help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, D.
Right arrow Articles by Simmen, R. C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, D.
Right arrow Articles by Simmen, R. C. M.
Endocrinology Vol. 143, No. 1 62-73
Copyright © 2002 by The Endocrine Society

RECEPTORS

Direct Interaction of the Krüppel-like Family (KLF) Member, BTEB1, and PR Mediates Progesterone-Responsive Gene Expression in Endometrial Epithelial Cells

Daying Zhang1, Xue-Lian Zhang1, Frank J. Michel, Jason L. Blum, Frank A. Simmen and Rosalia C. M. Simmen

Interdisciplinary Concentration in Animal Molecular and Cell Biology and Department of Animal Sciences, University of Florida, Gainesville, Florida 32611-0910

Address all correspondence and requests for reprints to: Dr. Rosalia C. M. Simmen, Department of Animal Sciences, Building 459, Shealy Drive, University of Florida, Gainesville, Florida 32611-0910; E-mail: simmen{at}animal.ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was undertaken to evaluate the underlying mechanism(s) by which PR and a Krüppel-like family member, basic transcription element binding protein (BTEB1), mediate endometrial epithelial expression of pregnancy-associated genes. Human endometrial carcinoma cell lines (Hec-1-A) expressing high and low levels of BTEB1 were transiently transfected with a human PR isoform (PR-B) expression construct and a luciferase reporter gene driven by the uteroferrin gene promoter that is responsive to both BTEB1 and the PR ligand progesterone. Unliganded PR inhibited luciferase activity in low and high BTEB backgrounds, and this effect was reversed by the synthetic progestin R5020 in both lines. Transactivation by PR of uteroferrin promoter activity (~4-fold) was maximal at lower R5020 concentrations (10 nM) in endometrial cells with higher BTEB1 expression, suggesting that nuclear BTEB1content influenced target gene promoter sensitivity to progesterone. BTEB1 and PR-B were found to physically interact in a progesterone-independent manner, using a coimmunoprecipitation assay that employed antibodies specific to either protein. Moreover, the formation of the BTEB1/PR complex, independent of progesterone, occurred within the context of uterine endometrial proteins and was diminished in late-pregnancy endometrium. Mammalian two-hybrid assays using the entire open reading frame of BTEB1 and/or PR-B fused to either the GAL4 DNA-binding domain or VP16 activation domain and a reporter gene (pG5CAT) under the control of GAL4-binding sites were used to evaluate the formation of functional PR-B/BTEB1 dimer in Cos-1 cells. GAL4/PR-B and VP16/PR-B induced (~3- to 4-fold) chloramphenicol acetyltransferase (CAT) activity in a progesterone-dependent manner, suggesting PR-dimer formation. By contrast, VP16/PR-B and GAL4/BTEB1 had no effect on basal CAT activity. The combination of VP16- and GAL4-PR-B fusion proteins with the BTEB1 expression construct, pCDNA3-BTEB1 enhanced ligand-bound PR-mediated CAT activity by ~3-fold. In transient cotransfection assays using the CAT reporter gene driven by the mouse mammary tumor virus-long terminal repeat promoter, which is responsive to ligand-bound PR but not BTEB1, BTEB1 increased PR-B-mediated CAT activity in a progesterone-dependent manner, consistent with a BTEB1/PR-dimer complex occurring independent of BTEB1 binding to DNA. Unliganded PR-B disrupted the DNA-binding activity of BTEB1 in gel retardation assays, and this effect was enhanced by the presence of PR ligand. Together, these findings support the conclusion that BTEB1 and PR-B are coregulatory proteins that mediate progesterone responsiveness of target genes by direct interactions, leading to the formation of a functional BTEB1/PR-dimer complex.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING MAMMALIAN PREGNANCY, progesterone exerts a critical influence over the status of the uterine endometrium in preparation for implantation and subsequent embryo development. In this tissue, progesterone in concert with estrogen and uterine-associated growth factors coordinates cellular proliferation, differentiation, and apoptosis in a temporal- and spatial-specific manner by binding to its nuclear receptor PR (1), which is expressed and regulated distinctly in the epithelial and stromal compartments of the uterus (2, 3). Recent studies (4) on the molecular biology of the PR in rodents and humans indicate that two PR isoforms, namely PR-A (94 kDa) and PR-B (114 kDa), mediate the effects of progesterone. These isoforms are transcribed from a single gene using distinct estrogen-inducible promoters and differ only by the presence of the most amino- terminal 164 amino acids in PR-B, relative to PR-A (5). Detailed structure/function studies on these PR isoforms indicate that PR-B in all cellular contexts in vitro, functions as a ligand-dependent transactivator of progesterone-responsive genes in contrast to PR-A, which in some contexts, acts as a ligand-dependent transcriptional repressor of PR-B as well as of other steroid hormone receptors (6, 7, 8, 9), especially when expressed in significant excess over PR-B (10). The latter observation has been attributed in part to the distinct conformations achieved by each isoform in the presence of ligand, resulting in their differential ability to bind coactivators or corepressors of transcription (11) as well as in their distinct susceptibility for phosphorylation on specific serine residues (12).

Recent studies (13, 14) using mouse mutants in which either PR-A or PR-B expression has been selectively ablated indicate that each PR isoform mediates distinct physiological responses to progesterone. Indeed, a subset of progesterone-target genes was identified to be transactivated selectively by PR-B, implying that the 164-amino acid region in the N-terminal portion of the protein mediates its specific interactions with numerous nuclear factors essential for transcriptional activity (15, 16). One recently described PR-interacting partner is the GC-box-binding transcription factor Sp1, a seemingly ubiquitous nuclear protein and a member of the Krüppel-like family (KLF) (17, 18). In that study, Sp1 was shown to mediate the transactivation of the cyclin-dependent kinase inhibitor p21 gene promoter, which lacks consensus progesterone-response elements, by forming a functional multiprotein complex with PR/progesterone and another transcription activator CBP/p300 (19). Sp1 has also been implicated in the regulation of estrogen-, retinoic acid-, and androgen-responsive genes via the formation of similar complexes (20, 21, 22, 23). Interestingly, Sp1 is generally considered a global, housekeeping transcription factor whose expression is more constitutive relative to other family members (24, 25). However, no evidence has yet been provided to indicate that other KLF members, which now total at least 18 and whose individual expression displays more spatial-, temporal-, and developmental-specificity than Sp1, can similarly mediate PR-dependent gene regulation in target cells.

The present study stems from previous work (26, 27) in which we showed that the uterine endometrial expression of basic transcription element-binding (BTEB1) protein, a KLF-family member (18, 24), is associated with transactivation of the gene encoding the progesterone-dependent uterine endometrial protein uteroferrin (UF). In those studies, we raised the possibility of functional interactions between BTEB1 and PR/progesterone, based on the model of Sp1/PR-progesterone interactions because: 1) like that of Sp1, endometrial BTEB1 expression is constitutive and preferential to epithelial cells (27, 28); 2) Sp1 regulation of UF promoter activity in vitro was altered by coexpression of BTEB1 (27); and 3) Sp1 and BTEB1 proteins are temporally coexpressed with PR in the early pregnancy endometrium (26, 27, 28, 29). In the present study, we show that progesterone sensitivity of the UF gene promoter to transactivation by PR-B is altered by cellular levels of BTEB1. We further show that this is likely a consequence of direct (physical) interaction between PR-B and BTEB1, possibly involving the formation of a ternary complex between the PR-dimer and this GC-box binding protein. Results implicate BTEB1 as a PR-interacting partner and suggest a novel mechanism by which progesterone regulates expression of its target genes in epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Reagents were obtained as follows: Restriction enzymes and Taq DNA polymerase from Roche Molecular Biochemicals (Indianapolis, IN); nick-translation kit from Amersham Pharmacia Biotech, Inc. (Piscataway, NJ); [{alpha}-32P]deoxycytidine triphosphate (3000 Ci/mmol) and Biotrans nylon membranes (0.2 µM) from ICN Radiochemicals (Irvine, CA); and cell culture media from Life Technologies, Inc. (Grand Island, NY). All molecular biology-grade chemicals and solvents, when not specified, were purchased from Fisher Scientific (Pittsburgh, PA).

Plasmids and expression constructs
The full-length human BTEB1 cDNA was isolated by RT-PCR from human endometrial Ishikawa cell RNA using sense (5'-ATGTCCGCGGCCGCCTACAT-3') and antisense (5'-TCACAAAGCGTTGGCCAGCG-3') primer sets corresponding to the reported human BTEB1 cDNA sequence (30), subcloned into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA), and its sequence and orientation confirmed by nucleotide sequence analysis at the Nucleotide Sequencing Facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. The pM and pVP16 chimeric constructs containing the entire coding region of human BTEB1 were generated by releasing the EcoRI insert from the hBTEB1 plasmid DNA and ligating this into the EcoRI site of pM and pVP16 cloning vectors, respectively (CLONTECH Laboratories, Inc., Palo Alto, CA). All resultant constructs were sequenced across the cloning junctions using appropriate primers to confirm the correct orientation and in-frame position of the inserted fragments. Plasmid DNAs were prepared for transfection studies using the Maxiprep system (QIAGEN, Valencia, CA). A luciferase (Luc) reporter construct containing the porcine UF gene promoter and 1143 bp of 5'-flanking sequence was used (GenBank accession number L23176). Rat BTEB1 cDNA cloned into pCDNA3 vector (pCDNA3-BTEB) and rat progesterone receptor-B cloned into pCMV5 vector (pCMV5-PR-B) were kindly provided by Drs. H. Imataka and B. Katzenellenbogen, respectively.

Transient transfections and reporter gene assays
The human endometrial carcinoma cell line Hec-1-A (a gift from the late Dr. P. G. Satyaswaroop, Hershey Medical Center, Hershey, PA) and stable transfectants derived from these cells that express high and low levels of BTEB1 (designated 4S and 2As cell lines, respectively) (31) were cultured as described previously (26). Twenty-four hours before transfection, cells were plated in six-well plates at a density of 6 x 105 cells per well. Cells were transfected by the polybrene method with UF promoter-Luc (UF-Luc) reporter plasmid (10 µg), in the presence or absence of expression vector for the full-length rat PR-B or corresponding empty vector (1 µg each), following previously described protocols (26). A synthetic progestin (R5020; 10 or 100 nM; NEN Life Science Products Inc., Boston, MA) was added immediately after transfection. Luc assays of resultant cell lysates were performed 48 h after transfection, following the manufacturer’s instructions (Promega Corp., Madison, WI). Luc activity was normalized to cellular extract protein amounts, which were determined by the Bradford dye-binding procedure (32). In a number of experiments, cells were cotransfected with pIND-LacZ reporter plasmid (2 µg), and ß-galactosidase activity was measured by a ß-galactosidase assay kit (Invitrogen) to evaluate potential variations in transfection efficiencies among the different cell lines.

Monkey kidney Cos-1 cells (American Type Culture Collection, Manassas, Va) were also used for transfection assays. Cells were propagated in DMEM containing 10% FBS and grown in six-well plates to 60–70% confluence. Cotransfections were carried out with the chloramphenicol acetyltransferase (CAT) reporter gene linked to mouse mammary tumor virus-long terminal repeat (MMTV-LTR) sequences representing -631 to +125 nt of the 5' regulatory region of the MMTV gene (33) (a gift of Dr. P. Chambon) and expression constructs (pCDNA3-BTEB and/or pCMV5-PR-B) or empty vectors (pCDNA3, PCMV5) in the presence or absence of R5020 (50 nM) by the lipofectAMINE method (31). Cells were harvested 24 h later and assayed for CAT activity using the CAT ELISA kit (Roche Diagnostics Corp., Indianapolis, IN). The CAT activity was measured by absorbance at 405 nm. The CAT concentration (pg/ml) corresponding to an absorbance reading was calculated using a standard calibration curve and normalized to cellular protein content. Transfection data are presented as least squares means (LSM) ± SEM from three independent experiments, in which each experiment was performed in triplicate.

Recombinant BTEB protein production and purification
The entire coding region of rat BTEB1 was generated by PCR (forward primer: 5'-ATGTCCGCGGCCGCCTACAT-3'; reverse primer: 5'-TCACAAGGGGCTGGCAAGAG-3'), using the pIND-BTEB1 plasmid DNA (31) as template for amplification. The PCR product was subcloned into the pCR T7/NT-TOPO vector (Invitrogen), and the ligation product was used for transformation of TOP10F' cells. Plasmid DNA containing the inserted fragment in the correct orientation, as determined by restriction enzyme digestion and nucleotide sequence analyses, was used to transform BL21(DE3)pLysS cells (Invitrogen). Transformed cells were used for large-scale preparation of the his-tagged BTEB1 fusion protein (his-BTEB1) and purified under denaturing conditions as detailed in the manufacturer’s manual (Invitrogen). The final purification step used the nickel-charged Sepharose resin (ProBond, Invitrogen) that bound the His-tag sequence present in the fusion protein. Bound protein was eluted from the column in low pH urea-containing PBS per the manufacturer’s instructions. Purified fractions were dialyzed overnight against Tris-buffer (10 mM Tris-HCl, pH 8.0, 0.1% Triton X-100), and then briefly centrifuged to remove any insoluble material. Aliquots were stored at -20 C until used.

Coimmunoprecipitation and immunoblotting
Purified rat his-BTEB1 (generated above), baculovirus-expressed His-tag-full-length human PR-B (34) (a generous gift of Dr. N. Weigel), and/or nuclear extracts prepared from pregnant pig endometrium as described previously (35) were used for in vitro pull-down interaction studies. In brief, purified his-BTEB1 was incubated with His-tag PR-B for 30 min at 4 C in 1x interaction buffer (20 mM Tris-HCl, pH 8.3, 150 mM NaCl, 1% Nonidet P-40, 0.1% Tween 20, 1 mg/ml BSA) in the presence or absence of R5020 (100 nM final concentration) in a total volume of 100 µl. An antibody (Anti-Xpress, Invitrogen) against a peptide segment present only in the his-BTEB1 protein was then added (2 µl) to each tube. After a 4-h incubation during which time the samples were rotated at 4 C, protein A-agarose slurry (50 µl; Sigma Biochemicals) was added, and the reaction was allowed to proceed overnight at the same temperature. The beads were then pelleted and washed four times with 500 µl PBS. Bound proteins were separated on an SDS-10% polyacrylamide gel and transferred onto nitrocellulose membranes. Blots were probed with anti-His-tag Ab (1:5000 dilution; SuperSignal HisProbe Western blotting kit, Pierce Chemical Co., Rockford, IL) and detected using the reagents included in the kit.

Anti-PR antibody (PgR Ab-8; NeoMarkers, Fremont, CA; 0.4–1 µg/tube) was also used, in conjunction with protein G-agarose (Sigma) to coimmunoprecipitate his-BTEB1 and his-tagged PR-B in the presence or absence of R5020 (100 nM). The precipitate was analyzed for presence of both PR-B and BTEB1, following the procedures described above using anti-His-tag Ab in Western blot analysis. Antibodies against PR (above; 1 µg/ml) and his-tag rat BTEB1 (generated by Research Genetics, Inc., Huntsville, AL; 1 µg/ml) were also used in Western blot analysis to identify coimmunoprecipitated proteins in endometrial nuclear extracts.

Mammalian two-hybrid assays
The propagation and culture of Cos-1 cells for the mammalian two-hybrid assays followed previously described protocols (36). Cells plated at an initial density of 6 x 105 cells per well were transfected at 60–70% confluence with pG5CAT reporter (5 µg) construct and specific combinations of pM-PR-B chimera (0.5 µg), pVP16-PR-B chimera (0.5 µg), pM-BTEB1 (0.5 µg), and pVP16-BTEB1 (0.5 µg), in DMEM containing 10% FBS, using lipofectAMINE, as described previously (31). The human PR-B expression constructs in pM and pVP16 vectors (36) were generously provided by Dr. D. Edwards. Twenty-four hours after transfection the medium was replenished with 10% FBS in DMEM containing vehicle alone (ethanol) or R5020 (100 nM) in ethanol. In other experiments, the full-length rat BTEB1 cDNA in pCDNA3 expression vector (37) was used in cotransfection experiments with pM-PR-B and pVP16-PR-B chimeric constructs in the presence or absence of R5020 (100 nM). Cells were harvested 24 h later and assayed for CAT activity. All transfection experiments were repeated three to four times, with each experiment carried out in triplicate wells.

Gel retardation assay
Gel retardation assays were performed using a double-stranded Sp1 oligomer (Promega Corp.), which was end labeled using T4 polynucleotide kinase (Roche Molecular Biochemicals, Indianapolis, IN) and [{gamma}32P]ATP. A typical binding reaction (in a final volume of 40 µl) consists of poly(dIdC) (2 µg), nuclear extract protein (10–20 µg), and labeled probe (8 x 104 cpm/tube) in binding buffer [10 mM Tris-HCl (pH 7.4), 60 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 10% glycerol (wt/vol), 4 µg of sonicated calf thymus DNA]. Incubation was carried out at 37 C for 15 min, using nuclear extract proteins isolated from endometrium of two different pigs at each indicated pregnancy day. In some experiments, unlabeled Sp1 oligomer (50x molar excess), His-tag PR-B (0.1 µg) or rabbit antipig BTEB peptide IgG (2 µg) (28) were added to the reaction mixture, which was incubated for 10 min at 4 C, before addition of labeled probe and further incubation for 15 min at 37 C. In studies using recombinant human Sp1 (Promega Corp.), incubation with labeled Sp1 probe was carried out at room temperature for 20 min. The resulting DNA-protein complexes were resolved from the free probe by electrophoresis in nondenaturing 5% polyacrylamide gels. The gels were dried and autoradiographed with intensifying screens at -80 C.

Statistical analysis
All numerical data were compared with appropriate controls as indicated for each experiment and analyzed using ANOVA following the general linear models procedure of the Statistical Analysis System (SAS) (38). Comparisons between groups were analyzed using predicted differences (pdiff) of the LSM. Luc and CAT values were normalized to the protein concentration of the cell extract. The statistical model included treatment and experiment, and only preplanned comparisons were made. Treatment means were considered significantly different at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transactivation of porcine UF gene transcription by PR-B ± progesterone in high and low BTEB1-expressing Hec-1-A cells
Our laboratories previously generated Hec-1-A sublines that have higher (4S) or lower (2As) BTEB1 expression levels than corresponding parent (untransfected) cells by stable transfection with BTEB1 expression constructs in the sense and antisense orientations (31). Evaluation of the promoter activity of the transiently transfected UF-Luc reporter construct in the generated stable sublines indicated increased stimulation of basal Luc activity in higher BTEB1-expressing cells (31), confirming previous observations that demonstrated transactivation of the UF gene promoter by BTEB1 (26). We have also previously ascertained that UF gene expression is induced by progesterone (39) at the level of its promoter (26, 40). To examine the effect of BTEB1 on PR-B-mediated progesterone stimulation of the UF gene promoter, PR-B expression construct or empty vector (pCMV5) and UF-Luc reporter construct were transiently cotransfected in 4S-BTEB and 2As-BTEB lines, in the presence or absence of the synthetic progestin R5020. In the absence of its ligand, PR-B decreased basal UF-promoter activity in both lines (Fig. 1Go). The PR-B inhibition of UF promoter activity was reversed by the addition of R5020, although the fold-induction achieved over basal activity was dependent on BTEB1 expression levels in transfected cells. Whereas ligand-bound PR-B elevated Luc activity to only slightly higher (P <= 0.05) than basal levels in low expressing BTEB cells, its effect was greater in high BTEB-expressing cells, in which a 4.5-fold increase over basal activity was observed. Interestingly, the ligand-dependent increase in Luc activity by PR-B was achieved only at higher R5020 concentration (100 nM but not 10 nM) in low BTEB1-expressing lines but was comparable at 10 and 100 nM R5020 in high BTEB1-expressing lines. Thus, the relative amounts of BTEB1 in progesterone-responsive cells appear to influence the sensitivity of the target gene promoter to transactivation by ligand-bound PR.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 1. Effect of BTEB1 on the transactivation of UF gene promoter by PR-B in the presence and absence of R5020. Stably transfected Hec-1-A cell lines expressing high (4S-BTEB) and low (2As-BTEB) levels of BTEB1 were cotransfected with empty vector (pCMV5) or pCMV5-PR-B expression vector and UF-Luc reporter construct, in the presence or absence of R5020 (10 or 100 nM). Results (Luc activity normalized to cellular protein content) are presented as LSM ± SEM of three independent experiments, each performed in triplicate. *, Statistical significance of P <= 0.05, compared with basal activity (pCMV5 alone) by ANOVA. Significant differences between treatment groups (indicated by brackets) were also identified by ANOVA.

 
Coimmunoprecipitation of PR and BTEB proteins
The increased progesterone sensitivity of the UF promoter to transactivation by PR-B, depending on cellular BTEB1 expression levels, suggested functional interactions between these nuclear proteins. To investigate the mechanism underlying this observation, we tested for a direct (physical) interaction between purified BTEB1 and PR-B by coimmunoprecipitation experiments. As a first step to generate sufficient amounts of BTEB1 protein for these studies, the entire coding region for rat BTEB1 (735 bp) was subcloned into the plasmid vector pCR T7/NT-TOPO for expression of the corresponding His-tagged fusion protein in bacterial cells. The protein was purified under denaturing conditions from bacterial cell lysates by passing extracts through a nickel-affinity Sepharose resin and subsequent elution of bound protein(s) with a series of buffers of progressively decreasing pH. In a Coomassie blue-stained gel, the purified protein was shown to migrate as a major band of ~39 kDa molecular mass owing to the addition of ~5 kDa from the vector sequence to the original rat BTEB1 protein of ~33 kDa molecular mass (Fig. 2Go). Two major immunoreactive bands, one of which has the same size as the major band seen in the Coomassie-stained gel and another of slightly lower molecular mass (36 kDa), were detected by Western blots using anti-His-tag antibody (Fig. 2Go). These two bands were shown to also react with a highly specific antibody (IgG fraction) raised against the 39-kDa his-BTEB1 fusion protein in rabbits (data not shown), confirming their identity as BTEB1.



View larger version (54K):
[in this window]
[in a new window]
  		Figure 2. Purification of recombinant His-tagged rat BTEB1 protein. The full-length rat BTEB1 cDNA was subcloned into the pCR T7/NT-TOPO expression vector, expressed in Escherichia coli, and purified using a His-tag affinity column. Aliquots of the initial bacterial sonicates (100 µg protein) and the final eluate (2 µg protein) were analyzed by SDS-PAGE, followed by staining with Coomassie-blue (left panel) or by immunoblotting with anti-His-tag antibody (right panel), following conditions described under Materials and Methods. Arrows indicate the positions of the expected intact recombinant His-tag BTEB1 protein (39 kDa) and a His-tag BTEB1 protein of slightly lower molecular mass (36 kDa).

 
The purified his-BTEB1 protein(s) was incubated with purified baculovirus-expressed human His-tagged PR-B, and any protein complexes formed were immunoprecipitated with anti-Xpress antibody that recognized a unique stretch of eight amino acids present only in the recombinant his-BTEB1 protein. Subsequent analysis of the immunoprecipitate by Western blot analysis using the anti-His-tag antibody that recognized the polyhistidine (6xHis) region present in both His-tagged PR-B and his-BTEB1, indicated that BTEB1 and PR-B formed a complex in vitro that was immunoprecipitated with the antibody directed against the his-BTEB1 protein (Fig. 3Go). The formation of the complex was independent of the presence of the PR ligand, R5020 in the interaction medium (Fig. 3AGo). Immunoprecipitation of the BTEB1/PR-B complex was also achieved using an anti-PR antibody (Fig. 3BGo). Increasing the ratio of PR-B to BTEB1 had no apparent effect on the formation of the complex. The specificity of the antibodies used for coimmunoprecipitation and Western blot analysis, respectively, was demonstrated by the lack of any detectable immunoprecipitable complex when: 1) an unrelated purified porcine protein, UF, was incubated with PR-B; 2) PR-B was incubated alone with anti-Xpress antibody; 3) BTEB was incubated with anti-Xpress antibody in the absence of PR-B; or 4) control rabbit serum was used for coimmunoprecipitation (Fig. 3AGo).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 3. Coimmunoprecipitation of BTEB1 and PR-B. His-BTEB1 and his-PR-B were incubated in the presence or absence of R5020 (progesterone, 100 nM) and immunoprecipitated (IP) by anti-Xpress antibody, which recognizes a region present only in the his-BTEB1 protein (A) or by anti-PR antibody and corresponding control antibody (B). The immunoprecipitates were subjected to Western blot analysis (WB) using anti-His-tag antibody, which recognizes both PR and BTEB1 proteins. The results shown are representative autoradiograms from two independent experiments. A, The component(s) present in the reaction mixture before immunoprecipitation with anti-Xpress antibody are indicated from left: BTEB1 + PR-B (5:1 molar ratio) + R5020; BTEB1 + PR-B (5:1 molar ratio); PR-B (0.1 µg); BTEB1 (2.5 µg); UF (2.5 µg) + PR-B (0.1 µg). B, BTEB1 and PR-B in the presence of R5020 were incubated at varying molar ratios (BTEB1/PR-B- 7:1, 4:1, 1:1) and immunoprecipitated with either anti-PR antibody or normal rabbit serum (NRS) IgG.

 
To determine whether BTEB1 and PR-B transcription factors interact within the context of other endometrial proteins, coimmunoprecipitation experiments were conducted in the presence of nuclear extracts prepared from pregnancy pig endometrium, which contain PR protein (26, 29). In these experiments, nuclear extract proteins (100 µg) were incubated with either his-BTEB1 (0.5 µg) or his-tag PR-B (0.5 µg) in interaction buffer, under the conditions described above, in the presence and absence of R5020 (100 nM). The formation of any protein complexes was monitored by immunoprecipitation with either anti-Xpress (Fig. 4AGo) or anti-PR (Fig. 4BGo) antibody, collection of the immunoprecipitated complex with protein A (for anti-Xpress)- or protein G (for anti-PR)-Sepharose, and analysis of the immunoprecipitate by Western blots using anti-PR or anti-BTEB1 antibodies, respectively. As shown in Fig. 4AGo, an immunoreactive protein, comigrating with purified baculovirus-expressed human PR-B, was coprecipitated with the anti-Xpress antibody, which was specific to the BTEB fusion protein, in the presence and absence of R5020 from nuclear extracts prepared from d12 pregnancy endometrium. Further, an immunoreactive protein corresponding to BTEB1 was coprecipitated by anti-PR-B antibody from nuclear extracts prepared from early- (d12), mid- (d60), and late- (d90) pregnancy endometrium in the presence and absence of R5020 (Fig. 4BGo). Interestingly, the amount of endogenous BTEB1 pulled down by anti-PR antibody in late pregnancy endometrium (d90) was less (by approximately greater than 50%) than that observed for early- and mid-pregnancy endometrium. Western blot analysis of endometrial nuclear extracts indicated that endogenous BTEB1 levels were comparable at these pregnancy days (Fig. 4CGo). The immunoreactive band of lower molecular mass likely represents a partially degraded form of the full-length protein. These findings suggest that PR-B can specifically form a complex with BTEB1 in vitro in the presence of other endometrial nuclear proteins and that formation of the complex is influenced by pregnancy status.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Endometrial PR associates with BTEB1. Nuclear extracts prepared from endometrium of two pigs at early (d 12) pregnancy stage (designated NE#1 and NE#2) (A) or from pigs at d 12, 60, and 90 of pregnancy (B) were incubated with his-BTEB1 (A) or his-PR-B (B), in the presence or absence of R5020 (100 nM), following conditions described under Materials and Methods. The resulting protein complexes were immunoprecipitated with anti-Xpress antibody that is specific to added his-BTEB1 (A) or anti-PR antibody (B), resolved by SDS-PAGE, and subsequently immunoblotted with an antibody directed against either PR (A) or BTEB1 (B). The migration positions of PR-B (A) or BTEB1 (B) are indicated by arrows. The hPR and NS represent his-tag human PR-B (positive control) and nonspecific binding, respectively. C, Nuclear extracts prepared from endometrium of two individual pigs per pregnancy (px) day were analyzed by Western blot, using rabbit antirat BTEB1. Each lane represents one endometrial sample at the indicated pregnancy day and contains 100 µg total protein.

 
Mammalian two-hybrid and transient transfection assays
To further confirm functional BTEB1/PR interactions, we used a two-hybrid assay in Cos-1 cells (36). These cells were used because the endometrial carcinoma cell line Hec-1-A exhibited very low CAT activity upon transient transfection with the CAT reporter gene pG5CAT, precluding reliable measurements of CAT enzymatic activity. We employed PR-B and BTEB1, each fused to either the GAL4-DNA-binding domain in pM vector or the activation domain in pVP16 vector. As positive controls for these experiments, we tested the functional interactions of pM-PR-B and pVP16-PR-B in inducing the expression of pG5CAT in the presence and absence of R5020. The PR-B constructs have been extensively characterized in a previous study (36), which demonstrated that PR-B fused to either GAL4 binding domain or VP16 activation domain displayed the same steroid binding activity as unfused PR-B. For negative controls, the stimulation of CAT activity by the empty vectors (pM, pVP16), the single PR-B hybrid (pM-PR-B, VP16-PR-B), or single BTEB hybrid (pM-BTEB1; pVP16-BTEB1), alone or in combination, upon cotransfection with the pG5CAT reporter, with and without added R5020 in the medium, was evaluated. Results shown in Fig. 5AGo indicated similar background activities of empty vectors, PR-B single hybrids, and BTEB single hybrids, in the presence or absence of R5020.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. Functional interactions of PR-B and BTEB1 by mammalian two-hybrid assay. Cos-1 cells were transiently cotransfected with the PG5CAT reporter and different expression constructs (A, B, C) in the presence or absence of R5020 (progesterone; 100 nM), following conditions described under Materials and Methods. CAT activity in transfected cells was normalized to protein content in cellular lysates, and the data are presented as LSM ± SEM from four independent experiments, each performed in triplicate. The components present in each transfection experiment, in addition to pG5CAT reporter (0.5 µg) are indicated by (+). *, Significant difference (P <= 0.05) from corresponding assays carried out in the absence of added R5020.

 
An exception is the CAT activity demonstrated by the pM-PR-B construct upon cotransfection with pVP-16 vector, which was increased by 1.8-fold from that of basal in response to R5020, an observation that was similarly reported by the original source of these constructs (36). Cotransfection of pM-PR-B and pVP16-PR-B increased reporter activity from basal levels by 3-fold in the presence of R5020. When BTEB1 was fused to the pM vector and PR-B was fused to the VP-16 vector, no R5020-dependent increase in reporter activity was observed (Fig. 5BGo). Interestingly, when BTEB1 was fused to the VP-16 vector, and PR-B was fused to the pM vector, a ligand-dependent increase (3-fold) in CAT activity was observed; the fold-increase with this combination was higher (3-fold vs.1.8-fold) than that observed when pM-PR-B was cotransfected with pVP16 vector alone. Addition of nonfused BTEB (as an expression construct in pCDNA3 vector) together with the pM-PR-B and pVP-16-PR-B fusion proteins, however, induced the activation of the CAT promoter by greater than 8-fold in a progesterone-dependent manner. Indeed, this fold-induction was approximately 3 times higher than that obtained with the PR-dimer alone (Fig. 5CGo). These results suggest that induction of progesterone-responsive gene activity is enhanced by the formation of a functional complex between BTEB1 and the PR-B dimer and that this occurs independent of BTEB1 binding to its DNA recognition sequences.

To further demonstrate that the ligand-bound PR dimer forms a functional complex with BTEB1, independent of BTEB1 DNA-binding activity, transient cotransfection experiments were carried out in Cos-1 cells with expression constructs for BTEB1 and/or PR-B in the presence or absence of added R5020, using a reporter gene MMTV-LTR-CAT, which contains recognition sequences for PR but not BTEB1 (33). As anticipated, BTEB1 had no effect on MMTV-LTR promoter activity, but PR increased this gene’s transcriptional activity in a progesterone-dependent manner (Fig. 6Go). Moreover, the combination of ligand-bound PR-B and BTEB1 further increased MMTV-LTR promoter activity over that achieved with PR-B dimer alone, confirming that a functional interaction between PR-dimer and BTEB1 can occur without BTEB1 binding to its canonical GC-rich DNA sequence.



View larger version (47K):
[in this window]
[in a new window]
  		Figure 6. Effect of BTEB1 on PR/P-mediated transactivation of the MMTV-LTR gene promoter. Cos-1 cells were transiently cotransfected with BTEB1 expression vector (BTEB-pCDNA; 0.5 µg), PR-B expression vector (PR-B-pCMV5; 0.5 µg), and corresponding empty vectors (pCMV5, pCDNA3; 0.5 µg each) and the MMTV-LTR CAT reporter construct (5 µg), as described under Materials and Methods. Cells with added PR were incubated in the presence or absence of R5020 (50 nM). Results are presented as LSM ± SEM of three independent experiments, with each experiment performed in triplicate. Means without a common superscript differ significantly (P <= 0.05).

 
Gel retardation assays
To evaluate the consequence of BTEB1/PR interactions on the affinity of BTEB1 for its DNA recognition sequence within the context of the pregnancy endometrium, gel retardation assays were performed with a [32P]-labeled GC-rich DNA fragment (Sp1 oligomer) that was previously shown to bind BTEB1 and the GC-box binding transcription factor Sp1 (28, 41) and that does not contain the consensus recognition sequence for PR protein. Four bands representing specific DNA complexes were formed upon incubation of endometrial nuclear proteins with the labeled probe (Fig. 7Go). Of these four, the fastest migrating complex (complex 4) was determined to represent BTEB1 because it was the only complex "supershifted" upon addition of a specific anti-BTEB1 antiserum to the reaction mixture (Fig. 7AGo). The slowest migrating complex (complex 1) likely represents Sp1, by virtue of its comigration to purified human Sp1 (Fig. 7BGo). To determine whether addition of PR had an effect on the formation of any of the four complexes, and specifically that involving BTEB1, nuclear extract proteins from early pregnancy pig endometrium (d 11 and 14; two different nuclear extract preparations per pregnancy day) were incubated with purified baculovirus-expressed His-tag PR-B (0.1 µg) in the presence and absence of R5020 (100 nM). Results shown (Fig. 7CGo) indicated that although unliganded PR-B diminished the formation of complex 4, ligand-bound PR-B (i.e., PR-B dimer) had a more dramatic effect. PR similarly inhibited the formation of the other three slower migrating DNA-protein complexes, an effect that was also enhanced by the presence of R5020. These results suggest that the interaction of PR-B with BTEB1 as well as other GC-box binding proteins including Sp1, which are expressed by the pregnancy endometrium, occurs independent of progesterone and is likely favored over that of these proteins’ respective binding to their DNA recognition sequences.



View larger version (73K):
[in this window]
[in a new window]
  		Figure 7. Effect of unliganded and ligand-bound PR-B on DNA-binding activity of endometrial GC-box binding proteins. Nuclear extracts prepared from endometrium of two pigs each at early pregnancy stages (d 11 or 14) or recombinant human Sp1 were incubated with 32P-labeled Sp1 oligomer in the presence or absence of 50-fold molar excess of unlabeled probe (designated as "cold"), antibody to BTEB1, or his-tagged PR-B (0.1 µg) in the presence or absence of R5020 (progesterone, 100 nM). DNA-bound proteins are designated as 1–4, with 1 and 4 likely representing Sp1 and BTEB1, respectively, and 2 and 3 of unknown identities. In panel C, each bracketed panel represents a different nuclear extract preparation from endometrium of individual pigs at the indicated pregnancy day (d 11 or 14).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The purpose of this study was to further analyze the nature of and the molecular mechanisms underlying putative functional interactions between PR and BTEB1 in the transactivation of the gene encoding UF, as a paradigm for pregnancy- associated, endometrial epithelial-specific gene transcription. Previous studies have demonstrated the UF gene to be regulated at the level of its promoter by the steroid hormone progesterone (26, 39, 40) as well as the GC-box binding transcription factors Sp1 and BTEB1 (26, 27). Because Sp1 has been functionally linked to several steroid hormone receptors, including PR, in the transactivation of a number of genes, each of which exhibits specific spatial and temporal patterns of expression (19, 20, 21, 22, 27) and because BTEB1 and Sp1 may mediate gene transcription by binding to the same recognition sequences within target genes (41, 42), the possibility that BTEB1, similar to Sp1, interacts with PR to functionally regulate endometrial gene expression during pregnancy seemed likely. In the absence of any well-characterized mammalian epithelial cell line from uterine endometrium of pregnancy, we used two lines derived from human endometrial carcinoma Hec-1-A cells, which were engineered to express high (4S) and low (2As) levels of BTEB1, respectively (31), and which were transiently cotransfected with PR-B to mimic the status of the pregnancy endometrium.

Using this in vitro model, we show that the sensitivity of endometrial epithelial cells to PR-B-mediated progesterone induction of UF gene promoter activity was influenced by the cellular expression levels of BTEB1. In particular, we observed that maximal transactivation of basal UF promoter activity by PR-B (an increase of at least 4.5-fold) was achieved at 10-fold lower R5020 concentration in a high BTEB1 background than in a background of low BTEB1. We further show that this effect of progesterone is likely a consequence of direct interactions between BTEB1 and PR-B. However, although unliganded PR and BTEB1 can form a complex in vitro, productive BTEB1/PR-B interaction requires progesterone. Our results suggest that a functional complex conferring progesterone-dependent transactivation likely consists of BTEB1 in a ternary complex with the PR-dimer, although we cannot exclude the possibility that the PR-dimer/BTEB1 complex is part of a multicomplex unit that also includes other coactivators previously implicated in PR action (43). These results identify BTEB1 as a novel PR- interacting partner and suggest that the transcriptional response of progesterone-responsive genes to progesterone in endometrial epithelial cells may involve BTEB1.

Our studies examined the functional interactions of PR and BTEB1 under no and high progesterone conditions, the latter simulating the status of early pregnancy, and their physical interactions using nuclear extracts prepared from endometrium at different pregnancy days to begin to provide biological context to this novel PR-transcriptional complex. Two things are particularly worth noting from these studies. First, the observed reduction of basal promoter activity of the progesterone-responsive UF gene in cells expressing both BTEB1 and PR in the absence of progesterone was associated with progesterone-independent physical interactions between these nuclear proteins. Second, the interaction of PR and BTEB1 within the context of the pregnancy endometrium was found to decrease at the late pregnancy stage. These findings suggest that: 1) progesterone is not required for PR-BTEB1 complex formation; 2) the interactions of these proteins alone can modify the promoter activity of various genes whose regulatory regions, like that of UF, may contain recognition sequences for both; and 3) changes in the amounts of BTEB/PR complex formed with pregnancy status may underlie the temporal expression of distinct endometrial genes during this period. Indeed, because endometrial expression of BTEB1 protein appears constitutive across pregnancy (28 , this study), the observed decrease may be a function of diminished BTEB1 activity and/or reduced concentrations of other proteins involved in the stabilization of the PR/BTEB complex. In this regard, numerous studies have now documented that the transactivation function of PR is governed by its ability to associate with transcriptional coactivators and its inability to recruit corepressors (11, 19, 43, 44). Thus, BTEB1, in the presence of progesterone, may enhance the biological activity of PR by facilitating the association of the PR-dimer with a number of coactivators, resulting in this receptor’s increased interaction with the general transcription machinery. Confirmation of this hypothesis awaits the identification of specific coactivators whose association with PR is increased in the presence of BTEB1.

Previous studies from other laboratories (37, 45) as well as our own (26) indicate that BTEB1 can act as a bonafide DNA-binding protein via its own recognition sequence within target gene regulatory regions. Thus, the possibility that this nuclear protein may also function as a PR-dimer interacting partner as suggested from the present studies raises questions as to how BTEB1 may perform the dual role of a transacting factor via binding to its specific cis-acting regulatory elements and a transcriptional coactivator, independent of DNA-binding activity. The model presented in Fig. 8Go suggests a possible mechanism by which BTEB1 might perform these roles, which may be dependent on cell as well as gene context. In cells coexpressing PR and BTEB1, the transcription of gene promoters containing recognition sites for both BTEB1 and PR is predicted to be inhibited, relative to those containing BTEB1 recognition sequences alone because of the likely formation of a nonproductive, transcriptionally inactive BTEB1/PR complex. In the presence of progesterone, however, the formation of the productive, transcriptionally active PR-dimer/BTEB1 complex is anticipated to enhance the activity of these gene promoters, above that predicted from the individual activities of BTEB1 and the PR-dimer, each of which bind to its respective recognition sequence. This model further presupposes that the formation of the PR/BTEB1 complex, under conditions of high or no progesterone, is favored over that in which BTEB1 binds to its own recognition sequence. The latter is consistent with results from gel shift assays demonstrating the diminished formation of the labeled BTE-BTEB1 DNA-protein complex when either unliganded or ligand-bound PR was included in the binding interaction. Together, these results suggest that BTEB1 can influence the direction of the transcriptional response of cells expressing endogenous PR, depending on the cellular progesterone status.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 8. Model of BTEB1 and PR-B interactions within a progesterone-responsive gene promoter. BTE and PRE represent DNA recognition sequences for BTEB1 and PR-dimer, respectively. Heat shock proteins that bind unliganded PR are designated as Hsp. X and Y represent nuclear coactivators that interact with ligand-bound PR-dimer to form the activated transcriptional complex. In the unliganded state, the interaction of PR-B with BTEB1 can result in the inhibition of BTEB1 binding to BTE element. When progesterone is present, the formation of a functional PR-dimer allows the subsequent formation of a complex involving BTEB1 and possibly other coactivators, likely leading to a stable and highly activated transcriptional state. (+), (++), and (++++) represent basal, moderate, and high promoter activity, respectively.

 
Recently, we demonstrated that increased expression levels of BTEB1 in the endocarcinoma cell line Hec-1-A, which lacks PR, were correlated with increased proliferative status in response to serum-derived factors (31). The increased proliferation seen in high BTEB1-expressing cells, compared with those with low BTEB1 expression, was associated with increased traversal of the cell cycle, consistent with these cells’ enhanced expression of cyclin D1 and other growth-associated proteins (31). Thus, BTEB1 can function to modulate cell growth, albeit the underlying mechanism is presently unclear. Interestingly, the findings reported herein that BTEB1 can productively partner with the PR-dimer, an inhibitor of cell proliferation through its transcriptional inhibition of growth-associated proteins (19, 46, 47) suggests that BTEB1 manifests versatility in its growth regulatory activity, depending on cell context. These results underscore the importance of further analysis of BTEB1 interacting partners for understanding the molecular basis of endometrial growth regulation.

The previously documented physical association of Sp1 with PR was shown to occur only in the presence of PR agonists (19). In the present study, however, disruption of Sp1 interaction with its cis-element occurred upon PR addition, irrespective of progesterone status, analogous to that observed for BTEB1, suggesting a similarity in the nature of the interaction of BTEB1 or Sp1 with PR. Although the difference in the results of the two studies may be a function of the distinct assays involved (coimmunoprecipitation in the earlier study vs. DNA binding in the present study), our results are consistent with those of a previous study that demonstrated a functional interaction of unliganded PR with the transcription factor AP-1 (16). In this regard, we have previously shown that coexpression of BTEB1 and Sp1 in endometrial cells resulted in the exclusion of the other’s DNA binding activity for a target gene promoter (27). Thus, BTEB1 and Sp1 may compensate for each other’s function under many cellular contexts, including that involving binding to PR, albeit demonstration of this will ultimately require comparison of the specific phenotypes generated by Sp1 (48) and BTEB1 null, mutant mouse models.

In conclusion, we demonstrate in the present study that BTEB1 likely plays an important role in PR-mediated gene transactivation by binding to PR. Given that BTEB1 can function as a positive growth regulator in some contexts and that PR-B mediated progesterone transactivation has been shown to contribute to both progesterone-dependent proliferative and antiproliferative responses (14, 47), a better understanding of the mechanisms underlying the formation of the PR-BTEB1 complex and respective BTEB1 and PR domains involved in this interaction could lead to the development of novel strategies for manipulating transcriptional events that control the balance of cellular proliferation and differentiation.


   Acknowledgments
 
The authors thank Drs. Benita Katzenellenbogen (University of Illinois, Urbana, IL), Nancy Weigel (Baylor College of Medicine, Houston, TX), Dean Edwards (University of Colorado, Denver, CO), Hiroaki Imataka (McGill University, Québec, Canada), and Pierre Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France) for generous provision of reagents as indicated in the text. We are also grateful to Ge Zhao for technical assistance and for help in formatting of figures.


   Footnotes
 
This work was supported by NIH Grant HD-21961 and by the Florida Agricultural Experiment Station and approved for publication as Journal Series R-08338.

1 Both authors contributed equally to this work. Back

Abbreviations: 2As-BTEB, Low levels of BTEB1; BTEB1, basic transcription element binding protein; CAT, chloramphenicol acetyltransferase; Hec-1-A, human endometrial carcinoma cell line; his-BTEB1, his-tagged BTEB1 fusion protein; KLF, Krüppel-like family; LSM, least squares means; Luc, luciferase; MMTV-LTR, mouse mammary tumor virus-long terminal repeat; 4S-BTEB, high levels of BTEB1; UF, uteroferrin; UF-Luc, UF promoter-Luc.

Received June 27, 2001.

Accepted for publication September 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  2. Tibbetts TA, Mendoza-Meneses M, O’Malley BW, Conneely OM 1998 Mutual and intercompartmental regulation of estrogen receptor and progesterone receptor expression in the mouse uterus. Biol Reprod 59:1143–1152[Abstract/Free Full Text]
  3. Kurita T, Lee KJ, Cooke PS, Taylor JA, Lubahn DB, Cunha GR 2000 Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract. Biol Reprod 62:821–830[Abstract/Free Full Text]
  4. Horwitz KB, Alexander PS 1983 In situ photolinked nuclear progesterone receptors of human breast cancer cell lines: subunit molecular weights after transformation and translocation. Endocrinology 113:2195–2201[Abstract]
  5. Kastner P, Krust A, Turcotte B, Stropp U, Tora L, Gronemeyer H, Chambon P 1990 Two distinct estrogen-regulated promoters generate transcripts encoding the two functionally different human progesterone receptor forms A and B. EMBO J 9:1603–1614[Medline]
  6. Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O’Malley BW, McDonnell DP 1993 Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function. Mol Endocrinol 7:1244–1255[Abstract]
  7. McDonnell DP, Goldman ME 1994 RU486 exerts antiestrogenic activities through a novel progesterone receptor A form-mediated mechanism. J Biol Chem 269:11945–11949[Abstract/Free Full Text]
  8. McDonnell DP, Shahbaz MM, Vegeto E, Goldman ME 1994 The human progesterone receptor A-form functions as a transcriptional modulator of mineralocorticoid receptor transcriptional activity. J Steroid Biochem Mol Biol 48:425–432[CrossRef][Medline]
  9. Giangrande PH, Pollio G, McDonnell DP 1997 Mapping and characterization of the functional domains responsible for the differential activity of the A and B isoforms of the human progesterone receptor. J Biol Chem 272:32889–32900[Abstract/Free Full Text]
  10. McGovern EM, Clarke CL 1999 Effect of overexpression of progesterone receptor A on endogenous progestin-sensitive endpoints in breast cancer cells. Mol Endocrinol 13:1657–1671[Abstract/Free Full Text]
  11. Giangrande PH, Kimbrel EA, Edwards DP, McDonnell DP 2000 The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding. Mol Cell Biol 20:3102–3115[Abstract/Free Full Text]
  12. Clemm DL, Sherman L, Boonyaratanakornkit V, Schrader WT, Weigel NL, Edwards DP 2000 Differential hormone-dependent phosphorylation of progesterone receptor A and B forms revealed by a phosphoserine site-specific monoclonal antibody. Mol Endocrinol 14:52–65[Abstract/Free Full Text]
  13. Lydon JP, DeMayo FJ, Funnk CR, Mani SK, Hughes AR, Montgomery Jr CA, Shymala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit pleiotropic reproductive abnormalities. Genes Dev 9:2226–2278
  14. Mulac-Jericevic B, Mullinax RA, DeMayo FJ, Lydon JP, Conneely OM 2000 Subgroup of reproductive functions of progesterone mediated by progesterone receptor-B isoform. Science 289:1751–1754[Abstract/Free Full Text]
  15. Schwerk C, Klotzbucher M, Sachs M, Ulber V, Klein-Hitpass L 1995 Identification of transactivation function in the progesterone receptor that interacts with the TAFII 110 subunit of the TFIID complex. J Biol Chem 270:21331–21338[Abstract/Free Full Text]
  16. Bamberger AM, Bamberger CM, Gellersen B, Schulte HM 1996 Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells. Proc Natl Acad Sci USA 93:6169–6174[Abstract/Free Full Text]
  17. Kadonaga JT, Carner KR, Masiarz FR, Tjian R 1987 Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51:1079–1090[CrossRef][Medline]
  18. Turner J, Crossley M 1999 Mammalian Krüppel-like transcription factors: more than just a pretty finger. Trends Biochem Sci 24:236–241[CrossRef][Medline]
  19. 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 273:10696–10701[Abstract/Free Full Text]
  20. Qin C, Singh P, Safe S 1999 Transcriptional activation of insulin-like growth factor-binding protein-4 by 17ß-estradiol in MCF-7 cells: role of estrogen receptor-Sp1 complexes. Endocrinology 140:2501–2508[Abstract/Free Full Text]
  21. Husmann M, Dragneva Y, Romahn E, Jehnichen P 2000 Nuclear receptors modulate the interaction of Sp1 and GC-rich DNA via ternary complex formation. Biochem J 352:763–772
  22. Petz LN, Nardulli AM 2000 Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter. Mol Endocrinol 14:972–985[Abstract/Free Full Text]
  23. Lu S, Jenster G, Epner DE 2000 Androgen induction of cyclin-dependent kinase inhibitor p21 gene: role of androgen receptor and transcription factor Sp1 complex. Mol Endocrinol 14:753–760[Abstract/Free Full Text]
  24. Philipsen S, Suske G 1999 A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 27:2991–3000[Abstract/Free Full Text]
  25. Treichel D, Becker M, Gruss P 2001 The novel transcription factor gene Sp5 exhibits a dynamic and highly restricted expression pattern during mouse embryogenesis. Mech Dev 101:175–179[CrossRef][Medline]
  26. Simmen RCM, Chung TE, Imataka H, Michel FJ, Badinga L, Simmen FA 1999 Trans-activation functions of the Sp-related nuclear factor, basic transcription element-binding protein, and progesterone receptor in endometrial epithelial cells. Endocrinology 140:2517–2525[Abstract/Free Full Text]
  27. Simmen RCM, Zhang XL, Zhang D, Wang Y, Michel FJ, Simmen FA 2000 Expression and regulatory function of the transcription factor Sp1 in the uterine endometrium at early pregnancy: implications for epithelial phenotype. Mol Cell Endocrinol 159:159–170[CrossRef][Medline]
  28. 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 57:707–714[Abstract]
  29. 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 2:253–262[Abstract]
  30. Ohe N, Yamasaki Y, Sogawa K, Inazawa J, Ariyama T, Oshimura M, Fujii-Kuriyama Y 1993 Chromosomal localization and cDNA sequence of human BTEB, a GC box binding protein. Somat Cell Mol Genet 19:499–503[CrossRef][Medline]
  31. Zhang X-L, Simmen FA, Michel FJ, Simmen RCM 2001 Increased expression of the Zn-finger transcription factor BTEB1 in human endometrial cells is correlated with distinct cell phenotype, gene expression patterns, and proliferative responsiveness to serum and TGF-ß1. Mol Cell Endocrinol 181:81–96[CrossRef][Medline]
  32. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  33. Cato ACB, Miksicek R, Schutz G, Arnemann J, Beato M 1986 The hormone regulatory element of mouse mammary tumour virus mediates progesterone induction. EMBO J 5:2237–2240[Medline]
  34. Allan GF, Leng X, Tsai SY, Weigel NL, Edwards DP, Tsai MJ, O’Malley BW 1992 Hormone and antihormone induce distinct conformational changes which are central to steroid receptor activation. J Biol Chem 267:19513–19520[Abstract/Free Full Text]
  35. 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 13:365–376[Medline]
  36. Leonhardt SA, Altmann M, Edwards DP 1998 Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay. Mol Endocrinol 12:1914–1930[Abstract/Free Full Text]
  37. Imataka H, Sogawa K, Yasumoto K, Kikuchi Y, Sasano K, Kobayashi A, Hayami M, Fujii-Kuriyama 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 11:3663–3671[Medline]
  38. SAS 1988 SAS/STAT User’s guide (release 6.03 ed). Cary, NC: SAS Institute Inc.
  39. 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 8:543–554[Medline]
  40. 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 Molec Biol 46: 439–450
  41. 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 108:51–65[CrossRef][Medline]
  42. Sogawa K, Kikuchi Y, Imataka H, Fujii-Kuriyama Y 1993 Comparison of DNA-binding properties between BTEB and Sp1. J Biochem 114:605–609[Abstract/Free Full Text]
  43. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor co-regulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  44. Xu Y, Klein-Hitpass L, Bagchi MK 2000 E1A-mediated repression of progesterone receptor-dependent transactivation involves inhibition of the assembly of a multisubunit coactivation complex. Mol Cell Biol 20:2138–2146[Abstract/Free Full Text]
  45. Chen A, Davis BH 2000 The DNA binding protein BTEB mediates acetaldehyde-induced, jun N-terminal kinase-dependent {alpha}I (I) collagen gene expression in rat hepatic stellate cells. Mol Cell Biol 20:2818–2826[Abstract/Free Full Text]
  46. Vazquez F, Rodriguez-Manzaneque JC, Lydon JP, Edwards DP, O’Malley BW, Iruela-Arispe ML 1999 Progesterone regulates proliferation of endothelial cells. J Biol Chem 274:2185–2192[Abstract/Free Full Text]
  47. Clarke CL, Sutherland RL 1990 Progestin regulation of cellular proliferation. Endocr Rev 11:266–301[Medline]
  48. Marin M, Karis A, Visser P, Grosveld F, Philipsen S 1997 Transcription factor Sp1 is essential for early development but dispensable for cell growth and differentiation. Cell 89:619–628[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
Z. Zeng, M. C. Velarde, F. A. Simmen, and R. C.M. Simmen
Delayed Parturition and Altered Myometrial Progesterone Receptor Isoform A Expression in Mice Null for Kruppel-Like Factor 9
Biol Reprod, June 1, 2008; 78(6): 1029 - 1037.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Bagamasbad, K. L. Howdeshell, L. M. Sachs, B. A. Demeneix, and R. J. Denver
A Role for Basic Transcription Element-binding Protein 1 (BTEB1) in the Autoinduction of Thyroid Hormone Receptor
J. Biol. Chem., January 25, 2008; 283(4): 2275 - 2285.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. C. Velarde, Z. Zeng, J. R. McQuown, F. A. Simmen, and R. C. M. Simmen
Kruppel-Like Factor 9 Is a Negative Regulator of Ligand-Dependent Estrogen Receptor {alpha} Signaling in Ishikawa Endometrial Adenocarcinoma Cells
Mol. Endocrinol., December 1, 2007; 21(12): 2988 - 3001.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
F. A. Simmen, R. Xiao, M. C. Velarde, R. D. Nicholson, M. T. Bowman, Y. Fujii-Kuriyama, S. P. Oh, and R. C. M. Simmen
Dysregulation of intestinal crypt cell proliferation and villus cell migration in mice lacking Kruppel-like factor 9
Am J Physiol Gastrointest Liver Physiol, June 1, 2007; 292(6): G1757 - G1769.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
X. Xu, J. Ding, G. Rao, J. Shen, R. A. Prinz, N. Rana, and W.P. Dmowski
Estradiol induces heparanase-1 expression and heparan sulphate proteoglycan degradation in human endometrium
Hum. Reprod., April 1, 2007; 22(4): 927 - 937.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
N. Matsumoto, A. Kubo, H. Liu, K. Akita, F. Laub, F. Ramirez, G. Keller, and S. L. Friedman
Developmental regulation of yolk sac hematopoiesis by Kruppel-like factor 6
B