Estradiol Represses Human T-Cell Leukemia Virus-type 1
Tax Activation of Tumor Necrosis Factor-α Gene
Transcription
Christina Tzagarakis-Foster1, Romas Geleziunas2, Abderrahim Lomri1,
Jinping An1 and Dale C. Leitman1*
1Department of Obstetrics, Gynecology and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, 513 Parnassus Avenue, San Francisco CA 94143, USA. 2 Merck Research Laboratories, West Point, PA 19486 Present address for Abderrahim Lomri: Hop Lariboisiere, INSERM, U349, F-75475 Paris, France
Running Title: E2 Repression of the TNF-α Gene Address correspondence to Dale C. Leitman: University of California, San Francisco Center for Reproductive Sciences, HSE 1619 P.O. Box 0556 San Francisco, CA 94143-0556 Tel. (415) 502-5261 FAX (415) 753-3271 email: [email protected]
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on September 16, 2002 as Manuscript M205355200 by guest on M
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Adult T-cell leukemia is caused by human T-Cell Leukemia Virus type I (HTLV-I).
HTLV-I Tax protein is essential for clinical manifestations because it activates viral and
cellular gene transcription. Tax enhances production of TNF-α, which may lead to bone
and joint destruction. Because estrogens might prevent osteoporosis by repressing TNF-α
gene transcription, we investigated whether estrogens inhibit the transcriptional effects of
Tax on the TNF-α promoter. Tax activated the –1044, -163 and –125 TNF-α promoters by
9-25-fold, but not the –82 promoter, demonstrating that Tax activation requires –125 and –
82 region, known as the TNF-RE. Three copies the TNF-RE upstream of the minimal
thymidine kinase promoter conferred a similar magnitude of activation by Tax. We
demonstrated that c-jun, NFκB p50 and p65 interact with and activate the TNF-RE by
using mutational analysis of the TNF-RE, Tax mutants that selectively activate NFκB or
CREB/ATF pathway, and gel shift assays with nuclear extracts. Estradiol markedly
repressed Tax activated transcription of the TNF-α gene with ERα or ERβ. Nuclear
extracts from U2OS cells stably transfected with ERα demonstrated that ERs interact with
the TNF-RE. Our studies provide evidence that ERs repress Tax activated TNF-α
transcription by interacting with a c-jun and NFκB platform on the TNF-RE. Estrogens
may ameliorate bone and inflammatory joint diseases in patients infected with HTLV-I by
repressing transcription of the TNF-α gene.
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INTRODUCTION
The human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell
leukemia (ATL), which is a fatal T-lymphoproliferative disorder (1,2), and a chronic progressive
disease of the central nervous system termed HTLV-1 associated myelopathy/tropical spastic
paraparesis (3). HTLV-1 infection is also associated with several autoimmune disorders, such as
Sjogren’s syndrome, and arthropathy, which is a chronic inflammatory disorder of joints similar
to idiopathic rheumatoid arthritis (4,5).
In addition to genes encoding retroviral structural proteins, such as gag, pol and env, HTLV-
1 also encodes for regulatory proteins, such as Tax (6,7). Tax is a 40 kDa zinc-finger protein
involved in the etiology of ATL and its associated diseases by stimulating viral and cellular gene
expression (8). Tax regulates gene expression mainly by activating a variety of transcription
factors that interact with promoters of target genes (9-14) (15,16). Tax likely participates in the
pathogenesis of diseases associated with HTLV-I infection by inducing multiple cytokine genes,
including IL-2, IL-2Rα, granulocyte-macrophage colony stimulating factor, IL-6, and tumor
necrosis factor-α (TNF-α) (17-24).
Transgenic mice expressing Tax display thymic aplasia, neurofibromas and skeletal
alterations, such as an increased number of osteoclasts (25-28). Tax expressing mice also exhibit
a bone phenotype similar to that observed in HTLV-1 infected humans. Tax promotes bone
diseases and hypercalcemia by increasing the expression of several cytokines. For example, T-
cells infected with HTLV-1 express constitutively high levels of TNF-α (29) leading to increased
serum levels of TNF-α (30). Because TNF-α acts as an inhibitory factor for proliferation of
osteoblasts and promotes the differentiation of precursor cells to mature osteoclasts (31),
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excessive TNF-α production leads to bone resorption and hypercalcemia (32), two characteristic
features of ATL.
Albrecht et al. reported that Tax could activate the TNF-α promoter in a region that binds
NFκB (33). We previously identified a region in the TNF-α promoter, the TNF-response element
(TNF-RE) that is activated by TNF-α (34). We also found that estradiol (E2) represses TNF-α
activation of the TNF-α promoter in the presence of estrogen receptor α (ERα) or β (ERβ) (35).
Intriguingly, repression by ERs does not require direct DNA binding, as deletion of the DNA
binding domain of ER does not prevent E2 from inhibiting TNF-α activation of the TNF-α gene
(35). This observation suggests that repression by ERs is not mediated through DNA binding, but
most likely through protein-protein interactions with other transcription factors at the TNF-α
promoter.
Several studies indicate that HTLV-1 infection exhibits gender specific differences in clinical
outcomes, which are thought to result from different levels of sex hormones. Women infected
with HTLV-1 have a lower level of Tax expression and viral load as well as a decreased incidence
of ATL compared to men (36). Furthermore, Hisada et al. reported that the mortality from ATL
is 4-fold higher in males relative to females (37). These observations suggest that higher levels of
estrogens in women may attenuate the clinical effects of HTLV-1 by inhibiting the action of Tax.
To test this hypothesis, we investigated if estrogens repress Tax-mediated stimulation of TNF-α
expression. The results shown here demonstrate that ERs repress Tax activation of TNF-α gene
transcription in the presence of E2 by interacting with c-jun and NFκB. E2 repression of Tax-
induced of TNF-α gene expression, suggests that estrogens may provide a potential therapeutic
approach to ameliorate HTLV-1 associated bone and inflammatory joint diseases.
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MATERIALS AND METHODS
Cell Culture, Transient Transfection and Luciferase Assays–U937 (human monocytic
leukemia cells) and U2OS (human osteosarcoma cells) were maintained as previously described
(38). For transient transfection assays, cells were collected, transferred to a cuvette and
electroporated using a Bio-Rad gene pulser as previously described (38,39). Following
electroporation, cells were resuspended in phenol red free DMEM/F-12 media containing 5%
fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin and
plated in 12-well tissue culture dishes. Cells were treated with 17β-estradiol (E2) for 24 hours
following transfection and then collected and lysed in 200 µl 1X lysis buffer (Promega Co.,
Madison, WI). Luciferase assays were performed according to the manufacture’s instructions
(Promega Co., Madison, WI) using a Monolight luminometer. All experiments presented in the
figures were performed at least three times, and the data were similar between experiments.
U20S cells stably transfected with a plasmid that expresses a Tet repressor were purchased from
Invitrogen (Invitrogen Life Technologies, Carlsbad, CA). These cells were then stably transfected
with full-length ERα cloned downstream of a CMV promoter that contains two Tet responsive
elements (pcDNA TO vector). The U20S-ERα cells were selected with hygromycin and zeocin.
Individual clones were screened for the presence of ERα by RT-PCR and western blotting.
Electrophoretic Mobility Shift Assays–Binding reactions were performed using purified NFκB
p50 or c-jun (Promega Co., Madison, WI). 1 µl undiluted c-jun or 1 µl diluted NFκB p50
[diluted 1:25 in buffer containing 20mM HEPES (pH 7.9), 400 mM KCl, 1 mM EDTA (pH 8.0),
1 mM EGTA (pH 8.0), 1 mM DTT, 1 mM PMSF, 10% glycerol] was added to 15 µl total of
binding buffer [10 mM HEPES, (pH 7.9), 50 mM KCl, 0.2 mM EDTA (pH 8.0), 0.6 mM EGTA
(pH 8.0), 10% glycerol, 2.5 mM DTT, 200 µg BSA, 2 µg poly (dI-dC)]. Binding reactions were
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incubated for 15 minutes at 4°C. Following binding, anti-c-jun (Cell Signaling Technologies) or
anti-NFκB p50 (gift from Dr. Warner Greene, Gladstone Institute of Virology and Immunology,
San Francisco, CA) were added to the reactions, which were incubated for an additional 15
minutes at 4°C. Radiolabeled wild-type TNF-RE (containing the –125 to –82 region of the TNF-
α promoter) probe was then added (40,000 cpm per reaction) and binding reactions were allowed
to incubate for an additional 15 minutes at room temperature. Resulting complexes were
electrophoresed through a 5% non-denaturing polyacrylamide gel with 1X TBE running buffer
(200 volts, 4°C). Following electrophoresis, gels were dried and exposed to film or examined
using a Storm Phosphorimager and analysis software (Molecular Dynamics).
Quantitative real-time PCR–U2OS-vector control or U2OS-ERα stable cells were transiently
transfected with 0.5 µg Tax expression plasmid (a gift from Dr. Warner Greene) and treated with
E2 or ethanol for 24 hours. Following treatment, total RNA was isolated using Trizol (Invitrogen
Life Technologies, Carlsbad, CA). Reverse transcription reactions were performed using 500 ng
total RNA, 250 ng random primers (Invitrogen Life Technologies, Carlsbad, CA), 200 units
MuLV Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA), 1X RT buffer, 1
mM dNTP mix, 7.5 mM MgCl2 and 40 units RNAse inhibitor (Boehringer Mannheim,
Indianapolis, IN). Reactions were incubated at 25 °C for 10 minutes, 48 °C for 40 minutes, and
95 °C for 5 minutes. Real-time PCR detection of TNF-α expression was performed using the
Pre-Developed TaqMan Assay Reagents Target Kit for TNF-α (PE Biosystems, Foster City, CA)
and the ABI PRISM® 7700 (Applied Biosystems, Foster City, CA). Control reactions were
performed using primers and a probe to detect β-glucuronidase (GUS). Gus primers were: (GUS
forward) CTCATTTGGAATTTTGCCGATT, (GUS reverse)
CCGAGTGAAGATCCCCTTTTTA and probe 6FAM-
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TGAACAGTCACCGACGAGAGTGCTGG-TAMRA (Operon, Alameda, CA). Average Ct
(threshold cycle) was calculated using the sequence detection software supplied with the ABI
PRISM® 7700.
RESULTS
Tax Activation and ERα and ERβ Repression of the TNF-α Promoter in U937 Cells–We used
the human monocytic leukemia cell line, U937 to identify regions within the TNF-α promoter
that may be responsive to HTLV-I Tax. This cell line is known to express the TNF-α gene in
response to cytokines (34). U937 cells were co-transfected with the luciferase reporter
containing several deletions of the TNF-α promoter (-1044 to -82) and a Tax expression vector.
Tax activated the –1044, –163 and –125 TNF-α promoter constructs by about 9-25-fold (Fig. 1).
Deleting the TNF-α promoter from –125 to -82 abolished Tax activation. The -125 to -82 region
of the TNF-α promoter was previously termed the TNF-response element, because it is activated
by TNF-α (34). Tax activated three copies of the TNF-RE upstream of the minimal thymidine
kinase promoter by a similar magnitude to the -1044 TNF-α promoter (Fig. 2A). This finding
demonstrates that the TNF-RE contains elements that confer responsiveness to Tax. Expression
of ERα or ERβ resulted in a marked repression (88% and 73%, respectively) of Tax-stimulated
TNF-RE activity in response to E2. The repression by E2 was dose-dependent with maximal
effect observed at 1 nM (Fig. 2B). E2 also repressed Tax activation of the TNF-RE in an adult
human osteoblastic (AHTO) cell line (40) that is immortalized by SV-40 Large T oncogene (Fig.
2C), demonstrating that the effect of E2 also occurs in bone cells.
ERα and ERβ Repress Endogenous TNF-α Expression in U2OS Cells–In order to investigate
if the repression by E2 is physiologically relevant, we performed quantitative real time-PCR
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analysis to determine if E2 also represses Tax-mediated activation of the endogenous TNF-α
gene. For these studies, we used U2OS-ERα cells, which are human osteosarcoma cells stably
transfected with a tetracycline inducible CMV promoter that drives the expression of the ERα
cDNA. The U2OS-ERα cells or a vector control cell line (U2OS-vector) were induced with
doxycycline, transfected with the Tax expression plasmid and treated with E2 for 12 hours.
Quantitative PCR analysis demonstrates that Tax activates the endogenous TNF-α gene by 3-fold
compared to the U2OS-vector control cells without Tax (Fig. 3). E2 produces a 50% reduction in
Tax induced TNF-α mRNA levels in the U2OS-ERα cells. These results demonstrate that
similar to transient transfection assays, ERα represses Tax activation of the endogenous TNF-α
gene expression in an E2-dependent manner.
c-jun/CRE and NFATp/NFκB Elements are Required for Tax Activation of the TNF-RE–We
next sought to explore the mechanism whereby Tax activates the TNF-α promoter. Several
studies showed that ETS, ATF-2, NFATp, NFκB and c-jun/CREB-related transcription factors
bind to the TNF-RE in the TNF-α promoter (34,41-43). NFATp/NFκB and c-jun/CRE are
apparently the most critical elements that regulate the TNF-α promoter function because previous
studies eliminated the ETS binding site as being central to TNF-α promoter activity (44). To
investigate the role of NFATp/NFκB (5’ GGGTTTCTCC 3’) and c-jun/CRE (5’ TGAGCTCA 3’)
elements in Tax activation of the TNF-α promoter, transfection assays were performed using
luciferase reporters containing the wild type TNF-RE or mutations of the c-jun/CRE or
NFATp/NFκB binding sites upstream of the tk promoter. As observed in previous experiments,
Tax activates the wild type TNF-RE approximately 10-fold (Fig. 4), whereas mutations in the c-
jun/CRE or NFATp/NFκB binding site severely diminished Tax activation. As expected,
mutations in both c-jun/CRE and NFATp/NFκB binding sites also dramatically impairs Tax
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activation. No differences were observed between the levels of Tax activation with the single
mutation reporters compared to the double mutation reporter constructs. Therefore, maximal Tax
activation of the TNF-α promoter requires both c-jun/CRE and NFATp/NFκB elements.
NFκB Activity is Necessary for Tax-mediated Activation of the TNF-RE–To further dissect the
pathways involved in Tax activation of the TNF-α promoter, we utilized two previously
characterized Tax mutants, Tax M22 and Tax M47. The Tax M22 mutant is unable to activate
NFκB while maintaining its ability to activate transcription factors of the CREB/ATF family
(45-47), whereas the Tax M47 mutant activates the NFκB pathway, but not the CREB/ATF
pathway (48). As shown in Fig. 5, wildtype and Tax M47 activated the TNF-RE, which was
inhibited by E2. In contrast, Tax M22 was ineffective at activating the TNF-RE and no repression
by E2 was observed. These results demonstrate that Tax has to activate NFκB, but not
CREB/ATF in order to stimulate the TNF-α promoter.
Purified NFκB p50 and c-jun Bind to the TNF-RE–We investigated the ability of c-jun and
NFκB to bind the TNF-RE by electrophoretic mobility shift assays. Binding reactions were done
with purified p50 and c-jun, the DNA binding component of NFκB and AP-1, respectively. As
shown in Fig. 6, both purified NFκB p50 and c-jun bind to the TNF-RE probe (lanes 1 and 2
respectively). When reactions containing both NFκB p50 and c-jun were incubated for 15
minutes or 60 minutes prior to binding to the TNF-RE probe, there are shifted complexes that
correspond to the individual proteins (compare lanes 3 and 4 with lanes 1 and 2). By using
selective antibodies it is clear that the slower migrating complex (marked by arrow) contains
both NFκB p50 and c-jun. (lanes 5 and 6). These results indicate that NFκB p50 and c-jun bind
simultaneously to the TNF-RE.
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ERα Binds to a c-jun/NFκB Complex in the TNF-RE–To begin to probe the mechanism
whereby E2 represses Tax-mediated activation of the TNF-α promoter, we investigated our
hypothesis that ERs bind to the TNF-α promoter indirectly via interactions with transcription
factors bound to the TNF-RE, because we previously reported that ERs do not bind directly to
the TNF-RE (35). For these studies, we used U20S cells stably transfected with ERα. Following
treatment with doxycycline to induce ERα expression, the cells were transfected with the Tax
expression plasmid and treated with E2 for 12 hours. Binding reactions were performed with
U20S-ERα nuclear extracts and specific antibodies to c-jun, NFκB p50, NFκB p65, ATF-2 and
ERα to identify candidate proteins that bind the TNF-RE promoter (lanes 2 – 6 respectively). A
predominant single shifted band is observed with the nuclear extract (Fig. 7). This band is
supershifted with antibodies to c-jun, NFκB p50, and NFκB p65, but not with an antibody to
ATF-2. This pattern demonstrates that a p50 and p65 heterodimer binds to the NFκB site in the
TNF-RE. Furthermore, c-jun binds to the TNF-RE, but not ATF-2, suggesting that the complex
that binds to the c-jun/CRE element is a homodimer of c-jun or more likely a heterodimer with
another transcription factor. There is also a strong supershift with the ERα antibody indicating
that it is also present in the complex bound to the TNF-RE. Taken together, these data suggest
that the complex that binds to the TNF-RE contains c-jun and NFκB, and that ERα represses the
TNF-α promoter by binding to these factors via protein-protein interactions.
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DISCUSSION
Our studies demonstrate that ERs repress Tax activation of TNF-α promoter in transient
transfection assays. The results from quantitative PCR analysis showed that the repression by E2
is not an artifact of reporter plasmids, because E2 produced a similar magnitude of repression of
the endogenous TNF-α gene. Deletion studies showed that the -125 to -82 region of the TNF-α
promoter is necessary for Tax activation. This region contains binding sites for ETS, c-jun, ATF-
2, NFATp and NFκB transcription factors that might mediate the Tax activation of the TNF-α
promoter. Tsai et al. reported that a c-jun/ATF-2 heterodimer binds to the c-jun/CRE in the TNF-
RE (42). The CREB/ATF defective M47 Tax mutant activated the TNF-RE more than wild-type
Tax, suggesting that CREB and ATF-2 are not the factors activated by Tax in these cells. Carter
et al. showed that M47 Tax is more stable then wildtype tax (49), which can explain the finding
that M47 Tax produced a greater activation of the TNF-RE, compared to wildtype Tax.
Activation of the NFκB element by Tax is essential for maximum activation of the TNF-α
promoter, because the M22 Tax mutant deficient in activating the NFκB pathway is unable to
stimulate the TNF-RE reporter. Our in vitro DNA binding studies using nuclear extracts from
Tax-stimulated human osteosarcoma cells showed that NFκB p50, NFκB p65, c-jun and ERα, but
not ATF-2 interact with TNF-RE element. These results indicate that the activation of the TNF-α
promoter by Tax requires c-jun and NFκB, but not ATF-2. Furthermore, c-jun and NFκB must
bind simultaneously to the TNF-RE to activate the TNF-α promoter, because a mutation in either
site abrogates activation by Tax. Other studies reported that related factors, such as c-fos do not
interact with the TNF-RE (34).
Our results suggest the following model whereby ER represses the activation by Tax at the
TNF-RE. After the activation of NFκB by Tax, the NFκB p50/p65 complex binds next to c-jun
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on the TNF-RE leading to the activation of the TNF-α promoter. ER is then recruited to the
promoter by binding to a platform comprised of c-jun and NFκB. Support for this model of ER
action is provided by evidence that ERα or ERβ do not directly bind to the TNF-α promoter (35)
and that NFκB p65 and ER can interact (50-53). Furthermore, ERα and c-jun proteins interact
directly (54) in glutathione S-transferase pull-down and mammalian two-hybrid assays (55).
Alternatively, ER could be recruited to the TNF-α promoter through a bridging protein in direct
contact with the c- jun/NFκB complex.
Examination of the sequence within the TNF-RE region of the TNF-α promoter reveals that
there is only 1 nucleotide separating the c-jun/CRE and NFATp/NFκB elements. Previous
experiments have found that nucleotide insertions resulting in either 0.5, 1 or 1.5 extra turns of the
DNA helix abolishes TNF-α activation (44). Therefore, it is likely that c-jun and NFκB on the
TNF-α promoter are critical for providing the initial platform for ER binding to the TNF-α
promoter, and disruption of the platform prevents the assembly of other factors necessary for
activation of the TNF-α gene by Tax.
One key question that remains is what are the additional proteins in the complex that mediate
repression. There is evidence suggesting that coactivators, such as glucocorticoid receptor
interacting protein-1 (GRIP1) are involved in steroid receptor repression of gene transcription.
GRIP1 potentiates ER-mediated repression of the TNF-α gene (35) and glucocorticoid receptor-
mediated repression of the collagenase gene (56). Another protein that may be part of the
repression complex is p300/CBP, because it is involved in Tax-mediated activation of HTLV-1
transcription in vitro (57). Whether these proteins are an integral part of the repression complex
at the TNF-α promoter or if other proteins participate in repression remains to be determined.
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Individuals infected with HTLV-1 suffer from various bone and joint diseases most likely
due to an increased expression of various cytokines, including TNF-α. Hypercalcemia, which
results from enhanced bone resorption, is a prevalent complication observed in patients with ATL,
and has been linked to early death (58). The cause of hypercalcemia in ATL patients is unknown,
but it has been suggested that TNF-α plays a role in this disease because it is associated with
increased serum levels of TNF-α (30). Tax is also strongly expressed in human synovial cells,
which leads to joint destruction. In a transgenic mouse model of HTLV-I infection, mice
expressing Tax exhibit skeletal alterations resembling Paget’s disease, a chronic disorder that
results in enlarged and deformed bones and have chronic inflammatory polyarthropathy (8,59).
Analysis of the joints in these transgenic mice demonstrated enhanced expression of several
cytokines including TNF-α (59).
Intriguingly, similar to HTLV-I infected patients, many postmenopausal women develop
bone and joint diseases that might occur from excessive TNF-α production. A prominent role for
TNF-α in the pathogenesis of osteoporosis is supported by animal studies. For example,
overexpression of TNF-α in mice produces profound hypercalcemia from enhanced bone
resorption (60). Furthermore, the loss of bone mineral density observed in mice after
oophorectomy can be prevented with TNF binding proteins (61) or a soluble TNF receptor that
prevents the action of TNF-α (62). TNF-α might cause osteoporosis by inducing several proteins
responsible for differentiating precursor monocytic cells into bone resorbing osteoclasts (63,64).
Estrogens are used extensively in postmenopausal women to prevent osteoporosis (65,66).
Several studies indicate that the bone sparing effect of estrogens is at least in part due to its ability
to repress TNF-α gene transcription and down regulate TNF-α levels (67,68). Other studies
indicate that TNF-α is involved in the destruction of articular cartilage that is observed in
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osteoarthritis (69). Osteoarthritis is a prevalent condition that is exacerbated by estrogen
deficiency in postmenopausal women and shows some improvement with estrogen replacement
(70), possibly by decreasing TNF-α levels. Whereas estrogens are useful in postmenopausal bone
and joint diseases, our study suggests that they also might be a potential therapeutic approach for
HTLV-1 associated bone and inflammatory joint diseases by directly targeting TNF-α gene
expression. Estrogens are known to exhibit anti-inflammatory properties (71), but it is not known
if this effect occurs through ERα, ERβ or both ERs. Our results demonstrating that ERβ is more
effective than ERα at inhibiting TNF-α and Tax-activation of TNF-α gene transcription, suggest
that ERβ may be the predominate receptor that mediates the anti-inflammatory effects of
estrogens. The finding that E2 is a potent repressor of Tax activation of TNF-α gene may also
account for the observations that females infected with HTLV-I exhibit less severe clinical
manifestations and lower mortality compared to males (37).
ACKNOWLEDGEMENTS
We thank P. Chambon, J-A Gustafsson, and W. Greene for providing plasmids. This
work was supported by a NIH postdoctoral training grant and Bank of America Giannini
postdoctoral fellowship to C. T-F, and grants to D. C. L. from the Paul Beeson Physician Faculty
Scholars in Aging Research Program (funded by Alliance for Aging Research, John A. Hartford
Foundation, Commonwealth Fund and Starr Foundation), National Institute of Child Health and
Human Development Women’s Reproductive Health Research Program and Susan B. Komen
Foundation.
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FIGURE LEGENDS
FIG. 1. A Tax-responsive element is localized to the –125 to –82 region in the TNF-α
promoter. U937 cells were transfected with plasmids containing a luciferase reporter fused to
various regions of the TNF-α promoter or three copies of the TNF-RE (-125 to -82) upstream of
the minimal thymidine kinase promoter in the presence or absence of a Tax expression plasmid
(1 µg). After 24 hours cells were harvested and luciferase activity was assayed. Each data point
is the average of triplicate determinations. The S.E. was < 10%.
FIG. 2. A, ERα and ERβ repress Tax-activated transcription of the TNF-RE in U937 cells.
U937 cells were transfected with TNF-RE-tk-luc plasmid (3 µg), a Tax expression vector (500
ng) and an expression vector for ERα (1 µg) or ERβ (1 µg). Cells were treated with 10 nM E2 for
24 hours and luciferase activity was measured. B, dose-dependent repression of ER mediated
repression of Tax activation of the TNF-RE. U937 cells were transfected as described above and
then treated with increasing concentrations (10–13 M to 10–6 M) of E2. After 24 hours cells were
harvested and luciferase activity was assayed. C, ERα and ERβ repress Tax-activated
transcription of the TNF-RE in human (AHTO) osteoblasts. AHTO cells were transfected with
TNF-RE-tk-luc plasmid (3 µg), a Tax expression vector (500 ng) and an expression vector for
ERα (1 µg) or ERβ (1 µg). Cells were treated with 10 nM E2 for 24 hours and then luciferase
activity was measured. Each data point is the average of triplicate determinations. The S.E was
< 10%.
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FIG. 3. Tax stimulates endogenous TNF-α gene expression. Tetracycline-inducible
U2OS-ERα cells were transfected with the Tax expression plasmid. The cells were treated with
doxycycline to induce ERα expression and maintained in the absence or presence of 10 nM E2
for 24 hours. Quantitative real time-PCR was performed for TNF-α mRNA, which was
normalized to β−glucoronidase mRNA.
FIG. 4. Tax activation of the TNF-α promoter requires c-jun/CRE and NFκB/NFATp
binding sites. Mutations were made in either the c-jun/CRE or NFκB/NFATp elements located
within the –125 to +93 TNF-α promoter. U937 cells were cotransfected with the Tax expression
plasmid (500 ng) and luciferase reporter constructs containing wildtype TNF-RE, c-jun/CRE
mutation, NFκB/NFATp mutation or c-jun/CRE – NFκB/NFATp double mutation upstream of
the tk promoter. Cells were treated with 10 nM E2 for 24 hours and luciferase activity was
measured. Each data point is the average of triplicate determinations. The S.E. was < 10%.
FIG. 5. Tax activation of the TNF-α promoter requires activation of NFκB. U937 cells
were cotransfected with TNF-RE tk-luc, ERα or ERβ expression plasmid (1 µg) and either a
wildtype Tax expression plasmid, a Tax M22 mutant (NFκB -/CREB-ATF +), or a Tax M47
mutant (NFκB +/CREB-ATF -). Cells were maintained in the absence or presence of 10 nM E2
for 24 hours and luciferase and renilla luciferase activity were measured. Renilla luciferase
activity was used to normalize for transfection efficiency. Each data point is the average of
triplicate determinations. The S.E. was < 10%.
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FIG. 6. Purified NFκB p50 and c-jun bind to the TNF-RE. Electrophoretic mobility shift
assays were performed using purified NFκB p50 or c-jun. Binding reactions containing either
purified proteins were allowed to incubate for either 15 minutes or 60 minutes at 4 °C prior to
the addition of NFκB p50 or p65 specific antibodies. The presence of c-jun and NFκB p50 is
confirmed by supershifting with specific antibodies (marked by the asterisk). The arrow
indicates supershifted complexes.
FIG. 7. ERα, NFκB and c-jun interact on the TNF-RE. Tetracycline-inducible U2OS-ERα
cells were transfected with the Tax expression plasmid and then treated with 10 nM E2 for 24
hours, and doxycycline to induce ERα expression. Nuclear extracts were prepared and then
incubated with c-jun, NFκB p50, NFκB p65, ATF-2 or ERα antibodies. Proteins or protein
complexes that bound to a radiolabeled TNF-RE probe were detected by autoradiography. The
arrow indicates supershifted complexes.
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C. LeitmanChristina Tzagarakis-Foster, Romas Geleziunas, Abderrahim Lomri, Jinping An and Dale
gene transcriptionαnecrosis factor-Estradiol represses human T-cell Leukemia virus-type 1 tax activation of tumor
published online September 16, 2002J. Biol. Chem.
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