Glyceraldehyde-3-phosphate Dehydrogenase Enhances Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells*

Androgen receptor (AR) functions as a transcriptional factor for genes involved in proliferation and differentiation of normal and cancerous prostate cells. Coactivators that bind to AR are required for maximal androgen action. Here we report that increasing the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in a prostate cancer cell line by as little as 1.8-fold enhances transcriptional activity of AR (but not the transcriptional activity of glucocorticoid receptor or estrogen receptor α) in a ligand-dependent manner and results in an increased expression of prostate-specific antigen. Small interference RNA-mediated knockdown of GAPDH significantly attenuated ligand-activated AR transactivation. Immunoprecipitation analysis revealed the presence of an endogenous protein complex containing GAPDH and AR in both the cytoplasm and nucleus. Addition of a nuclear localization signal (NLS) to GAPDH (GAPDH-NLS) completely abolished the ability of GAPDH to transactivate AR. Neither wild-type GAPDH nor GAPDH-NLS enhanced transcriptional activity of mutant AR (ARΔC-Nuc) that is a constitutively active form of AR in the nucleus, even though GAPDH-NLS formed a complex with wild-type AR or ARΔC-Nuc. AR transactivation was enhanced by a mutant GAPDH lacking dehydrogenase activity. GAPDH enhanced the transcriptional activity of AR(T875A) activated by an antagonist such as hydroxyflutamide or cyproterone acetate. These results indicate that GAPDH functions as a coactivator with high selectivity for AR and enhances AR transactivation independent of its glycolytic activity. Further, these data suggest that formation of a GAPDH·AR complex in the cytoplasm rather than nucleus is essential for GAPDH to enhance AR transactivation.

to act more specifically on AR. AR contains a characteristic polyglutamine (polyQ) tract, and the expansion of the polyQ tract results in a reduction in the ability of the AR to activate transcription (8), indicating that conformational changes of the polyQ tract per se have an influence on AR transactivation. Furthermore, ARA24 has been shown to interact with the polyQ tract of AR as a coactivator to enhance AR transactivation (9). ARA24 is identical to Ran, which is involved in nucleocytoplasmic transport (10). These results suggest that intramolecular and/or intermolecular interactions involving the polyQ tract play a critical role in modulation of AR transactivation.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that reversibly catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate in the cytosol. Recent studies have demonstrated that GAPDH serves as a multifunctional protein contributing to a number of nonglycolytic cellular processes such as nuclear tRNA transport, apoptosis, DNA repair, and DNA replication in the nucleus, vesicular transport, membrane fusion and microtubule bundling in the particulate fraction (11), and cell spreading in the extracellular space (12). Furthermore, in vitro experiments have shown that GAPDH binds to four gene products (AR, ataxin-1, atrophin, and huntingtin), each produced in a different neurodegenerative disorder (13,14). A common structural feature of these gene products is a polyQ tract. In fact, GAPDH binds to synthetic polyQ peptides, although binding of GAPDH to AR does not vary with the length of the polyQ tract. Thus, these results indicate that GAPDH interacts with AR through the polyQ tract in the NTD, although the physiological significance of the interaction between GAPDH and AR remains unclear. On the other hand, the expression level of GAPDH mRNA is correlated with the metastatic potential and cell motility of rat prostate cancer cells (15) and increased in late pathological stages of human prostate tumors (16). These results suggest that GAPDH regulates AR transactivation, especially when the expression of GAPDH is up-regulated in cells expressing AR. In the present study, we report that GAPDH enhances the transcriptional activity of AR in a ligand-dependent manner and that the role of GAPDH in AR transactivation is independent of glycolysis. Furthermore, we demonstrate that a protein complex containing GAPDH and AR exists in both the cytoplasm and nucleus.

EXPERIMENTAL PROCEDURES
Cell Culture-COS-7 cells and human prostate cancer PC-3 and LNCaP cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 g/ml streptomycin and maintained at 37°C in a 5% CO 2 /95% air atmosphere at 100% humidity unless otherwise indicated. COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA), and PC-3 and LNCaP cells were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Miyagi, Japan).
Plasmids-Human ER␣ mammalian expression vector, pCAGGS-ER␣, was kindly provided by Dr. T. Adachi (Kyoto University, Kyoto, Japan). The coding regions of the human GAPDH, AR, GR, ARA70, and ARA24/Ran cDNAs (Gen-Bank TM accession numbers M33197, M23263, X03225, NM_005437, and NM_006325, respectively) were amplified by two-sequential, nested PCR using human brain (cerebral cortex) Marathon-ready cDNA (Clontech Laboratories, San Jose, CA) as a template and specific primer combinations, followed by cloning into pCR2.1-TOPO vectors. Primers with a typical Kozak sequence and appropriate restriction enzyme sites were designed for the second PCR. The ARA70N cDNA encoding the N-terminal region (amino acids 1-401) of ARA70 was amplified by PCR using ARA70 cDNA as a template and the specific primers. For pcDNA3.1-GAPDH-Myc-His and pcDNA3.1-ARA70N-Myc-His plasmids expressing GAPDH and ANA70N with Myc and His tags at the C terminus, GAPDH and ARA70N cDNAs were subcloned into the EcoRI and BamHI sites and NotI and BamHI sites of pcDNA3.1-Myc-His, respectively. The GAPDH(C151S) cDNA encoding mutant GAPDH substituting Ser for Cys at position of 151 was amplified by PCR using specific primers and the EcoRI-digested fragments of pCR2.1-GAPDH(C151S) (12) as a template and cloned, followed by insertion into the EcoRI and BamHI sites of pcDNA3.1-Myc-His, termed pcDNA3.1-GAPDH(C151S)-Myc-His. The expression vector pcDNA3.1-GAPDH-NLS-Myc-His was constructed by insertion of the DNA fragment encoding three tandem repeat of simian virus 40 large T antigen nuclear localization signal (NLS) in-frame to the BamHI sites of pcDNA3.1-GAPDH-Myc-His, resulting in a fusion protein of GAPDH with NLS, containing Myc and His tags. The GAPDH(258 -261A) cDNA encoding GAPDH with a nuclear export signal (NES) containing alanine mutations was synthesized by site-directed mutagenesis of pcDNA3.1-GAPDH-Myc-His using the QuikChange II XL site-directed mutagenesis kit (Stratagene), followed by construction of pcDNA3.1-GAPDH(258 -261A)-Myc-His plasmid. For pcDNA3.1-AR and pcDNA3.1-GR plasmids expressing AR and GR with no tags, respectively, AR and GR cDNAs were subcloned into the NotI and BamHI sites of pcDNA3.1-Myc-His. The AR cDNA cloned in the present study contains sequences encoding 25 repetitive polyQ and 18 repetitive polyglycine regions, and the translated protein is composed of 917 amino acids. The AR⌬C-Nuc cDNA encoding mutant AR (amino acids 1-658) lacking the LBD was amplified by PCR using specific primers and the AR cDNA as a template, followed by construction of pcDNA3.1-AR⌬C-Nuc. AR(T877A) substituting Ala for Thr at position of 877 of AR is found in many prostate tumors, including LNCaP cells (17). Amino acid position 877 of AR(T877A) corresponds to amino acid position 875 of AR cloned in the present study, because AR is polymorphic in its polyQ and polyglycine regions. Therefore, we constructed the expression vector pcDNA3.1-AR(T875A) by site-directed mutagenesis of the pcDNA3.1-AR using the QuikChange II XL site-directed mutagenesis kit. For pCAGGS-ARA24/Ran plasmid expressing ARA24/Ran with no tags, ARA24/Ran cDNA was subcloned into EcoRI sites of pCAGGS (18), which was kindly provided by Dr. J. Miyazaki (Osaka University, Osaka, Japan). Luciferase reporter vectors (pARE2-TATA-Luc and p3xERE-TATA-Luc) in which the pGL3-basic vector has two tandem repeats of an androgen-response ele-ment and three tandem repeats of an estrogen-response element, together with an adenovirus E1b TATA sequence, were constructed according to the methods of Moilanen et al. (19) and Legler et al. (20), respectively.
Immunoprecipitation-For preparation of whole cell fraction, LNCaP or PC-3 cells were incubated for 15 h in the presence of 0.2 nM R1881, harvested, and resuspended in IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 10 mM sodium pyrophosphate, 2 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 g/ml leupeptin, and 1 g/ml aprotinin). COS-7 cells were co-transfected with pcDNA3.1-AR or pcDNA3.1-AR⌬C-Nuc and pcDNA3.1-GAPDH-NLS-Myc-His for 5 h, followed by incubation for 28 h. Cells were further incubated for 15 h in the presence of 0.2 nM R1881, followed by harvesting and resuspension in IP buffer. Cell suspension was incubated for 30 min on ice and centrifuged at 20,000 ϫ g for 20 min at 4°C. The supernatant is referred to as whole cell fraction. For preparation of nuclear and cytoplasmic fractions, LNCaP cells were incubated for 15 h in the presence of 0.2 nM R1881 and resuspended in hypotonic buffer (10 mM Hepes-NaOH, pH 7.5, containing 10 mM KCl, 1.5 mM MgCl 2 , 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 g/ml leupeptin, and 1 g/ml aprotinin) and homogenized by repeated passage through a 23-gauge needle. The homogenate was subjected to differential centrifugation as described previously (21), except for minor modifications. Nuclei were sonicated in IP buffer, followed by centrifugation at 20,000 ϫ g for 20 min. The supernatant is referred to as the nuclear fraction. The post-nuclear fraction was diluted with IP buffer and incubated for 30 min on ice, followed by centrifugation at 20,000 ϫ g for 20 min. The supernatant is referred to as the cytoplasmic fraction. The whole cell fraction (LNCaP or PC-3 cells, 1 mg; COS-7 cells, 500 g), nuclear fraction (333 g), or cytoplasmic fraction (1 mg) that had been precleared with protein G-Sepharose (Amersham Biosciences) for 1 h was incubated with monoclonal mouse anti-AR IgG (2 g, clone AR441, Santa Cruz Biotechnology) or control mouse IgG (2 g) for 1 h at 4°C, followed by addition of 60 l of protein G-Sepharose (50% slurry) pre-equilibrated with IP buffer. The mixture was incubated for 4 h at 4°C, and the resin was washed with IP buffer. Proteins bound to the resin were analyzed by Western blotting with monoclonal mouse anti-AR (1/1,000), anti-GAPDH (1/5,000, clone 6G5, Biogenesis, Poole, UK) and anti-Myc (1/3,000, clone 9B11, Cell Signaling Technology, Beverly, MA) antibodies and polyclonal rabbit anti-AR antibodies (1/1,000, N-20, Santa Cruz Biotechnology), followed by reaction with the horseradish peroxidase-conjugated goat antimouse IgG (1/3,000) and anti-rabbit IgG (1/3,000), respectively. Immunoreactive bands were detected by Super Signal Chemiluminescent substrate.
Statistics-Data are expressed as mean Ϯ S.D., and Student's t test for unpaired samples was applied for statistical analysis. Differences were considered significant when p Ͻ 0.05.

Enhancement of AR Transactivation by GAPDH-To
define the ability of GAPDH to modulate AR-mediated transcription, the human prostate cancer PC-3 cell line (an ARnegative cell line) was transiently co-transfected with the pARE2-TATA-Luc reporter plasmid, pcDNA3.1-AR and pcDNA3.1-GAPDH-Myc-His, and the transcriptional activity of AR was measured as luciferase activity. The transcriptional activity of AR was increased with an increasing dosage of pcDNA3.1-GAPDH-Myc-His, which resulted in an increase of recombinant GAPDH-Myc. In contrast, GAPDH had no influence on the AR transactivation in the absence of ligand (Fig.  1A). When the transcriptional activity of AR was determined at various concentrations of R1881 with PC-3 cells overexpressing GAPDH at the same level, the enhancing effect of GAPDH on AR transactivation was observed even in the presence of 0.05 nM R1881 (Fig. 1B). When similar experiments were performed in LNCaP cells (an AR-positive human prostate cancer cell line) or COS-7 cells (an AR-negative African green monkey kidney cell line), AR transactivation was enhanced by GAPDH in both cell lines (Fig. 1C). Prostate-specific antigen (PSA) was used as a biomarker for prostate cancer progression. Because PSA is an AR-mediated endogenous target gene with androgen-response elements, expression of PSA is enhanced by AR transactivation (22). To determine whether GAPDH activates the expression of PSA, GAPDH was overexpressed in LNCaP cells, and then a cell lysate was subjected to Western blot analysis with anti-PSA antibodies.
PSA was detected in the presence of R1881, and overexpression of GAPDH resulted in an increased expression level of PSA (Fig. 2). These results indicate that GAPDH enhances the transcriptional activity of AR.
To examine the effect of endogenous GAPDH on AR transactivation, PC-3 cells were treated with GAPDH-specific siRNA, and then the transcriptional activity of AR was determined. siRNA-mediated knockdown of GAPDH resulted in a significant decrease of ligand-induced AR transactivation Luciferase reporter assay and statistical analysis were performed as described in A. (Fig. 3). Western blot analyses showed that GAPDH siRNA specifically reduced endogenous GAPDH, but not endogenous ␣-tubulin. These data demonstrate that endogenous GAPDH contributes to AR transactivation.
Expression Level of Exogenous GAPDH-To assess the amount of exogenous GAPDH required to enhance AR transactivation, the ratio of exogenous GAPDH to endogenous GAPDH in the positively transfected cells was determined. Firstly, to determine the transfection efficiency, PC-3 cells were transfected with pcDNA3.1-GAPDH-Myc-His, fixed, and incubated with anti-Myc IgG. Bound antibodies were immunoreacted with Alexa Fluor 488-conjugated secondary antimouse IgG, and the nucleus was stained with DAPI (Fig. 4A). Random fields were observed by immunofluorescent microscopy, and the transfection efficiency was determined as the   A, cells were fixed, permeated, and incubated with anti-Myc IgG. Immunoreactive antibodies were visualized using Alexa Fluor 488-conjugated secondary anti-mouse IgG. The nucleus was stained with DAPI. Random fields were photographed, and quantification of cells stained by DAPI or immunoreacted with anti-Myc IgG was performed. The number of GAPDH-Myc-expressing cells was normalized to that of DAPI-stained cells. Values are calculated from data of ten random fields, and results are representative of two independent experiments. In B: Upper panel, whole cell lysates were prepared, and the indicated amounts of proteins were subjected to SDS-PAGE, followed by Western blot analysis with anti-GAPDH antibodies. Lower panel, the intensities of immunoreactive bands for endogenous or exogenous GAPDH were quantified by densitometry, and a standard curve was prepared by plotting the intensity of immunoreactive band for endogenous GAPDH. The intensity of immunoreactive band for exogenous GAPDH in 20 g of whole cell lysates is indicated by an arrow on the standard curve for endogenous GAPDH. Each graph is representative of two independent experiments. ratio of the number of recombinant GAPDH-Myc-expressed cells to that of DAPI-stained cells. Approximately 24% of the cells were calculated to express exogenous GAPDH (transfection efficiency ϭ 24%). Subsequently, to determine the ratio of exogenous GAPDH to endogenous GAPDH in whole cells, PC-3 cells were transfected with pcDNA3.1-GAPDH-Myc-His. Whole cell lysates were prepared, and the indicated amounts of proteins in whole cell lysates were subjected to SDS-PAGE, followed by Western blot analysis with anti-GAPDH antibodies (Fig. 4B, upper panel). Densitometry of the immunoreactive endogenous or exogenous GAPDH was quantified, and a standard curve was prepared by plotting the intensity of immunoreactive band for endogenous GAPDH (Fig. 4B, lower panel). Densitometry analysis showed that the intensity of immunoreactive endogenous GAPDH was proportional to the amount of whole cell lysates in the range of 2-10 g of protein.
The intensity of exogenous GAPDH-Myc in 20 g of whole cell lysates, when quantitated by using a standard curve, corresponded to that of endogenous GAPDH in 3.8 g of whole cell lysates. Because whole cells are composed of the positively and negatively transfected cells, the amount of lysates from positively transfected cells was calculated to be 4.8 g in 20 g of whole cell lysates by taking into account the transfection efficiency. Thus, the ratio of the exogenous GAPDH-Myc molecules to the endogenous GAPDH molecules in the positively transfected cells was 0.8:1 (3.8:4.8). These results indicate that increasing the expression of intracellular GAPDH by as little as 1.8-fold enhances AR transactivation.
Endogenous GAPDH⅐AR Complex in Vivo-In vitro experiments have demonstrated that recombinant AR physically interacts with GAPDH (13,14). To assess whether GAPDH is associated with AR in vivo, LNCaP cells were cultured in the presence or absence of ligand, and whole cell fraction was incubated with anti-AR IgG or control mouse IgG as a negative control. Protein complexes bound to IgG were separated by SDS-PAGE, followed by Western blot analysis. Anti-AR IgG co-immunoprecipitated GAPDH with AR in the presence or absence of ligand, whereas neither GAPDH nor AR was immunoprecipitated with control IgG (Fig. 5A). When whole cell fraction was prepared from PC-3 cells as a negative control, GAPDH was not immunoprecipitated by anti-AR IgG. These results indicate that a protein complex composed of GAPDH and AR is formed in vivo. The ligand-free AR is localized as an inactive form in the cytosol, and the ligand-bound AR is translocated into the nucleus as an active form. GAPDH is localized not only in the cytosolic fraction, but also in the nuclear and particulate fractions. Therefore, to determine the subcellular distribution of the GAPDH⅐AR complex, the cytoplasmic and nuclear fractions were prepared from LNCaP cells, followed by immunoprecipitation with anti-AR IgG or control mouse IgG. The anti-AR IgG immunoprecipitated endogenous GAPDH⅐AR complex in both the cytoplasmic and nuclear fractions (Fig. 5B). These results indicate that GAPDH forms a complex with AR in the cytoplasm and nucleus.
Nuclear GAPDH and AR Transactivation-To assess whether nuclear GAPDH plays a critical role in AR transactivation, we constructed a recombinant fusion protein with the simian virus 40 large T antigen nuclear localization signal between GAPDH and Myc tag (GAPDH-NLS-Myc). To evaluate the distribution of GAPDH-NLS-Myc in intact cells, PC-3 cells that had been transfected with GAPDH-NLS-Myc expression vector were immunostained using anti-Myc IgG, followed by Alexa Fluor-labeled secondary anti-mouse IgG. Using immunofluorescent microscopy, we observed that the fluorescent image of GAPDH-NLS-Myc overlaid the DAPI-stained nucleus, indicating a predominant localization of GAPDH-NLS-Myc in the nucleus (Fig. 6A, lower panels). On the other hand, GAPDH-Myc was distributed throughout the cells, although it is partially localized in the nucleus (Fig. 6A, upper  panels). To further define the subcellular distribution of GAPDH-NLS-Myc, the PC-3 cells overexpressing GAPDH-NLS-Myc were homogenized, and subcellular fractionation was performed by differential centrifugation. In addition, whole cell fraction was prepared and subjected to Western blot analysis. GAPDH-NLS-Myc was predominantly detected as a nuclear rather than cytosolic protein (Fig. 6B, middle panel). On the contrary, GAPDH-Myc was predominantly detected in the cytosol and partly in the nucleus (Fig. 6B, left panel). The expression patterns of GAPDH-Myc among fractions were consistent with those of endogenous GAPDH. When PC-3 cells were co-transfected with GAPDH-NLS-Myc expression vec- tor, AR expression vector and pARE2-TATA-luc, GAPDH-NLS-Myc had no stimulatory effect on the AR transactivation, at the same expression level as GAPDH-Myc (Fig. 6C, left  panel). To further study the role of nuclear GAPDH in AR transactivation, we constructed the NES-mutated GAPDH, GAPDH(258 -261A)-Myc, corresponding to the previously reported mutant GAPDH construct M3 (23). Compared with GAPDH-Myc, no additional enhancing effect of GAPDH(258 -261A)-Myc on AR transactivation was observed, even though more GAPDH(258 -261A)-Myc accumulated in the nucleus (Fig. 6, B and C, right panels). Because the cytosolic marker TPI and the nuclear marker lamin B1, respectively, were observed only in each corresponding fraction, there was no contamination between cytosolic and nuclear fractions. These results indicate that addition of NLS to GAPDH, which facilitates the nuclear translocation of GAPDH, completely abolishes the ability of GAPDH to transactivate AR and that disruption of NES in GAPDH, which results in more accumulation of GAPDH in the nucleus, causes no additional enhancing effect on AR transactivation.
Complexes of GAPDH-NLS and Mutant AR-Truncated mutant AR (AR⌬C-Nuc) corresponding to the previous reported AR1-660 (24) is constitutively localized in the nucleus even in the absence of ligand and exerts androgen action as a constitutively active form of AR. To determine the effect of GAPDH-NLS on the transcriptional activity of AR⌬C-Nuc, luciferase assay was determined. Indeed, AR⌬C-Nuc exhibited constitutive transcriptional activity similar to ligandactivated wild-type AR. Neither wild-type GAPDH nor GAPDH-NLS enhanced transcriptional activity of AR⌬C-Nuc (Fig. 7A). Next, the presence of a nuclear GAPDH⅐AR complex was studied using GAPDH-NLS and AR⌬C-Nuc. COS-7 cells were co-transfected with GAPDH-NLS and wild-type AR or AR⌬C-Nuc expression plasmids. Immunoprecipitation with anti-AR IgG revealed that GAPDH-NLS formed a complex with wild-type AR or AR⌬C-Nuc (Fig. 7B). These results support the presence of GAPDH⅐AR complex in the nucleus. Further, the data indicate that, although GAPDH-NLS forms a complex with wild-type AR or AR⌬C-Nuc in the nucleus, GAPDH-NLS enhances transcriptional activity of neither wildtype AR nor AR⌬C-Nuc.
Enhancement of AR Transactivation by Mutant GAPDH Lacking Glycolytic Function-We examined whether the glycolytic function of GAPDH is involved in GAPDH-enhanced AR transactivation. Because the dehydrogenase activity of GAPDH requires Cys-151 of the active center (25), a mutant replacing Ser for Cys at position 151 of GAPDH, GAPDH(C151S), is inactive (12). Therefore, to examine whether GAPDH(C151S) enhances the transcriptional activity of AR, a GAPDH(C151S) expression vector was co-transfected into cells with AR expression vector and reporter vectors. The ability of GAPDH(C151S) to enhance AR transactivation was comparable to that of wild-type GAPDH, indicating that AR transactivation by GAPDH is independent of its dehydrogenase activity (Fig. 8).
Effects of GAPDH and/or ARA24/Ran on the Transcriptional Activities of AR, GR, and ER␣-ARA24/Ran enhances AR transactivation through binding to the polyQ tract in the NTD of AR (9). To determine whether GAPDH exerts a competitive influence through its polyQ tract on ARA24/Ran-enhanced AR transactivation, reporter assays were performed with PC-3 cells overexpressing both GAPDH and ARA24/Ran. GAPDH and ARA24/Ran enhanced AR transactivation up to 1.6-and 2.2fold, respectively, whereas co-expression of GAPDH and ARA24/Ran increased AR transactivation up to 4.3-fold, indi- for 24 h. Cells were cultured in fresh medium for an additional 24 h. Cells were homogenized, and whole cell fraction (W) was prepared. Whole cell fraction was fractionated into cytosolic (C), nuclear (N), and particulate fractions (P) by differential centrifugation as described under "Experimental Procedures." Proteins (10 g) in each fraction were subjected to SDS-PAGE, followed by Western blot analyses with anti-Myc, anti-GAPDH, anti-TPI (a cytosolic marker), and anti-lamin B1 (a nuclear marker) antibodies. In C: Left panel, PC-3 cells were co-transfected and incubated in the presence of R1881 as described in A. Samples used in the luciferase reporter assay were analyzed by Western blotting with anti-Myc IgG. Right panel, PC-3 cells were co-transfected with pcDNA3.1-AR (0.5 g), pARE2-TATA-Luc (0.5 g), pRL-TK (0.05 g), and either pcDNA3.1-GAPDH-Myc-His (2.0 g) or pcDNA3.1-GAPDH(258 -261A)-Myc-His (2.5 g) for 24 h and further incubated in the presence of R1881. Luciferase activities were determined. Each graph is representative of three independent experiments. Statistically significant differences of RLU between the GAPDH plasmid-and mock plasmid-transfected cells are indicated by *p Ͻ 0.05.
cating that GAPDH and ARA24/Ran have additive effects on AR transactivation (Fig. 9A). Some of the coactivators that bind to the DBD or LBD of AR also affect the biological function of other steroid receptors because of the high degree of sequence homology among DBDs or LBDs of the steroid receptor family. To assess the effects of GAPDH on the transcriptional activities of other steroid receptors, the transcriptional activity of GR or ER␣ was determined in a luciferase reporter assay. COS-7 cells were co-transfected with either GR expression vector and pARE2-TATA-Luc or ER␣ expression vector and p3xERE-TATA-Luc together with GAPDH-Myc, ARA24/Ran, or ARA70N expression vector. GAPDH did not affect ligand-activated GR or ER␣ transactivation, although ARA24/Ran enhanced the transcriptional activity of GR, but not ER␣, in a ligand-dependent manner (Fig. 9, B and C). In contrast, ARA70N enhanced the transcriptional activities of GR and ER␣ in the presence of ligand. When the effects of GAPDH, ARA24/ Ran, or ARA70N on GR or ER␣ transactivation were tested in PC-3 cells, similar results were observed (data not shown). Although ARA70N is a deletion mutant of ARA70, ARA70N was used as a positive control, because it exhibits stronger transcriptional coactivator activity than full-length ARA70. These results indicate that GAPDH has a preferential selectivity for AR in comparison to ARA24/Ran or ARA70N.
Enhancement of Anti-androgen-activated AR(T875A) Transactivation by GAPDH-Mutation of Thr to Ala at position of 877 in AR, resulting in AR(T877A), enables anti-androgens such as hydroxyflutamide (HF) and cyproterone acetate (CPA) to function as AR agonists (17). In contrast, bicalutamide (Casodex) does not have AR agonist activity of wild-type and T877A mutant ARs (26). We examined the effects of GAPDH on the anti-androgen-activated transcriptional activities with the wild-type AR or AR(T877A). AR(T875A) in place of AR(T877A) was used in the present experiments as described under "Experimental Procedures." As shown in Fig. 10, when the effects of the anti-androgens on the AR transactivation were determined, CPA had marginal agonist activity (Ͻ2-fold) on wild-type AR, and HF and CPA had substantial agonist activity on AR(T875A). GAPDH substantially enhanced the  transcriptional activity of CPA-activated AR(T875A), and marginally enhanced the transcriptional activity of HF-activated AR(T875A), whereas GAPDH did not enhance the transcriptional activity of the wild-type AR in the presence of the antiandrogens. In contrast, when ARA70N as a positive control was tested, ARA70N enhanced the transcriptional activity of wildtype AR or AR(T875A) in the presence of HF, Casodex, or CPA. These results indicate that GAPDH activates the AR-signaling pathway when cells expressing AR(T877A) such as LNCaP cells (AR(T875A) in the present study) are treated with anti-androgens such as HF and CPA.

DISCUSSION
The AR is a ligand-activated transcription factor that mediates the biological responses of androgens, especially DHT. The AR not only mediates prostate development, but also serves as a master regulator of primary prostatic cancer growth. AR-mediated transactivation is enhanced by accessory coactivators (e.g. ARA group, p300, and p160 family) (6,7). Most of the coactivators bind to DBD or LBD, which have a high homology among members of the nuclear receptor superfamily and serve as a common positive regulator for the transactivation of multiple nuclear receptors. On the other hand, the NTD is variable among nuclear receptors, and AR contains a characteristic region composed of a highly polymorphic polyQ tract. Therefore, the association of AR-binding proteins such as coactivators with a polyQ tract may cause a conformational change that is optimal for the AR transactivation of NTD, thereby specifically enhancing the transactivation of AR. In vitro experiments demonstrate that GAPDH interacts with the  receptors, such as SRC-1 and TRAP220, are expressed at similar levels in normal and tumor prostate tissues (35). Contradictory results have been reported recently concerning the expression level of ARA70 in prostate tumor tissues (35,36). The overexpression of coactivators is predicted to contribute to the progression of diseases related to AR transactivation (e.g. prostate cancer), because coactivators enhance AR transactivation and enable AR to become active at lower concentrations of ligand. Therefore, we suggest that GAPDH as well as ARA24/ Ran and PIAS1 play more critical roles in tumorigenesis and progression of androgen-dependent prostate cancer cells than general coactivators for nuclear receptors (e.g. SRC-1 and ARA70).
Androgen deprivation by surgical or chemical castration results in a dramatic regression of prostate tumor growth, because prostate cancer cells are androgen-dependent at early stages. However, most tumors gradually become resistant to androgen deprivation or anti-androgens, and eventually the disease relapses. Although the recurrent cancer is androgenindependent, the mechanisms by which prostate tumors shift from being androgen-dependent to androgen-independent remain unclear. Some of the mechanisms may include mutations in the AR gene that allows the AR to respond to antiandrogens and involve aberrant expression of coactivators that enhance the transcriptional activity of AR even in the presence of low levels of androgens. In recurrent prostate cancer tissue during androgen deprivation therapy, androgens are present at sufficient levels (mean testosterone levels: 2.78 nM; mean DHT levels: 1.45 nM) to activate transcriptional activity of AR (37). Furthermore, an AR mutation, T877A, which is present in LNCaP cells, permits transcriptional responses to anti-androgens (e.g. HF and CPA), whereas wild-type AR is insensitive to anti-androgens. Our study demonstrates that overexpression of GAPDH stimulates AR transactivation in prostate cancer cells even at low concentrations of R1881 (0.05 nM Ͼ) and that GAPDH enables AR antagonists such as HF and CPA to behave as strong agonists for AR(T877A). Thus, increased expression of GAPDH in prostate cancer may be one of the mechanisms by which anti-androgens such as HF and CPA fail to suppress tumor growth and instead promote tumor growth.