Abstract
The UDP-glucuronosyltransferases (UGTs) 2B15 and 2B17 are the major UGTs involved in the inactivation and elimination of the active androgens, dihydrotestosterone and testosterone. Although regulation of these UGT genes by various endogenous and exogenous ligands, including steroid hormones and bile acids, is well documented, the mechanisms controlling their basal gene expression are poorly understood. We recently reported that Forkhead box protein A1 (FOXA1) regulates the basal expression of the UGT2B17 gene in prostate cancer cells. In this study, we show that FOXA1 also regulates basal expression of the UGT2B15 gene in the prostate cell line LNCaP (lymph node carcinoma of the prostate). FOXA1 binds to a site −208 to −217 base pairs relative to the UGT2B15 translation start site, as shown by electromobility shift and chromatin immunoprecipitation assays. Mutation of this site prevents binding and substantially decreases basal UGT2B15 promoter activity. Silencing of FOXA1 expression by small interfering RNA significantly reduced UGT2B15 transcript levels, further confirming a crucial role of FOXA1 in controlling UGT2B15 gene expression. Because local inactivation of active androgens by UGT2B15 and UGT2B17 has been shown to be a major determinant of androgen response and signaling activity, regulation of the UGT2B15 and UGT2B17 genes by FOXA1 may have an important role in the maintenance of androgen homeostasis within prostate cancer cells.
Introduction
Androgen signaling through the androgen receptor is crucial for human prostate growth and function; however, excessive androgen signaling within the prostate is implicated in prostate cancer development and progression (Kaarbo et al., 2007). The prostate has all the essential steroidogenic enzymes necessary to produce active androgens locally from circulating adrenal precursor sex steroids. For example, testosterone can be synthesized in the prostate from either androstenedione by 17β-hydroxysteroid dehydrogenase (HSD) or androst-5-ene-3β,17β-diol by 3β-HSD/(Δ5/Δ4 isomerase), and it can be further transformed into the most potent androgen, 5α-dihydrotestosterone (DHT), by 5α-reductase (Labrie et al., 2005). In addition, the prostate also expresses steroid-metabolizing enzymes, including DHT-inactivating enzymes (Barbier and Bélanger, 2008). The expression of 3α-HSD and 17β-HSD within the prostate permits the local interconversion between DHT and its metabolites androsterone (ADT), 5α-androstane-3α,17β-diol (3α-diol), and 5α-androstane-3β,17β-diol (Labrie et al., 2005). This pathway is considered to be reversible and is at least partially dependent on the redox status within the cellular context. The influence of this pathway on the intracellular levels of active androgens within the prostate has not yet been fully investigated. However, the expression of two major androgen-conjugating enzymes from the UDP-glucuronosyltransferase (UGT) superfamily, UGT2B15 and UGT2B17, leads to the irreversible inactivation of DHT and its metabolites within the prostate through glucuronidation (Chouinard et al., 2004).
UGT2B15 and UGT2B17 are 95% identical in amino acid sequence but possess unique substrate specificities toward 5α-reduced androgens (Turgeon et al., 2000). UGT2B17 conjugates both the 3- and 17-hydroxy positions of androgens, such as the 3-OH of ADT and the 17-OH of 3α-diol or DHT, whereas UGT2B15 specifically glucuronidates the 17-hydroxy position of androgens, including the 17-OH of 3α-diol and DHT (Dubois et al., 1999; Turgeon et al., 2001). In the prostate, UGT2B17 is expressed in the basal cells of the alveoli and is thought to be involved in glucuronidation of DHT, ADT, and 3α-diol in these cells, whereas UGT2B15 is exclusively expressed in the luminal cells, where it only inactivates DHT, because the enzymes for the formation of ADT and 3α-diol are not present in these cells (Barbier and Bélanger, 2008). As glucuronidation of androgens and their metabolites facilitates their excretion, UGT2B15 and UGT2B17 help reduce intracellular DHT concentrations and consequently prevent excessive androgen signaling within the prostate (Chouinard et al., 2008). Indeed, it has been shown recently that the glucuronidation of androgens by UGT2B15 and UGT2B17 is a major determinant of the androgen response in prostate cancer LNCaP cells, as evidenced by the reported enhanced expression of androgen target genes in UGT2B15-/UGT2B17-deficient cells compared with control cells in the presence of DHT (Chouinard et al., 2007).
Given the crucial role of UGT2B15 and UGT2B17 enzymes in the local inactivation of active androgens, it is important to decipher the molecular mechanism(s) that regulates their expression within the prostate. Accumulating evidence strongly suggests that the expression of the human UGT2B15 and UGT2B17 genes are negatively regulated at the transcriptional level by a number of endogenous and exogenous ligands in androgen receptor-positive human prostate cancer cell lines. These include androgen receptor-mediated suppression of UGT2B15 and UGT2B17 expression by natural (DHT) and synthetic (R1881) androgens (Chouinard et al., 2006; Bao et al., 2008); the farnesoid X receptor-mediated repression by farnesoid X receptor activators, such as chenodeoxycholic acid, GW4064, and androsterone (Kaeding et al., 2008b); and the vitamin D receptor-mediated down-regulation by calcitriol (1α,25-dihydroxyvitamin D3), the active metabolite of vitamin D (Kaeding et al., 2008a). However, in contrast to these effects of ligands, the mechanisms regulating basal UGT2B15 and UGT2B17 gene expression are poorly understood.
Forkhead box (FOX) proteins are highly conserved transcriptional regulators implicated in cancer development and progression. FOXA proteins consist of three isoforms, namely FOXA1 (HNF3α), FOXA2 (HNF3β), and FOXA3 (HNF3γ) (Mincheva et al., 1997; Myatt and Lam, 2007). FOXA1 appears to act as a transcriptional facilitator by binding to its cognate response elements as a monomer in the regulatory regions of target genes and enhancing the access of other transcription factors (Lupien et al., 2008).
We recently reported that FOXA1 is a major regulator of the basal expression of UGT2B17 gene in prostate cancer cell lines (Hu et al., 2010). In this study, we report that FOXA1 is also essential for the basal transcriptional activity of the UGT2B15 gene in LNCaP cells.
Materials and Methods
Generation of UGT2B15 Promoter Luciferase Reporter Deletion Constructs and Mutants.
Sixteen pGL3-derived luciferase reporter constructs carrying varying lengths of the UGT2B15 proximal promoter were used in transient transfection in this study. Fourteen of these constructs were previously reported, namely 2B15-2716/-3Luc, 2B15-747/-3Luc, 2B15-705/-3Luc, 2B15-595/-3Luc, 2B15-556/-3Luc, 2B15-458/-3Luc, 2B15-412/-3Luc, 2B15-361/-3Luc, 2B15-310/-3Luc, 2B15-253/-3Luc, 2B15-202/-3Luc, 2B15-154/-3Luc, 2B15-101/-3Luc, and 2B15-54/-3Luc (Hu and Mackenzie, 2009). The other two constructs were generated with the construct 2B15-747/-3Luc as the template. The UGT2B15 promoter regions from nucleotides −3 to −511 and −3 to −648 were amplified by polymerase chain reaction (PCR) using the forward primers 2B15-511For 5′-AGCCATGGTACCGTGAAAGTAAAATTCTGT-3′ and 2B15-648For 5′-AGCCATGGTACCAAGGGTCCAGAAAATGC-3′, respectively, and the common reverse primer as described previously (Hu and Mackenzie, 2009). Underlined are the KpnI restriction sites added to the 5′ end of these primers to facilitate subsequent cloning. The resultant amplicons were ligated into the KpnI and MluI sites of the pGL3-basic vector to create the promoter constructs 2B15-511/-3Luc and 2B15-648/-3Luc. Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and a complementary pair of primers. Using the forward primer 2B15-FOXA1-MT1 with the sequence 5′-GACTAGAGTAATTGCCCCCCTAAAAGAACACC-3′, the FOXA1 site was mutated from 5′-TGTAAACATAAA-3′ to 5′-TGCCCCCCTAAA-3′ (mutated nucleotides are underlined) on two constructs, 2B15-253/-3Luc and 2B15-458/-3Luc, to generate their respective mutants, 2B15-253/FOXA1-MT/-3Luc and 2B15-458/FOXA1-MT/-3Luc. The promoter sequences of all reporter constructs were confirmed by DNA sequencing.
Transient Transfection and Luciferase Reporter Assay.
The LNCaP human prostatic cancer cell line, one of the most frequently used in vitro models of human prostate cancer, was obtained from the American Type Culture Collection (Manassas, VA) and routinely maintained in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 5% (v/v) fetal bovine serum (FBS) at 37°C in a 5% CO2/95% atmosphere. All transfections were performed using Lipofectamine 2000 (Invitrogen). Exponentially growing LNCaP cells were trypsinized and subsequently plated in 24-well plates at a density of 2 × 105 cells per well in 800 μl of RPMI medium containing 5% FBS. Transfections were conducted when the culture reached approximately 50 to 60% confluence. In brief, 500 μl of medium was aspirated from each well and replaced with 300 μl of serum-free RPMI transfection mixture containing 0.5 μg of each reporter construct and 25 ng of pRL-null plasmid, which served as an internal control to normalize transfection efficiency. Sixteen hours post-transfection, 300 μl of medium was aspirated from each well and replaced with 500 μl of fresh RPMI medium supplemented with 5% FBS. Forty-eight hours post-transfection, cells were lysed in passive lysis buffer and analyzed for firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instructions and a Packard TopCount luminescence and scintillation counter (PerkinElmer Life and Analytical Sciences, Waltham, MA). Transfections were performed in triplicate, and all experiments were repeated at least twice.
Electrophoretic Mobility Shift and Supershift Assays.
The FOXA1 expression vector was prepared by cloning full-length FOXA1 cDNA into the EcoRI site of the mammalian expression vector pCMX-PL2. With this FOXA1 expression plasmid as template, recombinant FOXA1 proteins were synthesized using the TnT Quick Coupled Transcription/Translation kit according to the manufacturer's instructions (Promega). Supershift and electrophoretic mobility shift assays (EMSAs) were performed with 2B15 promoter-specific probes, which contained either a wild-type FOXA1 site (designated 2B15-FOXA1) with the sense sequence 5′-TAATTGTAAACATAAAAGAACACCAAACACACTAA-3′ (the FOXA1 site is underlined) or a mutated FOXA1 site (2B15-FOXA1-MT2) with the sense sequence 5′-TAATTGCGCACATAAAAGAACACCAAACACACTAA-3′ (mutated nucleotides are underlined). The anti-FOXA1 antibody (H-120) for supershift assays was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). EMSA experiments were carried out essentially as reported elsewhere (Hu and Mackenzie, 2009).
Chromatin Immunoprecipitation Assay and Quantitative Real-Time PCR.
Chromatin immunoprecipitation assay and quantitative real-time PCR (ChIP-qPCR) were performed as described previously (Hu and Mackenzie, 2009). In brief, LNCaP cells were grown in RPMI medium supplemented with 5% FBS. When the culture reached 90% confluence, cells were crossed-linked by 1% formaldehyde and subsequently quenched by 125 mM glycine solution. Cells were lysed, sonicated, and then subjected to immunoprecipitation with 10 μg of each specific antibody or equivalent amounts of the rabbit preimmune IgG control. The resulting chromatin precipitates were captured by Protein A Sepharose CL-4B beads (GE Healthcare, Chalfont St. Giles, UK) and subsequently were eluted from these beads after several washes in different buffers as described previously (Hu and Mackenzie, 2009). Cross-linking was reversed by heating the eluates at 65°C overnight. The resulting DNA/protein precipitates were digested with proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. The DNA pellets were dissolved in 50 μl of Tris-EDTA buffer. qPCR was performed with 2 μl of each of the resultant DNA samples as templates and locus-specific primers. qPCR primers for both the UGT2B15 promoter (Hu and Mackenzie, 2009) and the control locus (chromosome 7, 72,190,955–72,192,054) (Carroll et al., 2006) were the same as described previously. Antibodies against estrogen receptor α (ERα) (HC-20) or FOXA1 (H-120, the same as used in EMSAs), and the rabbit preimmune IgG control (sc-2027) were purchased from Santa Cruz Biotechnology, Inc.
Small Interfering RNA Knockdown Experiments.
ON-TARGETplus SMARTpool small interfering RNA (siRNA) against FOXA1 (NM_004496, designated anti-FOXA1 siRNA) and ON-TARGETplus nontargeting pool siRNA (nontarget siRNA) were purchased from Dharmacon RNA Technologies (Lafayette, CO). LNCaP cells were maintained in RPMI 1640 medium supplemented with 5% FBS. Cells grown at logarithmic phase were harvested and seeded into six-well culture plates at a density of 1 × 106 cells per well in 2 ml of RPMI medium, consisting of 1.5 ml of cell suspension in serum-containing RPMI medium and 0.5 ml of the serum-free transfection mixture, containing 8 μl of Lipofectamine (Invitrogen) and 400 nM either anti-FOXA1 siRNA or nontarget siRNA. Transfection was conducted for 24 to 48 h, followed by the addition of 2 ml of fresh RPMI medium with 5% FBS to each well. Cells were harvested 68 to 72 h post-transfection, and mRNA levels of target genes were measured by reverse transcription (RT)-qPCR as described previously (Hu and Mackenzie, 2009). RT-PCR primers were 5′-GAAGATGGAAGGGCATGAAACCA-3′ (forward) and 5′-TGGCATAGGACATGTTGAAGGACG-3′ (reverse) for FOXA1 and 5′-GAAGGTGAAGGTCGGAGTC-3′ (forward) and 5′-GAAGATGGTGATGGGATTTC-3′ (reverse) for GAPDH. Those for UGT2B15 and 18S rRNA were the same as previously reported (Congiu et al., 2002). Data from 18S rRNA transcripts were used as a reference to normalize the amount of total RNA amplified in each reaction.
Results and Discussion
The UGT2B15 and UGT2B17 genes share considerable sequence homology between their proximal promoters (Fig. 1), so it is likely that they are regulated by some of the same transcription factors. We have recently shown that FOXA1 regulates the basal activity of the UGT2B17 promoter in LNCaP cells (Hu et al., 2010). We have also shown that a single base change (the A/G SNP at −155 bp) within the UGT2B17 FOXA1 site can alter promoter activity up to 13-fold. Although the equivalent putative FOXA1 site of the UGT2B15 promoter does not contain this particular SNP, it does differ from that of UGT2B17 by a single base (Fig. 1). Because single base changes can diminish FOXA1 binding and substantially reduce promoter activity, it was necessary to confirm that the UGT2B15 promoter was active and was regulated by FOXA1. It was also important to determine whether other sites in the UGT2B25 proximal promoter were involved in control of basal UGT2B15 promoter activity.
For this purpose, a series of 16 luciferase reporter constructs carrying varying lengths of the UGT2B15 proximal promoter were generated. As shown in Fig. 2, reporter constructs with UGT2B15 promoter lengths of ≤202 bp possessed very low levels of activity relative to the background activity of the promoter-less pGL3-basic vector, when transfected into LNCaP cells. In contrast, constructs with UGT2B15 promoter lengths of >253 bp possessed activity levels that were approximately 15-fold higher than that of the construct 2B15-202-3Luc (p > 0.001). These results suggested that the UGT2B15 promoter between −202 and 253 bp harbors a positive regulatory cis-acting element(s), which is pivotal to its basal transcription activity in LNCaP cells, and that similar elements are not present further upstream (up to −2716 bp). Based on a comparison to the UGT2B17 promoter, this 52-bp region contains the putative UGT2B15 FOXA1 site (5′-TGTAAACATAAA-3′) located between nucleotides −218 and −207 relative to the translation start site of the UGT2B15 gene. To determine whether this FOXA1 site contributes to the high activity levels of UGT2B15 promoters >253 bp in length, we generated 2B15-253/FOXA1-MT/-3Luc bearing a mutated FOXA1 site (5′-TGCCCCCCTAAA-3′; mutated sequences are underlined). When transfected into LNCaP cells, this construct had greatly diminished promoter activity in comparison to its unmutated counterpart (p < 0.001) (Fig. 2). To further verify its functionality, we mutated this FOXA1 site in another construct 2B15-458/-3Luc to create the mutant 2B15-458/FOXA1-MT/-3Luc. When transfected into LNCaP cells, this mutation also resulted in a large reduction in promoter activity compared to its wild-type counterpart (p < 0.01) (Fig. 2). Altogether, these results clearly demonstrate that basal UGT2B15 promoter activity is dependent on an intact FOXA1 site and that transcription factors that bind outside this region do not have a major role in basal UGT2B15 expression in LNCaP cells, under our experimental conditions.
There are three FOXA proteins, A1, A2, and A3, which bind to the same consensus sequence (Lai et al., 1990; Lai et al., 1991). However, because only FOXA1 protein expression has been demonstrated in LNCaP cells (Mirosevich et al., 2006), we performed EMSAs to see whether FOXA1 bound to the UGT2B15 FOXA1 site using in vitro-transcribed/-translated recombinant FOXA1 protein.
Incubation of a UGT2B15 promoter probe (nucleotides −222 to −188) containing the putative FOXA1 site with recombinant FOXA1 protein led to the formation of a FOXA1-specific protein/DNA complex (labeled A in Fig. 3A, lane 2), as evidenced by the formation of the supershifted complex upon the addition of the anti-FOXA1 antibody (labeled SS in Fig. 3A, lane 4). Furthermore, the absence of complex A in the negative control (Fig. 3A, lane 1), as well as the disruption of this complex in competition assays in the presence of a 100-fold molar excess of unlabeled probe (Fig. 3A, lane 3), provide further evidence that FOXA1 binds to its cognate site in the UGT2B15 promoter. To prove the direct involvement of the putative FOXA1 site in the formation of complex A, further EMSAs were performed using probes with a mutated FOXA1 site. As shown in Fig. 3A, lane 5, mutating the putative FOXA1 site in the UGT2B15 promoter almost completely disrupted complex A formation, thereby confirming the necessity of this site in forming the FOXA1-specific protein/DNA complex A.
Having established that recombinant FOXA1 binds to the UGT2B15 promoter in vitro, we next performed ChIP/qPCR to see whether endogenous FOXA1 proteins in LNCaP cells could be constitutively present at the FOXA1 site within the UGT2B15 promoter. ChIP assays were performed with specific antibodies against FOXA1 or ERα and the rabbit preimmune IgG control for normalizing against possible nonselective background immunoprecipitation. FOXA1 occupancy on the UGT2B15 promoter was quantified with the FOXA1-containing region of the UGT2B15 promoter spanning nucleotides -381 to -135 relative to the translation start site. As shown in Fig. 3B, we observed a ∼5.5-fold enrichment of the UGT2B15 promoter in the samples precipitated from the anti-FOXA1 antibody over the IgG-precipitated controls (p < 0.001). By contrast, there was no enrichment of this promoter region in the samples precipitated from the anti-ERα antibody used as a negative control in these experiments. There was also no binding to the negative control locus (Carroll et al., 2006). Collectively, these ChIP assays provided evidence for the in vivo binding of FOXA1 to the UGT2B15 promoter region harboring the FOXA1 site in LNCaP cells.
The involvement of FOXA1 in UGT2B15 gene transcription was further investigated by siRNA silencing technologies. We transfected siRNAs targeting FOXA1 mRNA into LNCaP cells and subsequently measured the mRNA levels of target genes using quantitative real-time RT-PCR. As shown in Fig. 3C, transfection of LNCaP cells with anti-FOXA1 siRNA significantly decreased the levels of endogenous FOXA1 transcripts to 48% of that in control cells treated with nontargeting siRNAs (p < 0.01). This reduction of FOXA1 mRNA resulted in an approximately 3-fold decrease in UGT2B15 mRNA levels (p < 0.001), whereas there was no significant effect on the mRNA levels of the negative control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Taken together, these siRNA silencing experiments strongly suggest that FOXA1 has a positive regulatory role in UGT2B15 gene transcription in LNCaP cells.
In summary, this study identifies a functional FOXA1 site within the proximal UGT2B15 promoter that is important for basal UGT2B15 gene transcriptional activity in LNCaP cells. Although differing by one base to the equivalent site in the UGT2B17 gene, the UGT2B15 FOXA1 site is closer in sequence to the high-activity UGT2B17 promoter allele (-155A) and thus sustains significant promoter activity. Hence, our results show for the first time that FOXA1, a pioneering transcription factor (Lupien et al., 2008), plays a crucial role in controlling basal expression of UGT2B15 in prostate cancer cells. Together with our previous demonstration of the importance of FOXA1 in regulating basal UGT2B17 gene expression (Hu et al., 2010), these findings provide new insights into our understanding of the molecular mechanisms that determine androgen glucuronidation capacity and hence androgen homeostasis and signaling in the prostate. Because UGT2B15 and UGT2B17 are the major enzymes inactivating and eliminating active androgens by glucuronidation, the control of UGT2B15 gene expression by FOXA1 would be of particular significance for individuals homozygous for the UGT2B17 deletion, where UGT2B15 is the only enzyme in the prostate with the capacity to effectively glucuronidate active androgens (McCarroll et al., 2006).
Footnotes
The work was supported by the National Health and Medical Research Council of Australia [Grant 426705].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.035436.
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ABBREVIATIONS:
- HSD
- hydroxysteroid dehydrogenase
- DHT
- dihydrotestosterone
- ADT
- androsterone
- 3α-diol
- 5α-androstane-3α,17β-diol
- UGT
- UDP-glucuronosyltransferase
- R1881
- 17β-hydroxy-17α-methyl-19-norandrost-4,9,11-trien-3-one
- FOX
- Forkhead box
- PCR
- polymerase chain reaction
- FBS
- fetal bovine serum
- EMSA
- electrophoretic mobility shift assay
- ChIP
- chromatin immunoprecipitation
- RT-qPCR
- reverse transcription-quantitative real-time PCR
- HNF
- hepatocyte nuclear factor
- siRNA
- small interfering RNA
- bp
- base pair
- SNP
- single nucleotide polymorphism
- ERα
- estrogen receptor α
- rRNA
- ribosomal RNA
- LNCaP
- lymph node carcinoma of the prostate
- GW4064
- 3-(2,6-dichlorophenol)-4-(3′-carboxy-2-chlorostilben-4-yl-)oxymethyl-5-isopropyl-isoxazole.
- Received July 13, 2010.
- Accepted August 23, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics