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Endocrinology Vol. 147, No. 10 4599-4607
Copyright © 2006 by The Endocrine Society

Growth Inhibitory Concentrations of Androgens Up-Regulate Insulin-Like Growth Factor Binding Protein-3 Expression via an Androgen Response Element in LNCaP Human Prostate Cancer Cells

Lihong Peng, Peter J. Malloy, Jining Wang and David Feldman

Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5103

Address all correspondence and requests for reprints to: Dr. David Feldman, Division of Endocrinology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305-5103. E-mail: feldman{at}cmgm.stanford.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding protein-3 (IGFBP-3), the most abundant circulating IGF binding protein, inhibits cell growth and induces apoptosis by both IGF-I-dependent and -independent pathways. The ability of IGFBP-3 to inhibit tumor growth has been demonstrated in many cancers including prostate cancer (PCa). High concentrations of androgens, which inhibit the growth of the LNCaP human PCa cell line, have been shown to have both positive and negative effects on IGFBP-3 expression by different laboratories. To further explore the relationship between IGFBP-3 and androgens, we examined IGFBP-3 expression in LNCaP cells. We demonstrate that IGFBP-3 expression can be induced by 10 nM of the synthetic androgen R1881 or dihydrotestosterone. Transactivation assays show that the 6-kb IGFBP-3 promoter sequence directly responds to androgen treatment. In silico analysis identified a putative androgen response element (ARE) at –2879/–2865 in the IGFBP-3 promoter. A single point mutation in this ARE disrupted transactivation by R1881. Combining the data obtained from EMSA, chromatin immunoprecipitation and mutational analysis, we conclude that a novel functional ARE is present in the IGFBP-3 promoter that directly mediates androgen induction of IGFBP-3 expression. Furthermore, we found that the combination of androgens and calcitriol significantly potentiated the IGFBP-3 promoter activity, suggesting that enhanced induction of the expression of the endogenous IGFBP-3 gene may contribute to the greater inhibition of LNCaP cell growth by combined calcitriol and androgens. Because androgens are well known to stimulate PCa growth and androgen deprivation therapy causes PCa to regress, the stimulation by androgens of this antiproliferative and proapoptotic protein is paradoxical and raises interesting questions about the role of androgen-stimulated IGFBP-3 in PCa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF BINDING PROTEIN (IGFBP)-3 is a member of the IGFBP family that bind the potent mitogen IGF-I with high affinity and specificity (1, 2, 3). It is thought that IGFBPs serve to extend the half-life of IGF-I as well as transport and modulate the biological actions of IGFs on target cells. IGFBP-3 is the most abundant circulating carrier protein binding more than 75% of serum IGF-I (1, 2, 3). In addition to sequestering IGF-I to diminish the growth stimulating action of IGF-I in an IGF-dependent manner, IGFBP-3 also exhibits multiple IGF-independent actions, including inhibition of cell growth and induction of apoptosis (1, 2, 3, 4, 5, 6, 7). Other steps in the mechanism of IGFBP-3 action include binding to TGFß type V receptor (8), translocation to the nucleus via the importin ß-subunit (9), and interacting with the nuclear retinoid X receptor-{alpha} (10).

Epidemiological studies have suggested that high plasma levels of IGF-I and low concentrations of IGFBP-3 are associated with increased risk of prostate cancer (PCa) (11, 12, 13), suggesting that high IGF-I and low IGFBP-3 may be potential risk markers of PCa. IGFBP-3 has been shown to promote apoptosis and inhibit cellular proliferation in a variety of cell lines including PCa cells in an IGF-independent manner (1, 2, 3, 4, 5, 6, 7, 14, 15, 16). Recently Liu et al. (17) reported that the combination of IGFBP-3 with retinoid X receptor ligands resulted in a synergistic effect to suppress the growth of PCa xenografts by inducing apoptosis. Using a transgenic mouse model, Silha et al. (18) demonstrated that at the early stages of the development of prostate cancer, overexpression of IGFBP-3 attenuates tumor growth via IGF-dependent actions, and as the cancer advances, IGFBP-3 suppresses tumor growth entirely through IGF-independent mechanisms (3, 18). Both animal and preclinical studies provide evidence that IGFBP-3 is an important antitumor agent (1, 2, 3, 4, 5, 6, 7, 17, 18, 19).

Previously we found that calcitriol induction of IGFBP-3 importantly contributed to its antiproliferative actions (20). We identified a functional vitamin D response element (VDRE) in the IGFBP-3 promoter that directly mediates calcitriol actions to induce IGFBP-3 expression (21). p53 has also been shown to regulate IGFBP-3 synthesis via a direct protein/gene interaction (22). In LNCaP cells either positive or negative regulation of IGFBP-3 expression by androgens has been reported by several groups (23, 24, 25, 26, 27). Goossens et al. (23), Arnold et al. (25), and Kojima et al. (27) showed an inhibitory effect of androgens on expression of IGFBP-3. On the other hand, Martin and Pattison (24) observed a stimulatory effect of androgens on IGFBP-3. The reason for these conflicting results remains unclear.

Androgens are essential for the development of the normal prostate and the maintenance of prostate function (28). In addition to the well-known ability of androgens to stimulate prostate growth and androgen deprivation therapy to cause prostate cancer regression (29, 30, 31), a number of studies have demonstrated that androgens can exert antiproliferative and prodifferentiative actions on PCa cells (32, 33, 34, 35, 36, 37). Other studies in LNCaP cells demonstrate that low doses of androgen stimulate cell growth, whereas high concentrations result in inhibition of cell growth (33, 38, 39, 40). The mechanism for these divergent or biphasic actions remains unknown. Androgen actions are mediated by the androgen receptor (AR), a member of the steroid, thyroid, retinoid receptor superfamily of nuclear transcription factors (28). AR binds as a homodimer to androgen response elements (AREs) containing an inverted repeat of the core sequence (5'-TGTTCT-3') separated by a three-nucleotide spacer (41).

In the present study, we used the androgen-responsive LNCaP cells as a model to study the regulation of IGFBP-3 by growth inhibitory concentrations of androgens and determine the molecular mechanism involved. We provide the first evidence that the induction of IGFBP-3 by androgens is mediated through a novel functional ARE site in the distal region of the IGFBP-3 promoter and is possibly involved in the growth-inhibitory action of androgens. Furthermore, we demonstrate that the interaction of calcitriol and androgens leads to a substantial increase in IGFBP-3 promoter activity that is mediated through their respective response elements in the IGFBP-3 promoter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The LNCaP cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium containing 5% fetal bovine serum (FBS) at 37 C in a humidified atmosphere with 5% CO2. During all treatments, the serum concentration was reduced to 2% FBS.

Cell growth assay
LNCaP cells were plated at low density (5 x 104) in six-well plates and grown in 5% FBS-RPMI 1640 medium. After 24 h incubation, the cells were treated with vehicle or androgens in triplicate for 6 d. Fresh medium with androgen was replenished 72 h later in all experiments. At d 6, the cells were processed for DNA quantitation using the Burton reagent (42).

Flow cytometric analysis (FACS)
LNCaP cells were plated at 3 x 105 per well in six-well plates in 5% FBS-RPMI 1640 medium. On the next day, the cells were treated with ethanol or R1881 (at 0.1 or 10 nM). After 2 d of growth, the cells were trypsinized and harvested. The pellets were then washed in PBS buffer containing 5 mM EDTA and fixed in ice-cold 0% ethanol for at least 1 h at 4 C (43). RNA in the fixed cells was digested for 30 min at room temperature with the addition of 100 µg/ml RNase A. Then cells were stained with 50 µ/ml propidium iodide (Sigma, St. Louis, MO). The cell cycle distribution was determined by FACS and analyzed using the FlowJo software (Tree Star, Ashland, OR).

Preparation of RNA and quantitative real-time RT-PCR (qRT-PCR)
Total RNA was isolated using RNeasy minikit (QIAGEN, Valencia, CA). cDNA was synthesized from 2.5 µg of total RNA with random hexamer primers and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Real-time PCR analysis was performed using DNA Engine Opticon fluorescence detection system (MJ Research, Foster City, CA). The PCR was set up in 20 µl with 0.25 µM of each primer; 2 µl of 1:3 diluted cDNA template; and 10 µl of 2x Master Mix containing modified DNA polymerase, PCR buffer, SYBR Green I, 5 mM MgCl2, and 4 mM deoxynucleotide triphosphate mix including deoxyuridine 5-triphosphate supplied from the DyNAmo SYBR Green qRT-PCR kit (MJ Research). The cycling was performed using the following program: 95 C 5 min, 40 cycles of 94 C 30 sec, 58 C 30 sec, and 72 C 30 sec. Comparative cycle threshold method (2-{Delta}{Delta}CT) was used to semiquantitate the target signal change in treatment vs. control (44). The TATA-binding protein gene was used as an internal control to normalize the amount of cDNA added to the PCR. The following primers were used: IGFBP-3, 5'-AAGTTCCACCCCCTCCATTC-3' (sense), 5'-TCTTCCATTTCTCTACGGCAG-3' (antisense); TATA-binding protein, 5'-TGCTGAGAAGAGTGTGCTGGAG-3' (sense), 5'-TCTGAATAGGCTGTGGGGTC-3' (antisense).

Determination of IGFBP-3 secretion by LNCaP cells
IGFBP-3 levels in conditioned medium secreted by LNCaP cells were measured using an ELISA kit according to the manufacturer’s instructions (Diagnostic Systems Laboratories, Webster, TX).

Plasmid constructions
The IGFBP-3 promoter reporter constructs containing –5992/+55, –3590/+55, and –1901/+55 fragment in pGL3-Basic (Promega, Madison, WI) were previously reported (21). A 1.85-kb fragment from –3590 to –1753 was cloned into the heterologous thymidine kinase (TK) promoter-driven luciferase reporter (LUC) (45). Synthetic oligonucleotides for IGFBP-3 ARE (–2879/–2865) were annealed and cloned into MluI/XhoI sites in TK-LUC. Annealed BP3-VDRE oligonucleotides (21) were ligated to the BP3-ARE/TK construct to generate the BP3-ARE/VDRE construct. A single-point mutation (G to T) in the IGFBP-3 ARE site was introduced into the –3590/+55/pGL3-LUC reporter construct using the GeneEditor in vitro site-directed mutagenesis system (Promega) following the manufacturer’s instructions. Oligonucleotides containing point mutations in the BP3-ARE or BP3-VDRE sequence with MluI/XhoI site overhangs were synthesized and directly cloned into the TK-LUC vector. Positive clones were confirmed by sequencing.

Transfection analysis
LNCaP cells were plated at a density of 3–5 x 105 cells in six-well plates the day before transfection. At approximately 75% confluence, the cells were transfected with 1 µg DNA using 2 µl of TransIT-Insecta transfection reagent per well (Mirus, Madison, WI). Ten nanograms of the Renilla luciferase plasmid pRL-null (Promega) were included in each transfection to control for the transfection efficiency. After 20 h the cells were treated with ethanol or 10 nM R1881, a synthetic androgen, in the absence or presence of calcitriol and incubated for an additional 18 h. The cells were lysed using passive lysis buffer (Promega). Luciferase activity was determined using the dual-luciferase assay system (Promega) that was normalized to the Renilla luciferase activity. Activity of each construct was expressed as fold induction over control.

Isolation of nuclear extracts and EMSA
Nuclear extracts from LNCaP cells were isolated using the 1-h minipreparation technique (46). EMSAs were performed with modifications as described previously (21, 47). Annealed BP3-ARE oligonucleotides (5'-CCAAAGGCTCTTTCAGTTCTAGGA-3') were radiolabeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. DNA binding reactions were carried out in a total volume of 20 µl containing 4 mM HEPES (pH 7.9), 50 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 10% glycerol, 2 µg poly(dI:dC), and 5 µg BSA. Nuclear extracts (10 µg) were added along with 0.1–0.4 ng radiolabeled probes (50,000 cpm/reaction) and incubated for 20 min at room temperature. For competition assays, a 100-fold molar excess of unlabeled oligonucleotides was added to the binding reaction mixture 15 min before the addition of the labeled probe. The DNA-protein complexes were separated on 4% nondenaturing polyacrylamide gels (29:1) and processed as described previously (21).

Chromatin immunoprecipitation assay (ChIP)
Confluent LNCaP cells were treated with ethanol or 10 nM R1881 for 20 min and subjected to ChIP assay using AR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (21). The purified DNA fragments were subjected to PCR using primers (sense: 5'-AGATTTCCTGTCTCCACCAATACG-3'; antisense: 5'-TGCTTTTCTTCTGCTCAGCCC-3'), which amplify a 259-bp fragment spanning the BP3-ARE in the IGFBP-3 promoter. The PCR was carried out at 95 C for 5 min and then 30 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec with a final extension for 8 min at 72 C.

Western blot analysis
LNCaP cells were treated with vehicle or R1881 for 40 h. Extracts were prepared in radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA] containing a protease inhibitor tablet (Roche Molecular Biochemicals, Indianapolis, IN). Supernatants were subjected to SDS-PAGE and Western blot analysis using p21, p27, and ß-actin mouse monoclonal antibodies (Santa Cruz Biotechnology) as described previously (45).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of growth arrest and stimulation of cyclin-dependent kinase (CDK) inhibitors by androgens in LNCaP cells
Previous studies from different groups have demonstrated that LNCaP cells exhibit a biphasic growth pattern in response to androgens with low concentrations stimulating cell proliferation and high concentrations inducing growth arrest (33, 38, 39, 40, 48). In our hands, LNCaP cells have always shown this biphasic effect (40). As shown in Fig. 1AGo, 0.1 nM R1881 almost doubled the amount of DNA, whereas 1 and 10 nM R1881 caused an approximate 50% reduction in DNA content, compared with the vehicle control, reflecting a typical biphasic growth response of LNCaP cells to androgens, either R1881 or dihydrotestosterone. In concurrent experiments, we examined the effect of the growth-inhibitory dose of R1881 on the level of the CDK inhibitors p21 and p27 by Western blot, proteins associated with cell cycle arrest. Consistent with previous reports (49, 50), addition of 10 nM R1881 caused an increase in CDK inhibitors p21 and p27 (Fig. 1BGo). This increase resulted in about 11% more cells accumulating in the G1 phase of the cell cycle accompanied by a decrease in the S/G2/M fraction as shown in Table 1Go. In contrast, 0.1 nM R1881 with growth-stimulatory effect resulted in a small but significant decrease in the proportion of cells in G1 with an increase in G2/M/S phase. Our observations confirm previous reports that G1 arrest contributes to growth inhibition induced by high concentrations of androgens (48).


Figure 1
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  		FIG. 1. A growth-inhibitory dose of R1881 induces the levels of p21 and p27 in LNCaP cells. A, LNCaP cells were treated with increasing concentrations of R1881 (0–10 nM). Media were changed every 3 d and fresh hormones added. On d 6, the cells were harvested for DNA assay. The DNA content of each treatment was expressed as percentage of control, the mean ± SE of three experiments. *, P < 0.05, compared with the control group. B, LNCaP cells were treated with vehicle or 10 nM R1881 for 48 h. The cell pellets were subjected to protein extraction and then Western blot analysis of p21, p27, and ß-actin.

 

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  		TABLE 1. Effecs of R1881 treatment on cell cycle in LNCaP cells

 
A growth-inhibitory dose of androgen up-regulates IGFBP-3
To determine whether high concentrations of androgens affect the expression of IGFBP-3, an important growth regulator, we treated LNCaP cells with 10 nM of androgens and analyzed IGFBP-3 gene expression over time. As shown in Fig. 2AGo, R1881 treatment, relative to control, elicited a significant 2.5-fold increase in IGFBP-3 gene transcription after 6 h. The IGFBP-3 mRNA levels continued to increase with longer incubation times. IGFBP-3 protein levels were unchanged at 6 h but increased after 12 h and remained elevated after 24 and 48 h (Fig. 2BGo). Addition of cycloheximide, a protein synthesis inhibitor, failed to block IGFBP-3 induction by androgens (data not shown). These results suggest that R1881 may directly regulate the expression of IGFBP-3 in LNCaP cells. Similar findings were obtained with dihydrotestosterone (data not shown).


Figure 2
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  		FIG. 2. Growth-inhibitory doses of androgens increase IGFBP-3 mRNA and protein expression in LNCaP cells. Confluent LNCaP cells were treated with vehicle or 10 nM R1881 and samples taken over time (A and B). A, RNA was prepared for qRT-PCR analysis. B, The conditioned medium was collected for determining IGFBP-3 levels using an ELISA. C, LNCaP cells were treated with 10 nM R1881 in the absence or presence of 10 µM casodex (Cdx) for 16 h and then processed for IGFBP-3 mRNA and protein. Values are expressed as fold change over control. The data represent three separate experiments (mean ± SE). *, P < 0.05; **, P < 0.01, compared with the vehicle-treated group.

 
We next determined whether the increase in IGFBP-3 by R1881 was AR dependent using the androgen antagonist casodex. Addition of 10 µM casodex blocked IGFBP-3 induction by R1881 demonstrating that IGFBP-3 induction is mediated by the AR (Fig. 2CGo).

Transactivation of the IGFBP-3 promoter by androgens
To determine whether androgens directly regulate the transcription of IGFBP-3, we transfected LNCaP cells with IGFBP-3 promoter reporter constructs previously described by our laboratory (21). As shown in Fig. 3Go, the –1901/+55 construct was unresponsive to R1881 treatment, whereas the –3595/+55 construct exhibited approximately 1.9-fold induction by R1881. No further increase in transactivation was observed when fragments up to –5992 were tested. The results show that the region from –3595 to –1901 in the IGFBP-3 promoter contains a potential ARE.


Figure 3
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  		FIG. 3. Transcriptional activation of the human IGFBP-3 promoter by R1881. Fragments of the IGFBP-3 promoter sequence were cloned into the pGL3-basic reporter vector as previously described (21 ). Transfected LNCaP cells were treated with vehicle or 10 nM R1881 for 18 h. A Renilla luciferase expression vector was used to control for transfection efficiency. The activity of each construct is expressed as treatment vs. control that is set at 1. Each value represents the mean ± SE of three independent transfections, each performed in triplicate. *, P < 0.05, compared with the control group.

 
Identification of a functional ARE in the IGFBP-3 promoter
To determine whether the sequence between –3590 and –1901 can act as an enhancer element, we cloned a 1.85-kb fragment from –3590 to –1753 into a luciferase reporter driven by the TK-LUC promoter (45). As shown in Fig. 4AGo, in transactivation assays in LNCaP cells, the 1.85 kb sequence exhibited approximately 2-fold increase in luciferase activity after R1881 treatment. Addition of casodex blocked the induction by R1881 (Fig. 4AGo). The empty TK-LUC vector was unresponsive to R1881 (data not shown).


Figure 4
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  		FIG. 4. Characterization of an ARE in the IGFBP-3 promoter. A, The TK-LUC heterologous construct containing the 1.85-kb fragment from –3590 to –1753 of the IGFBP-3 promoter was transfected into LNCaP cells and then treated with 10 nM R1881 or 10 µM casodex (Cdx) or 10 nM R1881 plus 10 µM casodex (Cdx+R). The activity of the construct was expressed as fold increase over control. B, Comparison of a potential ARE site at –2879/–2865 in the IGFBP-3 promoter (BP3-ARE) to the ARE consensus and the well-characterized ARE in the secretory component promoter (SC ARE1.2). The consensus nucleotides for AR binding are illustrated in bold, and lower-case letters represent variations form the consensus. C, The potential ARE was cloned into the TK-LUC vector. The construct was transfected into LNCaP cells and treated with vehicle or 10 nM R1881. The activity of the construct is displayed as fold induction by R1881 over the control. All transfections were carried out in triplicate, and the data represent the mean ± SE of three experiments. *, P < 0.05, compared with the vehicle-treated group.

 
In silico analysis of this 1.85-kb fragment identified a potential ARE located between –2879 and –2865. As shown in Fig. 4BGo, the putative BP3-ARE (5'-GGCTCT TTC TGTTCT-3') contains two partial direct repeats separated by a three-nucleotide spacer. (The bold letters indicate the consensus sequence.) The BP3-ARE is 92% identical with the ARE of the secretory component promoter (SC ARE1.2) and 87% identical with the ARE consensus sequence (41, 51, 52). The SC ARE1.2 has been characterized as a high-affinity AR-specific ARE (51).

To determine whether the BP3-ARE sequence functions as an ARE, we subcloned the BP3-ARE sequence directly into the TK-LUC vector (BP3-ARE/TK). LNCaP cells were transfected with the BP3-ARE/TK heterologous reporter. Treatment with R1881 resulted in an approximately 2-fold induction of luciferase activity (Fig. 4CGo). To further confirm the BP3-ARE acts as an enhancer element, we created a G to T mutation (TGTTCT to TtTTCT) (Fig. 5AGo) in the BP3-ARE sequence in the –3590/+55 native promoter reporter construct as well as the BP3-ARE/TK heterologous construct. As shown in Fig. 5BGo, the mutation resulted in a loss of androgen inducibility for both constructs.


Figure 5
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  		FIG. 5. The BP3-ARE is a functional ARE. A, Sequences of wild-type BP3-ARE or mutated BP3-ARE (BP3-AREmt). The lower-case letter represents the mutated base. B, The BP3-AREmt mutation (A) was introduced into the BP3–3590/+55 promoter construct and the BP3-ARE heterologous construct. These constructs were transfected into LNCaP cells and treated with vehicle or 10 nM R1881 for 18 h. The activity of each construct is expressed as fold induction over control. Data represent three independent experiments (mean ± SE), each performed in triplicate. wt, Wild type; mt, BP3-AREmt. *, P < 0.05, compared with the vehicle-treated group.

 
Confirmation of a novel ARE in the IGFBP-3 promoter
EMSAs were used to determine whether AR binds to the putative IGFBP-3 ARE sequence. [32P]-labeled BP3-AREs were incubated with LNCaP nuclear extracts in the presence or absence of R1881. A strong specific DNA-protein complex was observed with the androgen-treated cell extract (Fig. 6AGo, lane 1). When a 100-fold molar excess of unlabeled BP3-ARE or SC ARE1.2 oligonucleotide was added, the shifted band signal was diminished (Fig. 6AGo, lanes 2–3). In contrast, the DNA binding was not competed when unlabeled mutant BP3-AREmt oligonucleotides were added (Fig. 6AGo, lane 4).


Figure 6
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  		FIG. 6. The AR binds to the BP3-ARE in vitro and in vivo. A, EMSA showing specific binding of the BP-3 ARE with LNCaP nuclear extracts. LNCaP cells were treated with vehicle or 10 nM R1881 for 2 h and nuclear extracts prepared. The nuclear extracts were incubated with radiolabeled BP3-ARE in the absence (lane 1) or presence of 100-fold molar excess of cold competitor oligonucleotides as indicated. B, ChIP assay. LNCaP cells were treated with vehicle or 10 nM R1881 for 20 min. The cells were collected and subjected to ChIP assay using an AR antibody. The 259-bp PCR product encompasses the BP3-ARE sequence in the IGFBP-3 promoter. Lanes 1–2, Input DNA; lanes 3–4, IgG, mouse IgG; lanes 5–6, AR, anti-AR antibody; lane 7, 100-bp DNA ladder.

 
ChIP assays were performed to determine whether R1881 recruits AR to the chromatinized BP3-ARE in vivo. LNCaP cells were treated with or without 10 nM R1881 and then subjected to ChIP assay. PCR was performed with the purified immunoprecipitated DNA using primers designed to amplify the IGFBP-3 promoter sequence spanning the BP3-ARE. As shown in Fig. 6BGo, in the AR antibody (lanes 5–6) immunoprecipitation reactions, a 259-bp DNA fragment was amplified only in the androgen-treated cells (lane 6), indicating that the AR interacts with the BP3-ARE in vivo in a ligand-dependent manner. The results further confirm the presence of a functional ARE in the natural chromatin structure in intact cells.

Taken together, our data strongly demonstrate the presence of a functional BP3-ARE in the human IGFBP-3 promoter that is activated by the AR in response to the androgens.

Androgens and calcitriol synergistically activate the IGFBP-3 promoter through their respective response elements
We previously demonstrated that calcitriol up-regulates IGFBP-3 through a VDRE in the IGFBP-3 promoter (21). The VDRE site is approximately 400 bp upstream of the ARE site in the IGFBP-3 promoter. The physical position of each response element in the native promoter is shown in Fig. 7AGo. To examine the combined effects of androgens and calcitriol on the IGFBP-3 promoter, a DNA fragment containing BP3-ARE and BP3-VDRE sequences in the IGFBP-3 promoter was cloned into the TK-LUC vector. Mutant constructs containing either a mutated ARE site or mutated VDRE site or both mutated sites were also examined. As shown in Fig. 7BGo, the intact sequence responded to each hormone individually and exhibited a greater activation in the presence of both ligands. Mutations in either the ARE or VDRE sites resulted in the loss of response to the corresponding hormone. Mutation of both response elements eliminated the response to both ligands.


Figure 7
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  		FIG. 7. IGFBP-3 regulation by R1881 and calcitriol is directly mediated through interaction with ARE and VDRE in the IGFBP-3 promoter. A, Schematic structure of the IGFBP-3 promoter showing positions of hormone response elements VDRE –3296/–3282 and ARE –879/–2865 relative to the transcription start site (+1). B, Single copies of the wild-type (wt) or mutant (mt) ARE and wt or mt VDRE oligonucleotide were cloned into the TK-LUC vector. The heterologous constructs were transfected into LNCaP cells and treated with vehicle, 10 nM R1881 (R), 10 nM calcitriol (1,25D), or 10 nM R1881 plus 10 nM calcitriol (R+D) for 18 h. The transactivation activity of each construct is expressed as treatment vs. control, which is set as 1. Each value represents the mean ± SE of three transfections, each performed in triplicate. *, P < 0.05; **, P < 0.01, when compared with the vehicle-treated group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we first verified that our LNCaP cell system displayed a biphasic growth response to androgens (Fig. 1AGo) as previously reported (33, 38, 39, 40). A growth-inhibitory dose of R1881 induced the levels of the CDK inhibitors p21 and p27 (Fig. 1BGo), thus causing accumulation of cells in G1 (Table 1Go). In contrast, the growth-stimulatory dose of R1881 triggered an increase in cells in the S/G2/M phase (Table 1Go). In our LNCaP cells, we found that growth-inhibitory doses of androgens up-regulated IGFBP-3 gene expression by a direct interaction of the AR with an ARE in the IGFBP-3 promoter. Based on the ability of casodex to block the induction, the androgen action on IGFBP-3 is ligand and AR dependent (Figs. 2CGo and 4AGo). The presence of a functional ARE motif is strongly supported by the fact that the BP3-ARE sequence confers androgen responsiveness in both its natural promoter setting and a heterologous promoter (Figs. 3Go and 4Go). Mutations in the BP3-ARE eliminate androgen induction by either heterologous or natural promoters (Fig. 5Go). EMSAs and ChIP assays demonstrate specific recognition and binding of AR to the ARE sequence in vitro and in vivo (Fig. 6Go). Furthermore, we find that the combination of calcitriol and androgens results in a greater induction in IGFBP-3 promoter activity than is individually mediated through the interaction of their receptors with their respective hormone response elements (Fig. 7Go).

The BP3-ARE sequence exhibits a high degree of similarity with the SC ARE1.2 (5'-GGCTCT TTC AGTTCT-3') located in the secretory component (sc) gene and the PB-ARE-2 (5'-GGTTCT TGG AGTACT-3') found in the probasin promoter. Each of these AREs has been demonstrated to be specifically recognized by the AR with high binding affinity (51, 53). There are high homologies among steroid nuclear receptors, the glucocorticoid receptor (GR), progesterone receptor, and AR so that they sometimes are capable of overlapping into the same response element. The ability of some response elements to be highly selective for the AR probably acts in concert with other mechanisms to generate androgen-specific regulation of transcription in vivo (41).

It is known that within the steroid nuclear receptor core binding site (5'-TGTTCT-3') guanine and cytosine at position 2 and 5, respectively, are essential for high-affinity binding of this family including GR and progesterone receptor as well as AR (28, 54). On the other hand, the residues at position 3 and 4 determine specific binding of the family members. Verrijdt et al. (51) demonstrated that in the SC ARE1.2 (5'-GGCTCT TTC AGTTCT-3'), the thymidine residue at position 4 in the 5' half-site is responsible for specific AR binding as opposed to GR binding. Therefore, the striking resemblance of the BP3-ARE and SC ARE1.2 and the presence of a thymidine at position 4 in the 5' half-site of the BP3-ARE (5'-GGCTCT TTC TGTTCT-3') suggest that the BP3-ARE may be an AR-selective element.

IGFBP-3 expression has been shown to be regulated by a variety of specific growth stimulators and inhibitors including TGFß (55, 56), retinoic acid (56), calcitriol (20, 21, 57), TNF{alpha} (58), the histone deacetylase inhibitors trichostatin A and sodium butyrate (59), and androgens (23, 24, 25, 26, 27) as well as p53 (22, 56, 57, 58). Calcitriol exerts its antiproliferative actions by promoting G0/G1 cell cycle arrest, inducing IGFBP-3 production and stimulating apoptosis (60). In the androgen-dependent LNCaP cell line, calcitriol up-regulates the expression of the IGFBP-3 gene through a VDRE located at –3296/–3282 in the distal region of the IGFBP-3 promoter (21). Calcitriol-induced IGFBP-3 expression leads to increased expression of p21, causing cell cycle arrest (20). Evidence has shown that up-regulation of IGFBP-3 expression plays an important role in the in vivo growth-inhibitory effects initiated by calcitriol and its analogs (60, 61). On the other hand, the effect of androgens on the regulation of the IGFBP-3 gene remains controversial (23, 24, 25, 26, 27). Here we show a direct stimulatory effect of androgens on IGFBP-3 expression via AR-dependent action on the IGFBP-3 promoter. This stimulatory effect is similar to data from Martin and Pattison (24). On the contrary, Goossens et al. (23), Arnold et al. (25), and Kojima et al. (27) reported a down-regulation of IGFBP-3 by androgens in LNCaP cells. The different observations between these groups may be a result of cell passage or differences in cell culture conditions or cell densities that can alter steroid responsiveness, even within the same cell line (62). However, our studies convincingly demonstrate that high concentrations of androgens directly up-regulate the expression of the IGFBP-3 gene through a functional ARE site in the IGFBP-3 promoter. These findings also reveal a cross-talk between the two cell growth regulators, IGFBP-3 and androgens, at the molecular level. The findings also raise an interesting possibility that induction of IGFBP-3 expression may be involved in the androgen mediated antiproliferative actions that have been reported in LNCaP cells (33, 34, 35, 40).

In addition, our observation of enhanced IGFBP-3 promoter activity by the interaction of androgens and calcitriol in transactivation assays suggests enhanced induction of the expression of the endogenous IGFBP-3 gene by these two hormone classes. The two regulatory elements, VDRE and ARE, are separated by approximately 400 bp and appear to interact or cooperate to regulate the level of IGFBP-3 expression. In a separate study we found that the two hormones in combination resulted in a more than 40-fold increase in IGFBP-3 mRNA and protein levels. Further detailed studies of the cross-talk between androgens and calcitriol on IGFBP-3 expression and the role of IGFBP-3 in regulating cell growth are described in that paper (Peng, L., J. Wang, P. J. Malloy, and D. Feldman, submitted for publication). This action may reflect the enhanced inhibition of LNCaP cell growth by combined calcitriol and androgens (63). These studies provide new insights into potential interactions between calcitriol and androgens, two regulators of IGFBP-3 expression.

The paradox raised by these studies is that androgens are well-known stimulators of prostate cancer growth (29, 64), yet they directly induce expression of IGFBP-3, an antiproliferative and proapoptotic protein (1, 2, 3). We speculate that some of the growth-inhibitory actions of androgens noted in some conditions (33, 34, 35, 37) may be related in part to IGFBP-3 induction. In a recent study, Hendriksen et al. (65) defined the AR pathway by selecting 200 genes from PCa xenografts, cell lines, and patient-derived tissues. Their conclusions were that in the well-differentiated state of early PCa, AR activity was involved in stimulating genes involved in growth promotion as well as growth inhibition and cell differentiation, resulting in a low growth rate. In high-grade PCa, there is a selective down-regulation of some AR target genes that inhibit proliferation or stimulate apoptosis or differentiation. Although the authors did not find IGFBP-3 in their arrays, their conclusions provide an attractive hypothesis to explain how androgens can induce a growth inhibiting prodifferentiating gene.


   Acknowledgments
 
We thank Dr. Larisa Nonn (Stanford University Medical Center, Stanford, CA) for her advice on setting up FACS experiments. We greatly appreciate Gu Zhang (Genentech, South San Francisco, CA) for her help on FACS data analysis.


   Footnotes
 
This work was supported by Department of the Army Grant DAMD17-02-1-0142 and National Institutes of Health Grant DK 42482.

Disclosure summary: the authors have nothing to disclose.

First Published Online July 6, 2006

Abbreviations: AR, Androgen receptor; ARE, androgen response element; CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation assay; FACS, flow cytometric analysis; FBS, fetal bovine serum; GR, glucocorticoid receptor; IGFBP, IGF binding protein; LUC, luciferase; PCa, prostate cancer; qRT-PCR, quantitative real-time RT-PCR; TK, thymidine kinase; VDRE, vitamin D response element.

Received April 27, 2006.

Accepted for publication June 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Paquette, B. Bessette, E. Ledru, and C. Deal
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