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. 2006 Dec 13;25(24):5703-15.
doi: 10.1038/sj.emboj.7601435. Epub 2006 Nov 30.

p53 mediates the negative regulation of MDM2 by orphan receptor TR3

Bi-xing Zhao  1 , Hang-zi Chen, Na-zi Lei, Gui-deng Li, Wen-xiu Zhao, Yan-yan Zhan, Bo Liu, Sheng-cai Lin, Qiao Wu
Affiliations

p53 mediates the negative regulation of MDM2 by orphan receptor TR3

Bi-xing Zhao et al. EMBO J. .

Abstract

MDM2 is an oncoprotein whose transforming potential is activated by overexpression. The expression level of MDM2 is negatively regulated by orphan receptor TR3 that mainly acts as a transcriptional factor to regulate gene expression. However, the underlying mechanism is largely unclear. Here, we present the first evidence that inhibition of TR3 on MDM2 is mediated by p53. We found that TR3 directly interacts with p53 but not MDM2, and such interaction is critical for TR3 to inhibit MDM2 expression. TR3 downregulates p53 transcriptional activity by blocking its acetylation, leading to a decrease on the transcription level of MDM2. Furthermore, TR3 binding to p53 obstructs its ubiquitination and degradation induced by MDM2, resulting in the MDM2 ubiquitination and degradation. In addition, TR3 could enhance p53-mediated apoptosis induced by UV irradiation. Taken together, our findings demonstrate that p53 mediates the suppression of TR3 on MDM2 at both transcriptional and post-transcriptional level and suggest TR3 as a potential target to develop new anticancer agents that restrict MDM2-induced tumor progression.

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Figures

Figure 1
Figure 1
TR3 inhibits cellular MDM2 expression mediated by p53. (A) TR3 inhibited MDM2 expression in a dose-dependent manner. Increasing amounts of Myc-TR3 were transfected into 293 cells. After transfection, cell lysates were prepared and analyzed by Western blotting using anti-MDM2 antibody to indicate endogenous MDM2 level. (B) Inhibition of MDM2 by TR3 required p53. Flag-TR3, Myc-p53, or Myc-p53R175H (a dominant-negative form of p53) expression vectors were transfected into H1299 or 293 cells as indicated, and then monitored for their expression by Western blotting using anti-Flag or anti-Myc antibody. Anti-MDM2 antibody was used to indicate endogenous MDM2 level. (C) Effect of VP-16 on endogenous TR3, p53, and MDM2 expression. HepG2 cells were transfected with siRNA-p53 or its control siRNA-Ctrl. Cells were treated with VP-16 for 6 h, and lysates were then subjected to Western blot analysis using anti-TR3, -MDM2, or -p53 antibody. (D) Effect of UV on the expressions of TR3, p53, MDM2, and p21waf1/cip1. HepG2 cells were transfected with GFP-TR3 expression vector. After transfection, cells were irradiated with UV (50 J/m2), and then harvested 8 h later. Cell lysates were prepared and analyzed for expressions of p53, MDM2, p21waf1/cip1, and TR3 by Western blot using their corresponding antibodies (for p53, MDM2, and p21waf1/cip1) or GFP antibody (for TR3). In each panel, Tubulin expression served as control for indicating the similar loading proteins in each lane.
Figure 2
Figure 2
TR3 interacts with p53. (A) In vitro Co-IP experiment in 293 cells. Flag-TR3, Myc-p53, or HA-MDM2 were transfected into 293 cells as indicated and cell lysates were then immunoprecipitated using anti-Flag or -HA antibody. The immunoprecipitates were examined by Western blotting using anti-Myc antibody. Input represented 10% of cell lysates used in the Co-IP experiment. (B) In vivo Co-IP experiment in HepG2 cells. Cell lysates from cells treated with or without VP-16 for 6 h were incubated with anti-TR3 antibody. For Western blotting of immunoprecipitates, anti-p53 antibody was used. (C) GST pull-down assay for determination of TR3–p53 interaction. GST-TR3, GST-p53, GST-MDM2, or GST control protein was incubated with related protein as described in Materials and methods. Bound proteins were analyzed by Western blotting. (D) TR3 interacted with p53 in yeast. TR3 or p53 cDNA was cloned into the yeast expression vector as described in Materials and methods. The resulting expression vectors were introduced into yeast cells. β-Gal activity was assayed. The result represented the average±mean from three independent experiments.
Figure 3
Figure 3
Interaction of TR3 with p53 is required for inhibition of MDM2. (A) Identification of TR3 sequence critical for p53 binding. Schematic diagrams depict different TR3 deletion constructs used in the domain mapping experiments (Top). 293 cells were transfected with Myc-p53 and GFP-TR3 truncation mutants. Cell lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates and cell lysates were then analyzed by Western blotting separately using anti-GFP-antibody for GFP-TR3 and its truncation mutants, and anti-Myc antibody for Myc-p53. GFP was used as a negative control. (B) Effect of different TR3 truncation mutants on MDM2 inhibition. GFP-TR3 and its truncation mutants were transfected into 293 cells. Endogenous MDM2 level was monitored by Western blotting by using anti-MDM2 antibody. GFP-TR3 and its mutants were revealed by anti-GFP antibody. (C) Interaction of TR3 with p53 and its deletion mutants. Structures of deletion mutants of p53 were shown on the top. 293 cells were transfected with HA-TR3 and different Myc-p53 deletion mutants as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates and cell lysates were then analyzed by Western blotting separately using anti-Myc-antibody for Myc-p53 and its deletion mutants, and anti-HA antibody for HA-TR3. (D) Effect of TR3 on MDM2 inhibition mediated by different p53 deletion mutants. GFP-TR3, together with Myc-p53 or its deletion mutants as indicated, was transfected into H1299 cells. Endogenous MDM2 level was monitored by Western blotting by using anti-MDM2 antibody. GFP-TR3, p53, and its deletion mutants were revealed by anti-GFP antibody or anti-Myc antibody.
Figure 4
Figure 4
Regulation of p53 transcriptional activity by TR3. (A–C) TR3 repressed p53 transcriptional activity. p53-Luciferase reporter and β-gal gene expression vector, together with different dose of PECE-TR3 expression vector as indicated, was transfected into 293, U2OS, and H1299 cells in the absence or presence of p53 expression vector. Reporter gene activity was determined and normalized in relation to the cotransfected β-gal activity. The bars represent the average±mean from three independent experiments. (D–F) Effect of TR3 truncation mutants on transcriptional activity of p53. p53-Luciferase reporter and β-gal gene expression vector, together with different combinations of TR3 truncation mutants in the absence or presence of p53 expression vector, were transfected into 293, H1299, and U2OS cells as indicated. Reporter gene activity was determined and normalized as described above. (G) TR3 inhibited p300-induced p53 acetylation. Myc-p53, HA-p300, and different amount of Flag-TR3 were transfected into 293 cells. After transfection, cell lysates were immunoprecipitated using anti-Myc antibody for p53. Acetylated p53 was determined by anti-acetylated Lys antibody in Western blot analysis. The expressions of TR3 and p300 were immunoblotted by anti-Flag and -HA antibody, respectively. (H) Effect of TR3 on p300-induced p53 transcriptional activity. TR3, TR3 mutants or p300 expression vector, p53-Luciferase reporter, and β-gal gene expression vector were transfected into U2OS cells as indicated. Reporter gene activity was determined and normalized as described above.
Figure 5
Figure 5
Effect of TR3 on p53-induced MDM2 mRNA expression. (A, B) TR3 regulated MDM2 mRNA expression through p53. 293 cells were transfected with different expression vectors, including Flag-TR3 and its mutants, Myc-p53, and SiRNA-p53 as indicated. After transfection, total RNA was prepared and analyzed for expression of MDM2 mRNA by RT–PCR and real-time PCR. Representatives of at least three independent experiments with similar results are shown. (C) TR3 decreased MDM2 promoter-Luc reporter activity. 293 and U2OS cells were transfected with different expression vectors, including PECE-TR3 and its deletion mutants as indicated. Measurement of MDM2 promoter-Luc activity and data presentation were as described in the legend to Figure 4A. (D) Effect of UV on regulation of apoptosis. HepG2 cells were transfected with TR3 expression vector. After transfection, cells were irradiated with UV (50 J/m2), and then harvested 8 h later. Cells were immunostained with PI as described in Materials and methods, then analyzed by using flow cytometer. The numbers indicate the apoptotic rate. Two independent experiments were carried our. (E) Effect of TR3 on p53-induced apoptosis in H1299 cells. TR3, with or without p53, was transfected into cells. Apoptotic rate was determined as described in Figure 5D.
Figure 6
Figure 6
(A, B) TR3 protects p53 from ubiquitination and degradation by MDM2. Different expression vectors as indicated were transfected into 293 cells. The expression of p53, MDM2, and TR3 was analyzed by Western blotting. For ubiquitination assay, His-ubiquitin and different expression vectors indicated were coexpressed in 293 cells. p53 ubiquitination was monitored in immunoblots performed on nickel–agarose beads-purified proteins, and then probed with antibody to GFP to identify any p53-associated ubiquitin.
Figure 7
Figure 7
Interaction of TR3 with p53 promotes MDM2 self-degradation. (A) Subcellular localization of TR3, p53, and MDM2 in 293 cells. Myc-TR3, GFP-p53, and/or HA-MDM2 were transfected into cells as indicated. Cells were immunostained for detecting TR3 by Myc antibody followed by Texas Red-conjugated secondary antibody and for detecting MDM2 by HA antibody followed by Texas Red or Alexa flour 350-conjugated secondary antibody. Stained cells were visualized with the confocal microscope. (B) The relative binding affinity of MDM2 and TR3 to p53 by Co-IP assay. 293 cells were transfected with Myc-p53, HA-TR3, and HA-MDM2. Western blot against the common HA tag was used to compare the expression levels of TR3 and MDM2. Binding affinity of TR3 and MDM2 to p53 was shown by using anti-HA antibody in p53 immunoprecipites. (C) Fluorescence emission spectra of MDM2 and TR3. GST-p53, in a final concentration of 2 μM, was incubated with 1 μM of GST-MDM2 or GST-TR3 protein (upper panel). Either 1 μM of MDM2 or TR3 protein was incubated with different concentration of p53 (from 26 to 182 nM). After incubation for 2 min at pH 8.0, 25°C, the fluorescence intensity was detected (down panel). All spectra were recorded at λex=275 nm. (D, E) TR3 binding to p53 resulted in MDM2 ubiquitination. Different expression vectors, including TR3 and p53 deletion mutants as indicated, were cotransfected into 293 cells and then treated with MG132 for 3 h. MDM2 ubiquitination was monitored in immunoblots performed on nickel resin-purified proteins, and then probed with antibody against HA to identify any MDM2-associated ubiquitin. To show the expression levels of MDM2 protein, the same cell lysates were subjected to Western blotting, probing with antibody against HA. Tubulin was used as a loading control. (F) Interaction among TR3, p53, and MDM2 point mutant. Myc-p53, HA-MDM2/C464A, and increasing Flag-TR3 expression vectors were transfected into 293 cells as indicated, then Co-IP assay was performed as described in Materials and methods. (G) TR3 inhibited exogenous MDM2 expression in a dose-dependent manner. Myc-TR3 and HA-MDM2 were transfected into 293 cells. MDM2 expression level was determined by Western blotting using anti-HA antibody. (H) Effect of CHX on MDM2 degradation. Myc-TR3 and HA-MDM2 were transfected into 293 cells, and then treated with CHX (100 μg/ml) for the indicated times. MDM2 expression level was determined by Western blotting using anti-HA antibody. The levels of MDM2 protein were quantified by densitometry. (I) Effect of CHX on p53 degradation. Myc-p53 and Flag-TR3 were transfected into 293 cells, and then treated with CHX (100 μg/ml) for the indicated times. p53 expression level was determined by Western blotting using anti-Myc antibody. The levels of p53 protein were quantified by densitometry.
Figure 7
Figure 7
(F) Interaction among TR3, p53, and MDM2 point mutant. Myc-p53, HA-MDM2/C464A, and increasing Flag-TR3 expression vectors were transfected into 293 cells as indicated, then Co-IP assay was performed as described in Materials and methods. (G) TR3 inhibited exogenous MDM2 expression in a dose-dependent manner. Myc-TR3 and HA-MDM2 were transfected into 293 cells. MDM2 expression level was determined by Western blotting using anti-HA antibody. (H) Effect of CHX on MDM2 degradation. Myc-TR3 and HA-MDM2 were transfected into 293 cells, and then treated with CHX (100 μg/ml) for the indicated times. MDM2 expression level was determined by Western blotting using anti-HA antibody. The levels of MDM2 protein were quantified by densitometry. (I) Effect of CHX on p53 degradation. Myc-p53 and Flag-TR3 were transfected into 293 cells, and then treated with CHX (100 μg/ml) for the indicated times. p53 expression level was determined by Western blotting using anti-Myc antibody. The levels of p53 protein were quantified by densitometry.
Figure 8
Figure 8
A proposed model for p53 to mediate the negative regulation of MDM2 by TR3.
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