typically bind to corepressors in the absence of hormone; on addition of hormone agonists, these nuclear receptors physically release from the corepressors and recruit coactivators (23, 33, 44, 47). Intriguingly, however, nonligand signal transduction pathways also play important roles in modulating the interaction of nuclear receptors with corepressors and coactivators. Particularly notable is the ability of protein kinase signaling pathways, such as those represented by the epidermal growth factor (EGF) receptor or by protein kinase A, to interfere with the SMRT-nuclear receptor interaction and to counteract transcriptional repression (19, 30, 62). Activation of the EGF receptor, for example, virtually abolishes the ability of SMRT to interact with T3Rs and eliminates T3R-mediated repression, even in the absence of thyroid hormone (19). The inhibitory effects of EGF receptor signaling on SMRT function are also observed with RARs and are mediated, at least in part, through a mitogen-activated protein (MAP) kinase cascade that culminates in phosphorylation of the SMRT protein, dissociation of SMRT from the nuclear receptor partner, and a relocalization of SMRT out of the nucleus and into the cytoplasm of the cell (20).

Aberrations in the interaction of nuclear receptors with corepressors can result in endocrine and neoplastic disorders. For example, human acute promyelocytic leukemia (APL) is associated with chromosomal translocations that fuse ectopic open reading frames to the DNA and hormone-binding domains of RAR (8, 11, 29, 41, 45). The most common form of translocation in APL results in the synthesis of a PML (promyelocytic leukemia)-RAR chimeric polypeptide. The PML-RAR chimera requires significantly higher retinoid concentrations to release from corepressor than does the wild-type RAR (15-17, 34, 35, 42). This defect in retinoid signaling plays an important role in generating the leukemic phenotype, and treatment of PML-RAR leukemic cells with supraphysiological levels of retinoic acid leads to release of corepressor from the PML-RAR and differentiation of the leukemic cell (15-17, 18, 35).

The ability of high concentrations of retinoic acid to induce differentiation in PML-RAR leukemias has been employed clinically to treat human APL (8, 11, 29, 41, 45). Recently, it has been recognized that arsenic trioxide acts synergistically with retinoic acid to induce long-term remissions in APL and can be effective in retinoid-resistant cases of APL (5, 14, 28, 53, 58). The precise molecular mechanisms behind the effects of arsenite in APL cells are not fully understood but are thought to involve both an apoptotic and a differentiation response (4, 14, 26, 28, 52, 59, 63, 70). Notably, arsenic trioxide is a strong inducer of MAP kinase signaling cascades in many cell contexts (2, 9, 36, 37, 40, 48). Given the ability of growth factor receptors to abolish SMRT function through a MAP kinase cascade (20), we tested whether arsenic trioxide treatment might affect SMRT function in a similar fashion. We report here that treatment with arsenic trioxide results in a profound inhibition in the ability of SMRT to interact with its transcription factor partners, such as T3Rs and RARs. At least one component of this inhibition of SMRT function appears to be mediated by an arsenite-mediated activation of a MAP kinase cascade, resulting in hyperphosphorylation of the corepressor and an alteration in its subcellular distribution. Notably, arsenite induces a similar and rapid inhibition of the ability of SMRT to interact with the PML-RAR oncoprotein in APL cells, and the ability of SMRT to induce differentiation and apoptosis in these cells is antagonized by inhibitors of MAP kinase cascade signaling. We propose that the effects of arsenite on normal cells and in APL are, at least in part, conferred through a MAP kinase cascade-mediated inhibition of corepressor function.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmid constructs. The pCMV-SMRT-C vectors were constructed by inserting EcoRI fragments from the previously described pSG-5 TRAC-1 constructs into a pCR3.1 vector (Invitrogen, Carlsbad, Calif.) (50). Construction of the mammalian two-hybrid vectors for various SMRT and T3R derivatives was as previously described (19, 20, 64). The pSG5-GAL4-activation domain (AD)-RAR and retinoid X receptor  (RXR) vectors were constructed by inserting EcoRV and XhoI fragments, generated by PCR, into the pSG5-GAL4-AD vector. The green fluorescent protein (GFP)-SMRT vector was constructed by inserting the BsrGI-to-EcoRI(blunt) fragment from pSG5-SMRT into the BsrGI-HindIII(blunt) sites on a CMV-GFPTYGBH vector (20). Expression vectors for full-length mitogen-elevated kinase kinase 1 (MEKK-1) and mitogen-elevated kinase 1 (MEK-1) expression plasmids were obtained from Chris Jamieson (University of California, San Francisco).

Transient transfections. CV-1 cells transfections were performed by a Lipofectin-mediated method using the general protocol recommended by the manufacturer (GIBCO/BRL Life Technologies, Rockville, Md.). Approximately 4 × 10⁵ cells were transfected with 50 ng of pSG5-T3R or pSG5-v-erb A plasmid (designated T3R-vA), 100 ng of pCMV-lacZ or pCH110 as an internal control, and 100 ng of a ptk-luc-TRE reporter, together with expression vectors for the various signal transducers tested here (equal quantities of the equivalent empty vectors were substituted where appropriate) (20). The cells were transferred to serum-free Dulbecco's modified Eagle's medium (DMEM) 24 h after transfection and were harvested 24 h later. The luciferase and -galactosidase assays were performed as previously described, and the relative luciferase activity (expressed as the ratio of luciferase to -galactosidase activity) was determined (20).

Mammalian two-hybrid assays. Exponentially growing CV-1 cells (7 × 10⁴ cells per well in 12-well culture plates) were transiently transfected by the Lipofectin method using 25 ng of the appropriate pSG5-GAL4DBD vector, 100 ng of the appropriate pSG5-GAL4AD vector, 100 ng of the pGL2-GAL4(17-mer)-luciferase reporter, 100 ng of the pCMV-lacZ internal control, and an appropriate expression vector for the various signal transducers tested here (or equal quantities of the equivalent empty vector where appropriate) (19, 20). The cells were transferred to serum-free DMEM 24 h after transfection and were harvested an additional 24 h later. The luciferase and -galactosidase assays were performed as previously described, and the relative luciferase activity (expressed as the ratio of luciferase to -galactosidase activity) was determined for each sample (19, 20).

Immunoblotting. CV-1 cells (7 × 10⁴ per well) were transfected with the appropriate expression vectors as indicated for each experiment, harvested 48 h after transfection by scraping, and lysed by mixing the cells with sodium dodecyl sulfate (SDS)-electrophoresis sample buffer. The lysates were then sonicated to reduce viscosity, boiled for 5 min, and loaded into an SDS-7.5% polyacrylamide-0.3% bis-acrylamide gel. After electrophoresis, the proteins were electrophoretically transferred to a nitrocellulose membrane. The membrane was incubated in blocking buffer (2.5% nonfat dry milk in TBST [0.1% Tween 20, 150 mM NaCl, 10 mM Tris-Cl; pH 7.6]) for 1 h and then incubated with appropriate primary antibodies (diluted in 2.5% bovine serum albumin in TBST) for 1 h. The membranes were next washed extensively with TBST and incubated with appropriate secondary antibodies (either horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody, diluted 1:2,000; Affinity BioReagents, Golden, Colo.). After extensive washing with TBST, the chemiluminescent Western detection system was employed for visualization of the immunoreactive proteins as specified by the manufacturer (NEN Life Science, Boston, Mass.).

Phosphorylation-dephosphorylation assay. CV-1 cells (2.5 × 10⁵) were transfected with the pCMV-SMRT-C and arsenite or with the MEKK-1 expression vector, as indicated for the individual experiments. The cells were harvested 48 h later by scraping and centrifugation in 150 µl of whole-cell extraction buffer (25 mM HEPES [pH 7.5], 300 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1% Triton X-100, 0.1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail [Boehringer-Mannheim, Mannheim, Germany]). Lysates were then incubated in the presence or absence of 0.5 U of calf intestinal alkaline phosphatase (New England Biolabs [NEB], Beverly, Mass.) for 30 min at 30°C. The incubations were terminated by mixing the samples with SDS sample buffer; the samples were boiled for 5 min, loaded into an SDS-7.5% polyacrylamide-0.3% bis acrylamide gel, and subjected to electrophoresis and immunoblotting as described above.

Coimmunoprecipitation assay. Approximately 10⁷ NB-4 cells were treated with either 1 µM all-trans retinoic acid or 0.1 or 1 µM As₂O₃ for various times. Whole-cell lysates were prepared by gently sonicating the cells in 1 ml of immunoprecipitation (IP) buffer (1× phosphate-buffered saline, 1 mM EDTA, 1.5 mg of iodoacetamide per ml, 100 µM Na₃VO₄, 0.5% Triton X-100, 20 mM -glycerolphosphate, 0.2 mM phenylmethylsulfonyl fluoride, and 1× complete protease inhibitor cocktail). After clarification by a 15-min centrifugation in a microcentrifuge at 4°C, the resulting supernatant was incubated for an additional 3 h at 4°C with either 2.5 µl of anti-SMRT antibody (PA1-843, diluted 1:400; Affinity Bioreagent), 2.5 µl of anti-RAR antibody, or 10 µl of anti-PML antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Fifteen microliters of either protein A-Sepharose (as a 50% slurry in IP buffer; Sigma Chemical Co., St. Louis, Mo.) or protein G-Sepharose was then added, and the samples were incubated with continuous mixing for 4 h more at 4°C. The protein A-Sepharose matrix was extensively washed with IP buffer, and any proteins remaining bound to it were eluted with SDS sample buffer and detected by Western analysis.

Laser-scanning confocal microscopy. Approximately 8 × 10⁴ CV-1 cells were seeded in a chambered cover glass cell culture system (Nalge-Nunc, Rochester, N.Y.). The cells were then transfected with the pCMV-GFP-SMRT vector together with MEKK-1 expression vector (or an equivalent empty vector as a control) using the Effectene procedure (Qiagen, Hilden, Germany). A day after transfection the cells were transferred into serum-free DMEM containing 15 µM As₂O₃ and were incubated for an additional 24 h. The subcellular location of the GFP-SMRT fusion polypeptide was visualized using a Leica TCS-SP UV/Ar/Kr laser-scanning confocal microscope, employing excitation at 488 nm and detection at 500 to 540 nm. The effect of arsenite on the subcellular location of TFIIB, employed as a control, was analyzed in a similar fashion but using an indirect immunofluorescence protocol: CV-1 cells were treated with arsenite for 24 h (or left untreated) as described above, washed, fixed with 3% formaldehyde, permeabilized with 0.5% Triton X-100 in 1× PBS, and visualized using anti-TFIIB antiserum (sc-225; Santa Cruz Biotechnology) and rhodamine-conjugated anti-rabbit immunoglobulin G (Cappel, West Chester, Pa.).

Analysis of level of phosphorylated MEK-1/2. CV-1 cells were grown in a 37°C humidified atmosphere containing 5% CO₂ in DMEM supplemented with 10% fetal bovine serum. Cells were maintained in serum-free medium for at least 16 h prior to treatment with As₂O₃. As₂O₃ was added to the medium to a final concentration of 20 µM. Cells were harvested by adding hot SDS sample buffer, and the amount of phosphorylated MEK-1/2 was examined by Western analysis using a PhosphoPlus MEK-1/2 (Ser217/221) antibody kit (NEB).

Analysis of cell surface marker, apoptosis, and DNA content. A fluorescence-activated cell sorting (FACS) flow cytometer was calibrated with CaliBRITE beads prior to use (Becton Dickinson, Mountain View, Calif.). The amount of myeloid cell surface marker CD11c on the NB-4 cells was determined by flow cytometry analysis using phycoerythrin-conjugated antibody from Becton Dickinson. The percent positive cells was quantified by FACScan analyzer (CellQuest program; Becton Dickinson). A terminal deoxynucleotidyl transferase labeling (TUNEL) reaction was performed on the NB-4 cells according to the manufacturer's instructions (Roche Molecular Biochemical Company, Indianapolis, Ind.) to measure apoptosis-associated DNA fragmentation. Cells were analyzed by flow cytometry using a FACScan. All data were collected and analyzed by CellQuest software. Cells were also analyzed for DNA content by flow cytometry analysis of propidium iodide-stained nuclei.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Arsenic trioxide strongly and specifically interferes with the ability of SMRT to interact with nuclear receptors. We wished to determine if arsenic trioxide could alter the ability of SMRT to interact with its nuclear receptor partners. To address this question, we first employed a mammalian two-hybrid assay. For this assay, a GAL4DBD-SMRT fusion construct, a GAL4-AD-T3R fusion construct, and a luciferase reporter bearing GAL4(17-mer) binding sites were introduced separately or in combination into CV-1 cells. A strong activation of the GAL(17-mer) luciferase reporter was observed when all three constructs were introduced together, reflecting the ability of the SMRT and T3R sequences to interact and thereby reconstitute a functional GAL4 transcription factor (Fig. 1A). This two-hybrid assay appeared to be a valid measure of the SMRT-T3R interaction: (i) each of the fusion constructs was transcriptionally inactive when introduced separately; (ii) T3 thyroid hormone abolishes the T3R interaction with SMRT both in vitro and in the mammalian two-hybrid system; (iii) mutants of T3R (e.g., P156R) or other nuclear receptors, such as vitamin D3 receptor, that do not interact with SMRT in vitro do not interact in the two-hybrid system; (iv) unrelated proteins or SMRT mutants (SMRT RID) that fail to interact with T3R in vitro do not interact with T3R in the two-hybrid system; and (v) mutants of T3R that bind SMRT in a hormone-independent fashion in vitro (e.g., T3R-vA) exhibit a hormone-independent interaction with SMRT in the two-hybrid assay (Fig. 1A).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Effect of kinase signaling and As2O3 on the interaction between T3R and SMRT. (A) Effect of hormone on SMRT interaction with nuclear hormone receptors in a mammalian two-hybrid assay. A pSG5 plasmid containing either GAL4DBD alone (empty DBD), a GAL4DBD fused with the receptor interaction domain of SMRT (amino acids 751 to 1495; DBD-SMRT), or a GAL4DBD fused with a region of SMRT lacking the receptor interaction domain (amino acids 751 to 1074, DBD-SMRT RID) were cotransfected into CV-1 cells with a GAL4(17-mer) thymidine kinase promoter-luciferase reporter and a pSG5-GAL4AD construct by a calcium phosphate precipitation method. Cotransfected GAL4AD constructs are indicated below the panels (empty, GAL4AD domain alone; T3R, GAL4-AD fused with T3R ligand-binding domain; P156R, GAL4-AD fused with a T3R construct with a mutation that disrupts SMRT association; T3R-vA, GAL4-AD fused with the analogous region of the v-Erb A mutant form of T3R, v-Erb A; VDR, GAL4-AD fused with a vitamin D3 receptor ligand-binding domain). The cells were incubated in the absence or presence of 1 µM cognate hormone (T3), the cells were harvested, and luciferase activity was determined relative to -galactosidase activity of pCH110 plasmid introduced as an internal control. Data are averages and standard deviations from duplicate experiments. (B) Inhibition of two-hybrid SMRT-T3R interaction by v-Erb B, MEKK-1, and MEK-1. A pSG5 plasmid expressing a GAL4DBD alone (empty DBD) or a GAL4DBD fused with the receptor interaction domain of SMRT (DBD-SMRT) was transiently cotransfected into CV-1 cells by Lipofectin, with either an empty pSG5-GAL4AD vector, the pSG5-GAL4AD fused to the ligand-binding domain of T3R, or T3R-vA. In addition, the cells were cotransfected with 100 ng of expression plasmid for either v-Erb B, v-Ras, a full-length MEKK-1, or a MEK-1 clone, as indicated at the bottom. The cells were incubated in the absence of T3, and the luciferase activity was determined and normalized to -galactosidase activity for each sample. (C) Inhibition of the mammalian two-hybrid interaction between SMRT and T3R by As2O3. The protocol for panel B was repeated, but in the absence or presence or absence of 20 µM As2O3, as indicated at the bottom. A control two-hybrid interaction between a DBD-SMRT construct and an AD-T3R construct was assayed under the same conditions to detect any nonspecific effects of arsenite on the two-hybrid system (see the text). The scale for the v-Erb A interaction with SMRT (right axis) differs from that for the other three panels (left axis).

View larger version (25K): [in this window] [in a new window]   FIG. 2.   Effect of As2O3 on the interaction between SMRT and RAR and between SMRT and PLZF. (A) Inhibition of the mammalian two-hybrid interaction between SMRT and RAR by As2O3. A protocol similar to that for Fig. 1C was used, but with pSG5-GAL4AD fused to the ligand-binding domain of RAR in place of the T3R construct. The cells were left untreated or treated with 15 µM As2O3 or with all-trans retinoic acid (ATRA). (B) Inhibition of the interaction between SMRT and PLZF by As2O3. The same protocol was used, but with pSG5-GAL4AD fused to the PLZF open reading frame in the absence or presence of 15 µM As2O3. Data are averages and standard deviations of duplicate experiments.

The inhibitory effects of arsenic trioxide on SMRT function in CV-1 cells are mediated, in part, through activation of a MAP kinase cascade and result in phosphorylation of the corepressor. In many cell types, arsenic trioxide acts as a strong inducer of MAP kinase cascade signaling (2, 9, 36, 37, 40, 48). Given the overall similarity between the inhibitory effects of arsenic trioxide and the inhibitory effects of MEKK-1 or MEK-1, we next explored whether the effects of arsenic trioxide on SMRT function were, in fact mediated through this (or an analogous) MAP kinase cascade. Consistent with this hypothesis, arsenic trioxide treatment resulted in a rapid increase in the phosphorylation state of MEK-1/2, suggestive of an activation of MAP kinase cascade signaling in these cells (Fig. 3A). We have reported that chemical inhibitors of MEK-1/2 counteract the effects of MAP kinase cascade signaling and stabilize the SMRT-nuclear receptor interaction (20). Significantly, the inhibitory effects of arsenite in the SMRT two-hybrid assay were also counteracted by MEK-1/2 inhibitors, such as PD98059 (Fig. 3B), whereas MEK-1/2 inhibitors had little or no effect on the SMRT-T3R two-hybrid interaction in the absence of arsenite (reference 20 and data not shown) and only a very small effect on reporter gene expression in the empty-vector system (Fig. 3B). These results suggest that the arsenite antagonizes SMRT function, at least in part, through a MAP kinase cascade involving MEK-1 or a comparable MAP kinase kinase intermediate.

View larger version (21K): [in this window] [in a new window]   FIG. 3.   Effect of As2O3 treatment on phosphorylation of MEK-1/2 (A) and effect of a MEK-1 inhibitor on SMRT-T3R interaction (B). (A) CV-1 cells were treated with As2O3 for the specified times. The cells were subsequently lysed, and the extracts were resolved by SDS-PAGE and analyzed by immunoblotting using antibody specific for bulk MEK-1/2 or for phosphorylated MEK-1/2 (Ser217/221). (B) The effect of As2O3 on the SMRT-T3R two-hybrid interaction was tested as described for Fig. 1C, but in the absence or presence of 5 µM PD98059, as indicated at the bottom.

View larger version (44K): [in this window] [in a new window]   FIG. 4.   Phosphorylation of SMRT induced by As2O3. (A) SMRT immunoblot of CV-1 cells after MEKK-1 and As2O3 treatments. pCMV-SMRT-C (a construct limited to expressing amino acids 751 to 1495 of SMRT) was introduced into the CV-1 cells together with an empty vector (None) or a MEKK-1 expression vector; alternatively, the cells were treated with 20 µM As2O3. Whole-cell lysates were prepared from the cells, and the extract was analyzed by Western blotting using antibody specific for SMRT protein. (B) Phosphatase treatment of SMRT reverses the change in electrophoretic mobility induced by arsenite. CV-1 cells were transfected together with pCMV-SMRT-C (amino acids 751 to 1495) and either empty vector or the MEKK-1 expression vector. After As2O3 treatment as in panel A, whole-cell lysates were prepared and were incubated without or with 1 U of calf intestinal alkaline phosphatase (CIAP) for 30 min. (C) SMRT shifts in mobility rapidly after arsenite treatment. The experiment used for panel A was repeated, with samples taken and analyzed at the time points listed.

View larger version (59K): [in this window] [in a new window]   FIG. 5.   Effect of As2O3 treatment on subcellular location of SMRT proteins. CV-1 cells were transfected with GFP-SMRT alone, cotransfected with MEKK-1, or treated with 15 µM As2O3 after transfection. The subcellular location of the GFP-SMRT was subsequently visualized by confocal fluorescent microscopy. As a control experiment, the subcellular location of TFIIB under the same conditions was analyzed by immunofluorescence. (A) GFP-SMRT alone; (B) GFP-SMRT plus MEKK-1 expression vector; (C and D) GFP-SMRT plus As2O3 treatment; (E) TFIIB without As2O3 treatment; (F) TFIIB after treatment with 15 µM As2O3.

Arsenite can induce both apoptosis and an incomplete differentiation response in APL-derived NB-4 cells. The ability of arsenite to antagonize SMRT function in CV-1 cells led us to examine the effects of arsenite in APLs. We employed NB-4 cells, which are derived from a human APL and which synthesize the PML-RAR chimeric oncoprotein. NB-4 cells can be induced to differentiate by treatment with high levels of all-trans retinoic acid, and this response is thought to be mediated by a hormone-mediated release of corepressor from the PML-RAR protein (15-17, 35). Consistent with these prior observations, addition of all-trans retinoic acid to our NB-4 cell cultures resulted in a strong differentiation response which could be detected using FACS analysis with an antibody directed against CD-11c, an early differentiation marker (Fig. 6A), or a colorimetric assay for leukocyte alkaline phosphatase, a late differentiation marker (data not shown) (4, 14). In contrast to these robust differentiation responses, all-trans retinoic acid treatment induced little or no apoptosis in the NB-4 cells, as determined by TUNEL assay (Fig. 6B).

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Apoptosis and differentiation of NB-4 cells upon As2O3 treatment. (A) Effect of As2O3 and a MEK-1 inhibitor on the differentiation of NB-4 cells. NB-4 cells were left untreated (None), treated with all-trans retinoic acid (ATRA), or treated with As2O3 in the presence or absence of the MEK-1 inhibitor U0126 for 72 h. Cells were subjected to flow cytometry after staining with phycoerythrin-conjugated anti-CD11c antibody. (B) Effect of As2O3 and MEK-1 inhibitor on the apoptosis of NB-4 cells. NB-4 cells were treated with a 1 µM concentration of either ATRA or As2O3 in the presence or absence of the MEK-1 inhibitor U0126 for 72 h. Cells were subjected to flow cytometry after TUNEL reaction. (C) Induction of MEK-1 signaling by arsenite in NB-4 cells treated with As2O3. The cells were lysed, and the lysate was analyzed by SDS-PAGE and immunoblotting using anti-phospho-MEK-1/2 (Ser217/221) antibody as for Fig. 3A.

The effects of arsenic trioxide on APL cell differentiation and apoptosis are mediated, in part, through a MEK-1 intermediate and may operate by disrupting the interaction between SMRT and the PML-RAR oncoprotein. PML-RAR-mediated leukemogenesis is closely linked to the ability of PML-RAR to bind corepressor and to act as a dominant negative repressor of transcription; release of corepressor from PML-RAR in response to supraphysiological concentrations of all-trans retinoic acid results in differentiation of the APL cells and remission of disease (15-17, 35). Might the ability of arsenite to induce APL cell differentiation reflect a corresponding ability of arsenite to inhibit the interaction of SMRT with PML-RAR? We first tested whether the effects of arsenite on the NB-4 cells involve an activation of a MAP kinase cascade similar to that observed in CV-1 cells. Consistent with this hypothesis, arsenic trioxide induced a transient increase in MAP kinase cascade signaling in the NB-4 cells, as demonstrated by an increase in phosphorylation of MEK-1/2 (Fig. 6C); MEK-1/2 phosphorylation in NB-4 cells peaked somewhat more rapidly than that in CV-1 cells and then returned to baseline (Fig. 3), presumably reflecting differences in signal induction and attenuation in the two different cell lines. Is this activation of this MAP kinase cascade involved in mediating the effects of arsenite on NB-4 cell differentiation and apoptosis? Indeed, treatment of the NB-4 cells with the MEK-1/2 inhibitor U0126 significantly impaired the ability of arsenite to induce differentiation and, to a lesser degree, apoptosis in these cells (Fig. 6A and B). In contrast, U0126 did not inhibit the differentiation of NB-4 cells in response to all-trans retinoic acid treatment (Fig. 6A).

View larger version (31K): [in this window] [in a new window]   FIG. 7.   Effect of As2O3 on the physical association of PML-RAR with SMRT protein in NB-4 cells. (A) Physical association of SMRT protein with PML-RAR in NB-4 cells. NB-4 cells were treated with all-trans retinoic acid (ATRA) or As2O3 for 24 h. Whole-cell extracts were prepared and subjected to immunoprecipitation with SMRT or PML antibody. The immunoblots were subsequently probed with the antibodies indicated on the right. (B) Loss of the physical association of SMRT with PML-RAR occurs before degradation of PML-RAR protein in NB-4 cells. NB-4 cells were treated with 0.1 µM As2O3. Whole-cell extracts were prepared at the indicated times and subjected to immunoprecipitation with SMRT, RAR, or PML antibody. The immunoprecipitates were then analyzed by SDS-PAGE, and the immunoblot was probed with anti-PML antibodies. The positions of SMRT, PML-RAR, and the product of the untranslocated PML locus are indicated. (C) Quantification of data in panel B.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Arsenic trioxide is a potent inhibitor of SMRT function. The ability of nuclear receptors to modulate gene expression is mediated through the recruitment of auxiliary proteins, referred to as corepressors and coactivators (7, 10, 22, 54, 60, 66). The interactions of nuclear receptors with these auxiliary proteins are tightly regulated and play a central role in determining the transcriptional properties, positive or negative, displayed by a given receptor. Hormone ligand is one important modulator of this corepressor-coactivator exchange; binding of cognate hormone is thought to induce a specific change in the conformation of the nuclear receptors that leads to a release of the corepressor complex and an acquisition of the coactivator complex (23, 33, 44, 47). However, the association of these auxiliary proteins with nuclear receptors can also be regulated in a ligand-independent manner through the effects of protein kinases (19, 62). We have previously shown that SMRT is phosphorylated in response to a MAP kinase cascade that is initiated by the EGF receptor and propagated through the kinase hierarchy by MEKK-1 and MEK-1 (20). As a result of these phosphorylation events, the ability of SMRT to interact with T3Rs, RARs, or PLZF is severely impaired.

Arsenite results in dissociation of SMRT from PML-RAR and induces a mixed differentiation and apoptotic response in APL-derived cells that is counteracted by inhibitors of MEK-1 signaling. APL in humans is associated with chromosomal translocations that lead to the synthesis of aberrant x-RAR chimeric proteins (8, 11, 29, 41, 45). These x-RAR chimeras retain the ability to bind to retinoid-responsive target genes but are impaired in hormone-mediated corepressor release, and they are thought to function as dominant negative inhibitors of normal retinoid signaling. Consistent with this proposal, supraphysiological levels of retinoic acid that induce release of corepressor from PML-RAR also induce differentiation in PML-RAR APL cells in culture and result in clinical remission in APL patients bearing the PML-RAR translocation (10, 15-17, 34, 35, 42).

Arsenite and cancer. Arsenic is an element that occurs in nature in many forms and exerts many pleiotropic effects on biological systems. In addition to its widely recognized, acutely toxic actions, arsenic produces many chronic effects on organisms. Somewhat paradoxically, in light of its antineoplastic effects in APL, arsenic trioxide is also known to be a powerful tumor promoter in human beings, and arsenic-mediated oncogenicity represents a significant health issue both for personnel in the chemical industry and for the human population as a whole as a result of arsenic contamination of ground water (55). Arsenic operates in biological systems through multiple mechanisms. Based on the results presented here, we suggest that a previously unrecognized effect of arsenic, the ability of arsenite to inhibit corepressor function, must be added to the mechanisms by which arsenic can perturb biological systems and should be considered in interpreting the toxic, oncogenic, and antineoplastic effects of the trivalent metalloids.