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Endocrinology Vol. 147, No. 4 1969-1978
Copyright © 2006 by The Endocrine Society

Progesterone Receptor Transactivation of the Secretory Leukocyte Protease Inhibitor Gene in Ishikawa Endometrial Epithelial Cells Involves Recruitment of Krüppel-Like Factor 9/Basic Transcription Element Binding Protein-1

Michael C. Velarde, Mary Iruthayanathan, Renea R. Eason, Daying Zhang, Frank A. Simmen and Rosalia C. M. Simmen

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Nutrition Center, Little Rock, Arkansas 72202

Address all correspondence and requests for reprints to: Dr. Rosalia C. M. Simmen, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Arkansas Children’s Nutrition Center, 1120 Marshall Street, Little Rock, Arkansas 72202. E-mail: simmenrosalia{at}uams.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone receptor (PR), a ligand-inducible transcription factor, mediates the physiological actions of progesterone (P) through two distinct isoforms, PR-A and PR-B, and numerous nuclear coregulators. We previously demonstrated that basic transcription element binding protein-1 (BTEB1), a transcription factor of the Krüppel-like family, is a functional PR-interacting protein, based on the subfertility phenotype and reduced P sensitivity of uterine PR target genes, on BTEB1 null mutation. Here we examined the role of BTEB1 in PR-mediated signaling in endometrial epithelial cells using Ishikawa human endocarcinoma cells and the P-responsive secretory leukocyte protease inhibitor (SLPI) gene. Treatment of Ishikawa cells with P for 24 h increased SLPI and BTEB1 transcript levels without similar effects on PR expression. P induction was abolished by the PR antagonist RU486, whereas knockdown of BTEB1 with short interfering RNA reduced P-responsive BTEB1 but not SLPI expression to basal levels. Forced expression of BTEB1, either by stable or transient transfections of BTEB1 expression constructs in endometrial carcinoma cells, enhanced SLPI promoter activity. Chromatin immunoprecipitation with anti-BTEB1 antibody demonstrated BTEB1 recruitment to the proximal GC-rich containing SLPI promoter region (–97 to –86) in human endometrial carcinoma (Hec1A) cells overexpressing BTEB1. In Ishikawa cells, recruitment of BTEB1 to the proximal, GC-rich region and the distal, progesterone-responsive element-like containing region (–635 to –514) was P dependent and was accompanied by corecruitment of PR and the PR coactivator cAMP-response element binding protein-binding protein. PR-B, rather than PR-A isoform, preferentially associated with BTEB1 in the GC-rich region, whereas both PR isoforms were recruited to the progesterone-responsive element-like site along with BTEB1. Our findings define a novel pathway for BTEB1/PR cross-talk to facilitate P-dependent gene transcription in endometrial epithelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE STEROID HORMONE progesterone (P) plays essential roles in uterine endometrial growth and differentiation (1, 2). It classically acts through two progesterone receptor (PR) isoforms, PR-A and PR-B, which in the human and mouse are encoded by a single gene (3, 4). Binding of P to PR leads to the formation of PR homo- or heterodimers, which influence transcription by either direct interaction with progesterone-responsive elements (PRE) or forming functional complexes with other nuclear proteins that directly bind DNA, within the promoter and regulatory regions of target genes (5, 6). PR-regulated gene transcription also involves the participation of numerous steroid receptor coregulators, whose interaction with PR is dependent on whether the latter is bound to an agonist (for coactivator) or an antagonist (for corepressor). Coactivators increase whereas corepressors decrease the transcriptional activity of PR by influencing the stability of the preinitiation complex formed by PR with the basal transcriptional machinery and altering the acetylation status of nucleosomal core histones, leading to an open or closed chromatin structure (7). Gene ablation of several steroid receptor coactivators [e.g. steroid receptor coactivator (SRC)-1, SRC-2, SRC-3] in the mouse has revealed their importance in PR-dependent biological functions (8, 9, 10). However, because these proteins may be coexpressed and similarly regulated in the same cells, the criteria by which they are selected as PR nuclear partner(s) for activation of specific target genes remain unclear. In a recent study using a novel PR activity indicator mouse model (11), the distinct coactivator roles of SRC-1 in the uterus and SRC-3 in the mammary gland were demonstrated (12). Interestingly, whereas SRC-1 was shown to function as a PR coactivator in endometrial stromal (ST) and myometrial cells, it acted as a coinhibitor of PR target genes in luminal (LE) and glandular (GE) epithelium (12). These data suggest that tissue- and cell-type contexts are important modifiers of PR-dependent transcriptional response. Other nuclear receptors including estrogen receptor-{alpha} can bind PR coactivators, thus potentially limiting the latter’s availability for interaction with PR (13). Given that aberrant PR signaling leading to dysregulated gene expression may underlie failure of normal pregnancy as well as uterine oncogenesis (14, 15, 16, 17), the question of what coactivators are used by PR and how their interactions modify transactivation of specific genes remains relevant for understanding P-mediated physiological responses.

A novel PR coactivator, termed basic transcription element binding protein-1 (BTEB1), was recently described by our group, based on cumulative studies carried out in vivo and in vitro. BTEB1 is one of 25 known members of the Krüppel-like family of transcription factors, with postulated dual roles as a transactivator or a transrepressor, depending on cell context (18, 19). In previous studies, we found that BTEB1 is expressed in early pregnancy endometrium, coincident with that of PR (20, 21, 22); BTEB1 functionally interacts with ligand-activated PR-B, resulting in enhanced PR-B transactivity in human endometrial epithelial cells (22); and female mice null for the BTEB1 gene are subfertile due to decreased numbers of implanting embryos and partial uterine insensitivity to P-stimulation (23). In studies to evaluate the mechanism underlying the subfertility phenotype, we found that absence of the BTEB1 gene altered the spatiotemporal patterns of cell proliferation and apoptosis in the endometrium as well as the abundance of PR and ER{alpha} transcripts in ST cells, leading to developmental asynchrony between the uterine endometrium and the implantation-ready embryo (24). Taken together, these data denote the biological significance of BTEB1 in PR-mediated physiological events in the uterus.

Because BTEB1 is differentially expressed in uterine endometrial compartments (ST>GE; undetectable in LE) during early pregnancy in the mouse (23), it is important to study BTEB1/PR interactions in individual cell types to fully discern BTEB1’s role in influencing the genomic consequences of PR action. In the present study, we used the well-differentiated human endometrial epithelial cell line Ishikawa, whose regulated expression of ER{alpha} and PR resembles that of normal uterine epithelial cells (25), to elucidate the mechanisms of PR-mediated transcription involving BTEB1. Moreover, we used the gene for secretory leukocyte protease inhibitor (SLPI) (26) as a paradigm for P-mediated, endometrial epithelial-specific gene transcription because: 1) SLPI is predominantly expressed in endometrial GE cells, with low and absent expression in LE and ST cells, respectively (27, 28, 29); 2) uterine SLPI expression is regulated by P (30, 31); 3) epithelial overexpression of SLPI is associated with increased proliferation and promotion of metastatic potential (32, 33, 34); and 4) null mutation of BTEB1 was associated with uterine hypoplasia coincident with decreased SLPI expression (23). Our results show that whereas BTEB1 can induce SLPI gene expression independent of P, PR/P transactivation of the SLPI gene occurs, in part, through P induction of BTEB1 gene expression and P/PR-mediated corecruitment of BTEB1 and the PR coactivator cAMP-response element binding protein-binding protein (CBP) to the SLPI promoter. Results define a novel pathway for BTEB1/PR cross-talk that facilitates P-dependent gene transcription in endometrial epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Reagents, laboratory supplies, and antibodies were obtained from the following vendors: cell culture media and supplies and TriZol reagent, antibiotic/antimycotic solution and glutamine from Invitrogen Life Technologies (Carlsbad, CA); and oligonucleotides from Integrated DNA Technologies, Inc. (Coralville, IA). All other reagents, except when noted, were purchased from Fisher Scientific (Pittsburgh, PA).

Cell culture
The human endometrial epithelial adenocarcinoma cell line, Ishikawa, was routinely grown at 37 C in an atmosphere of 5% CO2-95% air in MEM supplemented with 10% (vol/vol) fetal bovine serum (FBS) and 1% antibiotic/antimycotic, following previously described protocols (34). The clonal lines Hec1A-4S and Hec1A-2AS, which were derived from the human endometrial adenocarcinoma cell line Hec1A by stable transfection of sense and antisense BTEB1 expression vectors, respectively, were cultured in McCoy’s 5A medium containing 10% FBS as previously described (35). Medium was replaced every 2–3 d, and cells were split when confluent. For P addition, cells were seeded in 60-mm plates at a density of 6 x 105 cells/well. Subconfluent cells (50%) were treated with vehicle (PBS) or 100 nM Promegestone (R5020; NEN Life Science Products, Inc., Boston, MA) in the presence or absence of the PR antagonist mifepristone (RU486, 100 nM; Sigma, St. Louis, MO). All treatments were performed in charcoal-stripped serum-containing medium (10% FBS). For gene expression analyses, cells were collected at 1, 3, and 7 d after P treatment. Experiments were carried out in quadruplicate and independently performed on three separate occasions.

RNA isolation and quantitative RT-PCR
Total RNA was prepared from Ishikawa cells or whole uterus using TriZol reagent (Invitrogen). Total RNA was quantified and analyzed for integrity using the Agilent 2100 Bioanalyzer and RNA 6000 NanoLabChip kit (Agilent Biotechnologies, Palo Alto, CA). RNA samples were reverse transcribed using random primers and a cDNA synthesis kit following the manufacturer’s protocols (Applied Biosystems, Foster City, CA). mRNA levels were determined by real-time quantitative RT-PCR (QPCR). Each primer set was designed to flank an intron to prevent the amplification of genomic DNA, using PrimerExpress (Applied Biosystems). The forward and reverse primers, respectively, for the human genes and resultant PCR product sizes (in parentheses) were: SLPI, 5'-GCT GTG GAA GGC TCT GGA AA-3' and 5'-TGC CCA TGC AAC ACT TCA AG-3' (297 bp); BTEB1, 5'-TGG CTG TGG GAA AGT CTA TGG-3' and 5'-CTC GTC TGA GCG GGA GAA CT-3' (124 bp); PR-A/B, 5'-GGT GGC ATG GTC CTT GGA-3' and 5'-GCT TAG GGC TTG GCT TTC ATT-3' (123 bp); PR-B, 5'-CGA CCC AGG AGG TGG AGA T-3' and 5'-GAG GGA AAA GGG AAG GAG GAG-3'; (105 bp), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CAA CGG ATT TGG TCG TAT TGG-3' and 5'-TGA CGG TGC CAT GGA ATT T-3' (158 bp); CBP, 5'-GGG AAG CAG CTG TGT ACC ATT C-3' and 5'-GTC GTC ACC CAG GGT CAC AT-3' (120 bp); and 18S, 5'-TCT TAG CTG AGT GTC CCG CG-3' and 5'-ATC ATG GCC TCA GTT CCG AA-3' (151 bp). cDNA samples (1/25 of reverse transcription reaction) were amplified using the SYBR Green PCR Master Mix under conditions recommended by the manufacturer (Bio-Rad Laboratories, Hercules, CA): 1) preincubation at 50 C for 2 min; 2) DNA polymerase activation at 95 C for 2 min; and 3) 40 PCR cycles of 95 C for 15 sec and 60 C for 1 min. Samples were assayed in duplicate using the ABI Prism 7000 detection system (Applied Biosystems). For each primer set, a standard curve was generated by serial dilution of pooled cDNAs. The threshold cycle, which represents the fractional cycle number where the fluorescent signal exceeds background was obtained for each reaction and used to calculate the mean RNA quantity. The melting points of all products were routinely determined to confirm the absence of primer-dimers. For each sample, relative gene expression was calculated with 18S rRNA as internal reference.

Transient transfection and luciferase assays
Ishikawa cells were seeded at a density of 5 x 105 cells/well in 6-well dishes in serum-containing medium and incubated for 24 h to allow cells to adhere. Cells (70% confluent) were cotransfected with SLPI promoter-reporter plasmid or empty vector plasmid (pGL2; Promega, Madison, WI) (10 µg DNA per well) and rat BTEB1 (pcDNA-BTEB1) or empty expression constructs (1 µg DNA per well) using 5 µl of lipofectAMINE reagent (Invitrogen) in OPTI-MEM I reduced serum medium (Invitrogen) for 6 h (34). The SLPI reporter-promoter plasmid, which contains 120 bp of the 5' regulatory region of the porcine SLPI gene, exhibits more than 75% homology to human and mouse promoter sequences within this region (36, 37, 38). After transfection, cells were incubated in serum-containing medium for an additional 18 h. Cell lysates were collected 48 h later, and luciferase activity (measured as relative light units) was assessed by using a luciferase assay system (Promega) and a MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, VA). Similar procedures were performed for Hec1A clonal lines 4S and 2AS, except that only the SLPI reporter-promoter plasmid (above) or empty vector plasmid (10 µg DNA per well) were transfected. In a number of experiments, ß-galactosidase expression plasmid pSV-GAL (5 µg per well; Promega) was cotransfected with SLPI-Luc-reporter constructs to evaluate transfection efficiency. Protein concentration of extracts was determined by the Lowry method using BSA as standard. Results were normalized to the protein content of each sample and are presented as least-squares means ± SEM. Two independent transfection experiments were performed, with each experiment carried out in triplicate (n = 6 samples per treatment group).

Western blot analyses
Western blotting was carried out as previously described (34), using rabbit antirat BTEB1 antibody (21). Immunoreactive bands were detected by sequential incubation with antirabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and Western lightning chemiluminescence reagents (Pierce Biotechnology, Rockford, IL). Signals were captured and quantified with the Bio-Rad molecular analyst detection system and Quantity One software.

RNA interference
Reverse transfection of Ishikawa cells plated at a density of 1 x 105 in 12-well dishes used siPORT NeoFX lipid-based agent (Ambion, Austin, TX) in OPTI-MEM-reduced serum medium (Invitrogen). Double-stranded small interfering (si)RNA targeting human BTEB1 mRNA (catalog no. 16708), a negative control siRNA (catalog no. 4615), and a positive control GAPDH siRNA (catalog no. 4605) were obtained from Ambion Inc. and were used at 50 nM final concentration. After 8 h, the cells were transferred to fresh MEM-10% FBS and further incubated for 16 h to allow the cells to recover from the transfection procedure. Treatment with R5020 (100 nM) was carried out for 24 h in fresh MEM containing 10% charcoal-stripped FBS, with the corresponding control cells receiving only vehicle (PBS). Total RNA was collected and assayed for relative abundance of BTEB1, PR, and SLPI transcripts by QPCR as described above. Three independent experiments were performed, each in quadruplicate.

Chromatin immunoprecipitation (ChIP) assays
Hec1A-4S cells were grown in 10-cm plates until 70% confluent before ChIP assay. Ishikawa cells were grown to 50% confluence and then treated with vehicle or 100 nM R5020 in 10% charcoal-stripped FBS medium for 24 h. Cells were processed for ChIP following the manufacturer’s recommendations (Upstate Biotechnology, Inc., Lake Placid, NY). Proteins were cross-linked to DNA by adding 10% formaldehyde to plated cells. Cells were washed in PBS containing protease inhibitors (Protease Inhibitor Cocktail Set III; Calbiochem, La Jolla, CA), and lysed in the same buffer with added sodium dodecyl sulfate. The lysates were sonicated on ice for 15 pulses in 10-sec cycles, with a 20-sec pause between each cycle, using a Sonic Dismembrator 50 (Fisher Scientific) at a setting of 6 to generate sheared DNA of approximately 400 bp in length. Aliquots of the lysate were set aside to quantify DNA present (DNA input); the remainder was diluted 1:10 in ChIP dilution buffer and cleared in a solution containing Protein A/G PLUS-agarose (Santa Cruz Biotechnology) and sonicated salmon sperm DNA (Invitrogen) for 30 min with rotation. Supernatants were collected by centrifugation and then immunoprecipitated using 5 µg each of anti-BTEB1 goat polyclonal antibody (sc-12996; Santa Cruz Biotechnology), anti-CBP mouse monoclonal antibody (sc-7300; Santa Cruz Biotechnology), anti-PR(A/B) mouse monoclonal antibody (PgR Ab-3; NeoMarkers, Fremont, CA), or anti-PR-B mouse monoclonal antibody (PgR Ab-6, Neomarkers). For the preimmune control, an equal amount of normal serum was substituted for the test antibody. Immune complexes were recovered by incubation with salmon sperm DNA/Protein A/G PLUS agarose for 1 h at 4 C, successively washed with a series of salt solutions, and then eluted with buffer containing 1% sodium dodecyl sulfate and 0.1 M NaHCO3. The formaldehyde cross-links were reversed by the addition of 0.2 M NaCl and Rnase (20 µg/ml) and incubation for 4 h at 65 C. The ChIP-captured DNA was purified for PCR by treatment with Proteinase K, followed by phenol-chloroform extraction and precipitation with isopropyl alcohol. RT-PCR using specific primers corresponding to human SLPI promoter BTEB1-binding site region (forward: 5'-GTG TTG GCC TCA TAG CCT TAC C-3'; reverse: 5'-GCC TCT CCC AGC TAC TAT TGC A-3'; product size of 112 bp) and PRE-like site region (forward: 5'-CGG AGC TCT TCT TCA GCT TTC A-3'; reverse: 5'-TCA GCT CGT TCA ACC AAC TGA T-3'; product size of 122 bp) were carried out under the following conditions: 1) 94 C for 2 min; 2) 33 cycles of 94 C for 45 sec, 56 C for 1 min, and 72 C for 30 sec; and 3) final extension of 72 C for 7 min. PCR products were resolved on a 2% agarose gel containing ethidium bromide and visualized under UV light. Band intensities were quantified using the Bio-Rad molecular analyst detection system.

ChIP-captured DNA was analyzed for recruitment of PR-(A/B) and PR-B proteins by QPCR under conditions described above. As a negative control, recruitment of PR-(A/B) and PR-B to a sequence (+273 to +373 nt; 101 bp) within intron I of the SLPI gene was evaluated using forward (5'-CTC TAT GCA GCC ATG CTG TCA-3') and reverse (5'-AGA GTC GCA GCA AGG TGA AGA-3') primers.

Data analysis
Data, presented as least-square means ± SEM, were subjected to analysis by Student’s t test or two-way ANOVA, as indicated under each figure legend. Differences between means in two-way ANOVA were further analyzed by Tukey test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P effects on SLPI gene expression in Ishikawa cells
Previous studies with a number of tissues and cell types have shown that SLPI transcript levels are increased by P (30, 31, 39). To determine whether P increases SLPI gene expression in Ishikawa cells, we examined the levels of SLPI transcripts in response to P treatment by QPCR. Cells treated with the synthetic progestin R5020 for 1 and 3 d showed increased SLPI transcript levels (Fig. 1AGo); this was not observed with P treatment for 7 d (Fig. 1AGo) or for shorter duration (6 and 12 h; data not shown). The addition of the PR antagonist RU486 30 min before treatment of cells with R5020 completely abolished P-enhanced SLPI gene expression (Fig. 1BGo). P induction of SLPI transcript levels was not accompanied by changes in PR-A/B or PR-B transcripts (Fig. 1Go, C and D). At 7 d, a decrease in PR-B, but not PR-A/B, transcript levels was observed coincident with loss of P-induced SLPI expression.


Figure 1
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  		FIG. 1. Regulation of SLPI gene expression by progesterone. Ishikawa cells were incubated in medium containing 10% charcoal-stripped FBS ± R5020 (100 nM) for the indicated days (1, 3, or 7), and analyzed for mRNA expression by QPCR. A, Induction of SLPI by R5020 at 1, 3, and 7 d. B, Inhibition of R5020-induced SLPI transcript levels by the PR antagonist RU486. Cells were incubated in medium ± R5020 in the presence or absence of RU486 for 24 h before isolation of RNA for analysis. Levels of total PR-A/B (C) and PR-B (D) transcripts were quantified in cells treated with R5020 for 1, 3, and 7 d. Results (least squares means ± SEM) are from three independent experiments and are expressed as fold induction over the control value (–R5020). Differences were identified by two-way ANOVA, followed by Tukey test. Means with different superscripts differ at P < 0.05.

 
BTEB1 effects on SLPI gene expression
Because BTEB1 is a PR-interacting protein (21, 22, 23), we sought to determine whether cross-talk between BTEB1 and PR is involved in P-regulated SLPI gene expression. To address this, we performed siRNA knockdown to diminish the expression of BTEB1 in control (vehicle) and P-treated Ishikawa cells. Basal expression of BTEB1 was not affected by BTEB1 siRNA duplex (50 nM) in non-P-treated cells (Fig. 2AGo); this effect was similarly observed with 100 nM of siRNA (data not shown) and suggests very low endogenous levels of BTEB1 in Ishikawa cells because use of the same siRNA in a human endometrial stromal cell line HESC (40) resulted in BTEB1 knockdown (data not shown). Treatment with P for 24 h increased BTEB1 gene expression by 2-fold, which was abrogated by addition of BTEB1 siRNA, suggesting a role for BTEB1 in mediating its own P-dependent expression. Consistent with the absence of BTEB1 siRNA effect on BTEB1 transcript levels in non-P-treated cells, SLPI transcript levels did not differ in cells without or with added BTEB1 siRNA (Fig. 2BGo). SLPI transcript levels were increased by P (Fig. 2BGo), similar to that shown above (Fig. 1AGo); this induction was diminished by the addition of BTEB1 siRNA, albeit not to basal levels shown for BTEB1 (Fig. 2AGo). The decrease in P-induced SLPI transcript levels by BTEB1 siRNA occurred in the absence of similar effects on total PR-A/B or PR-B levels (Fig. 2Go, C and D). The specificity of the siRNA was demonstrated by the lack of effects of BTEB1 siRNA on GAPDH mRNA and the negative control (scrambled sequence) siRNA on BTEB1 and SLPI mRNAs, respectively (data not shown).


Figure 2
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  		FIG. 2. Effects of BTEB1 on SLPI and PR expression in control and R5020-treated Ishikawa cells. Ishikawa cells were mock transfected or transfected with BTEB1 siRNA and incubated for an additional 24 h in charcoal-stripped FBS-containing medium in the presence or absence of R5020 (100 nM). Harvested cells were analyzed for BTEB1 (A), SLPI (B), PR-A/B (C), or PR-B (D) transcript levels by QPCR. Results (least squares means ± SEM) are from three independent experiments and are expressed as fold induction over the control (–R5020). Differences were identified by two-way ANOVA, followed by Tukey test. Means with different superscripts differ at P < 0.05.

 
P effects on BTEB1 gene expression
To determine whether P-induced BTEB1 expression (Fig. 2AGo) is temporally coincident with that of SLPI, we evaluated BTEB1 mRNA levels in Ishikawa cells treated with P for 1, 3, and 7 d. BTEB1 mRNA abundance was increased by P to almost the same magnitude (1.5- to 2-fold) as for SLPI (Fig. 3AGo). The temporal induction of BTEB1 mRNA by P occurred for a longer duration (up to 7 d) than SLPI (Fig. 3AGo), although P did not influence its expression at shorter (6 and 12 h) exposure times (data not shown). The induction of BTEB1 transcript levels by P at 24 h was similarly observed at the protein level (Fig. 3BGo). RU486 antagonized the P-dependent induction of BTEB1 (Fig. 3CGo), suggesting PR-mediated regulation of BTEB1 expression by P.


Figure 3
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  		FIG. 3. Regulation of BTEB1 gene expression by progesterone. A, Ishikawa cells were incubated in charcoal-stripped FBS-containing medium ± R5020 (100 nM) for 1, 3, or 7 d. Total RNA was isolated and BTEB1 mRNA levels were analyzed by QPCR. Results (least squares means ± SEM) are from three independent experiments and are expressed as fold induction over the control value (–R5020). B, Ishikawa cells were incubated in charcoal-stripped FBS-containing medium ± R5020 (100 nM) and 24 h later were collected for analyses of BTEB1 protein levels by Western blots. A representative blot (100 µg total protein loaded per lane) and graphical representation of immunoreactive BTEB1 (molecular mass 36 kDa) levels in cells treated with or without R5020 for 24 h are shown. Results (least squares means ± SEM) from three independent experiments are expressed as fold induction over the control (–R5020). C, Inhibition of R5020-induced BTEB1 transcript levels by the PR antagonist RU486. Cells were incubated in medium ± R5020 in the presence or absence of RU486 for 24 h. Results (least squares means ± SEM) from three independent experiments are expressed as fold induction over the control (–R5020). Significant differences were identified by two-way ANOVA, followed by Tukey test (A and C) or t test (B). Means with different superscripts or indicated with * differ at P < 0.05.

 
BTEB1 enhances SLPI promoter activity
Next, we asked whether the increase in SLPI expression by P is a consequence of P-induction of BTEB1. To address this, we mimicked the P increase in BTEB1 levels using cells that overexpress BTEB1 and evaluated SLPI promoter activity. In the first experiment, Ishikawa cells were cotransfected with a BTEB1 expression construct (pCDNA3-BTEB1) containing the full-length rat BTEB1 cDNA, which shares 96% homology with human BTEB1 (18) or empty construct and the SLPI promoter-reporter construct (–120SLPI) or control promoterless (Luc) construct. Ishikawa cells cotransfected with pCDNA3-BTEB1 (+BTEB1) showed higher SLPI promoter activity than those cotransfected with empty expression construct (-BTEB1) or those transfected with the control promoterless construct (Luc) (Fig. 4AGo). In the second experiment, SLPI promoter activity was evaluated in high (4S) and low (2AS) BTEB1-expressing, PR-negative Hec1A clonal lines, respectively (35), on transfection with either SLPI promoter-reporter (–120SLPI) or control promoterless (Luc) constructs. 4S displayed higher SLPI promoter activity than 2AS, with the latter showing activity similar to the promoterless construct (Fig. 4BGo).


Figure 4
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  		FIG. 4. BTEB1 regulation of SLPI promoter activity in endometrial epithelial cells. A, Ishikawa cells were cotransfected with SLPI promoter-reporter plasmid (containing 120 bp of the 5' regulatory region of the porcine SLPI gene) or empty vector (Luc) plasmid and BTEB1 expression construct (pcDNA-BTEB1) or empty expression vector. B, Hec1A 4S cells were transfected with SLPI promoter-reporter plasmid (above) or empty vector plasmid. Cell lysates were analyzed for luciferase activity. Results of two independent experiments (with each experiment carried out in triplicate) were normalized to the protein content and are shown as least squares means ± SEM. Significant differences were identified by two-way ANOVA, followed by Tukey test. Means with different superscripts differ at P < 0.05. RLU, Relative light unit.

 
BTEB1 is recruited to the SLPI promoter
To further determine whether BTEB1 directly regulates SLPI transcription independent of PR/P, we examined whether the 120-bp region located immediately upstream of the translation initiation site (+1, ATG) and the mRNA start site (–22 nt) of the human SLPI promoter contains GC-boxes that serve as BTEB1 binding sites (31, 36). A single GC-rich sequence was found between –97 and –86 nt upstream of the human SLPI promoter (Fig. 5AGo), which was also located in the same region for the mouse and pig SLPI promoters (37, 38). To evaluate the binding of BTEB1 to this GC-box independent of P, we performed ChIP on Hec1A-4S cells, which express high levels of BTEB1 (35). Antibody against BTEB1 precipitated the SLPI promoter region containing the BTEB1 binding site; this was not observed on omission of the antibody in the reaction (Fig. 5Go, B and C). Thus, BTEB1 binds to the GC-box of the SLPI promoter.


Figure 5
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  		FIG. 5. Recruitment of BTEB1 to the SLPI promoter in Hec1A 4S cells. A, The location of the GC-rich sequence within the proximal region of the human SLPI promoter was determined by ClustalW alignment program. The bracketed area (–200 to –89 nt) indicates the SLPI promoter region amplified by the PCR primers used for ChIP analysis; +1 is the ATG translation initiation site. B, Cross-linked, sheared chromatin from Hec1A cells stably transfected with sense BTEB1 expression construct (4S) was immunoprecipitated with antibody against BTEB1. DNA was analyzed by PCR, using primers described in A. A representative PCR amplification is shown from an ethidium bromide-stained agarose gel. C, Results of PCR amplifications from four independent experiments (least squares means ± SEM) are expressed as the fold change relative to control (–R5020). Significant difference (*) at P < 0.05 was identified by t test.

 
BTEB1 recruitment to the SLPI promoter is enhanced by P
We evaluated the binding of BTEB1 and PR to the SLPI promoter in Ishikawa cells incubated for 24 h in medium containing vehicle alone (–R5020) or with added R5020 (100 nM). In non-P-treated cells (–R5020), neither preimmune nor anti-BTEB1 antibody precipitated the SLPI promoter region containing the GC-box (Fig. 6Go, A and B). In P-treated cells (+R5020), however, DNA-bound BTEB1 was detected with anti-BTEB1 but not preimmune antibody. Interestingly, the association of BTEB1 to the GC-box of the SLPI promoter in P-treated cells was accompanied by the precipitation of the same SLPI promoter region with antibodies to PR-A/B isoforms or PR-B alone (Fig. 6Go). The PR coactivator CBP, which we have shown to functionally interact with BTEB1 and PR in vitro (22), was also recruited to the same region in P-treated cells (Fig. 6Go). Unlike PR but similar to BTEB1 (Figs. 1CGo and 3AGo), the recruitment of CBP was accompanied by P induction of its mRNA (Fig. 6CGo).


Figure 6
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  		FIG. 6. Recruitment of BTEB1, PR, and CBP to the proximal SLPI promoter region. Ishikawa cells were incubated in charcoal-stripped FBS-containing medium ± R5020 (100 nM) for 24 h. Cross-linked, sheared chromatin was immunoprecipitated with preimmune serum or antibodies against BTEB1, PR-(A/B), PR-B, and CBP. DNA was analyzed by PCR, using primers spanning the GC-box region of the SLPI promoter. A, Representative ChIP PCR gels. B, ChIP PCR results (least squares means ± SEM) from four independent experiments are expressed as the fold change relative to control (–R5020). Significant difference (*) at P < 0.05 was identified by t test. C, Ishikawa cells were incubated in charcoal-stripped FBS-containing medium ± R5020 (100 nM) for 24 h and analyzed for levels of CBP mRNA by QPCR. Results (least squares means ± SEM) from three independent experiments are expressed as fold induction over the control (–R5020). Significant difference (*) at P < 0.05 was determined by t test.

 
To examine whether PR binds to PRE-like sites distinct from its association with GC-box bound BTEB1, the recruitment of PR to the –635 to –514 nt SLPI promoter region containing these sequences (Fig. 7AGo) was examined by ChIP. Without P-treatment (–R5020), no PR was present in this region of the promoter. However, after P treatment, PR and its coactivator CBP were recruited to this region. Interestingly, BTEB1 was also found to associate with this same region, which lacks GC-rich sequences (Fig. 7Go, B and C).


Figure 7
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  		FIG. 7. Recruitment of BTEB1, PR, and CBP to the PRE-like sequence of the SLPI promoter. A, PRE-like site is located at –591 to –564 nt upstream of the SLPI promoter; +1 is the ATG translation start site. The bracketed area (–635 to –514 nt) indicates the SLPI promoter region amplified by the PCR primers. B and C, Ishikawa cells were incubated in 10% charcoal-stripped FBS-containing medium ± R5020 (100 nM) for 24 h. Cross-linked, sheared chromatin was immunoprecipitated with normal serum (preimmune) or antibodies against BTEB1, PR-(A/B), PR-B, and CBP. DNA was analyzed by PCR using primers described in A. B, Representative ChIP PCR gels. C, ChIP PCR results (least squares means ± SEM) from four independent experiments are expressed as fold change relative to control (–R5020). Significant difference (*) at P < 0.05 was identified by t test.

 
To determine whether either PR isoform preferentially interacts with GC-box bound BTEB1 or binds to PRE-like sequences of the SLPI promoter, we performed quantitative measurement of PR bound to distal (PRE-like containing) and proximal (GC-box containing) SLPI promoter regions using QPCR. Recruitment of PR to a sequence within intron I of the SLPI gene was analyzed in parallel as negative control. Consistent with previous results (Fig. 7Go, B and C), cells treated with P had increased recruitment of total PR (PR-A/B) and PR-B to the distal and proximal regions of the SLPI promoter, with no recruitment observed to intron I (Fig. 8Go). For the proximal region, PR-A/B and PR-B antibodies yielded a 2-fold-change in recruitment with P than without P (Fig. 8Go, A and B). By contrast, in the distal promoter region, there was an approximately 50% higher P-dependent recruitment observed with PR-A/B antibody than PR-B antibody. These results suggest that PR-B is preferentially recruited over PR-A to the proximal SLPI promoter region containing the BTEB1 GC-rich sequence binding site.


Figure 8
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  		FIG. 8. Recruitment of PR-A/B and PR-B to the proximal and distal SLPI promoter regions and intron I of the SLPI gene. Ishikawa cells were incubated in 10% charcoal-stripped FBS-containing medium ± R5020 (100 nM) for 24 h. Cross-linked, sheared chromatin was immunoprecipitated with antibodies against PR-(A/B) and PR-B, and recruitment of PR-A/B and PR-B proteins in specific genomic regions was analyzed by QPCR. ChIP QPCR results (least squares means ± SEM) from four independent experiments using anti-PR-A/B (A) or anti-PR-B (B) antibody are expressed as fold change relative to control (–R5020). Significant differences were identified by two-way ANOVA, followed by Tukey test. Means with different superscripts differ at P < 0.05 except for c (with ^), P = 0.07.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we used ChIP and RNA interference to gain further insights into the role of BTEB1 as a PR coactivator in P-regulated transactivation of the endometrial epithelial gene SLPI. We report here that in P-stimulated Ishikawa cells: 1) BTEB1 forms functional complexes with both PR isoforms and the PR coactivator CBP; 2) the assembly of the BTEB1/PR/CBP complex occurs by binding of BTEB1 to either its GC-box recognition motif or PRE-bound PR; 3) PR-A and PR-B, possibly as homo- or heterodimers directly bind the SLPI promoter PRE-sequences; and 4) PR-B, rather than PR-A isoform, preferentially associates with GC-box bound BTEB1. These data indicate that BTEB1 is an important participant in P/PR transcriptional regulation of the epithelial-expressed gene SLPI and corroborate our previous descriptions of uterine phenotypes in BTEB1 female null mice, in which P-dependent responses were compromised with loss of BTEB1 expression (23, 24). Moreover, these findings provide further support to the notion that BTEB1 functions in nonredundant pathways with other PR coactivators to facilitate uterine sensitivity to P.

Our studies used the SLPI gene as a paradigm for P-regulated transactivation in Ishikawa endometrial epithelial cells because P induction of SLPI gene expression in vivo and in vitro has been demonstrated in this cell type for humans and primates (30, 31). In addition, we previously demonstrated a graded loss of SLPI responsiveness to P as a function of BTEB1 genotype (23); ovariectomized, P-treated BTEB1+/– female mice had 2-fold higher and lower levels of uterine SLPI transcripts, respectively, than BTEB1-null and wild-type counterparts. Moreover, because SLPI expression in uterine endometrium is localized predominantly to GE and is undetectable in ST cells (29, 31, 32, 39), whereas BTEB1 is expressed in both cell compartments (23), data obtained provide a framework for evaluating cell context-dependent BTEB1/PR interactions. We found that whereas P-induction of SLPI gene expression in Ishikawa cells is temporally coincident with that of BTEB1 and that PR mediates P-induced expression of both genes, PR transactivation of SLPI involves two distinct but likely concurrent mechanisms. One pathway requires the corecruitment of BTEB1 and another PR coactivator, CBP, to the SLPI promoter. A second pathway does not involve BTEB1 because knock-down of P-induced BTEB1 levels using BTEB1 siRNA did not completely abrogate P-induced SLPI expression, a finding consistent with the absence of complete loss of P-regulated expression of uterine SLPI in BTEB1 null mice (23). The present results highlight the participation of other PR coactivators in PR-mediated transactivation of SLPI. Moreover, given that BTEB1 alone, when present at sufficiently high levels (Hec1A-4S cells), can induce SLPI promoter activity, the existence of BTEB1-mediated signaling pathways, independent of P/PR, is also suggested.

Although the criterion used by PR to select its functional partners in endometrial epithelial cells remains to be defined, data presented here and in another study (22) suggest that BTEB1 and CBP may associate with PR in tandem. CBP exhibits intrinsic histone acetyltransferase activity (41) and may be requisite for attaining an open chromatin structure to facilitate binding of other transcription factors for optimal transactivation. In a previous study using HeLa cell nuclear extracts (42), the sequential recruitment of SRC-1 and then p300/CBP by PR in enhancing PR-dependent transactivation was demonstrated in vitro, a finding that raises the question of whether with a different cell type or target gene, BTEB1 can substitute for SRC-1. Indeed, the absence of SRC-1 and PR interaction in the P-dominated uterus had been reported (43); complex formation between p300/CBP and PR, but not between SRC-1 and PR, was observed in the human endometrium at the secretory phase of the menstrual cycle, despite the concurrent expression of SRC-1 and CBP. Whereas the recruitment of SRC-1 to the SLPI promoter was not evaluated in the present study, it is possible that the observed BTEB1-independent pathway of PR activation of the SLPI gene may involve SRC-1 in association with CBP.

The PR/P induction of BTEB1 in Ishikawa cells suggests that BTEB1 is both a downstream target as well as a coactivator of PR/P signaling. Regulation of BTEB1 expression by P has been previously suggested from gene microarray analysis of mouse uteri during P-induced delayed implantation (44) and mammary T47D cells treated with P (45); however, the present study represents the first report on the P/PR-mediated induction of BTEB1 expression in endometrial epithelial cells. Moreover, whereas we have suggested that BTEB1 regulates PR transactivation in both endometrial ST (24) and GE (22) cells, the present study provides the first mechanistic insight on PR/BTEB1 cross-talk for any endometrial cell type using a natural promoter. Interestingly, up- (with P treatment) or down (with BTEB1 siRNA)-regulation of BTEB1 expression in Ishikawa cells was not correlated with changes in expression levels of PR isoforms at shorter (1 and 3 d) P exposure. However, prolonged exposure to P (7 d) resulted in decreased levels of PR-B transcripts with no effect on total PR, suggesting increased expression of PR-A isoform relative to PR-B. These results likely reflect ligand-mediated down-regulation of PR-B, independent of BTEB1. In a previous study (24), we showed that in BTEB1 null mice, there was a reduction in PR mRNA abundance in endometrial ST cells of the P-dominated uteri (pregnancy d 3.5 and 4.5), when compared with those of wild-type counterparts. By contrast, endometrial GE cells in wild-type and BTEB1 null mice did not differ in their PR expression levels at these pregnancy days (data not shown). Thus, BTEB1 might have more global functions in uterine ST cells, consistent with its higher level of expression in these relative to GE cells in the early pregnancy endometrium (23). Alternatively or in addition, these findings could be related to the relative levels of PR-A vs. PR-B present in each cell type and the specific PR isoform that mediates P-dependent transactivation of the PR gene.

The existence of PR-A and PR-B and their distinct roles in different endometrial cell compartments have raised the important question of which PR isoform preferentially interacts with BTEB1 as a function of cell context. We have suggested on the basis of the reproductive phenotypes of PR-A null mice (15) and the ST phenotype of the early pregnant BTEB1 null mice (23, 24) that PR-A is the preferential BTEB1-interacting partner in ST cells (24). Furthermore, PR-B likely interacts with BTEB1 in endometrial epithelial cells, based on the coimmunoprecipitation of these nuclear proteins and the superactivation of PR-B, but not of PR-A, transactivity by BTEB1 in Hec1A cells, on cotransfection with expression constructs for these proteins (22). To address this question in the present study, we carried out ChIP analysis of the SLPI promoter using antibodies that recognized PR-A/B or only PR-B. Using QPCR that enabled quantitative measurements of PR-B bound to the distal (PRE containing) and proximal (GC-box containing) SLPI promoter regions, we found preferential recruitment of PR-B to the proximal relative to the distal promoter region. This suggests that BTEB1, when bound to its recognition GC-box motif, predominantly forms a functional complex with the PR-B dimer and CBP but associates with either PR-A and PR-B isoforms, when the latter are bound to their own recognition sequences. Results demonstrate the functional interdependence of BTEB1 and PR in P/PR-mediated transcriptional activity and raise two interesting mechanistic questions, elucidation of which will provide further support for its physiological relevance in other P-target cells. The first relates to the relative contribution of BTEB1, when bound in either context, to the formation of a functional transcriptional complex with PR, and second, to whether the predominance of one PR isoform over another in BTEB1-expressing cells alters the outcome of P-regulated gene networks.

In conclusion, the present results identify BTEB1 as a functional PR-A and PR-B interacting partner in the P-mediated transcriptional activation of SLPI gene in Ishikawa endometrial epithelial cells. We further show that in gene promoters with regulatory regions that contain noncanonical PRE and GC-box motifs, the mechanism of transcriptional regulation by PR may involve the participation of both cis-elements via their ability to bind PR, either directly (PRE) or indirectly (through BTEB1), resulting in a functional complex that also involves CBP. Given that the development of endometrial carcinoma is associated with discordant expression of PR isoforms (46); that P inhibition of endometrial cancer growth and invasiveness is mediated by PR-B (47); and that epithelial cell proliferation (32) as well as tumorigenesis and metastatic potential (33) are induced by SLPI, the disruption of BTEB1-mediated PR signaling in epithelial cells may have significant physiological implications in the control of P-dependent growth and differentiation of normal and abnormal endometrial cells.


   Acknowledgments
 
The authors thank Dr. Bhuvanesh Dave for help with siRNA studies.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD21961 (to R.C.M.S. and F.A.S.) and the University of Arkansas for Medical Sciences Graduate Student Research Fund (to M.C.V.).

The authors have nothing to declare.

First Published Online December 29, 2005

Abbreviations: BTEB1, Basic transcription element binding protein-1; CBP, cAMP-response element binding protein-binding protein; ChIP, chromatin immunoprecipitation; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GE, glandular epithelium; LE, luminal epithelium; nt, nucleotide; P, progesterone; PR, progesterone receptor, PRE, progesterone-responsive element; QPCR, quantitative RT-PCR; si, small interfering; SLPI, secretory leukocyte protease inhibitor; SRC, steroid receptor coactivator; ST, stromal.

Received November 8, 2005.

Accepted for publication December 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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