Direct Regulation of Androgen Receptor-Associated Protein 70 by

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Endocrinology 148(7):3485–3495 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1239

Direct Regulation of Androgen Receptor-Associated Protein 70 by Thyroid Hormone and Its Receptors Pei-Ju Tai,* Ya-Hui Huang,* Chung-Hsuan Shih, Ruey-Nan Chen, Chi-De Chen, Wei-Jan Chen, Chia-Siu Wang, and Kwang-Huei Lin Department of Biochemistry (P.-J.T., Y.-H.H., C.-H.S., R.-N.C., C.-D.C., K.-H.L.), Chang-Gung University, and First Cardiovascular Division (W.-J.C.), Chang Gung Memorial Hospital, Taoyuan, Taiwan 333, Republic of China; and Department of General Surgery (C.-S.W.), Chang Gung Memorial Hospital at Chiayi, Taiwan 613, Republic of China Thyroid hormone (T3) regulates multiple physiological processes during development, growth, differentiation, and metabolism. Most T3 actions are mediated via thyroid hormone receptors (TRs) that are members of the nuclear hormone receptor superfamily of ligand-dependent transcription factors. The effects of T3 treatment on target gene regulation was previously examined in TR␣1-overexpressing hepatoma cell lines (HepG2-TR␣1). Androgen receptor (AR)-associated protein 70 (ARA70) was one gene found to be up-regulated by T3. The ARA70 is a ligand-dependent coactivator for the AR and was significantly increased by 4- to 5-fold after T3 treatment by Northern blot analyses in the HepG2-TR␣1 stable cell line. T3 induced a 1- to 2-fold increase in the HepG2-TR␤1 stable cell line. Both stable cell lines attained the highest fold expression after 24 h treatment with 10 nM T3. The ARA70 protein was increased up to 1.9-fold after T3 treatment in HepG2-TR␣1

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HE THYROID HORMONE T3 has an important role in eukaryotic cell development, differentiation, and metabolism (1). Lack of T3 hormone in early human development reduces growth and may cause mental retardation; T3 plays a major role in metabolic balance in later life (2, 3). These activities are mediated by nuclear thyroid hormone receptors (TRs), of which two principal TR types have been identified. TRs are ligand-dependent transcription factors comprised of modular functional domains that mediate hormone binding (ligands), DNA binding, receptor homo- and heterodimerization, and interaction with other transcription factors and cofactors (2–5). The principal function of a TR as a transcription factor is to regulate target gene expression by binding directly to specific DNA elements called thyroid hormone response elements (TREs) in the promoter regions of these genes. The ability to bind to specific sequences in target genes is crucial for TR function. In the absence of the First Published Online April 5, 2007 * The contributions of P.-J.T. and Y.-H.H. were equal. Abbreviations: AR, Androgen receptor; ARA70, AR-associated protein 70; CHX, cycloheximide; ER, estrogen receptor; GST, glutathioneS-transferase; Lap, lysozyme; ME, malic enzyme; NR, nuclear receptor; SD, Sprague Dawley; Pal, palindrome; PPAR␣, peroxisome proliferatoractivated receptor-␣; Q-RT-PCR, quantitative RT-PCR; SMRT, silencing mediator of retinoic acid and TR; TBS, Tris-buffered saline; TR, thyroid hormone receptor; TRE, thyroid hormone response element; TX, thyroidectomy; VDR, vitamin D receptor. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

cells. Similar findings were obtained in thyroidectomized rats after T3 application. Cycloheximide treatment did not suppress induction of ARA70 transcription by T3, suggesting that this regulation is direct. A series of deletion mutants of ARA70 promoter fragments in pGL2 plasmid were generated to localize the thyroid hormone response element (TRE). The DNA fragments (ⴚ234/ⴚ190 or ⴙ56/ⴙ119) gave 1.55- or 2-fold enhanced promoter activity by T3. Thus, two TRE sites exist in the upstream-regulatory region of ARA70. The TR-TRE interaction was further confirmed with EMSAs. Additionally, ARA70 could interfere with TR/TRE complex formation. Therefore, the data indicated that ARA70 suppresses T3 signaling in a TRE-dependent manner. These experimental results suggest that T3 directly up-regulates ARA70 gene expression. Subsequently, ARA70 negatively regulates T3 signaling. (Endocrinology 148: 3485–3495, 2007)

T3 ligand, TRs suppress expression of target genes, a phenomenon known as transcriptional silencing. This process is believed to be mediated by interaction, via the ligand binding domain, between the receptor and transcriptional corepressors such as silencing mediator of retinoic acid and TR (SMRT) (6). Ligand binding induces dissociation of TRs from corepressors, causing recruitment of transcriptional coactivators such as steroid receptor coactivator (SRC) and subsequent activation of target gene expression (7). cDNA microarrays have been performed to identify the mechanism of target gene regulation after T3 treatment in a TR␣1-overexpression hepatoma cell line (HepG2-TR␣1). Studies have demonstrated that 148 of the 7597 genes represented were positively regulated by T3, including fibrinogen (8), fibronectin (9), transferrin (10), and several other coagulation factor system components. These studies indicated that the hepatoma cell line HepG2 stably expressed functional TR␣1 or TR␤1 and directly regulated fibrinogen expression (8). This regulation can also be observed in thyroidectomized rats. Additionally, several of these genes from microarray are transcription factors that are not traditionally associated with thyroid hormone function. This study thus focused on these genes and further verified their response to T3 treatment at both the RNA and protein level. Particular attention was paid to androgen receptor (AR)-associated protein 70 (ARA70) because the control of ARA70 expression by T3 and its significance have not been elucidated in a cellular context. This study demonstrates that T3 up-regulates ARA70 ex-

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Tai et al. • Regulation of ARA70 by T3


pression in HepG2-TR␣1 and -TR␤1 cells. Additionally, the effect of T3 on the degree of ARA70 expression does not require de novo synthesis of cellular proteins. Similar regulation was observed in vivo. This study demonstrates TR protein binding to ARA70 5⬘-flanking regions. Finally, the experimental data indicated that ARA70 suppressed thyroid hormone signaling in a TRE-dependent manner. Materials and Methods Cell culture The human hepatoma cell line HepG2 and CV-1 (African green monkey kidney fibroblast) obtained from the American Type Culture Collection (Manassas, VA), were routinely grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum. Both TR␣1- and TR␤1-overexpressing cell lines have been characterized previously (11). In this study, HepG2-TR␣1#1, HepG2-TR␣1#2, and HepG2-TR␤1 overexpressing and HepG2-Neo clones were used. Serum was depleted of T3 as described elsewhere (12). Cells were cultured at 37 C in a humidified atmosphere of 95% air and 5% CO2.

Immunoblot analysis Cell lysates were fractionated using SDS-PAGE on a 10% gel, and separated proteins were transferred to a nitrocellulose membrane (Amersham, Piscataway, NJ). The membrane was gently shaken for 2 h at room temperature in 5% (wt/vol) nonfat dried milk in Tris-buffered saline (TBS), washed three times with TBS, and then incubated for 1 h with goat polyclonal antibodies to ARA70 (1:1000 dilution in TBS) (Santa Cruz Biotechnology, Santa Cruz, CA) or with mouse monoclonal antibody C4 to TR protein (1:2000 dilution in TBS, kindly provided by S.-Y. Cheng, National Cancer Institute, National Institutes of Health, Bethesda, MD) (13). After additional washing, the membrane was incubated for 1 h with horseradish peroxidase conjugated to affinity-purified antibodies to either rabbit (1:2000 dilution in TBS) or mouse (1:2000 dilution in TBS) Ig (Santa Cruz Biotechnology). Immune complexes were then visualized by chemiluminescence with an ECL detection kit (Amersham). Intensities of immunoreactive bands were quantitated via Image Gauge software (Fuji Film, Tokyo, Japan).

Northern blot analysis Total RNA was extracted from cells by TRIzol reagent. Equal amounts of total RNA (20 ␮g) were analyzed on a 1.2% agarose-formaldehyde gel as described elsewhere (11). This gel was then blotted onto a nitrocellulose membrane and subjected to Northern blot analysis as described previously (11). A full-length ARA70 cDNA fragment (1845 bp) was amplified, labeled with [␣-32P]dCTP (3000 Ci/mmol) (Amersham) by PCR, and employed as a probe. The membrane was subsequently reprobed with ␣-32P-labeled 18S rDNA fragment to confirm equal application of RNA to each lane. In some experiments, cells were treated with T3 and 10 ␮g/ml cycloheximide (CHX) (Sigma-Aldrich, St. Louis, MO) simultaneously for 12 or 24 h, followed by total RNA isolation and Northern blot analysis.

Cloning the ARA70 promoter fragments and assay of their activities Fragments of the ARA70 promoter were amplified by PCR on the basis of the published nucleotide sequence (International Human Genome Sequencing Consortium, DNA sequence of Homo sapiens, NT008583), which was then inserted into the pGL2 vector (Promega


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Corp., Madison, WI). Series deletion mutants of the ARA70 promoter were generated by PCR amplification. Sequences of all promoter constructs were verified by automated DNA sequencing. To determine the transactivation activity of TREs in the ARA70 promoter, CV-1 cells were transfected (5 ⫻ 104 cells per 12-well dish) with 0.66 ␮g DNA of pGL2 vector containing ARA70 promoter sequences with the use of Lipofectamine (Life Technologies, Inc., Rockville, MD). Cells were also transfected with 0.33 ␮g pcDNA3-TR␣1 expression vectors and with 0.33 ␮g ␤-galactosidase expression vector, pSV␤ plasmid (Clontech Laboratories, Inc., Palo Alto, CA). Twenty-four hours after transfection, cells were incubated in the absence or presence of 10 nm T3 for 24 h and then lysed to measure luciferase and ␤-galactosidase activities (14).

EMSA The ␣-32P-labeled ARA70 oligonucleotides (⫹56/⫹119, ⫺234/⫺190) were prepared by PCR as described previously (11). The TR proteins were prepared by using the glutathione-S-transferase (GST)-TR fusion protein as described elsewhere (15). For EMSA, equal amounts of GST-TR proteins were incubated with the labeled probes in the absence or presence of increasing concentrations of wild-type cold probes or mutant probes in the binding buffer and electrophoresed at 4 C for 2–3 h at a constant voltage of 250 V. The gel was dried and autoradiographed. The C4 antibody (against TR) was used to confirm the identity of the protein complex.

Quantitative RT-PCR (Q-RT-PCR) Total RNA was extracted from cells using TRIzol as described previously. Subsequently, cDNA was synthesized using the Superscript II kit for RT-PCR (Life Technologies) as described previously (8). Rat ARA70 primers were as follows: forward, 5⬘-AGAAAGGAGAGTAGCAGGATTGATCT-3⬘; reverse, 5⬘-TAGCATAGGCA ACTCAGAGG CTTAA-3⬘. Real-time Q-RT-PCR was conducted in a 15-␮l reaction mixture containing 50 nm forward and reverse primers, 1⫻ Sybr Green reaction mix (Applied Biosystems, Foster City, CA), and varying quantities of template as described previously (8). Sybr Green fluorescence was measured with the ABI PRISM 7000 sequence detection system (Applied Biosystems) as described previously (8, 16).

Animals Male Sprague Dawley (SD) rats received thyroidectomies (TX) at 6 wk of age (developmentally mature) according to methods employed in previous reports (8, 17). Each animal was given 1% calcium lactate in drinking water after surgery. Two weeks after surgery, each rat was injected with T3 at 10 ␮g/100 g body weight or a control vehicle (2.5 mm NaOH in PBS) daily for two additional weeks. T3 and TSH from serum were assayed by RIA (18) or immunoradiometric assay (19), respectively. All animal experiments in this study were performed in accordance with National Institutes of Health guidelines and the Chang-Gung Institutional Animal Care and Use Committee Guide for Care and Use of Laboratory Animals.

Results Effects of T3 on expression of ARA70 mRNA and protein in HepG2-TR␣1 and -TR␤1 cell lines

ARA70 was up-regulated by T3 determined via previous cDNA microarray analysis in this laboratory. After verification, ARA70 RNA was induced approximately 4- to 5-fold with 10 nm T3 based on Q-RT-PCR assays (data not shown).

FIG. 1. Effect of T3 on the abundance of ARA70 mRNA in HepG2 cell lines. A, TR levels in four cell lines; B–E, HepG2-TR␣1#1 (B), HepG2-TR␣1#2 (C), HepG2-TR␤1 (D), and HepG2-Neo (E) cells were incubated for 12, 24, or 48 h in the absence or presence of 1–100 nM T3, after which total RNA was isolated and subjected (20 ␮g per lane) to Northern blot analysis with ␣-32P-labeled ARA70 or 18S rDNA probes. The positions of 3.5-kb ARA70 mRNA and 1.9-kb 18S rRNAs are indicated. Intensities of the ARA70 mRNA bands on blots similar to that shown in B–E were quantified, and the amount of the T3-induced increase and abundance of ARA70 transcripts was determined at each time point. Quantitation results are fold inductions using 0 nM T3 at 12 h as 1-fold. Data are means ⫾ SE of values from three independent experiments. Student’s t test: **, P ⬍ 0.01; *, P ⬍ 0.05, 100 nM T3-treated vs. 12 h, 0 nM T3-treated.

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The four HepG2 cell lines used in this study are referred to as HepG2-TR␣1#1, -TR␣1#2, -TR␤1, and -Neo where the TR protein was overexpressed approximately 10-, 3-, and 3-fold, respectively, compared with the HepG2-Neo control cell line (Fig. 1A). Northern blot analysis was used to further examine the response of ARA70 mRNA expression to the exogenous addition of T3. In all three cell lines investigated, a 3.5-kb ARA70 transcript was detected (Fig. 1, B–E). Exposure to 10 nm T3 for 12 h significantly (P ⬍ 0.01) induced the expression of ARA70 mRNA in HepG2-TR␣1#1, HepG2-TR␣1#2, and HepG2-TR␤1 cells with increases of 3.4-, 2.14-, and 1.8-fold, respectively (Fig. 1, B–D). This increased expression indicated that the increased expression of ARA70 mRNA induced by T3 corresponds to the amount of TR in individual cell lines. The highest levels of T3 (100 nm) or extended treatment (24 or 48 h) by T3 used in this experiment further increased the level of ARA70 expression (up to ⬃5-fold) above that already measured in the 10 nm T3 experiment in HepG2TR␣1 cells (Fig. 1, B and C). These experimental results also imply that ARA70 gene expression (up to 2.7-fold, Fig. 1B) is stimulated by 1 nm T3 induction in the medium. Applying 10 nm T3 for 24 or 48 h to the HepG2-TR␤1 cell line significantly (P ⬍ 0.01) increased ARA70 levels 1.88- and 1.63-fold, respectively (Fig. 1D). However, in cells treated with 100 nm T3 for 24 or 48 h, ARA70 mRNA expression did not further increase in TR␤1 cells (Fig. 1D). Additionally, T3 had a minimal effect on the expression of ARA70 mRNA in HepG2-Neo cells (Fig. 1E). Thus, the effect of T3 on ARA70 mRNA expression is apparently mediated, at least partly, at the mRNA level. To further analyze time-dependent induction of ARA70 at the mRNA level, HepG2-TR␣1#1 and -TR␤1 cells were cultured with or without 10 nm T3 and analyzed at various time points. As early as 6 h after T3 treatment, ARA70 mRNA expression increased roughly 1.4-fold in HepG2-TR␣1#1 cells by Northern blot analysis. Thereafter, significant (P ⬍ 0.05 or P ⬍ 0.01) increases in ARA70 mRNA expression were observed (1.9-, 2.4-, 2.5-, and 2.5-fold at 12, 24, 36, and 48 h, respectively) (Fig. 2A). In the HepG2-TR␤1 cells expressing lower TR protein, ARA70 mRNA was induced about 1.4-fold by T3 as early as 6 h. Thereafter, increases of 1.6-, 1.66-, 1.7-, and 1.73-fold were observed in ARA70 mRNA expression at similar time points analyzed by Q-RT-PCR (Fig. 2B). Thus, induction of ARA70 expression in these cells is sensitive and responds quickly to T3 treatment. The effect of TRs on the level of ARA70 protein expression was determined when HepG2-TR isogenic cell lines were incubated in media containing 100 nm T3 across different time points. The T3 significantly increased the level of ARA70 in the HepG2-TR␣1#1 and -TR␤1 stable cell lines in comparison with that of the HepG2-Neo control cell line (Fig. 3). Levels of ARA70 increased approximately 1.7- to 1.8-fold after incubation of HepG2-TR␣1#1 and HepG2-TR␤1 cells with 100 nm T3 for 24 h. Moreover, 100 nm T3 for 48-h generated a slightly higher induction (up to 1.9-fold) of ARA70 in TR␣1 but not in TR␤1 cells (Fig. 3, A and B). These experimental results suggest that the effect of T3 on the level of ARA70 in cells overexpressing TR␣1 was time dependent. Additionally, Western blot analysis indicated that exposure of control HepG2-Neo cells, expressing endogenous levels of


FIG. 2. Time-dependent induction of ARA70 by T3. Expression of ARA70 in HepG2-TR␣#1 analyzed by Northern blot (A) or HepG2TR␤1 analyzed by Q-RT-PCR (B) was determined at 3, 6, 12, 24, 36, and 48 h in the absence or presence of 10 nM T3. Quantitation results for A and B are fold inductions using 0 nM T3 at 12 h as 1-fold. ARA70 was induced up 2- to 3-fold by T3 after 48-h treatment. Data are means ⫾ SE of values from three independent experiments. Student’s t test: **, P ⬍ 0.01; *, P ⬍ 0.05, T3-treated vs. 12 h, 0 nM T3-treated.

TR proteins, to 100 nm T3 had a minor effect on ARA70 expression (Fig. 3C). Thus, the extent of ARA70 induced by T3 is positively associated with the level of TR protein expression. Effects of T3 and CHX on the abundance of ARA70 mRNA

To elucidate further the regulatory action of T3 on ARA70 expression, a protein synthesis inhibitor, CHX, was applied. Induction of ARA70 mRNA expression by T3 in the presence or absence of CHX was observed for the indicated time periods in HepG2-TR␣1#1 and -TR␤1 cells. The transcriptional response of ARA70 mRNA to T3 over 12- and 24-h periods was not significantly reduced in the presence of CHX-treated HepG2-TR␣1#1 cells (Fig. 4A). Similar results were observed for TR␤1-overexpressing cell lines (Fig. 4B). These experimental data suggest that suppressing protein synthesis did not inhibit T3-induced ARA70 transcription. This lack of inhibiting effect suggests de novo protein syn-



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FIG. 3. Effect of T3 on ARA70 protein expression in HepG2 cells. The ARA70 expression levels in HepG2-TR␣1#1 (A), HepG2-TR␤1 (B), or HepG2-Neo (C) cells were determined by incubating with T3-depleted medium in the absence or presence of 100 nM T3 for 12, 24, or 48 h, after which cell lysates (50 ␮g protein) were subjected to immunoblot analysis with polyclonal antibodies for ARA70. The position of the 70-kDa protein is indicated on the left side of each blot. Intensities of the ARA70 band were quantified, and the degree of T3-induced activation was determined at each time point. Quantitation results are fold inductions using 0 nM T3 at 12 h as 1-fold. Data are means ⫾ SE of values from three independent experiments. Student’s t test: **, P ⬍ 0.01; *, P ⬍ 0.05, T3-treated vs. 12 h, 0 nM T3-treated.

thesis may not be essential to activation of ARA70 and that regulation may be direct. Expression of ARA70 induced by T3 at the transcriptional level

Promoter activity was assayed to confirm further that T3 regulation of ARA70 expression occurred at the transcriptional level. The ARA70 5⬘-flanking region encompassing nucleotides ⫺3319/⫹119 was cloned and then placed upstream of the luciferase reporter gene in pGL2. Various deletion mutants were also prepared to localize the potential TREs. Using these reporter constructs, the effect of transactivation of TR by T3 on the ARA70 5⬘-flanking regions was

FIG. 4. CHX did not suppress the response of ARA70 to T3 activation. HepG2-TR␣1#1 (A) or HepG2-TR␤1 (B) cells were treated as described in Fig. 1 with or without 1 ␮g/ml or 10 ␮g/ml CHX. After T3 activation for various lengths of time, total RNA was isolated and subjected (20 ␮g per lane) to Northern blot analysis. Intensities of the ARA70 and 18S RNA bands on blots were quantified, and the increase in abundance of ARA70 mRNA transcripts was determined at each time point. Experimental results are fold inductions as compared with those for control 12-h, 0 nM T3 conditions. Data are means ⫾ SE of values from three independent experiments. Student’s t test: **, P ⬍ 0.01; *, P ⬍ 0.05, T3-treated vs. 12 h, 0 nM T3-treated.

determined. Northern blot analyses indicated that T3 induced an approximate 2- to 5-fold increase in ARA70 mRNA transcription (Figs. 1 and 2). Figure 5A illustrates that transactivational activity of the ⫺3319/⫹119 (⫹1 is relative to the transcriptional initiation site) containing the reporter construct was increased 1.62-fold (TR␣1) or 1.8-fold (TR␤1) in the presence of T3 in CV-1 cells. When the 5⬘ end of the ⫺3319/ ⫹119 was truncated to yield the ⫺1826/⫹119, ⫺976/⫹119, ⫺415/⫹119, ⫺261/⫹119, and ⫺234/⫹119 constructs, the T3-induced activation was roughly 2-fold. Cells transfected with the ⫺190/⫹119 construct still resulted in a 2.14-fold (TR␣1) increase in transcriptional activity. These experimental data further indicate that the TR interaction site was

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FIG. 5. The T3-dependent trans-activity of TR in ARA70 promoter. The CV-1 cells were transfected with a series of luciferase reporter plasmids containing the ARA70 5⬘flanking region encompassing nucleotides ⫺3319 to ⫹119, its deletion mutants, and TR with a ␤-galactosidase plasmid to control for transfection efficiency. The ARA70 promoter constructs were placed in pGL2-basic vector (A) or in pA3TK-Luc vector (B). The constructs were subsequently incubated for 24 h in T3-depleted (Td) medium containing the indicated concentrations of T3, after which the activities of luciferase and ␤-galactosidase in cell lysates were measured. The activity of luciferase was normalized based on the activity of ␤-galactosidase. Results are presented as means ⫾ SE of data from three independent experiments performed in triplicate. TK, Thymidine kinase. C, The wildtype (WT) TRE and mutant (MT) sequences in the ARA70 5⬘-flanking promoter regions.

localized in the ⫺190/⫹119 region. The localization of the TR interaction site in this region was further confirmed using a different reporter construct containing a minimum TK promoter upstream of the Luc reporter (pA3TK-Luc) (Fig. 5B). To further localize the promoter element responsible for T3induced transactivation activity, ⫺234/⫹119 was divided into two fragments with sequences encompassing ⫺234/-190 and ⫺190/⫹119; the transactivation activities of these two constructs was compared. However, the activities exhibited by ⫺234/⫺190 was less than those in ⫺190/⫹119. Both can be activated roughly 1.55- and 2.41-fold by T3 in the TR␣1 cell, respectively. The two elements may contain a potential TRE. The ⫺190/⫹119 was further deleted from the 5⬘ end to become ⫺149/⫹119, ⫺31/⫹119, ⫺1/⫹119, and ⫹56/⫹119. The transactivation activities of these constructs were all roughly a 2-fold activation by T3 (Fig. 5B). However, when the fragment from ⫹56 to ⫹119 was deleted to become ⫺1/ ⫹65, transactivation activity was decreased. Additionally, if both ⫺234/⫺190 and ⫹56/⫹119 fragments were deleted, the transcriptional activities of ⫺190/⫹65 were greatly reduced. These effects demonstrated that the two positive TREs were located in the ⫺234/⫺190 and ⫹56/⫹119 regions (Fig. 5C). Notably, the transcriptional activity of the latter was stronger than the former. Examining the nucleotide sequences in ⫺234/

⫺190 and ⫹56/⫹119 regions of the ARA70 promoter revealed atypical TREs with sequences of ⫺211(AGGTGC)catccg(AGGTCC)-194 and ⫹99(TGGTGA)gtcgg(TGACCT)⫹115, respectively (Fig. 5C). The above sequences are homologous to the TRE consensus sequence (AGGTCA)n6(AGGTCA) direct repeat or (AGGTCA)n5(TGACCT) atypical palindrome reported previously (20). Finally, the transcriptional activity of ⫺234/ ⫺190 and ⫹56/⫹119 was significantly reduced where TRE sequences were changed to AAAAAA, as Fig. 5C (MT1 and MT2) illustrates. Binding of TR to the ⫺234/⫺190 and ⫹56/⫹119 fragments on the ARA70 promoter

To confirm that ⫺211(AGGTGC)catccg(AGGTCC)-194 and ⫹99(TGGTGA)gtcgg (TGACCT)⫹115 function as a TRE in the ARA70 promoter, EMSA was conducted using both fragments as probes. Chicken lysozyme genes containing two inverted repeated half-site motifs were separated with six nucleotides (Lys) and used as a control (Fig. 6A) (21). Reactions performed with ␣-32P-labeled ⫹56/⫹119 fragment and TR␣1 protein produced one prominent (indicated by D) and specific band supershift (SS) by the C4 antibody (Fig. 6B, lanes 3 and 9) but not the control antibody (Fig. 6B, lane 8).



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FIG. 6. Analysis by EMSA of the binding of TR proteins to the ⫹56/⫹119 and ⫺234/⫺190 fragments of the ARA70 promoter. The GST-TR (100 ng for ⫹56/ ⫹119 probe and 400 ng for ⫺234/⫺190 probe) was incubated in a final volume of 20 ␮l with approximately 20,000 cpm of ␣-32P-labeled oligonucleotide probes of the ARA70 promoter for 40 min at room temperature. ARA70 protein was in vitro translated by TNT lysate (Promega, Rockville, MD). Panel A, Lys-TRE probe as a positive control; panels B and D, ARA70 ⫹56/⫹119-TRE probe; panel C, ARA70 ⫺234/ ⫺190-TRE probe. C4 is a mouse monoclonal antibody against TR proteins, and MOPC21 is a nonspecific mouse IgG1 MOPC21 (Sigma-Aldrich). The positions of probes complexed with TR are indicated. D, Dimer; HD, heterodimer; M, monomer; non-SC, nonspecific competition; NS, nonspecific; SC, specific competition; SS, TR complex supershifted by C4 antibody.

The specific TR␣1/TRE complex could be competed by adding 10⫻ and 50⫻ molar excess of specific cold competitors (Fig. 6B, lanes 4 and 5) but not by nonspecific cold competitors (Fig. 6B, lanes 6 and 7). The sequences for specific and nonspecific competitors were shown in Fig. 5C (WT2 or MT2, respectively). Only the GST protein is unable to bind to the TRE (Fig. 6B, lane 2). Similar data were obtained when TR␤1 was used (data not shown). Additionally, the ␣-32P-labeled ⫺234/ ⫺190 fragment reacted with TR␤1 protein and yielded one specific band (indicated by D, Fig. 6C, lane 3) that could be supershifted by the C4 antibody (Fig. 6C, lane 9). However,

TR proteins could not be supershifted by the control antibody (MOPC21, Fig. 6C, lane 8). The specific TR␤1/TRE complex could be competed by adding 10⫻ and 50⫻ molar excess of specific cold competitors (Fig. 6C, lanes 4 and 5) but not by nonspecific cold competitors (Fig. 6C, lanes 6 and 7). The sequences for specific and nonspecific competitors were shown in Fig. 5C (WT1 or MT1, respectively). Only the GST protein could not bind to the TRE (Fig. 6C, lane 2). Similar data were obtained when TR␣1 was used (data not shown). These experimental results demonstrate that the DNA-bound TR bands (Fig. 6, B and C) were specific because the TR/TR complexes can be supershifted by the

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TR-specific antibody (C4) but not by control antibody. However, the binding affinity of ⫹56/⫹119 fragment is much stronger than that in ⫺234/⫺190 because a 4-fold increase in TR protein was used for EMSA analysis of the ⫺234/⫺190 fragment. Combined, these analytical findings suggest that TR bound to the two TRE binding motifs that were functional in the promoter assay (Fig. 5). T3 induced ARA70 mRNA expression in vivo

Two groups of 6-wk-old male SD rats underwent thyroidectomies (TX) to elucidate in vivo response of ARA70 to T3 treatment. One group (n ⫽ 10 per group) was injected with T3 daily for 2 wk (TX⫹T3), whereas the second group received no T3 injections (TX). The third group was shamoperated. Rats were killed upon completion of the experiment, and their serum was collected to test for T3 and TSH. The livers were removed for Q-RT-PCR or Western blot analysis. The T3 serum levels in the TX group were approximately 0.022-fold (12.3 vs. 548.1 ng/dl) those in the group that underwent T3 treatment (TX⫹T3). Levels of TSH in the TX group were approximately 67.2-fold (2.42 vs. 0.036 ng/ ml) those in the T3-treated group. The T3 and TSH serum levels in the sham group were approximately 48.1 ng/dl and 0.197 ng/ml, respectively. The experimental results indicated that ARA70 mRNA levels in the TX⫹T3 rat were approximately 1.66-fold higher than those in the TX group (Fig. 7A). The ARA70 bands in the three groups after Q-RT-PCR were analyzed by agarose gel (Fig. 7B). Furthermore, ARA70 protein levels in the TX⫹T3 rat were approximately 1.9-fold higher than those in the TX group (Fig. 7, C and D). The RNA and protein levels in sham-operated animals were approximately 1.34- and 1.42-fold, respectively, higher than those in the TX group (Fig. 7).

FIG. 7. Induction of ARA70 expression by thyroid hormone in rat liver. A, Expression of ARA70 in TX, TX⫹T3, and sham male SD rat liver was determined by Q-RT-PCR as described in Materials and Methods. Data are values of means ⫾ SE from the experiment (three groups per experiment; n ⫽ 10 per group). These results are displayed as fold activation compared with those in the control group (TX). B, ARA70 expression determined by Q-RTPCR and analyzed by the agarose gel. C, ARA70 protein expression determined by Western blot. D, Intensities of the ARA70 protein in C were quantified, and the degree of T3-induced activation was determined in each group by using TX as 1. Student’s t test: *, P ⬍ 0.05, TX⫹T3 or sham vs. TX.


ARA70 suppression of T3-dependent TR transcriptional activity

After being induced by T3, the effect of ARA70 on the TR signaling pathway mediated by the three TREs [palindrome (Pal), malic enzyme (ME), and lysozyme (Lap)] were measured by reporter assays. The HepG2-TR␣1#1 or CV-1 cells were cotransfected simultaneously with a positive reporter containing TRE and the ARA70-expressing vector. Cotransfection results demonstrated that h-TR␣1 transcriptional activity mediated by Pal-, ME-, or Lap-TRE (22) was suppressed significantly (P ⬍ 0.05 or P ⬍ 0.01) by ARA70 (0.2 or 0.3 ␮g) roughly 15, 46, and 42%, respectively, using 10 nm T3 for 24 h in CV-1 cells (Fig. 8, A–C). Transfection of increased amounts of ARA70 (0.6 or 0.9 ␮g) increased suppression of TR transactivation activities (Fig. 8, A and B). A similar result was also obtained using HepG2-TR␣1 cells (Fig. 8, D–F). However, increased ARA70 (0.9 ␮g) did not significantly affect T3 signaling in this cell line (Fig. 8, D–F). Furthermore, what is the possible mechanism for ARA70 to suppress TR transcriptional activities? The EMSA experiments indicated that TR/TRE complex decreased as ARA70 protein increased (Fig. 6D, lanes 6 – 8). Discussion

Regulation of ARA70 by T3 was previously identified by cDNA microarrays. Several of these genes from microarray analysis are transcription factors, believed to be unassociated with thyroid hormone function. Therefore, this study investigated the molecular regulation of ARA70 by T3 in isogenic HepG2 cell lines. The regulation of ARA70 was significantly increased after T3 treatment at mRNA and protein levels. This regulation was direct, and two TREs were identified. Similar regulation was noted in TX rats after T3 application.



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FIG. 8. Repression of T3-dependent transcriptional activity in CV-1 and HepG2-TR cells by ARA70. CV-1 (A–C) and HepG2-TR␣1 (D–F) cells were cotransfected individually with various TRE reporter plasmids, Pal-TRE (A and D), ME-TRE (B and E), and Lap-TRE (C and F) (22) and ␤-galactosidase using the lipofectamine transfection technique. CV-1 cells were cotransfected with TR␣1 expression plasmid. In some experiments, cells were simultaneously cotransfected with increasing amounts of ARA70 plasmid. Subsequently, cells were further incubated at 37 C for 24 h in the absence (data not shown) or presence of 10 nM T3. Luciferase activities are expressed as fold increases of T3 induction and normalized for protein concentrations and ␤-galactosidase activity. Data (means ⫾ SE) are expressed as fold activation (compared with 0 ␮g ARA70 condition) from three independent experiments performed in triplicate. Student’s t test: **, P ⬍ 0.01; *, P ⬍ 0.05, 0 ␮g ARA70vs. ARA70-transfected.

Subsequently, ARA70 suppressed T3 signaling in a TREdependent manner. The liver is clearly recognized as a target organ for thyroid hormones. In fact, Chamba et al. (23) reported roughly equal quantities of TR␣1 and TR␤1 protein in human hepatocytes. Reported physiological concentrations of T3 vary. Kansara et al. (24) reported a physiological T3 concentration of 10 nm. Lenoir et al. (25) reported a physiological concentration of T3 ranging from 0.1–2.5 nm. Kato et al. (26) reported a physio-

logical T3 concentration of 1–10 nm. In this study, 1 nm T3 induced ARA70 mRNA expression; therefore, regulation of ARA70 by T3 can be observed both physiologically and pathologically if the target cell expresses detectable TR. Additionally, many TR-regulated mechanisms also have been studied in CV-1 for its high transfection efficiency and yielded convincing results (27–29). The ARA70 protein, also known as a nuclear receptor coactivator, has been isolated from the human brain (30) and

3494 Endocrinology, July 2007, 148(7):3485–3495

prostate cDNA libraries (31). The protein ARA70 increases transcriptional activity of AR cotransfection assays. Alen et al. (32) demonstrated that ARA70 binds to transcription factor IIB and p300/CBP (cAMP-associated binding proteinbinding protein)-associated factor in vitro, suggesting that ARA70 acts as a coregulator between steroid receptors and transcriptional machinery. Notably, studies of laboratory rats indicated ARA70 (30) likely plays some role in Sertoli cell development. Moreover, postnatal Sertoli cell maturation is characterized by steadily increasing AR (33, 34). Furthermore, T3 stimulates production of AR and AR mRNA in vitro (34), another process considered important for normal maturation of Sertoli cells (33). Experimental results suggest that T3 stimulates ARA70 expression in coordination with AR to regulate Sertoli cell maturation. AR plays a key reproductive role in males (35). Hyperthyroidism appears to alter spermatogenesis and fertility (36). This study provides molecular evidence that T3/TR may be mediated by ARA70 and may influence the male reproductive system. Notably, after administering T3 to TX rats for 14 d, ARA70 mRNA was detectable in liver by Q-RT-PCR assay regardless of T3 level. Additionally, ARA70 protein was detectable in all three groups of rats. Although, Yeh and Chang (30) reported that ARA70 was undetectable in normal rat liver. The Q-RTPCR method used to analyze expression of ARA70 mRNA in this study is more sensitive than Northern blot used by Yeh and Chang (30). The binding affinity of TR to the ⫹56/⫹119 is much stronger than that in ⫺234/⫺190 fragment. These results were supported by the EMSA experiment and promoter assay. The 4-fold GST-TR proteins used in the ⫺234/⫺190 fragment had even lower intensities of TR/TRE complexes in the EMSA experiments (Fig. 6, B vs. C). The difference may be due to the three mismatches between the ⫺211/⫺194 and the TRE consensus sequence, whereas only two mismatches appear in the ⫹99/⫹115 site compared with the n5-spaced palindrome. The nuclear receptors (NRs) clearly prefer specific NR boxes. One factor driving this specificity is the amino acid residues immediately flanking the NR box (37–39). Moore et al. (40) employed a homogeneous equilibrium binding assay to demonstrate the binding affinity between TR and ARA70. Their experimental data suggested that ARA70 was associated with TR (Kd ⫽10 –30 ␮m). Apparently, TR regulates ARA70, and ARA70 subsequently impacts the TR signaling via interaction with ARA70. The participation of ARA70 in T3-dependent signaling has significant implications in the biology of steroid signaling. Notably, ARA70 dramatically enhances AR transcriptional activity in prostate cancer cells (41). Whether ARA70 aberrant expression occurs in hyperthyroidism or hypothyroidism patients is an interesting question. Experimental results also demonstrate that ARA70 is a negative regulator of TR functions. Numerous TR-interacting proteins have recently been identified (42– 46). Among these TR-interacting proteins, nuclear receptor corepressor (N-CoR) (46), SMRT (45) and short heterodimer partner (SHP) (43) have been described. The ARA70 likely repression mechanism is that TR/ARA70 interaction and ARA70 prevents TR from binding to its cognate response elements. This theory is supported by the EMSA experiment and also by


Moore et al. (40). However, the ARA70 repression mechanism requires additional study. Overexpression of ARA70 reversed AR down-regulation of estrogen receptor (ER)␣ signaling. However, with a progressive increase of transfected AR, ARA70 enhanced the inhibitory effect of AR on ER␣ signaling (47). The experimental results in this study demonstrate that ARA70 inhibits TR signaling. However, an increase in transfected TR did not alter ARA70 inhibitory activity (data not shown). Moreover, T3 increased AR levels in a prostatic carcinoma cell line (48) and in rat Sertoli and peritubular cells (34). Additionally, ARA70 is a coactivator for AR and may be a functional link between modulation of TR/AR cross-talk in T3-signaling models. Furthermore, both ARA70 and ARA54 modulate vitamin D receptor (VDR) transactivation, and the competition for ARA70 mediates the suppressive effect of androgen-AR on VDR transactivation. Suppression of VDR transactivation by AR signaling was restored via ARA70 overexpression but not via ARA54 overexpression (49). In addition to TR, AR, and ER interacting with ARA70, ARA70 demonstrated the strongest enhancement of peroxisome proliferator-activated receptor-␣ (PPAR␣) transcription among coactivators examined in prostate cancer cells. Mutation of the N terminal of the PPAR␣ ligand-binding domain dramatically reduced the ability of ARA70 to promote PPAR␣:RXR transcription (50). However, the interacting domain within TR with ARA70 is currently unknown. Experimental results of this study demonstrated that T3 has a crucial role in ARA70 expression at the transcriptional and translational level. Induction of ARA70 by T3 was direct, as an absolute not requirement for de novo protein synthesis. Finally, ARA70-inhibited TR signaling is involved in TR/ ARA70 interaction. Experimental data presented here further elucidates the action of TR in hepatoma cell lines. Of significant importance is the elucidation of T3 control of numerous genes related to transcription regulation. Further study is required to investigate tissue or cell-specific T3 target genes and the physiological role of T3 induction. Acknowledgments Received September 12, 2006. Accepted March 23, 2007. Address all correspondence and requests for reprints to: Dr. KwangHuei Lin, Department of Biochemistry, Chang-Gung University, 259 Wen-hwa 1 Road, Taoyuan, Taiwan 333, Republic of China. E-mail: [email protected]. This work was supported by grants from Chang-Gung University, Taoyuan, Taiwan (CMRP 1332, CMRPD 34013, and NMRP 1074), Chang-Gung Molecular Medicine Research Center, Taoyuan, Taiwan (CMRP 140041), and the National Science Council of the Republic of China (NSC 91-2320-B-182-041). Disclosure Statement: The authors have nothing to declare.

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