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. 2000 Feb;20(3):957-70.
doi: 10.1128/MCB.20.3.957-970.2000.

Orphan receptor COUP-TF is required for induction of retinoic acid receptor beta, growth inhibition, and apoptosis by retinoic acid in cancer cells

B Lin  1 , G Q Chen, D Xiao, S K Kolluri, X Cao, H Su, X K Zhang
Affiliations

Orphan receptor COUP-TF is required for induction of retinoic acid receptor beta, growth inhibition, and apoptosis by retinoic acid in cancer cells

B Lin et al. Mol Cell Biol. 2000 Feb.

Abstract

Retinoic acid receptor beta (RARbeta) plays a critical role in mediating the anticancer effects of retinoids. Expression of RARbeta is highly induced by retinoic acid (RA) through a RA response element (betaRARE) that is activated by heterodimers of RARs and retinoid X receptors (RXRs). However, RARbeta induction is often lost in cancer cells despite expression of RARs and RXRs. In this study, we provide evidence that orphan receptor COUP-TF is required for induction of RARbeta expression, growth inhibition, and apoptosis by RA in cancer cells. Expression of COUP-TF correlates with RARbeta induction in a variety of cancer cell lines. In addition, stable expression of COUP-TF in COUP-TF-negative cancer cells restores induction of RARbeta expression, growth inhibition, and apoptosis by RA, whereas inhibition of COUP-TF by expression of COUP-TF antisense RNA represses the RA effects. In a transient transfection assay, COUP-TF strongly induced transcriptional activity of the RARbeta promoter in a RA- and RARalpha-dependent manner. By mutation analysis, we demonstrate that the effect of COUP-TF requires its binding to a DR-8 element present in the RARbeta promoter. The binding of COUP-TF to the DR-8 element synergistically increases the RA-dependent RARalpha transactivation function by enhancing the interaction of RARalpha with its coactivator CREB binding protein. These results demonstrate that COUP-TF, by serving as an accessory protein for RARalpha to induce RARbeta expression, plays a critical role in regulating the anticancer activities of retinoids.

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Figures

FIG. 1
FIG. 1
Correlation between COUP-TF expression and RARβ induction by RA in human breast cancer (A), bladder cancer (B), and lung cancer (C) lines. Total RNAs were prepared from the indicated cancer cell lines treated with or without all-trans-RA (10−6 M) for 24 h and analyzed for the expression of COUP-TFI and RARβ. For a control, the expression of β-actin is shown.
FIG. 2
FIG. 2
Stable expression of COUP-TF in COUP-TF-negative, RA-resistant MDA-MB231 cells enhances the effect of RA on RARβ induction, growth inhibition, and apoptosis induction. (A) Expression of transfected COUP-TFI in MDA-MB231 cells. COUP-TFI was stably transfected into MDA-MB231 cells, and expression of transfected COUP-TFI was determined by Northern blotting. For comparison, cells transfected with the empty vector (MB231/vector) were used. (B) Expression of RARβ gene in MDA-MB231 cells and MDA-MB231/COUP-TF stable clones. Total RNAs were prepared from MDA-MB231, its COUP-TFI stable clones (MB231/COUP#16 and MB231/COUP#10), and MDA-MB231 transfected with the empty vector (MB231/vector). Cells were treated with or without all-trans-RA (10−6 M) for 24 h and analyzed for the expression of RARβ by Northern blotting. In the control, the expression of β-actin is shown. (C) Growth-inhibitory effect of RA in MDA-MB231 and MDA-MB231/COUP-TF stable clones. Cells were seeded at 1,000 cells per well in a 96-well plate and treated with the indicated concentrations of all-trans-RA for 6 days. The numbers of viable cells were determined by the MTT assay. (D) Effect of RA on apoptosis of MDA-MB231 cells and MDA-MB231/COUP-TF stable clones. Cells were treated with or without all-trans-RA (10−6 M) for 48 h, and DNA fragmentations were then determined by the TdT assay. Representative histograms show relative apoptotic cell numbers.
FIG. 3
FIG. 3
Inhibition of COUP-TF expression by stable expression of COUP-TF anti-sense RNA in COUP-TF-positive J82 cells represses effect of RA on RARβ expression, growth inhibition, and apoptosis induction. (A) Expression of transfected COUP-TFI antisense RNA in J82 cells. COUP-TFI cDNA was stably transfected into J82 cells, and expression of transfected COUP-TFI antisense RNA in a selected stable clone (J82/A-COUP) was analyzed by Northern blotting. For comparison, the parental J82 cells and J82 cells stably transfected with the empty vector (J82/vector) were used. (B) Inhibition of RARβ induction by RA by stable expression of COUP-TFI antisense RNA in J82 cells. Total RNAs were prepared from J82, J82/vector, and J82/A-COUP cells treated with or without all-trans-RA (10−6 M) for 24 h and analyzed for the expression of RARβ by Northern blotting. The expression of β-actin is shown for the control. (C) Inhibition of RA-induced growth inhibition by stable expression of COUP-TF antisense RNA in J82 cells. Cells were seeded at 1,000 cells per well in a 96-well plate and treated with the indicated concentrations of all-trans-RA for 6 days. The numbers of viable cells were determined by the MTT assay. (D) Inhibition of RA-induced apoptosis of J82 cells by COUP-TF antisense RNA. Cells were treated with or without all-trans-RA (10−6 M) for 48 h, and DNA fragmentations were then determined by the TdT assay. Representative histograms show relative apoptotic cell numbers.
FIG. 4
FIG. 4
Inhibition of anchorage-independent growth of MDA-MB231 cells by COUP-TF gene expression. (A) Visualization of colonies formed in the soft agar by parental MDA-MB231, MB231/COUP#10, MB231/COUP#16, and MB231/vector cells in the presence or absence of RA (10−7 M). (B) Quantitation of colonies formed by parental MDA-MB231, MB231/COUP#10, MB231/COUP#16, and MB231/vector cells. Colonies formed by MB231/COUP#10, MB231/COUP#16, MB231/vector, and parental MDA-MB231 cells in the presence or absence of all-trans-RA (10−7 M) were scored and expressed as percentages of colonies formed by cells treated with control solvent.
FIG. 5
FIG. 5
COUP-TF enhances RARβ promoter activity in a RARα- and RA-dependent manner. (A) Activation of RARβ promoter activity by COUP-TF in CV-1 cells. RARβ promoter reporter (−745RARβCAT; 700 ng) was cotransfected with the indicated amounts of expression vector for COUP-TFII and RARα into CV-1 cells. Cells were treated with or without all-trans-RA (10−6 M) for 24 h and assayed for CAT activity. (B) Activation of RARβ promoter in HT-1376 bladder cancer cells by COUP-TF. Cells were transfected with 1,500 ng of −745RARβCAT reporter gene together with expression vectors for RARα (300 ng) and/or COUP-TFII (300 ng). Cells were treated with or without all-trans-RA (10−6 M) and 24 h later were assayed for CAT activity. Data shown represent the means of three independent experiments.
FIG. 6
FIG. 6
Identification of COUP-TF response element in the RARβ promoter. (A) Schematic representation of the RARβ promoter deletion mutants. The Sp-1 binding site, βRARE, and the TATA box are indicated. (B) Effect of COUP-TFII on RA-induced RARα transactivation activity of various RARβ promoter deletion mutants. CV-1 cells were transfected with 700 ng of CAT reporter gene containing the indicated RARβ promoter fragment together with expression vectors for COUP-TFII (50 ng) and RARα (20 ng). Cells were then treated with or without all-trans-RA (10−6 M) and 24 h later were assayed for CAT activity. −, without receptor cotransfection. Data shown represent the means of three independent experiments.
FIG. 7
FIG. 7
Analysis of COUP-TF binding on the COUP-TF-RE in the RARβ promoter. (A) Sequence of the COUP-TF-RE. Arrows indicate the AGGTCA-like core motifs. (B) Binding of COUP-TF on the COUP-TF-RE. In vitro-synthesized COUP-TFI or COUP-TFII was incubated with 32P-labeled oligonucleotide containing the COUP-TF-RE and analyzed by a gel retardation assay. For comparison, the binding of RARα, RXRα, and the RARα-RXRα heterodimer was analyzed. For the competition assay, a 50-fold excess amount of the indicated oligonucleotide was used. −, without competitor; solid arrowheads, specific COUP-TF binding complex; open arrowheads, nonspecific binding complex.
FIG. 8
FIG. 8
COUP-TF-RE is required for positive regulation of RARβ promoter activity by COUP-TF. (A) Mutations of the COUP-TF-RE. Depicted are the COUP-TF-RE sequence and its mutations (boldfaced). (B) The mutated COUP-TF-REs failed to bind to the COUP-TF protein. The indicated COUP-TF-RE mutant was synthesized and analyzed for its binding to COUP-TF by the gel retardation assay. In vitro-synthesized COUP-TF protein was incubated with 32P-labeled COUP-TF-RE or the mutated COUP-TF-RE and analyzed by a gel retardation assay. Solid arrowhead, specific COUP-TAF binding complex; open arrowhead, nonspecific binding. (C) Mutations of COUP-TF-RE abolished the effect of COUP-TF on the RARα transactivation function in the RARβ promoter. The COUP-TF-RE in the RARβ promoter was mutated by PCR. The resulting RARβ promoter mutants (−745RARβCAT/COUP-TF-RE/M1 and −745RARβCAT/COUP-TF-RE/M2) were analyzed by transient transfection assay in CV-1 cells for their effect on COUP-TF activity. CV-1 cells were transfected with the 700-ng reporter gene together with the indicated amounts of expression vectors for RARα and COUP-TFII. Cells were treated with or without all-trans-RA (10−6 M) and 24 h later were assayed for CAT activity. CAT activity was normalized for transfection efficiency to the corresponding β-Gal activity. Data shown represent the means of three independent experiments.
FIG. 9
FIG. 9
DNA binding of COUP-TF is required for its enhancing effect on RARα activity in the RARβ promoter. (A) Schematic representation of COUP-TF mutants. The DNA binding domain and the ligand domain of COUP-TFII as well as the A/B, C, D, and E/F domains are indicated. (B) Binding of the COUP-TFII mutants to COUP-TF-RE and TREpal. In vitro-synthesized COUP-TFII or the indicated COUP-TFII deletion mutant was incubated with 32P-labeled COUP-TF-RE or TREpal as indicated and analyzed by a gel retardation assay. The arrowheads indicate the specific COUP-TF binding complexes. (C) Effect of the COUP-TFII mutants on RARα activity in the RARβ promoter. CV-1 cells were transfected with 700 ng of −745RARβCAT reporter gene together with the expression receptor for RARα (20 ng) and the indicated amount of COUP-TFII or COUP-TFII deletion mutants. Cells were treated with or without all-trans-RA (10−6 M) for 24 h and were assayed for CAT activity. Data shown represent the means of three independent experiments.
FIG. 10
FIG. 10
COUP-TF-RE alone is not sufficient to confer the activation function of COUP-TF. COUP-TF-RE was cloned into pBLCAT2, which contained the TK promoter linked with the CAT gene. The resulting reporter construct (COUP-TF-RE-tk-CAT) was analyzed for its response to the COUP-TF effect by transient transfection assay. The reporter gene (100 ng) was cotransfected with the indicated amounts of expression receptor for RARα and COUP-TFII into CV-1 cells. After transfection, cells were treated with or without all-trans-RA (10−6 M) and 24 h later were assayed for CAT activity. Data shown represent the means of three independent experiments.
FIG. 11
FIG. 11
βRARE in the RARβ promoter is required for the enhancing effect of COUP-TF on RARα activity in the RARβ promoter. (A) Schematic representation of the βRARE mutations. Depicted are wild-type βRARE in the RARβ promoter and its mutations (boldface). Arrows indicate the care motifs of the βRARE. (B) Effect of βRARE mutations on retinoid receptor binding. In vitro-synthesized RARα and RXRα were incubated with 32P-labeled βRARE or the mutated βRARE and analyzed by a gel retardation assay. The arrowhead indicates specific RAR-RXR heterodimer binding. (C) Mutations of the βRARE impair the enhancing effect of COUP-TF on RARα activity in the RARβ promoter. Mutations in the βRARE of the RARβ promoter were introduced by PCR as described in Materials and Methods. The resulting RARβ promoter mutants (−745RARβCAT/βRARE/M1 and −745RARβCAT/βRARE/M2) were analyzed by transient transfection assay for the effect of COUP-TF. CV-1 cells were transfected with 700 ng of reporter gene together with the indicated amounts of expression vectors for COUP-TFII and RARα. Cells were then treated with or without all-trans-RA (10−6 M) and 24 h later were assayed for CAT activity. Data shown represent the means of three independent experiments.
FIG. 12
FIG. 12
COUP-TF enhances recruitment of CBP by RARα. (A) COUP-TF enhances interaction of RAR and CBP. COUP-TFI or RARα protein was synthesized in bacteria with pGEX-2T (Pharmacia). The GST–COUP-TFI or GST-RARα fusion protein was immobilized on glutathione-Sepharose beads, while the same amount of GST was also immobilized on beads as a control. 35S-labeled CBP was then mixed with beads and in vitro-synthesized RARα (for GST–COUP-TFI) or in vitro-synthesized COUP-TFI (for GST-RARα) in the presence of 10−6 M all-trans-RA. After extensive washing, the bound proteins were analyzed by SDS-PAGE. The input proteins are shown for comparison. (B) COUP-TFI interacts with RARα. To analyze the interaction between COUP-TF and RARα, 35S-labeled RARα was mixed with GST–COUP-TFI fusion protein on beads. After extensive washing, the bound proteins were analyzed by SDS-PAGE. As a comparison, the input proteins are shown. (C) COUP-TF increases the effect of CBP on RARα activity. The indicated amounts of expression vector for RARα, COUP-TFII, and CBP were cotransfected with RARβ promoter into CV-1 cells. Cells were treated with or without all-trans-RA (10−6 M) for 24 h and then were assayed for CAT activity. The corresponding β-Gal activity was normalized as a control.
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