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Cancer Research 68, 8173, October 1, 2008. doi: 10.1158/0008-5472.CAN-08-2290
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

Androgenic Control of Transforming Growth Factor-β Signaling in Prostate Epithelial Cells through Transcriptional Suppression of Transforming Growth Factor-β Receptor II

Kyung Song1, Hui Wang1,2, Tracy L. Krebs1, Seong-Jin Kim3,4 and David Danielpour1,2

1 Case Comprehensive Cancer Center Research Laboratories, The Division of General Medical Sciences-Oncology, 2 Department of Pharmacology, and 3 Department of Pediatrics, Rainbow Babies and Children's Hospital, Case Western Reserve University, Cleveland, Ohio; and 4 Lee Gil Ya Cancer Center and Diabetes Institute, Gachon University of Medicine and Science, Songdo, Korea

Requests for reprints: David Danielpour, Wolstein Research Building, Room 3-532, 2103 Cornell Avenue, Cleveland, OH 44106. Phone: 216-368-5670; Fax: 216-368-8919; E-mail: dxd49{at}po.cwru.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The androgen receptor cross-talks with transforming growth factor-β (TGF-β) through mechanisms that remain poorly understood. Here we provide strong evidence that 5{alpha}-dihydrotestosterone (DHT) intercepts the ability of prostate epithelial cells to undergo TGF-β–induced apoptosis, and present a new model for this androgenic effect. We report that DHT decreases the level of TGF-β receptor II (TβRII) through a transcriptional mechanism, leading to suppression of the ability of TGF-β to down-regulate expression of Bcl-xL and cyclin Ds, activate caspase-3, and induce apoptosis. Promoter analysis, DNA pulldown, and electrophoretic mobility shift assays support that transcriptional down-regulation of TβRII by DHT occurs through Sp1/Sp3 response elements, with the binding of Sp1 to the TβRII promoter being suppressed by DHT, largely driven by loss of Sp1 protein and/or activity. These results provide fresh insight on the mechanism of growth control by androgens and the progression of prostate cancer to androgen independence. [Cancer Res 2008;68(19):8173–82]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Transforming growth factor-β (TGF-β), which is a multifunctional cytokine with an important role as a potent tumor suppressor in a variety of tissues including the prostate (1–7), propagates signals through two transmembrane serine/threonine receptor kinases, TGF-β receptor type I (TβRI) and type II (TβRII), which directly activate Smad2 and Smad3 by phosphorylating their two COOH-terminal serine residues (8–10). TGF-β is well recognized to induce growth arrest or/and apoptosis of prostate epithelium, occurring through mechanisms that seem to be intricately controlled by epidermal growth factor (EGF), insulin-like growth factor I, phosphatidylinositol 3-kinase/Akt, and androgen receptor (AR; refs. 11–13).

AR is a member of the nuclear receptor superfamily of transcription factors residing predominantly in the cytoplasm as inactive complexes with molecular chaperones, particularly heat shock proteins 70 and 90 (14, 15). Binding of androgen frees AR from its chaperones, thereby promoting the translocation of AR to the nucleus where this receptor functions in the transcriptional control of numerous genes involved in development, growth, and function of androgen target tissues such as the prostate. Although transcriptional responses of AR occur mainly through the direct binding of AR to DNA at AR response elements, the function of AR is influenced by its association with numerous coregulators that also serve as junctions of cross-talk with other signaling pathways (16–19).

One of the important pathways AR cross-talks with is the TGF-β pathway (11, 20–23). In rodents, androgen deprivation leads to rapid elevation in levels of TGF-β, TβRI, and TβRII and activation of Smad2 and Smad3, concomitant with the onset of apoptosis (24–27). Further in vivo and in vitro studies support that androgens promote cell survival, in part, through blocking TGF-β–induced responses, although the underlying mechanisms remain poorly investigated (11, 28–30).

AR has been shown to physically interact with Smad3 in the absence of androgen or TGF-β stimulation (11, 21, 22). We previously reported that the physical interaction of AR with Smad3 confers the ability of 5{alpha}-dihydrotestosterone (DHT) to suppress TGF-β– or Smad3-induced transcription (11). In our model, AR physically interacts with Smad3, and DHT then blocks the association of Smad3 to the Smad binding element (11). Those results suggested that overactivation of AR during prostate tumor progression may lead to loss of TGF-β–induced responses, particularly the ability of TGF-β to function as a tumor suppressor. However, several important issues remained unresolved, principally whether androgens could actually reverse TGF-β–promoted growth arrest or/and apoptosis, the spectrum of TGF-β–regulated genes affected by androgens, and the subset of those gene changes involved in growth control or apoptosis. Here we showed that androgen-bound AR significantly protects cells from TGF-β–induced apoptosis, likely by reversing TGF-β–promoted down-regulation of Bcl-xL and cyclin Ds and activation of caspase-3 in human and rat prostate cell lines. We also provide evidence that androgen broadly influences TGF-β responses by intercepting an additional step in the TGF-β signaling pathway, i.e., by suppressing TβRII expression through a transcriptional mechanism mediated partly by down-regulation of Sp1 activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Materials. Sources were as follows: recombinant human TGF-β1 (R&D Systems), PNGase F (New England Biolab), pGL3-basic-luciferase (Promega), Sp1 consensus (sc-2502) and mutant (sc-2503) oligonucleotides (Santa Cruz Biotechnology), characterized fetal bovine serum (FBS) and dextran-charcoal–stripped FBS (DC, HyClone), and CellTiter 96 Non-Radioactive Cell Proliferation Assay Kit (Promega). For Western blot antibodies, see Supplementary data.

Cell culture. NRP-154 and NRP-152 prostatic epithelial cell lines (31, 32) were maintained in GM2.1 culture medium as described previously (33). The DU145 human prostate cancer cell line was maintained in DMEM/F12 supplemented with 5% FBS. LNCaP, C4-2B, and VCaP human prostate cancer cell lines were maintained in DMEM/F12 containing 10% FBS in poly-D-lysine–coated 75-cm2 culture flask (11). Unless indicated, all experiments in NRP-154 and NRP-152 cells were done in 1% DC-GM3 medium, and experiments involving DU145, LNCaP, C4-2B, and VCaP cells were done in DMEM/F12 supplemented with 1% or 10% DC-stripped FBS and 15 mmol/L HEPES. Experiments in LNCaP and C4-2B cells transiently infected with Admax-TβRII were done in DMEM/F12 supplemented with 1% DC-stripped FBS, 15 mmol/L HEPES, and 20 ng/mL EGF.

Cell number and cell viability assays. CellTiter 96 nonradioactive cell proliferation assay kit (Promega) was used to assess cell viability. In brief, NRP-154/AR4 cells were plated at a density of 5 x 103/100 µL/well in 96-well plates with 1% DC-GM3 medium ±2 µg/mL doxycycline and ±DHT. Cells were incubated for overnight followed by treatment with TGF-β1 for 72 h. Cell viability was expressed as absorbance relative to that of untreated control. For cell number assay, NRP-154 cells in 12-well plates (5 x 104/1 mL/well) were infected with AdMax-AR (1:500) for 2 h and cultured overnight in 1% DC-GM3 ± DHT. TGF-β1 was added and cells were incubated for an additional 48 h. Cell number was assayed using a Coulter Electronics counter as before (34).

Crystal violet nuclei staining. Cells were fixed with 2% formalin/PBS and incubated with 0.2 mg/mL of crystal violet solution for nuclei staining. Cells were then washed twice with 1x PBS and dye was eluted by adding 1% Triton X-100/PBS. The eluted dye was subjected to spectrophotometry at 550 nm.

Northern blot analysis. Northern blot analysis was done essentially as described (35). In brief, 10 µg of total RNA were electrophoresed, and equal loading and even transfer were assessed by visualization of the 18S rRNA. mRNA was detected with cDNA probes labeled with [32P]dCTP using Prime-It RmT Random Primer labeling Kit (Stratagene).

Reverse transcription-PCR. Reverse transcription was done as described (13). Taq Polymerase Master Mix (Promega) was used for PCR amplification of rat TβRII, using 32 or 28 cycles, respectively, of the following temperature gradients: 95°C for 15 s, 60°C for 30 s, and 72°C for 2 min. β-Actin, amplified as above for 21 cycles, served as an internal control. Refer to Supplementary data for primer sequences.

Transient transfection and luciferase assay. Cells were transfected using either the calcium phosphate coprecipitation method (NRP-154) or Invitrogen Lipofectamine plus reagent (LNCaP and DU145) as before (11). Luciferase activity was measured using Promega Dual Luciferase Assay Kit and a ML3000 Microtiter Plate Luminometer. The Sp1 response element reporter construct, Sp1-luc, was made by inserting four copies of a consensus Sp1 response element upstream of the TATA transcription start site (in HindIII and SalI of MCS) of the basic luciferase cloning vector, pCIS-CK (Stratagene).

Adenoviruses. Adenovirus vectors that direct the expression of HA-TβRII (AdMax-HA-TβRII WT; ref. 12) were constructed using the AdMax system (Microbix Biosystems) as described previously (12). For AdMax-AR, the corresponding region to the coding sequence of AR was subcloned from pCDNA3-AR (11) into the adenovirus shuttle vector, pDC515. HEK293 cells in six-well plates were cotransfected with pDC515-AR (1 µg) and 1 µg of the genomic vector, pBHGfrtDE1,3FLP, by calcium phosphate–mediated transfection (11). Virus preparations were done as previously described (12).

DNA fragmentation assay. All of these procedures were done essentially as described (36, 37). DNA was purified and internucleosomal DNA fragmentation was detected using TACS apoptosis DNA ladder kit (Trevigen) according to the manufacturer's instructions.

DNA pulldown assay. Biotin-labeled Sp1 oligonucleotides (WT and mutant, 22-mer) were dimerized with its complements. For each reaction, 1.5 µg of dimer were incubated for 15 min at room temperature with 50 µL of Dynabeads M-280 streptavidin (Invitrogen) washed twice with 2x B&W buffer [10 mmol/L Tris-Cl (pH 7.5), 1 mmol/L EDTA, 2 mol/L NaCl]. After conjugation in 1x B&W buffer, oligo-conjugated beads were washed thrice with 1x B&W buffer to remove unconjugated oligonucleotides and resuspended with ice-cold DNAP buffer [10 mmol/L HEPES (pH 7.9), 100 mmol/L KCl, 5 mmol/L MgCl2, 10% glycerol, 0.5% NP40, 1 mmol/L EDTA] with 1 mmol/L DTT added freshly. Nuclear protein (100 µg) was incubated with oligo-conjugated beads, and reaction volume was adjusted up to 500 µL with 1x DNAP containing Complete EDTA-free Protease inhibitor Mixture (Roche), 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, and 1 mmol/L DTT. Polydeoxyinosinic-deoxycytidylic acid (5 µg) was added to the reaction tube, which was then incubated for 4 h at 4°C with gentle mixing on a rotator. Beads were washed thrice on ice with DNAP containing 1 mmol/L DTT, eluted with 45 µL of 1x SDS buffer by treating for 5 min at 85°C. Eluates were subjected to Western blot analysis.

AR-inducible cell lines, Western blots, and preparation of nuclear and cytosolic extract. See Supplementary data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Androgen protects NRP-154 cells from TGF-β–induced apoptosis. We previously reported that androgens can intercept TGF-β–induced changes in gene expression through a physical interaction of AR with Smad3 in LNCaP and NRP-154 cells transfected with TβRII and AR, respectively. Our electrophoretic mobility shift assay results indicated that AR blocked Smad3 binding to Smad3-binding element (11). However, the effect of androgens on growth suppression and apoptosis was undefined due to lack of a suitable prostate carcinoma cell line that expressed AR and responded to TGF-β by growth suppression and/or apoptosis. To resolve this barrier, we generated an adenoviral system (AdMax-AR) to efficiently express AR (>90% infection) in the NRP-154 cell line, which is exquisitely sensitive to TGF-β–induced apoptosis (37). The AR- or control virus (AdMax-cont)–infected cells were treated with DHT (1 or 10 nmol/L) 24 hours before the addition of TGF-β, and changes in apoptosis and cell morphology were observed 48 hours later (Fig. 1A ). Forty-eight hours of TGF-β1 treatment killed essentially all cells infected with the control or AR virus, whereas treatment with 1 or 10 nmol/L DHT significantly protected AR-expressing cells against killing by TGF-β1 (Fig. 1A, Supplementary Fig. S1A, and data not shown). Such changes were also reflected at the level of total internucleosomal DNA fragmentation (Fig. 1B), a hallmark of apoptosis. We confirmed that ligand-bound AR can also protect against TGF-β–induced death in a doxycycline-inducible, AR-expressing NRP-154 clonal cell line (NRP-154/AR4; Fig. 1C, left) and in nontumorigenic NRP-152 cells infected with AdMax-AR (Supplementary Fig. S1B).


Figure 1
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  		Figure 1. Androgen protects cells from TGF-β–induced cell death in the NRP-154 cell line. A, NRP-154 cells were transiently infected with either AdMax-cont or AdMax-AR (1:500) and cultured ±DHT (1 or 10 nmol/L) for 24 h before 48 h of TGF-β1 (10 ng/mL) treatment. B, the internucleosomal fragmentation was measured following treatment with ±TGF-β1, ±DHT, and ±AdMax-AR in NRP-154. C, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in NRP-154/AR4 cells stably expressing AR and treated as in B (left). DHT was added to NRP-154 cells transiently overexpressing AR for 24 h before TGF-β1 treatment. Five days later, the live cells were cultured in GM2.1 medium for additional 10 d and cell number was assayed using a Coulter Electronics counter (right). D, NRP-154 cells transiently overexpressing AR were cultured in ±DHT for 24 h before treatment with TGF-β1. After 5 d of TGF-β1 treatment, medium was replaced with GM2.1 and cells were cultured for 4 d before crystal violet staining to evaluate cell viability. Cell viability was quantified by spectrophotometry at 550 nm, measuring the dye eluted from cells. Columns, average of triplicate independent experiments; bar, SD. Representative of two to three independent experiments.

 
To examine the duration of such protection, we next assessed changes in total viable cells after allowing the TGF-β ± DHT treated cultures (5 days) to recover in their normal growth medium without exogenous TGF-β or DHT for 4 to 10 days. Our data supported that as little as 0.1 nmol/L DHT enormously protected the cells from TGF-β1–induced apoptosis, as no cells survived the effects of TGF-β unless protected by DHT, giving rise to >104-fold increases in total cell number with DHT versus without DHT [Fig. 1C (right) and D]. Taken together, these data support that AR signaling very effectively suppresses TGF-β–induced apoptosis in prostate epithelial cells.

DHT suppresses the ability of TGF-β1 to induce the expression of plasminogen activator inhibitor-1, activate caspase-3, and suppress the expression of Bcl-xL and cyclin Ds. The above results suggest that androgen-bound AR may interrupt the ability of TGF-β to control the expression of or activate proteins involved in apoptosis or cell cycle control. We previously reported that ligand-bound AR inhibits TGF-β–induced transcriptional responses, using various response element luciferase reporter constructs and the plasminogen activator inhibitor-1 (PAI-1) promoter construct, 3TP-lux (11). To confirm those results at the level of endogenous PAI-l, effects of DHT on TGF-β–induced expression of PAI-1 mRNA and protein were examined in NRP-154 + AR and in LNCaP + TβRII. TGF-β1 strongly induced PAI-1 mRNA and protein levels, occurring in a manner that was abolished by pretreatment with DHT (Fig. 2A ). A bone metastastic derivative of LNCaP, C4-2B, which are androgen refractory likely through having constitutively active AR (38), failed to induce PAI-1 protein expression in response to TGF-β1 despite enforced expression of TβRII (Fig. 2A).


Figure 2
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  		Figure 2. DHT reverses the expression of genes regulated by TGF-β1 in NRP-154 and LNCaP cells. A, NPR-154 ± AR cells were treated with DHT for 24 h before TGF-β1. LNCaP and C4-2B cells were incubated with DHT for 24 h before TGF-β1. PAI-1 was determined by either RT-PCR (left) or Western blot analysis (right). B, NRP-154 ± AR cells were pretreated with DHT for 24 h followed by incubation for additional 48 h (left) or 24 h (right) with TGF-β1. NRP-154 ± AR cells (C) and LNCaP and C4-2B cells (D) were treated with ±DHT and ±TGF-β1. Whole cell lysates (50 µg protein) were subjected to Western blot analysis (B–D). Representative of two to three different experiments or treatments (A–D).

 
We showed that TGF-β1 induces apoptosis, activates caspase-3, down-regulates the antiapoptotic factor Bcl-xL (39), induces cell cycle arrest at G1 and G2-M, and down-regulates cyclin D2 in NRP-154 and NRP-152 (derived from preneoplastic rat prostate). To understand how androgen prevents TGF-β–induced growth suppression and apoptosis, we examined whether DHT could affect the ability of TGF-β to control the expression of antiapoptotic or proapoptotic factors and cyclin Ds. Either 1 or 10 nmol/L DHT significantly blocked TGF-β–induced activation of caspase-3 (Fig. 2B), whereas 10 nmol/L DHT was necessary to reverse the TGF-β1–induced loss of Bcl-xL in NRP-154 ± AR (Fig. 2B), suggesting that the reversal of caspase-3 activation was independent of that of Bcl-xL. We next tested the possibility that androgen treatment can reverse the ability of TGF-β1 to reduce cyclin D expression. In the case of NRP-154 + AR cells, TGF-β1 treatment down-regulated cyclin D3 levels at 24 hours, and D1 and D3 by 48 hours. Such down-regulation of cyclin D3 was reversed by DHT (Fig. 2C). Similar experiments were done in LNCaP and C4-2B cells infected with AdMax-TβRII. DHT slightly increased the expression of all three cyclin Ds in LNCaP cells. In contrast, C4-2B cells expressed high basal levels of all three cyclin Ds, consistent with the expression of constitutively active AR in those cells, and showed no induction by DHT (Fig. 2D). As expected, DHT completely reversed the TGF-β1–mediated down-regulation of cyclin D1, cyclin D2, and cyclin D3 at both 24 hours (Fig. 2D) and 48 hours (data not shown) in LNCaP cells. However, C4-2B cells having constitutively active AR were refractory to the down-regulation of cyclin Ds by TGF-β1 (Fig. 2D). Taken together, the above data suggest that distinct mechanisms control the ability of TGF-β to down-regulate each of the D cyclins and that such differential regulation is cell type specific.

DHT down-regulates the expression of TβRII, but not TβRI. We previously reported that androgens disrupt the binding of Smad3 to Smad3-binding element through a direct association of AR with Smad3 (11). However, further investigation in our group suggested that additional mechanisms may be involved in the disruption of TGF-β responses by androgens. Our study suggested that TGF-β1–activated Smad3 was markedly repressed by androgen.5 Thus, to better understand the mechanism by which AR suppresses TGF-β1 responses, we examined whether DHT may also alter the levels of TGF-β receptors. NRP-154 + AR cells were preincubated with DHT for 48 hours before TGF-β1 treatment, and protein levels of TβRI/II were determined. DHT substantially down-regulated the levels of TβRII, but not TβRI, irrespective of TGF-β1 treatment (Fig. 3A ). We further performed time course experiments of DHT on TβRII expression. TβRII protein levels were substantially down-regulated as early as 3 hours of DHT addition and persisted at the same level up to 48 hours, whereas no change in TβRI protein levels was observed at any of the time points tested (Fig. 3B). Similar to NRP-154, in DU145, ligand-stimulated AR abolished TβRII protein expression, and neither DHT nor AR alone inhibited TβRII expression (Fig. 3C). We were not able to assess the effect of DHT on the regulation of TβRII in LNCaP and C4-2B because their level of endogenous TβRII was undetectable. We therefore used another AR-positive prostate cancer cell line, VCaP, to assess the role of endogenous AR on endogenous TβRII levels. Consistently, 48-hour treatment with DHT repressed the level of TβRII in VCaP (Fig. 3D). Taken together, our data suggest that androgen suppresses TGF-β signaling partly by decreasing TβRII.


Figure 3
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  		Figure 3. Suppression by androgen of the level of TβRII, not TβRI. A and B, effect of androgen on protein levels of TGF-β receptors. NRP-154 ± AR cells were pretreated ±DHT for 48 h before TGF-β1 at the indicted concentrations (A) or for the indicated times before TGF-β1 (10 ng/mL, 1 h; B). C and D, effect of DHT on the level of TβRII in DU145 ± AR (C) and VCaP (D) cells, stimulated with DHT for 48 h. Samples deglycosylated were subjected to Western blotting. Representative of two to three different experiments (A–D).

 
Transcriptional repression of TβRII by DHT. The relatively rapid (3 hours) down-regulation of TβRII protein by DHT (Fig. 3B) suggested a transcriptional mechanism. We therefore examined whether DHT also suppressed the levels of TβRII mRNA. Consistent with the above results, a time course experiment revealed that DHT significantly decreased the mRNA levels of TβRII, with noticeable changes occurring between 4 and 12 hours of treatment (Fig. 4A, left ). DU145 + AR also showed robust loss of TβRII expression following 24 hours of DHT treatment (Fig. 4A, right). Together, these data suggest that DHT controls TβRII protein levels through mRNA stability or transcriptional control rather than protein stability or translational control.


Figure 4
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  		Figure 4. Transcriptional regulation of TβRII by DHT in NRP-154, LNCaP, DU145, and VCaP cells. A, RT-PCR for TβRII in NRP-154 cells and effect of DHT on the level of TβRII mRNA in DU145 cells, by Northern blot analysis. B and C, cells were transfected with 1 µg of total DNA including TβRII promoter-luciferase (pTβRII-luc) constructs and CMV-renilla, followed by DHT treatment for 48 h or the indicated time. For LNCaP cells (B) with pTβRII(–216/+35)-luc, cells were incubated with 1 µmol/L MS275 for an additional 24 h following DHT treatment. Relative values of firefly luciferase normalized to renilla luciferase. Columns, average of triplicate determinations; bars, SE.

 
To determine if DHT can inhibit expression of TβRII through a transcriptional mechanism, we cotransfected various prostate cell lines with ±pCDNA3-AR, TβRII promoter constructs (pTβRII-luc) along with cytomegalovirus (CMV)-renilla, followed by 24 hours ±10 nmol/L DHT, and monitored changes in relative luciferase activity (Fig. 4B). The pTβRII(–216/+35)-luc construct, which has two positive response elements (PRE1 and PRE2), shows optimal promoter activity (40). DHT significantly inhibited this promoter activity by ~85% in NRP-154 + AR and by >90% in DU145 + AR cells (Fig. 4B), consistent with protein and mRNA levels. The full-length TβRII promoter construct, pTβRII(–1670/+38)-luc, although less active, was almost completely (>90%) suppressed by DHT under the same conditions in DU145 + AR cells (Supplementary Fig. S2A). Because LNCaP cells express low to undetectable levels of TβRII and have very low pTβRII-luc activity, we used the histone deacetylase inhibitor MS-275 to enhance this promoter activity, as before (13). DHT (24 hours) effectively suppressed the expression of this promoter in LNCaP treated with 1 µmol/L MS-275. About 50% inhibition of this promoter activity was observed by DHT in VCaP (Fig. 4B).

To identify the specific promoter region responsible for suppression by DHT, cells were transiently transfected with various 5'-end truncations of TβRII promoter-luciferase constructs (ref. 41; Fig. 4C). Surprisingly, the effect of inhibition by androgen was observed in all the truncated TβRII-promoter constructs despite differences in the magnitude of repression between cell lines (Fig. 4C). This unexpected observation led us to examine the involvement of transcription factors essential for transcription or initiation of transcription by TATA-less promoters. In the case of TβRII promoter, Sp1 has been shown to be critical in transcriptional initiation of TβRII, as it is TATA-less. All deletion constructs of this promoter tested (Fig. 4C) contain either one or two Sp1 sites at –143 and –25. We introduced mutations between –63 and +2 at sites corresponding to putative transcription factor binding elements including Sp1 site at –25 (Supplementary Fig. S2B) and tested whether any of those mutants would ablate suppression by DHT. As expected, mutation of the –25 Sp1 site abolished promoter activity in both DU145 and NRP-154 cells, suggesting this Sp1 site functions as a transcriptional start site. Other mutations substantially lowered the basal activity of this promoter but failed to reverse the inhibitory effect of DHT (Supplementary Fig. S2C). Taken together, these data strongly implicate Sp1 as a target of androgen-mediated suppression of the TβRII promoter.

DHT inhibits Sp1 expression. To study the potential role of Sp1 in mediating the suppression of the TβRII promoter by DHT, we measured the protein levels of Sp1 in NRP-154 + AR cells following treatment with DHT for various times. DHT significantly lowered the level of Sp1 by 6 hours of treatment and continued through 48 hours (Fig. 5A ), correlating with levels of TβRII (Fig. 3). Sp1 levels were similarly decreased by DHT in DU145 + AR cells (Fig. 5A).


Figure 5
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  		Figure 5. Sp1 is a mediator for transcriptional regulation of TβRII by DHT. A, effect of DHT pretreatment (48 h) on Sp1 and Sp3 levels in NRP-154 + AR. Time course experiments of DHT in NRP-154 + AR. Levels of Sp1 and Sp3 in DU145 ± AR cells treated with DHT. Sp1 protein levels in whole lysates, nuclei (Nc), and cytosol (Cyt) after DHT treatment (1 nmol/L) in NRP-154 ± AR cells. B, NRP154 + AR, DU145, and VCaP cells were transiently transfected with CMV-renilla and either control vector (pCIS-CK) or Sp1 response element reporter (Sp1-luc). C, DNA pulldown assay; biotin-labeled consensus Sp1 oligonucleotides (WT and mutant) were incubated with nuclear lysates from NRP-154 ± AR cells (input, bottom; left). Effect of overexpressed WT-Sp1 on Sp1-luc activity inhibited by DHT in NRP-154 transiently expressing AR (right). Data shown are relative values of firefly luciferase normalized to renilla luciferase. Columns, average of triplicate determinations; bars, SE. D, schematic representation of our proposed model.

 
Although Sp3 is known to function mainly as a transcriptional suppressor, it may also function as a transcriptional activator. Sp3 binds to the same Sp1 response elements but may either activate or repress transcription, depending on the context of other response elements or transcription regulators. Therefore, we also examined changes in Sp3 expression on the same blots. In contrast to Sp1, DHT did not affect Sp3 expression in NRP-154 + AR; however, DHT down-regulated Sp3, especially the 60-kDa Sp3 isoform in DU145 + AR (Fig. 5A). Collectively, these data suggest that the transcriptional suppression of TβRII by androgen may occur through down-regulation of Sp1/Sp3.

Sp1 is activated by posttranslational modifications that promote its rapid nuclear translocation. We further studied whether the loss of Sp1 protein by androgen in whole cell lysates reflects its level in the nuclear compartment and influences the activity of Sp1. As in Fig. 5A and Supplementary Fig. S3A, no Sp1 was detected in the cytosolic fraction and a markedly low level of Sp1 was measured in the nuclear fraction of DHT-treated cells, showing similar Sp1 levels in parallel whole cell lysates. We next tested the effect of DHT on the transcriptional activity of Sp1 alone by using an Sp1 reporter construct (Sp1-luc), composed of four tandem consensus Sp1 binding elements inserted upstream of the TATA transcription start site of the promoter-less luciferase reporter pCIS-CK. As expected, DHT strikingly inhibited the activity of Sp1-luc (>90%) in DU145, supporting the loss of Sp1 activity caused by DHT (Fig. 5B), reflecting the loss of Sp1 levels in the nucleus (Fig. 5A). Similar results were obtained with NRP-154 + AR and VCaP cells (Fig. 5B). Loss of Sp1 activity was next tested by measuring the physical association of endogenous Sp1 from NRP-154-AR cells to biotinylated WT or mutant Sp1 consensus oligonucleotides that were pulled down by strepavidin-agarose resin (Fig. 5C). Our results showed that Sp1 constitutively binds consensus WT but not mutant Sp1 oligos, and that DHT abolished binding of Sp1 to Sp1 oligo only in cells expressing AR (Fig. 5C). We also performed electrophoretic mobility shift assay using Sp1 consensus oligonucleotides (WT and mutant) and Sp1 binding site in the TβRII promoter region –25 (Supplementary Fig. S3B and C). Androgen-stimulated AR significantly diminished DNA binding to WT but not mutant Sp1 oligonucleotide (Supplementary Fig. S3B). Furthermore, when a 32-mer oligonucleotide designed from TβRII (–25p) was subjected to electrophoretic mobility shift assay, DHT clearly suppressed DNA binding of Sp1 (Supplementary Fig. S3C).

To confirm in our model that loss of Sp1 activity by DHT plays a role in the ability of DHT to suppress Sp1-dependent promoter activity, we attempted to reverse the DHT-dependent loss of Sp1 activity by overexpressing WT-Sp1 in NRP-154-AR cells. As shown in Fig. 5C, the DNA binding activity of Sp1, which was inhibited by ligand-activated AR, was completely restored by exogenously expressed WT-Sp1. Moreover, expression of exogenous Sp1 partially reversed the ability of DHT to diminish TβRII promoter activity in NRP-154 + AR (P < 0.001; Supplementary Fig. S3D). Taken together, the above results strongly support that DHT blocks the transcription of TβRII, at least partly, through down-regulation of Sp1 protein expression and inhibition of total DNA binding resulting from its reduced expression (Fig. 5D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
Here we provide the first evidence supporting that DHT, functioning through AR, suppresses the TGF-β signaling pathway controlling apoptosis and growth arrest. Moreover, we show that DHT stimulation interrupts TGF-β signaling by shutting down the production of newly made TβRII through a transcriptional mechanism. This mechanism is likely to function cooperatively with another mechanism we previously described (11), involving the direct binding of active Smad3 to AR, which blocks the interaction of Smad3 with Smad3-binding element on target genes. Here we report the first observation that DHT down-regulates the expression and activity of Sp1/Sp3, and provide evidence that DHT-induced transcriptional repression of TβRII is, at least in part, mediated by down-regulation of Sp1 levels, leading to reduced association of nuclear Sp1 to Sp1 response elements in the TβRII promoter. Previous studies have clearly established the importance of Sp1 and Sp3 in transcriptional controls of TβRII (42). Further efforts in our laboratory are under way to understand the underlying implications of the DHT-mediated loss of Sp1 activity in the regulation of other androgenic responses and to delineate the mechanism by which androgens down-regulate Sp1 protein levels or its biological activity. These issues are likely to be of fundamental importance in the regulation of androgenic responses, considering the broad range of TATA-less genes that may be influenced by such changes in Sp1 activity. Preliminary reverse transcription-PCR (RT-PCR) data from NRP-154-AR cells showed that levels of Sp1 mRNA were not significantly altered by DHT (Supplementary Fig. S4), suggesting that down-regulation of Sp1 by DHT occurs at the level of protein stability or translational control, rather than mRNA stability or transcriptional control. Although our data support a role for Sp1 as a mediator of transcriptional control of TβRII by DHT, we believe that other transcriptional factors such as CBF-A and YY-1, which are slightly suppressed by ligand-bound AR (preliminary study and data not shown), may also play a role in such regulation. Therefore, we speculate that androgen regulation of TβRII is more complex, involving multiple transcriptional factors.

Our data here are consistent with in vivo studies on the rat prostate where androgen ablation induced by castration was shown to activate the TGF-β signaling not only by inducing the expression of TGF-βs but also by elevating the levels of TβRII and Smad3 activation (24, 26, 27). We thus suggest that androgens may protect against apoptosis of prostate epithelial cells through multiple mechanisms, involving loss of TGF-β1 and TβRII and the direct association of Smad3 with AR. The latter mechanism is more likely to account for suppression of TGF-β responses in LNCaP-TβRII, where TβRII expression is under the control of CMV promoter (not controlled by androgen).

It is well known that prostate cancers ultimately fail antiandrogen therapy as they progress toward the state of androgen independence. Although the mechanisms behind this are yet to be resolved, growing evidence supports that the AR signaling pathway is constitutively activated rather than suppressed, most likely through AR mimicking the conformation of ligand-activated AR but without androgen (18). AR silencing studies show that androgen-independent prostate cancer cells require AR for survival, parallel to the requirement of androgen in androgen-dependent epithelial cells (43). Regardless of the mechanisms involved, our study suggests that constitutive activation of the AR pathway may suppress TGF-β signaling through down-regulation of TβRII expression. Consistent with our observation is the C4-2B cell line bearing constitutively active AR; these cells are resistant to TGF-β even when TβRII is overexpressed. In fact, even exogenous TβRII was difficult to detect in C4-2 cells. Moreover, EGF, which enhances expression of TβRII in LNCaP cells (13), failed to elevate TβRII levels in C4-2B cells or sensitize them to TGF-β. Our preliminary data showed no differences between LNCaP and C4-2B cells in the efficiency of transfection or infection. This result therefore indicates that constitutively activated AR in C4-2B cells may promote loss of exogenous TβRII, suggesting that an additional mechanism may be operating in androgen refractory prostate cancer that leads to loss of TβRII. Such a mechanism may contribute to the overall loss of TβRII expression found in human prostate cancer and correlate with poor clinical outcome. Based on these observations and ideas, we propose that constitutive activation of AR during prostate cancer progression may cause loss of the tumor suppressor function of TGF-β in prostate carcinoma cells, thus aiding in tumor progression.

A recent report shows that loss of the tumor suppressor PTEN, occurring at high incidence in late-stage prostate cancers and leading to constitutive activation of Akt and mammalian target of rapamycin, promotes androgen independence (44). We previously showed that the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway can suppress TGF-β signaling through suppression of the activation of Smad3 (33). Thus, loss of PTEN may promote androgen independence, at least partly, by suppressing TGF-β signaling through an AR-independent mechanism or/and through a mechanism involving the activation of AR by the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway. Regardless of the specific mechanisms involved, loss of growth suppression or apoptosis by TGF-β is likely to enhance tumor growth. Thus, restoring TGF-β responses in androgen-independent epithelial cells, perhaps by intercepting the ability of AR to disrupt TGF-β signaling, is likely to have important therapeutic implications.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: National Cancer Institute grants R01CA092102 and R01CA102074 (D. Danielpour).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Joan Massagué for 3TP-lux plasmid and Dr. Harvey Lodish for pCMV5-TβRII.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

5 Unpublished data. Back

Received 6/17/08. Revised 7/16/08. Accepted 7/21/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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