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1 /99/$03.00/0 Vol. 140, No. 6 Endocrinology Printed in U.S.A. Copyright 1999 by The Endocrine Society Trans-Activation Functions of the Sp-Related Nuclear Factor, Basic Transcription Element-Binding Protein, and Progesterone Receptor in Endometrial Epithelial Cells* ROSALIA C. M. SIMMEN, TRICIA E. CHUNG, HIROAKI IMATAKA, FRANK J. MICHEL, LOKENGA BADINGA, AND FRANK A. SIMMEN Interdisciplinary Concentration in Animal Molecular and Cell Biology (R.C.M.S., T.E.C., F.J.M., L.B., F.A.S.), and Departments of Animal Science (R.C.M.S., T.E.C., F.J.M., L.B.) and Dairy and Poultry Sciences (F.A.S.), University of Florida, Gainesville, Florida ; and the Department of Biochemistry, McGill University (H.I.), Montréal, Québec, Canada H3G 1Y6 THE STEROID hormones estrogen (E) and progesterone (P) are recognized to play predominant roles in pregnancy-associated events by regulating the expression of uterine endometrial genes associated with this tissue s development and growth. However, the modes by which these hormones, acting through their respective nuclear receptors (1), modify uterine gene expression in a cell type- and pregnancy-dependent context are not well understood. Novel mechanisms for E- and P-regulated gene expression have recently emerged from studies demonstrating a lack of correspondence between steroid inducibility of gene expression and the presence of consensus recognition sequences for steroid hormone receptors within gene regulatory regions. In Received August 20, Address all correspondence and requests for reprints to: Rosalia C. M. Simmen, Ph.D., Department of Animal Science, Building 459, Shealy Drive, University of Florida, Gainesville, Florida * This work was supported by NIH Grant HD and USDA Grants and This manuscript is published as Journal Series R from the Florida Agricultural Experiment Station. ABSTRACT The present study examined the trans-activation potential of basic transcription element-binding protein (BTEB), a recently identified member of the Sp family of GC box-binding transcription factors, on the expression of the gene encoding the pregnancy-associated, epithelial-specific, and progesterone (P)-induced porcine uterine endometrial secretory protein, uteroferrin (UF). Endometrial expression of BTEB, P receptor (PR), and UF genes was analyzed by RT-PCR as a function of pregnancy stage and cell type and was correlated with the levels of endometrial BTEB that were quantified by Western blot and/or electrophoretic mobility shift assay. PR, BTEB, and UF messenger RNAs (mrnas) were present in early (day 12) and mid(day 60) pregnancy pig endometrium, although expression levels varied for each mrna (UF, day 12 day 60; PR and BTEB, day 12 day 60). Within the endometrium, glandular epithelial (GE) cells manifested higher amounts of UF mrna than stromal fibroblastic cells, whereas both cell types had comparable amounts of BTEB and PR mrnas. Expression of BTEB, however, was limited to endometrial GE cells. A BTEB expression vector (pcdna-3bteb) was used to examine the effect of increased BTEB protein on UF gene expression and promoter activity in primary cultures of pig endometrial GE cells. Cells transiently transfected with pcdna-3bteb had 2-fold higher UF mrna levels than those transfected with the empty expression vector (pcdna-3). Further, cells cotransfected with a UF promoter-luciferase ( 1935UF-Luc) reporter gene and the BTEB expression vector had 2-fold higher Luc activity than those cotransfected with reporter gene and pcdna-3. This effect of BTEB was not observed in transfected endometrial stromal fibroblastic cells, but was apparent in the human endometrial epithelial carcinoma cell lines ECC-1 and Hec- 1-A, which exhibit low levels of BTEB protein and low or undetectable PR mrna levels, respectively. The respective contributions of BTEB and PR to the modulation of UF promoter activity were examined by cotransfection of Hec-1-A and ECC-1 cells with expression plasmids for BTEB and PR and one of two UF promoter constructs ( 831UF- Luc or 1935UF-Luc) in the absence or presence of P. The increase in UF promoter activity with BTEB was mimicked by PR in a P- dependent manner in both cell lines. The combined effect of PR/P and BTEB appeared additive in Hec-1-A cells and was synergistic in ECC-1 cells. These results highlight the cell context dependence of the trans-activation potential of BTEB and suggest its unique role, in concert with PR, in directing the temporal expression of endometrial epithelial genes of pregnancy. (Endocrinology 140: , 1999) one such study (2), the effect of P on transcription of the p21 gene was found to be dependent, not on the presence of canonical recognition sequences for the P receptor (PR), but on those that bind an unrelated nuclear factor, namely Sp1. Further, E induction of the c-fos gene required the presence of GC-rich motifs within its promoter region that bind the E receptor (ER)/Sp1 complex, rather than the presence of an ER recognition sequence (3). These findings suggest that transcriptional activation by E and P may involve in part interactions of their cognate receptors with other nuclear proteins in addition to or in place of their direct binding to DNA. The extent of these interactions and the physiological context under which they occur, however, remain unclear. The findings that Sp1, a member of the C 2 H 2 zinc finger family that also includes Sp2, Sp3, Sp4, basic transcription element-binding protein (BTEB), and BTEB2 (4 8), mediates E- and P-regulated gene expression (2, 3, 9, 10) raised the question of whether this function is specific to this GC boxbinding protein or is shared by other family members. In the rabbit uteroglobin gene, whose pregnancy-associated endometrial expression is mediated by E and P, a functional role 2517

2 2518 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR Endo 1999 Vol 140 No 6 for Sp1 has been suggested in its basal (11, 12) as well as E-induced (10) transcription. Other Sp family members function either in place of (e.g. Sp4) or as a repressor (e.g. Sp3) of Sp1 by binding to common recognition sites within the uteroglobin gene promoter (11). It has not been demonstrated whether the newly identified GC box-binding proteins BTEB and BTEB2 exert similar effects as Sp1. Whereas expression of Sp1 is ubiquitous (4 7), that of BTEB or BTEB2 appears to be more restricted and varies with tissue and cell type (8, 13, 14). One mammalian tissue that exhibits a high level of BTEB expression is the uterine endometrium of pregnancy (14). In the pig, uterine endometrial expression of BTEB is epithelial cell type specific (14), which correlates with the expression of other P-regulated genes of pregnancy (15). This suggested the potential for BTEB to functionally modulate the pregnancy-associated expression of a number of these endometrial genes. The gene encoding uteroferrin (UF), a porcine transplacental iron transport protein (16) that belongs to a family of tartrate-resistant acid phosphatases in mammals (17), is a likely candidate for BTEB regulation, as sequences that have been shown to bind BTEB are located in its 5 regulatory region in close proximity to those that bind PR (18, 19). However, no studies have previously examined the trans-activation of the endometrial UF gene by this or a related nuclear factor(s). In the present study, we have used the UF gene as a model to evaluate the possibility that the temporal and highly regulated expression of epithelial-specific, pregnancy-associated uterine genes involves the actions of PR and BTEB. Toward this end, we examined whether a functional association exists between coexpression of BTEB and PR and the epithelial-specific temporal induction of the UF gene during pregnancy. Further, we tested whether BTEB, alone or in combination with the PR/P complex, trans-activates the UF gene promoter specifically in endometrial epithelial cells. Cell culture Materials and Methods Glandular epithelial (GE) and stromal (ST) cells were isolated from early (day 12) pregnancy pig endometrial tissue and cultured as previously described (20). These cells were used immediately upon reaching 80% confluence and were not further subcultured. The human endometrial carcinoma cell line Hec-1-A (American Type Culture Collection, Manassas, VA) was cultured in McCoy s 5A medium containing 10% FBS and phenol red. ECC-1, a human endometrial carcinoma cell line (Dr. P. G. Satyaswaroop, Hershey Medical Center, Hershey, PA) (21) was propagated in DMEM-Ham s F-12 containing 10% FBS. Ishikawa cells were propagated and cultured as previously described (22). Cells were maintained at 37 C in an atmosphere containing 5% CO 2-95% air. Plasmid constructs Chimeric UF-luciferase (Luc) reporter gene constructs were generated by subcloning 5 -genomic fragments of the UF gene (23) representing 1935 to 23 ( 1935UF-Luc) and 831 to 23 ( 831UF-Luc), respectively, into the HindIII site of the pgl3-basic vector (Promega Corp., Madison, WI). The orientations of the subcloned fragments were confirmed by DNA sequencing. The BTEB expression construct (pcdna-3bteb) consisted of the entire coding region of human BTEB (24), but lacked the 5 -untranslated sequences of the messenger RNA (mrna) that were identified to be involved in translational repression (13). Previous studies have shown that Hepa1 cells, whose endogenous expression of BTEB is normally undetectable by Western blot analysis, exhibited detectable levels of this protein when transfected with this expression vector (Imataka, H., unpublished data). The expression construct encoding the full-length rat PR B (PR B ) complementary DNA (cdna) was a gift from Dr. Benita Katzenellenbogen (University of Illinois, Urbana, IL) (25). Transfection and luciferase assays Transient transfections were performed on primary cultures of pig GE and ST cells and human endometrial carcinoma cell lines, when 80% confluent, by the polybrene method using CsCl density gradient purified plasmid DNAs (20). Plasmid DNA (10 g for each construct or expression vector) was added to cells plated on six-well culture dishes and incubated for 6 h. Cells were then washed with HBSS (ph 7.4) and treated with dimethylsulfoxide (DMSO) in HBSS (ECC-1, 20% DMSO for 3 min; Hec-1-A and primary cells, 25% DMSO for 4 min) to increase cell permeability. Cells were washed with HBSS twice and then incubated in fresh medium containing 10% FBS with or without added P (10 nm). After 48 h, cells were washed twice with ice-cold HBSS and lysed in single strength lysis buffer (200 l; Promega Corp., Madison, WI). The experiments were performed at least three times using independent DNA preparations of each construct and for primary cultures, cells that were isolated from endometrium of at least three individual pigs. The protein concentration of cell lysate (5 l) was determined using the Bio-Rad Laboratories, Inc., protein assay reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Quantitative determination of Luc activity (measured as relative light units) in cell lysates was carried out using an Autolumat Luminometer (EG&G Berthold, Bad Wildbad, Germany). Luciferase activity was normalized to the protein concentration of cell lysates. Analyses of RNA and protein Total RNA was isolated from pregnant pig endometrial tissues, primary cultures of pig endometrial GE or ST cells and from human endometrial cell lines by use of TriZol (Life Technologies, Grand Island, NY) following the manufacturer s instructions. The integrity of the RNA was assessed by inspection of 28S and 18S band intensities after agarose gel electrophoresis. The relative amounts of PR, BTEB, UF, and 2 - microglobulin ( 2 M) mrnas in the indicated samples were evaluated by RT-PCR (26) or by RNA dot blot analysis. The RT-PCR analyses to detect PR, UF, and 2 M mrnas were subjected to preliminary validation and optimization steps using a pool of day 60 pregnant pig endometrial cdna to ensure that amplification of the products was in the exponential phase and the assay was linear relative to the amount of input RNA. These steps included varying the amount of cdna template used for a fixed number of PCR cycles, adjusting the number of cycles for a fixed template amount, and determining the optimal Mg 2 concentration and ph for each cdna template-primer combination using the PCR Optimizer kit (Invitrogen, San Diego, CA). The optimal conditions and primer pairs for detecting BTEB transcript by RT-PCR have been previously described (14). The primer pairs for PCR amplification of UF, PR, and 2 M transcripts were designed from previously published porcine cdna sequences (27 29) and were synthesized by the DNA synthesis core facility of the Interdisciplinary Center for Biotechnology Research at the University of Florida, Gainesville. Optimal thermal cycling parameters were the following: PR: 95 C, 1 min; 55 C, 1 min; 72 C, 1 min, with 1 l cdna template in 2 mm Mg 2 and ph 9.0 (30 cycles); BTEB: same as PR, except that 0.5 l cdna template was used; UF: 95 C, 2 min; 55 C, 2 min; 72 C, 30 sec, with 0.2 l cdna template in2mm Mg 2 and ph 9.0 (30 cycles); and 2 M: same as PR, except that 0.1 l cdna template was used (30 cycles). DNA fragments of 416, 290, 812, and 287 bp were generated by PCR of cdnas for BTEB, PR, UF, and 2 M, respectively, and were verified by nucleotide sequence analysis. For analysis of PCR band intensities, photographs were scanned at high resolution, and the integrated density of the band was calculated using the Alpha Imager 2000 Documentation and Analysis System (Alpha Innotech Co., San Leandro, CA). The intensities of the PR, UF, and BTEB signals were normalized to that of the 2 M internal control. Dot blot analysis for UF mrna abundance was performed as previously described (30), using a 32 P-labeled porcine UF gene cdna insert as probe (27), and resultant hybridization intensities were quantified by PhosphorImage analysis (Molecular Dynamics, Inc., Sunnyvale, CA).

3 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR 2519 Nuclear extracts (NE) were prepared from endometrial tissues and cells, following previously described procedures (14, 23). The levels of BTEB protein in NE were examined by Western immunoblot analysis, using rabbit antisera generated against either porcine BTEB fusion protein (for pig tissues) (14) or rat BTEB (for human endometrial cell lines) (13) at a dilution of 1:500 in TBS buffer (1 mm Tris and 160 mm NaCl, ph 7.4) containing 1% Blotto (nonfat dry milk), as previously described (14). The immunoreactive BTEB band was subsequently visualized by further incubation of the filter with [ 125 I]protein A (Amersham, Arlington Heights, IL) in binding buffer, followed by autoradiography. Electrophoretic mobility shift assays Approximately 10 g NE protein were used for each gel mobility shift assay. A double stranded oligonucleotide probe containing the GC-rich region located at 768 to 749 bp (termed GC box 1) (19) of the UF gene 5 -flanking region was end-labeled using [ - 32 P]ATP and T4 polynucleotide kinase and used as probe for the binding reactions. This fragment specifically binds BTEB protein, as previously determined from competition assays with wild-type GC box 1 and unrelated oligomers (19) and by gel supershift with anti-bteb antiserum (14). The NE was preincubated with antirat BTEB antiserum (1 l) or preimmune serum (1 l) for 30 min at 4 C before addition of the labeled probe. The binding reactions were performed at 37 C for 30 min, and DNA-protein complexes were resolved in a 6% nondenaturing polyacrylamide gel as described previously (19). Statistical analysis All numerical data obtained from densitometric analysis of PCR bands or hybridization blots and from luciferase assays were subjected to least squares ANOVA using the general linear models procedures of the Statistical Analysis System (SAS Institute, Inc., Cary, NC) (31). Significant effects due to day of pregnancy, cell type, or treatment (promoter construct, expression construct, presence or absence of P) were separated by orthogonal contrasts or the Student-Newman-Keuls sequential range test (31). Values were considered significant at P 0.05 and are presented as the least squares mean (LSM) sem. Results BTEB, PR, and UF genes are coexpressed in pregnancy endometrium We previously demonstrated the presence of BTEB protein in the endometrium during pregnancy (14), suggestive of a role for this nuclear factor in pregnancy-associated endometrial gene expression. Further, we showed that endometrial UF gene expression is induced by P at the level of mrna (32) and gene promoter activity (18). To confirm the relationship among PR, BTEB, and UF gene activity, the levels of BTEB mrna and protein and PR mrna in endometrium at early (day 12) and mid (day 60) pregnancy were evaluated and correlated with those of UF mrna. RT-PCR analyses of endometrial RNAs demonstrated the presence of PR and BTEB transcripts on days 12 and 60 of pregnancy, coincident with expression of UF mrna and BTEB protein (Figs. 1 and 2). On day 12, only one of three endometrial tissues analyzed expressed the UF mrna, and this level of expression was lower than that exhibited by all three endometrial tissues on day 60, consistent with previous results (33). The levels of PR mrna, when normalized to 2 M, whose expression was constant during this period, did not differ (PR/ 2, FIG. 1. Expression of UF, PR, and 2 M genes in pregnant pig endometrium. Relative steady state mrna levels for UF, PR, and 2 M were determined by RT-PCR, as described in Materials and Methods. Endometria from early (D12) and mid (D60) pregnancy pig (n 3 animals/day of pregnancy) were used as a tissue source for RNA isolation. Equal aliquots (15 l) from each PCR reaction were electrophoresed in a 1.5% agarose gel, which was stained with ethidium bromide to visualize the PCR products. The sizes of the expected products for UF, PR, and 2 M transcripts are 812, 290, and 287 bp, respectively. FIG. 2. Relative BTEB mrna and protein levels in pregnant pig endometrium. Endometrium from early (D12) and mid (D60) pregnancy pig (n 3 animals/day of pregnancy) was used as a tissue source for RNA isolation and for preparation of nuclear extracts. A, Steady state levels of BTEB mrna were determined by RT-PCR, as described in Materials and Methods. Equal aliquots (15 l) from each PCR reaction were subjected to Southern hybridization, using a 32 P- labeled fragment of porcine BTEB cdna as probe. B, Equal amounts of nuclear extract protein (30 g) were separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot using polyclonal antiserum raised against pig BTEB fusion protein, as described in Materials and Methods. The immunoreactive band of 32 kda representing pig BTEB is shown, with the migration positions of the mol wt markers indicated by arrows on the left.

4 2520 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR Endo 1999 Vol 140 No 6 vs ; P 0.05) between days 12 and 60 of pregnancy. BTEB mrna and protein levels also appeared to be similar at these pregnancy days, in agreement with the results of a previous study (14). Endometrial GE and ST cells isolated in early pregnancy (day 12) exhibited differential expression of the UF gene, with GE and ST cells exhibiting high (from four of four animals) and undetectable (from four of four animals) amounts, respectively, of the UF transcript (Fig. 3). To examine whether the endometrial epithelial cell type expression of the UF gene is correlated with PR and/or BTEB, GE and ST cells were evaluated for expression of their respective mrnas. BTEB mrnas were present in both GE (BTEB/ 2, ) and ST (BTEB/ 2, ) at comparable levels, although only GE cells demonstrated detectable BTEB protein as evaluated by electrophoretic mobility shift assay (Fig. 4). This complex has been previously confirmed to represent endometrial BTEB bound to the GC-rich motif by its ability to be supershifted by a specific BTEB antiserum (14). Similarly, the levels of PR mrna, when normalized to those of 2 M, were comparable (P 0.05) in GE (PR/ 2, ) and ST (PR/ 2, ) cells. Effect of BTEB on UF gene promoter and mrna abundance in endometrial GE cells To demonstrate that BTEB can functionally trans-activate the UF gene promoter in an epithelial cell context, primary cultures of GE and ST cells isolated from early pregnancy pig endometrium (day 12) were transiently cotransfected with the 1935UF-Luc reporter construct and the BTEB expression construct (pcdna-3bteb) or the corresponding empty FIG. 4. Expression levels of BTEB mrna and protein in primary cultures of GE and ST cells of early pregnancy pig endometrium. A, GE and ST cells (isolated as described in Fig. 3) were analyzed for expression of BTEB mrna by RT-PCR. Total RNAs for the analysis were prepared from different primary cultures of GE and ST cells, with each culture isolated from endometrium of an early pregnancy (D12) pig (n 4 pigs). Equal aliquots (15 l) of each PCR reaction were electrophoresed on a 1.5% agarose gel, which was transferred to a nylon membrane and hybridized with a 32 P-labeled pig BTEB cdna fragment. B, Different preparations of nuclear extracts from primary cultures of endometrial GE and ST cells, representing four early pregnancy (D12) pigs were analyzed for presence of BTEB protein by electrophoretic mobility shift assay, using a 32 P-labeled oligonucleotide probe containing a GC-rich region previously shown to bind BTEB (14). The top and bottom arrows represent bound and free oligonucleotide probes, respectively. FIG. 3. Expression of UF, PR, and 2 M mrnas in primary cultures of GE and ST cells of early pregnancy pig endometrium. GE and ST cells were isolated from endometrium of four individual early pregnancy (D12) pigs, separately grown to confluence, and analyzed for expression of the indicated mrnas by RT-PCR, as described in Materials and Methods. The sizes of the transcripts for UF, PR, and 2 M are 812, 290, and 287 bp, respectively. expression vector (pcdna-3). In these studies, the transfection efficiency of ST cells was consistently higher (by at least 5-fold) than that of GE cells, as measured by parallel transfection with the pgl2 control plasmid and subsequent assay for Luc activity (data not shown). The basal promoter activity of the 1935UF-Luc reporter gene in GE cells was increased by cotransfection of the BTEB expression plasmid, whereas that in ST cells was relatively unaffected (Fig. 5, A and B). The effect of BTEB on UF promoter activity in epithelial cells was further evaluated by measuring the steady state levels of endogenous UF mrna in primary cultures of endometrial GE cells transiently transfected with empty expression vector or BTEB expression vector. Cells were har-

5 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR 2521 FIG. 5. Effect of BTEB on UF promoter activity as a function of endometrial cell type and on UF gene expression in GE cells. Primary cultures of GE and ST cells, isolated from early pregnancy (D12) pig endometrium were transiently transfected with a UF-Luc reporter gene construct ( 1935UF-Luc) and either the BTEB expression vector (pcdna-3bteb) or empty vector (pcdna-3), as described in Materials and Methods. A and B, Transcription from the UF promoter was measured by luciferase assay (expressed as relative light units) of whole cell lysates. Results (LSM SEM) were normalized to cell extract protein concentration and were from four independent experiments, with each experiment representing isolated cells from a distinct D12 pregnancy pig endometrium and, within each experiment, three separate transfections. Bars with different superscripts differ (P 0.05); those without superscripts do not differ (P 0.05). C, Endometrial GE cells, transiently transfected with empty vector (pcdna-3) or BTEB expression vector (pcdna-3bteb), were analyzed 24 h later for steady state UF mrna levels by dot blot analysis of total RNA, as described in Materials and Methods. Hybridization intensities were quantified by phosphorimage analysis. Results are graphically presented as the LSM SEM from three independent experiments, where each experiment represents transfection of GE cells independently isolated from three animals. Bars with different superscripts differ (P 0.05). vested 24 h after transfection, cellular RNA was isolated, and relative levels of UF mrna were quantified by dot blot analysis, followed by phosphorimaging of hybridization intensity. A significant (2-fold; P 0.05) induction of steady state UF mrna levels was observed with increased BTEB content (Fig. 5C), consistent with that observed at the level of UF gene promoter activity (Fig. 5A). Expression of BTEB in human endometrial cell lines The human endometrial cell lines ECC-1, Ishikawa, and Hec-1-A support the activity of the UF gene promoter to direct Luc gene expression (23) (this study) despite undetectable endogenous UF transcripts (data not shown). These observations suggest that endometrial epithelial-derived cells

6 2522 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR Endo 1999 Vol 140 No 6 FIG. 6. Expression of BTEB mrna and protein in human endometrial carcinoma cell lines. A, Total cellular RNA isolated from the endometrial cell lines ECC-1, Hec-1-A (HEC-1A), and Ishikawa and from an early pregnancy pig endometrium (d12px) were analyzed for the presence of BTEB transcript by RT-PCR. Equal aliquots (15 l) from each PCR reaction were electrophoresed on an agarose gel, which was stained with ethidium bromide to visualize the PCR products. B, Nuclear extracts prepared from ECC-1 and Hec- 1-A cells were subjected to electrophoretic mobility shift assays using a 32 P-labeled double stranded oligonucleotide probe containing a GC-rich region that binds BTEB, as described in Materials and Methods. The BTEB-oligo complex (indicated by the bottom arrow) was shifted to a slower migrating complex (indicated by the top arrow) with the addition of antirat BTEB antiserum, but not of control (preimmune) serum. may have in common nuclear factors that partly support the expression of genes associated with the epithelial phenotype. To evaluate the possibility that one such regulatory factor is BTEB, the expression of BTEB gene in these cells was examined at the levels of its mrna and protein. ECC-1, Hec-1-A, and Ishikawa cells express the BTEB gene at levels comparable to those in pregnant pig endometrium, as detected by RT-PCR (Fig. 6A). The presence of BTEB protein in NE prepared from ECC-1 and Hec-1-A cells was evaluated by Western blot and gel retardation antibody supershift assays. Western blot analysis using rabbit antirat BTEB antiserum failed to detect BTEB in NE prepared from either cell line (data not shown). However, the more sensitive gel shift assay using a 32 P-labeled oligonucleotide fragment corresponding to a GC-rich region within the UF gene promoter and that was previously shown to bind BTEB (14, 19) demonstrated the presence of a protein/dna complex in ECC-1 NE (Fig. 6B). The electrophoretic mobility of this complex was retarded by the addition of rabbit antirat BTEB antiserum, but not by preimmune rabbit serum. Nuclear extracts prepared from Hec-1-A cells exhibited the formation of a similar, albeit weaker intensity, protein/dna complex that was also supershifted by anti-bteb antiserum, but not by preimmune serum (Fig. 6B). BTEB and PR in the trans-activation of the UF gene The lower levels of BTEB protein in ECC-1 and Hec-1-A cell lines, as indicated by its inability to be detected by Western blot analysis, in contrast to the pregnancy endometrium in the pig (Ref. 14 and this study), suggested that the absence of endogenous UF gene expression in these cells may in part be a consequence of their diminished BTEB content. To test

7 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR 2523 FIG. 7. Effects of BTEB and PR on UF promoter activity in the human endometrial carcinoma cell line Hec-1-A. Cells were transfected with BTEB expression vector, PR expression vector, or both and one of two UF-Luc reporter gene constructs ( 831UF-Luc, 1935Uf-Luc), as described in Materials and Methods. Ten micrograms of each expression vector and/or reporter gene were added per well. The total amount of DNA per well was equalized to 30 g by the addition of appropriate amounts of the empty vector (pcdna-3). Cells with added PR were incubated in the absence or presence of P (10 nm), which was added to the fresh culture medium subsequent to DMSO shock. Cells were harvested after 48 h of incubation, and cell lysates were analyzed for Luc activity as described in Materials and Methods. Results are graphically presented as the LSM SEM of four independent transfection experiments, with each experiment performed in triplicate. whether increased levels of BTEB protein can enhance basal UF promoter activity in endometrial epithelial-derived cell lines, ECC-1 and Hec-1-A cells were transiently transfected with the 1935UF-Luc reporter plasmid along with the empty vector or BTEB expression vector. Cells were harvested 48 h after transfection and assayed for Luc activity. BTEB significantly increased (P 0.05) the transcriptional activity of 1935UF-Luc relative to that in the presence of empty expression vector in both cell lines (Figs. 7 and 8). To evaluate whether BTEB and PR additively or synergistically trans-activate the UF gene promoter in endometrial epithelial cells, Hec-1-A and ECC-1 cells, which also exhibit undetectable PR transcripts (34) (data from this laboratory, not shown) were cotransfected with 1935UF-Luc and/or 831UF-Luc plasmids, which contain three and two P-responsive sequences (18), respectively; expression plasmids for BTEB and/or PR; and for those transfected with PR in the presence or absence of added P (10 nm) in the culture medium. Extracts isolated from these cells were assayed for Luc activity. Similar to BTEB, PR in the presence of P increased transcription from the long and short UF constructs in Hec- 1-A cells (Fig. 7). Cotransfection of BTEB and PR (plus P) had additive effects on the activity of both promoter constructs (Fig. 7). However, although the effect of BTEB on UF promoter activity was more pronounced for the longer construct (P 0.05), that of PR/P did not differ between constructs (P 0.107). In ECC-1 cells, cotransfection of BTEB and PR (in the presence of P) resulted in a significant trans-activation of the 1935UF-Luc promoter construct at a level that was higher (P 0.05) than the additive effects of the two expression vectors when tested separately (Fig. 8). Discussion In the present study, we demonstrate that the Sp-related nuclear factor BTEB trans-activates the UF gene promoter, alone and in combination with the PR/P complex. This action of BTEB is specific to endometrial epithelial-derived cells and probably underlies in part the endogenous expression of the UF gene in GE cells of pregnancy. Moreover, coexpression of PR with BTEB in epithelial cells appears requisite for UF gene expression, because a relative lack of either or both proteins is associated with undetectable UF transcripts. These results are the first to identify BTEB as an important regulatory component in the pregnancy-associated transcription of an endometrial epithelial gene. Primary cultures of GE and ST cells from early pregnancy pig endometrium and the human endometrial transformed (carcinoma) cell lines that exhibit the epithelial phenotype were used to examine the cell context dependence of BTEB

8 2524 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR Endo 1999 Vol 140 No 6 FIG. 8. Effects of BTEB and PR on UF promoter activity in the human endometrial carcinoma cell line ECC-1. Cells were transfected with BTEB expression vector, PR expression vector, or both and with a UF-Luc reporter gene construct ( 1935Uf-Luc), as described in Fig. 7. Results are graphically presented as the LSM SEM of four independent transfection experiments, with each experiment performed in triplicate. action for a number of reasons. First, isolated ST cells do not manifest BTEB protein, either endogenously or upon transient transfection with the BTEB expression construct, despite the presence of the BTEB transcript. Second, ECC-1 and Hec-1-A cells have diminished BTEB protein levels relative to primary cultures of endometrial GE cells; hence, a temporal association between increased expression of this protein and induction of UF promoter activity could be readily established. Finally, isolated GE cells represent a more physiologically relevant environment for UF gene expression. Using these models, we obtained several lines of evidence to support the involvement of BTEB in the transcriptional regulation of the UF gene: 1) BTEB, in the absence or presence of PR, increased UF promoter activity in endometrial epithelial cells; 2) UF mrna levels in pig endometrial GE cells were induced by increased BTEB levels; 3) endometrial carcinoma cells exhibiting diminished BTEB protein content, relative to endometrial epithelial cells of pregnancy, do not express the endogenous UF gene; and 4) ST cells, which express PR at a level comparable to that of GE cells, but not BTEB, have undetectable expression of UF. Collectively, these results indicate that BTEB, similar to its family member Sp1 (2, 9, 10, 12), mediates the trans-activation of an endometrial epithelial gene. In support of previous studies (13), the data presented here demonstrate the cell context-dependent translational control of BTEB expression in two cell systems, namely the porcine endometrial stromal fibroblastic cells and human endometrial carcinoma cells exhibiting the epithelial phenotype. Thus, although BTEB transcripts in these cells are readily detectable, expression of the corresponding protein is minimal (ECC-1, Hec-1-A) or undetectable (ST cells). Interestingly, transient transfection of a human BTEB expression construct that lacked specific 5 -untranslated sequences previously identified to mediate translational repression of BTEB mrna in HeLa cells (13) increased the expression of corresponding BTEB protein in ECC-1 and Hec-1-A cells, as measured by increased transcription from the UF gene promoter. By contrast, similarly transfected ST cells did not exhibit the anticipated increase in UF promoter activity. These results suggest that additional factors, unrelated to those involved in translational repression, might modulate the synthesis and/or functional activity of BTEB protein in ST cells. The apparent requirement for coexpression of BTEB and P-dependent PR for the high level transcriptional activity of UF, a gene preferentially expressed by uterine epithelial cells of pregnancy (Ref. 20 and this study), raises the interesting possibility that uterine differentiation associated with pregnancy events may involve a threshold concentration of each of these factors, below which the functional signaling mechanism(s) responsible for activation of specific gene targets is not achieved. Indeed, GE cells isolated from early pregnancy pig endometrium showed a modest, but consistently higher, level of UF mrna upon transfection with BTEB expression construct, relative to control transfected cells. As GE cells express functional PR (20), this finding suggests that increasing the ratio of BTEB to PR might favor UF gene expression. However, day 60 pregnancy endometrium, which manifests a similar BTEB to PR ratio as day 12 pregnancy endometrium in vivo, exhibits higher expression of the UF gene (Ref. 33 and this study). This lack of correlation between relative expression of UF and the BTEB/PR ratio could be due to the presence of other factors or hormonal influences that act in synergy with or modulate the effects of BTEB and PR/P. In this regard, we have previously shown that an 80-kDa nuclear protein that binds a negatively acting cis element upstream of the UF gene promoter is expressed at higher levels in ECC-1 cells and in the early pregnancy pig endometrium than in the midpregnancy pig endometrium (19, 23). A similar pattern of expression was observed for the nuclear protein Sp1 in ECC-1 cells and in early vs. midpregnancy pig endometrium (unpublished data from this laboratory). As Sp1 binds to GC-rich sequences with the same affinity as BTEB (6) and, hence, can compete for BTEB-binding sites associated with the UF gene promoter, Sp1 may function as a trans-repressor of BTEB action. Taken together, these results are consistent with the idea that the magnitude of BTEB and PR/P activities, as assessed by their effects on UF gene expression, is dependent on endometrial cell context and may constitute a critical determinant of epithelialassociated gene transcription. The localization of binding sites for PR and BTEB in close proximity to each other within the UF gene promoter region (18, 19) suggested the possibility that a functional PR/BTEB complex might mediate the induction of UF gene transcription in endometrial epithelial cells. Indeed, the formation of transcriptionally active ER or PR complexes with Sp1 that then bind to GC-rich enhancer elements has been implicated in the steroid hormone induction of gene transcription (3, 9, 35). The data presented here cannot directly address this possibility; however, our observations that BTEB increased UF promoter activity in the absence of PR or any added cofactor and that PR/P enhanced UF promoter activity in the absence of BTEB suggest that trans-activation by BTEB or PR occurs mainly via their direct binding to respective recognition sequences on the UF promoter. The functional relevance of the additive vs. synergistic effects of BTEB and activated PR shown here on the activation of endometrial epithelial-associated genes remains to be ascertained. It is interesting to note, however, that ECC-1 cells exhibit higher levels of endogenous BTEB protein than Hec-1-A cells, sug-

9 ENDOMETRIAL EPITHELIAL GENE REGULATION BY BTEB AND PR 2525 gesting that, at least in vitro, a higher BTEB/PR ratio might favor enhanced UF promoter activity. Alternatively, the extent of the interactions between BTEB and the PR/P complex might require the participation of cell-specific cofactors. In a previous study (18), three P-responsive sequences within the 2-kb 5 -flanking region of the UF gene were identified by their ability to bind the PR protein and to exhibit P-dependent enhancer activity within the context of homologous and heterologous promoters in a rabbit endometrial cell line, HRE-H9, that endogenously expresses functional PR. The present study shows that in Hec-1-A cells cotransfected with a PR expression vector, the effects of PR/P were largely mediated by sequences within the 831 construct that contained two of the three identified P-responsive sequences. In contrast, additional BTEB-responsive sequences appear to be located more upstream of the initially reported BTEB-binding sites within 768 to 749 nucleotides (19), as the effect of BTEB on the short construct was increased with additional flanking sequence. A closer examination of sequences between 831 to 1935, however, did not reveal the presence of consensus elements known to bind Sp-related family members (23). Thus, BTEB effects may also be mediated by noncanonical sequences or by interaction with other as yet unknown nuclear proteins. In conclusion, the present study demonstrates a functional role for coexpression of P-dependent PR and BTEB in mediating the high level promoter activity of an epithelialspecific, pregnancy-associated gene. The cell context dependence of BTEB expression as well as of its trans-activation potential predict a unique role for BTEB in the regulation of endometrial epithelial gene transcription during pregnancy. Acknowledgments We thank Dr. P. G. Satyaswaroop (Hershey Medical Center, Hershey, PA) for helpful discussions during the course of this study, Logan Graddy and Ge Zhao for help with densitometry and statistical analysis, Inseok Kwak for critique of the manuscript, and other members of our laboratories for help with animal breeding and tissue collection. References 1. Baniahmad A, Burris TP, Tsai M-J 1994 The nuclear hormone receptor superfamily. In: Tsai M-J, O Malley BW (eds) MBIU: Mechanism of Steroid Hormone Regulation of Gene Transcription. Landes, Austin, pp Owen GI, Richer JK, Tung L, Takimoto G, Horwitz KB 1998 Progesterone regulates transcription of the p21 (WAF1) cyclin-dependent kinase inhibitor gene through Sp1 and CBP/p300. J Biol Chem 273: Duan R, Porter W, Safe S 1998 Estrogen-induced c-fos protooncogene expression in MCF-7 human breast cancer cells: role of estrogen receptor-sp1 complex formation. Endocrinology 139: Kadonaga JT, Carner KR, Masiarz FR, Tjian R 1987 Isolation of cdna encoding transcription factor Sp1 and analysis of the DNA binding domain. Cell 51: Hagen G, Muller S, Beato M, Suske G 1992 Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes. Nucleic Acids Res 20: Imataka H, Sogawa K, Yasumoto K-I, Kikuchi Y, Sasano K, Kobayashi A, Hayami M, Fujii-Kurayama Y 1992 Two regulatory proteins that bind to the basic transcription element (BTE), a GC box sequence in the promoter region of the rat P-4501A1 gene. EMBO J 11: Kingsley C, Winoto A 1992 Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol Cell Biol 12: Sogawa K, Imataka H, Yamasaki Y, Kusume H, Abe H, Fujii-Kuriyama Y 1993 cdna cloning and transcriptional properties of a novel GC box-binding protein, BTEB2. Nucleic Acids Res 21: Krishnan V, Wang X, Safe S 1994 Estrogen receptor-sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells. J Biol Chem 269: Scholz A, Truss M, Beato M 1998 Hormone-induced recruitment of Sp1 mediates estrogen activation of the rabbit uteroglobin gene in endometrial epithelium. J Biol Chem 273: Hagen G, Muller S, Beato M, Suske G 1994 Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J 13: Dennig J, Hagen G, Beato M, Suske G 1995 Members of the Sp transcription factor family control transcription from the uteroglobin promoter. J Biol Chem 270: Imataka H, Nakayama K, Yasumoto K-I, Mizuno A, Fujii-Kuriyama Y, Hayami M 1993 Cell-specific translational control of transcription factor BTEB expression. The role of an upstream AUG in the 5 -untranslated region. J Biol Chem 269: Wang Y, Michel FJ, Wing A, Simmen FA, Simmen RCM 1997 Cell-type expression, immunolocalization, and deoxyribonucleic acid-binding activity of basic transcription element binding transcription factor, an Sp-related family member, in porcine endometrium of pregnancy. Biol Reprod 57: Simmen RCM, Simmen FA 1990 Regulation of uterine and conceptus secretory activity in the pig. J Reprod Fertil [Suppl] 40: Roberts RM, Bazer FW 1980 The properties, hormonal control and synthesis of uteroferrin, the purple protein of the pig uterus. In: Beato M (ed) Steroid- Induced Proteins. Elsevier-North Holland, Amsterdam, pp Drexler HG, Gignac SM 1994 Characterization and expression of tartrateresistant acid phosphatase (TRAP) in hematopoietic cells. Leukemia 8: Lamian V, Gonzalez BY, Michel FJ, Simmen RCM 1993 Non-consensus progesterone-response elements mediate the progesterone-regulated endometrial expression of the uteroferrin gene. J Steroid Biochem Mol Biol 46: Gonzalez BY, Perera OP, Michel FJ, Simmen RCM 1995 Multiple upstream promoter elements of the gene for the pregnancy-associated tartrate-resistant acid phosphatase, uteroferrin bind human endometrial nuclear proteins. Mol Cell Endocrinol 108: Reed KL, Badinga L, Davis DL, Chung TE, Simmen RCM 1996 Porcine endometrial glandular epithelial cells in vitro: transcriptional activities of the pregnancy-associated genes encoding antileukoproteinase and uteroferrin. Biol Reprod 55: Satyaswaroop PG, Tabibzadeh SS 1991 Extracellular matrix and the patterns of differentiation of human endometrial carcinomas in vitro and in vivo. Cancer Res 51: Nishida M, Kasahara K, Kaneko M, Iwasaki H, Hayashi K 1985 Establishment of a new human endometrial adenocarcinoma cell line, Ishikawa cells, containing estrogen and progesterone receptors. Acta Obstet Gynaecol Jpn 37: Gonzalez BY, Michel FJ, Simmen RCM 1994 A regulatory element within the uteroferrin gene 5 -flanking region binds a pregnancy-associated uterine endometrial protein. DNA Cell Biol 13: Ohe N, Yamasaki Y, Sogawa K, Inazawa J, Ariyama T, Oshimura M, Fujji- Kuriyama Y 1993 Chromosomal localization and cdna sequence of human BTEB, a GC box binding protein. Somat Cell Mol Genet 19: Kraus WL, Weis KE, Katzenellenbogen BS 1995 Inhibitory cross-talk between steroid hormone receptors: differential targeting of estrogen receptor in the repression of its transcriptional activity by agonist- and antagonist-occupied progestin receptors. Mol Cell Biol 15: Green ML, Simmen RCM, Simmen FA 1995 Developmental regulation of steroidogenic enzyme gene expression in the periimplantation porcine conceptus: a paracrine role for insulin-like growth factor-i. Endocrinology 136: Simmen RCM, Srinivas V, Roberts RM 1989 cdna sequence, gene organization, and progesterone induction of mrna for uteroferrin, a porcine uterine iron transport protein. DNA 8: Iwai M, Yasuda K, Fukuoka M, Iawai T, Takakura K, Taii S, Nakanishi S. Mori T 1991 Luteinizing hormone induces progesterone receptor gene expression in cultured porcine granulosa cells. Endocrinology 129: Reddy NR, Wilkie BN, Mallard BA 1996 Construction of an internal control to quantitate multiple porcine cytokine mrnas by RT-PCR. BioTechniques 21: Simmen RCM, Simmen FA, Hofig A, Farmer SJ, Bazer FW 1990 Hormonal regulation of insulin-like growth factor gene expression in pig uterus. Endocrinology 127: Barr AJ, Goodnight JH, Sall JP, Blair WH, Chilko DM 1979 General linear models procedure of the statistical analysis system. In: SAS User s Guide. Statistical Analysis Systems Institute, Cary, NC, pp Simmen RCM, Simmen FA, Bazer FW 1991 Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression. Biol Reprod 44: Simmen RCM, Baumbach GA, Roberts RM 1988 Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein. Mol Endocrinol 2: Bamberger AM, Erdmann I, Jenatsekke S, Barberger CM, Schulte HM 1997 Regulation of the human leukaemia inhibitory factor (LIF) promoter in HEC-1B endometrial carcinoma cells. Mol Hum Reprod 3: Sun G, Porter W, Safe S 1998 Estrogen-induced retinoic acid receptor 1 gene expression: role of estrogen receptor-sp1 complex. Mol Endocrinol 12: