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NEOPLASIA
From the Department of Preventive Medicine and AIDS
Research and the Department of Biochemistry, Institute of Tropical
Medicine, Nagasaki University; the Departments of Anatomy and Surgery
II, Nagasaki University School of Medicine, Japan; the Department of
Infectious Disease and Immunology, Okinawa-Asia Research Center of
Medical Science, Faculty of Medicine, University of the Ryukyus,
Nishihara, Okinawa, Japan; and the Department of Microbiology and
Immunology, Uniformed Services University of the Health Sciences,
Bethesda, MD.
Human T-cell leukemia virus type I (HTLV-I) Tax is a potent
transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to
leukemogenesis in adult T-cell leukemia. Previously,
HTLV-I- infected T-cell clones were found to be resistant to growth
inhibition by transforming growth factor (TGF)- Human T-cell leukemia virus type I (HTLV-I) is an
etiologic agent of an acute malignancy of CD4+ T
lymphocytes called adult T-cell leukemia (ATL).1,2 The virus-encoded regulatory protein, Tax, is critical for HTLV-I replication and is thought to contribute to ATL development. Several experimental observations indicate that Tax mediates the oncogenic activity of HTLV-I. For example, Tax immortalizes primary human T
lymphocytes and transforms rodent fibroblasts in vitro.3-5 In addition, transgenic mice expressing Tax develop mesenchymal tumors
or large granular lymphocytic leukemia in vivo.6,7
The exact mechanism through which Tax exerts its oncogenic potential is
still unknown. Tax was originally identified as a transcriptional
activator for viral gene expression and then was shown to activate the
expression of a number of cellular genes, many of which either encode
proteins involved in the regulation of cellular proliferation (ie,
interleukin [IL]-2,8 IL-2 receptor Proliferation and differentiation of cells are tightly regulated by a
delicate balance of growth factors and growth-inhibitory factors.
Transforming growth factor (TGF)- The Smad family proteins are critical components of the TGF- Previously, it was reported that HTLV-I-infected T-cell clones were
resistant to growth inhibition by TGF- Plasmid constructions
Cell lines, transfections, and luciferase assays
Assay for cell proliferation To generate stable Mv1Lu cell lines overexpressing Tax, cells were transfected with pH2R40M using lipofectamine. Cells were selected with G418 (800 µg/mL) for 2 to 3 weeks and then cloned using a cloning cylinder. The expression of Tax mRNA in cell clones was examined by reverse transcriptase-polymerase chain reaction as previously described.54 For cell proliferation studies, Tax-expressing clones and control clone were plated at a density of 5 × 103 cells/well in 96-well microtiter plates. After 24-hour plating, cells were treated with increasing concentrations of TGF- for 48 hours and then assayed for cell growth with the use of a
Cell Counting kit (Wako Chemical, Osaka, Japan) based on an MTT assay. Each experiment was performed at least 3 times, and typical results are shown.
Immunoprecipitation and Western blot analysis COS7 cells transiently transfected with the indicated constructs were washed and lysed in TNE buffer. Whole-cell extracts or immunoprecipitates produced with anti-Flag, anti-HA, or anti-Tax (Lt-4)55 were visualized by immunoblotting with anti-Tax, anti-Flag, or anti-HA antibodies.
Tax inhibits TGF- -mediated
transcriptional responses using transient cotransfection assays. We
first used p3TP-Lux, a TGF--responsive reporter plasmid that
contains 3 repeats of a 12-O-tetradecanoylphorbol 13-acetate response
element and a fragment from positions  636 to 740 of the human
plasminogen activator inhibitor-1 (PAI-1)
promoter.51,56-58 This construct has been shown to be
efficiently stimulated by TGF- through its receptors in a variety of
cell lines. p3TP-Lux was transiently transfected together with either
empty (pH2Rneo) or Tax expression vector (pH2R40M), and luciferase
activity was measured in the extracts from untreated cells or cells
treated with 10 ng/mL TGF- for 24 hours. As a model for these
experiments, we used HepG2 and Mv1Lu cells, which are frequently used
for studies of TGF--induced transactivation because they express
TGF- receptors and are highly responsive to TGF- and contain
endogenous Smad2, Smad3, and Smad4. When p3TP-Lux alone was transfected
into HepG2 or Mv1Lu cells, a significant increase in luciferase
activity was observed in the presence of TGF- (Figure
1). These transactivations were repressed
almost to control levels when Tax was transfected with p3TP-Lux.
Similar repression occurred when we used another TGF--responsive reporter, p800neoLuc,52 which contained the PAI-1 promoter
alone, and p15P113-Luc,53 which contained the p15 promoter
(Figure 1). These results indicate that Tax may repress TGF-
signaling by interrupting intracellular signaling pathways.
To determine whether Tax could affect the antiproliferative effects of
TGF-
Tax inhibits Smad-induced responses to TGF- -induced
transcriptional activation of downstream genes. To investigate the
effects of Tax on Smad signaling, we assayed transcriptional responses
in the presence of various Smad proteins. As shown in Figure
3, Tax inhibits Smad2-, Smad3-, Smad4-,
or a combination of Smad2 and Smad4 (Smad2/4)- or Smad3 and Smad4
(Smad3/4)-induced transcriptional activation of p3TP-Lux. Therefore,
Tax inhibited TGF- signaling by blocking the ability of the
Smad2/Smad4 and Smad3/Smad4 complex to activate the transcription of
TGF--responsive genes.
Tax inhibits transcriptional activation induced by the
constitutively active T RI, associate with Smad4, and are subsequently translocated into
the nucleus.28,29 To determine whether the activated
TRI-induced transcription is affected by Tax, we used TRI-T204D
(a constitutively active TGF- type I kinase receptor). TRI-T204D
induced transcription, which is likely mediated by endogenous Smad
proteins. On the other hand, elevation of luciferase activity by
TRI-WT (wild-type TGF- type I receptor) did not occur with the
p3TP-Lux construct because TRI-WT could not signal in the absence of
ligand. TRI-T204D induced transcription was significantly reduced by
Tax (Figure 4A).
TGF- or activin signaling, which induces assembly of a DNA-binding
complex that is composed of Smad2, Smad4, and a member of the FAST
family of forkhead DNA-binding proteins. Because HepG2 cells do not
have endogenous FAST activity, in the absence of overexpressed FAST,
the ARE-Lux reporter construct was not stimulated by the overexpression
of a combination of Smad2 and Smad4, either in the presence or absence
of TGF- (Figure 4B). In the presence of overexpressed FAST, the
reporter gene activity was induced by TGF-, which may be explained
by an activation mediated by endogenous Smad2 and Smad4 proteins.
Furthermore, the coexpression of Smad2, Smad4, and FAST resulted in an
activation of the ARE-Lux construct, indicating that these 3 different
proteins form a transcriptionally active complex. Enhancement of these
transcription by TGF- occurred. We investigated the effect of Tax on
transcriptional activation of the ARE. Cotransfection of Tax markedly
inhibited transcriptional activation of the ARE-Lux, mediated by a
complex of Smad2, Smad4, and FAST (Figure 4B).
Lack of interaction of Tax with Smad proteins Because Tax was shown to associate with DNA-binding proteins in transactivation,59,60 it was suspected that Tax might physically interact with Smad proteins. To identify the target through which Tax represses TGF- signaling, we examined whether Tax could
interact with the Smad proteins. To this end, we transfected Smad2 and Smad3 tagged with the Flag peptide (Flag-Smad2 and Flag-Smad3) and
Smad4 tagged with the HA peptide (Smad4-HA) into COS7 cells in the
absence or the presence of Tax. Whole extracts from these cells were
immunoprecipitated with the anti-Flag and anti-HA antibodies, and the
precipitates were analyzed by immunoblotting with the anti-Tax
antibody. As shown in Figure 5A, we could
not observe that Tax was coimmunoprecipitated. Smad proteins were
expressed efficiently along with Tax in the transfected cells, as can
be seen by immunoblotting with the anti-Flag and anti-HA or the
anti-Tax antibody (Figure 5A, middle and bottom, lanes 3-8). Whole cell extracts from COS7 cells transfected were also immunoprecipitated using
anti-Tax. However, Smad proteins were not detected in the immunoprecipitates with anti-Tax (data not shown). These results suggest that Tax possibly antagonizes TGF- signaling through an
indirect mechanism that does not involve binding to Smad
proteins.
Tax does not inhibit receptor-dependent formation of heteromers containing Smad2 and Smad4 Smad2 or Smad3 is directly phosphorylated by the activated TRI,
associates with Smad4, and is subsequently translocated into the
nucleus.28,29 To determine which of these processes is affected by Tax, we first examined ligand-induced formation of heteromers containing Smad2 and Smad4. As shown in Figure 5B, Smad2
associates with Smad4 by the constitutively activated TRI (TRI-T204D) in the presence of Tax as strongly as it is in the absence of Tax, indicating that Tax does not inhibit receptor-dependent heteromers formation of Smad2 and Smad4. Tax did not immunoprecipitate with heteromers containing Smad2 and Smad4 (data not shown).
Tax mutant defective in CBP/p300 binding fails to repress the Smad-dependent transcriptional activation To analyze further the pathways through which Tax inhibited TGF- signaling, we examined several previously characterized Tax
mutants to see which failed to inhibit Smad transactivation. After an
initial screen of multiple Tax mutants, we obtained data for 2 Tax
mutants, Tax703 (M47), which contains amino acid substitutions at
positions 319 and 320, and M22, which contains amino acid substitutions at positions 130 and 131.48 An HTLV-I LTR luciferase
reporter that contains 3 unique CRE-containing 21-bp repeats was used
to assay for the effects of Tax on the CREB pathway. HepG2 cells were
transfected with the HTLV-I LTR-LUC or  B-LUC reporter plasmids, together with the control pHAPr-1-neo or plasmid expressing the wild-type Tax or the Tax mutants M22 and Tax703. These studies demonstrated that wild-type Tax could activate gene expression from
both the HTLV-I LTR and the NF-B (Figure
6A). In contrast, the Tax mutant Tax703
was defective in the activation of gene expression from the HTLV-I LTR
but not the NF-B, whereas the Tax mutant M22 activated gene
expression from the HTLV-I LTR but not the NF-B. The relative
ability of each Tax mutant to repress the PAI-1 promoter luciferase was
then compared in transient transfection assays in HepG2 cells. Both
wild-type Tax and M22 were able to significantly repress transcription
from the PAI-1 promoter (Figure 6A). In contrast, Tax mutant Tax703
failed to inhibit Smad function. These results indicate that
Tax-mediated activation of CREB pathway is essential for the repression
of Smad transactivation function. Interaction of Tax with CBP/p300 is
essential for transactivation of the viral LTR.49,61
Tax703 showed a decreased binding of CBP.62 To further
demonstrate that Tax interaction with CBP/p300 was necessary for the
repression of the PAI-1 promoter, we used a Tax mutant defective for
CBP/p300 interaction.49 Tax K88A carries a single point
mutation within the CBP/p300 binding domain, and this protein does not
interact with the amino-terminal KIX domain of CBP/p300.49
Tax K88A activated NF-B but not HTLV-I LTR promoter activity,
whereas wild-type Tax activated both promoter activities in HepG2 cells
(Figure 6B). Using this mutant Tax, we analyzed the effect on the
transactivation functions of Smad protein. As expected, Tax K88A failed
to repress transcription from the PAI-1 promoter (Figure 6B),
indicating that the CBP/p300 binding domain of Tax is involved in the
suppression of Smad transactivation function. Taken together, our data
demonstrate that Tax repression of the PAI-1 promoter activity
correlates with the ability of Tax to interact with the coactivators
CBP or p300.
Coexpression of CBP and p300 recovers repression of Smad3-mediated transactivation by Tax Recently, association of various Smads with the coactivators CBP and p300 for the potentiation of TGF--induced transcriptional activity has been demonstrated.42-46 Tax was also shown to
bind to CBP and p300.62,63 The observations described
above suggested that the suppression of Smad transactivation by Tax
might occur through sequestration of a limiting pool of common
transcriptional coactivators, such as CBP and p300, and thus may be
reversed by the expression of additional amounts of these coactivators.
First, we showed that cotransfection of HepG2 cells with Smad3 and with a CBP or p300 expression plasmid, together with the p3TP-Lux, led to an
increase of Smad3 transcriptional activity (Figure
7A). Next, a CBP or p300 expression
plasmid was cotransfected with a p3TP-Lux reporter plasmid, together
with Tax and Smad3 expression plasmids. As observed previously (Figure
3), Tax inhibited Smad3 transcriptional activity in HepG2 cells (Figure
7A). Significantly, coexpression of CBP or p300 reversed the inhibition
of Smad3 by Tax (Figure 7A). These results confirm that CBP/p300
potentiated Smad3-dependent transcription. They also indicate these
coactivators counter-inhibited the Tax trans-repressing effect on
Smad3-dependent transcription.
Reciprocal repression between Tax and Smad3 If repression of Smad3 by Tax occurs as a consequence of competition for CBP/p300, then overexpression of Smad3 should similarly repress Tax function. To test this possibility, we performed the reciprocal experiment using a reporter plasmid HTLV-I LTR-LUC. Cotransfection of the Smad3 expression plasmid repressed Tax transcriptional activation of the HTLV-I LTR (Figure 7B). The reciprocal repression observed with these 2 transcription factors shows that a cross-coupling mechanism is operating between Tax and Smad. Tax might compete with Smad in binding with CBP/p300, thereby repressing its transactivation function (Figure 8A). However, Tax has been shown to interact with the amino-terminal KIX domain, whereas the Smad proteins interact with a carboxy-terminal region of CBP/p300.62-64 Alternatively, either Tax or Smad3 directly interacts with CBP/p300, and this interaction leads to a change in conformation or stability of the complex comprising the other factor and CBP/p300 (Figure 8B). Taken together, these results indicate that the corepression of transcriptional activity by Tax and Smad is consistent with the sequestration of a limiting pool of CBP/p300.
In the current study, we show that the HTLV-I Tax functions as a
negative regulator in TGF- Tax is known to target cellular proteins, such as I Although definitive involvement of Smad proteins in hematologic
malignancies remains to be determined, defects in the TGF-
We thank Dr M. Hatanaka for pH2R40M and pH2Rneo; Dr K. Matsumoto for pH
Submitted October 2, 2000; accepted December 6, 2000.
Supported in part by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Naoki Mori, Department of Preventive Medicine and AIDS Research, Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan; e-mail: n-mori{at}net.nagasaki-u.ac.jp.
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signaling through
interaction with CREB-binding protein/p300
Top
Abstract
Introduction
chain,
, 
,
+Tax). Cells were incubated for 24 hours in the presence or absence of
10 ng/mL TGF-
], Tax-10 [
], and Tax-15 [
]) and
unmodified (clone Neo-10 [
p
