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First published online 19 October 2004
doi: 10.1242/jcs.01474

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RXR acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells

Xiao-Feng Lin^*, Bi-Xing Zhao^*, Hang-Zhi Chen, Xiao-Feng Ye, Chao-Yi Yang, Hai-Ying Zhou, Ming-Qing Zhang, Sheng-Cai Lin and Qiao Wu

Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, Fujian Province, 361005, China

View larger version (37K): [in a new window]   Fig. 1. RXR shuttles between the nucleus and the cytoplasm. (A) NIH3T3 cells were transfected with GFP-RXR expression vector and HeLa cells were then seeded onto the same coverslip. Before being fused by PEG to form heterokaryons, cells were cultured in medium containing cycloheximide (CHX) to inhibit new protein synthesis. Fused cells were treated with or without 9-cis retinoic acid (9-cis-RA, 1 µM) for 1 hour, then immunostained with Hoechst 33258 and tubulin antibody followed by Cy3-conjugated secondary antibody to display the nuclei and the whole cells simultaneously. The murine NIH3T3 nuclei gave a characteristic staining of intranuclear bodies (speckles) and the human HeLa nuclei displayed a diffuse pattern indicated by the arrows. (B) Subcellular localization of RXR in response to 9-cis retinoic acid. MGC80-3 cells were treated with or without 9-cis retinoic acid for the indicated times, fixed and immunostained with anti-RXR antibody followed by FITC-conjugated secondary antibody. Cells were also stained with propidium iodide (PI, Sigma, 50 µg/ml) to visualize nuclei. Images were visualized using a confocal microscope and the two images were merged (Overlay). (C) 9-cis retinoic acid-induced redistribution of RXR shown by western blotting in MGC80-3 cells. Cells were treated with 9-cis retinoic acid for the times indicated with nuclear and cytoplasmic fractions prepared as described in Materials and Methods. Different protein portions were then subjected to western blotting with anti-RXR antibody. Lamin B1 and -tubulin were used to quantify the amount of protein loaded.

View larger version (33K): [in a new window]   Fig. 2. Import and export pathways of RXR. (A) Effects of temperature and sodium azide on shuttling of RXR. Heterokaryons were prepared and visualized as described in Fig. 1A. Fused cells were separately incubated at 37°C or 4°C or with sodium azide (1 mM) for 2 hours. Arrows indicates the HeLa nuclei. (B) Effect of wheat germ agglutinin (WGA) on RXR import. WGA (100 µg/ml) was transfected into MGC80-3 cells by means of the Chariot protein delivery system for 2 hours and then harvested. Cell lysate, nuclear and cytoplasmic fractions were prepared as described in Materials and Methods, and were then subjected to western blotting with anti-RXR antibody. Lamin B1 and tubulin were used as loading controls. (C) Effects of leptomycin (LMB) on shuttling of RXR. NIH3T3 cells were transfected with GFP-RXR, then fused with HeLa cells. The heterokaryons were treated with or without LMB (100 µg/ml) for 2 hours. GFP-p53 was used as a positive control. The HeLa nuclei are indicated by arrows.

View larger version (33K): [in a new window]   Fig. 3. Intact DBD domain in RXR is responsible for NES function. (A) Schematic representation of RXR and its mutants. (B) Subcellular localization of RXR mutants. RXR and its mutants were fused to GFP, then transfected into NIH3T3 cells. After transfection, cells were stained with Hoechst 33258 to visualize nuclei. Localizations of GFP-RXR protein and its mutants were visualized using fluorescent microscope. (C) Shuttling behavior of RXR and its different mutants. GFP-RXR and different GFP-RXR mutants were separately transfected into NIH3T3 cells as indicated, then fused with HeLa cells. The heterokaryons were visualized as described in Fig. 1A. Arrows indicate HeLa nuclei.

View larger version (48K): [in a new window]   Fig. 4. Translocation of TR3 and RXR in response to 9-cis retinoic acid. (A) Colocalization of endogenous RXR and TR3 in the mitochondria. MGC80-3 cells were treated with or without 9-cis retinoic acid (1 µM) for 12 hours and then immunostained with anti-TR3, anti-RXR and anti-Hsp60 antibodies followed with corresponding FITC-, Cy3-and Cy5-conjugated secondary antibodies to show endogenous TR3, RXR and Hsp60 proteins simultaneously. The fluorescent images were visualized with a confocal microscope, and the images were merged as indicated (Overlay). (B) MGC80-3 cells were treated with or without 9-cis retinoic acid (1 µM) for the indicated times. Cell extracts were prepared and immunoprecipitated with anti-RXR antibody, then subjected to SDS-PAGE, blotted, and probed with anti-TR3 antibody. The immunoprecipitated RXR used in each lane was quantified by western blotting with anti-RXR antibody. The same extract was applied to ascertain the position and expression of TR3 by western blotting with antibodies against TR3 (Input). IgG was used as negative control where no signal band was detected. (C) Distribution of TR3 and RXR in MGC80-3 cells. Nuclear, cytosolic and mitochondrial fractions were prepared as described in Materials and Methods, and the expression of RXR and TR3 in response to 9-cis retinoic acid at different time points was determined by western blotting. Mitochondrial Hsp60, nuclear protein Lamin B1 and cytosolic tubulin were detected as protein loading controls.

View larger version (42K): [in a new window]   Fig. 5. RXRa serves as a carrier for TR3 translocation initiated by 9-cis retinoic acid. (A) Detection of TR3 translocation mediated by RXR in living MGC80-3 cells. Living cells transfected with GFP-TR3 alone or together with RXR were treated with 9-cis retinoic acid at different time points as indicated. The GFP-TR3 translocation from the nucleus to the cytoplasm was visualized under a fluorescent microscope. To detect the effect of LMB on TR3 translocation, transfected cells were pre-treated with LMB for 2 hours, followed by treatment of 9-cis retinoic acid for the indicated times. (B) Repression of endogenous RXR and TR3 by antisense RXR, antisense TR3 or TR3-siRNA. Cells were stably transfected with different expression vectors or transiently transfected with siRNA as described in Materials and Methods. Expression level of endogenous RXR or TR3 was detected by western blotting. Empty vector and scrambled-siRNA were used as controls. tubulin was used to quantify the amount of protein loaded in each lane. (C) Effects of antisense RXR, antisense TR3 and TR3-siRNA on the translocation of RXR and TR3. MGC80-3 cells were transfected with different expression vectors or siRNAs as described in B and then treated with 9-cis retinoic acid (1 µM) for 6 hours. Nuclear (N) and cytoplasmic (C) fractions were subjected to western blotting, probed with anti-TR3 or anti-RXR antibody as indicated. tubulin and lamin B1 were used as loading controls. Scrambled-siRNA was used as positive control.

View larger version (38K): [in a new window]   Fig. 6. Mitochondrial TR3 contributes to apoptosis of MGC80-3 cells. (A) Colocalization of mitochondrial TR3 with apoptotic cells. MGC80-3 cells were transfected with GFP-RXR alone or GFP-TR3 together with RXR, and then treated with 9-cis retinoic acid (1 µM) for 48 hours. The nuclear morphology stained by DAPI was visualized using a fluorescence microscope and the two images were merged. Apoptotic cells were indicated by arrows. (B) Effect of TR3 and its mutants on apoptosis induction. MGC80-3 cells were transfected with RXR either alone or with GFP-TR3, GFP-TR3N or GFP-TR3C, and then treated with or without 9-cis-RA (1 µM) for 48 hours. The cells were stained with DAPI to show nuclear morphology. Apoptotic cells that displayed nuclear condensation and fragmentation were scored by examination of 300 transfected cells.

View larger version (22K): [in a new window]   Fig. 7. TR3 with a deletion of its DBD is able to induce apoptosis. (A) Schematic representation of TR3 and its mutants. (B) Cellular localization of GFP-TR3DBD and GFP-TR3 in MGC80-3 cells. Cells were transfected with GFP-TR3DBD or GFP-TR3 respectively and immunostained with anti-Hsp60 antibody followed by Cy3-conjugated secondary antibody to visualize mitochondria. GFP-TR3DBD or GFP-TR3 and Hsp60 were visualized with a confocal microscope. (C) Effect of TR3DBD on apoptosis induction. MGC80-3 cells were transfected with GFP-TR3DBD or GFP-TR3, and then stained with DAPI to show nuclear morphology. Apoptotic cells were determined as described in Fig. 6B. GFP was used as a control.

View larger version (32K): [in a new window]   Fig. 8. RXR-TR3 interaction correlates well with their ability to target to the mitochondria. (A) Cellular localization of GFP-TR3N and GFP-TR3C in response to 9-cis-RA. GFP-TR3N or GFP-TR3C together with RXR was transfected into MGC80 cells. Cells were treated with or without 9-cis-RA (1 µM) for 12 hours, then immunostained with anti-Hsp60 antibody. GFP-TR3N, GFP-TR3C and mitochondria (Hsp60) were visualized using a confocal microscope. (B) Interaction of RXR with different TR3 mutants. GFP-TR3N or GFP-TR3C together with Myc-RXR was cotransfected into HEK293 cells. Cell extracts were prepared and immunoprecipitated with anti-Myc antibody. The immunoprecipitate was subjected to SDS-PAGE, blotted and probed with anti-GFP antibody. The same membrane was also blotted with anti-Myc antibody to determine immunoprecipitation (IP) specificity and efficiency. Input represents 5% of cell lysates used in the IP western blot assay. The empty vector was used as a control.

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