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Originally published In Press as doi:10.1074/jbc.M205355200 on September 16, 2002

J. Biol. Chem., Vol. 277, Issue 47, 44772-44777, November 22, 2002
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Estradiol Represses Human T-cell Leukemia Virus Type 1 Tax Activation of Tumor Necrosis Factor-alpha Gene Transcription*

Christina Tzagarakis-FosterDagger , Romas Geleziunas§, Abderrahim LomriDagger , Jinping AnDagger , and Dale C. LeitmanDagger ||

From the Dagger  Department of Obstetrics, Gynecology and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143 and § Merck Research Laboratories, West Point, Pennsylvania 19486

Received for publication, May 30, 2002, and in revised form, August 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Adult T-cell leukemia is caused by human T-cell leukemia virus type I (HTLV-I). The HTLV-I Tax protein is essential for clinical manifestations because it activates viral and cellular gene transcription. Tax enhances production of tumor necrosis factor-alpha (TNF-alpha ), which may lead to bone and joint destruction. Because estrogens might prevent osteoporosis by repressing TNF-alpha gene transcription, we investigated whether estrogens inhibit the transcriptional effects of Tax on the TNF-alpha promoter. Tax activated the -1044, -163, and -125 TNF-alpha promoters by 9-25-fold but not the -82 promoter, demonstrating that Tax activation requires the -125 to -82 region, known as the TNF response element (TNF-RE). Three copies of the TNF-RE upstream of the minimal thymidine kinase promoter conferred a similar magnitude of activation by Tax. We demonstrated that c-Jun, NFkappa B, p50, and p65 interact with and activate the TNF-RE by using mutational analysis of the TNF-RE, Tax mutants that selectively activate NFkappa B or the cAMP-response element binding protein/activating transcription factor pathway, and gel shift assays with nuclear extracts. Estradiol markedly repressed Tax-activated transcription of the TNF-alpha gene with estrogen receptor (ER) alpha  or beta . Nuclear extracts from U2OS cells stably transfected with ERalpha demonstrated that ERs interact with the TNF-RE. Our studies provide evidence that ERs repress Tax-activated TNF-alpha transcription by interacting with a c-Jun and NFkappa B 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-alpha gene.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The human T-cell leukemia virus type 1 (HTLV-1)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 such as gag, pol, and env encoding retroviral structural proteins, HTLV-1 also encodes for regulatory proteins such as Tax (6, 7). Tax is a 40-kDa zinc finger protein that is 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 (9-16). Tax likely participates in the pathogenesis of diseases associated with HTLV-I infection by inducing multiple cytokine genes including interleukin-2, interleukin-2Ralpha , granulocyte-macrophage colony stimulating factor, interleukin-6, and tumor necrosis factor-alpha (TNF-alpha ) (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-alpha (29), leading to increased serum levels of TNF-alpha (30). Because TNF-alpha acts as an inhibitory factor for the proliferation of osteoblasts and promotes the differentiation of precursor cells to mature osteoclasts (31), excessive TNF-alpha production leads to bone resorption and hypercalcemia (32), two characteristic features of ATL.

Albrecht et al. (33) reported that Tax could activate the TNF-alpha promoter in a region that binds NFkappa B. We previously identified a region in the TNF-alpha promoter, the TNF-response element (TNF-RE), that is activated by TNF-alpha (34). We also found that estradiol (E2) represses TNF-alpha activation of the TNF-alpha promoter in the presence of estrogen receptor alpha  (ERalpha ) or beta  (ERbeta ) (35). Intriguingly, repression by ERs does not require direct DNA binding, because deletion of the DNA binding domain of ER does not prevent E2 from inhibiting TNF-alpha activation of the TNF-alpha 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-alpha promoter.

Several studies (36, 37) 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 with men (36). Furthermore, Hisada et al. (37) reported that the mortality from ATL is 4-fold higher in males relative to females. 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 whether estrogens repress Tax-mediated stimulation of TNF-alpha expression. The results shown here demonstrate that ERs repress Tax activation of TNF-alpha gene transcription in the presence of E2 by interacting with c-Jun and NFkappa B. E2 repression of Tax-induced TNF-alpha gene expression suggests that estrogens may provide a potential therapeutic approach to ameliorate HTLV-1-associated bone and inflammatory joint diseases.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture, Transient Transfection, and Luciferase Assays-- U937 (human monocytic leukemia cells) and U2OS (human osteosarcoma cells) were maintained as described previously (38). For transient transfection assays, cells were collected, transferred to a cuvette, and electroporated using a Bio-Rad Gene Pulser as described previously (38, 39). Following electroporation, cells were resuspended in phenol red-free Dulbecco's modified Eagle's medium/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 17beta -estradiol for 24 h following transfection and then collected and lysed in 200 µl of 1× lysis buffer (Promega, Madison, WI). Luciferase assays were performed according to the manufacturer's instructions (Promega) using a Monolight luminometer. All experiments presented in the Figs. 1-7 legends were performed at least three times, and the data were similar between experiments. U20S cells stably transfected with a plasmid that expresses a tetracycline repressor were purchased from Invitrogen. These cells were then stably transfected with full-length ERalpha cloned downstream of a cytomegalovirus promoter that contains two tetracycline-responsive elements (pcDNA TO vector). The U20S-ERalpha cells were selected with hygromycin and zeocin. Individual clones were screened for the presence of ERalpha by reverse transcription PCR and Western blotting.

Electrophoretic Mobility Shift Assays-- Binding reactions were performed using purified NFkappa B p50 or c-Jun (Promega). 1 µl of undiluted c-Jun or 1 µl of diluted NFkappa B p50 (diluted 1:25 in buffer containing 20 mM HEPES (pH 7.9), 400 mM KCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol) was added to 15 µl of total 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 dithiothreitol, 200 of µg bovine serum albumin, and 2 µg of poly(dI-dC)). Binding reactions were incubated for 15 min at 4 °C. Following binding, anti-c-Jun (Cell Signaling Technologies) or anti-NFkappa B 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 min at 4 °C. Radiolabeled wild-type TNF-RE (containing the -125 to -82 region of the TNF-alpha promoter) probe was then added (40,000 cpm per reaction), and binding reactions were allowed to incubate for an additional 15 min at room temperature. The resulting complexes were electrophoresed through a 5% nondenaturing polyacrylamide gel with 1× 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 (Amersham Biosciences).

Quantitative Real Time PCR-- U2OS-vector control or U2OS-ERalpha stable cells were transiently transfected with 0.5 µg of Tax expression plasmid (a gift from Dr. Warner Greene) and treated with E2 or ethanol for 24 h. Following treatment, total RNA was isolated using Trizol (Invitrogen). Reverse transcription reactions were performed using 500 ng of total RNA, 250 ng of random primers (Invitrogen), 200 units of MuLV reverse transcriptase (Invitrogen), 1× reverse transcriptase buffer, 1 mM dNTP mix, 7.5 mM MgCl2, and 40 units of RNase inhibitor (Roche Molecular Biochemicals). Reactions were incubated at 25 °C for 10 min, 48 °C for 40 min, and 95 °C for 5 min. Real-time PCR detection of TNF-alpha expression was performed using the pre-developed TaqMan assay reagents target kit for TNF-alpha (Applied Biosystems, Foster City, CA) and the ABI PRISM® 7700 (Applied Biosystems). Control reactions were performed using primers and a probe to detect beta -glucoronidase (GUS). GUS primers were GUS forward, 5'-CTCATTTGGAATTTTGCCGATT-3'; GUS reverse, '-CCGAGTGAAGATCCCCTTTTTA-3'; and probe 6FAM-TGAACAGTCACCGACGAGAGTGCTGG-TAMRA (Operon, Alameda, CA). Average threshold cycle was calculated using the sequence detection software supplied with the ABI PRISM® 7700.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tax Activation and ERalpha and ERbeta Repression of the TNF-alpha Promoter in U937 Cells-- We used the human monocytic leukemia cell line U937 to identify regions within the TNF-alpha promoter that may be responsive to HTLV-I Tax. This cell line is known to express the TNF-alpha gene in response to cytokines (34). U937 cells were co-transfected with the luciferase reporter containing several deletions of the TNF-alpha promoter (-1044 to -82) and a Tax expression vector. Tax activated the -1044, -163, and -125 TNF-alpha promoter constructs by about 9-25-fold (Fig. 1). Deleting the TNF-alpha promoter from -125 to -82 abolished Tax activation. The -125 to -82 region of the TNF-alpha promoter was previously termed the TNF-response element, because it is activated by TNF-alpha (34). Tax activated three copies of the TNF-RE upstream of the minimal thymidine kinase promoter by a similar magnitude to the -1044 TNF-alpha promoter (Fig. 2A). This finding demonstrates that the TNF-RE contains elements that confer responsiveness to Tax. Expression of ERalpha or ERbeta 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 the SV-40 large T oncogene (Fig. 2C), demonstrating that the effect of E2 also occurs in bone cells.


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  		Fig. 1.   A Tax-responsive element is localized to the -125 to -82 region in the TNF-alpha promoter. U937 cells were transfected with plasmids containing a luciferase reporter fused to various regions of the TNF-alpha 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 h cells were harvested, and luciferase activity was assayed. Each data point is the average of triplicate determinations. The S.E. was <10%.


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  		Fig. 2.   A, ERalpha and ERbeta repress Tax-activated transcription of the TNF-RE in U937 cells. U937 cells were transfected with the TNF-RE-tk-luc plasmid (3 µg), a Tax expression vector (500 ng), and an expression vector for ERalpha (1 µg) or ERbeta (1 µg). Cells were treated with 10 nM E2 for 24 h, 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 (10-13-10-6 M) of E2. After 24 h cells were harvested, and luciferase activity was assayed. C, ERalpha and ERbeta repress Tax-activated transcription of the TNF-RE in human (AHTO) osteoblasts. AHTO cells were transfected with the TNF-RE-tk-luc plasmid (3 µg), a Tax expression vector (500 ng), and an expression vector for ERalpha (1 µg) or ERbeta (1 µg). Cells were treated with 10 nM E2 for 24 h, and then luciferase activity was measured. Each data point is the average of triplicate determinations. The S.E. was < 10%.

ERalpha and ERbeta Repress Endogenous TNF-alpha Expression in U2OS Cells-- To investigate whether the repression by E2 is physiologically relevant, we performed quantitative real time PCR analysis to determine whether E2 also represses Tax-mediated activation of the endogenous TNF-alpha gene. For these studies, we used U2OS-ERalpha cells, which are human osteosarcoma cells stably transfected with a tetracycline-inducible cytomegalovirus promoter that drives the expression of the ERalpha cDNA. The U2OS-ERalpha 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 h. Quantitative PCR analysis demonstrates that Tax activates the endogenous TNF-alpha gene by 3-fold compared with the U2OS-vector control cells without Tax (Fig. 3). E2 produces a 50% reduction in Tax-induced TNF-alpha mRNA levels in the U2OS-ERalpha cells. These results demonstrate that, similarly to transient transfection assays, ERalpha represses Tax activation of the endogenous TNF-alpha gene expression in an E2-dependent manner.


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  		Fig. 3.   Tax stimulates endogenous TNF-alpha gene expression. Tetracycline-inducible U2OS-ERalpha cells were transfected with the Tax expression plasmid. The cells were treated with doxycycline to induce ERalpha expression and maintained in the absence or presence of 10 nM E2 for 24 h. Quantitative real time PCR was performed for TNF-alpha mRNA, which was normalized to beta -glucuronidase mRNA.

c-Jun/CRE and NFATp/NFkappa B Elements Are Required for Tax Activation of the TNF-RE-- We next sought to explore the mechanism whereby Tax activates the TNF-alpha promoter. Several studies showed that ETS-, ATF-2-, NFATp-, NFkappa B-, and c-Jun/CREB-related transcription factors bind to the TNF-RE in the TNF-alpha promoter (34, 41-43). NFATp/NFkappa B and c-Jun/CRE are apparently the most critical elements that regulate the TNF-alpha promoter function, because previous studies eliminated the ETS binding site as being central to TNF-alpha promoter activity (44). To investigate the role of NFATp/NFkappa B (5'-GGGTTTCTCC-3') and c-Jun/CRE (5'-TGAGCTCA-3') elements in Tax activation of the TNF-alpha promoter, transfection assays were performed using luciferase reporters containing the wild type TNF-RE or mutations of the c-Jun/CRE or NFATp/NFkappa B binding sites upstream of the thymidine kinase promoter. As observed in previous experiments, Tax activates the wild type TNF-RE ~10-fold (Fig. 4), whereas mutations in the c-Jun/CRE or NFATp/NFkappa B binding site severely diminished Tax activation. As expected, mutations in both c-Jun/CRE and NFATp/NFkappa B binding sites also dramatically impaired Tax activation. No differences were observed between the levels of Tax activation with the single mutation reporters compared with the double mutation reporter constructs. Therefore, maximal Tax activation of the TNF-alpha promoter requires both c-Jun/CRE and NFATp/NFkappa B elements.


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  		Fig. 4.   Tax activation of the TNF-alpha promoter requires c-Jun/CRE and NFkappa B/NFATp binding sites. Mutations were made in either the c-Jun/CRE or NFkappa B/NFATp elements located within the -125 to +93 TNF-alpha promoter. U937 cells were cotransfected with the Tax expression plasmid (500 ng), and luciferase reporter constructs containing wild type TNF-RE, c-Jun/CRE mutation, NFkappa B/NFATp mutation, or c-Jun/CRE-NFkappa B/NFATp double mutation. Cells were treated with 10 nM E2 for 24 h, and luciferase activity was measured. Each data point is the average of triplicate determinations. The S.E. was < 10%.

NFkappa B Activity Is Necessary for Tax-mediated Activation of the TNF-RE-- To further dissect the pathways involved in Tax activation of the TNF-alpha promoter, we utilized two previously characterized Tax mutants, Tax M22 and Tax M47. The Tax M22 mutant is unable to activate NFkappa B while maintaining its ability to activate transcription factors of the CREB/ATF family (45-47), whereas the Tax M47 mutant activates the NFkappa B pathway but not the CREB/ATF pathway (48). As shown in Fig. 5, wild type 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 NFkappa B but not CREB/ATF to stimulate the TNF-alpha promoter.


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  		Fig. 5.   Tax activation of the TNF-alpha promoter requires activation of NFkappa B. U937 cells were cotransfected with TNF-RE tk-luc, ERalpha , or ERbeta expression plasmid (1 µg) and either a wild type Tax expression plasmid, a Tax M22 mutant (NFkappa B-/CREB-ATF+), or a Tax M47 mutant (NFkappa B+/CREB-ATF-). Cells were maintained in the absence or presence of 10 nM E2 for 24 h, and luciferase and renilla luciferase activities 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%.

Purified NFkappa B p50 and c-Jun Bind to the TNF-RE-- We investigated the ability of c-Jun and NFkappa B to bind the TNF-RE by electrophoretic mobility shift assays. Binding reactions were done with purified p50 and c-Jun, the DNA binding components of NFkappa B and AP-1, respectively. As shown in Fig. 6, both purified NFkappa B p50 and c-Jun bind to the TNF-RE probe (lanes 1 and 2, respectively). When reactions containing both NFkappa B p50 and c-Jun were incubated for 15 or 60 min prior to binding to the TNF-RE probe, there were 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 the arrow) contains both NFkappa B p50 and c-Jun. (lanes 5 and 6). These results indicate that NFkappa B p50 and c-Jun bind simultaneously to the TNF-RE.


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  		Fig. 6.   Purified NFkappa B p50 and c-Jun bind to the TNF-RE. Electrophoretic mobility shift assays were performed using purified NFkappa B p50 or c-Jun. Binding reactions containing either purified protein were allowed to incubate for either 15 or 60 min at 4 °C prior to the addition of NFkappa B p50- or p65-specific antibodies. The presence of c-Jun and NFkappa B p50 is confirmed by supershifting with specific antibodies (marked by the asterisk). The arrow indicates supershifted complexes.

ERalpha Binds to a c-Jun/NFkappa B Complex in the TNF-RE-- To begin to probe the mechanism whereby E2 represses Tax-mediated activation of the TNF-alpha promoter, we investigated our hypothesis that ERs bind to the TNF-alpha promoter indirectly via interactions with transcription factors bound to the TNF-RE, because we reported previously that ERs do not bind directly to the TNF-RE (35). For these studies, we used U20S cells stably transfected with ERalpha . Following treatment with doxycycline to induce ERalpha expression, the cells were transfected with the Tax expression plasmid and treated with E2 for 12 h. Binding reactions were performed with U20S-ERalpha nuclear extracts and specific antibodies to c-Jun, NFkappa B p50, NFkappa B p65, ATF-2, and ERalpha to identify candidate proteins that bind the TNF-RE (Fig. 6, 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, NFkappa B p50, and NFkappa B p65, but not with an antibody to ATF-2. This pattern demonstrates that a p50 and p65 heterodimer binds to the NFkappa B 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 ERalpha 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 NFkappa B and that ERalpha represses the TNF-alpha promoter by binding to these factors via protein-protein interactions.


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  		Fig. 7.   ERalpha , NFkappa B, and c-Jun interact on the TNF-RE. Tetracycline-inducible U2OS-ERalpha cells were transfected with the Tax expression plasmid and then treated with 10 nM E2 for 24 h and doxycycline to induce ERalpha expression. Nuclear extracts were prepared and then incubated with c-Jun, NFkappa B p50, NFkappa B p65, ATF-2, or ERalpha antibodies. Proteins or protein complexes that bound to a radiolabeled TNF-RE probe were detected by autoradiography. The arrow indicates supershifted complexes.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our studies demonstrate that ERs repress Tax activation of the TNF-alpha 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-alpha gene. Deletion studies showed that the -125 to -82 region of the TNF-alpha promoter is necessary for Tax activation. This region contains binding sites for ETS, c-Jun, ATF-2, NFATp, and NFkappa B transcription factors that might mediate the Tax activation of the TNF-alpha promoter. Tsai et al. (42) reported that a c-Jun/ATF-2 heterodimer binds to the c-Jun/CRE in the TNF-RE. 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. (49) showed that M47 Tax is more stable then wild type Tax, which can explain the finding that M47 Tax produced a greater activation of the TNF-RE compared with wild type Tax. Activation of the NFkappa B element by Tax is essential for maximum activation of the TNF-alpha promoter, because the M22 Tax mutant deficient in activating the NFkappa B 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 NFkappa B p50, NFkappa B p65, c-Jun, and ERalpha but not ATF-2 interact with the TNF-RE element. These results indicate that the activation of the TNF-alpha promoter by Tax requires c-Jun and NFkappa B but not ATF-2. Furthermore, c-Jun and NFkappa B must bind simultaneously to the TNF-RE to activate the TNF-alpha 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 NFkappa B by Tax, the NFkappa B p50/p65 complex binds next to c-Jun on the TNF-RE, leading to the activation of the TNF-alpha promoter. The ER is then recruited to the promoter by binding to a platform comprised of c-Jun and NFkappa B. Support for this model of ER action is provided by evidence that ERalpha or ERbeta do not directly bind to the TNF-alpha promoter (35) and that NFkappa B p65 and ER can interact (50-53). Furthermore, ERalpha 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-alpha promoter through a bridging protein in direct contact with the c-Jun/NFkappa B complex.

Examination of the sequence within the TNF-RE region of the TNF-alpha promoter reveals that there is only one nucleotide separating the c-Jun/CRE and NFATp/NFkappa B 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-alpha activation (44). Therefore, it is likely that c-Jun and NFkappa B on the TNF-alpha promoter are critical for providing the initial platform for ER binding to the TNF-alpha promoter, and disruption of the platform prevents the assembly of other factors necessary for activation of the TNF-alpha 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-alpha 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-alpha promoter or whether other proteins participate in repression remains to be determined.

Individuals infected with HTLV-1 suffer from various bone and joint diseases most likely due to an increased expression of various cytokines, including TNF-alpha . 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-alpha plays a role in this disease because it is associated with increased serum levels of TNF-alpha (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-alpha (59).

Intriguingly, similarly to HTLV-I infected patients, many postmenopausal women develop bone and joint diseases that might occur from excessive TNF-alpha production. A prominent role for TNF-alpha in the pathogenesis of osteoporosis is supported by animal studies. For example, overexpression of TNF-alpha in mice produces profound hypercalcemia from enhanced bone resorption (60). Furthermore, the loss of bone mineral density observed in mice after an oophorectomy can be prevented with TNF-binding proteins (61) or a soluble TNF receptor that prevents the action of TNF-alpha (62). TNF-alpha 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-alpha gene transcription and down-regulate TNF-alpha levels (67, 68). Other studies indicate that TNF-alpha is involved in the destruction of the articular cartilage that is observed in 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-alpha 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-alpha gene expression. Estrogens are known to exhibit anti-inflammatory properties (71), but it is not known if this effect occurs through ERalpha , ERbeta , or both of these ERs. Our results demonstrating that ERbeta is more effective than ERalpha at inhibiting TNF-alpha and Tax-activation of TNF-alpha gene transcription suggest that ERbeta 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 the TNF-alpha gene may also account for the observation that females infected with HTLV-I exhibit less severe clinical manifestations and lower mortality compared with males (37).

    ACKNOWLEDGEMENTS

We thank P. Chambon, J.-A. Gustafsson, and W. Greene for providing plasmids.

    FOOTNOTES

* This work was supported by a National Institutes of Health postdoctoral training grant and a Bank of America Giannini postdoctoral fellowship (to C. T.-F.) and grants from the Paul Beeson Physician Faculty Scholars in Aging Research Program (funded by the Alliance for Aging Research, the John A. Hartford Foundation, the Commonwealth Fund, and the Starr Foundation), the NICHD National Institutes of Health Women's Reproductive Health Research Program, and the Susan B. Komen Foundation (to D. C. L.).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.

Present address: Hop Lariboisiere, INSERM, U349, F-75475 Paris, France.

|| To whom correspondence should be addressed: 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; E-mail: leitmand@obgyn.ucsf.edu.

Published, JBC Papers in Press, September 16, 2002, DOI 10.1074/jbc.M205355200

    ABBREVIATIONS

The abbreviations used are: HTLV-1, human T-cell leukemia virus type 1; ATL, adult T-cell leukemia; TNF, tumor necrosis factor; TNF-RE, TNF-response element; NFkappa B, nuclear factor kappa B; NFATp, nuclear factor of T cells; E2, estradiol; ER, estrogen receptor; GUS, beta -glucuronidase; CRE, cAMP-response element; CREB, CRE-binding protein; ATF, activating transcription factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D., and Gallo, R. C. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7415-7419[Abstract/Free Full Text]
2. Yoshida, M., Miyoshi, I., and Hinuma, Y. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2031-2035[Abstract/Free Full Text]
3. Gessain, A., Barin, F., Vernant, J. C., Gout, O., Maurs, L., Calender, A., and de The, G. (1985) Lancet 2, 407-410[CrossRef][Medline] [Order article via Infotrieve]
4. Terada, K., Katamine, S., Eguchi, K., Moriuchi, R., Kita, M., Shimada, H., Yamashita, I., Iwata, K., Tsuji, Y., Nagataki, S., et al.. (1994) Lancet 344, 1116-1119[CrossRef][Medline] [Order article via Infotrieve]
5. Watanabe, T. (1997) Int. J. Hematol. 66, 257-278[CrossRef][Medline] [Order article via Infotrieve]
6. Lenzmeier, B. A., Baird, E. E., Dervan, P. B., and Nyborg, J. K. (1999) J. Mol. Biol. 291, 731-744[CrossRef][Medline] [Order article via Infotrieve]
7. Johnson, J. M., Harrod, R., and Franchini, G. (2001) Int. J. Exp. Pathol. 82, 135-147[CrossRef][Medline] [Order article via Infotrieve]
8. Ruddle, N. H., Li, C. B., Horne, W. C., Santiago, P., Troiano, N., Jay, G., Horowitz, M., and Baron, R. (1993) Virology 197, 196-204[CrossRef][Medline] [Order article via Infotrieve]
9. Zhao, L. J., and Giam, C. Z. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11445-11449[Abstract/Free Full Text]
10. Fujii, M., Tsuchiya, H., Chuhjo, T., Akizawa, T., and Seiki, M. (1992) Genes Dev. 6, 2066-2076[Abstract/Free Full Text]
11. Hirai, H., Suzuki, T., Fujisawa, J., Inoue, J., and Yoshida, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3584-3588[Abstract/Free Full Text]
12. Franklin, A. A., Kubik, M. F., Uittenbogaard, M. N., Brauweiler, A., Utaisincharoen, P., Matthews, M. A., Dynan, W. S., Hoeffler, J. P., and Nyborg, J. K. (1993) J. Biol. Chem. 268, 21225-21231[Abstract/Free Full Text]
13. Wagner, S., and Green, M. R. (1993) Science 262, 395-399[Abstract/Free Full Text]
14. Caron, C., Rousset, R., Beraud, C., Moncollin, V., Egly, J. M., and Jalinot, P. (1993) EMBO J. 12, 4269-4278[Medline] [Order article via Infotrieve]
15. Suzuki, T., Hirai, H., Fujisawa, J., Fujita, T., and Yoshida, M. (1993) Oncogene 8, 2391-2397[Medline] [Order article via Infotrieve]
16. Beraud, C., Sun, S. C., Ganchi, P., Ballard, D. W., and Greene, W. C. (1994) Mol. Cell. Biol. 14, 1374-1382[Abstract/Free Full Text]
17. Siekevitz, M., Feinberg, M. B., Holbrook, N., Wong-Staal, F., and Greene, W. C. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5389-5393[Abstract/Free Full Text]
18. Ballard, D. W., Bohnlein, E., Lowenthal, J. W., Wano, Y., Franza, B. R., and Greene, W. C. (1988) Science 241, 1652-1655[Abstract/Free Full Text]
19. Miyatake, S., Seiki, M., Yoshida, M., and Arai, K. (1988) Mol. Cell. Biol. 8, 5581-5587[Abstract/Free Full Text]
20. Ruben, S., Poteat, H., Tan, T. H., Kawakami, K., Roeder, R., Haseltine, W., and Rosen, C. A. (1988) Science 241, 89-92[Abstract/Free Full Text]
21. Leung, K., and Nabel, G. J. (1988) Nature 333, 776-778[CrossRef][Medline] [Order article via Infotrieve]
22. Hoyos, B., Ballard, D. W., Bohnlein, E., Siekevitz, M., and Greene, W. C. (1989) Science 244, 457-460[Abstract/Free Full Text]
23. Mori, N., Shirakawa, F., Shimizu, H., Murakami, S., Oda, S., Yamamoto, K., and Eto, S. (1994) Blood 84, 2904-2911[Abstract/Free Full Text]
24. Cowan, E. P., Alexander, R. K., Daniel, S., Kashanchi, F., and Brady, J. N. (1997) J. Virol. 71, 6982-6989[Abstract]
25. Hinrichs, S. H., Nerenberg, M., Reynolds, R. K., Khoury, G., and Jay, G. (1987) Science 237, 1340-1343[Abstract/Free Full Text]
26. Hausmann, S., Biddison, W. E., Smith, K. J., Ding, Y. H., Garboczi, D. N., Utz, U., Wiley, D. C., and Wucherpfennig, K. W. (1999) J. Immunol. 162, 5389-5397[Abstract/Free Full Text]
27. Ozden, S., Coscoy, L., and Gonzalez-Dunia, D. (1996) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13, S154-61[Medline] [Order article via Infotrieve]
28. Coscoy, L., Gonzalez-Dunia, D., Tangy, F., Syan, S., Brahic, M., and Ozden, S. (1998) Virology 248, 332-341[CrossRef][Medline] [Order article via Infotrieve]
29. Tschachler, E., Bohnlein, E., Felzmann, S., and Reitz, M. S., Jr. (1993) Blood 81, 95-100[Abstract/Free Full Text]
30. Shimamoto, Y., Funai, N., Watanabe, M., and Suga, K. (1996) Int. J. Hematol. 64, 111-118[CrossRef][Medline] [Order article via Infotrieve]
31. Centrella, M., McCarthy, T. L., and Canalis, E. (1988) Endocrinology 123, 1442-1448[Abstract/Free Full Text]
32. Beutler, B., and Cerami, A. (1989) Annu. Rev. Immunol. 7, 625-655[Medline] [Order article via Infotrieve]
33. Albrecht, H., Shakhov, A. N., and Jongeneel, C. V. (1992) J. Virol. 66, 6191-6193[Abstract/Free Full Text]
34. Leitman, D. C., Ribeiro, R. C., Mackow, E. R., Baxter, J. D., and West, B. L. (1991) J. Biol. Chem. 266, 9343-9346[Abstract/Free Full Text]
35. An, J., Ribeiro, R. C., Webb, P., Gustafsson, J. A., Kushner, P. J., Baxter, J. D., and Leitman, D. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 15161-15166[Abstract/Free Full Text]
36. Hisada, M., Okayama, A., Tachibana, N., Stuver, S. O., Spiegelman, D. L., Tsubouchi, H., and Mueller, N. E. (1998) Int. J. Cancer 77, 188-192[CrossRef][Medline] [Order article via Infotrieve]
37. Hisada, M., Okayama, A., Spiegelman, D., Mueller, N. E., and Stuver, S. O. (2001) Int. J Cancer 91, 497-499[CrossRef][Medline] [Order article via Infotrieve]
38. An, J., Tzagarakis-Foster, C., Scharschmidt, T. C., Lomri, N., and Leitman, D. C. (2001) J. Biol. Chem. 276, 17808-17814[Abstract/Free Full Text]
39. Leitman, D. C., Mackow, E. R., Williams, T., Baxter, J. D., and West, B. L. (1992) Mol. Cell. Biol. 12, 1352-1356[Abstract/Free Full Text]
40. Lomri, A., Fromigue, O., Hott, M., and Marie, P. J. (1999) Calcif. Tissue Int. 64, 394-401[CrossRef][Medline] [Order article via Infotrieve]
41. Goldfeld, A. E., McCaffrey, P. G., Strominger, J. L., and Rao, A. (1993) J. Exp. Med. 178, 1365-1379[Abstract/Free Full Text]
42. Tsai, E. Y., Jain, J., Pesavento, P. A., Rao, A., and Goldfeld, A. E. (1996) Mol. Cell. Biol. 16, 459-467[Abstract]
43. Tsai, E. Y., Falvo, J. V., Tsytsykova, A. V., Barczak, A. K., Reimold, A. M., Glimcher, L. H., Fenton, M. J., Gordon, D. C., Dunn, I. F., and Goldfeld, A. E. (2000) Mol. Cell. Biol. 20, 6084-6094[Abstract/Free Full Text]
44. Yao, J., Mackman, N., Edgington, T. S., and Fan, S. T. (1997) J. Biol. Chem. 272, 17795-17801[Abstract/Free Full Text]
45. Smith, M. R., and Greene, W. C. (1990) Genes Dev. 4, 1875-1885[Abstract/Free Full Text]
46. Sun, S. C., Elwood, J., Beraud, C., and Greene, W. C. (1994) Mol. Cell. Biol. 14, 7377-7384[Abstract/Free Full Text]
47. Kanno, T., Brown, K., Franzoso, G., and Siebenlist, U. (1994) Mol. Cell. Biol. 14, 6443-6451[Abstract/Free Full Text]
48. Yin, M. J., Christerson, L. B., Yamamoto, Y., Kwak, Y. T., Xu, S., Mercurio, F., Barbosa, M., Cobb, M. H., and Gaynor, R. B. (1998) Cell 93, 875-884[CrossRef][Medline] [Order article via Infotrieve]
49. Carter, R. S., Geyer, B. C., Xie, M., Acevedo-Suarez, C. A., and Ballard, D. W. (2001) J. Biol. Chem. 276, 24445-24448[Abstract/Free Full Text]
50. Stein, B., and Yang, M. X. (1995) Mol. Cell. Biol. 15, 4971-4979[Abstract]
51. Galien, R., and Garcia, T. (1997) Nucleic Acids Res. 25, 2424-2429[Abstract/Free Full Text]
52. Galien, R., Evans, H. F., and Garcia, T. (1996) Mol. Endocrinol. 10, 713-722[Abstract/Free Full Text]
53. McKay, L. I., and Cidlowski, J. A. (1998) Mol. Endocrinol. 12, 45-56[Abstract/Free Full Text]
54. Doucas, V., Spyrou, G., and Yaniv, M. (1991) EMBO J. 10, 2237-2245[Medline] [Order article via Infotrieve]
55. Teyssier, C., Belguise, K., Galtier, F., and Chalbos, D. (2001) J. Biol. Chem. 276, 36361-36369[Abstract/Free Full Text]
56. Rogatsky, I., Zarember, K. A., and Yamamoto, K. R. (2001) EMBO J. 20, 6071-6083[CrossRef][Medline] [Order article via Infotrieve]
57. Jiang, H., Lu, H., Schiltz, R. L., Pise-Masison, C. A., Ogryzko, V. V., Nakatani, Y., and Brady, J. N. (1999) Mol. Cell. Biol. 19, 8136-8145[Abstract/Free Full Text]
58. Kiyokawa, T., Yamaguchi, K., Takeya, M., Takahashi, K., Watanabe, T., Matsumoto, T., Lee, S. Y., and Takatsuki, K. (1987) Cancer 59, 1187-1191[CrossRef][Medline] [Order article via Infotrieve]
59. Iwakura, Y., Saijo, S., Kioka, Y., Nakayama-Yamada, J., Itagaki, K., Tosu, M., Asano, M., Kanai, Y., and Kakimoto, K. (1995) J. Immunol. 155, 1588-1598[Abstract]
60. Johnson, R. A., Boyce, B. F., Mundy, G. R., and Roodman, G. D. (1989) Endocrinology 124, 1424-1427[Abstract/Free Full Text]
61. Kimble, R. B., Bain, S., and Pacifici, R. (1997) J. Bone Miner. Res. 12, 935-941[CrossRef][Medline] [Order article via Infotrieve]
62. Ammann, P., Rizzoli, R., Bonjour, J. P., Bourrin, S., Meyer, J. M., Vassalli, P., and Garcia, I. (1997) J. Clin. Invest. 99, 1699-1703[Medline] [Order article via Infotrieve]
63. Riggs, B. L. (2000) J. Clin. Invest. 106, 1203-1204[Medline] [Order article via Infotrieve]
64. Teitelbaum, S. L. (2000) Science 289, 1504-1508[Abstract/Free Full Text]
65. Johnson, S. R. (1998) Med. Clin. North Am. 82, 297-320[CrossRef][Medline] [Order article via Infotrieve]
66. Kenny, A. M., and Prestwood, K. M. (2000) Rheum. Dis. Clin. North Am. 26, 569-591[CrossRef][Medline] [Order article via Infotrieve]
67. Pacifici, R., Brown, C., Puscheck, E., Friedrich, E., Slatopolsky, E., Maggio, D., McCracken, R., and Avioli, L. V. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5134-5138[Abstract/Free Full Text]
68. Pacifici, R. (1996) J. Bone Miner. Res. 11, 1043-1051[Medline] [Order article via Infotrieve]
69. Goldring, M. B. (2000) Curr. Rheumatol. Rep. 2, 459-465[Medline] [Order article via Infotrieve]
70. Dennison, E. M., Arden, N. K., Kellingray, S., Croft, P., Coggon, D., and Cooper, C. (1998) Br. J. Rheumatol. 37, 1198-1202[Abstract/Free Full Text]
71. Bruce-Keller, A. J., Keeling, J. L., Keller, J. N., Huang, F. F., Camondola, S., and Mattson, M. P. (2000) Endocrinology 141, 3646-3656[Abstract/Free Full Text]

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