Activation of the Androgen Receptor N-terminal Domain by Interleukin-6 via MAPK and STAT3 Signal Transduction Pathways
2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês
10.1074/jbc.m108255200
ISSN1083-351X
AutoresTakeshi Ueda, Nicholas Bruchovsky, Marianne D. Sadar,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoThe androgen receptor (AR) is a ligand-activated transcription factor that mediates the biological responses of androgens. However, non-androgenic pathways have also been shown to activate the AR. The mechanism of cross-talk between the interleukin-6 (IL-6) and AR signal transduction pathways was investigated in LNCaP human prostate cancer cells. IL-6 induced several androgen-response element-driven reporters that are dependent upon the AR, increased the phosphorylation of mitogen-activated protein kinase (MAPK), and activated the AR N-terminal domain (NTD). Inhibitors to MAPK and JAK decreased the IL-6-induced phosphorylation of MAPK and activation of the AR NTD. Immunoprecipitation and transactivation studies showed a direct interaction between amino acids 234–558 of the AR NTD and STAT3 following IL-6 treatment of LNCaP cells. These results demonstrate that activation of the human AR NTD by IL-6 was mediated through MAPK and STAT3 signal transduction pathways in LNCaP prostate cancer cells. The androgen receptor (AR) is a ligand-activated transcription factor that mediates the biological responses of androgens. However, non-androgenic pathways have also been shown to activate the AR. The mechanism of cross-talk between the interleukin-6 (IL-6) and AR signal transduction pathways was investigated in LNCaP human prostate cancer cells. IL-6 induced several androgen-response element-driven reporters that are dependent upon the AR, increased the phosphorylation of mitogen-activated protein kinase (MAPK), and activated the AR N-terminal domain (NTD). Inhibitors to MAPK and JAK decreased the IL-6-induced phosphorylation of MAPK and activation of the AR NTD. Immunoprecipitation and transactivation studies showed a direct interaction between amino acids 234–558 of the AR NTD and STAT3 following IL-6 treatment of LNCaP cells. These results demonstrate that activation of the human AR NTD by IL-6 was mediated through MAPK and STAT3 signal transduction pathways in LNCaP prostate cancer cells. Interleukin-6 (IL-6) 1IL-6interleukin-6NTDN-terminal domainMAPKmitogen-activated protein kinaseGAPDHglyceraldehyde-3-phosphate dehydrogenaseARandrogen receptorAREandrogen-response elementsJAKJanus kinaseSTATsignal transducers and activators of transcriptionDBDDNA-binding domainPSAprostate-specific antigenFBSfetal bovine serumEGFepidermal growth factorIGFinsulin-like growth factorKGFkeratinocyte growth factorMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideRTreverse transcriptasePKAprotein kinase APIphosphatidylinositolgpglycoproteintkthymidine kinasePBprobasinMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinase was originally identified as a T cell-derived cytokine that induces terminal differentiation of B cells into antibody-producing cells (1Teranishi T. Hirano T. Arima N. Onoue K. J. Immunol. 1982; 128: 1903-1908PubMed Google Scholar,2Hirano T. Yasukawa K. Harada H. Taga T. Watanabe Y. Matsuda T. Kashiwamura S. Nakajima K. Koyama K. 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The AR can also be activated in the absence of its cognate ligand by signaling pathways initiated by various growth factors (23Culig Z. Hobisch A. Cronauer M.V. Radmayr C. Trapman J. Hittmair A. Bartsch G. Klocker H. Cancer Res. 1994; 54: 5474-5478PubMed Google Scholar, 24Hobisch A. Eder I.E. Putz T. Horninger W. Bartsch G. Klocker H. Culig Z. Cancer Res. 1998; 58: 4640-4645PubMed Google Scholar, 25Craft N. Shostak Y. Carey M. Sawyers C.L. Nat. Med. 1999; 5: 280-285Crossref PubMed Scopus (849) Google Scholar, 26Chen T. Wang L.H. Farrar W.L. Cancer Res. 2000; 60: 2132-2135PubMed Google Scholar) and stimulation of protein kinase pathways (27Nazareth L.V. Weigel N.L. J. Biol. Chem. 1996; 271: 19900-19907Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The AR has been suggested to regulate the expression of at least 60 genes in the rat prostate (29Wang Z. Tufts R. Haleem R. Cai X. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12999-13004Crossref PubMed Scopus (93) Google Scholar). An example of expression of a human gene that is up-regulated by androgens in the prostate is prostate-specific antigen (PSA). PSA is a member of the serine protease family and is expressed in the epithelium of "normal" prostate tissue and benign prostatic hyperplasia, prostate cancer specimens, and the LNCaP prostate cancer cell line (30Purnell D.M. Heatfield B.M. Trump B.F. Cancer Res. 1984; 44: 285-292PubMed Google Scholar). The expression of the PSA gene is regulated at the transcriptional level by androgen through several well characterized AREs (31Schuur E.R. Henderson G.A. Kmetec L.A. Miller J.D. Lamparski H.G. Henderson D.R. J. Biol. Chem. 1996; 271: 7043-7051Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). However, in the absence of androgens, PSA has been shown to become elevated in prostate cancer cells maintained in monolayer andin vivo (for a review see Ref. 32Sadar M.D. Hussain M. Bruchovsky N. Endocr. Relat. Cancer. 1999; 6: 487-502Crossref PubMed Scopus (140) Google Scholar). The mechanisms that regulate the gene expression of PSA in the absence of androgen are still unclear. Based upon these findings, we examined previously suspected growth factors for their ability to induce PSA gene expression prior to delineating a possible underlying mechanism. Here we identify for the first time that the human AR NTD is activated by IL-6 by a mechanism that is dependent upon mitogen-activated protein kinase (MAPK) and STAT3 signal transduction pathways in LNCaP prostate cancer cells. Human prostate cancer LNCaP cells were maintained in RPMI 1640 supplemented with 5% (v/v) fetal bovine serum (FBS) (Invitrogen), penicillin (100 units/ml), and streptomycin (100 μg/μl) at 37 °C in an atmosphere of 5% CO2 in the air. All chemicals were purchased from Sigma, unless stated otherwise. Insulin-like growth factor (IGF)-I, keratinocyte growth factor (KGF), bovine serum albumin, and protease inhibitor mixture tablets (CompleteTM) were obtained fromRoche Molecular Biochemicals. Epidermal growth factor (EGF) was purchased from Invitrogen. IL-6 was obtained from R & D Systems (Minneapolis, MN). The nonsteroidal antiandrogen bicalutamide was kindly supplied by Dr. Mark Zarenda (Zeneca).R p-(8-Br-cAMPs) and AG490 were obtained from Calbiochem. U0126 was from Promega (Madison, WI). The human AR cDNA was a kind gift from A. O. Brinkman (Erasmus University, Rotterdam, The Netherlands). The following plasmids have been described previously: PSA (−630/+12)-luciferase (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 33Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 34Sadar M.D. Gleave M.E. Cancer Res. 2000; 60: 5825-5831PubMed Google Scholar), PB-luciferase (35Snoek R. Bruchovsky N. Kasper S. Matusik R.J. Gleave M. Sato N. Mawji N.R. Rennie P.S. Prostate. 1998; 36: 256-263Crossref PubMed Scopus (40) Google Scholar), ARR3-tk-luciferase (36Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Crossref PubMed Scopus (37) Google Scholar), AR-(1–558)-Gal4DBD, Gal4DBD (the control vector), and p5×Gal4UAS-TATA-luciferase (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). AR-(1–233)-Gal4DBD, AR-(234–390)-Gal4DBD, and AR-(391–558)-Gal4DBD plasmids were constructed by PCR of the nucleotides 363–1062, 1063–1533, 1534–2037, respectively, of the human AR cDNA using primers 5′-AAA AGG ATC CGG ATG GAA GTG CAG TTA GGG CT and 5′-TTT GGA TCC TCA GTT GTC AGA AAT GGT CGA AGTBGCC, or 5′-AAA AGG ATC CGG GCC AAG GAG TTG TGT AAG GCA GT and 5′-AAA AGG CTT CAG GTC TTC TGG GGT GGA AAG TAA TAG as described previously (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). PSA (6.1 kb)-luciferase was kindly provided by Dr. J.-T. Hsieh (the University of Southwestern Medical Center, Dallas, TX). The expression vectors for dominant-negative STATs (pCAGGS-Neo-HA-STAT3F, pCAGGS-Neo-HA-STAT3D, and pCAGGS-Neo-HA-STAT1F), wild-type STAT3 (pCAGGS-Neo-HA-STAT3), and the control vector (pCAGGS-Neo) were kindly provided by Dr. M. Hibi and Dr. T. Hirano (Osaka University Graduate School of Medicine, Japan). LNCaP cells (3 × 105/well) were plated on 6-well plates and incubated with RPMI 1640 containing 5% FBS for 24 h. Transfection was performed by using LIPOFECTIN® Reagent (5 μl/well) (Invitrogen) according to the methods published previously (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 33Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 34Sadar M.D. Gleave M.E. Cancer Res. 2000; 60: 5825-5831PubMed Google Scholar). The total amount of plasmid DNA was prepared to 3 μg/well by addition of control plasmid that encoded the luciferase gene but lacked the promoter insert. After 24 h, the medium was replaced with serum-free RPMI 1640 containing 1 mg/ml bovine serum albumin with R1881 or growth factors. Cells were collected after 24 or 48 h of incubation using the lysis buffer provided in the luciferase kit (Promega). Luciferase activities were measured by using Dual Luciferase Assay System (Promega) with the aid of a multiplate luminometer (EG & G Berthold, Germany). Luciferase activities were normalized by the protein concentration of the samples as measured by the method of Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). The results are presented as the fold induction that is the relative luciferase activity of the treated cells divided by that of the control. All transfection experiments were carried out in triplicate wells and repeated no less than 4 times using at least 2 sets of plasmids that were prepared separately. LNCaP cells (5 × 105) were plated in 60-mm plates in RPMI containing 5% FBS. Immediately prior to experiments, the media were changed to RPMI containing 5% dextran-charcoal-stripped serum for 48 h. The cells were treated with IL-6 in RPMI containing 5% dextran-charcoal-stripped serum for 16 h. Total RNA was extracted with Trizol (Invitrogen) and fractionated by electrophoresis before blotting onto Hybond N+ filters (Amersham Biosciences). The 1.4-kb EcoRI fragments and 1-kbBamHI GAPDH fragments were labeled with [α-32P]dCTP by Random Primers DNA labeling kit (Invitrogen). Hybridizations were performed according to the method described previously (33Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The mRNA bands were quantified with the STORM 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Semiquantitative RT-PCR was performed as described previously (38Lin D.L. Whitney M.C. Yao Z. Keller E.T. Clin. Cancer Res. 2001; 7: 1773-1781PubMed Google Scholar) with minor modifications. PSA and GAPDH primers were as follows: PSA 418/21 sense 5′-GGCAGGTGCTTGTAGCCTCTC-3′; PSA 939/21 antisense, 5′-CACCCGAGCAGGTGCTTTTGC-3′; GAPDH, sense, 5′-CCGAGCCACATCGCTCAGAENDASH-3′ and GAPDH antisense, 5′-CCCAGCCTTCTCCTGGTG-3′. For quantitation 5 μm PSA primers (1 μl) were mixed with 2.5 μm GAPDH primers (1 μl) and then resolved on 1.3% agarose gel, and the bands were analyzed with ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA). The PSA fragments were normalized to GAPDH. LNCaP cells (1 × 104) were plated in 96-well plates in RPMI containing FBS (0.5%) in a final volume of 0.1 ml. The next day the cells were treated with R1881, forskolin, IL-6, or mixtures of the compounds. After 3 days in culture, cell proliferation was assessed by adding 50 μl of MTT dye (1 mg/ml) in serum-free media to the cells. After 4 h of incubation, the cells were solubilized in Me2SO (150 μl per well) prior to reading the absorbance at 570 nm using a microplate reader (Dynex Technologies). LNCaP cells (2 × 106) were plated on dishes (10 cm diameter) in RPMI 1640 containing 5% FBS. Twenty four hours later, the medium was removed and replaced with RPMI 1640 (e.g. serum-free media) for 24 h prior to the addition of IL-6, forskolin, or inhibitors. After incubation with these compounds, whole-cell lysates were prepared as described previously (39Antras J. Lasnier F. Pairault J. J. Biol. Chem. 1991; 266: 1157-1161Abstract Full Text PDF PubMed Google Scholar). Equal amounts of protein (40 μg) from each sample were electrophoresed on SDS-PAGE (8 or 10%) followed by transfer to a nitrocellulose membrane for Western blot analysis. Immunoblots were blocked for 1 h in 5% nonfat dry milk (w/v) in TBST containing 20 mm Tris-HCl (pH 7.6), 150 mm NaCl, and 0.1% Tween 20. Blots were incubated overnight with anti-phospho-p44/42 MAPK (Thr-202/Tyr-204) antibody (1:500), anti-phospho-Stat3 (Tyr-705) antibody (1:1000) (Cell Signaling Technology, Inc., Beverly, MA), or anti-phospho-STAT3 (Ser-727) antibody (1:1000) (Upstate Biotechnology, Inc., Lake Placid, NY), washed with TBST three times, and incubated for 1 h with the second antibody (1:2000; Cell Signaling Technology, or Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibodies were diluted with 5% nonfat dry milk in TBST. The protein bands were detected by the enhanced chemiluminescence kit (Cell Signaling Technology or Amersham Biosciences). Densitometric analyses of protein bands from scanned x-ray films were performed using the Personal Densitometer (Molecular Dynamics). LNCaP cells (2 × 106) were plated on 10-cm dishes in RPMI containing 5% FBS for 24 h before transfecting with expression vectors encoding STAT3 (2 μg/dish) and either His-tagged AR-(1–558), AR-(1–233), AR-(234–391), or AR-(392–558) using LIPOFECTIN® reagent (Invitrogen). After 24 h the cells were treated with IL-6 (50 ng/ml) or vehicle for 6 h before harvesting. Harvested cells were lysed in Soft RIPA buffer (PBS, 1% sodium deoxycholate, 20 mm sodium molybdate, 50 mm NaF, 25 mm β-glycerophosphate, 1 mm EDTA, 1% Nonidet P-40 and protease inhibitors). Cell lysates were passed several times through a 30½-gauge needle to disrupt the nuclei. Immunoprecipitations were performed using anti-His antibody conjugated to agarose (Santa Cruz Biotechnology). Immune complexes were analyzed by SDS-PAGE/immunoblot assay with anti-STAT3 (Upstate Biotechnology, Inc., Lake Placid, NY). EGF, KGF, IGF-I, IGF-II, and IL-6 have been suspected to play a role in the progression of prostate cancer to androgen independence, which is clinically determined by elevating levels of PSA. Therefore, these compounds were screened using expression of PSA as an end point in the well differentiated LNCaP human prostate cancer cell line to identify whether any of these growth factors may be used as a model to delineate a possible mechanism of action. LNCaP cells were transiently transfected with the PSA (−630/+12)-luciferase reporter plasmid. This region of the PSA promoter has been partially characterized and contains several AREs that are required for androgen induction (31Schuur E.R. Henderson G.A. Kmetec L.A. Miller J.D. Lamparski H.G. Henderson D.R. J. Biol. Chem. 1996; 271: 7043-7051Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 40Cleutjens K.B.J.M. van Eekelen C.C.E.M. van der Korput H.A.G.M. Brinkmann A.O. Trapman J. J. Biol. Chem. 1996; 271: 6379-6388Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). Maximum induction (6-fold) of PSA-luciferase reporter activity by the synthetic androgen, R1881, was obtained at 1 nm and remained elevated at 10 nm. These results are consistent with previous reports (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 33Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 34Sadar M.D. Gleave M.E. Cancer Res. 2000; 60: 5825-5831PubMed Google Scholar). All studies measuring activities of the PSA promoter were performed in parallel with saturating concentrations of R1881 (10 nm), which was included as a positive control. Exposure of transiently transfected LNCaP cells to IL-6 (50 ng/ml) resulted in a 12-fold increase in PSA-reporter activity relative to the control (Fig.1). EGF, KGF, IGF-I, and IGF-II yielded negligible to no effect on PSA-luciferase activities. Our results with EGF are consistent with the report showing that this growth factor has no effect in the absence of androgen on the secretion of PSA in LNCaP cells (41Henttu P. Vihko P. Cancer Res. 1993; 53: 1051-1058PubMed Google Scholar). These results show that IL-6 causes androgen-independent increases in PSA-luciferase activity, whereas other previously suspected growth factors did not affect the activity of this reporter in LNCaP cells. Because IL-6 caused an increased in PSA reporter activity, we next examined whether PSA mRNA levels were increased in LNCaP cells exposed to IL-6. Northern blot analysis showed that IL-6 caused a 2-fold increase in PSA mRNA levels compared with control values (Fig.2 A, compare 2nd and3rd lanes). R1881 was used as a positive control and as expected caused a robust increase in PSA mRNA levels (1st lane). Increased levels of PSA mRNA were also detected by semiquantitative RT-PCR using RNA isolated from LNCaP cells exposed to two different concentrations of IL-6 (1 and 10 ng/ml) (Fig.2 B). IL-6 (10 ng/ml) caused a greater than 2-fold increase in PSA mRNA levels in LNCaP cells. These results are consistent with two previous reports (24Hobisch A. Eder I.E. Putz T. Horninger W. Bartsch G. Klocker H. Culig Z. Cancer Res. 1998; 58: 4640-4645PubMed Google Scholar, 38Lin D.L. Whitney M.C. Yao Z. Keller E.T. Clin. Cancer Res. 2001; 7: 1773-1781PubMed Google Scholar) using semiquantitative PCR to detect a 2-fold increase in levels of PSA mRNA in LNCaP cells exposed to IL-6. The effect of IL-6 on the proliferation of LNCaP cells is controversial with reports of increases (5Okamoto M. Lee C. Oyasu R. Cancer Res. 1997; 57: 141-146PubMed Google Scholar, 42Giri D. Ozen M. Ittmann M. Am. J. Pathol. 2001; 159: 2159-2165Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 43Smith P.C. Keller E.T. Prostate. 2001; 48: 47-53Crossref PubMed Scopus (101) Google Scholar) and decreases (6Chung T.D., Yu, J.J. Spiotto M.T. Bartkowski M. Simons J.W. Prostate. 1999; 38: 199-207Crossref PubMed Scopus (196) Google Scholar, 24Hobisch A. Eder I.E. Putz T. Horninger W. Bartsch G. Klocker H. Culig Z. Cancer Res. 1998; 58: 4640-4645PubMed Google Scholar, 44Richie C.K. Andrews L.R. Thomas K.G. Tindall D.J. Fitzpatrick L.A. Endocrinology. 1997; 138: 1145-1150Crossref PubMed Scopus (85) Google Scholar, 45Levesque E. Beaulieu M. Guillemette C. Hum D.W. Belanger A. Endocrinology. 1998; 139: 2375-2381Crossref PubMed Google Scholar). To place our results in context with other reports, we examined the effects of IL-6 on the proliferation of LNCaP cells in comparison to androgen (R1881) and forskolin. IL-6, forskolin, and R1881 increased proliferation of LNCaP cells as compared with control levels after 3 days (Fig.3). IL-6 and forskolin were comparable in promoting proliferation at the concentrations and conditions used in this experiment. A mixture of IL-6 with R1881 did not promote further increases in proliferation over that observed with R1881 alone. A mixture of IL-6 and forskolin also did not increase the proliferation of LNCaP cells over that obtained with each of the individual compounds. To determine whether the induction of PSA by IL-6 is through a similar mechanism as R1881, LNCaP cells were treated with a saturating concentration of R1881 to examine whether any further increases in PSA could be induced in the presence of the two compounds. The induction of PSA-luciferase activity by IL-6 was dose-dependent both in the absence or presence of R1881 (Fig. 4 A). Maximum induction of PSA reporter activity by IL-6 was obtained at the concentration of 100 ng/ml. Combined treatments with IL-6 and R1881 resulted in synergistic increases in the activation of the PSA reporter construct over that achieved solely with a saturating concentration of R1881. Recently, we reported that activation of the protein kinase A (PKA) pathway using forskolin resulted in androgen-independent increases in the expression of the PSA gene (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). The optimal concentration of forskolin required to achieve maximum induction of the PSA-luciferase reporter was 50 μm (28Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). To provide insight into the mechanism of androgen-independent induction of PSA by IL-6, the effects of both IL-6 and forskolin treatments on the activity of the PSA-luciferase reporter were examined. At the optimal concentration of forskolin (50 μm), PSA-luciferase activity was increased 53-fold (Fig. 4 B). Combined treatment of LNCaP cells with an increasing concentration of IL-6 (1–100 ng/ml) and a constant concentration of forskolin (50 μm) resulted in synergic increases in PSA-luciferase activities that were dependent upon the concentration of IL-6. The PSA (6.1 kb)-reporter gene construct that contains both the enhancer and promoter regions has been reported to be highly inducible by androgens when compared with the reporter containing only the promoter region (46Cleutjens K.B.J.M. van der Korput H.A.G.M. van Eekelen C.C.E.M. van Rooij H.C.J. Faber P.W. Trapman J. Mol. Endocrinol. 1997; 11: 148-161Crossref PubMed Scopus (294) Google Scholar). We used this longer PSA (6.1 kb) reporter to determine whether IL-6 would also have a greater effect on this reporter. As shown in Fig. 5 A, the PSA (6.1 kb)-luciferase reporter was induced 70-fold by R1881, 8-fold by IL-6, and 113-fold by a mixture of R1881 and IL-6. These results suggest that the induction of PSA by IL-6 is mediated primarily through the −630 to +12 region of the PSA promoter and does not encompass DNA elements upstream in the enhancer region. To determine whether other androgen-responsive reporter constructs that contain AREs could be induced by IL-6, two additional reporters were evaluated in LNCaP cells. The first of these constructs was the probasin (PB)-promoter (−286/+28) which is a naturally occurring androgen-regulated promoter from the rat that contains ARE1 and ARE2 (35Snoek R. Bruchovsky N. Kasper S. Matusik R.J. Gleave M. Sato N. Mawji N.R. Rennie P.S. Prostate. 1998; 36: 256-263Crossref PubMed Scopus (40) Google Scholar). As shown in Fig. 5 B, the PB-luciferase reporter construct was induced 146-fold by R1881, 8-fold by IL-6, and 218-fold by a mixture of R1881 and IL-6. These results show that IL-6 induces androgen-independent increases of PB-luciferase activity when used solely, and synergistic increases when used in combination with R1881. These results are consistent with those obtained using the PSA-luciferase reporters. The second of these reporters was the ARR3-thymidine kinase (tk)-luciferase, which is an artificial reporter construct that contains three tandem repeats of the rat PB ARE1 and ARE2 regions upstream of a luciferase reporter (36Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Crossref PubMed Scopus (37) Google Scholar). The effect of IL-6 on the ARR3-tk-reporter construct was unique compared with those obtained with PSA and PB. Although R1881 induced ARR3-tk-luciferase activity by 627-fold, IL-6 proved to be a very poor inducer of this construct (less than 2-fold) (Fig.5 C). However, a synergistic induction of the ARR3-tk-luciferase reporter was observed in the presence of both IL-6 and R1881, consistent with the results obtained with PSA and PB reporters. The differences in the induction of these three reporter constructs between R1881 and IL-6 demonstrate promoter-specific responses. AREs are present in all four of the above reporters that respond to IL-6 either in the presence or absence of androgen, suggesting a role for the AR in the underlying mechanism. To test this hypothesis
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