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. 2011 Aug 18;118(7):1865-76.
doi: 10.1182/blood-2010-12-326199. Epub 2011 Jun 24.

HTLV-1 bZIP factor enhances TGF-β signaling through p300 coactivator

Tiejun Zhao  1 , Yorifumi Satou, Kenji Sugata, Paola Miyazato, Patrick L Green, Takeshi Imamura, Masao Matsuoka
Affiliations

HTLV-1 bZIP factor enhances TGF-β signaling through p300 coactivator

Tiejun Zhao et al. Blood. .

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-β (TGF-β)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-β signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-β response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-β. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-β signaling. Our results suggest that HBZ, by enhancing TGF-β signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence.

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Figures

Figure 1
Figure 1
HBZ activated TGF-β signaling. In 12-well plates, HepG2 cells were cotransfected with 2 ng of phRL-TK, 0.5 μg of reporter plasmid TARE-Luc (A), 3TP-Lux (B), or 9 × CAGA-Luc (C), and 0.5 μg of pcDNA3.1-mycHis-sHBZ. At 24 hours after transfection, the cells were treated with TGF-β (10 ng/mL). After 24 hours, the cells were harvested and analyzed for luciferase activity. Expression of sHBZ was detected by Western blot (middle panel). Coomassie brilliant blue (CBB) staining was shown as the loading control (botom panel). (D) CTLL-2 cells were transfected with 3TP-Lux (2 μg), phRL-TK (10 ng), and pME18Sneo-sHBZ (0.4 μg) by electroporation. Luciferase activity was measured 24 hours after stimulation by TGF-β. (E) In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), and pcDNA3.1-mycHis-sHBZ (0, 5, 10, 20, 50, 100, 200, 1000, and 4000 ng). At 24 hours after transfection, the cells were treated with or without TGF-β. After 24 hours, the cells were harvested and analyzed for luciferase activity. mycHis-sHBZ was detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel).
Figure 2
Figure 2
HBZ interacts with Smad proteins. (A) HBZ interacted with Smad proteins. COS7 cells were cotransfected with mycHis-sHBZ (6 μg) and FLAG-Smad2, Smad3, Smad4, and Smad7 (6 μg). At 24 hours after transfection, the cells were treated with or without TGF-β (5 ng/mL). Cell lysates were subjected to immunoprecipitation using anti-c-Myc followed by immunoblotting using anti-FLAG for detection of Smad proteins. (B) sHBZ colocalized with Smad3 in the cell nucleus. COS7 cells were transfected with mycHis-sHBZ (0.6 μg) together with (v-viii) or without (iii-iv) FLAG-Smad3 (0.4 μg). sHBZ was detected using anti-MYC Cy3 antibody (iv,vi). Smad3 was detected using anti–Flag-biotin and secondary streptavidin-Alexa-488 antibody (ii,v). The overlay of sHBZ and Smad3 is shown (vii-viii). DAPI (4,6-diamidino-2-phenylindole) was used to counterstain the nucleus (i,iii). (C) HBZ did not influence the Smad3/Smad4 interaction. COS7 cells were transfected with the indicated expression vectors (3 μg each). Cell lysates were subjected to immunoprecipitation using anti-FLAG followed by immunoblot (IB) using anti–c-Myc.
Figure 3
Figure 3
HBZ activates TGF-β signaling dependent on p300. (A) E1A repressed HBZ-induced activation of TGF-β. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pME18Sneo-sHBZ (20 ng), and pCS2+-E1A or pCS2+-E1A-ΔNT (2 ng). Luciferase activity was measured 24 hours after stimulation by TGF-β (0, 10 ng/mL). (B) HBZ synergized with Smad3 and p300 to enhance TGF-β. HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pcDNA3.1-mycHis-sHBZ (200 ng), FLAG-Smad3 (50 ng), and pCMV-p300 (2, 5 μg). At 24 hours after transfection, the cells were treated with or without TGF-β (10 ng/mL). Luciferase activity was measured after 24 hours. Expression of sHBZ, Smad3, and p300 was detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel). (C) HBZ, Smad3, and p300 could form a ternary complex. mycHis-sHBZ (4 μg), FLAG-Smad3 (4 μg), and HA-p300 (4 μg) were cotransfected into COS7 cells, which were subsequently treated with TGF-β (5 ng/mL). Ternary complexes were detected by sequential immunoprecipitation with anti-FLAG agarose affinity gel and anti-HA antibody, followed by immunoblotting with the His antibody. (D) HBZ enhanced the interaction between Smad3 and p300. COS7 cells were cotransfected with mycHis-sHBZ (4 μg), FLAG-Smad3 (4 μg), and HA-p300 (4 μg). Cell lysates (samples from the experiment of Figure 4E) were subjected to immunoprecipitation using anti-HA followed by immunoblotting with anti-FLAG. (E) sHBZ, Smad3, and p300 bind to the Smad-responsive promoter. After transfection with mycHis-sHBZ, FLAG-Smad3, and HA-p300, and treatment with 5 ng/mL of TGF-β for 24 hours, HepG2 cells were chromatin immunoprecipitated by each indicated antibody. The precipitated DNAs and 1% of the input cell lysates were amplified by the 3TP promoter specific primers. Expression of sHBZ, Smad3, and p300 was detected by Western blot (bottom panel).
Figure 4
Figure 4
Domains of HBZ responsible for the activation of TGF-β signaling. (A) Comparison of the effect of sHBZ and usHBZ on TGF-β activation. (Left panel) Schematic diagram of sHBZ and usHBZ. (Right panel) HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pcDNA3.1-mycHis-sHBZ, or pcDNA3.1-mycHis-usHBZ (200 ng). Luciferase activity was measured 24 hours after 10 ng/mL TGF-β stimulation. mycHis tagged sHBZ and usHBZ were detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel). (B) Schematic diagram of sHBZ and its mutants used in this study. Characteristic domains of sHBZ are indicated as follows: AD, CD, and basic leucine zipper domain (bZIP). (C) Analysis of sHBZ deletion mutants for their effect on TGF-β–mediated signaling. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), and pME18Sneo-sHBZ mutants (0, 5, 20, 100, 200, and 500 ng). Luciferase activity was measured 24 hours after stimulation by TGF-β (10 ng/mL). (D) The N-terminal LXXLL1 motif of HBZ is important in enhancing TGF-β–induced luciferase expression. (Top panel) Schema of sHBZ-ΔbZIP and its deletion mutants. The locations of the LXXLL motifs are indicated. The mutated residues in the LXXLL1 motif are in bold and underlined. (Middle panel) In 24-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.25 μg), phRL-TK (1 ng), and mycHis-sHBZ-ΔbZIP or its mutants (1 μg). At 24 hours after transfection, the cells were stimulated with or without 10 ng/mL TGF-β. Cell lysates were subjected to luciferase assay 24 hours after stimulation. sHBZ-ΔbZIP and its mutants were detected by Western blot. CBB staining was shown as the loading control (bottom panel). (E) Determination of the region of HBZ responsible for the interaction with Smad3. COS7 cells were transfected with the indicated mycHis-sHBZ mutants along with the FLAG-Smad3 and HA-p300 vectors. Cell lysates were subjected to immunoprecipitation using anti–c-Myc followed by immunoblotting using anti-FLAG. (F) Schematic drawing of Smad3 and its deletion mutants. The locations of the MH1 domain, MH2 domain, and the linker domain are indicated. (G) Mapping the region of the Smad3 protein necessary for interaction with sHBZ. COS7 cells were transfected with HA-p300, mycHis-sHBZ, and full-length or mutant FLAG-Smad3. At 48 hours after transfection, total cell lysates were subjected to immunoprecipitation using anti–c-Myc followed by IB using anti-FLAG.
Figure 5
Figure 5
Higher expression of HBZ partially suppressed activation of TGF-β signaling via AP-1. (A) HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pCDNA3-c-Fos (0, 0.1 μg), FLAG-Smad3 (0, 0.1 μg), and pcDNA3.1-mycHis-sHBZ (0, 20, 200, and 4000 ng). At 24 hours after transfection, the cells were treated with or without TGF-β (10 ng/mL). After 24 hours, the cells were harvested, and luciferase activity was determined. Expression of sHBZ, Smad3, and c-Fos was detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel). (B) HepG2 cells were cotransfected with 9 × CAGA-Luc (0.5 μg), phRL-TK (2 ng), and pME18Sneo-sHBZ (0, 2, 5, 10, 20, 50, 100, and 200 ng). Luciferase activity was measured 24 hours after 10 ng/mL TGF-β stimulation. (C) sHBZ inhibited AP-1 signaling via its bZIP domain. Jurkat cells were cotransfected with AP-1-Luc (1 μg), phRL-TK (10 ng), pCG-Tax (1 μg), and pME18Sneo-sHBZ mutants (1 μg). After 48 hours, luciferase activity was measured.
Figure 6
Figure 6
Physiologic level of HBZ overcame the Tax-mediated suppression of TGF-β signaling. (A) Comparing the level of HBZ protein in HBZ-transfected HepG2 cells with the level of HBZ in ATL and HTLV-1–immortalized cell lines. Total protein was extracted from sHBZ-transfected HepG2 cells (samples from supplemental Figure 1) and the indicated cell lines, and subjected to immunoblotting using HBZ antibody. (B) Endogenous HBZ interacted with Smad3. ATL-55T cells were treated with 10 ng/mL TGF-β. After 10 hours, whole cell lysate was subjected to immunoprecipitation with anti-Smad3 or control IgG, and immunoprecipitates were probed with anti-HBZ antibody. (C) HBZ overcame the repression of TGF-β signaling induced by Tax. In 12-well plates, HepG2 cells were cotransfected with 3TP-Lux (0.5 μg), phRL-TK (2 ng), pCG-Tax (0, 0.2 μg), and pcDNA3.1-mycHis-sHBZ (0, 0.2 μg). At 24 hours after transfection, the cells were treated with or without 10 ng/mL TGF-β. After 24 hours, the cells were harvested and analyzed for luciferase activity. sHBZ and Tax were detected by Western blot (middle panel). CBB staining was shown as the loading control (bottom panel).
Figure 7
Figure 7
HBZ induced Foxp3 expression in naive T cells through Smad3. (A) HBZ modulated the expression of selected TGF-β target genes. (Left panel) Mouse naive T cells were transduced with pMXs-IG vector encoding sHBZ or empty vector. Forty-eight hours after viral infection, total RNA was extracted from sorted green fluorescent protein-positive cells. The level of Pdgfb, Sox4, Cdkn1a, Cdkn2b, Ctgf, Foxp3, Runx1, Myc, Tsc22d1, Id2, β-actin, and HBZ mRNA was analyzed by semiquantitative RT-PCR. (Right panel) Schema of the effect of TGF-β and sHBZ on TGF-β target gene transcription. ↑ indicates up-regulation; ↓, down-regulation; and -, no effect. (B) CTLL-2/pME18Sneo and CTLL-2/sHBZ, cells were plated in 96-well plates. Cells were treated with increasing concentrations of TGF-β for 72 hours. Proliferation of each cell was examined by methyl thiazolyl tetrazolium assay. Expression of HBZ was detected by RT-PCR (bottom panel). (C) SB431542, an inhibitor of the TGF-β receptor, could not inhibit the induction of Foxp3 by HBZ. Mouse CD4+CD25− T cells were transduced with pGCDNsamI/N vector encoding sHBZ, or with empty vector. Three days after TGF-β (0.2 ng/mL) and SB431542 (5μM) treatment, cells were stained with anti-Foxp3 in addition to anti-NGFR and then analyzed by flow cytometry. Numbers indicate the percentage of Foxp3-positive cells among NGFR-positive cells. (D) HBZ induced FoxP3 in human naive T cells. Human CD4+CD25− T cells were transfected with lentiviral vectors expressing sHBZ, or with empty vector. Two days after stimulating with TGF-β (0.1 ng/mL), cells were stained with antibodies for CD4, NGFR, and Foxp3 and then analyzed by flow cytometry. (E) SIS3 inhibited the HBZ-induced Foxp3 induction. Mouse CD4+CD25− T cells were transduced with pGCDNsamI/N vector encoding sHBZ or empty vector. Fifteen hours after viral infection, SIS3 (5μM) and TGF-β (1 ng/mL) were added. Thirty-six hours after treatment, Foxp3 expression was detected by flow cytometry. Numbers indicate the percentage of Foxp3-positive cells among NGFR-positive cells. Representative data from 3 independent experiments are shown. (F) HBZ activated transcription of the Foxp3 promoter through its Smad site of enhancer. EL4 cells were transfected with the Foxp3 reporter plasmid or its mutants with or without the sHBZ-expressing plasmid (pcDNA3.1-mycHis-sHBZ). Luciferase activity was measured 48 hours after stimulation by TGF-β. Expression of sHBZ was detected by Western blot. CBB staining was shown as the loading control. (G) HBZ formed complex with Smad3/p300 in FoxP3 enhancer. MT-2 cells treated with 5 ng/mL of TGF-β for 2 hours, and chromatin immunoprecipitated by each indicated antibody. The precipitated DNAs and 1% of the input cell lysates were amplified by the specific primers for FoxP3 enhancer.
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