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J. Biol. Chem., Vol. 277, Issue 37, 33766-33775, September 13, 2002

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Human T-cell Lymphotropic Virus Type 1 Tax Inhibits Transforming Growth Factor- Signaling by Blocking the Association of Smad Proteins with Smad-binding Element^*

Dug Keun Lee, Byung-Chul Kim, John N. Brady^§, Kuan-Teh Jeang^¶, and Seong-Jin Kim

From the  Laboratory of Cell Regulation and Carcinogenesis, ^§ Basic Science Laboratory, NCI, and the ^¶ Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, January 7, 2002, and in revised form, May 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The human T-cell lymphotropic virus type 1 (HTLV-1) oncoprotein Tax is implicated in various clinical manifestations associated with infection by HTLV-1, including an aggressive and fatal T-cell malignancy. Because many human HTLV-1-infected T-cell lines are resistant to the growth inhibitory activity of transforming growth factor  (TGF-), we examined the possibility that the HTVL-1-Tax oncoprotein regulates TGF- signaling. We show that Tax significantly decreases transcriptional activity and growth inhibition in response to TGF-. Tax inhibits TGF--induced plasminogen activator inhibitor-1 expression and Smad2 phosphorylation. Competitive interaction studies show that Tax inhibits TGF- signaling, in part, by disrupting the interaction of the Smads with the transcriptional co-activator p300. Tax directly interacts with Smad2, Smad3, and Smad4; the Smad MH2 domain binds to Tax. Furthermore, Tax inhibits Smad3·Smad4 complex formation and its DNA binding. These results suggest that suppression of Smad-mediated signaling by Tax may contribute to HTLV-1-associated leukemogenesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Human T-cell lymphotropic virus type 1 (HTLV-1)¹ is a human retrovirus that causes both adult T-cell leukemia (ATL) and the degenerative neuromuscular disease tropical spastic paraparesis, or HTLV-1-associated myelopathy (1, 2). The mechanism of HTLV-1 pathogenesis is still unclear, but it has been postulated that a 40-kDa Tax protein, which HTLV-1 pro-viral DNA encodes, is involved in the hyper-proliferation and transformation of T-cells in ATL. The viral Tax protein not only regulates HTLV-1 gene expression but also stimulates the transcription of several cellular genes including activating transcription factors/CRE-binding proteins (CREB) (3), NFB-IB complex (4-6), p67^SRF (7, 8), Ets1 (9), NF-Y (10), and Sp1 (11). In addition, Tax binds to the basal transcription factors TFIIA (12) and TFFIIB, and to TFIID through the TATA-binding protein (13) and TATA-binding protein-associated factor TAF_II28 (14). Moreover, Tax interacts with CREB-binding protein (CBP) (15, 16), a cofactor facilitating transcriptional activation by CREB. Through these interactions, Tax enhances the expression of a variety of target genes related to cellular activation and growth. These include genes for transforming growth factor-1 (TGF-1), interleukin-2, interleukin-6, granulocyte-macrophage colony-stimulating factor, Fra-1, c-Myc, c-Fos, and c-Jun (17-24). Aberrant expression of these proteins may be involved in the dysregulated proliferation of HTLV-1-infected cells.

Tax also induces cell cycle progression through direct interaction with cell cycle regulators. Tax binds and inactivates p16^INK4a, a negative regulatory molecule of the cell cycle (25). Tax may also directly associate with cyclin D, which is important in cell cycle transition from the G₁ to S phase (26). Recent studies suggest that the mechanism of Tax-mediated cellular transformation is a failure to repair DNA damage. As a consequence, Tax-expressing cells accumulate aneuploidogenic and clastogenic lesions which are postulated to lead to a transformed phenotype (27, 28).

TGF- inhibits the growth of most epithelial and lymphoid cells, and this negative regulation of cellular proliferation by TGF- has been shown to constitute a tumor suppressor pathway (29, 30). Smad2 and Smad3 have been identified as direct downstream mediators of TGF- signaling (31). Receptor-mediated phosphorylation of these Smads induces their association with the shared partner Smad4 followed by translocation into the nucleus where these complexes activate transcription of specific genes (32, 33). Smad proteins contain a conserved amino-terminal domain (MH1) that binds DNA (34), and a conserved carboxyl-terminal domain (MH2) that binds receptors, partner Smads, and transcriptional coactivators (35). These two domains are separated by a more divergent linker region.

A previous report demonstrated that HTLV-1-infected T-cell lines became resistant to TGF- growth inhibitory activity (2). We hypothesized that this TGF- resistance results from the HTLV-1 Tax protein, and we examined whether Tax alters TGF- signaling. In this study, we demonstrate that Tax inhibits the transcriptional activation and growth inhibition responses to TGF-. Tax inhibits TGF- signaling, in part, by competitive interactions with both Smad proteins and p300. We also show that Tax binds to Smad2, Smad3, and Smad4 directly. Furthermore, we demonstrate that Tax prevents binding of the Smad complex to its target sequence, and thereafter inhibits TGF- signal transduction. These results suggest that the inhibition of TGF- signaling by Tax may lead to the HTLV-1-associated leukemogenesis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Constructs-- FLAG-tagged Smad2, -3, and -4 deletion constructs were generated by polymerase chain reaction using a proofreading polymerase and subcloned EF-FLAG vector. All polymerase chain reaction-generated products were sequenced using the dideoxynucleotide method.

Generation of Mv1Lu Cell Lines Expressing Tax-- The Tax of a human T-cell lymphoma virus was PCR amplified, restriction-digested, and purified to be subcloned into the MFG vector (36). A IRES-NEO cassette was also subcloned into the constructs to obtain stable transfectants. The control vector, MFG-CAT, was described previously (37). For cell proliferation assay, Tax-expressing cells were plated in 24-well dishes at a density of 5 × 10⁴ cells per well in 0.5 ml of assay medium (Dulbecco's modified Eagle's medium, 0.2% fetal bovine serum). After incubating for 22 h in the presence or absence of TGF-, cells were pulse-labeled with 0.5 µCi of [³H]thymidine for 2 h at 37 °C. Cells were fixed, trypsinized, solubilized, and transferred to scintillation vials to measure radioactivity as described previously (18).

Cell Culture, Transfection, and Reporter Assays-- Cell lines were maintained in Dulbecco's modified Eagle's medium, minimal essential medium, or RPMI supplemented with 10% fetal bovine serum. HepG2 and Mv1Lu stable cells were transfected with 3TP-Lux (38), 4xSBE-luc (39), in six-well plates using Lipofectin (Invitrogen) according to the manufacturer's instructions. After transfection, cells were treated with 5 ng/ml TGF- for 24 h in media. All assays were performed in triplicate, and are represented as the mean ± S.E. of three independent transfections.

Western Blots, GST Pull-down Assay, and Immunoprecipitation-- HepG2 cells were transiently transfected with the indicated plasmids. After 24 h, cells were switched to 0.2% serum overnight, and treated with 5 ng/ml TGF-1 for 2 h and then whole cell extracts were prepared as described (40). Extracts were separated by SDS-PAGE followed by electrotransfer to nitrocellulose membranes and probed with polyclonal or monoclonal antisera followed by horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG, respectively, and visualized by chemiluminescence according to the manufacturer's instructions (Pierce). Immunoprecipitation was carried out by incubation with antibody for 1 h. After immunoprecipitates were washed with the buffer containing 100 mM NaCl and 75 mM KCl, Western blots were prepared. C81 and Jurkat cells were treated using the same method as HepG2 cells.

GST Pull-down Assay-- The coding region for Smad2, Smad3, or Smad4 was PCR-amplified and subcloned into the TOPO vector (Invitrogen Corp.). These plasmids were used as templates for RNA synthesis by T7 RNA polymerase followed by translation in rabbit reticulocyte extracts (Promega Corp., Madison, WI). The GST-Tax fusion protein expressed in Escherichia coli was grown and partially purified by adsorption to glutathione-Sepharose beads in the presence of the detergents N-laurylsarcosine (Sarkosyl) and Triton X-100. Samples of each protein (0.5-1.0 µg) bound to Sepharose were preincubated with ethidium bromide (40 µg/ml) for 30 min. Then the samples were shaken for 1 h at room temperature with 5-10 µl of [³⁵S]methionine-labeled in vitro translated Smad proteins. The beads were washed four times in NETN buffer and boiled for 3 min in 2× SDS-electrophoresis loading buffer before fractionation on 4-20% Tris glycine gels (Invitrogen). The gels were rinsed in 10% acetic acid, dried, and exposed to x-ray film for autoradiography.

Binding of the Smad-containing Complex to Biotinylated DNA-- DNA binding using biotinylated oligonucleotides was performed as described (41). Cells were treated with 5 ng/ml TGF- for 2 h. After preclearing with streptavidin-agarose for 1 h, cell lysates were incubated with 30 pmol of biotinylated double stranded 3xCAGA oligonucleotides and 12 µg of poly(dI-dC) for 1 h. Proteins were precipitated with streptavidin-agarose for 30 min, washed, and detected by immunoblotting.

Gel Shift Assay-- Gel mobility shift assay was performed as described previously (42). To prepare the nuclear extracts from CV-1-neo and CV-1-Tax cells, cells were incubated in the presence or absence of TGF-1 for 24 h and lysed and used in a gel shift assay. Briefly, the cells were harvested by scraping, washed in cold phosphate-buffered saline, and incubated in 2 packed cell volumes of buffer A (10 mM HEPES, pH 8.0, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, 200 mM sucrose, 0.5 mM phenylmethanesulfonyl fluoride, 1 µg of both leupeptin and aprotinin per ml, 0.5% Nonidet P-40) for 5 min at 4 °C. The crude nuclei released by lysis were collected by microcentrifugation (15 s), rinsed once in buffer A, and resuspended in 2/3 packed cell volume of buffer C (20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 1.0 mM dithiothreitol, 1.0 µg of both leupeptin and aprotinin per ml). Nuclei were incubated on a rocking platform at 4 °C for 30 min and clarified by microcentrifugation for 5 min. The resulting supernatants were diluted 1:1 with buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, 1 µg of both leupeptin and aprotinin per ml). Nuclear extracts were frozen on dry ice and stored at 80 °C. The extract (30 µg) was incubated with the oligonucleotide probe (41) labeled with [³²P] (2 × 10⁵ cpm) in 20 µl of reaction buffer at room temperature for 20 min, and the reaction mixture was analyzed by electrophoresis on a 4% nondenaturing polyacrylamide gel and run in 0.5× Tris borate-EDTA buffer. After electrophoresis the gel was dried and autoradiographed.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transcriptional Repression of a TGF--responsive Gene by Tax-- To examine the role of Tax in TGF--induced transcriptional activation, we co-transfected HepG2 cells with a Tax expression construct and either the TGF--responsive 3TP-lux reporter construct or SBE4-luc, which contains four SBE (Smad-binding element) sites in tandem (39). Introduction of Tax repressed the TGF--dependent activities of these reporter gene constructs (Fig. 1, A and B) suggesting that Tax represses TGF--induced transactivation. The repression of the SBE4-luc reporter activity by Tax suggests that it may directly inhibit the transcriptional activation of Smad complexes. To confirm that Tax is directly involved in Smad-mediated transcriptional activation, we used a heterologous reporter assay in which the Gal4 DNA-binding domain was fused to various Smad proteins. Gal4-Smad2, Gal4-Smad3, or Gal4-Smad4 expression constructs were cotransfected with a luciferase reporter construct (G5E1b-lux), which contained five Gal4-binding sites upstream of the AdE1b TATA box. As expected, TGF- treatment did not induce transcription by the minimal Gal4-DNA binding domain, and Tax did not have any effect on its transcription. However, cotransfection of a Tax expression vector with Gal4-Smad2, Gal4-Smad3, or Gal4-Smad4 decreased the TGF--dependent activation of these constructs (Fig. 1C), demonstrating that Tax can directly diminish Smad-mediated transcriptional activation.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Tax represses TGF--induced transcriptional activation. Tax was co-transfected into HepG2 cells with either 3TP-Lux (A) or SBE4-luc (B). Luciferase activity was measured 24 h after TGF-1 stimulation. C, GAL4 fusion constructs were transfected into HepG2 cells, along with the reporter plasmid, then cells were incubated in the presence or absence or TGF-1. Data shown are means of triplicate measurements from one representative transfection.

To examine whether Tax renders resistance to the TGF- growth inhibitory activity, we generated Mv1Lu mink lung epithelial cells stably expressing Tax. TGF- inhibited the proliferation of control Mv1Lu cell, whereas overexpression of Tax abrogated TGF- growth inhibitory activity (Fig. 2A). We also examined the inhibitory effect of Tax on the TGF--induced induction of the endogenous target gene. An asynchronous population of either control CV-1 or Tax-expressing CV-1 cells (Fig. 2C) was treated with TGF-1 for 20 h, and Western blot analysis of p21 was performed. TGF-1 treatment resulted in an ~9-10-fold increase in the level of p21 protein in control CV-1 cells, whereas the induction of the p21 protein level by TGF-1 was markedly decreased in CV-1-Tax cells (Fig. 2D). We next examined whether Tax inhibits phosphorylation of Smad2 in response to TGF-1. As shown in Fig. 2E, TGF-1 treatment increased phosphorylation of Smad2 in control CV-1 cells, but the level of Smad2 phosphorylation was significantly reduced in CV-1-Tax cells.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Tax block the TGF- growth activity. A, Tax-expressing Mv1Lu cells and control cells were treated with varying concentrations of TGF-1 as indicated. After 22 h of TGF-1 treatment for the cells, the cells were pulsed with [3H]thymidine and harvested 2 h later. The experiments were repeated three times. All values represent the averages of the three determinations mean ± 1 S.D. Expression of Tax protein was analyzed by Western blots using anti-Tax antibody in Tax-expressing Mv1Lu cells (B) and CV-1 monkey kidney cells (C). D, effect of Tax on the level of p21 protein induced by TGF-. Asynchronously growing CV-1-neo and CV-1-Tax cells were incubated in the presence or absence of TGF-1 for 24 h. p21 protein levels on the whole cell lysate were examined by Western blot analysis. -Actin protein levels were examined for the loading control. E, Western blot analysis of Smad2 activation in CV-1-neo and CV-1-Tax cells. After treatment for 30 min with TGF-1 (5 ng/ml), a band of 58 kDa representing phosphorylated Smad2 (Smad2P) was detected in cell extracts by immunoblotting using rabbit anti-Smad2P antibody. TGF-1 induced phosphorylation of Smad2 in CV-1-neo cells, but the level of Smad2 phosphorylation was markedly decreased in CV-1-Tax cells.

Competition between Tax and Smad for Binding to p300-- To address the mechanism whereby Tax suppresses the transcriptional activation of the TGF- signal transduction pathway, cells were co-transfected with Tax and an increasing dose of either p300 or p300/CBP-associated factor (PCAF) (Fig. 3A). A previous study has demonstrated that Tax inhibits the ability of the Smads to mediate TGF--induced transcriptional activation by interfering with the recruitment of CBP/p300 (43). We confirmed this observation as well. As shown in Fig. 3A, Tax inhibited the TGF--induced transcriptional activity, but p300 restored the suppression of the TGF--induced transcriptional activation by Tax in a dose-dependent manner. PCAF, however, failed to restore the suppressed activity. Tax also suppressed, in a dose-dependent manner, the potentiation of the TGF- transcriptional activity by p300 (Fig. 3B). These results suggest that Tax may block the interaction between the Smads and p300. To confirm this hypothesis, we assessed the interaction between transfected Smad2 and endogenous p300 in HepG2 cells in the presence or absence of Tax. Immunoprecipitation of endogenous p300, followed by Western blotting with FLAG antibody to detect Smad2, showed that overexpression of Tax interferes with the interaction of p300 with Smad2. Increasing the amount of transfected Smad2 overcame the Tax-mediated inhibition and restored the Smad2/p300 association (Fig. 3C). These data suggest that Tax inhibits Smad signaling by competing with Smad2 for binding to p300.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Tax inhibits the interaction of Smad2 with p300. A, HepG2 cells were transiently transfected with SBE4-luc with or without Tax, or together with an increasing amount of the p300 expression construct (0.5, 1.0, and 1.5 µg) or PCAF expression construct (0.5, 1.0, and 1.5 µg). Luciferase activity was measured 24 h after TGF-1 stimulation. B, HepG2 cells were transiently transfected with SBE4-luc with or without the p300 expression construct, or together with an increasing amount of the Tax expression construct (0.5, 1.0, and 1.5 µg). Luciferase activity was measured 24 h after TGF-1 stimulation. Data shown are means of triplicate measurements from one representative transfection. C, HepG2 cells were transfected with the FLAG-tagged Smad2 and Tax expression construct. Interaction between p300 and Smad2 was analyzed by immunoblotting with the anti-FLAG antibody after immunoprecipitating with anti-p300 antibody. The expression of transfected constructs or endogenous protein was monitored by immunoblotting with antibodies against p300, FLAG for Smad2, or Tax.

Tax Interacts with Smads-- To examine the possibility that Tax interacts directly with Smad proteins, we used HepG2 cells transfected with a Tax expression vector and FLAG/Myc-tagged Smad expression constructs. There was a ligand-independent interaction between Tax and Smad2, Smad3, or Smad4 (Fig. 4, A-C). Immunoprecipitation assays were also performed using SW480 cell extracts. We had the same results as in HepG2 cells (data not shown). The interaction between these Smad proteins and Tax was also studied by GST pull-down assays in vitro using ³⁵S-labeled Smad2, -3, and -4 proteins. Tax interacted with ³⁵S-labeled Smad2, -3, or -4 (Fig. 3D). These results demonstrated that Tax binds to Smad2, -3, or -4 directly.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Tax interacts with the Smad2, -3, and -4. Tax was transfected into HepG2 cells with the FLAG-tagged Smad2 and -3, and Myc-tagged Smad4 constructs. Cells were treated with TGF-1 for 2 h. Cell extracts were subjected to immunoprecipitation using an anti-Tax or anti-FLAG antibody and Gamma bind beads (Amersham Biosciences), followed by immunoblotting with anti-FLAG, anti-Myc, or anti-Tax antibody (A-C). The expression of Tax and Smads was monitored as indicated. The interaction between Smads and Tax was examined by a in vitro GST pull-down assay. Bacterially expressed GST-Tax and GST alone were incubated with [35S]methionine-labeled Smad proteins (D). 25% of [35S]methionine-labeled Smad proteins used for the assay were applied as controls (Input).

To demonstrate that Tax interacts with Smad2, -3, and -4 in vivo, C81 cells, an HTLV-1-transformed T-cell line that constitutively expresses Tax, were used (44). Whole cell extracts were prepared from C81 cells and from Jurkat cells for control. Expression of endogenous Smad2, -3, and -4 was confirmed by Western blot analysis using rabbit polyclonal antibodies against these proteins (Fig. 4). Tax was only detected in C81 cells (Fig. 5A, the bottom panel of lane 2). Total cell extracts were prepared from Jurkat and C81 cells and immunoprecipitated with anti-Tax antibody or Smad3 antibody. The resulting Western blots demonstrate that Tax was specifically co-immunoprecipitated with endogenous Smad2, -3, or -4 (Fig. 5B).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Tax interacts with Smads in vivo. Nuclear extracts were prepared from Jurkat (lane 1 of A and B) and C81 (lane 2 of A and B) cells, and immunoprecipitated with an anti-Tax antibody (Smad2 or -4) or with anti-Smad3 antibody (Smad3). Western blotting with anti-Smad2, anti-Smad4, or Tax antibody was used to analyze the components in the immunoprecipitates. Expression of endogenous Smad2, -3, -4, and Tax were also confirmed by Western blotting.

Tax Interacts with the MH2 Domain of Smads-- Immunoprecipitation assays were performed using various FLAG-tagged Smad2, Smad3, or Smad4 expression constructs along with a full-length Tax to determine the domain of Smad2, -3, or -4 interacting with Tax. Tax was found to associate with the carboxyl-terminal MH2 domain of Smad2, -3, or -4, but not with the amino-terminal MH1 or middle linker domain of this molecule (Fig. 6, B, D, and F), demonstrating that the MH2 domain contained the Tax interaction domain.

View larger version (50K): [in this window] [in a new window]   Fig. 6.   Mapping of domains that Tax interacts with Smads. A, schematic drawings of Smad2 truncation mutants. B, FLAG-tagged full-length and truncated Smad2 expression constructs were cotransfected into HepG2 cells together with Tax, and Smad2 proteins were isolated by immunoprecipitation with anti-FLAG antibody. The Smad2-bound Tax was detected by protein immunoblotting with an anti-Tax antibody (top). Cell lysates were blotted with anti-FLAG to confirm expression of full-length and FLAG-Smad2-deletion mutants (middle). The expression of Tax protein in the lysates was detected using anti-Tax antibody (bottom). C, schematic drawings of Smad3 deletion constructs. D, the Smad3-bound Tax was detected by protein immunoblotting with an anti-Tax antibody (top). Cell lysates were blotted with anti-FLAG to confirm expression of full-length FLAG-Smad3 and deletion mutants, respectively (middle). The expression of Tax protein in the lysates was detected using anti-Tax antibody (bottom). E, schematic drawings of Smad4 deletion constructs. F, the Smad4-bound Tax was detected by protein immunoblotting with an anti-Tax antibody (top). Cell lysates were blotted with anti-Myc or anti-FLAG to confirm expression of full-length Myc-Smad4 and deletion mutants, respectively (middle). The expression of Tax protein in the lysates was detected using anti-Tax antibody (bottom).

Because Tax interacts with the MH2 domains of the Smads, which are the R-Smad/Smad4 interaction domains, we examined whether Tax inhibits this complex formation. The Myc-tagged Smad4 expression construct was co-transfected with the FLAG-tagged Smad3 expression construct together with or without the Tax expression construct into HepG2 cells. 24 h after transfection, cells were incubated in the presence or absence of TGF-1 for 30 min and whole cell extracts were prepared. To investigate Smad3·Smad4 complex formation, total cell extracts were immunoprecipitated with anti-Myc antibody and FLAG-Smad3 bound to Myc-Smad4 was examined using anti-FLAG antibody by Western blot analysis. As shown in Fig. 7, Tax expression markedly decreased the level of Smad3 bound to Smad4, demonstrating that Tax inhibits R-Smad·Smad4 complex formation.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Tax inhibits Smad3·Smad4 complex formation. FLAG-tagged Smad3 and Myc-tagged Smad4 expression constructs were co-transfected into HepG2 cells together with Tax. After treatment for 1 h with TGF-1 (5 ng/ml), Smad4 protein was isolated by immunoprecipitation with anti-Myc antibody. The Smad4-bound Smad3 was detected by protein immunoblotting with an anti-FLAG antibody. Cell lysates were blotted with anti-FLAG antibody to confirm expression of Smad3 and with anti-Myc antibody to check expression of Smad4. The expression of Tax protein in the lysates was detected using anti-Tax antibody.

Tax Mutant M47 Failed to Repress TGF--induced Transcription because of Defective Interaction with Smad3-- To confirm the mechanism of Tax blocking on TGF- signaling, we examined the effect of Tax mutants M22 and M47 on TGF--induced transcription. M22 (T130A,L131S) is a mutant that is partially defective in dimerization (45, 46). In contrast, M47 (L319R,L329S) is a COOH-terminal mutant that retains the ability to form dimers and bind CREB (45, 46). In the luciferase assays using 3TP-Lux, a TGF- responsive reporter plasmid, both wild-type Tax and M22 were able to significantly repress the TGF--dependent activities of this reporter gene construct. However, Tax mutant M47 failed to repress TGF--induced transactivation (Fig. 8A). Using these Tax mutant constructs together with a wild-type Tax, we performed immunoprecipitation assays with anti-FLAG antibody for Smad3. As shown in Fig. 8B, wild-type Tax and M22 showed interaction with Smad3, however, the ability of M47 to interact with Smad3 was markedly decreased (Fig. 8B). This result suggests that M47 cannot repress TGF- transcription activity because of its inability to interact with Smad3.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Tax mutant M47 does not repress TGF--induced transcriptional activation and does not interact with Smad3. A, HepG2 cells were transfected with the 3TP-Lux reporter construct along with wild-type Tax, M47 mutant, and M22 mutant. After transfection, cells were stimulated with 5 ng/ml TGF-1 for 24 h, and luciferase activity was measured. B, FLAG-tagged Smad3 proteins were co-transfected into HepG2 cells together with wild-type Tax, M47, or M22 mutants, and isolated by immunoprecipitation with anti-FLAG antibody. The Smad3-bound Tax was detected by protein immunoblotting with an anti-Tax antibody (top). Cell lysates were blotted with anti-FLAG to confirm expression of FLAG-Smad3 (middle). The expression of wild-type Tax, M47, and M22 mutants in the lysates was detected using anti-Tax antibody (bottom).

Tax Inhibits the Formation of the Smad3-containing Complex-- Using M22 and M47 Tax mutants together with a wild-type Tax, we performed the CAGA binding assay (41) after transient transfection into HepG2 cells. In Fig. 9A, TGF- treatment showed Smad3 binding to the CAGA element (upper panel, lane 2), and this binding is significantly diminished in the wild-type Tax-transfected HepG2 cells (lane 3). However, Tax mutant M47 failed to block this binding (Fig. 9A, lane 4). In contrast, M22 showed the inhibitory activity on this binding as much as wild-type Tax (Fig. 9A, lane 5).

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Tax inhibits the formation of the Smad containing complex. A, the FLAG-tagged Smad3 construct was co-transfected with either wild-type Tax, or M47, or M22 mutants into HepG2 cells. After transfection, cells were stimulated with 5 ng/ml TGF-1 for 2 h, and Smad3 binding to CAGA biotinylated DNA was examined. B, Tax expression inhibits the formation of the Smad-containing complex in a gel shift assay. CV-1-neo and CV-1-Tax cells were treated with 5 ng/ml TGF-1 for 24 h and nuclear extracts were prepared and processed as described under "Materials and Methods."

To examine whether Tax inhibits the formation of the Smad-containing complex, we also performed a gel shift assay using an oligonucleotide encompassing a TGF--responsive element in the plasminogen activator inhibitor-1 promoter (586 ~ 551). Previous studies have shown that co-transfection of a constitutively active TGF- type 1 receptor (TRI-T204D) together with Smad3 and Smad4 generates a Smad-containing complex visualized in a gel-shift assay and this complex can be supershifted with either Smad3 or Smad4 antibodies (31). Nuclear extracts were prepared from the CV-1-neo and CV-1-Tax cells after TGF-1 treatment. TGF-1 treatment markedly increased the formation of the Smad-containing complex in CV-1-neo cells, whereas expression of Tax in CV-1-Tax cells prevented the formation of the Smad-containing complex (Fig. 9B). These data strongly indicate a direct inhibitory role of Tax on the formation of the Smad-containing complex.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The pathogenesis of ATL is still not understood, but it has been postulated that the viral Tax protein is involved in the proliferation and transformation of T cells in ATL. Tax is known to modulate cellular proliferative responses through two broad mechanisms. First, Tax directly targets specific transcriptional regulators including E2F, CREB, NFB, SRF, Ib, and CBP/P300. The regulation of E2F is a key target for oncoviruses. Binding of pRB and the other pocket proteins to viral proteins, such as adenovirus E1A, simian virus large T antigen, and papillomavirus E7, leads to a stimulation of E2F-dependent transcription. The induction of the E2F DNA binding activity in HTLV-1-infected T-cell lines and in leukemic cells obtained from ATL patients suggests that the activation of E2F-dependent transcription by HTLV-1 could be involved in the proliferative response during HTLV-1 infection (44). Second, in addition to transcriptional regulation, Tax modifies cell cycle regulators, primarily by affecting inhibitors of cyclin-dependent kinases. Tax binds to and inactivates the p16^INK4a protein, which belongs to the INK4 family of cyclin-dependent kinase inhibitors (25), thus resulting in the activation of cyclin-dependent kinase 4. This effect of Tax relieves cells from p16^INK4a-induced growth arrest and may also contribute to the cellular immortalization and transformation induced by HTLV-1 infection.

Our studies have suggested yet another mechanism by which Tax may promote cellular proliferation and transformation. Hollsberg et al. (47) have previously shown that HTLV-1-infected T-cell lines become resistant to TGF--induced growth arrest. We suspected that the source of this TGF- resistance resides in the HTLV-1 Tax protein, and that, given the numerous studies that have documented that loss of TGF- signaling promotes tumor formation, this TGF- resistance may provide an important means by which these cells become oncogenic.

Therefore, to investigate the carcinogenic mechanism of HTLV-1, we explored the influence of Tax on TGF- signaling. 3TP-Lux and SBE reporter assays, which test the integrity of the entire TGF- signaling pathway, were substantially inhibited by Tax, clearly indicating that Tax affects some portion of the TGF- signal transduction cascade. We next localized the point within the pathway at which this blockade occurs by demonstrating that Tax associates with Smad proteins, directly for Smad2, Smad3, and Smad4.

During the preparation of this manuscript, Mori et al. (43) reported that Tax inhibits TGF- signaling, but does not bind to Smad proteins, a conclusion that is contrary to the results of the present study. The precise reason for this discrepancy is not clear at the present time. Interaction between Tax and Smad proteins may be cell-type specific. One potential argument is that the observed interaction between Tax and Smad in HepG2 cells is an artifact of overexpression of these proteins. However, the results obtained from C81 cells, a cell line that was established from a patient with HTLV-1-induced acute T-cell leukemia (44), unequivocally demonstrate that Tax binds to Smads proteins (Fig. 5). We have also shown the direct interaction between Smads and Tax by in vitro GST pull-down assays using ³⁵S-labeled Smad2, -3, and -4 proteins (Fig. 4D). To confirm our findings, we examined the effect of Tax mutants, M22 and M47, on TGF--induced transcription. In luciferase assays using 3TP-lux, M47 failed to show inhibitory activity on the TGF--induced transcription. In contrast, M22 showed significant inhibition (Fig. 8A). This result confirms the finding by Mori et al. (43). They concluded that the differential effect of M22 and M47 on TGF- transcription might be because of their ability to interact with CBP. However, in another study, it has been shown that M47, defective in the COOH-terminal transactivation domain, continued to interact with CBP/p300 (48). They also used a Tax mutant (K88A) defective for the CBP/p300-binding domain, and a KID-like domain in Tax is responsible for the recruitment of CBP/p300 (48). Tax K88A failed to repress transcription from the plasminogen activator inhibitor-1 promoter (43). They suggested that the CBP/p300-binding domain of Tax is involved in the suppression of Smad transactivation function. Because it is known that Smad2, -3, and -4 bind to CBP/p300, and these interactions are promoted by treatment with TGF- (49-52), Tax may inhibit TGF- signaling by competing for Smad-CBP/p300 interaction as suggested by Mori et al. (43). However, the competition for Smad-CBP/p300 by Tax cannot explain why Tax M47 fails to repress the Smad transactivation activity even though it still interacts with CBP/p300. In this study, we have shown that M47 does not interact with Smad3 and could not block binding of the Smad complex to its target sequence (Figs. 8 and 9). These results may explain why M47 failed to repress TGF- transcription activity. Taken together, our observations clearly demonstrate that Tax interacts with Smads directly and specifically. Our present study and the published literature suggest that Tax may inhibit TGF- transcription activity through two different mechanisms, competing for Smad-CBP/p300 interaction and binding to Smads directly.

This study demonstrates that HTLV-1 Tax inhibits TGF- signaling, in part, by interaction with Smad proteins and through blocking binding of the Smad complex to its target DNA sequence. Tax inhibits TGF- signaling by competitive interactions with both Smad proteins and p300. This decrease in TGF- signaling likely provides the optimal conditions for tumorigenesis in a way that complete abrogation of signaling could not. Significantly, we have shown that inhibition is sufficient to disrupt the growth inhibitory control that TGF- normally exercises over T lymphocytes. Without one of the most important brakes on their proliferation, these infected cells can then multiply at high rates. But this inhibition of TGF- signaling is not complete. In particular, our group has previously shown that cell lines overexpressing Tax still respond to TGF-1 stimulation (18). Treated cells show increased mRNA production for both Tax and, through an autoregulatory loop, TGF-1 itself. This excess serum TGF-1, which has been documented in HTLV-1-infected patients (17), then has the potential to significantly alter the function of the remaining normal lymphocytes, most pertinently by diminishing tumor surveillance. With the immune system hampered in its ability to recognize and destroy emerging malignant clones, the increased serum TGF-1 can enhance tumorigenesis. This considerable inhibition of TGF- signaling by Tax thus provides a uniquely favorable environment for the development of T-cell leukemia.

Inhibition of Smad-mediated signaling has been suggested as one of the critical mechanisms for leukemogenesis induced by oncoproteins. An oncoprotein Evi-1 has been shown to interact with Smad3 through its first zinc finger motif and to antagonize the growth inhibitory effect of TGF- (53). Evi-1 is overexpressed in human myeloid leukemias and myelodysplastic syndromes by chromosomal rearrangements involving 3p26, to which Evi-1 is mapped (54, 55). These findings, together with our finding, suggest a new paradigm that suppression of Smad-mediated signaling may contribute to leukemogenesis associated with oncoproteins.

	ACKNOWLEDGEMENTS

We thank Anita Roberts and Sejal Patel for helpful discussions and critical review of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 301-496-8350; Fax: 301-496-8395; E-mail: kims@mail.nih.gov.

Published, JBC Papers in Press, July 3, 2002, DOI 10.1074/jbc.M200150200

	ABBREVIATIONS

The abbreviations used are: HTLV-1, human T-cell lymphotropic virus type 1; ATL, adult T-cell leukemia; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; TGF-, transforming growth factor-; GST, glutathione S-transferase.

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