Modulation of retinoid signaling by a cytoplasmic viral protein via sequestration of Sp110b, a potent transcriptional corepressor of retinoic acid receptor, from the nucleus - PubMed (original) (raw)

FIG. 1.

FIG. 1.

HCV core sensitized ATRA-induced cell death. (A) Production levels of the core in each clone of MCF-7 cells stably transfected with pLXSH-core (MCF-7-core 6 and -core 21) or the empty vector pLXSH (MCF-7-vec) were analyzed by immunoblot analysis with an anti-core (right, upper panel) and an anti-α-tubulin (right, lower panel) antibody as an internal control. MCF-7-core 6, MCF-7-core 21, and MCF-7-vec cells (5 × 104 each) were incubated in the absence (no treatment) or presence (ATRA) of 1 μM ATRA for 96 h. Living and dead cells were quantitated by staining with trypan blue. The average percentage of cell death from three independent experiments is presented. Open (bars 1 and 4), solid (bars 2 and 5), and hatched (bars 3 and 6) bars, MCF-7-vec, -core 6, and -core 21, respectively. (B) MCF-7 cells (5 × 104) transfected with 2.5 μg of pMACS-Kk and each expression plasmid given below were treated with (solid bars) or without (open bars) 1 μM ATRA for 96 h after the concentration of transfected cells by using the MACSelect system (see Materials and Methods). For bars 11 and 12, 50 μM monodansylcadaverine (MDC) was added simultaneously with ATRA. The percentage of cell death was estimated as described for panel A. Bars 1 and 2, empty vector; bars 3, 4, 11, and 12, pCMV-core (3 μg); bars 5 and 6, pCMV-core (6 μg); bars 7 and 8, pCMV-core (3 μg) and pCMV-Sp110b(389-453) (CBR fragment; 4.5 μg); bars 9 and 10, pCMV-core(6162M) (3 μg). (C) Enhancement of ATRA-induced tTGase expression by the core. MCF-7 cells (2 × 105) transfected with pMACS-Kk and each expression plasmid given below were treated for 48 h with (lanes 4 to 6) or without (lanes 1 to 3) 1 μM ATRA after cell concentration as described for panel B. Following the extraction of total RNA from these cells, mRNA levels of tTGase (upper panel) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (lower panel) were semiquantified by RT-PCR as described in Materials and Methods. RTase(−), experimental control treated like other samples, but without reverse transcriptase. Lanes: 1 and 4, empty vector; 2 and 5, pCMV-core; 3 and 6, pCMV-core and pCMV-Sp110b(389-453) (CBR fragment).

FIG. 2.

FIG. 2.

Activation of RARα-mediated transcription in cells expressing the core. (A) COS-7 cells were transfected with 25 ng of pRARE-Luc, pCMV-core at the doses indicated, and the empty vector for a total amount of 400 ng of plasmids. Cells were then treated with (solid bars) or without (open bars) 1 μM ATRA. At 24 h posttransfection, luciferase activities of whole-cell lysates were measured. Data are means of the relative luciferase activities in three independent experiments. Error bars, standard deviations. (B) MCF-7-core 6, MCF-7-core 21, and MCF-7-vec cells were transfected with 25 ng of the pRARE-Luc reporter plasmid. (C) Identification of the region within the core essential for activation of RARα-mediated transcription. COS-7 cells were transfected with 25 ng of pRARE-Luc and 350 ng of a series of truncated core expression plasmids [core full, pCMV-core; coreΔ(101-191), pCMV-coreΔ(101-191); coreΔ(1-20 + 81-191), pCMV-coreΔ(1-20 + 81-191); coreΔ(21-80)-p7, pCMV-coreΔ(21-80)-p7] or control plasmids (control, pKS+/CMV; p7, pCMV-p7). Experimental conditions and presentation of data for panels B and C were the same as those described for panel A.

FIG. 3.

FIG. 3.

Interaction of the core with Sp110b. (A) The 35S-labeled in vitro transcription-translation product of full-length core (top panel) or the core(6162M) mutant (center panel) was incubated with recombinant Sp110 or Sp110b fused to GST (GST-Sp110 or GST-Sp110b) or with GST as a negative control. The GST pulldown assay was performed as described in Materials and Methods. 1/10 input, signal for 1/10 the amount of 35S-labeled product used in the pulldown assay. Coomassie brilliant blue (CBB) staining patterns of pulled-down proteins are shown in the bottom panel. Arrowhead, circle, and square indicate the bands corresponding to GST, GST-Sp110, and GST-Sp110b, respectively. (B) Mapping of the region interacting with the core in Sp110b by deletion analysis. (Left) Schematic representations of the full-length and truncated mutants of Sp110 and Sp110b are shown. Numbers above the diagrams indicate the amino acid positions from the amino terminus of Sp110 or Sp110b. The Sp100-like domain (Sp), SAND domain (S), PHD (P), and bromodomain (B) are indicated. (Right) Designations of the GST fusion protein and 35S-labeled derivatives of Sp110 and Sp110b are given above and to the left of the autoradiograms (a through k), respectively. GST-core80, GST fused with the region of the core from aa 1 to 80. CBB staining patterns of pulled-down proteins are shown in the bottom panel. Arrow and arrowhead indicate bands corresponding to GST and GST-core80, respectively. Two dots indicate apparent degradation products of GST-core80. (C) Interaction between the core and Sp110b produced in the cells. Lysates from COS-7 cells transfected with 1 μg of pCMV-Sp110b and/or pCMV-core were used for coimmunoprecipitation, followed by immunoblot analysis. The combinations of plasmids used for the transfection are indicated at the top. “IP” designates the antibodies used for immunoprecipitation, either the anti-FLAG antibody (FL) or normal mouse IgG (used as a negative control). Coimmunoprecipitated core with FLAG-tagged Sp110b was detected with an anti-core antibody (top panel). Center and bottom panels show results of experiments in which the core and FLAG-tagged Sp110b in total-cell lysates from transfectants were detected by anti-core and anti-FLAG antibodies, respectively.

FIG. 4.

FIG. 4.

Expression of Sp110 and Sp110b mRNAs in human tissues and cell lines. (A) Northern blot analysis detecting Sp110 and Sp110b mRNAs in human tissues. (Upper panel) Ten-microgram portions of total RNAs from human brain, heart, liver, lung, kidney, bone marrow, placenta, thymus, testis, and spleen were analyzed by using a 32P-labeled Sp110b fragment as a probe. Positions of molecular size markers are indicated on the left. (Lower panel). Ethidium bromide staining of 28S rRNA served as a loading control. (B) Semiquantitative RT-PCR analysis of Sp110 and Sp110b mRNAs in human tissues and cell lines. (Upper panel) cDNA fragments of Sp110 and Sp110b mRNAs were simultaneously amplified by RT-PCR using a common sense primer and two antisense primers specific to Sp110 and Sp110b mRNAs, respectively. To evaluate the quantifying ability of this system, mixtures of in vitro-synthesized Sp110 and Sp110b RNAs at various concentrations (given above the gel, in femtograms) were used as templates (lanes 1 to 9). Total RNAs from human kidneys (lane 10) and spleens (lane 11) and from MCF-7 (lane 12), Huh-7 (lane 13), Jurkat (lane 14), and HeLa (lane 15) cells were examined. (Lower panel) As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was detected by a similar protocol (lanes 10 to 15). RTase(−), experimental control treated like other samples, but without reverse transcriptase.

FIG. 5.

FIG. 5.

Sp110b is a potent transcriptional corepressor of RARα. (A through C) Activation and suppression of RARα-mediated transcription by Sp110 and Sp110b, respectively. COS-7 cells were transfected with 25 ng of pRARE-Luc with pCA-Sp110 (A), pCMV-Sp110b (B), or pCMV-Sp110b with pSG5-RARα (C) at the indicated doses for a total of 400 ng of plasmids by adjusting with the empty vector. Additional conditions were as described in the legend to Fig. 2A. RARα protein production levels for each condition were examined by immunoblot analysis (C, lower panel). (D) Interaction of Sp110 and Sp110b with RARα. A GST pulldown assay was performed as described in Materials and Methods. 35S-labeled RARα was incubated with GST-Sp110, GST-Sp110b, or GST in the absence [ATRA(−)] or presence [ATRA (+)] of 1 μM ATRA. The Coomassie brilliant blue (CBB) staining pattern of pulled-down products is shown in the bottom panel. Positions of molecular size markers are given on the left. Arrowhead, circle, and square indicate bands corresponding to GST, GST-Sp110, and GST-Sp110b, respectively. (E) Coimmunoprecipitation assay detecting the interaction between Sp110 and RARα. Lysates from ATRA-treated 293T cells overproducing FLAG-tagged Sp110b (FL-Sp110b) and/or RXRα with HA-tagged RARα (HA-RARα) were used for coimmunoprecipitation followed by immunoblot analysis. Data are presented essentially as described in the legend to Fig. 3C. IgG, normal mouse IgG; HA, anti-HA antibody. FL-Sp110b coimmunoprecipitated (co-IP) with HA-RARα was detected by using an anti-FLAG antibody (upper panel). Center and lower panels show HA-RARα and FL-Sp110b in total-cell lysates detected by anti-HA and anti-FLAG antibodies, respectively. (F) DNA-protein complex immunoprecipitation assay detecting the RARα-Sp110b-RARE complex. 293T cells overproducing either RXRα with HA-RARα or FL-Sp110b, or both, and carrying either pRARE-Luc (RARE +) or the p-55BLuc reporter plasmid lacking RARE (RARE −), were treated with ATRA. Formaldehyde-cross-linked DNA-protein complexes were then immunoprecipitated with anti-HA (HA), anti-FLAG (FL), or normal mouse IgG (IgG). The DNA extracted from the respective immunoprecipitates was amplified by PCR as described in Materials and Methods.

FIG. 6.

FIG. 6.

The RARα activation capacity of HCV core was reduced by RNAi elimination of endogenous Sp110b. (A) RNAi elimination of endogenous Sp110b protein in HeLa cells. Extracts from cells treated with 300 U of gamma interferon/ml after transfection with either an siRNA specific for Sp110 and Sp110b [Sp110(b)-siRNA] or a randomized siRNA (control-siRNA) was analyzed with an antibody recognizing both Sp110 and Sp110b (upper panel) or anti-α-tubulin (lower panel). As a positive control, small amounts of extracts from cells overproducing FLAG-tagged Sp110 or Sp110b were also analyzed. Positions of molecular size markers (in kilodaltons) are given on the right. (B) HeLa cells were transfected with control-siRNA (bars 1 and 2) or Sp110(b)-siRNA (bars 3 and 4). At 24 h posttransfection, plasmid transfection with 25 ng of pRARE-Luc and 300 ng of either pKS+/CMV (bars 1 and 3) or pCMV-core (bars 2 and 4) was carried out. An additional 24 h later, the cells were harvested; luciferase activity was measured as described in the legend to Fig. 2A. Data are means of the relative luciferase activities in three independent experiments. Comparisons of luciferase activities (bars 1 versus 2 [×2.69] and bars 3 versus 4 [×1.30]) are shown above the graph. The combinations of siRNAs and plasmids used are indicated below the graph.

FIG. 7.

FIG. 7.

The subcellular localization of Sp110b was altered from the nucleus to an area around the cytoplasmic surface of ER membranes by core expression. (A) Indirect immunofluorescence analysis was performed on COS-7 cells transfected with pCMV-core (panel 1), pCA-Sp110 (panel 2), pCA-Sp110 with pSG5-RARα (panels 3 to 5), pCA-Sp110 with pCMV-core (panels 6 to 8), pCMV-Sp110b (panel 9), pCMV-Sp110b with pSG5-RARα (panels 10 to 12), pCMV-Sp110b with pCMV-core (panels 13 to 15), pCMV-core(6162 M) (panel 16), pCMV-Sp110b with pCMV-core(6162 M) (panels 17 to 19), pCMV-Sp110b(1-276 + 454-539) (panel 20), pCMV-Sp110b(1-276 + 454-539) with pCMV-core (panels 21 to 23), or pCMV-Sp110b and pCMV-core with pcDNA-Sp110b(389-453) (myc-CBR fragment) (panels 24 to 29) and on HeLa cells transfected with pKS+/CMV (panel 30) or pCMV-core (panels 31 to 33) following treatment with 300 U of gamma interferon/ml to allow the detection of endogenous Sp110b. The primary antibodies used were anti-FLAG (panels 2, 3, 6, 9, 10, 13, 17, 20, 21, and 27) (green), anti-Myc (panel 24) (green), anti-Sp110(b) (panels 30 and 31) (green), anti-core (panels 1, 7, 14, 16, 18, 22, 25, 28, and 32) (red), and anti-RARα (panels 4 and 11) (red). Merged images of green and red signals are shown in panels 5, 8, 12, 15, 19, 23, 26, 29, and 33. DAPI was used to visualize nuclear staining (right panels). (B) Subcellular fractionation was performed on cells transfected with 1 μg of pCMV-Sp110b (lanes 1 to 3), 1 μg of pCMV-core (lanes 4 to 6), and 1 μg each of pCMV-Sp110b and pCMV-core (lanes 7 to 9). The transfectants were homogenized and separated into nuclear (N), microsomal-membrane (MM), and cytosolic (C) fractions by centrifugation as described in Materials and Methods. The FLAG-tagged Sp110b, core, α-tubulin, and SC-35 proteins in those fractions were detected by immunoblot analysis.

FIG. 8.

FIG. 8.

Sequestration of Sp110b from the nucleus plays a significant role in the activation of RARα-mediated transcription by core expression. (A) A reporter assay was performed using COS-7 cells transfected with a total of 400 ng of plasmids including 25 ng of pRARE-Luc, the effector plasmids pCMV-Sp110b and pCMV-core, and empty vector. Other conditions were the same as those described in the legend to Fig. 2A. The amounts of the effector plasmids (in nanograms) in each experiment are given below the graph. (B) A reporter assay was performed using COS-7 cells transfected with a total of 400 ng of plasmids including 25 ng of pRARE-Luc, the effector plasmids pCMV-core, pCMV-Sp110b(389-453) (CBR fragment), and pCMV-core(6162M), and empty vector. Data are means of the relative luciferase activities from three independent experiments.

FIG. 9.

FIG. 9.

Schematic representation of the mechanistic model of activation of RARα-mediated transcription by the core. (i) In the absence of the core, Sp110b is located in the nucleus, playing a suppressive role in RARα-mediated transcription. (ii) In the presence of the core, Sp110b is sequestered to ER membranes through interaction with the core, which is located on the cytoplasmic surfaces of ER membranes. This sequestration results in a reduction of the transcriptional suppressive effect of Sp110b in the nucleus. Consequently, ATRA-induced transcription and expression of RARα-responsive genes, such as tTGase, are enhanced.