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J. Biol. Chem., Vol. 277, Issue 9, 7076-7085, March 1, 2002

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Activation of the Androgen Receptor N-terminal Domain by Interleukin-6 via MAPK and STAT3 Signal Transduction Pathways^*

Takeshi Ueda, Nicholas Bruchovsky, and Marianne D. Sadar

From the Department of Cancer Endocrinology, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada

Received for publication, August 27, 2001, and in revised form, December 17, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Interleukin-6 (IL-6)¹ was originally identified as a T cell-derived cytokine that induces terminal differentiation of B cells into antibody-producing cells (1, 2). IL-6 is a multifunctional cytokine that plays an important role in the regulation of hematopoiesis, immune response, inflammation, bone metabolism, and neural development (3). IL-6 is produced by lymphoid, non-lymphoid cells, and cancer cells (4). IL-6 can increase the proliferation of some cancer cells, including prostate (5, 6), and is considered to be an autocrine or a paracrine growth factor (7-9).

The receptor for IL-6 is composed of an IL-6 subunit ( chain or gp80) and a signal transducer, gp130 ( chain) (10, 11). The mRNA encoding IL-6 subunit is detectable in both cell lines and specimens of prostate cancer (12, 13). Upon binding of IL-6 to its receptor, a homodimer of gp130 is formed that results in activation of Janus kinases (JAKs) (14-16). Once gp130 is tyrosine-phosphorylated, it recruits signal-transducing molecules, SHP-2 (protein tyrosine phosphatase 2), and signal transducers and activators of transcription 3 (STAT3) (for a review see Ref. 17). Monomers of STAT3 protein exist in the cytoplasm of non-stimulated cells, and when activated by tyrosine phosphorylation by JAK in response to IL-6 they dimerize through SH2-phosphotyrosyl interactions that lead to nuclear translocation and DNA binding to initiate transcription (18-20).

The androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of nuclear receptors (21). These receptors have similar structures that are composed of an N-terminal domain (NTD) that is involved in transcriptional activation, a DNA-binding domain (DBD), a hinge region, and a ligand-binding domain. After the ligand binds to AR, the ligand-receptor complex translocates to the nucleus and binds specific androgen-response elements (AREs) on the chromosome (22). The AR can also be activated in the absence of its cognate ligand by signaling pathways initiated by various growth factors (23-26) and stimulation of protein kinase pathways (27, 28). The AR has been suggested to regulate the expression of at least 60 genes in the rat prostate (29). 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 (30). The expression of the PSA gene is regulated at the transcriptional level by androgen through several well characterized AREs (31). However, in the absence of androgens, PSA has been shown to become elevated in prostate cancer cells maintained in monolayer and in vivo (for a review see Ref. 32). 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.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and Materials-- 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 (Complete^TM) were obtained from Roche 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).

Plasmids-- 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 (28, 33, 34), PB-luciferase (35), ARR₃-tk-luciferase (36), AR-(1-558)-Gal4DBD, Gal4DBD (the control vector), and p5×Gal4UAS-TATA-luciferase (28). 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 (28). 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).

Transfection and Luciferase Assay-- 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 (28, 33, 34). 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 (37). 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.

Northern Blot Analysis-- 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-kb BamHI GAPDH fragments were labeled with [-³²P]dCTP by Random Primers DNA labeling kit (Invitrogen). Hybridizations were performed according to the method described previously (33). The mRNA bands were quantified with the STORM 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Semiquantitative RT-PCR-- Semiquantitative RT-PCR was performed as described previously (38) 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.

MTT Assays-- 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).

Immunoblots-- 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 (39). 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).

Co-immunoprecipitations-- 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 -glycerophosphate, 1 mM EDTA, 1% Nonidet P-40 and protease inhibitors). Cell lysates were passed several times through a 301/2-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).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Androgen-independent Induction of the PSA Promoter Gene by IL-6-- 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 (31, 40). 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 (28, 33, 34). 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 (41). 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.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effects of R1881 and growth factors on PSA-luciferase activity in LNCaP cells. LNCaP cells were transiently transfected with PSA (630/+12)-luciferase (1 µg/well) for 24 h and then incubated with R1881 (10 nM), growth factors (50 ng/ml), or vehicle for an additional 48 h under serum-free conditions. The total amount of plasmid DNA transfected was normalized to 3 µg/well by addition of the empty vector. The error bars represent the mean ± S.E. of three independent experiments.

IL-6 Increases PSA mRNA Levels 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. 2A, compare 2nd and 3rd 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. 2B). 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 (24, 38) using semiquantitative PCR to detect a 2-fold increase in levels of PSA mRNA in LNCaP cells exposed to IL-6.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   IL-6 increases PSA mRNA levels in LNCaP cells. A, Northern blot analysis of PSA mRNA levels in LNCaP cells exposed to R1881 (10 nM) and IL-6 (5 ng/ml); and B, semiquantitative RT-PCR for the measurement of PSA mRNA levels in LNCaP cells exposed to IL-6 (1 and 10 ng/ml) in RPMI containing 5% dextran-charcoal-stripped serum for 16 h prior to isolation of total RNA (described under "Materials and Methods").

IL-6 Increases Proliferation of LNCaP Cells-- The effect of IL-6 on the proliferation of LNCaP cells is controversial with reports of increases (5, 42, 43) and decreases (6, 24, 44, 45). 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.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   IL-6 increases the proliferation of LNCaP cells. Cells were treated with R1881 (10 nM), forskolin (FSK, 1 µM), IL-6 (50 ng/ml), IL-6 plus R1881 (50 ng/ml and 10 nM, respectively), or IL-6 plus FSK (50 ng/ml and 1 µM, respectively). After 3 days in culture, cell proliferation was assessed by the MTT assay. Fold induction represents the mean of the treated value for absorbance divided by the mean value for the control. Student's t test, two-tailed: ***, p < 0.0001; **, p < 0.05. Error bars signify the mean ± S.E., n = 5.

Synergistic Increases in the Induction of PSA Promoter Gene by IL-6 with Androgen or Activation of the PKA Pathway-- 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. 4A). 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.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effects of IL-6 and R1881 or forskolin on PSA-luciferase activity in LNCaP cells. LNCaP cells were transiently transfected with PSA (630/+12)-luciferase (1 µg/well) for 24 h and then pre-treated with IL-6 (ng/ml) for 2 h before the addition of R1881 (10 nM) (A) or vehicle or forskolin (50 µM) (B) or vehicle and then incubated for an additional 48 h under serum-free conditions. The error bars represent the mean ± S.E. of three independent experiments.

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 (28). The optimal concentration of forskolin required to achieve maximum induction of the PSA-luciferase reporter was 50 µM (28). 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. 4B). 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.

Induction of Other Androgen-responsive Reporters by IL-6 in LNCaP Cells-- 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 (46). 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. 5A, 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.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of IL-6 on the activities of androgen-inducible reporters in LNCaP cells. LNCaP cells were transiently transfected with PSA (6.1 kb) (A), PB (286/+28) (B), or ARR3-tk-luciferase (C) (1 µg/well) for 24 h and treated with R1881 (10 nM), IL-6 (50 ng/ml), a mixture of R1881 (10 nM) and IL-6 (50 ng/ml), or vehicle for an additional 48 h under serum-free conditions. The error bars represent the mean ± S.E. of three independent experiments.

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 (35). As shown in Fig. 5B, 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 (36). 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. 5C). 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.

Bicalutamide Inhibits the Induction of PSA-Luciferase Activity by IL-6-- 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, we employed the non-steroidal antiandrogen, bicalutamide, that specifically inhibits the AR while having no effect on other steroid hormone receptors. LNCaP cells were transiently transfected with the PSA (630/+12)-luciferase reporter and treated with bicalutamide and IL-6. Expectedly, bicalutamide blocked the induction of the PSA-luciferase activity by R1881 by 84%. Bicalutamide also blocked the induction of PSA-luciferase activities by IL-6 by 70% (Fig. 6). These results suggest that the induction of PSA promoter activity by IL-6 is dependent upon a functional AR.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Inhibitory effect of bicalutamide on the induction of PSA-luciferase by IL-6. LNCaP cells were transiently transfected with PSA (630/+12)-luciferase (1 µg/well) for 24 h and then pre-treated with bicalutamide (10 µM) or vehicle for 2 h before the addition of IL-6 (50 ng/ml) and R1881 (10 nM) and then incubated for an additional 48 h under serum-free conditions. The error bars represent the mean ± S.E. of three independent experiments.

IL-6 Activates the Human AR NTD-- Ligand-independent activation of the estrogen receptor has been shown to involve the NTD for its activation by EGF (47) and IGF-I (48). Therefore, we sought to determine whether IL-6 targeted the AR NTD to activate this receptor. To do this, the yeast Gal4 system was employed using a construct of the AR NTD fused to the Gal4DBD (AR-(1-558)-Gal4DBD). LNCaP cells were co-transfected with the expression vector encoding the AR-(1-558)-Gal4DBD and a reporter gene containing the Gal4-binding site (p5×Gal4UAS-TATA-luciferase). Because R1881 binds to the ligand-binding domain of the AR, which is absent in our construct, it was included as a negative control. Forskolin, which increases the intracellular levels of cAMP, was previously shown to activate a similar system (28) and was included here as a positive control.

As expected, R1881 had no effect, whereas forskolin resulted in approximately a 6-fold increase in the activation of the AR NTD measured as increased Gal4-luciferase activity in co-transfected LNCaP cells (Fig. 7). Exposure of cells to IL-6 resulted in a 9-fold increase in Gal4-luciferase activity. A mixture of IL-6 with R1881 did not significantly alter the activity of this reporter compared with the activity achieved with IL-6 alone. However, a mixture of IL-6 and forskolin resulted in a synergistic increase (greater than 60-fold) in activity of the Gal4-luciferase reporter. These data suggest that the AR NTD is a target of the IL-6 signaling pathway and that simultaneous stimulation of the PKA pathway results in synergistic activation of the AR NTD.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effect of IL-6 on the activity of the human AR NTD. Transactivation assays were performed in LNCaP cells co-transfected with the 5×Gal4UAS-TATA-luciferase (1 µg/well) and AR-(1-558)-Gal4DBD (50 ng/well) or Gal4DBD for 24 h prior to incubation with R1881 (10 nM), forskolin (50 µM), IL-6 (50 ng/ml), a mixture of IL-6 and R1881, a mixture of IL-6 and forskolin, or vehicle for an additional 24 h. The total amount of plasmid DNA transfected was normalized to 3 µg/well by addition of the empty vector. The error bars represent the mean ± S.E. of three independent experiments and are normalized to respectively treated Gal4DBD (vector alone) values.

Activation of the AR NTD by IL-6 Is Mediated through the MAPK Pathway-- Signaling to the nucleus by IL-6 has been reported to involve various pathways in LNCaP cells including PKA, protein kinase C, phosphatidylinositol (PI) 3'-kinase, MAPK, and JAK-STAT (24, 49-51). The AR NTD contains a number of putative phosphorylation sites for serine-proline-directed kinase, DNA-dependent kinase, protein kinase C, casein kinase I and II, PKA, MAPK, and calmodulin kinase II. This raises the possibility that IL-6 may activate the AR NTD via a phosphorylation event involving one or more of these pathways. To identify the signal transduction pathway involved in activation of the AR NTD by IL-6, we employed a series of inhibitors of various protein kinases and included forskolin as a control in these studies. The concentration of each of the inhibitors was evaluated by MTT assay, and no cytotoxic effects were observed (data not shown). To determine the role of PKA in IL-6 activation of the AR NTD, LNCaP cells were pre-treated with the PKA inhibitor, R_p-(8-bromo-cAMPs). This inhibitor blocked the activation of the AR NTD by forskolin but had no effect on the activation of the AR NTD by IL-6 (Fig. 8; compare 6th with 7th and 11th with 12th lanes) or a mixture of IL-6 and forskolin (13th and 14th lanes). These results suggest that the PKA pathway is not involved in the activation of the AR NTD by IL-6.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effects of inhibitors on the activity of the AR NTD. Transactivation assays were performed as described in Fig. 7 with LNCaP cells pre-treated for 2 h with each inhibitor, Rp-(8-bromo-cAMPs) (RP, 200 µM), wortmannin (WM, 100 nM), U0126 (10 µM), and AG490 (50 µM), prior to the addition of IL-6 (50 ng/ml), forskolin (FSK, 50 µM), or vehicle and then incubated for an additional 24 h. The error bars represent the mean ± S.E. of three independent experiments.

The PI 3'-kinase pathway has been reported to be a major contributor in the signaling of IL-6 (52, 53). The role of PI 3'-kinase in the activation of the AR NTD by IL-6 was examined using the PI 3'-kinase inhibitor, wortmannin. A 30% decrease in the activation of the AR NTD by IL-6 was observed in cells exposed to wortmannin (Fig. 8, compare 6th with 8th lane). This inhibitor had no effect on the activation of the AR NTD by forskolin (Fig. 8, compare 11th with 13th lane). Thus, the PI 3'-kinase pathway may play a role in the activation of the AR NTD by IL-6.

To evaluate the role of the MAPK pathway in the activation of the AR NTD by IL-6, LNCaP cells were pre-treated with the MAPK kinase (MEK) inhibitor, U0126. Interestingly, both IL-6 and forskolin activation of the AR NTD was blocked by this inhibitor (Fig. 8, compare 6th with 9th and 11th with 14th lanes). These results suggest that both IL-6 and forskolin activate the AR NTD by a pathway requiring the MAPK pathway.

The JAK-STAT pathway is an important signal transduction pathway for IL-6. STAT3 is a transcription factor involved in IL-6 signal transduction. The transcriptional activity of STAT3 is regulated by phosphorylation at two sites, tyrosine 705 and serine 727 by JAK and MAPK, respectively (54, 55). Therefore, we investigated whether inhibition of JAK would affect activation of the AR NTD by IL-6. The JAK inhibitor, AG490, has been shown to block the activation of STAT3 in mycosis fungus-derived T cell lymphoma cells (56). AG490 reduced IL-6 activation of the AR NTD by 85% (Fig. 8, compare 6th with 10th lane). These results demonstrate that JAK is an important pathway for activation of the AR NTD in response to IL-6.

Stimulation of the PKA Pathway Causes a Synergistic Increase in the Activation of MAPK by IL-6-- Results presented in Fig. 8 suggest that the MAPK and the JAK-STAT3 pathways are involved in the IL-6 activation of the AR NTD. To confirm that IL-6 activates the MAPK and STAT3 pathways, time course studies were performed. Western blot analyses showed that phosphorylation of MAPK (isoforms p44 and p42) and STAT3 at tyrosine 705 were maximum after 15 min of exposure of LNCaP cells to IL-6 (Fig. 9, A and B). At this time point, IL-6 caused an increase in phosphorylation of MAPK that was comparable with that achieved with forskolin (Fig. 9C, compare 3rd with 5th lane). Simultaneous treatment of cells with IL-6 and forskolin resulted in a synergistic increase in the phosphorylation of MAPK (Fig. 9C, compare 3rd and 5th lanes with 7th lane). The MEK inhibitor U0126 completely blocked the phosphorylation of MAPK in cells exposed to IL-6, forskolin, or the combined treatment of IL-6 and forskolin (compare 3rd with 4th, 5th with 6th, and 7th with 8th lanes). U0126 did not block the phosphorylation of STAT3 at tyrosine 705 (Fig. 9D, compare lane 3 with 4). Forskolin had no effect on the phosphorylation of STAT3 at tyrosine 705 (Fig. 9D, lane 5). Thus, exposure of LNCaP cells to IL-6 results in increased phosphorylation of MAPK and STAT3-tyrosine 705, whereas forskolin only appears to increase the phosphorylation of MAPK.

View larger version (25K): [in this window] [in a new window]   Fig. 9.   Effect of IL-6 on phosphorylation of MAPK and STAT3 in LNCaP cells. Time course studies in LNCaP cells showing IL-6-induced phosphorylation of MAPK (A) and STAT3 (B). LNCaP cells were incubated in serum-free conditions for 24 h and then treated for the indicated times with IL-6 (50 ng/ml). Effect of an inhibitor of MEK on IL-6-induced phosphorylation of MAPK (C) and STAT3 (D). LNCaP cells were incubated in serum-free conditions for 24 h and then treated with IL-6 (50 ng/ml), forskolin (50 µM), or vehicle for 15 min, with or without pretreatment with U0126 (10 µM; 30 min) or vehicle. Phosphorylation of MAPK or STAT3 was detected by Western blot analysis using anti-phospho-MAPK or STAT3 antibodies (P-MAPK or P-STAT3[Tyr-705]). Membranes were stripped and re-blotted with anti-MAPK or STAT3 antibodies that detected total MAPK (T-MAPK) or total STAT3 (T-STAT3).

JAK Inhibitor Blocks Phosphorylation of STAT3 by IL-6-- Because transactivation of STAT3 is regulated by phosphorylation at tyrosine 705 and serine 727 by JAK and MAPK (54, 55), we examined phosphorylation levels of STAT3 by Western blot analysis. Application of a specific inhibitor of JAK, AG490, inhibited the activation of the AR NTD by IL-6 (Fig. 8) and blocked phosphorylation of tyrosine 705 of STAT3 (Fig. 10A, compare lane 3 with 4). This inhibitor also blocked the phosphorylation of MAPK in LNCaP cells treated with IL-6 as shown in Fig. 10B (compare lane 3 with 4). By using an antibody to phosphorylated STAT3 at serine 727, IL-6 was shown to increase the phosphorylation of STAT3 at this serine, whereas forskolin had no effect (Fig. 10C, compare lane 2 with 3). These results indicate that activation of the JAK pathway by IL-6 is involved in the phosphorylation of both STAT3 and MAPK in LNCaP cells.

View larger version (16K): [in this window] [in a new window]   Fig. 10.   Effect of a JAK inhibitor on IL-6-induced phosphorylation of STAT3-tyrosine 705 (A) and MAPK (B) in LNCaP cells. LNCaP cells were incubated in serum-free conditions for 24 h and then treated with IL-6 (50 ng/ml) or vehicle for 15 min, with or without pretreatment with AG490 (50 µM) or vehicle. C, IL-6 induced phosphorylation of STAT3 at serine 727. LNCaP cells were incubated in serum-free conditions for 24 h and then treated with IL-6 (50 ng/ml), forskolin (50 µM), or vehicle for 15 min. Western blots were prepared as described in Fig. 9.

Dominant Negatives of STAT3 Inhibit the Activation of the AR NTD by IL-6-- The results above suggest that the activation of the AR NTD by IL-6 can be blocked by inhibition of JAK. This implies a possible role for STAT3 in this mechanism. To explore this possibility, we employed expression vectors encoding various dominant negatives of STAT3 such that we could specifically block STAT3 (17, 26, 57, 58). The STAT3F mutant carries a tyrosine to phenylalanine substitution at codon 705 that reduces tyrosine phosphorylation of wild-type STAT3, thereby inhibiting both dimerization and DNA binding of STAT3. The STAT3D mutant can be phosphorylated by IL-6 but has no STAT3 DNA binding activity. STAT1F has a tyrosine to phenylalanine substitution at residue 701 and inhibits the activation of transcription of the reporter genes by interferon- (57). To evaluate the role of STAT3 in the activation of the AR NTD by IL-6, the activation of the AR-(1-558)-Gal4DBD with the Gal4-luciferase reporter was examined in LNCaP cells transiently co-transfected with these dominant-negative STAT3 constructs. Overexpression of STAT3F and STAT3D (Fig. 11) suppressed activation of the AR NTD by IL-6. Dominant-negative STAT1 (STAT1F) did not reduce activation of the AR NTD by IL-6. These results indicate that STAT3 plays a role in the activation of the AR NTD by IL-6.

View larger version (16K): [in this window] [in a new window]   Fig. 11.   Effects of dominant-negative STATs on the activity of AR NTD induced by IL-6. Transactivation assays were performed in LNCaP cells co-transfected with 0.5 µg of plasmid DNA encoding dominant negatives of STAT3 (STAT3F or STAT3D), a dominant-negative STAT1 (STAT1F), or Neo (vector alone) as described in Fig. 7. After 24 h, serum-starved cells were treated with IL-6 (50 ng/ml) or vehicle and then incubated for an additional 24 h. The error bars represent the mean ± S.E. of three independent experiments.

Interaction between STAT3 and Amino Acids 234-558 of the AR NTD-- The above results suggest that STAT3 may interact with the AR NTD. To investigate this possibility further, we first determined the region on the AR NTD to which IL-6-induced activity could be mapped. To do expression vectors, amino acids 1-558, 1-233, 234-390, and 391-558 of the AR NTD fused to Gal4DBD (AR-(1-558)-Gal4DBD, AR-(1-233)-Gal4DBD, AR-(234-390)-Gal4DBD, and AR-(391-558)-Gal4DBD, respectively) were used. As shown in Fig. 12A, IL-6 had no effect on the activation of Gal4-luciferase activity in cells co-transfected with AR-(1-233)-Gal4DBD compared with the AR NTD encompassing amino acids 1-558, 234-390, and 391-558 (compare 4th with 2nd, 3rd, and 5th lanes). Thus amino acids 234-558 of the AR NTD are required for IL-6-induced activity. However, the most active construct was AR-(234-390)-Gal4DBD. Next we tested whether STAT3 could be immunoprecipitated with this region of the AR NTD. As shown in Fig. 12B, IL-6 induced protein-protein interactions between STAT3 and the AR NTD in LNCaP cells that were transfected with AR-(1-558)-His tag, AR-(234-390)-His tag, and AR-(391-558)-His tag. Consistent with transactivation studies, IL-6 did not induce protein-protein interactions in LNCaP cells that were transfected with AR-(1-233)-His tag. As a further control, IL-6 did not induce protein-protein interactions in LNCaP cells that were transfected with the expression vector for His tag (data not shown). No interaction was detected in the absence of IL-6. These data suggest that STAT3 interacts with amino acids 234-558 of the AR NTD in LNCaP cells exposed to IL-6. The fact interaction with STAT3 is observed with all three fragments that contain all or part of the AF-1 site (AR-(1-558), AR-(234-390), and AR-(391-558)) of the AR suggests that STAT3 may interact with the AF-1 site as part of a large multiprotein complex or alternatively STAT3 may have multiple binding sites on both the AR-(234-390) and AR-(391-558).

View larger version (28K): [in this window] [in a new window]   Fig. 12.   Effect of IL-6 on the interaction between amino acids 234 and 558 of the AR NTD and STAT3. A, mapping of the AR NTD required for IL-6-induced activity. Transactivation assays were performed in LNCaP cells co-transfected with the 5×Gal4UAS-TATA-luciferase (1 µg/well) and AR-(1-558)-Gal4DBD (50 ng/well), AR-(1-233)-Gal4DBD (50 ng/well), AR-(234-390)-Gal4DBD (50 ng/well), and AR-(391-558)-Gal4DBD (50 ng/well) or Gal4DBD (50 ng/well) for 24 h prior to incubation with IL-6 (50 ng/ml) or vehicle for an additional 24 h. The total amount of plasmid DNA transfected was normalized to 3 µg/well by addition of empty vector. The error bars represent the mean ± S.E. of three independent experiments. RLU, relative luminescent unit(s). B, physical interaction between the AR NTD and STAT3 in LNCaP cells exposed to IL-6. LNCaP cells co-transfected with an expression vector for STAT3 (2 µg/dish) and either His-tagged AR-(1-558), AR-(1-233), AR-(234-391), and AR-(392-558) prior to exposure to IL-6 (50 ng/ml) or vehicle for 6 h. Extracts were immunoprecipitated (IP) with anti-His antibody. Immune complexes were analyzed by SDS-PAGE/immunoblot assay with anti-STAT3.

	DISCUSSION

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Ligand-mediated activation of the AR results in its conformation becoming more compact upon ligand binding, dissociation of heat-shock proteins, homodimerization, and phosphorylation prior to DNA binding to AREs on androgen-regulated genes such as PSA (22). The mechanisms underlying ligand-independent activation of the AR via cross-talk with other signaling pathways (23, 25, 27, 28) have not been elucidated. The present studies investigating the ability of IL-6 to induce PSA gene expression via cross-talk with the AR revealed the following. 1) IL-6 induced PSA gene expression by an AR-dependent pathway in LNCaP cells, whereas EGF, KGF, IGF-I, and IGF-II had no effect. 2) IL-6 induction of PSA reporter activity was synergistic in the presence of either androgen or forskolin, which stimulates the PKA pathway. 3) IL-6 induction of androgen-responsive reporter genes was gene-specific. 4) IL-6 activated the AR NTD, and a synergistic activation was achieved with a mixture of IL-6 plus forskolin. 5) Activation of the AR NTD by IL-6 was blocked by inhibitors to MAPK, JAK, and dominant negatives of STAT3. 6) Both IL-6 and forskolin increased phosphorylation of MAPK and when used together synergistic increases were observed. 7) Phosphorylation of MAPK by IL-6 was blocked by inhibitors to MAPK and JAK. 8) STAT3 co-immunoprecipitated with the AR NTD.

PSA is an androgen-regulated gene in the prostate, and its promoter and enhancer regions contains several well characterized AREs to which the ligand-activated AR binds to initiate transcription (31, 40, 59) and does not contain a STAT-binding site as determined by sequence analysis. We show that IL-6 induced the activity of the PSA (630/+12)-luciferase reporter by a mechanism dependent upon the AR in human LNCaP prostate cancer cells, whereas previously suspected growth factors, EGF, KGF, IGF-I, and IGF-II, had no effect. These results are in conflict to a previous report (23) that IGF-I, KGF, and EGF can activate the AR to increase an ARE-driven reporter gene construct in prostate cancer cell lines. A possible explanation for these disparities may include promoter or cell specificity because the earlier studies (23) generally used an artificial reporter containing two AREs in Du145 cells transfected with an expression vector for the AR. Support for this hypothesis can be drawn from our studies showing that the ARR₃-tk-luciferase reporter was poorly induced by IL-6, whereas the PSA and PB reporters were strongly induced. Such promoter-specific responses via a non-ligand-activated AR versus the ligand-activated AR have been reported previously in prostate cancer cells exposed to butyrate and compounds that stimulate the PKA pathway (28, 34). A possible mechanism underlying promoter-specific responses to the ligand-activated AR versus the non-ligand-activated AR may involve recruitment of different co-activators. Ligand-independent activation of the AR may cause the AR to adopt a different conformation to that assumed by the ligand-bound AR. This could result in the exposure of different regions of the AR for alternative protein-protein interactions. Alternatively, the discrepancy with the reported results may be due to cell specificity, because the previous studies (51, 60) were performed in poorly differentiated human prostate Du145 cells, rather than the well differentiated LNCaP cells used here, and differences in MAPK and STAT3 signaling have been noted between these two cell lines.

Synergistic or additive increases in the transcription of ARE-driven reporters have been reported in the presence of androgen and compounds that activate protein kinase pathways (34, 61-64). We observed a synergistic induction of the ARE-driven reporters in response to a saturating concentration of R1881 and IL-6, which may suggest that an additional mechanism to that used solely by R1881 is involved in the IL-6 pathway. Whole-cell levels of AR were not increased by IL-6 (data not shown), therefore negating increased AR levels as a possible mechanism for the synergistic increases observed. In addition, a synergistic activation of the AR by IL-6 and forskolin was shown for the first time. Our data are consistent in the fact that synergistic increases were measured for the following: 1) the induction of PSA reporter activity; 2) the induction of Gal4-luciferase reporter driven by the AR NTD fused to Gal4DBD; and 3) the levels of phosphorylated MAPK in cells exposed to a mixture of IL-6 and forskolin. A possible mechanism for these enhanced increases observed for PSA-luciferase activity is that IL-6 and forskolin signaling converges and amplifies the MAPK pathway leading to synergistic activation of the AR NTD.

The AR NTD interacts with co-activators (65-67), contains the AF-1 site required for transactivation (68-73), and has numerous putative phosphorylation sites that have been proposed to be targeted by compounds that stimulate protein kinase pathways (28, 74). Indeed, cross-talk between the AR NTD and PKA signal transduction pathways in LNCaP cells was recently shown by Sadar (28). Here we demonstrated that IL-6 activates the AR NTD, and this activation was independent of the PKA pathway (Fig. 8). Activation of the AR NTD by IL-6 and forskolin was effectively blocked by an inhibitor of MAPK kinase. This suggests that the MAPK pathway plays an important role not only in cross-talk between IL-6 but also between PKA and the AR signal transduction pathways. These data are the first to show the importance of the MAPK pathway in the activation of the AR NTD. Whether the underlying mechanism of MAPK involves a change in the phosphorylation state of the AR NTD or a change in the phosphorylation state of a co-activator such as steroid receptor co-activator-1, which is phosphorylated by MAPK (75) and interacts with this region of the AR (65), is currently under investigation in our laboratory.

There has been disparity in the literature concerning forskolin activation of the MAPK pathway in LNCaP cells for reasons that remain unknown (60, 76). Our results are consistent with those of Chen et al. (76) showing that forskolin increases MAPK activity. The notion that these compounds may increase PSA gene expression via cross-talk between the MAPK and AR signal transduction pathway is also consistent with the following reports. 1) MAPK activity is increased in androgen-independent prostate cancer, a condition that is clinically defined by increased expression of serum PSA levels in patients treated with androgen withdrawal therapy (77, 78). 2) The MAPK cascade has been shown to activate the AR in prostate cancer cells (50, 79).

The tyrosine kinases JAK1, JAK2, and Tyk2 are constitutively associated with the gp130 (14, 15) and are activated in response to IL-6. The JAK-STAT pathway appears to play an important role for ligand-independent activation of the AR by IL-6 as indicated in the studies presented here and illustrated in our present working model (Fig. 13). Our results demonstrated that an inhibition of JAK blocked activation of the AR NTD, tyrosine phosphorylation of STAT3, and phosphorylation of MAPK in cells exposed to IL-6. These results suggest that activation of JAK by IL-6 is involved in both STAT3 and MAPK pathways in LNCaP cells. Cross-talk between the IL-6-JAK-STAT3 and ligand-bound glucocorticoid receptor pathways have revealed that STAT3 can act as a transcriptional co-activator without it binding to its respective DNA-binding motif (80). Here we show for the first time that STAT3 directly interacts with the amino acids 234-558 of the AR as suggested in co-immunoprecipitation studies and shown in transactivation experiments. These results are consistent with a recent report (26) that STAT3 is required for AR-mediated gene activation in response to IL-6 in LNCaP cells.

View larger version (29K): [in this window] [in a new window]   Fig. 13.   A hypothetical working model of signal transduction pathways leading to ligand-independent activation of the AR by IL-6 in LNCaP cells. IL-6-mediated and forskolin-mediated signals are illustrated. The IL-6 signaling cascade induces up-regulation of AR-regulated genes such as PSA by ligand-independent activation of the AR, which is stimulated by both phosphorylation of STAT3 and MAPK. Stimulation of the PKA pathway using forskolin results in phosphorylation of MAPK. Therefore, the IL-6 and forskolin signaling pathways may converge and amplify the MAPK pathway leading to synergistic activation of the AR NTD.

In summary, our results suggest that in the absence of androgens the AR NTD can be activated by IL-6 by a pathway dependent upon MAPK and STAT3 in prostate cancer cells. Because activation of STAT3 signaling has been shown to be important for the progression of prostate cancer cells (51) and to induce transformation of cells (81), we propose that STAT3 plays an important role in androgen-independent growth and progression of prostate cancer that is clinically determined by androgen-independent increases in PSA gene expression (77). Further support for this hypothesis can be drawn from the detection of elevated levels of IL-6 in the serum of patients with androgen-independent prostate cancer (82) and the fact that IL-6 increases the proliferation of prostate cancer cells as shown here (Fig. 3) and by others (5, 6, 42, 43).

	ACKNOWLEDGEMENTS

We thank N. R. Mawji for excellent technical assistance, Dr. R. Snoek for helpful discussions, and Dr. T. S.-Y. Kim for critical review of the manuscript.

	FOOTNOTES

^* This work was supported by the National Institutes of Health, George M. O'Brien Research Center Grant P50 DK47656, and the United States Army Medical Research and Materiel Command DOD Prostate Research Program Grant DAM17-98-1-8577.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Cancer Endocrinology, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, British Columbia V5Z 4E6, Canada. Tel.: 604-877-6036; Fax: 604-877-6011; E-mail: msadar@bccancer.bc.ca.

Published, JBC Papers in Press, December 19, 2001, DOI 10.1074/jbc.M108255200

	ABBREVIATIONS

The abbreviations used are: IL-6, interleukin-6; NTD, N-terminal domain; MAPK, mitogen-activated protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; AR, androgen receptor; ARE, androgen-response elements; JAK, Janus kinase; STAT, signal transducers and activators of transcription; DBD, DNA-binding domain; PSA, prostate-specific antigen; FBS, fetal bovine serum; EGF, epidermal growth factor; IGF, insulin-like growth factor; KGF, keratinocyte growth factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT, reverse transcriptase; PKA, protein kinase A; PI, phosphatidylinositol; gp, glycoprotein; tk, thymidine kinase; PB, probasin; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES