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Molecular Endocrinology, doi:10.1210/me.2005-0240
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*Prostate Cancer
Molecular Endocrinology 20 (5): 984-995
Copyright © 2006 by The Endocrine Society

The CCAAT Enhancer-Binding Protein-{alpha} Negatively Regulates the Transactivation of Androgen Receptor in Prostate Cancer Cells

Soma Chattopadhyay1, Eun-Yeung Gong1, Miok Hwang, Eunsook Park, Hyun Joo Lee, Cheol Yi Hong, Hueng-Sik Choi, Jae-Hun Cheong, Hyuk Bang Kwon and Keesook Lee

Hormone Research Center (S.C., E.-Y.G., M.H., E.P., H.J.L., C.Y., H.-S.C., H.B.K., K.L.) School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea; and Department of Molecular Biology (J.-H.C.), College of Natural Science, Pusan National University, Busan 609-735, Republic of Korea

Address all correspondence and requests for reprints to: Keesook Lee, Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: klee{at}chonnam.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The basic leucine zipper transcription factor, CCAAT enhancer-binding protein-{alpha} (C/EBP{alpha}), negatively regulates cell proliferation and induces terminal differentiation of various cell types. C/EBP{alpha} is expressed in the prostate, but its potential role in the tissue is unknown. Herein, we show that C/EBP{alpha} is highly expressed at the stage of growth arrest during prostate development. Furthermore, overexpression of C/EBP{alpha} decreases the rate of DNA synthesis in LNCaP prostate cancer cells. Investigation of the potential cross-talk between C/EBP{alpha} and androgen receptor (AR) that is responsible for androgen-dependent prostate proliferation demonstrates that androgen-dependent transactivation of AR is strongly repressed by C/EBP{alpha}. C/EBP{alpha} directly binds AR in vitro and forms a complex with AR in vivo. C/EBP{alpha} neither prevents the nuclear translocation of AR nor disrupts the N/C-terminal interaction of AR, which are both necessary for its proper transactivation activity upon ligand binding. To modulate AR transactivation, however, C/EBP{alpha} does compete with AR coactivators for AR binding. Additionally, C/EBP{alpha} is recruited onto AR-target promoters with AR and is further able to inhibit the expression of endogenous prostate-specific antigen in prostate cancer cells. Our results suggest C/EBP{alpha} as a potent AR corepressor and provide insight into the role of C/EBP{alpha} in prostate development and cancer.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NORMAL DEVELOPMENT, maintenance, and function of the prostate gland are largely regulated by androgens, which act through androgen receptor (AR) (1, 2). Androgens and AR also play an integral role in the growth of prostate tumors (3). AR function can be modulated by intracellular signaling pathways, transcription factors, cell cycle proteins, and other factors, which modify AR transcriptional activity or provide means for cross-talk between androgen and other signals. Thus, AR activity in the prostate is a complex sum of its own transcriptional activity, input from signal-transducing systems, and combinatorial activity of other transcriptional coregulators (4). AR is expressed in secretory epithelial cells, periacinar stromal cells, and perivascular smooth muscle of the prostate (5). When AR activity is inhibited by any means, the prostate undergoes involution due to apoptosis and atrophy in both the stromal and epithelial layers (5).

AR is a ligand-dependent transcription factor that directs the expression of target genes when activated by androgens (1). AR consists of three separate functional domains: the N-terminal activating domain, the middle DNA-binding domain, and the C-terminal ligand-binding domain (6). The N terminus has been shown to directly interact with the C terminus in a ligand-dependent manner, which is required for the full transcriptional potential of AR (7). Upon ligand binding, AR translocates into the nucleus, thereby binding to specific DNA sequences, referred to as androgen response elements (AREs), in the regulatory region of target genes (7, 8). The function of AR is modulated by other proteins called "coactivators" and "corepressors." Coactivators potentiate ligand-dependent transactivation of receptors with diverse modes of action, including direct interaction with basal transcription factors and covalent modification of histones and other proteins (9, 10, 11, 12). In contrast, corepressors may recruit histone deacetylase (HDAC) activity or block the association of a coactivator to the receptor complex (13). They may also inhibit the receptor complex from binding a DNA response element to suppress gene expression (14).

The CCAAT/enhancer binding protein-{alpha} (C/EBP{alpha}) is a transcription factor that belongs to the basic leucine zipper protein family. The factor consists of three structural components, including the N-terminal transactivation region, the middle basic DNA-binding region, and the C-terminal leucine zipper region. Several lines of evidence have suggested that C/EBP{alpha} plays a crucial role in regulating the balance between cell proliferation and differentiation (15). In many cell types, including liver and myeloid, C/EBP{alpha} is an important negative regulator of cell proliferation. For example, liver quiescence is mediated by the growth-inhibitory activity of C/EBP{alpha}, and liver cells from C/EBP{alpha} knockout mice show an increased rate of proliferation, expressing higher levels of cell cycle proteins (16, 17). The mechanism by which C/EBP{alpha} inhibits cell proliferation differs among cell types. For instance, C/EBP{alpha} inhibits liver cell proliferation by protein-protein interactions (16, 17, 18, 19), whereas inhibitory action of C/EBP{alpha} in proliferating myeloid cells involves the transcriptional activity of C/EBP{alpha} (20, 21, 22). Apart from its role in cell proliferation, C/EBP{alpha} also regulates terminal differentiation of the various cell types, including hepatocytes, adipocytes, myelomonocytes, keratinocytes, and epithelial cells of the gut (23, 24, 25, 26, 27). Thus, it has been hypothesized that abnormalities in C/EBP{alpha} expression and function may contribute to the development of malignancies in a variety of tissues (28).

In the present study, we demonstrate that C/EBP{alpha}, the expression of which is the highest at the exit stages of proliferation in prostate epithelial cells, represses the transactivation of AR in prostate cancer cells. C/EBP{alpha} directly binds AR and competes with other histone acetyltransferase and nonhistone acetyltransferase coactivators for AR binding. Furthermore, C/EBP{alpha} is recruited onto AR-target promoters to inhibit the expression of AR-target genes. As a whole, our results suggest C/EBP{alpha} as a potent AR corepressor and provide insight into the role of C/EBP{alpha} in prostate development and cancer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
C/EBP{alpha} Is Highly Expressed at the Exit Stages of Cell Proliferation in Rat Prostate
C/EBP{alpha} is expressed in human prostate (29), whereas it is known to block cell proliferation and regulate terminal differentiation in several circumstances (23, 24, 25, 26, 27). To gain insight into the role of C/EBP{alpha} in the prostate, we first investigated the expression pattern of C/EBP{alpha} at different developmental stages of rat prostate. C/EBP{alpha} messages were abundantly expressed in 3- and 4-wk-old prostate and very weakly expressed in 2- and 8-wk-old prostate (Fig. 1AGo). In contrast, the messages were detected only in 2-wk-old testis, which is consistent with the previous report of undetectable or very low levels of C/EBP{alpha} expression in human testis (29). Additionally, we investigated cell types in which C/EBP{alpha} is expressed by immunohistochemistry of 3- and 4-wk-old rat prostate. As shown in Fig. 1BGo, C/EBP{alpha} protein was expressed in epithelial cells of rat prostate, being localized in the nucleus.


Figure 1
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  		Fig. 1. Expression of C/EBP{alpha} May Correlate with the Proliferation Arrest of the Prostate

A, C/EBP{alpha} expression during development of the prostate and testis in rats. Northern blot analysis of total RNAs from the prostate and testis at different developmental stages was performed using 32P-labeled C/EBP{alpha} cDNA as a probe. The expression of 18S rRNA was used as an internal control. B, Immunolocalization of C/EBP{alpha} protein in rat prostate. Immunohistochemistry was performed on paraffin sections of 3-wk-old rat prostate using an anti-C/EBP{alpha} antibody. The control section treated with only the secondary antibody was counterstained with hematoxylin. C, C/EBP{alpha} decreases the rate of DNA synthesis in AR-positive LNCaP, but not AR-negative PPC-1 prostate cancer cells. LNCaP and PPC-1 cells were transfected with either C/EBP{alpha} expression construct (+) or the empty vector (–). C/EBP siRNA or scrambled siRNA was also transfected along with C/EBP{alpha} expression plasmid where indicated. DNA synthesis rates were analyzed by [3H]thymidine incorporation assay. Expression of C/EBP{alpha} was examined by using anti-CEBP{alpha} antibody in Western blot analysis. ß-gal, ß-Galactosidase; kD, kilodalton.

 
The proliferation rate of rat prostate epithelial cells increases from birth to 3 wk of age, after which it sharply decreases to approximately 20% of the peak at 4 wk of age (30). The correlation between the high level of C/EBP{alpha} expression and the proliferation exit of the epithelial cells in the 3- to 4-wk-old prostate signifies that C/EBP{alpha} expression may cause proliferation arrest of prostate epithelial cells. To analyze the effect of C/EBP{alpha} expression on prostate cell proliferation, we transfected LNCaP and PPC-1 cells (31) with C/EBP{alpha} expression construct and checked the cell proliferation rate by [3H]thymidine incorporation assay. LNCaP and PPC-1 cells are AR-positive and AR-negative cancer cells, respectively, originated from human prostate epithelial cells. Interestingly, overexpression of C/EBP{alpha} significantly decreased the rate of DNA synthesis in LNCaP cells, but not in PPC-1 cells (Fig. 1CGo). A C/EBP{alpha} mutant, which has a deletion (amino acids 263–269) at the C terminus and is known to have a dominant-negative effect on wild-type C/EBP{alpha} (32), was unable to inhibit DNA synthesis. Furthermore, targeting C/EBP{alpha} expression with C/EBP{alpha} small interfering RNA (siRNA) abolished such C/EBP{alpha}-mediated inhibition of DNA synthesis. These results suggest that C/EBP{alpha} blocks proliferation of prostate cells and possibly in an AR-dependent manner.

C/EBP{alpha} Represses AR Transactivation
AR plays a pivotal role in prostate cell proliferation by acting primarily as a transcription factor (33), whereas C/EBP{alpha} is highly expressed at the exit stages of cell proliferation in rat prostate and causes proliferation arrest in AR-positive prostate cancer cells (Fig. 1Go). Although C/EBP{alpha} may act in various ways to regulate cell proliferation, we initially investigated the possibility of cross-talk between C/EBP{alpha} and AR, which may affect the AR function. We coexpressed C/EBP{alpha} and AR in the PPC-1 cells and accessed the effect on the transactivation potential of AR. As shown in Fig. 2AGo, C/EBP{alpha} strongly inhibited androgen-dependent AR transactivation. Interestingly, the C/EBP{alpha} mutant was unable to fully repress the AR transactivation (Fig. 2AGo). Furthermore, C/EBP{alpha} suppressed AR transactivation in a dose-dependent manner, again suggesting the specificity of C/EBP{alpha} action (Fig. 2BGo). We also observed similar repression of AR transactivation by C/EBPß, which has a high degree of homology with C/EBP{alpha} (data not shown).


Figure 2
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  		Fig. 2. C/EBP{alpha} Represses the Transactivation of AR

A, Repression of AR transactivation by C/EBP{alpha}. PPC-1 cells were cotransfected with 350 ng ARE2-TATA-luc reporter and 50 ng AR expression plasmid along with 500 ng C/EBP{alpha} or C/EBP{alpha} dominant-negative mutant (C/EBP{alpha} DN). Cells were treated with or without 10 nM DHT for 24 h. At least three independent experiments were combined, and values represent the mean ± SEM. B, Dose-dependent inhibition of AR transactivation by C/EBP{alpha}. PPC-1 cells were transfected with increasing concentrations (50, 100, 250, and 500 ng) of C/EBP{alpha} and others as in panel A. C, C/EBP{alpha}-mediated repression of AR transactivation with natural AR-target promoters. PPC-1 cells were transfected as in panel A, with PSA-luc or MMTV-luc reporter. D, C/EBP{alpha}-mediated repression of endogenous AR transactivation. LNCaP cells were transfected as in panel C, without the AR expression plasmid. E, No effect of C/EBP{alpha} expression on AR protein levels. PPC-1 cells were cotransfected with 200 ng AR and 100 ng ß-galactosidase expression plasmids together with or without 600 ng C/EBP{alpha} plasmid. Western blot analyses were performed using anti-AR, anti-C/EBP{alpha}, and anti-ß-gal antibodies. ß gal, ß-Galactosidase.

 
To establish the importance of C/EBP{alpha}-mediated AR repression, we examined the C/EBP{alpha} effect on natural AR-target promoters such as mouse mammary tumor virus (MMTV) and prostate-specific antigen (PSA). In PPC-1 cells, coexpression of C/EBP{alpha} with AR repressed AR transactivation on both MMTV and PSA promoters (Fig. 2CGo). Furthermore, C/EBP{alpha} also represses the endogenous AR transactivation on MMTV and PSA promoters in LNCaP cells that express the mutated, but functional, AR (Fig. 2DGo). Overexpression of C/EBP{alpha} had little effect on AR protein levels (Fig. 2EGo), which indicates that the C/EBP{alpha} suppression of AR activity is not due to the inhibition of AR expression. Taken together, these results suggest that the basic leucine zipper transcription factor C/EBP{alpha} represses AR transactivation in prostate cancer cells.

C/EBP{alpha} Physically Interacts with AR in Vitro and in Vivo
Glutathione-S-transferase (GST) pull-down assays were performed to examine whether AR repression by C/EBP{alpha} is mediated through direct protein-protein interaction. Interactions of AR with C/EBP{alpha}, as well as AR domains responsible for the interaction, were investigated using different AR deletion mutants fused to the GST protein (Fig. 3AGo). As shown in Fig. 3BGo, the in vitro translated C/EBP{alpha} interacted strongly with GST-AR DNA-binding domain and hinge region (DBDh), and less so with GST-AF1. However, no interaction was observed between C/EBP{alpha} and GST alone or GST-AR ligand-binding domain (LBD), demonstrating that C/EBP{alpha} interaction with AR is specific. C/EBP{alpha} domains responsible for its interaction with AR were then investigated using GST fusion protein of C/EBP{alpha} deletion mutants (Fig. 3CGo). The in vitro translated AR interacted with the full-length C/EBP{alpha}, but to a lesser extent with either the N-terminal or C-terminal region, indicating that full-length C/EBP{alpha} is probably necessary for proper interaction with AR (Fig. 3DGo).


Figure 3
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  		Fig. 3. In Vitro and in Vivo Interaction between C/EBP{alpha} and AR

A, Schematic representation of the full-length AR and its different domain deletion mutants used in GST pull-down assay. B, C/EBP{alpha} directly interacts with AR via its DBDh region in GST pull-down assays. A relatively weak interaction with the AF-1 domain of AR is also noted. [35S] methionine-labeled C/EBP{alpha} was allowed to bind with bacterially expressed GST alone or with different domain deletion mutants of AR (GST-AF-1, GST-DBDh, GST-LBD). Reactions were carried out with the equivalent amount of each protein as determined by Coomassie blue staining (data not shown). Labeled protein (5%) used in the binding reaction was loaded as input. C, Schematic representation of full-length C/EBP{alpha} and its deletion mutants. D, [35S] methionine-labeled AR was allowed to bind with GST alone, the full-length (GST-C/EBP{alpha}fl), or different deletion mutants of C/EBP{alpha} (GST-C/EBP{alpha}NT, GST-C/EBP{alpha}CT). E and F, C/EBP{alpha} is coimmunoprecipitated with AR. Cos-7 cells (panel E) were transfected with AR and C/EBP{alpha} expression plasmids and then treated with (+) or without (–) 10 nM DHT for 24 h post transfection. RWPE-1 cells (panel F) endogenously expressing both AR and C/EBP{alpha} were treated with (+) or without (–) 10 nM DHT for 24 h before harvest. Coimmunoprecipitations were conducted with anti-C/EBP{alpha} antibody. Western blot analyses of immunoprecipitated materials were performed using anti-AR or anti-C/EBP{alpha} antibody. Input blots are shown for the expression level of each protein. AD, Activation domain; AF1, activation function 1; CT, C terminus; NT, N terminus; IP, Immunoprecipitation; kD, kilodalton; LBD, ligand-binding domain; WB, Western blot; BR, basic region; LZIP, leucine zipper protein; DBDh, DNA-binding domain and hinge region.

 
To examine in vivo interaction between C/EBP{alpha} and AR, we performed coimmunoprecipitation assays with Cos-7 cells, which were cotransfected with AR and C/EBP{alpha} expression plasmids as well as RWPE-1 cells, human normal prostate cells that endogenously express both AR and C/EBP{alpha}. Immunoprecipitations performed using anti-C/EBP{alpha} antibody, followed by Western blot analysis of the immunoprecipitated complexes for AR and C/EBP{alpha}, revealed that AR was efficiently coprecipitated with C/EBP{alpha} in a ligand-dependent manner in both cases (Fig. 3Go, E and F). C/EBP{alpha} mRNA gives rise to two different protein forms (42 and 30 kDa), which are translated from different in-frame initiation codons by the leaky ribosome-scanning mechanism (34). We also observed the ligand-dependent association of AR with C/EBP{alpha} in immunoprecipitations conducted using anti-AR antibody (data not shown). These results suggest that C/EBP{alpha} forms a specific complex with AR in a ligand-dependent manner in vivo.

C/EBP{alpha} Neither Inhibits the N/C-Terminal Interaction nor the Nuclear Translocation of AR
Upon ligand binding, AR dissociates from heat shock proteins and translocates into the nucleus, thereby binding to its target gene promoters as a homodimer that is formed by the intermolecular N/C-terminal interaction of two AR molecules. Because some AR corepressors interfere with the steps involved in androgen-dependent AR activation, consequently repressing AR transactivation potential (14), the ability of C/EBP{alpha} to inhibit any of the AR activation steps, such as the N/C-terminal interaction and the nuclear translocation of AR, was investigated. In the GST pull-down experiments, the in vitro-translated AR bound the AF1-DBDh region of AR (GST-ARAF1DBDh) as expected, and this interaction was not affected by coincubation with C/EBP{alpha} (Fig. 4AGo). Moreover, in mammalian two-hybrid assays, C/EBP{alpha} did not affect interaction between GAL4-AR C terminus and VP16-AR N terminus (Fig. 4BGo), whereas a positive control, DAX-1, inhibited the interaction as previously reported (35). These results indicate that C/EBP{alpha} does not interfere with N/C-terminal interaction of AR.


Figure 4
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  		Fig. 4. C/EBP{alpha} Inhibits neither the N/C-Terminal Interaction nor the Nuclear Translocation of AR

A, No effect of C/EBP{alpha} on the N/C-terminal interaction of AR in GST pull-down assays. The AF1-DBDh region of AR (GST-ARAF1DBDh) was incubated with [35S]methionine-labeled AR and an increasing amount of [35S]methionine-labeled C/EBP{alpha}. B, No effect of C/EBP{alpha} on the N/C-terminal interaction of AR in mammalian two-hybrid assays. Cos-7 cells were transfected with GAL4-AR C-term and GAL4-tk-luc plasmids together with or without VP16-AR N-term and C/EBP{alpha} plasmids. DAX-1 served as a positive control. C, No effect of C/EBP{alpha} on the subcellular localization of AR. PPC-1 cells were transfected with GFP-AR and C/EBP{alpha} expression plasmids independently (top panel) or together (bottom panel), treated with or without 10 nM DHT, and processed for immunocytochemistry using anti-C/EBP{alpha} antibody. Fluorescence was analyzed with a laser scanning confocal microscope. C-term, C terminus; N-term, N terminus.

 
The effect of C/EBP{alpha} on AR nuclear translocation was accessed by coexpressing green fluorescent protein (GFP)-tagged AR and C/EBP{alpha} in Cos-7 cells and by analyzing potential subcellular colocalization by double-fluorescence staining. As shown in Fig. 4CGo, when cells were transfected with either GFP-AR or C/EBP{alpha} alone, AR protein was located predominantly in the cytoplasm in the absence of ligand, but, in the presence of 10 nM DHT, AR protein translocated into the nucleus. C/EBP{alpha}, a nuclear protein, was predictably located in the nucleus, and the presence of 10 nM DHT did not affect its localization. When GFP-AR and C/EBP{alpha} were coexpressed, neither AR nor C/EBP{alpha} protein was mislocalized in the presence or absence of ligand. These results suggest that AR repression by C/EBP{alpha} may not be due to nuclear exclusion of AR.

C/EBP{alpha} Competes with Other Coregulators for the Modulation of AR Transactivation
To explore the mechanism by which C/EBP{alpha} functions as a corepressor of AR, we assessed the involvement of HDACs using the HDAC inhibitor trichostatin A (TSA). In PPC-1 cells, the transactivation of AR was inhibited by C/EBP{alpha} coexpression, but the repressed AR transactivation was not significantly recovered after TSA treatment (Fig. 5AGo), suggesting that HDACs have no involvement in the C/EBP{alpha}-mediated suppression of AR transactivation.


Figure 5
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  		Fig. 5. C/EBP{alpha} Competes with Other Coregulators for the Modulation of AR Transactivation

A, HDAC activity is not involved in the C/EBP{alpha} repression of AR transactivation. PPC-1 cells were transfected as in Fig. 2AGo. The cells were treated with or without 100 nM TSA at the same time with 10 mM DHT treatment 24 h before harvesting. B, AR coactivators relieve the C/EBP{alpha}-mediated repression of AR transactivation. PPC-1 cells were cotransfected with 50 ng AR, 100 ng C/EBP{alpha}, and 500 ng AR coactivators. The expression level of the proteins was monitored by Western blot analyses. C, GRIP1 relieves C/EBP{alpha} repression of AR transactivation in a dose-dependent manner. D, C/EBP{alpha} represses GRIP1-elevated AR transactivation in a dose-dependent manner. SRC-1, Steroid receptor coactivator 1.

 
We then examined the possibility that C/EBP{alpha} interferes with the interaction between AR and its coactivators. PPC-1 cells were transfected with plasmids encoding AR, ARE2-TATA-luc reporter, and C/EBP{alpha} in the presence or absence of a specific coactivator, and we investigated whether coexpression of a specific coactivator derepresses the C/EBP{alpha} suppression of AR transactivation. As shown in Fig. 5BGo, ARA70, steroid receptor coactivator 1, p300, and glucocorticoid receptor-interacting protein 1 (GRIP1), all known to be AR coactivators (36, 37, 38, 39, 40), relieved the C/EBP{alpha} suppression to a certain extent, suggesting that C/EBP{alpha} and AR coactivators compete for the modulation of AR transactivation. Furthermore, GRIP1 was able to release the C/EBP{alpha}-mediated AR repression, and C/EBP{alpha} was able to repress the GRIP1-mediated AR transactivation, both in a dose-dependent manner (Fig. 5Go, C and D). This finding suggests that this coactivator competition with C/EBP{alpha} is quite specific. We also observed a similar competition between C/EBP{alpha} and p300 coactivator for the modulation of AR transactivation (data not shown). Together, these results suggest that C/EBP{alpha} may inhibit AR transactivation by interfering with the interaction between AR and its coactivators.

C/EBP{alpha} Is Recruited by AR onto AR Target Promoters in Vivo
Chromatin immunoprecipitation (ChIP) assays were performed to determine whether C/EBP{alpha} is recruited by AR onto an androgen-regulated promoter (Fig. 6AGo). Cos-7 cells were cotransfected with AR and C/EBP{alpha} expression plasmids together with linearized ARE2-TATA-Luc reporter and treated with 10 nM DHT. Cross-linked DNA fragments produced by sonication were immunoprecipitated with anti-AR or anti-C/EBP{alpha} antibody. Using pairs of specific primers spanning the ARE region of the reporter, the immunoprecipitates were analyzed by PCR. Occupancy of ARE promoter by AR was detected irrespective of C/EBP{alpha} expression in the presence of the ligand. C/EBP{alpha} was also associated with the ARE promoter when it was expressed, but only when it was coexpressed with AR. Neither AR nor C/EBP{alpha} was recruited onto the ARE promoter in the absence of the ligand. No signal was detected from the control PCR for nonspecific immunoprecipitation with primers specific to the luciferase-coding region, which is about 3.3 kb upstream of the ARE promoter in the pARE2-TATA-Luc reporter that was linearized at the SphI site located between the ARE promoter and luciferase-coding region.


Figure 6
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  		Fig. 6. C/EBP{alpha} Is Recruited by AR onto AR-Target Promoters

A, C/EBP{alpha} is recruited by AR onto an AR-target promoter. Cos-7 cells were transfected with an indicated combination of AR and C/EBP{alpha} expression plasmids together with linearized pARE2-TATA-Luc reporter in the presence or absence of 10 nM DHT. B, C/EBP{alpha} is recruited onto the endogenous AR-target PSA promoter. LNCaP cells were transfected with or without C/EBP{alpha} expression plasmid in the presence of 10 nM DHT. C, C/EBP{alpha} inhibits the recruitment of the AR coactivator GRIP1 to the PSA promoter. LNCaP cells were transfected with HA-GRIP1 and increasing amounts of C/EBP{alpha} expression plasmid. Cross-linked DNA fragments were immunoprecipitated with indicated antibodies and analyzed by PCR using pairs of specific primers spanning AREs of the promoters. A control PCR for nonspecific immunoprecipitation was performed with primers specific for the luciferase- or actin-coding region. HA, Hemagglutinin; IP, immunoprecipitation.

 
The recruitment of C/EBP{alpha} onto the endogenous PSA promoter, an AR-target promoter, was also accessed using LNCaP cells transfected with C/EBP{alpha} expression plasmid in the presence of androgen (Fig. 6BGo). AR occupied the PSA promoter irrespective of C/EBP{alpha} expression. C/EBP{alpha} was also associated with the PSA promoter when expressed.

Transient transfection analyses (Fig. 5Go, B–D) suggest that C/EBP{alpha} may inhibit AR transactivation by interfering with the interaction between AR and its coactivators. Therefore, we investigated the effect of C/EBP{alpha} expression on the recruitment of AR coactivators to the ARE of endogenous PSA promoter using LNCaP cells transfected with GRIP1 and increasing amounts of C/EBP{alpha} expression plasmid (Fig. 6CGo). C/EBP{alpha} inhibited the recruitment of GRIP1 to the ARE in a dose-dependent manner, with its increased recruitment to the promoter. These results suggest that C/EBP{alpha} is recruited to the androgen-regulated promoter by AR and blocks the association of AR coactivators in vivo.

C/EBP{alpha} Represses the Expression of the AR-Target PSA Gene
The best characterized androgen-responsive gene in the prostate is the gene that encodes PSA. PSA has been used as a prostate-specific tumor marker for monitoring prostate cancer and is a model gene for the study of mechanisms of AR-mediated transactivation. The results thus far allowed us to examine the effect of C/EBP{alpha} on the expression of endogenous PSA in AR-positive LNCaP cells. The prostate cancer cells were transfected with C/EBP{alpha} expression vector alone or together with C/EBP{alpha} siRNA. The level of PSA mRNA was determined by quantitative RT-PCR using PSA-specific primers. As shown in Fig. 7AGo, C/EBP{alpha} was capable of significantly down-regulating the expression of endogenous PSA gene in LNCaP cells, which was reversed by targeting C/EBP{alpha} expression with C/EBP{alpha} siRNA. The expressions of C/EBP{alpha} and AR proteins were confirmed by Western blot analysis (Fig. 7BGo). Together, these results suggest that C/EBP{alpha} is recruited to the androgen-regulated promoters by AR and inhibits AR target gene expression in vivo.


Figure 7
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  		Fig. 7. C/EBP{alpha} Represses the Expression of the AR-Target Gene, PSA, in Prostate Cancer Cells

A, C/EBP{alpha} repression of PSA expression in prostate cancer cells. LNCaP cells were transfected with C/EBP{alpha} expression plasmid alone or together with C/EBP{alpha} siRNA or scrambled siRNA as a negative control in the presence of 10 nM DHT. Transfected cells were analyzed for the expression of PSA mRNA by real time RT-PCR. B, The expression of C/EBP{alpha} and AR proteins was confirmed by Western blot analyses. ß-gal, ß-Galactosidase; WB, Western blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this paper, we demonstrate that C/EBP{alpha} is highly expressed at the exit stages of cell proliferation during prostate development, and its expression is capable of inhibiting the DNA synthesis in prostate cancer cells. These findings suggest that C/EBP{alpha} may be responsible for the developmental growth arrest of the prostate. A number of studies have demonstrated the critical role played by C/EBP{alpha} as a negative regulator of tissue growth (41). In addition, reports have emerged regarding the occurrence of C/EBP{alpha} mutations and polymorphism, or its loss of function in various malignant tissues (28, 42). Thus, C/EBP{alpha} is likely a key inhibitor for cell proliferation and growth in many tissues, including the prostate. Regarding the role of a negative regulator of cell proliferation, the regulation of C/EBP{alpha} gene expression in different tissues may be worth investigating to better understand the signaling for growth arrest.

It has been well established that AR plays an important role in prostate growth, as well as tumor development (1, 2, 3). In this report, we demonstrate that C/EBP{alpha} cross-talks with AR and down-regulates AR transactivation, suggesting that suppression of AR transactivation may be one mechanism by which C/EBP{alpha} inhibits cell proliferation in the prostate. Protein-protein interaction has been also implicated in the C/EBP{alpha}-mediated inhibition of liver cell growth (16, 17, 18, 19). C/EBP{alpha} directly interacts with cyclin-dependent kinases to inhibit their activity by blocking their association with cyclins. C/EBP{alpha} may also form a complex with RB-E2F4 to repress E2F transcription. On the other hand, the inhibitory action of C/EBP{alpha} in proliferating myeloid cells involves the transcriptional activity of C/EBP{alpha} (20, 21, 22). Thus, the mechanism by which C/EBP{alpha} inhibits cell proliferation seems to differ from cell type to cell type, although it acts as a negative regulator in many cell types.

In this report, we demonstrate that C/EBP{alpha} down-regulates AR transactivation by acting as a corepressor. Interestingly, the protein level of C/EBP{alpha} was previously reported to be down-regulated in the presence of androgens in differentiating adipocytes (43), suggesting that a negative feedback control loop may exist. Some androgen-target gene products, such as ARR19 and calreticulin, have also been shown to act as androgen corepressors (14, 44). The existence of such a negative feedback loop implicates an additional level of control for the fine tuning of cellular responses mediated by AR.

AR-mediated transcriptional regulation requires several protein complexes (45), which may interact in sequence, in parallel, or in combination. In vitro studies have shown that altered expression of AR coregulators may significantly modify the transcriptional activity of AR, suggesting that these coregulators could also contribute to the proliferation and progression of prostate cancer. Although many coactivators of AR have been identified, only a few corepressors have been well established and adequately characterized. The ability of C/EBP{alpha} to repress the expression of PSA, a clinical marker in the diagnosis and progression of prostate cancer, makes C/EBP{alpha} a new candidate as a therapeutic target. Detailed in vivo study of C/EBP{alpha} conditional knockout for the prostate may shed light on the development of new drugs for prostate cancers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids
Mouse AR expression and pARE2-TATA-Luc and PSA-Luc reporter plasmids have been previously described (44, 46, 47). VP16-AR N terminus containing AR amino acids 1–655 and GAL-AR C terminus containing AR amino acids 656–994 were constructed by inserting the corresponding regions of AR into pCMX-VP16 and pCMX-GAL4-N vector in frame, respectively. pcDNA3-C/EBP{alpha}, pcDNA3-C/EBPß, pGEX4T-1-C/EBP{alpha}, and its deletion mutants have been previously described (48). A dominant-negative mutant of C/EBP{alpha} was a kind gift from Dr. C. Stocking (University of Hamburg, Hamburg, Germany). pcDNA3-ARA70, pcDNA3-p300, pcDNA3-HA-DAX-1, and pcR3.1 steroid receptor coactivator 1 have been previously described (49, 50), and pSG5HA GRIP1 was kindly provided by Dr. M. Stallcup (University of Southern California, Los Angeles, CA).

Cell Culture and Transient Transfection Assay
Cos-7 and PPC-1 cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. LNCaP cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% FBS, 100 U/ml of penicillin/streptomycin, and 2 mM L-glutamine. RWPE-1 cells were maintained in keratinocyte-serum-free medium (Life Technologies) supplemented with 50 µg/ml bovine pituitary extract, 5 ng/ml epidermal growth factor, and antibiotic/antimycotic mixture (referred to as complete medium).

Cells were plated in 24-well plates 24 h before transfection and transfected with the indicated amount of expression plasmids and the reporter pARE2-TATA-Luc or GAL4-tk-Luc using Superfect (QIAGEN, Chatsworth, CA). Each transfection included the lacZ expression plasmid pCMVß as a control for transfection efficiency. Total amounts of expression vectors were kept constant by adding appropriate amounts of the empty vector. After transfection (24 h), the medium was replaced with fresh medium containing 10% charcoal-stripped serum and either 10 nM DHT or vehicle. Cells were harvested 24 h after the addition of hormone, and luciferase and ß-galactosidase activities were assayed as previously described (44). The levels of luciferase activity were normalized to the lacZ expression.

GST Pull-Down Assay
GST, GST-AR domain mutants, and GST-C/EBP{alpha} deletion mutants were expressed in Escherichia coli cells and isolated with glutathione-Sepharose-4B beads (Pharmacia Biotech AB, Piscataway, NJ). Immobilized GST fusion proteins were then incubated with [35S]methionine-labeled C/EBP{alpha} or AR proteins produced by in vitro translation using the TNT-coupled transcription-translation system (Promega Corp., Madison, WI). The binding reactions were carried out in 250 µl of GST-binding buffer (20 mM Tris-HCl at pH 7.9, 150 mM NaCl, 10% glycerol, 0.05% Nonidet P-40, 5 mM MgCl2, 0.5 mM EDTA, 1 mM dithiothreitol, and 1.5% BSA) overnight at 4 C. The beads were washed five times with 1 ml of GST-binding buffer. Bound proteins were eluted by the addition of 20 µl SDS-PAGE sample buffer and were analyzed by SDS-PAGE and autoradiography (44).

Coimmunoprecipitation and Western Blot Analysis
In vivo coimmunoprecipitation assays were performed with Cos-7 cells transfected with 1 µg AR and 3 µg C/EBP{alpha} expression plasmids or RWPE-1 cells. The cells were treated with or without 10 nM DHT for 24 h post transfection and harvested with RIPA cell lysis buffer (50 mM Tris-HCl at pH 7.5, 50 mM NaCl, 2.5 mM EGTA, 1% Triton X-100, 50 mM NaF, 10 mM Na4P2O7, 10 mM Na3VO4, 1 µg/ml aprotinin, 0.1 µg/ml leupeptin, 1 µg/ml pepstatin, 0.1 mM phenylmethylsulfonylfluoride, and 1 mM dithiothreitol). Whole-cell lysate (800 µg) was incubated with 20 µl protein A agarose bead slurry (Invitrogen, San Diego, CA) to exclude nonspecific binding and was then centrifuged. The supernatant was incubated with 2 µg anti-C/EBP{alpha} antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C. Each portion was further incubated for an additional 2 h after the addition of 20 µl protein A agarose bead slurry (Invitrogen). Agarose beads were washed six times each with RIPA buffer at 4 C, and bound proteins were separated by SDS-PAGE. Proteins on the gels were transferred to Protran nitrocellulose transfer membrane (Schleicher & Schuell Bioscience, Keene, NH), and subjected to Western blot analysis with anti-AR and anti-C/EBP{alpha} antibodies. Signals were then detected with an ECL kit (Amersham Biosciences, Piscataway, NJ).

Immunofluorescence
The day before transfection, PPC-1 and LNCaP cells were plated onto gelatin-coated coverslips. GFP-AR alone or together with C/EBP{alpha} was transiently transfected using Superfect reagent (QIAGEN). After 4 h, fresh medium was added to the cells. The next day cells were fed with fresh media with or without 10 nM DHT and incubated for an additional 24 h. Cells were then washed three times with cold PBS and fixed with 2% paraformaldehyde for 15 min. Fixed cells were mounted on glass slides and observed under laser scanning confocal microscope (Olympus Corp., Lake Success, NY). For detection of C/EBP{alpha}, cells mounted on glass slides were permeabilized with 2 ml PBS containing 0.1% Triton X-100 and 0.1 M glycine for 15 min, washed three times with 1x PBS, and blocked with freshly made blocking buffer (0.1% saponin and 0.05% BSA in PBS, pH 7.4) for 30 min at room temperature. Cells were incubated with primary anti-C/EBP{alpha} antibody (Santa Cruz Biotechnology) for 2 h at room temperature, washed three times with 1x PBS, and incubated for an additional hour with rhodamine-conjugated antirabbit secondary antibody.

Immunohistochemistry
The prostate dissected from a 3-wk-old rat was fixed in Bouin’s fixative (Sigma Chemical Co., St. Louis, MO) and embedded in paraffin by standard procedures. Sections were deparaffinized in Histoclear and rehydrated in an ethanol series. After the final wash in PBS, the sections were treated overnight with rabbit anti-C/EBP{alpha} antibody (Santa Cruz Biotechnology). Biotinylated goat antirabbit IgG, streptavidin conjugated to horseradish peroxidase, and peroxidase substrate-chromogen were used to detect signals according to the instructions of the manufacturer (Zymed Laboratories, Inc., South San Francisco, CA). Sections were counterstained with hematoxylin. Slides were then washed in distilled water, mounted with GVA mounting solution (Zymed), and observed under a light microscope with bright-field illumination.

Thymidine Incorporation
LNCaP and PPC-1 cells were cultured in 96-well plates at a density of 2 x 104 cells per well and transfected with either C/EBP{alpha} expression plasmid alone or the empty vector in 10% charcoal-stripped serum-supplied media. C/EBP siRNA or scrambled siRNA was also transfected along with C/EBP{alpha} expression plasmid where indicated. Each transfection included the lacZ expression plasmid pCMVß as a control for transfection efficiency. After sitting overnight, the cells were treated with 10 nM DHT for 24 h and then pulse labeled with [3H]thymidine (10 µCi/ml, specific activity 80 Ci/mmol, PerkinElmer Life Sciences, Norwalk, CT) for 4 h. Cells were harvested onto a glass microfiber filter (Whatman, Inc., Florham Park, NJ) and intensively washed with distilled water. Incorporation of thymidine into DNA is measured by counting the filters with a scintillation counter.

Transfection of siRNA
siRNAs specifically targeting C/EBP{alpha} (sense, 5'-GUCGGCCAGGAACUCGUCGTT-3'; and antisense, 5'-CGACGAGUUCCUGGCCGACTT-3') were custom designed (51, 52). LNCaP cells were transfected using the Oligofectamine Reagent (Invitrogen Life Technologies). Briefly, cells were grown in 35-mm dishes and overlaid with the transfection mixture containing siRNA (50 nM) in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% FBS and 2 mM L-glutamine. After 4 h incubation, complete medium without antibiotics was added and cells were incubated for 1 d. C/EBP was then transiently transfected as previously described. Scrambled siRNA (sense, 5'-GUAGUCCAUGGACCCGUAGTT-3'; and antisense, 5'-CUACGGGUCCAUGGACUACTT-3') was used as a negative control.

Northern Blot Analysis
Total RNA was extracted from the prostate with Tri reagent solution (Molecular Research Center, Inc., Cincinnati, OH). Total RNA (20 µg) was separated on a 1.2% denaturing agarose gel, transferred onto nylon membrane in 10x standard sodium citrate (SSC), and then immobilized under UV light. After prehybridization, the membrane was hybridized at 42 C in a solution containing 50% formamide, 10% dextran sulfate, 5x SSC, 1 mM EDTA, and 10 µg/ml of denatured salmon sperm DNA. After washing three times at 42 C for 20 min in 0.2x SSC and 0.1% sodium dodecyl sulfate as a final stringency, the membrane was exposed on Kodak x-ray film at –70 C.

ChIP Assay
Cos-7 cells were transfected with a combination of 1 µg pcDNA3-AR and 3 µg pcDNA3-C/EBP{alpha} expression plasmids as indicated. Cells were then treated 16 h after transfection with or without 10 nM DHT for 24 h and cross-linked with 1% formaldehyde for 10 min at room temperature. ChIP assays were performed as previously described (44). Anti-AR (Santa Cruz Biotechnology) or anti-C/EBP{alpha} (Santa Cruz Biotechnology) antibody was used for immunoprecipitation. Immunoprecipitated DNA and input-sheared DNA were subjected to PCR using ARE primer pairs (sense, 5'-CAGGTGCCAGAACATTTCTC-3'; and antisense, 5'-GAGTTTTCACTGCATACGACG-3'), which amplify an approximately 430-bp region spanning the ARE promoter of the reporter. As a negative control, PCRs were performed using Luc primer pairs (sense, 5'-GAAGGTTGTGGATCTGGATAC-3'; and antisense, 5'-TTTCCGTCATCGTCTTTCCG-3'), which amplify an approximately 370-bp region spanning the C-terminal part of the luciferase-coding region of the reporter.

LNCaP cells were transfected with C/EBP{alpha} expression vector with or without hemagglutinin-GRIP1 expression construct and processed for ChIP assays. Anti-AR, anti-C/EBP{alpha}, or antihemagglutinin antibody was used for immunoprecipitation. Immunoprecipitated DNA and input-sheared DNA were subjected to PCR using PSA primer pairs (forward, 5'-TGAGAAACCTGAGATTAGGA-3'; and reverse, 5'-ATCTCTCTCAGATCCAGGCT-3'), which amplify a 229-bp region (–4271 to ~–4043) spanning the ARE of PSA promoter. As a negative control, PCRs were performed using actin primer pairs (forward, 5'-GAGACCTTAACACCCCAGCC-3'; and reverse, 5'-CCGTCAGGCAGCTCATAGCTC-3'), which amplify a 362-bp region spanning exon 4 of the ß-actin gene.

Real-Time RT-PCR
Total RNAs were obtained from LNCaP cells transfected with C/EBP{alpha} expression plasmid together with or without C/EBP{alpha} siRNA or scrambled siRNA. Quantitative RT-PCR was performed using a real-time PCR machine (Corbett Research, Sydney, Australia) and the QuantiTect SYBR Green RT-PCR kit according to the protocol of the manufacturer. PCRs were performed with PSA-specific primers (forward, 5'-GGCCAGGTATTTCAGGTCAG-3'; and reverse, 5'-CCACGATGGTGTCCTTGATC-3'), which amplify a 570-bp fragment spanning open reading frame. As an internal control, PCRs were also performed using ß-actin-specific primers (forward, 5'-GAGACCTTCAACACCCCAGCC-3'; and reverse, 5'-CCGTCAGGCAGCTCATAGCTC-3'), which amplify a 362-bp region spanning exon 4.


   ACKNOWLEDGMENTS
 
We thank Drs. C. Stocking and M. Stallcup for graciously providing the C/EBP{alpha} dominant-negative mutant and GRIP1 expression vector, respectively.


   FOOTNOTES
 
This work was supported by a grant (A050287) from the Korea Health 21 Research and Development Project, Ministry of Health and Welfare, Republic of Korea.

All authors have nothing to declare.

First Published Online February 2, 2006

1 S.C. and E.-Y.G. contributed equally to the work. Back

Abbreviations: AR, Androgen receptor; ARE, androgen response element; C/EBP{alpha}, CCAAT enhancer-binding protein-{alpha}; ChIP, chromatin immunoprecipitation; FBS, fetal bovine serum; GFP, green fluorescent protein; GST, glutathione-S-transferase; GRIP1, glucocorticoid receptor-interacting protein 1; HDAC, histone deacetylase; MMTV, mouse mammary tumor virus; PSA, prostate-specific antigen; siRNA, small interfering RNA; SSC, standard sodium citrate; TSA, trichostatin A.

Received for publication June 20, 2005. Accepted for publication January 24, 2006.


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NURSA Molecule Pages Link:

Nuclear Receptors:   AR
Coregulators:   p300  |  SRC-1  |  GRIP1  |  ARA70
Ligands:   Dihydrotestosterone



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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals