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Endocrinology, doi:10.1210/en.2003-0777
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Endocrinology Vol. 145, No. 3 1096-1105
Copyright © 2004 by The Endocrine Society

A Role for CCAAT/Enhancer-Binding Protein ß in the Basal Regulation of the Distal-Less 3 Gene Promoter in Placental Cells

Manja P. Holland, Stuart P. Bliss, Kathie A. Berghorn and Mark S. Roberson

Department of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York 14853

Address all correspondence and requests for reprints to: Dr. Mark S. Roberson, T3-004d Veterinary Research Tower, Department of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The homeodomain protein Distal-less 3 (Dlx3) is essential for normal placental development in mice. Dlx3-null mice die by embryonic day 10.0 due to placental failure. The aim of our studies was to examine the transcriptional regulation and expression of Dlx3 in choriocarcinoma cell lines and primary trophoblasts from human placenta. A Dlx3 promoter fragment coupled to a luciferase reporter gene was sufficient to increase luciferase activity more than 11-fold over a luciferase control vector in choriocarcinoma cells, but not in a heterologous gonadotrope cell line. A 5' deletion series of the Dlx3 promoter revealed that a 13-nucleotide CCAAT box-containing element was required for basal expression in choriocarcinoma cell lines. Mutation of the CCAAT box within the context of the full-length promoter resulted in reduced basal activation of the Dlx3 reporter gene, suggesting that the CCAAT box was required for full basal expression. Western blot analysis revealed that Dlx3, CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}), and C/EBPß were present in choriocarcinoma cells and isolated trophoblasts from term human placentas. Electrophoretic mobility shift assays revealed the formation of a specific complex between choriocarcinoma cell nuclear extracts and the Dlx3 CCAAT box sequence. Competition and antibody electrophoretic mobility shift assays revealed that CCAAT/enhancer-binding protein ß (C/EBPß) binds the Dlx3 CCAAT box sequence. Overexpression of C/EBPß was sufficient to increase basal expression of a Dlx3 reporter gene in a dose-dependent manner. These studies provide the first insight into the mechanism(s) of Dlx3 gene expression in placental cells and suggest a role for C/EBPß in the basal regulation of the Dlx3 gene.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DISTAL-LESS 3 (Dlx3) is one of six members (Dlx1–6) of the Distal-less family of homeodomain proteins (1). The Distal-less family of proteins are all related to the Drosophila Distal-less homeodomain gene (Dl1), which regulates the formation of ventral appendages (2). In mammals, Dlx3 expression is limited to the branchial arches, dental tissues, epithelial derivatives, and placenta (1). Humans afflicted with tricho-dento-osseous syndrome exhibit abnormal hair, teeth, and bone development as a result of a four-nucleotide deletion in the coding region of the Dlx3 gene (3). This deletion causes a frameshift mutation that results in a premature stop codon and ultimately the deletion of the carboxyl-terminal portion of the Dlx3 protein. In a transgenic mouse model, targeted overexpression of Dlx3 in the epidermis results in severe abnormalities, including premature or accelerated epidermal differentiation and cessation of proliferation (4). These reports collectively provide convincing evidence of a key role for Dlx3 in the development and differentiation of epidermal derivatives.

In addition to its role in epidermal development, Dlx3 is required for normal placental development using a mouse knockout approach. Dlx3-null mice die in utero between embryonic d 9.5 (e9.5) and e10 as a result of placental defects that involve abnormal labyrinth layer development, including apparent defects in vascularization (5). The molecular mechanisms underlying placental failure in Dlx3-null mice have not been determined. Several studies (6, 7) indicate that Dlx3 acts as a transcriptional regulator of hormones and hormone synthesis in placental cell models. Using choriocarcinoma cells, Dlx3 was identified as a factor required for trophoblast-specific expression of the 3ß-hydroxysteroid dehydrogenase type VI (3ßHSDVI) gene (6). 3ßHSD exists as two isoforms in humans and six isoforms in mice and is an enzyme essential for the biosynthesis of all active steroid hormones. 3ßHSDI is expressed in the human placenta and is essential for the biosynthesis of placental progesterone and thus the maintenance of pregnancy. 3ßHSDVI is the murine orthologue of human 3ßHSDI. Dlx3 binds to the 3ßHSDVI promoter and is required for trophoblast-specific expression of this gene (6).

Dlx3 plays a key role in regulating cell-specific expression of the {alpha}-subunit of human chorionic gonadotropin (hCG) in choriocarcinoma cells (7). The major function of hCG during gestation is to serve as a luteotropin and aid in the maintenance of progesterone secretion from the corpus luteum until the placenta can produce sufficient quantities of progesterone to maintain pregnancy. Dlx3 is expressed in human placental trophoblasts at 8 wk gestation coincident with peak hCG production (7). Further, Dlx3 binds to the junctional regulatory element (JRE) within the complex pentameric array of cis elements known to be critical for expression of the glycoprotein hormone {alpha}-subunit in placental cells (7, 8, 9, 10, 11). Mutations within the JRE that block Dlx3 binding markedly reduce basal expression of the {alpha}-subunit (7). Interestingly, the combined actions of Dlx3 and activator protein-2{gamma} (AP-2{gamma}) appear to be necessary for the transcriptional regulation of both the glycoprotein hormone {alpha}-subunit and 3ßHSDVI genes (6, 7, 12, 13). In addition, AP-2 protein family members have been linked to the expression of both the ß-subunit of hCG (12) and the placental lactogen genes (14) in placental trophoblasts. These observations support the hypothesis that these two transcriptional regulators represent a unique trophoblast-specific combinatorial code that helps to define cell type-specific expression. In combination, Dlx3 and AP-2{gamma} may share common or overlapping interactions with coactivator/corepressor complexes that regulate these target genes.

Although Dlx3 has been shown to be a critical regulator of cell-specific expression of important placental proteins, the mechanism(s) associated with the regulation of the Dlx3 promoter in placental cells has not been elucidated. Our current studies presented here indicate that a CCAAT box located within the 5'-flanking sequences of the Dlx3 promoter is required for regulation of basal expression of Dlx3 in choriocarcinoma cells. The Dlx3 CCAAT box specifically binds CCAAT box/enhancer-binding protein ß (C/EBPß). Both Dlx3 and C/EBPß are coexpressed in human primary trophoblasts from normal term pregnancy, consistent with observations made in cell lines. These studies suggest a central role for C/EBPß in the basal regulation of the Dlx3 promoter in placental trophoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of reporter constructs and expression vectors
The reporter plasmid for the glycoprotein hormone {alpha}-subunit promoter fused with the luciferase reporter was a gift from Dr. Richard Maurer (Oregon Health Sciences University, Portland, OR) (7). Expression vectors for C/EBP{alpha} and C/EBPß were provided by Drs. Richard Day (University of Virginia Health Sciences Center, Charlottesville, VA) and Peter Johnson (National Cancer Institute, Frederick, MD). A portion of the 5'-flanking region of the Dlx3 gene from -1214 to +60 relative to the transcription start site was amplified from mouse genomic DNA by PCR. To facilitate cloning into a luciferase reporter, SstI and HindIII restriction sites were added to the forward and reverse primers, respectively. The two primers used in cloning Dlx3 were: forward -1214, 5'-GAGCTCATTACCCGGGAACTGGAC-3'; and reverse +60, 5'-AAGCTTCGCCGGCTGTCGG TCAGTCG-3'. The Dlx3 5'-flanking sequence was verified by DNA sequence analysis. The Dlx3 promoter fragment was cloned into pGL3-basic luciferase vector (Promega Corp., Madison, WI). The resulting reporter construct was designated Dlx3-luc and was used in transient transfection studies.

A series of successive deletions of the 5'-flanking region of the Dlx3 gene was constructed by PCR. The forward primers used in these reactions were: forward -608, 5'-GAGCTCGTGTCATTAAGATAA-3'; forward -303, 5'-GAGCTCGATTAGTAGATCCTG-3'; forward -121, 5'-GAGCTCCAGTGAGAAAGCGCG-3'; forward -90, 5'-GAGCTCAGGCAAGACTTGCAG-3'; forward -77, 5'-GAGCTCCAGCCAATCAGCGC-3'; and forward -64, 5'-GAGCTCGCAGGAGCCTCCCT-3'. The reverse primer used in all of these reactions was the reverse +60 primer described above. PCR products were cloned initially into the pGEM T Easy vector (Promega Corp.). Cloned sequences were verified through DNA sequence analysis. These fragments were then subcloned into the pGL3-basic vector.

PCR-based mutagenesis
PCR was used to create a block substitution of the CCAAT box sequence in the 5'-flanking region of Dlx3. The primer sequences containing the mutation are indicated with the regions of mutation underlined: MUTfw, 5'-AAGACTTGCAGGCGGCCGCCGCGCAGGAGCCTCCCTCGGCGACTCCAACATTG-3'; and MUTrev, 5'GCTCCTGCGCGGCGGC CGCCTGCAAGTCTTGCCTTCGCGGCAAAACACGCTCG-3'.

The 5'-flanking region of Dlx3 was used as a template for two PCR reactions containing the following primer pairs: forward -1214/MUTrev and reverse +60/MUTfw. The resulting PCR products were purified from agarose gel and used as a template for a PCR reaction with the forward -1214/reverse +60 primer pair. The product of this PCR reaction was purified, cloned into the pGEM T Easy vector, and subjected to DNA sequence analysis. The mutant Dlx3 promoter fragment was cloned into pGL3-basic as described above. The final product of this PCR-based mutagenesis, designated CCAAT mutant, consists of the flanking region of the Dlx3 gene from -1214 to +60 with a block substitution for the CCAAT box.

Cell culture, transient transfection, and luciferase assay
JEG3 and {alpha}T3–1 cells were cultured in DMEM (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS). BeWo cells were cultured in Waymouth’s MB 752/1 medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS. Before transfection, JEG3 and {alpha}T3–1 cells were split into fresh medium and cultured to approximately 50% confluence. JEG3 and {alpha}T3–1 cells were transfected by electroporation using a single electrical pulse at 220 V and 950 µFA. BeWo cells were plated to fresh medium at approximately 50% confluence and transfected with Lipofectamine for 6 h according to the manufacturer’s instructions (Invitrogen). All transfected cells were harvested 20–24 h after transfection. Cell lysates were prepared from transfected cells using three freeze-thaw cycles. Cellular debris was removed by centrifugation, and standardized amounts of cell protein were assayed for luciferase activity as previously described (15, 16, 17). All transfection studies were conducted independently at least three times with triplicate observations within each experiment. The data are presented as the mean ± SE and are taken from a single representative experiment (n = 3).

Isolation of cytotrophoblasts from human placenta
Studies using human term placentas were approved by the Cornell University institutional review board for the use of human subjects. Cytotrophoblast cells were isolated using a CD-9-negative selection method described by Richards and co-workers (18) with minor modifications. Briefly, term human placentas were processed within 1 h of collection by elective cesarean section. Villous tissue was sectioned into 1-cm3 portions and washed twice in RPMI A [RPMI 1640 (Invitrogen) containing 2.5 U/ml heparin, 50 U/ml penicillin/ streptomycin, and 5 µg/ml amphotericin B]. Tissue was then divided into 30-g portions, and each portion was suspended in 40 ml digestion medium [RPMI A containing pancreatin (Sigma-Aldrich Corp.; 55 mg/30 g tissue) and protease (Sigma-Aldrich Corp.; 0.5 mg/30 g tissue)] and digested at 37 C for 15 min with continuous agitation. Tissue was strained through 150-µm pore size Nitex mesh (Sefar-America, Kansas City, MO), washed once with RPMI A, resuspended in fresh digestion medium, and agitated at 37 C for an additional 15 min. Tissue was then strained again through 150-µm pore size Nitex mesh with vigorous agitation, and all strained media were pooled. Cells were collected by centrifugation and washed once in RPMI A. Cells were then resuspended in 100 ml erythrocyte lysis buffer (0.15 mM NH4Cl, 10 mM NaHCO3, and 0.1 mM Na2EDTA) and immediately repelleted. Cell pellets were resuspended in 20 ml RPMI B [RPMI A containing 50 U/ml deoxyribonuclease I (Invitrogen)] and strained through 50-µm pore size Nitex mesh. Cells were washed once in RPMI B, resuspended in 16 ml RPMI B, and counted using the trypan blue exclusion method. Goat serum (Sigma-Aldrich Corp.) was added to the cells to a final concentration of 20%, and the cells were incubated on ice for 30 min with occasional mixing. Cells were then repelleted, washed once with cold Dulbecco’s PBS (DPBS) containing 2% FBS, and resuspended to a final concentration of approximately 107 cells/ml in DPBS containing 2% FBS and 50 U/ml deoxyribonuclease I (DNase I). Mouse antihuman CD9 antibody (The Binding Site Ltd., San Diego, CA) was added to the suspension at a concentration of 15 µg/100 x 106 cells, and the suspension was incubated on ice for 30 min with occasional mixing. Cells were then collected by centrifugation, washed once in DPBS containing 2% FBS and 50 U/ml DNase I, and resuspended to a final concentration of 5 x 106 cells/ml in DPBS with 2% FBS and 50 U/ml DNase I. Goat antimouse immunomagnetic beads (Qiagen, Chatsworth, CA) were washed three times in cold DPBS with 2% FBS and added to the cell suspension to a final concentration of 25 x 108 beads/100 x 106 cells. The suspension was incubated on ice for 15 min with occasional mixing. CD9-positive cells were magnetically separated from the cytotrophoblasts at room temperature for 15 min. The cytotrophoblast-enriched supernatant was collected and subjected to two additional rounds of magnetic separation. The cytotrophoblasts were then collected by centrifugation; resuspended in DMEM containing 10% FBS, 50 U/ml penicillin/streptomycin, and 5 µg/ml amphotericin B; and plated for studies. The cells were plated on 60-mm culture dishes. At the time of collection, dishes of cells were placed on ice and washed with cold HEPES-buffered saline [20 mM HEPES (pH 7.5), 137 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, and 0.1% dextrose]. Cells were lysed and collected in a radioimmunoprecipitation assay buffer [20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 2 mM EDTA, 5 mM sodium vanadate, 0.2 mM phenylmethylsulfonylfluoride, and 5 mM benzamidine]. The cell lysates were then centrifuged to remove cellular debris, and protein content was determined by Bradford assay.

Cytokeratin immunocytochemistry
Purified cytotrophoblast cells from term human placenta were plated at a density of approximately 2 x 105/cm2 on glass slides. Slides were rinsed once in DPBS, and cells were fixed in 4% paraformaldehyde in DPBS for 15 min at room temperature. Slides were washed seven times in cold 150 mM NaCl, 40 mM K2HPO4, and 10 mM KH2PO4, pH 7.4 (KPBS) and were incubated at 4 C overnight in mouse anti-pan-cytokeratin IgG (Dako, Inc., Carpenteria, CA). Primary antibody was used at a dilution of 1:150 in KPBS with 0.4% Triton X-100. Control slides were prepared using normal rabbit serum at the same dilution in place of primary antibody. Slides were then washed seven times in KPBS and incubated for 1 h at room temperature in biotinylated rabbit antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) at a 1:1000 dilution in KPBS with 0.4% Triton X-100. After an additional seven washes, slides were developed using a commercial immunoperoxidase method according to the manufacturer’s instructions (Vectastain, Vector Laboratories, Inc.). The chromogen used was 0.015% 3,3-diaminobenzidine in Tris buffer, pH 7.2, with 0.002% H2O2. Cells were lightly counterstained with eosin, dehydrated by passage through an ascending ethanol series, cleared with xylene, and mounted. To clarify cell morphology, additional cells were fixed and stained with Wright’s stain according to the manufacturer’s instructions.

Preparation of nuclear and mouse skin extracts
JEG3 cells were cultured in 150-mm dishes to approximately 60–70% confluence. Nuclear extracts were prepared as described previously (19). Briefly, cells were washed and scraped into HEPES-buffered saline and then pelleted by centrifugation. Cells were resuspended in a hypotonic buffer and lysed in a Dounce homogenizer. The nuclei were isolated by centrifugation through a sucrose cushion. Nuclei were resuspended in EMSA binding buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 5% glycerol, 1 mM EDTA, and 1 mM dithiothreitol], and NaCl was added to a final concentration of 450 mM to extract nuclear proteins. After incubation at 4 C for 30 min with constant rocking, nuclear debris was removed by centrifugation at 75,000 x g for 30 min. The protein content of the nuclear extracts was determined by Bradford assay. The extracts were aliquoted and stored at -80 C. Mouse skin (chin pads) extracts were prepared in radioimmunoprecipitation assay buffer. After tissue disruption by Dounce homogenization (Kontes Co., Vineland, NJ), cellular debris was removed by centrifugation, and the protein concentration of the skin extracts was determined by Bradford assay.

Western blot analysis
JEG3 cell nuclear extracts and trophoblast whole cell lysates were suspended in an equal volume of 2x sodium dodecyl sulfate loading buffer [100 mM Tris (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, and 200 mM dithiothreitol]. Samples were boiled for 5 min, chilled on ice, and then resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat dried milk (NFDM)/Tris-buffered saline with Tween 20 [TBST; 10 mM Tris (pH 7.5), 150 mM sodium chloride, and 0.1% Tween 20]. Membranes were probed with C/EBP{alpha} antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at a 1:500 dilution in 2.5% NFDM/TBST, C/EBPß antibody (Santa Cruz Biotechnology) at 1:1000 in 2.5% TBST/NFDM, NF-YA antibody (Rockland Immunochemicals, Gilbertsville, PA) at 1:1000 in 2.5% TBST/NFDM, or actin antibody (Santa Cruz Biotechnology, Inc.) at 1:500 in 2.5% TBST/NFDM. After incubation in primary antibody, membranes were washed and probed with horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Hercules, CA) at a titer of 1:5000 in TBST containing 2.5% NFDM. Proteins were detected with enhanced chemiluminescence reagents (NEN Life Science Products-DuPont, Boston, MA).

EMSA and preparation of recombinant C/EBP{alpha} using wheat germ lysates
EMSAs were conducted as described previously (20). Oligonucleotides containing the CCAAT box sequence from the 5'-flanking region of Dlx3 were annealed: EMSAfw, 5'-CAGCCAATCAGCGCGCAGGA-3'; and EMSArev, 5'-TCCTGCGCGCTGATTGGCTG-3'. The oligonucleotides were subsequently radiolabeled with polynucleotide kinase and [{gamma}-32P]ATP. Poly(dI-dC) was used as a nonspecific competitor DNA (1 µg/reaction), and binding reactions were incubated at room temperature for 60 min in the absence of radiolabeled probe. Radiolabeled probe was then added to the reaction, and the reaction was incubated for another 60 min at room temperature. Binding complexes were resolved on 6% native polyacrylamide gels in 0.25x TBE (22.5 mM Tris, 22.5 mM boric acid, and 0.5 mM EDTA) at 4 C. Gels were dried, and DNA-protein interactions were visualized by autoradiography. In competition studies using EMSA, DNA competitors included the CCAAT box sequence from the Dlx3 promoter and the consensus cAMP response element (CRE) from secretogranin II promoter (21). The antisera used in DNA binding studies included C/EBP{alpha}, C/EBPß, and early growth response factor-1 (Egr-1; purchased from Santa Cruz Biotechnology). Recombinant C/EBP{alpha} was prepared according to the manufacturer’s instructions using a coupled transcription and translation synthesis reaction in commercially available wheat germ lysates (Promega Corp.). Initially, the control protein luciferase and C/EBP{alpha} were prepared using 35S-labeled methionine to confirm the production of proteins of appropriate molecular mass. Nonradioactive proteins were synthesized in a similar manner and used in the EMSA studies. In some experiments a consensus CCAAT DNA-binding site (5'-TGCAGATTGCGCAATCTGCA-3'; Santa Cruz Biotechnology, Inc.) was used as a positive control for C/EBP{alpha} binding.

Statistical analysis
Transfection data for experiments with more than two treatment groups were analyzed by ANOVA, and treatment differences were determined by a Duncan’s multiple range test. Transfection data for experiments with two treatment groups were analyzed by a one-tailed unpaired t test. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A Dlx3 reporter gene is expressed in JEG3 and BeWo choriocarcinoma cells, but not in pituitary {alpha}T3–1 cells
The 5'-flanking sequence of the Dlx3 gene (-1214 to +60) was cloned into a luciferase reporter vector (referred to as Dlx3-Luc) and transiently transfected into JEG3 and BeWo human choriocarcinoma cells, and the murine pituitary gonadotrope cell line, {alpha}T3–1. Both choriocarcinoma cell lines (BeWo cells not shown) and {alpha}T3–1 cells express the glycoprotein hormone {alpha}-subunit gene (Fig. 1Go, A and C). However, {alpha}-subunit promoter activity appears to be Dlx3 dependent only in choriocarcinoma cell lines, because in {alpha}T3–1 cells, Dlx3 is not detectable by Western blot analysis (7), and mutations within the Dlx3-binding site (JRE) did not alter basal expression (10). Consistent with this, Dlx3-Luc expression in JEG3 cells was approximately 11-fold greater (P < 0.05) than the expression level of the parent luciferase vector (Fig. 1BGo). Similar results were obtained using the BeWo choriocarcinoma cell line (not shown). In contrast, the expression of the Dlx3-Luc reporter and the parent luciferase vector were not different in {alpha}T3–1 cells (Fig. 1DGo). These experiments provide evidence that the fragment of the Dlx3 5'-flanking sequences used supports basal transcriptional activity in choriocarcinoma cells, but not in a gonadotrope cell line.



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  		FIG. 1. Dlx3 is expressed in choriocarcinoma cell lines, but not in cells of the gonadotrope lineage. In each of the two cell lines (A and B, JEG3 choriocarcinoma; C and D, {alpha}T3–1 gonadotrope), the expression levels of a human glycoprotein hormone {alpha}-subunit reporter gene (-880 to +46; h{alpha}-Luc) and a Dlx3 reporter gene (-1214 to +60; Dlx3-Luc) were compared with those of the empty luciferase vector (Luc vector). Cells were transiently transfected with 1 µg reporter construct by electroporation, and luciferase activity was examined approximately 24 h later. Each transient transfection study was conducted in triplicate on three separate occasions, all with similar results. Data shown are from a representative experiment and are reported as the mean ± SE The asterisks denote a significant difference from pairwise comparisons (P < 0.05).

 
A 13-nucleotide element within the Dlx3 promoter is required for basal expression in choriocarcinoma cell lines
A series of 5' deletions of the Dlx3 promoter were generated by PCR to identify a region(s) of the promoter required for mediating basal expression. The individual deletions were cloned into a luciferase reporter vector and transiently transfected into both JEG3 and BeWo cells. Cells were collected 24 h after transfection. In JEG3 cells, the expression of full basal activity of the Dlx3 promoter was dependent upon nucleotide sequences between -77 and -64 (Fig. 2AGo). Similar results were observed in transfection studies using BeWo cells (Fig. 2BGo). A CCAAT box motif was identified within nucleotide sequences between -77 and -64 in the Dlx3 promoter (Fig. 3AGo). To determine whether the CCAAT box was required for basal expression, a substitution mutation encompassing the entire CCAAT box was created in the context of the -1214 to +60 promoter fragment (Fig. 3AGo). Transfection studies revealed that mutation of the CCAAT motif resulted in a reduction (P < 0.05) in basal expression of the Dlx3 promoter (Fig. 3BGo). These data support the conclusion that the CCAAT box is required for basal expression of the Dlx3 promoter. Subsequent studies focused on the CCAAT box-binding complex in JEG3 cells.



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  		FIG. 2. A 13-nucleotide element within the Dlx3 promoter is required for basal expression in choriocarcinoma cells. PCR-based mutagenesis was used to create a series of sequential deletions from the 5' end of the 1214-bp Dlx3 5'-flanking sequence. Each of the reporters was transiently transfected via electroporation into JEG3 cells (A) or via lipofection into BeWo cells (B). The 5' terminus of the Dlx3 promoter present in each reporter construct is represented on the left of the figure. Each transfection study was conducted in triplicate on three separate occasions, all with similar results. Data shown are from a representative experiment and are reported as the mean ± SE. Bars with differing letters denote significant differences from ANOVA (P > 0.05). The asterisk denotes a significant difference from pairwise comparisons between the -77 and -64 Dlx3 reporters (P < 0.05). Over all experiments, deletion of the Dlx3 promoter to -64 (compared with the -1214 nucleotide promoter fragment) resulted in basal transcriptional activity of 22.9 ± 6.06% of the control value (±SEM).

 


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  		FIG. 3. A mutation in a CCAAT box reduced basal expression of the Dlx3 promoter in JEG3 cells. A, The CCAAT sequence was mutated using PCR in the context of the 1214-bp Dlx3-Luc promoter construct. The mutant sequence is shown beneath the sequence of the wild-type Dlx3 promoter. The arrowheads denote sites of deletions shown in Fig. 2Go. B, Both the mutant and wild-type Dlx3-Luc constructs were transiently transfected by electroporation into JEG3 cells, and luciferase activity was determined 24 h later. The asterisk indicates a difference between the mutant and wild-type Dlx3-Luc (P < 0.05). Transient transfection studies were conducted in triplicate on three separate occasions with similar results. Data shown are from a representative experiment and are reported as the mean ± SE. Over all experiments, the CCAAT box mutation resulted in transcriptional expression levels of 28.3 ± 4.1% of control (-1214 Dlx3 promoter; ±SEM) levels.

 
Several CCAAT box-binding proteins are present within choriocarcinoma cells and human trophoblasts from term placentas
Several transcription factors have been shown to bind to CCAAT box motifs, including C/EBPs and the A-subunit of NF-Y. Western blot analysis demonstrated that C/EBP{alpha} and C/EBPß were both present in JEG3 cells (Fig. 4AGo). The A-subunit of NF-Y was not detectable in Western blots of JEG3 cell nuclear extracts, but was readily detectable in mouse skin lysates (Fig. 4BGo). These studies suggested that NF-Y is not a likely candidate for regulation of the Dlx3 promoter at the CCAAT box in JEG3 placental cells. We next prepared human trophoblast cells for primary culture using a negative CD9 selection process that led to more than 90% purification of cytokeratin-positive cytotrophoblasts (Fig. 5AGo). Importantly, C/EBP{alpha}, C/EBPß, and Dlx3 are all coexpressed in primary cytotrophoblasts examined on d 1 of culture, suggesting that C/EBPs may play a functional role in the regulation of Dlx3 in primary trophoblasts (Fig. 5BGo).



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  		FIG. 4. C/EBP{alpha} and C/EBPß (but not NF-Y A-subunit) are present in JEG3 choriocarcinoma cells. A, Nuclear extracts were collected from JEG3 cells, and C/EBP{alpha} and C/EBPß were detected by Western blot analysis. The 50-kDa molecular mass standard is denoted on the right of the blots. B, Nuclear extracts were collected from JEG3 cells, and NF-YA was examined by Western blot analysis. Whole mouse skin lysate was used as a positive control for the NF-YA antibody. The blot was stripped and reprobed with actin antibody to control for lane loading.

 


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  		FIG. 5. C/EBP{alpha}, C/EBPß, and Dlx3 are coexpressed in purified human trophoblasts in primary culture. A, Immunocytochemistry was used to examine the expression of cytokeratins in cultured human trophoblasts. Primary human trophoblasts were cultured on coverslips for 24 h, followed by fixation and exposure to Wright’s stain for cell morphology or a pan-cytokeratin antibody with light eosin staining or NRS as a control. Greater than 90% of the cells examined were positive for cytokeratins. B, C/EBP{alpha}, C/EBPß, and Dlx3 were detected by Western blot analysis in lysates collected from two different preparations (samples 1 and 2) of human primary cytotrophoblasts isolated from term placenta. Human cytotrophoblasts were cultured for 24 h. Molecular weight standards are denoted on the right side of the blots.

 
The Dlx3 promoter CCAAT box binds C/EBPß, but not C/EBP{alpha}
To date, our studies have demonstrated that C/EBP{alpha} and C/EBPß are present in choriocarcinoma cells, and both proteins are coexpressed with Dlx3 in term placental trophoblasts in primary culture. EMSAs were performed with radiolabeled CCAAT box sequence and JEG3 nuclear extract to determine the characteristics and composition of the transcription factors that bind to the Dlx3 CCAAT box motif in placental cells. Dose-response binding studies using EMSA with nuclear extracts from JEG3 cells revealed a CCAAT box-binding complex (Fig. 6AGo). Substituting the mutant CCAAT box (see Fig. 3AGo) probe for the wild-type probe in EMSA resulted in a dramatic reduction in the shifted complex (Fig. 6AGo), suggesting DNA binding was dependent upon the central CCAAT motif of this element. Competition studies using a 100-fold molar excess of homologous (the CCAAT oligonucleotides) or heterologous oligonucleotides for a consensus CRE from the secretogranin II promoter (21) provided additional evidence for the specificity of the CCAAT-binding complex. The CCAAT-binding complex was abolished with a 100-fold molar excess of the homologous competitor. In contrast, a 100-fold molar excess of the consensus CRE did not alter CCAAT binding (Fig. 6BGo).



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  		FIG. 6. The Dlx3 promoter CCAAT box element forms specific binding complexes with JEG3 cell nuclear extract. EMSAs were performed using 32P-labeled CCAAT box probe and JEG3 nuclear extract. The CCAAT complex and free CCAAT probe are indicated by arrows to the left of each EMSA. A, Dose-response experiment with increasing amounts of JEG3 cell nuclear extract. The protein concentration of the nuclear extract was 1.5 mg/ml. B, A competition EMSA was carried out to determine the specificity of JEG3 nuclear extract binding to the CCAAT box. The binding reaction for the lane labeled Control contained 4 µg JEG3 nuclear extract protein along with radiolabeled CCAAT probe. The binding reactions for the lanes labeled CCAAT - 100 x and CRE - 100 x contained a 100-fold molar excess of unlabeled CCAAT and CRE oligonucleotides, respectively.

 
Specific antisera were used to determine the composition of the CCAAT box-binding complex from JEG3 cell nuclear extract. DNA-binding reactions were carried out with the incorporation of normal rabbit serum (NRS) and specific antibodies directed against C/EBP{alpha}, C/EBPß, and the inducible zinc finger transcription factor Egr-1. NRS and Egr-1 antisera served as negative controls. These EMSAs support the conclusion that an epitope similar to C/EBPß, but not C/EBP{alpha}, bound the Dlx3 CCAAT box motif (Fig. 7AGo). Addition of the C/EBPß antiserum blocked the formation of the CCAAT box-binding complex, reducing the shifted complex dramatically. Moreover, NRS and Egr-1 controls did not alter CCAAT box binding. These studies provide evidence that C/EBPß (or a similar epitope) is a strong candidate as a key regulator of basal Dlx3 promoter activity via this CCAAT box element in choriocarcinoma cells.



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  		FIG. 7. C/EBPß is present in a binding complex formed by the CCAAT box and JEG3 cell nuclear extract. JEG3 cell nuclear extracts, recombinant proteins, and 32P-labeled CCAAT box probes were used in an EMSA. A, The binding reaction for the lane labeled No protein did not contain JEG3 cell nuclear extract, whereas the rest of the binding reactions contained 4 µg JEG3 nuclear extract protein. The binding reaction for the lane labeled Control did not contain antisera. JEG3 nuclear extract was incubated in binding reactions in the presence of equivalent amounts of NRS, C/EBP{alpha}, C/EBPß, or Egr-1 antibodies as indicated. B, Recombinant radiolabeled C/EBP{alpha} and luciferase (as control protein) were prepared by coupled transcription and translation in wheat-germ lysates and examined using SDS-PAGE to confirm molecular mass. Equivalent, nonradiolabeled proteins were then used in EMSA (C). Binding reactions were prepared and resolved on native PAGE using radiolabeled probes for a consensus CCAAT box-binding site or the Dlx3 CCAAT box probe (see Materials and Methods). The binding reaction for the lane labeled No protein did not contain wheat-germ extract, whereas the rest of the binding reactions contained either 2.5 µl recombinant luciferase or C/EBP{alpha}. Some binding reactions were incubated in the presence of either NRS or C/EBP{alpha} antiserum. The C/EBP{alpha} complex and the supershift are indicated at the left of the panel.

 
In an effort to more clearly delineate a potential role for C/EBP{alpha} in the basal regulation of the Dlx3 promoter, C/EBP{alpha} and the control protein luciferase were prepared in a coupled transcription and translation reaction using wheat-germ lysate. Figure 7BGo depicts the expression of both proteins in this system. Nonradioactive C/EBP{alpha} and luciferase were then prepared and used in EMSA using a consensus CCAAT-binding site as well as the Dlx3 CCAAT box (Fig. 7CGo). C/EBP{alpha} bound to the consensus CCAAT probe, as confirmed by the use of antiserum specific for C/EBP{alpha}, which was effective in supershifting the binding complex. In contrast, recombinant C/EBP{alpha} failed to bind to the Dlx3 CCAAT box in the same experiment. These studies provide evidence to discount a probable role for C/EBP{alpha} in basal regulation of the Dlx3 promoter in choriocarcinoma cells.

Overexpression of C/EBPß is sufficient to activate the Dlx3 promoter
Overexpression studies were then conducted to determine whether C/EBPß could function as a transcriptional regulator of Dlx3 reporter expression in JEG3 cells. Cells were transiently cotransfected with either Dlx3-Luc (wild-type) or Dlx3-Luc containing the mutated CCAAT box (CCAAT mutant) and increasing doses of C/EBPß expression vectors. Luciferase activity was determined approximately 24 h after transfection. Overexpression of C/EBPß increased (P < 0.05) activity from the wild-type Dlx3-Luc reporter gene in a dose-dependent manner (Fig. 8Go). The response to C/EBPß overexpression using the Dlx3-CCAAT mutant was greatly reduced, but not completely abolished. This was probably due to additional C/EBPß-responsive elements putatively present upstream of the mutated CCAAT box within the Dlx3 promoter. Overall, reporter gene activity induced by C/EBPß was greatly reduced with the CCAAT box mutation, suggesting that the -77 to -64 CCAAT box element was necessary for C/EBPß-induced Dlx3 promoter activity.



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  		FIG. 8. Overexpression of C/EBPß results in increased expression of a Dlx3 reporter in JEG3 cells. Increasing doses of expression vector for C/EBPß and 1 µg of either Dlx3-Luc or the Dlx3-Luc CCAAT mutant reporter (Fig. 3AGo) were cotransfected by electroporation into JEG3 cells. Cells were collected approximately 24 h after electroporation for luciferase assay. Each transient transfection study was conducted in triplicate on three separate occasions with similar results. Data shown are from a representative experiment and are reported as the mean ± SE. Bars with different letters indicate a statistical difference in means at P < 0.05. Over all experiments, overexpression of C/EBPß (at the highest dose of expression vector compared with control) induced Dlx3 promoter activity 14.9 ± 4.0-fold (±SEM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vivo gene disruption studies demonstrate that Dlx3 expression is required for normal placental development and the successful completion of gestation in the mouse (5). It has also been demonstrated that Dlx3 plays an important role in the regulation of expression of the human glycoprotein hormone {alpha}-subunit (7) and murine 3ßHSDVI (6) genes in placental cells. Taken together, these studies support the conclusion that Dlx3 is a key transcriptional regulator of several genes linked to progesterone production during gestation. hCG serves as a luteotropin, whereas 3ßHSDVI is an obligatory enzyme required for the conversion of pregnenolone to progesterone in placental cells. The studies presented here provide important insight into the regulation of Dlx3 gene expression in cells of placental origin. Consistent with the regulation of key placental genes, we show that the Dlx3 promoter is activated in choriocarcinoma cells, but not in a gonadotrope cell line ({alpha}T3–1) that also expresses the glycoprotein hormone {alpha}-subunit gene. This expression pattern is consistent with our previous studies demonstrating the expression of Dlx3 protein in choriocarcinoma, but not in {alpha}T3–1 cells (7), and is consistent with the conclusion that the Dlx3 promoter fragment examined was adequate to confer transcriptional regulation, but not in all cell types.

Full basal expression of the Dlx3 promoter in choriocarcinoma cells required a 13-nucleotide CCAAT box located within the 5'-flanking sequence. DNA binding studies with JEG3 nuclear extracts indicated that an immunological epitope similar to C/EBPß, but not C/EBP{alpha}, was present within the CCAAT box-binding complex on the Dlx3 promoter. Interestingly, recombinant C/EBP{alpha} was clearly able to form a complex with a consensus CCAAT-binding site, providing evidence for the efficacy of the C/EBP{alpha} antiserum used in these DNA binding studies. Moreover, recombinant C/EBP{alpha} was unable to form a detectable complex with the Dlx3 CCAAT box within the context and constraints of the EMSA, suggesting that sequences surrounding the core CCAAT portion of this binding site may play a critical role in determining the specificity of C/EBP binding in placental cells. Consistent with our EMSA, overexpression of C/EBPß was sufficient to trans-activate the Dlx3 luciferase reporter via the CCAAT box. C/EBPß has been implicated as a basal transcriptional regulator of several genes, including promoters regulating the expression of the prolactin receptor (22), IGF-I (23), fibrinogen (24), and the steroidogenic acute regulatory protein (25, 26). Interestingly, C/EBPß has also been implicated in the expression of the prostaglandin H synthase-2 gene in a human cell line of amnion origin (27), suggesting a diverse role for C/EBPß in regulating genes within extraembryonic membranes.

The C/EBP family of transcription factors contains a highly conserved basic leucine zipper (bZIP) domain near the carboxyl-terminus. C/EBPs must form dimers to bind DNA. The leucine zipper motif is essential for dimerization, whereas DNA binding requires the presence of the basic domain adjacent to the leucine zipper (28). C/EBPs can form both homo- and heterodimers and may interact with other non-bZIP transcription factors (22, 29, 30, 31, 32, 33, 34). In the rat liver during postnatal development, the zinc finger transcription factor Sp1 is required for C/EBP-dependent transcriptional activation of the CYP2D5 p450 gene (31). In addition, it has been demonstrated that C/EBPß can associate with the bZIP proteins Fos and Jun, which results in a DNA binding specificity that differs from that of C/EBPß in its homodimeric form (33). C/EBPs play an important role in many cellular processes, including metabolism, differentiation, and inflammatory response, and are specifically involved in the differentiation of adipocytes, myeloid cells, hepatocytes, mammary epithelial cells, intestinal epithelial cells, keratinocytes, and ovarian luteal cells (35). The putative role of a heterodimeric binding partner(s) of C/EBPß in the context of the Dlx3 CCAAT box or the downstream role of Dlx3 in trophoblast differentiation remains to be elucidated.

Knockout mice have been useful in identifying the in vivo role of C/EBPs in a variety of cellular processes (36, 37, 38, 39, 40). C/EBPß-deficient mice exhibit two major phenotypes. Some C/EBPß-null mice die soon after birth as a result of severe hypoglycemia, whereas others survive to adulthood, but have fasting hypoglycemia, hypolipidemia, and impaired glucose production (41). Adult female C/EBPß-deficient mice are infertile due to impaired ovarian function, resulting in a functional lack of corpora lutea probably due to a failure in the signaling pathway induced by LH (42). Based upon the Dlx3-null mouse, one prediction might be that the C/EBPß-null mouse would have compromised trophoblast function. However, as C/EBPß-null embryos do not exhibit apparent placental defects and are maintained in utero until birth, the in vivo association between C/EBPß, Dlx3 expression, and placental function during early gestation remains unknown. The possibility exists that in C/EBPß-null embryos, other CCAAT box-binding proteins are capable of rescuing a potential placental phenotype in the C/EBPß-null animals. Further, although C/EBPß may play an important role in mediating basal expression, the possibility exists that locally controlled, inducible Dlx3 expression independent of the CCAAT box may prove important to the mechanisms underlying Dlx3-dependent trophoblast function during early gestation.

Interesting similarities and differences exist in the transcriptional regulation of the Dlx3 promoter in keratinocytes and placental cells. Dlx3 is expressed in keratinocytes and is required for normal epidermal development. Mutational analysis of the 5'-flanking region of the Dlx3 promoter in keratinocytes revealed a reduction in basal activity with mutations within the CCAAT box (43). Although keratinocytes and choriocarcinoma cells share the requirement for an intact CCAAT box for full basal expression of Dlx3, the binding factors that interact with this CCAAT box differ with cell type. Unlike choriocarcinoma cells, the Dlx3 CCAAT-binding complex in keratinocytes includes the CCAAT box-binding protein NF-Y, but does not appear to include C/EBPs (43). In our studies we were able to detect the NF-YA subunit in a mouse skin lysate, but NF-YA was not readily detectable in JEG3 cell nuclear extract (Fig. 5Go). In addition, overexpression of NF-YA in choriocarcinoma cells was not sufficient to stimulate Dlx3 promoter activity (data not shown). Overexpression of NF-YA has been shown to induce transcription from the SP-1 gene promoter (44). Although we cannot completely discount a possible role for NF-Y in placental cells, our expression data are consistent with the possibility that C/EBPs play a more prominent role in placental Dlx3 expression compared with NF-Y. In keratinocytes, C/EBPß is expressed and appears to play a prominent role in keratinocyte differentiation (45) and survival (46), presumably independent of Dlx3-induced differentiation. Thus, although similar cis elements within the Dlx3 promoter are required for basal expression in keratinocytes and choriocarcinoma cells, the transcriptional regulators contributing to basal expression from this cis element vary markedly with cell type.

Our studies support the conclusion that C/EBPß is involved in basal regulation of the Dlx3 gene promoter in choriocarcinoma cell lines. Importantly, we also demonstrate that Dlx3 and C/EBPß are present in preparations of trophoblast from normal term human placenta. These observations are consistent with the idea that molecular mechanisms defined in choriocarcinoma cell models have fidelity with potential mechanisms at play in nontransformed trophoblasts from normal human placentas. Our current studies extend our previous findings demonstrating Dlx3 expression in trophoblasts in 8-wk-old human placental microvilli coincident with high levels of hCG production (7). Those studies supported the possibility that Dlx3 expression may be initiated in cytotrophoblasts and subsequently maintained in syncytialized trophoblasts in human placental microvilli (7). The presence of Dlx3 in term human placenta suggests that Dlx3 may be expressed throughout gestation. The coordinate expression of C/EBPß with Dlx3 in human trophoblasts in term placentas supports the hypothesis that Dlx3 expression in fully differentiated trophoblasts may involve C/EBPß in vivo.


   Acknowledgments
 
We thank Richard A. Maurer, Peter Johnson, Roberto Mantovani, Richard Day, and Maria Morasso for generously providing reagents or helpful discussion. A special thanks to Stuart Handwerger and his lab members for their generous help with training in the methods for purification and culturing cytotrophoblasts from term human placenta. Appreciation is extended to Robert Cushman for aiding with statistical analyses.


   Footnotes
 
This work was supported by NIH Grant R01-HD-39354 (to M.S.R.).

Abbreviations: AP-2, Activator protein-2; bZIP, basic leucine zipper; C/EBP, CCAAT/enhancer-binding protein; CRE, cAMP response element; DNase, deoxyribonuclease; DPBS, Dulbecco’s PBS; e10.0, embryonic day 10.0; Egr-1, early growth response factor-1; FBS, fetal bovine serum; hCG, human chorionic gonadotropin; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; JRE, junctional regulatory element; NFDM, nonfat dried milk; NRS, normal rabbit serum; TBST, Tris-buffered saline with Tween 20.

Received June 24, 2003.

Accepted for publication December 3, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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