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Protein arginine methyltransferase 10 is required for androgen-dependent proliferation of LNCaP prostate cancer cells a
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Naoki Harada , Toshiki Takagi , Yoshihisa Nakano , Ryoichi Yamaji & Hiroshi Inui a
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan b
Osaka Women’s Junior College, Fujiidera, Japan Published online: 23 Mar 2015.
Click for updates To cite this article: Naoki Harada, Toshiki Takagi, Yoshihisa Nakano, Ryoichi Yamaji & Hiroshi Inui (2015): Protein arginine methyltransferase 10 is required for androgen-dependent proliferation of LNCaP prostate cancer cells, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1025035 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1025035
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Bioscience, Biotechnology, and Biochemistry, 2015
Protein arginine methyltransferase 10 is required for androgen-dependent proliferation of LNCaP prostate cancer cells Naoki Harada1,*, Toshiki Takagi1, Yoshihisa Nakano2, Ryoichi Yamaji1 and Hiroshi Inui1 1
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan; 2Osaka Women’s Junior College, Fujiidera, Japan Received January 7, 2015; accepted February 20, 2015
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http://dx.doi.org/10.1080/09168451.2015.1025035
Androgen receptor (AR) signaling is the master regulator of prostate cell growth. Here, to better understand AR signaling, we searched for AR-interacting proteins by yeast two-hybrid screening and identified protein arginine methyltransferase 10 (PRMT10) as one of the interacting proteins. PRMT10 was highly expressed in reproductive tissues, such as prostate. Immunostaining showed that PRMT10 was expressed in the nucleus of both epithelia and stroma of rat prostate. In human prostate cancer LNCaP cells, PRMT10 co-immunoprecipitated with AR in both the presence and absence of dihydrotestosterone (DHT). Knockdown of PRMT10 by siRNA decreased DHT-dependent LNCaP cell growth and induction of prostate-specific antigen, an AR-target gene, without apparent loss of AR. DHT decreased PRMT10 at both the mRNA and protein levels. The decrease in PRMT10 was canceled by knockdown of AR or an AR antagonist. These results indicate that PRMT10 plays an important role in androgen-dependent proliferation of prostate cancer cells. Key words:
androgen receptor; protein arginine methyltransferase 10; prostate cancer; LNCaP cell
Androgen receptor (AR) is a member of the steroidal hormone receptor superfamily of ligand-activated transcription factors and is composed of an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD).1–3) The male hormones testosterone and dihydrotestosterone (DHT) serve as endogenous ligands for AR. AR forms a protein complex with chaperone proteins (e.g. HSP90 and HSP70) under ligand-free conditions. Ligand-bound AR accumulates in the nucleus and regulates the expression of target genes by recruitment of co-activators.3,4) AR has a relatively strong activation function-1 region in the NTD and a relatively weak activation function-2 region in the LBD.3,5) The
NTD of AR shares little sequence similarity with other steroidal hormone receptors. Although the NTD of AR is considered to have unique characteristics for controlling its transactivation, little is known about co-activators that act by binding to the NTD of AR. AR plays a crucial role in the development and maintenance of the prostate gland. Prostate cancer is the most diagnosed malignant carcinoma in the United States,6) and most prostate cancers are adenocarcinomas derived from epithelial cells that highly express AR.7) Thus, understanding how AR stimulates the proliferation of prostate epithelial cells might shed some light on the development of prostate cancer. Identifying and characterizing genes that are up- or down-regulated by AR are essential for understanding androgen signaling. Prostate-specific antigen (PSA) is used as a diagnostic marker of prostate cancer and its expression is tightly controlled by AR.8) In the present study, to better understand AR signaling, we searched for AR-interacting proteins by yeast two-hybrid screening. We identified protein arginine methyltransferase 10 (PRMT10) as a novel AR-interacting partner and demonstrate that it is abundantly expressed in prostate. Furthermore, we show that PRMT10 is required for the proliferation of AR-dependent prostate cancer LNCaP cells.
Materials and methods Yeast two-hybrid assay. Proteins that bind to the NTD of AR were screened with the Matchmaker 3 yeast two-hybrid system (Clontech, Palo Alto, CA, USA) to screen proteins that bind to the NTD of AR. The amino acid number of AR is based on the GenBank Accession No. AAA51729. Although GAL4DBD is generally fused to the N-terminal side of the bait protein, we designed a C-terminal GAL4DBDfused bait protein. To construct a C-terminus GAL4DBD-fused AR (amino acids 1–98) expression vector, two of the three Hind III sites of pGBKT7 vector (nt 1606 and nt 6544) were sequentially mutated by directed mutagenesis using sense primer 5′-CTACCGG-
*Corresponding author. Email: [email protected] Abbreviations: AD, activation domain; AR, androgen receptor; DBD, DNA-binding domain; DHT, dihydrotestosterone; LBD, ligand-binding domain; NTD, N-terminal domain; PRMT, protein arginine methyltransferase; PSA, prostate-specific antigen. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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CATGCAACGTTGGCGTAATCATG-3′ and antisense primer 5′-CATGATTACGCCAACGTTGCATGCCGGTAG-3′ for mutation of the nt 1606 site and sense primer 5′-GAGCCCCGAAAGTCTACATTTTATGTTAGC-3′ and antisense primer 5′-GCTAACATAAAATGTAGACTTTCGGGGCTC-3′ for mutation of the nt 6544 site. A cDNA encoding AR(1–98) was inserted between the third Hind III site (nt 738), which is located immediately upstream of encoding region of GAL4DBD, and the EcoR I site, which is located at the multiple cloning site. Then, GAL4DBD cDNA was amplified and reinserted into the BamH I sites of the multiple cloning site. The resulting vector expresses AR(1–98)-GAL4DBD as a bait protein in yeast. Yeast AH109 that had been transformed by the bait vector was fused to yeast Y187 which had been pretransformed with normalized Matchmaker human universal cDNA libraries (Clontech). The transformants (~1 × 106 clones) were grown on triple or quadruple dropout selective medium containing 5 mM 3-amino-1,2,4-triazole. Prey plasmids were isolated from two-hybrid positive clones and sequenced. For growth and β-galactosidase assays, both bait and prey vectors were retransformed into AH109 and Y187, respectively, and the yeast was grown on selective medium. β-Galactosidase activities were determined according to manufacturer’s instrument using o-nitrophenyl-β-Dgalactopyranoside as a substrate. The absorbances at 405 and 595 nm were measured using microplate reader (Bio-Rad, Hercules, CA, USA). The number of units of β-galactosidase activity was calculated as 1000 × OD405/(OD595 × t × v). Cell culture. Human androgen-sensitive and -insensitive prostate cancer cells, LNCaP and PC-3 cells, respectively, were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Dextran-coated charcoal-treated fetal bovine serum and phenol red-free RPMI1640 medium were used for preparation of steroid-free medium.9) Cells were maintained at 37 °C in a 5% CO2/95% air atmosphere at 98% humidity unless otherwise indicated. Modified mammalian two-hybrid assay. A cDNA fragment containing PRMT10(796–845) (GenBank: NM_138364) was transferred from pACT2-PRMT10 (796–845) to pBIND vector (Clontech). AR expression vector, pcDNA3.1-AR, was described previously.9) PC-3 cells that had been cultured in a 48-well plate were transiently transfected with each 0.1 μg of pG5Luc, pBIND-PRMT10(796–845), and pcDNA3.1-AR using 0.5 μL of HilyMax reagent (Dojindo, Kumamoto, Japan). The amount of DNA was kept constant by the addition of pBIND or pcDNA3.1-Myc-His empty vector. Then, cells were incubated in the presence or absence of 10 nM DHT for 24 h. Luciferase reporter assay was performed as described previously.9) siRNA. LNCaP cells that had been cultured in steroid-free medium were transiently transfected with 10
nM of control siRNA (Mission_Negative control SIC-001, Sigma-Aldrich, St. Louis, MO, USA), human PRMT10 siRNA (siPRMT10#1 sense: 5′-CUUUGUAGUGCCCUCGCUA(dTdT)-3′ or siPRMT10#2 sense: 5′-GUGUAUGCCUGUGAGUUAU(dTdT)-3′), or human AR siRNAs (AR#1 sense: 5′-GACUCAGCUGCCCCAUCCA(dTdT)-3′ or AR#2 sense: using 5′-ACCAUGCAGAAUACAAAU(dTdT)-3′10)) Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) and Opti-MEM (Invitrogen) for 6 h (for Western blotting) or 12 h (for cell growth assay). The medium was then exchanged for fresh steroid-free RPMI 1640 medium. After 24 h of transfection, cells were treated with 10 nM DHT for an additional 24 h for Western blotting. Cell growth assay. LNCaP cells were seeded onto a 48-well plate using steroid-free RPMI1640 medium at 1.0 × 104 cells/well. After 24 h, cells were transfected with 10 nM of siRNA for 12 h. Medium was exchanged for steroid-free fresh RPMI1640 medium, and the cells were incubated in the presence or absence of 10 nM DHT for an additional 96 h. The medium was exchanged with fresh steroid-free medium containing 5% AlamarBlue (Trek Diagnostic Systems, Cleveland, OH, USA), followed by incubation for a further 4 h. Fluorescence was measured with Fluoroscan Ascent FL (excitation at 544 nm and emission at 590 nm, Labsystems, Helsinki, Finland). RT-PCR analyses. Male Wistar rats (10 weeks old) were obtained from Kiwa Laboratory Animals (Wakayama, Japan). Total RNA was extracted with Sepasol-RNA I super (Nacalai Tesque, Kyoto, Japan) from pooled (n = 5) tissue samples of rats or LNCaP cells. After DNase I-treatment, cDNA was synthesized from total RNA. PCR was performed with AmpliTaq Gold 360 (Applied Biosystems, Foster City, CA, USA) for tissue samples or Green Taq polymerase (GenScript, Piscataway, NJ, USA) for cell line samples using the primers listed in Table 1. PCR was performed as follows: denaturation at 94 °C for 1 min followed by 22–35 cycles at 94 °C for 30 s, 55–65 °C for 30 s, and 72 °C for 30 s. After agarose gel electrophoresis, PCR products were visualized using ethidium bromide and UV transilluminator (TOYOBO, Osaka, Japan). Animal experiments were approved by the Animal Care and Use Committee of Osaka Prefecture University in which veterinarian participated and were performed in compliance with its guidelines. Western blotting. LNCaP cells that had been cultured in steroid-free medium were treated for 10 nM DHT for 24 h. The AR antagonist bicalutamide (10 μM) was added 30 min prior to the treatment of DHT. Cells were lysed 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 phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and 1 μg/mL aprotinin) and sonicated. After centrifugation at 20,000 × g, the supernatant was subjected to SDS-PAGE followed by
Role of protein arginine methyltransferase 10 in prostate Table 1.
Primers for amplification of genes by RT-PCR.
Gene
Sequences of primers
rat PRMT1
5′-TTGACTCCTATGCCCACT-3′ 5′-CCACATCCAGCACCACC-3′ 5′-ATTACACCGCCACCGAC-3′ 5′-GAAGTTTCAGAGTCCCATAGC-3′ 5′-ATGCCTACTGCCTACGACCT-3′ 5′-CGGGTGTTCACGCTTCTC-3′ 5′-TATGTGGATTGGGACTGG-3′ 5′-TGAGGGAGGTAGGTGGG-3′ 5′-GCTGCTGTGAAGATTGTGGA-3′ 5′-CCAATCAGCTCTGTGTCAAA-3′ 5′-TTCATGCATGGGAGCATTTA-3′ 5′-ATGGTTCAACCGTTTCTTCG-3′ 5′-GACCAGATGGCAGTCATTCAG-3′ 5′-CAGCTCTCTTGCAATAGGCTG-3′ 5′-TTGCTGACAGGATGCAGAAG-3′ 5′-GTACTTGCGCTCAGGAGGAG-3′ 5′-GTGGAACGCTGGCACTTTAT-3′ 5′-TGCTTCCATCTTGTTTGCTG-3′ 5′-TGGAGTCCTGTGGCATCCACGAAA-3′ 5′-TGTAACGCAACTAACTAAGTCATAGTCCG-3′
rat PRMT2 rat PRMT4 rat PRMT6 rat PRMT7 rat PRMT10 rat AR rat β-actin human PRMT10 human β-actin
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Western blotting with anti-PRMT10 (Abgent, San Diego, CA, USA), anti-AR (N20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-α-tubulin (DM1A, Santa Cruz Biotechnology), or anti-PSA (C-19, Santa Cruz Biotechnology) antibodies. After immunoreaction with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad), the immunoreactive bands were developed as described previously.11) The band intensities were quantified using Image J (ver. 1.48, National Institutes of Health, Bethesda, MD, USA) and normalized to that of α-tubulin. Immunoprecipitation analysis. For expression of C-terminal 6xHis-tagged amino acid 1–293 of AR, AR (1–293)-His, PCR was performed using sense primer (5′-GCATATGGAAGTGCAGTTAGGGCTGG-3′) and antisense primer (5′-GGATCCTAATGATGATGATGATGATGTAGCAGAGAACCTTTGCATTCGG-3′), and pcDNA3.1-AR9) as a template, and the PCR product was inserted into Nde I and BamHI sites of pET30a(+) vector. His-HA-PRMT10(796–845) expression vector was constructed by inserting oligonucleotides encoding HA-tag and cDNA encoding PRMT10(796–845) into pET30 b(+) vector. These expression vectors were transformed into Escherichia coli BL21 (DE3) and the recombinant proteins were expressed by stimulating AR(1–293)-His isopropyl-β-D-thiogalactopyranoside. and His-HA-PRMT10(796–845) were affinity purified using Ni-Sepharose 6 Fast Flow resin (GE Healthcare, Piscataway, NJ, USA), followed by dialysis against phosphate-buffered saline (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4, pH7.4) containing 10% glycerol. Immunoprecipitation analysis was performed by incubating each 1 μg of AR (1–293)-His, His-HA-PRMT10(796–845), and protein G-Sepharose (GE Healthcare) with 1 μg control rabbit IgG or anti-AR(N20) IgG in IP buffer. After washing the resin, the proteins bound to the resin were analyzed by Western blotting with anti-HA (3F10, Roche
Anneal temp (°C)
Cycles
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58
35
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60
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Applied Science, Indianapolis, IN, USA) and anti-AR antibodies. LNCaP cells that had been grown on a 100 mm dish with steroid-free medium were incubated in the presence of 10 nM DHT for 3 h. Immunoprecipitation was performed as described previously.12) Briefly, cells were lysed in IP buffer on ice. Cell lysates were incubated with 1 μg of control rabbit IgG or anti-AR IgG, followed by addition of protein G-Sepharose. Proteins bound to the resin were analyzed by Western blotting using anti-AR or anti-PRMT10 (Betyl Laboratories, Montgomery, TX, USA) antibodies. Immunohistochemistry. Immunohistochemistry was performed as described previously.10) Briefly, prostate tissue of male Wistar rat (8 weeks old, Kiwa Laboratory Animals, n = 3) was fixed with 10% neutralized formaldehyde (Wako Pure Chemical Industries, Osaka, Japan) for 24 h at 4 °C. The prostate tissue was embedded in paraffin (TissueTek, Sakura Finetek Japan, Tokyo, Japan) and sliced into microtome sections at 4-μm thick, followed by attachment to slide glasses (S9901, Matsunami Glass, Osaka, Japan). The sections were dewaxed in Tissue-Clear (Sakura Finetek Japan), incubated in phosphate-buffered saline containing 5% H2O2 to inactivate endogenous peroxidase, heated by microwave exposure at 600 W for 10 min in 10 mM citrate buffer (pH6.0) for antigen retrieval, blocked by incubating with phosphate-buffered saline containing 6% skim milk, incubated with anti-PRMT10 (1/500, HPA036844, Sigma-Aldrich) or anti-AR (1/1000, N-20) antibody overnight at 4 °C, reacted with Histofine Simple Stain rat MAX-PO (Multi) (Nichirei, Tokyo, Japan), stained with a 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, USA), counterstained with hematoxylin, coverslipped with EUKIT (As One, Osaka, Japan), and observed using fluorescence microscopy (BZ9000, Keyence, Osaka, Japan).
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Statistical analysis. Statistical analysis was performed using JMP statistical software (version 8.01, SAS Institute, Cary, NC). Data were subjected to oneor two-way analysis of variance with Tukey’s post hoc testing. The threshold for statistical significance was set at p < 0.05.
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Results Identification of PRMT10 as an AR-interacting protein in yeast AR-interacting proteins were identified by yeast twohybrid screening. We used AR(1–98)-GAL4DBD, which consists of amino acids 1–98 of AR at the N-terminus of GAL4DBD as bait because this region is needed for the activation of AR.5) As a result of screening, we obtained PRMT10 (amino acids region 796–845) as a candidate for an AR-binding partner by sequencing of prey plasmid. Selective interaction between the bait and the prey protein was confirmed by β-galactosidase assay using freshly transformed yeast Y187. An increase in β-galactosidase activity, which indicates an interaction of the bait protein and prey proteins, was observed when both AR(1–98)GAL4DBD and GAL4 activation domain (AD)PRMT10(796–845) were expressed in yeast Y187 (Fig. 1A). Similarly, yeast AH109 grew on a plate with quadruple dropout medium when AR(1–98)GAL4DBD and GAL4AD-PRMT10(796–845) were expressed together (Fig. 1B). The association between both purified AR(1–293)-His and His-HA-PRMT10 (796–845) was assessed by immunoprecipitation. AntiAR IgG, but not control IgG, co-immunoprecipitated AR(1–293)-His with His-HA-PRMT10(796–845) (Fig. 1C). These results suggest that AR directly interacts with PRMT10 in vitro. Tissue distribution of PRMT10 PRMT10 was highly expressed in reproductive tissues (e.g. prostate, epididymis, and testis), brain (e.g. cerebrum and cerebellum), and adrenal gland, which closely resembled to the tissue distribution of AR (Fig. 2A). On the other hand, PRMT1, PRMT2, PRMT4, PRMT6, and PRMT7 were also preferentially expressed in brain and reproductive tissues. The doublet bands of PRMT4 are derived from splicing variants.13) In rat prostate, PRMT10 was highly expressed in the nucleus of both epithelial and stromal cells, whereas AR was predominantly expressed in the nucleus of epithelial cells (Fig. 2B). These results indicate that PRMT10 and AR are abundantly expressed in prostate epithelial cells. Interaction of AR and PRMT10 in LNCaP prostate cells In LNCaP prostate cancer cells, anti-AR IgG, but not control IgG, co-immunoprecipitated AR together with PRMT10 both in the presence and absence of DHT (Fig. 3A), indicating that PRMT10 and AR interacted with each other. Subsequently, we used a modified mammalian two-hybrid assay in which a steroidal hormone receptor ligand-dependently transactivates a
Fig. 1. Interaction between AR and PRMT10 in yeast. Notes: Bait protein (GAL4DBD-p53 or AR(1–98)-GAL4DBD) and prey protein (GAL4AD-PRMT10(796–845), GAL4AD-large Tantigen, or GAL4AD) were co-expressed in yeast. (A) β-Galactosidase assay of yeast Y187 strain. The graph is the representative of three independent experiments with three replicates. (B) Growth assay of yeast AH109 strain using plates with selective dropout medium. The photographs are representative of three independent experiments. (C) Immunoprecipitation of AR(1–293)-His and His-HA-PRMT10 (796–845) in vitro. Data are representative of three independent experiments.
luciferase reporter gene when the receptor binds to the bait. When GAL4DBD-PRMT10(796–845) was used as bait, the luciferase reporter was markedly activated by wild-type AR in a DHT-dependent manner (Fig. 3B).
Role of PRMT10 in AR-dependent proliferation in LNCaP cells Each of the siRNAs against PRMT10 effectively knocked down the expression of PRMT10 in LNCaP cells (Fig. 4A). Knockdown of PRMT10 suppressed DHT-stimulated LNCaP cell growth (Fig. 4B) and attenuated the DHT-induced expression of PSA without apparent loss of AR (Fig. 4C). These results suggest that PRMT10 regulates prostate cancer cell growth by regulating the transactivation of AR.
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Fig. 2. Tissue distribution and prostate localization of PRMT10. Notes: (A) mRNA expressions of PRMT10 in tissues of Wistar male rats. The PCR products were analyzed by agarose gel electrophoresis followed by visualization with a UV transilluminator. Data are representative of two independent experiments with similar results. (B) A prostate section of a Wistar male rat stained with anti-PRMT10 antibodies (left panel) or anti-AR antibodies (right panel). The stainings of these sections are representative of sections from three rats.
Regulation of PRMT10 expression by AR DHT suppressed PRMT10 protein in a dose-dependent manner, and the decrease was inhibited by the androgen antagonist bicalutamide in LNCaP cells (Fig. 5A). Similarly, the reduction of PRMT10 by DHT was observed at the mRNA level, and the suppression of PRMT10 mRNA was also reversed by bicalutamide (Fig. 5B). Moreover, DHT did not decrease PRMT10 when AR was knocked down with siRNAs against AR (Fig. 5C). These results indicate that the expression of PRMT10 was suppressed by DHT in an AR-dependent manner.
Discussion Androgen signaling is the master regulator of proliferation of both normal and cancerous prostate epithelial
cells. Therefore, understanding the mechanism underlying the activation of AR is crucial for understanding the development of cancerous and noncancerous prostate epithelial cells. The PRMT family consists of 11 different proteins.14) PRMT1–9 possess methyltransferase activity that catalyzes the methylation of arginine residues in target proteins using S-adenosylmethionine as a substrate. Although PRMT10 and 11 are identified as homologs of PRMT7 and PRMT9, respectively, their physiological roles and functions as methyltransferases are unclear.14,15) Here, we showed that PRMT10 is abundantly expressed in the prostate and is needed for androgen-dependent proliferation of prostate cancer cells. PRMT10 interacted with AR, and knockdown of PRMT10 attenuated DHT-dependent PSA expression and LNCaP cell growth. On the other hand,
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Fig. 3. Interaction of PRMT10 with AR in prostate cancer cells. Notes: (A) Binding of PRMT10 to AR in androgen-sensitive LNCaP cells. The cells were incubated with steroid-free medium and then stimulated with 10 nM DHT for 3 h. The cell lysate was immunoprecipitated with control or anti-AR antibodies. Proteins bound to the resin were analyzed by Western blotting with anti-PRMT10 or anti-AR antibodies. Each graph is the representative of three independent experiments. (B) Binding of steroidal hormone receptors to PRMT10 in androgen-insensitive PC-3 cells. The cells were transiently transfected with pG5-Luc and pBIND-PRMT10(796–845) or pBIND with pcDNA3.1-AR or pcDNA3.1 (mock). Cells were incubated with 10 nM DHT for 24 h. Luciferase activity in the absence of ligand was set to 1. Fold increase by ligand is indicated above the bar. The graph is the representative of three independent experiments. Data are expressed as means ± SD (n = 4). Different letters indicate statistical differences (p < 0.05).
knockdown of PRMT10 did not affect the growth of AR-negative prostate cancer DU145 (data not shown). These results indicate that PRMT10 is required for AR-dependent prostate cancer cell growth. Five other PRMT family proteins are considered to have been associated with AR function.16–20) PRMT1 is involved in the transactivation of AR,16) although it is unclear whether it directly interacts with AR. PRMT2 directly binds to AR (amino acids 325–919) and enhances AR transactivation.17) PRMT4/CARM1 regulates AR activity by association with a family of p160 co-activators (e.g. SRC-1 and GRIP1) and CBP/p300.18,19) Recent report shows that PRMT2, PRMT6, and PRMT7 form protein complexes with AR.20) Moreover, PRMT6 bound to the LBD of AR and is responsible for methylation of arginine residues of AR (R210, R212, R629, R787, and R789).20) Although PRMT10 was named after its homology to PRMT7, preliminary data cast doubt on its capacity to act as a methyltransferase.15) These PRMTs preferentially expressed in brain and reproductive tissues. In the present study, we found that PRMT10 bound to the NTD of AR. Although it is unclear whether other PRMTs bind to this region, they could not compensate for the loss of PRMT10 because
Fig. 4. Effects of PRMT10 knockdown on PRMT10 mRNA (A), cell growth (B), and expressions of PSA and AR proteins (C) in LNCaP cells. Notes: The cells were cultured in the steroid-free medium and transiently transfected with 10 nM control or PRMT10 siRNAs. (A) Cells were incubated in the presence or absence of DHT for 12 h and lysed. mRNA expression was measured by RT-PCR. (B) Cells were incubated in the presence or absence of 10 nM DHT for 96 h. Cell viability was determined using AlamarBlue dye. Data are expressed as means ± SD (n = 3). Fold increase by DHT is indicated above the bar. Different letters mean statistical differences (p < 0.05). (C) Cells were incubated in the presence or absence of DHT for 24 h. Cell lysates were analyzed by Western blotting. Expression levels of PSA and AR are normalized to that of α-tubulin and are shown below the panels as relative values. Data are representative of three independent experiments.
knockdown of PRMT10 significantly decreased PSA expression and androgen-dependent LNCaP cell growth. These results indicate that PRMT10 is essential for AR function. The tissue distribution of PRMT10 was similar to that of AR. Moreover, expression of PRMT10 was negatively regulated by AR at the mRNA level. According to the Genomatix software (https://www.genomatix.de),
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Author contribution N.H., T.T., R.Y., and H.I. designed the study and discussed the results. T.T. and N.H. performed the experiments. N.H., T.T., and Y.N. analyzed the data. N.H. wrote the manuscript with the help of all the other authors. All authors reviewed the manuscript.
Disclosure statement The authors have no conflict of interest.
Funding
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This work was supported by Grants-in-Aid for scientific research from the Japan Society for the Promotion of Science [grant number 25450176] to N.H.; Agricultural Chemical Research Foundation [grant number 1-308] to N.H.
References
Fig. 5. Regulation of PRMT10 expression in LNCaP cells. Notes: (A,B) Effects of DHT on the expression of PRMT10 protein (A) and the mRNA (B) levels. LNCaP cells that had been cultured in steroid-free medium were incubated with DHT (0.1, 1, or 10 nM) and/or bicalutamide (10 μM) for 24 h (for protein analysis) or for 12 h (for mRNA analysis). (C) Involvement of AR in PRMT10 expression. LNCaP cells were transiently transfected with 10 nM control or AR siRNAs. Cells were incubated in the presence or absence of 10 nM DHT for 24 h, and cell lysates were analyzed by Western blotting. Expression levels of PRMT10 and AR are normalized to that of α-tubulin and are shown below the panels as relative values. Data are representative of three independent experiments.
there are six putative androgen-response elements within 5 kb upstream of the translational start site of human PRMT10. However, it remains unclear whether AR directly binds to the elements and suppresses PRMT expression. Ligand-activated AR mainly acts as a positive regulator for the transcription of its target genes. However, AR acts as a negative regulator of some target genes.21) The expressions of Tip60 and CBP, both of which acetylate AR and enhance AR transactivation, are also suppressed by AR signaling.22,23) Our results strengthen the notion that prostate cells have a negative-feedback mechanism in which AR transcription is controlled by modulating the expression of AR co-regulators. The present results show that PRMT10 is abundantly expressed in the prostate and contributes to AR-dependent prostate cancer cell growth. PRMT10 expression was suppressed by AR signaling, suggesting the existence of a negative-feedback mechanism for controlling AR transcription. Our results indicate that PRMT10 has an important role in controlling AR function.
[1] Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr. Rev. 2004;25:276–308. [2] Chang CS, Kokontis J, Liao ST. Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science. 1988;240:324–326. [3] Shen HC, Coetzee GA. The androgen receptor: unlocking the secrets of its unique transactivation domain. Vitam. Horm. 2005;71:301–319. [4] Heinlein CA, Chang C. Androgen receptor (AR) coregulators: an overview. Endocr. Rev. 2002;23:175–200. [5] Jenster G, van der Korput HA, Trapman J, Brinkmann AO. Identification of two transcription activation units in the N-terminal domain of the human androgen receptor. J. Biol. Chem. 1995;270:7341–7346. [6] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J. Clin. 2012;62:10–29. [7] Prins GS, Birch L. The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology. 1995;136:1303–1314. [8] Oesterling JE. Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. J. Urol. 1991;145:907–923. [9] Harada N, Yasunaga R, Higashimura Y, Yamaji R, Fujimoto K, Moss J, Inui H, Nakano Y. Glyceraldehyde-3-phosphate dehydrogenase enhances transcriptional activity of androgen receptor in prostate cancer cells. J. Biol. Chem. 2007;282: 22651–22661. [10] Harada N, Katsuki T, Takahashi Y, Masuda T, Yoshinaga M, Adachi T, Izawa T, Kuwamura M, Nakano Y, Yamaji R, Inui H. Androgen receptor silences thioredoxin-interacting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic β-cells. J. Cell. Biochem. 2015. doi:10.1002/ jcb.25054. [11] Harada N, Inoue K, Yamaji R, Nakano Y, Inui H. Androgen deprivation causes truncation of the C-terminal region of androgen receptor in human prostate cancer LNCaP cells. Cancer Sci. 2012;103:1022–1027. [12] Harada N, Yokoyama T, Yamaji R, Nakano Y, Inui H. RanBP10 acts as a novel coactivator for the androgen receptor. Biochem. Biophys. Res. Commun. 2008;368:121–125. [13] Wang L, Charoensuksai P, Watson NJ, Wang X, Zhao Z, Coriano CG, Kerr LR, Xu W. CARM1 automethylation is controlled at the level of alternative splicing. Nucleic Acids Res. 2013;41:6870–6880. [14] Jahan S, Davie JR. Protein arginine methyltransferases (PRMTs): role in chromatin organization. Adv. Biol. Regul. 2014;57: 173–184.
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[15] Krause CD, Yang ZH, Kim YS, Lee JH, Cook JR, Pestka S. Protein arginine methyltransferases: evolution and assessment of their pharmacological and therapeutic potential. Pharmacol. Ther. 2007;113:50–87. [16] Bissinger EM, Heinke R, Spannhoff A, Eberlin A, Metzger E, Cura V, Hassenboehler P, Cavarelli J, Schule R, Bedford MT, Sippl W, Jung M. Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1. Bioorg. Med. Chem. 2011;19:3717–3731. [17] Meyer R, Wolf SS, Obendorf M. PRMT2, a member of the protein arginine methyltransferase family, is a coactivator of the androgen receptor. J. Steroid Biochem. Mol. Biol. 2007;107:1–14. [18] Koh SS, Chen D, Lee YH, Stallcup MR. Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J. Biol. Chem. 2001;276:1089–1098. [19] Koh SS, Li H, Lee YH, Widelitz RB, Chuong CM, Stallcup MR. Synergistic coactivator function by coactivator-associated arginine methyltransferase (CARM) 1 and beta-catenin with two different classes of DNA-binding transcriptional activators. J. Biol. Chem. 2002;277:26031–26035.
[20] Scaramuzzino C, Casci I, Parodi S, Lievens PM, Polanco MJ, Milioto C, Chivet M, Monaghan J, Mishra A, Badders N, Aggarwal T, Grunseich C, Sambataro F, Basso M, Fackelmayer FO, Taylor JP, Pandey UB, Pennuto M. Protein arginine methyltransferase 6 enhances polyglutamine-expanded androgen receptor function and toxicity in spinal and bulbar muscular atrophy. Neuron. 2015;85:88–100. [21] Jiang F, Wang Z. Identification of androgen-responsive genes in the rat ventral prostate by complementary deoxyribonucleic acid subtraction and microarray. Endocrinology. 2003;144: 1257–1265. [22] Halkidou K, Gnanapragasam VJ, Mehta PB, Logan IR, Brady ME, Cook S, Leung HY, Neal DE, Robson CN. Expression of Tip60, an androgen receptor coactivator, and its role in prostate cancer development. Oncogene. 2003;22:2466–2477. [23] Comuzzi B, Nemes C, Schmidt S, Jasarevic Z, Lodde M, Pycha A, Bartsch G, Offner F, Culig Z, Hobisch A. The androgen receptor co-activator CBP is up-regulated following androgen withdrawal and is highly expressed in advanced prostate cancer. J. Pathol. 2004;204:159–166.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Protein arginine methyltransferase 10 is required for androgen-dependent proliferation of LNCaP prostate cancer cells a
a
b
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a
Naoki Harada , Toshiki Takagi , Yoshihisa Nakano , Ryoichi Yamaji & Hiroshi Inui a
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan b
Osaka Women’s Junior College, Fujiidera, Japan Published online: 23 Mar 2015.
Click for updates To cite this article: Naoki Harada, Toshiki Takagi, Yoshihisa Nakano, Ryoichi Yamaji & Hiroshi Inui (2015): Protein arginine methyltransferase 10 is required for androgen-dependent proliferation of LNCaP prostate cancer cells, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1025035 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1025035
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Bioscience, Biotechnology, and Biochemistry, 2015
Protein arginine methyltransferase 10 is required for androgen-dependent proliferation of LNCaP prostate cancer cells Naoki Harada1,*, Toshiki Takagi1, Yoshihisa Nakano2, Ryoichi Yamaji1 and Hiroshi Inui1 1
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan; 2Osaka Women’s Junior College, Fujiidera, Japan Received January 7, 2015; accepted February 20, 2015
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http://dx.doi.org/10.1080/09168451.2015.1025035
Androgen receptor (AR) signaling is the master regulator of prostate cell growth. Here, to better understand AR signaling, we searched for AR-interacting proteins by yeast two-hybrid screening and identified protein arginine methyltransferase 10 (PRMT10) as one of the interacting proteins. PRMT10 was highly expressed in reproductive tissues, such as prostate. Immunostaining showed that PRMT10 was expressed in the nucleus of both epithelia and stroma of rat prostate. In human prostate cancer LNCaP cells, PRMT10 co-immunoprecipitated with AR in both the presence and absence of dihydrotestosterone (DHT). Knockdown of PRMT10 by siRNA decreased DHT-dependent LNCaP cell growth and induction of prostate-specific antigen, an AR-target gene, without apparent loss of AR. DHT decreased PRMT10 at both the mRNA and protein levels. The decrease in PRMT10 was canceled by knockdown of AR or an AR antagonist. These results indicate that PRMT10 plays an important role in androgen-dependent proliferation of prostate cancer cells. Key words:
androgen receptor; protein arginine methyltransferase 10; prostate cancer; LNCaP cell
Androgen receptor (AR) is a member of the steroidal hormone receptor superfamily of ligand-activated transcription factors and is composed of an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region, and a C-terminal ligand-binding domain (LBD).1–3) The male hormones testosterone and dihydrotestosterone (DHT) serve as endogenous ligands for AR. AR forms a protein complex with chaperone proteins (e.g. HSP90 and HSP70) under ligand-free conditions. Ligand-bound AR accumulates in the nucleus and regulates the expression of target genes by recruitment of co-activators.3,4) AR has a relatively strong activation function-1 region in the NTD and a relatively weak activation function-2 region in the LBD.3,5) The
NTD of AR shares little sequence similarity with other steroidal hormone receptors. Although the NTD of AR is considered to have unique characteristics for controlling its transactivation, little is known about co-activators that act by binding to the NTD of AR. AR plays a crucial role in the development and maintenance of the prostate gland. Prostate cancer is the most diagnosed malignant carcinoma in the United States,6) and most prostate cancers are adenocarcinomas derived from epithelial cells that highly express AR.7) Thus, understanding how AR stimulates the proliferation of prostate epithelial cells might shed some light on the development of prostate cancer. Identifying and characterizing genes that are up- or down-regulated by AR are essential for understanding androgen signaling. Prostate-specific antigen (PSA) is used as a diagnostic marker of prostate cancer and its expression is tightly controlled by AR.8) In the present study, to better understand AR signaling, we searched for AR-interacting proteins by yeast two-hybrid screening. We identified protein arginine methyltransferase 10 (PRMT10) as a novel AR-interacting partner and demonstrate that it is abundantly expressed in prostate. Furthermore, we show that PRMT10 is required for the proliferation of AR-dependent prostate cancer LNCaP cells.
Materials and methods Yeast two-hybrid assay. Proteins that bind to the NTD of AR were screened with the Matchmaker 3 yeast two-hybrid system (Clontech, Palo Alto, CA, USA) to screen proteins that bind to the NTD of AR. The amino acid number of AR is based on the GenBank Accession No. AAA51729. Although GAL4DBD is generally fused to the N-terminal side of the bait protein, we designed a C-terminal GAL4DBDfused bait protein. To construct a C-terminus GAL4DBD-fused AR (amino acids 1–98) expression vector, two of the three Hind III sites of pGBKT7 vector (nt 1606 and nt 6544) were sequentially mutated by directed mutagenesis using sense primer 5′-CTACCGG-
*Corresponding author. Email: [email protected] Abbreviations: AD, activation domain; AR, androgen receptor; DBD, DNA-binding domain; DHT, dihydrotestosterone; LBD, ligand-binding domain; NTD, N-terminal domain; PRMT, protein arginine methyltransferase; PSA, prostate-specific antigen. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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CATGCAACGTTGGCGTAATCATG-3′ and antisense primer 5′-CATGATTACGCCAACGTTGCATGCCGGTAG-3′ for mutation of the nt 1606 site and sense primer 5′-GAGCCCCGAAAGTCTACATTTTATGTTAGC-3′ and antisense primer 5′-GCTAACATAAAATGTAGACTTTCGGGGCTC-3′ for mutation of the nt 6544 site. A cDNA encoding AR(1–98) was inserted between the third Hind III site (nt 738), which is located immediately upstream of encoding region of GAL4DBD, and the EcoR I site, which is located at the multiple cloning site. Then, GAL4DBD cDNA was amplified and reinserted into the BamH I sites of the multiple cloning site. The resulting vector expresses AR(1–98)-GAL4DBD as a bait protein in yeast. Yeast AH109 that had been transformed by the bait vector was fused to yeast Y187 which had been pretransformed with normalized Matchmaker human universal cDNA libraries (Clontech). The transformants (~1 × 106 clones) were grown on triple or quadruple dropout selective medium containing 5 mM 3-amino-1,2,4-triazole. Prey plasmids were isolated from two-hybrid positive clones and sequenced. For growth and β-galactosidase assays, both bait and prey vectors were retransformed into AH109 and Y187, respectively, and the yeast was grown on selective medium. β-Galactosidase activities were determined according to manufacturer’s instrument using o-nitrophenyl-β-Dgalactopyranoside as a substrate. The absorbances at 405 and 595 nm were measured using microplate reader (Bio-Rad, Hercules, CA, USA). The number of units of β-galactosidase activity was calculated as 1000 × OD405/(OD595 × t × v). Cell culture. Human androgen-sensitive and -insensitive prostate cancer cells, LNCaP and PC-3 cells, respectively, were cultured in RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Dextran-coated charcoal-treated fetal bovine serum and phenol red-free RPMI1640 medium were used for preparation of steroid-free medium.9) Cells were maintained at 37 °C in a 5% CO2/95% air atmosphere at 98% humidity unless otherwise indicated. Modified mammalian two-hybrid assay. A cDNA fragment containing PRMT10(796–845) (GenBank: NM_138364) was transferred from pACT2-PRMT10 (796–845) to pBIND vector (Clontech). AR expression vector, pcDNA3.1-AR, was described previously.9) PC-3 cells that had been cultured in a 48-well plate were transiently transfected with each 0.1 μg of pG5Luc, pBIND-PRMT10(796–845), and pcDNA3.1-AR using 0.5 μL of HilyMax reagent (Dojindo, Kumamoto, Japan). The amount of DNA was kept constant by the addition of pBIND or pcDNA3.1-Myc-His empty vector. Then, cells were incubated in the presence or absence of 10 nM DHT for 24 h. Luciferase reporter assay was performed as described previously.9) siRNA. LNCaP cells that had been cultured in steroid-free medium were transiently transfected with 10
nM of control siRNA (Mission_Negative control SIC-001, Sigma-Aldrich, St. Louis, MO, USA), human PRMT10 siRNA (siPRMT10#1 sense: 5′-CUUUGUAGUGCCCUCGCUA(dTdT)-3′ or siPRMT10#2 sense: 5′-GUGUAUGCCUGUGAGUUAU(dTdT)-3′), or human AR siRNAs (AR#1 sense: 5′-GACUCAGCUGCCCCAUCCA(dTdT)-3′ or AR#2 sense: using 5′-ACCAUGCAGAAUACAAAU(dTdT)-3′10)) Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) and Opti-MEM (Invitrogen) for 6 h (for Western blotting) or 12 h (for cell growth assay). The medium was then exchanged for fresh steroid-free RPMI 1640 medium. After 24 h of transfection, cells were treated with 10 nM DHT for an additional 24 h for Western blotting. Cell growth assay. LNCaP cells were seeded onto a 48-well plate using steroid-free RPMI1640 medium at 1.0 × 104 cells/well. After 24 h, cells were transfected with 10 nM of siRNA for 12 h. Medium was exchanged for steroid-free fresh RPMI1640 medium, and the cells were incubated in the presence or absence of 10 nM DHT for an additional 96 h. The medium was exchanged with fresh steroid-free medium containing 5% AlamarBlue (Trek Diagnostic Systems, Cleveland, OH, USA), followed by incubation for a further 4 h. Fluorescence was measured with Fluoroscan Ascent FL (excitation at 544 nm and emission at 590 nm, Labsystems, Helsinki, Finland). RT-PCR analyses. Male Wistar rats (10 weeks old) were obtained from Kiwa Laboratory Animals (Wakayama, Japan). Total RNA was extracted with Sepasol-RNA I super (Nacalai Tesque, Kyoto, Japan) from pooled (n = 5) tissue samples of rats or LNCaP cells. After DNase I-treatment, cDNA was synthesized from total RNA. PCR was performed with AmpliTaq Gold 360 (Applied Biosystems, Foster City, CA, USA) for tissue samples or Green Taq polymerase (GenScript, Piscataway, NJ, USA) for cell line samples using the primers listed in Table 1. PCR was performed as follows: denaturation at 94 °C for 1 min followed by 22–35 cycles at 94 °C for 30 s, 55–65 °C for 30 s, and 72 °C for 30 s. After agarose gel electrophoresis, PCR products were visualized using ethidium bromide and UV transilluminator (TOYOBO, Osaka, Japan). Animal experiments were approved by the Animal Care and Use Committee of Osaka Prefecture University in which veterinarian participated and were performed in compliance with its guidelines. Western blotting. LNCaP cells that had been cultured in steroid-free medium were treated for 10 nM DHT for 24 h. The AR antagonist bicalutamide (10 μM) was added 30 min prior to the treatment of DHT. Cells were lysed 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 phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and 1 μg/mL aprotinin) and sonicated. After centrifugation at 20,000 × g, the supernatant was subjected to SDS-PAGE followed by
Role of protein arginine methyltransferase 10 in prostate Table 1.
Primers for amplification of genes by RT-PCR.
Gene
Sequences of primers
rat PRMT1
5′-TTGACTCCTATGCCCACT-3′ 5′-CCACATCCAGCACCACC-3′ 5′-ATTACACCGCCACCGAC-3′ 5′-GAAGTTTCAGAGTCCCATAGC-3′ 5′-ATGCCTACTGCCTACGACCT-3′ 5′-CGGGTGTTCACGCTTCTC-3′ 5′-TATGTGGATTGGGACTGG-3′ 5′-TGAGGGAGGTAGGTGGG-3′ 5′-GCTGCTGTGAAGATTGTGGA-3′ 5′-CCAATCAGCTCTGTGTCAAA-3′ 5′-TTCATGCATGGGAGCATTTA-3′ 5′-ATGGTTCAACCGTTTCTTCG-3′ 5′-GACCAGATGGCAGTCATTCAG-3′ 5′-CAGCTCTCTTGCAATAGGCTG-3′ 5′-TTGCTGACAGGATGCAGAAG-3′ 5′-GTACTTGCGCTCAGGAGGAG-3′ 5′-GTGGAACGCTGGCACTTTAT-3′ 5′-TGCTTCCATCTTGTTTGCTG-3′ 5′-TGGAGTCCTGTGGCATCCACGAAA-3′ 5′-TGTAACGCAACTAACTAAGTCATAGTCCG-3′
rat PRMT2 rat PRMT4 rat PRMT6 rat PRMT7 rat PRMT10 rat AR rat β-actin human PRMT10 human β-actin
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Western blotting with anti-PRMT10 (Abgent, San Diego, CA, USA), anti-AR (N20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-α-tubulin (DM1A, Santa Cruz Biotechnology), or anti-PSA (C-19, Santa Cruz Biotechnology) antibodies. After immunoreaction with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad), the immunoreactive bands were developed as described previously.11) The band intensities were quantified using Image J (ver. 1.48, National Institutes of Health, Bethesda, MD, USA) and normalized to that of α-tubulin. Immunoprecipitation analysis. For expression of C-terminal 6xHis-tagged amino acid 1–293 of AR, AR (1–293)-His, PCR was performed using sense primer (5′-GCATATGGAAGTGCAGTTAGGGCTGG-3′) and antisense primer (5′-GGATCCTAATGATGATGATGATGATGTAGCAGAGAACCTTTGCATTCGG-3′), and pcDNA3.1-AR9) as a template, and the PCR product was inserted into Nde I and BamHI sites of pET30a(+) vector. His-HA-PRMT10(796–845) expression vector was constructed by inserting oligonucleotides encoding HA-tag and cDNA encoding PRMT10(796–845) into pET30 b(+) vector. These expression vectors were transformed into Escherichia coli BL21 (DE3) and the recombinant proteins were expressed by stimulating AR(1–293)-His isopropyl-β-D-thiogalactopyranoside. and His-HA-PRMT10(796–845) were affinity purified using Ni-Sepharose 6 Fast Flow resin (GE Healthcare, Piscataway, NJ, USA), followed by dialysis against phosphate-buffered saline (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.47 mM KH2PO4, pH7.4) containing 10% glycerol. Immunoprecipitation analysis was performed by incubating each 1 μg of AR (1–293)-His, His-HA-PRMT10(796–845), and protein G-Sepharose (GE Healthcare) with 1 μg control rabbit IgG or anti-AR(N20) IgG in IP buffer. After washing the resin, the proteins bound to the resin were analyzed by Western blotting with anti-HA (3F10, Roche
Anneal temp (°C)
Cycles
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Applied Science, Indianapolis, IN, USA) and anti-AR antibodies. LNCaP cells that had been grown on a 100 mm dish with steroid-free medium were incubated in the presence of 10 nM DHT for 3 h. Immunoprecipitation was performed as described previously.12) Briefly, cells were lysed in IP buffer on ice. Cell lysates were incubated with 1 μg of control rabbit IgG or anti-AR IgG, followed by addition of protein G-Sepharose. Proteins bound to the resin were analyzed by Western blotting using anti-AR or anti-PRMT10 (Betyl Laboratories, Montgomery, TX, USA) antibodies. Immunohistochemistry. Immunohistochemistry was performed as described previously.10) Briefly, prostate tissue of male Wistar rat (8 weeks old, Kiwa Laboratory Animals, n = 3) was fixed with 10% neutralized formaldehyde (Wako Pure Chemical Industries, Osaka, Japan) for 24 h at 4 °C. The prostate tissue was embedded in paraffin (TissueTek, Sakura Finetek Japan, Tokyo, Japan) and sliced into microtome sections at 4-μm thick, followed by attachment to slide glasses (S9901, Matsunami Glass, Osaka, Japan). The sections were dewaxed in Tissue-Clear (Sakura Finetek Japan), incubated in phosphate-buffered saline containing 5% H2O2 to inactivate endogenous peroxidase, heated by microwave exposure at 600 W for 10 min in 10 mM citrate buffer (pH6.0) for antigen retrieval, blocked by incubating with phosphate-buffered saline containing 6% skim milk, incubated with anti-PRMT10 (1/500, HPA036844, Sigma-Aldrich) or anti-AR (1/1000, N-20) antibody overnight at 4 °C, reacted with Histofine Simple Stain rat MAX-PO (Multi) (Nichirei, Tokyo, Japan), stained with a 3,3′-diaminobenzidine substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, USA), counterstained with hematoxylin, coverslipped with EUKIT (As One, Osaka, Japan), and observed using fluorescence microscopy (BZ9000, Keyence, Osaka, Japan).
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Statistical analysis. Statistical analysis was performed using JMP statistical software (version 8.01, SAS Institute, Cary, NC). Data were subjected to oneor two-way analysis of variance with Tukey’s post hoc testing. The threshold for statistical significance was set at p < 0.05.
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Results Identification of PRMT10 as an AR-interacting protein in yeast AR-interacting proteins were identified by yeast twohybrid screening. We used AR(1–98)-GAL4DBD, which consists of amino acids 1–98 of AR at the N-terminus of GAL4DBD as bait because this region is needed for the activation of AR.5) As a result of screening, we obtained PRMT10 (amino acids region 796–845) as a candidate for an AR-binding partner by sequencing of prey plasmid. Selective interaction between the bait and the prey protein was confirmed by β-galactosidase assay using freshly transformed yeast Y187. An increase in β-galactosidase activity, which indicates an interaction of the bait protein and prey proteins, was observed when both AR(1–98)GAL4DBD and GAL4 activation domain (AD)PRMT10(796–845) were expressed in yeast Y187 (Fig. 1A). Similarly, yeast AH109 grew on a plate with quadruple dropout medium when AR(1–98)GAL4DBD and GAL4AD-PRMT10(796–845) were expressed together (Fig. 1B). The association between both purified AR(1–293)-His and His-HA-PRMT10 (796–845) was assessed by immunoprecipitation. AntiAR IgG, but not control IgG, co-immunoprecipitated AR(1–293)-His with His-HA-PRMT10(796–845) (Fig. 1C). These results suggest that AR directly interacts with PRMT10 in vitro. Tissue distribution of PRMT10 PRMT10 was highly expressed in reproductive tissues (e.g. prostate, epididymis, and testis), brain (e.g. cerebrum and cerebellum), and adrenal gland, which closely resembled to the tissue distribution of AR (Fig. 2A). On the other hand, PRMT1, PRMT2, PRMT4, PRMT6, and PRMT7 were also preferentially expressed in brain and reproductive tissues. The doublet bands of PRMT4 are derived from splicing variants.13) In rat prostate, PRMT10 was highly expressed in the nucleus of both epithelial and stromal cells, whereas AR was predominantly expressed in the nucleus of epithelial cells (Fig. 2B). These results indicate that PRMT10 and AR are abundantly expressed in prostate epithelial cells. Interaction of AR and PRMT10 in LNCaP prostate cells In LNCaP prostate cancer cells, anti-AR IgG, but not control IgG, co-immunoprecipitated AR together with PRMT10 both in the presence and absence of DHT (Fig. 3A), indicating that PRMT10 and AR interacted with each other. Subsequently, we used a modified mammalian two-hybrid assay in which a steroidal hormone receptor ligand-dependently transactivates a
Fig. 1. Interaction between AR and PRMT10 in yeast. Notes: Bait protein (GAL4DBD-p53 or AR(1–98)-GAL4DBD) and prey protein (GAL4AD-PRMT10(796–845), GAL4AD-large Tantigen, or GAL4AD) were co-expressed in yeast. (A) β-Galactosidase assay of yeast Y187 strain. The graph is the representative of three independent experiments with three replicates. (B) Growth assay of yeast AH109 strain using plates with selective dropout medium. The photographs are representative of three independent experiments. (C) Immunoprecipitation of AR(1–293)-His and His-HA-PRMT10 (796–845) in vitro. Data are representative of three independent experiments.
luciferase reporter gene when the receptor binds to the bait. When GAL4DBD-PRMT10(796–845) was used as bait, the luciferase reporter was markedly activated by wild-type AR in a DHT-dependent manner (Fig. 3B).
Role of PRMT10 in AR-dependent proliferation in LNCaP cells Each of the siRNAs against PRMT10 effectively knocked down the expression of PRMT10 in LNCaP cells (Fig. 4A). Knockdown of PRMT10 suppressed DHT-stimulated LNCaP cell growth (Fig. 4B) and attenuated the DHT-induced expression of PSA without apparent loss of AR (Fig. 4C). These results suggest that PRMT10 regulates prostate cancer cell growth by regulating the transactivation of AR.
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Fig. 2. Tissue distribution and prostate localization of PRMT10. Notes: (A) mRNA expressions of PRMT10 in tissues of Wistar male rats. The PCR products were analyzed by agarose gel electrophoresis followed by visualization with a UV transilluminator. Data are representative of two independent experiments with similar results. (B) A prostate section of a Wistar male rat stained with anti-PRMT10 antibodies (left panel) or anti-AR antibodies (right panel). The stainings of these sections are representative of sections from three rats.
Regulation of PRMT10 expression by AR DHT suppressed PRMT10 protein in a dose-dependent manner, and the decrease was inhibited by the androgen antagonist bicalutamide in LNCaP cells (Fig. 5A). Similarly, the reduction of PRMT10 by DHT was observed at the mRNA level, and the suppression of PRMT10 mRNA was also reversed by bicalutamide (Fig. 5B). Moreover, DHT did not decrease PRMT10 when AR was knocked down with siRNAs against AR (Fig. 5C). These results indicate that the expression of PRMT10 was suppressed by DHT in an AR-dependent manner.
Discussion Androgen signaling is the master regulator of proliferation of both normal and cancerous prostate epithelial
cells. Therefore, understanding the mechanism underlying the activation of AR is crucial for understanding the development of cancerous and noncancerous prostate epithelial cells. The PRMT family consists of 11 different proteins.14) PRMT1–9 possess methyltransferase activity that catalyzes the methylation of arginine residues in target proteins using S-adenosylmethionine as a substrate. Although PRMT10 and 11 are identified as homologs of PRMT7 and PRMT9, respectively, their physiological roles and functions as methyltransferases are unclear.14,15) Here, we showed that PRMT10 is abundantly expressed in the prostate and is needed for androgen-dependent proliferation of prostate cancer cells. PRMT10 interacted with AR, and knockdown of PRMT10 attenuated DHT-dependent PSA expression and LNCaP cell growth. On the other hand,
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Fig. 3. Interaction of PRMT10 with AR in prostate cancer cells. Notes: (A) Binding of PRMT10 to AR in androgen-sensitive LNCaP cells. The cells were incubated with steroid-free medium and then stimulated with 10 nM DHT for 3 h. The cell lysate was immunoprecipitated with control or anti-AR antibodies. Proteins bound to the resin were analyzed by Western blotting with anti-PRMT10 or anti-AR antibodies. Each graph is the representative of three independent experiments. (B) Binding of steroidal hormone receptors to PRMT10 in androgen-insensitive PC-3 cells. The cells were transiently transfected with pG5-Luc and pBIND-PRMT10(796–845) or pBIND with pcDNA3.1-AR or pcDNA3.1 (mock). Cells were incubated with 10 nM DHT for 24 h. Luciferase activity in the absence of ligand was set to 1. Fold increase by ligand is indicated above the bar. The graph is the representative of three independent experiments. Data are expressed as means ± SD (n = 4). Different letters indicate statistical differences (p < 0.05).
knockdown of PRMT10 did not affect the growth of AR-negative prostate cancer DU145 (data not shown). These results indicate that PRMT10 is required for AR-dependent prostate cancer cell growth. Five other PRMT family proteins are considered to have been associated with AR function.16–20) PRMT1 is involved in the transactivation of AR,16) although it is unclear whether it directly interacts with AR. PRMT2 directly binds to AR (amino acids 325–919) and enhances AR transactivation.17) PRMT4/CARM1 regulates AR activity by association with a family of p160 co-activators (e.g. SRC-1 and GRIP1) and CBP/p300.18,19) Recent report shows that PRMT2, PRMT6, and PRMT7 form protein complexes with AR.20) Moreover, PRMT6 bound to the LBD of AR and is responsible for methylation of arginine residues of AR (R210, R212, R629, R787, and R789).20) Although PRMT10 was named after its homology to PRMT7, preliminary data cast doubt on its capacity to act as a methyltransferase.15) These PRMTs preferentially expressed in brain and reproductive tissues. In the present study, we found that PRMT10 bound to the NTD of AR. Although it is unclear whether other PRMTs bind to this region, they could not compensate for the loss of PRMT10 because
Fig. 4. Effects of PRMT10 knockdown on PRMT10 mRNA (A), cell growth (B), and expressions of PSA and AR proteins (C) in LNCaP cells. Notes: The cells were cultured in the steroid-free medium and transiently transfected with 10 nM control or PRMT10 siRNAs. (A) Cells were incubated in the presence or absence of DHT for 12 h and lysed. mRNA expression was measured by RT-PCR. (B) Cells were incubated in the presence or absence of 10 nM DHT for 96 h. Cell viability was determined using AlamarBlue dye. Data are expressed as means ± SD (n = 3). Fold increase by DHT is indicated above the bar. Different letters mean statistical differences (p < 0.05). (C) Cells were incubated in the presence or absence of DHT for 24 h. Cell lysates were analyzed by Western blotting. Expression levels of PSA and AR are normalized to that of α-tubulin and are shown below the panels as relative values. Data are representative of three independent experiments.
knockdown of PRMT10 significantly decreased PSA expression and androgen-dependent LNCaP cell growth. These results indicate that PRMT10 is essential for AR function. The tissue distribution of PRMT10 was similar to that of AR. Moreover, expression of PRMT10 was negatively regulated by AR at the mRNA level. According to the Genomatix software (https://www.genomatix.de),
Role of protein arginine methyltransferase 10 in prostate
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Author contribution N.H., T.T., R.Y., and H.I. designed the study and discussed the results. T.T. and N.H. performed the experiments. N.H., T.T., and Y.N. analyzed the data. N.H. wrote the manuscript with the help of all the other authors. All authors reviewed the manuscript.
Disclosure statement The authors have no conflict of interest.
Funding
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This work was supported by Grants-in-Aid for scientific research from the Japan Society for the Promotion of Science [grant number 25450176] to N.H.; Agricultural Chemical Research Foundation [grant number 1-308] to N.H.
References
Fig. 5. Regulation of PRMT10 expression in LNCaP cells. Notes: (A,B) Effects of DHT on the expression of PRMT10 protein (A) and the mRNA (B) levels. LNCaP cells that had been cultured in steroid-free medium were incubated with DHT (0.1, 1, or 10 nM) and/or bicalutamide (10 μM) for 24 h (for protein analysis) or for 12 h (for mRNA analysis). (C) Involvement of AR in PRMT10 expression. LNCaP cells were transiently transfected with 10 nM control or AR siRNAs. Cells were incubated in the presence or absence of 10 nM DHT for 24 h, and cell lysates were analyzed by Western blotting. Expression levels of PRMT10 and AR are normalized to that of α-tubulin and are shown below the panels as relative values. Data are representative of three independent experiments.
there are six putative androgen-response elements within 5 kb upstream of the translational start site of human PRMT10. However, it remains unclear whether AR directly binds to the elements and suppresses PRMT expression. Ligand-activated AR mainly acts as a positive regulator for the transcription of its target genes. However, AR acts as a negative regulator of some target genes.21) The expressions of Tip60 and CBP, both of which acetylate AR and enhance AR transactivation, are also suppressed by AR signaling.22,23) Our results strengthen the notion that prostate cells have a negative-feedback mechanism in which AR transcription is controlled by modulating the expression of AR co-regulators. The present results show that PRMT10 is abundantly expressed in the prostate and contributes to AR-dependent prostate cancer cell growth. PRMT10 expression was suppressed by AR signaling, suggesting the existence of a negative-feedback mechanism for controlling AR transcription. Our results indicate that PRMT10 has an important role in controlling AR function.
[1] Heinlein CA, Chang C. Androgen receptor in prostate cancer. Endocr. Rev. 2004;25:276–308. [2] Chang CS, Kokontis J, Liao ST. Molecular cloning of human and rat complementary DNA encoding androgen receptors. Science. 1988;240:324–326. [3] Shen HC, Coetzee GA. The androgen receptor: unlocking the secrets of its unique transactivation domain. Vitam. Horm. 2005;71:301–319. [4] Heinlein CA, Chang C. Androgen receptor (AR) coregulators: an overview. Endocr. Rev. 2002;23:175–200. [5] Jenster G, van der Korput HA, Trapman J, Brinkmann AO. Identification of two transcription activation units in the N-terminal domain of the human androgen receptor. J. Biol. Chem. 1995;270:7341–7346. [6] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J. Clin. 2012;62:10–29. [7] Prins GS, Birch L. The developmental pattern of androgen receptor expression in rat prostate lobes is altered after neonatal exposure to estrogen. Endocrinology. 1995;136:1303–1314. [8] Oesterling JE. Prostate specific antigen: a critical assessment of the most useful tumor marker for adenocarcinoma of the prostate. J. Urol. 1991;145:907–923. [9] Harada N, Yasunaga R, Higashimura Y, Yamaji R, Fujimoto K, Moss J, Inui H, Nakano Y. Glyceraldehyde-3-phosphate dehydrogenase enhances transcriptional activity of androgen receptor in prostate cancer cells. J. Biol. Chem. 2007;282: 22651–22661. [10] Harada N, Katsuki T, Takahashi Y, Masuda T, Yoshinaga M, Adachi T, Izawa T, Kuwamura M, Nakano Y, Yamaji R, Inui H. Androgen receptor silences thioredoxin-interacting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic β-cells. J. Cell. Biochem. 2015. doi:10.1002/ jcb.25054. [11] Harada N, Inoue K, Yamaji R, Nakano Y, Inui H. Androgen deprivation causes truncation of the C-terminal region of androgen receptor in human prostate cancer LNCaP cells. Cancer Sci. 2012;103:1022–1027. [12] Harada N, Yokoyama T, Yamaji R, Nakano Y, Inui H. RanBP10 acts as a novel coactivator for the androgen receptor. Biochem. Biophys. Res. Commun. 2008;368:121–125. [13] Wang L, Charoensuksai P, Watson NJ, Wang X, Zhao Z, Coriano CG, Kerr LR, Xu W. CARM1 automethylation is controlled at the level of alternative splicing. Nucleic Acids Res. 2013;41:6870–6880. [14] Jahan S, Davie JR. Protein arginine methyltransferases (PRMTs): role in chromatin organization. Adv. Biol. Regul. 2014;57: 173–184.
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[15] Krause CD, Yang ZH, Kim YS, Lee JH, Cook JR, Pestka S. Protein arginine methyltransferases: evolution and assessment of their pharmacological and therapeutic potential. Pharmacol. Ther. 2007;113:50–87. [16] Bissinger EM, Heinke R, Spannhoff A, Eberlin A, Metzger E, Cura V, Hassenboehler P, Cavarelli J, Schule R, Bedford MT, Sippl W, Jung M. Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1. Bioorg. Med. Chem. 2011;19:3717–3731. [17] Meyer R, Wolf SS, Obendorf M. PRMT2, a member of the protein arginine methyltransferase family, is a coactivator of the androgen receptor. J. Steroid Biochem. Mol. Biol. 2007;107:1–14. [18] Koh SS, Chen D, Lee YH, Stallcup MR. Synergistic enhancement of nuclear receptor function by p160 coactivators and two coactivators with protein methyltransferase activities. J. Biol. Chem. 2001;276:1089–1098. [19] Koh SS, Li H, Lee YH, Widelitz RB, Chuong CM, Stallcup MR. Synergistic coactivator function by coactivator-associated arginine methyltransferase (CARM) 1 and beta-catenin with two different classes of DNA-binding transcriptional activators. J. Biol. Chem. 2002;277:26031–26035.
[20] Scaramuzzino C, Casci I, Parodi S, Lievens PM, Polanco MJ, Milioto C, Chivet M, Monaghan J, Mishra A, Badders N, Aggarwal T, Grunseich C, Sambataro F, Basso M, Fackelmayer FO, Taylor JP, Pandey UB, Pennuto M. Protein arginine methyltransferase 6 enhances polyglutamine-expanded androgen receptor function and toxicity in spinal and bulbar muscular atrophy. Neuron. 2015;85:88–100. [21] Jiang F, Wang Z. Identification of androgen-responsive genes in the rat ventral prostate by complementary deoxyribonucleic acid subtraction and microarray. Endocrinology. 2003;144: 1257–1265. [22] Halkidou K, Gnanapragasam VJ, Mehta PB, Logan IR, Brady ME, Cook S, Leung HY, Neal DE, Robson CN. Expression of Tip60, an androgen receptor coactivator, and its role in prostate cancer development. Oncogene. 2003;22:2466–2477. [23] Comuzzi B, Nemes C, Schmidt S, Jasarevic Z, Lodde M, Pycha A, Bartsch G, Offner F, Culig Z, Hobisch A. The androgen receptor co-activator CBP is up-regulated following androgen withdrawal and is highly expressed in advanced prostate cancer. J. Pathol. 2004;204:159–166.
