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Interdisciplinary Concentration in Animal Molecular and Cell Biology (R.C.M.S., T.E.C., F.J.M., L.B., F.A.S.), and Departments of Animal Science (R.C.M.S., T.E.C., F.J.M., L.B.) and Dairy and Poultry Sciences (F.A.S.), University of Florida, Gainesville, Florida 32611-0910; and the Department of Biochemistry, McGill University (H.I.), Montréal, Québec, Canada H3G 1Y6
Address all correspondence and requests for reprints to: Rosalia C. M. Simmen, Ph.D., Department of Animal Science, Building 459, Shealy Drive, University of Florida, Gainesville, Florida 32611-0910.
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Introduction |
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The findings that Sp1, a member of the C2H2 zinc finger family that also includes Sp2, Sp3, Sp4, basic transcription element-binding protein (BTEB), and BTEB2 (4, 5, 6, 7, 8), mediates E- and P-regulated gene expression (2, 3, 9, 10) raised the question of whether this function is specific to this GC box-binding protein or is shared by other family members. In the rabbit uteroglobin gene, whose pregnancy-associated endometrial expression is mediated by E and P, a functional role for Sp1 has been suggested in its basal (11, 12) as well as E-induced (10) transcription. Other Sp family members function either in place of (e.g. Sp4) or as a repressor (e.g. Sp3) of Sp1 by binding to common recognition sites within the uteroglobin gene promoter (11). It has not been demonstrated whether the newly identified GC box-binding proteins BTEB and BTEB2 exert similar effects as Sp1. Whereas expression of Sp1 is ubiquitous (4, 5, 6, 7), that of BTEB or BTEB2 appears to be more restricted and varies with tissue and cell type (8, 13, 14).
One mammalian tissue that exhibits a high level of BTEB expression is the uterine endometrium of pregnancy (14). In the pig, uterine endometrial expression of BTEB is epithelial cell type specific (14), which correlates with the expression of other P-regulated genes of pregnancy (15). This suggested the potential for BTEB to functionally modulate the pregnancy-associated expression of a number of these endometrial genes. The gene encoding uteroferrin (UF), a porcine transplacental iron transport protein (16) that belongs to a family of tartrate-resistant acid phosphatases in mammals (17), is a likely candidate for BTEB regulation, as sequences that have been shown to bind BTEB are located in its 5'-regulatory region in close proximity to those that bind PR (18, 19). However, no studies have previously examined the trans-activation of the endometrial UF gene by this or a related nuclear factor(s). In the present study, we have used the UF gene as a model to evaluate the possibility that the temporal and highly regulated expression of epithelial-specific, pregnancy-associated uterine genes involves the actions of PR and BTEB. Toward this end, we examined whether a functional association exists between coexpression of BTEB and PR and the epithelial-specific temporal induction of the UF gene during pregnancy. Further, we tested whether BTEB, alone or in combination with the PR/P complex, trans-activates the UF gene promoter specifically in endometrial epithelial cells.
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Materials and Methods |
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Plasmid constructs
Chimeric UF-luciferase (Luc) reporter gene constructs were
generated by subcloning 5'-genomic fragments of the UF gene (23)
representing -1935 to +23 (-1935UF-Luc) and -831 to +23
(-831UF-Luc), respectively, into the HindIII site of the
pGL3-Basic vector (Promega Corp., Madison, WI). The
orientations of the subcloned fragments were confirmed by DNA
sequencing. The BTEB expression construct (pcDNA-3BTEB) consisted of
the entire coding region of human BTEB (24), but lacked the
5'-untranslated sequences of the messenger RNA (mRNA) that were
identified to be involved in translational repression (13). Previous
studies have shown that Hepa1 cells, whose endogenous expression of
BTEB is normally undetectable by Western blot analysis, exhibited
detectable levels of this protein when transfected with this expression
vector (Imataka, H., unpublished data). The expression construct
encoding the full-length rat PR B (PRB) complementary DNA
(cDNA) was a gift from Dr. Benita Katzenellenbogen (University of
Illinois, Urbana, IL) (25).
Transfection and luciferase assays
Transient transfections were performed on primary cultures of
pig GE and ST cells and human endometrial carcinoma cell lines, when
80% confluent, by the polybrene method using CsCl density gradient
purified plasmid DNAs (20). Plasmid DNA (10 µg for each construct or
expression vector) was added to cells plated on six-well culture dishes
and incubated for 6 h. Cells were then washed with HBSS (pH 7.4)
and treated with dimethylsulfoxide (DMSO) in HBSS (ECC-1, 20% DMSO for
3 min; Hec-1-A and primary cells, 25% DMSO for 4 min) to increase cell
permeability. Cells were washed with HBSS twice and then incubated in
fresh medium containing 10% FBS with or without added P (10
nM). After 48 h, cells were washed twice with ice-cold
HBSS and lysed in single strength lysis buffer (200 µl; Promega Corp., Madison, WI). The experiments were performed at least
three times using independent DNA preparations of each construct and
for primary cultures, cells that were isolated from endometrium of at
least three individual pigs. The protein concentration of cell lysate
(5 µl) was determined using the Bio-Rad Laboratories, Inc., protein assay reagent (Bio-Rad Laboratories, Inc., Richmond, CA). Quantitative determination of Luc activity
(measured as relative light units) in cell lysates was carried out
using an Autolumat Luminometer (EG&G Berthold, Bad Wildbad, Germany).
Luciferase activity was normalized to the protein concentration of cell
lysates.
Analyses of RNA and protein
Total RNA was isolated from pregnant pig endometrial tissues,
primary cultures of pig endometrial GE or ST cells and from human
endometrial cell lines by use of TriZol (Life Technologies, Grand Island, NY) following the manufacturer’s
instructions. The integrity of the RNA was assessed by inspection of
28S and 18S band intensities after agarose gel electrophoresis. The
relative amounts of PR, BTEB, UF, and ß2-microglobulin
(ß2M) mRNAs in the indicated samples were evaluated by
RT-PCR (26) or by RNA dot blot analysis. The RT-PCR analyses to detect
PR, UF, and ß2M mRNAs were subjected to preliminary
validation and optimization steps using a pool of day 60 pregnant pig
endometrial cDNA to ensure that amplification of the products was in
the exponential phase and the assay was linear relative to the amount
of input RNA. These steps included varying the amount of cDNA template
used for a fixed number of PCR cycles, adjusting the number of cycles
for a fixed template amount, and determining the optimal
Mg2+ concentration and pH for each cDNA template-primer
combination using the PCR Optimizer kit (Invitrogen, San
Diego, CA). The optimal conditions and primer pairs for detecting BTEB
transcript by RT-PCR have been previously described (14). The primer
pairs for PCR amplification of UF, PR, and ß2M
transcripts were designed from previously published porcine cDNA
sequences (27, 28, 29) and were synthesized by the DNA synthesis core
facility of the Interdisciplinary Center for Biotechnology Research at
the University of Florida, Gainesville. Optimal thermal cycling
parameters were the following: PR: 95 C, 1 min; 55 C, 1 min; 72 C, 1
min, with 1 µl cDNA template in 2 mM Mg2+ and
pH 9.0 (30 cycles); BTEB: same as PR, except that 0.5 µl cDNA
template was used; UF: 95 C, 2 min; 55 C, 2 min; 72 C, 30 sec, with 0.2
µl cDNA template in 2 mM Mg2+ and pH 9.0 (30
cycles); and ß2M: same as PR, except that 0.1 µl cDNA
template was used (30 cycles). DNA fragments of 416, 290, 812, and 287
bp were generated by PCR of cDNAs for BTEB, PR, UF, and
ß2M, respectively, and were verified by nucleotide
sequence analysis. For analysis of PCR band intensities, photographs
were scanned at high resolution, and the integrated density of the band
was calculated using the Alpha Imager 2000 Documentation and Analysis
System (Alpha Innotech Co., San Leandro, CA). The intensities of
the PR, UF, and BTEB signals were normalized to that of the
ß2M internal control. Dot blot analysis for UF mRNA
abundance was performed as previously described (30), using a
32P-labeled porcine UF gene cDNA insert as probe (27), and
resultant hybridization intensities were quantified by PhosphorImage
analysis (Molecular Dynamics, Inc., Sunnyvale, CA).
Nuclear extracts (NE) were prepared from endometrial tissues and cells, following previously described procedures (14, 23). The levels of BTEB protein in NE were examined by Western immunoblot analysis, using rabbit antisera generated against either porcine BTEB fusion protein (for pig tissues) (14) or rat BTEB (for human endometrial cell lines) (13) at a dilution of 1:500 in TBS buffer (1 mM Tris and 160 mM NaCl, pH 7.4) containing 1% Blotto (nonfat dry milk), as previously described (14). The immunoreactive BTEB band was subsequently visualized by further incubation of the filter with [125I]protein A (Amersham, Arlington Heights, IL) in binding buffer, followed by autoradiography.
Electrophoretic mobility shift assays
Approximately 10 µg NE protein were used for each gel mobility
shift assay. A double stranded oligonucleotide probe containing the
GC-rich region located at -768 to -749 bp (termed GC box 1) (19) of
the UF gene 5'-flanking region was end-labeled using
[
-32P]ATP and T4 polynucleotide kinase and used as
probe for the binding reactions. This fragment specifically binds BTEB
protein, as previously determined from competition assays with
wild-type GC box 1 and unrelated oligomers (19) and by gel supershift
with anti-BTEB antiserum (14). The NE was preincubated with antirat
BTEB antiserum (1 µl) or preimmune serum (1 µl) for 30 min at 4 C
before addition of the labeled probe. The binding reactions were
performed at 37 C for 30 min, and DNA-protein complexes were resolved
in a 6% nondenaturing polyacrylamide gel as described previously
(19).
Statistical analysis
All numerical data obtained from densitometric analysis of PCR
bands or hybridization blots and from luciferase assays were subjected
to least squares ANOVA using the general linear models procedures of
the Statistical Analysis System (SAS Institute, Inc.,
Cary, NC) (31). Significant effects due to day of pregnancy, cell type,
or treatment (promoter construct, expression construct, presence or
absence of P) were separated by orthogonal contrasts or the
Student-Newman-Keuls sequential range test (31). Values were considered
significant at P 
0.05 and are presented as the least
squares mean (LSM) ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2).
On day 12, only one of three endometrial tissues analyzed expressed the
UF mRNA, and this level of expression was lower than that exhibited by
all three endometrial tissues on day 60, consistent with previous
results (33). The levels of PR mRNA, when normalized to
ß2M, whose expression was constant during this period,
did not differ (PR/ß2, 0.69 ± 0.17 vs.
0.50 ± 0.17; P > 0.05) between days 12 and 60 of
pregnancy. BTEB mRNA and protein levels also appeared to be similar at
these pregnancy days, in agreement with the results of a previous study
(14).
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). To examine whether the endometrial
epithelial cell type expression of the UF gene is correlated with PR
and/or BTEB, GE and ST cells were evaluated for expression of their
respective mRNAs. BTEB mRNAs were present in both GE
(BTEB/ß2, 0.51 ± 0.11) and ST
(BTEB/ß2, 0.36 ± 0.11) at comparable levels,
although only GE cells demonstrated detectable BTEB protein as
evaluated by electrophoretic mobility shift assay (Fig. 4). This complex has been previously
confirmed to represent endometrial BTEB bound to the GC-rich motif by
its ability to be supershifted by a specific BTEB antiserum (14).
Similarly, the levels of PR mRNA, when normalized to those of
ß2M, were comparable (P > 0.05) in GE
(PR/ß2, 0.97 ± 0.22) and ST (PR/ß2,
1.09 ± 0.22) cells.
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, A and
B).
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), consistent with that observed at the level of UF gene promoter
activity (Fig. 5A).
Expression of BTEB in human endometrial cell lines
The human endometrial cell lines ECC-1, Ishikawa, and
Hec-1-A support the activity of the UF gene promoter to direct Luc gene
expression (23) (this study) despite undetectable endogenous UF
transcripts (data not shown). These observations suggest that
endometrial epithelial-derived cells may have in common nuclear factors
that partly support the expression of genes associated with the
epithelial phenotype. To evaluate the possibility that one such
regulatory factor is BTEB, the expression of BTEB gene in these cells
was examined at the levels of its mRNA and protein. ECC-1, Hec-1-A, and
Ishikawa cells express the BTEB gene at levels comparable to those in
pregnant pig endometrium, as detected by RT-PCR (Fig. 6A). The presence of BTEB protein in NE
prepared from ECC-1 and Hec-1-A cells was evaluated by Western blot and
gel retardation antibody supershift assays. Western blot analysis using
rabbit antirat BTEB antiserum failed to detect BTEB in NE prepared from
either cell line (data not shown). However, the more sensitive gel
shift assay using a 32P-labeled oligonucleotide fragment
corresponding to a GC-rich region within the UF gene promoter and that
was previously shown to bind BTEB (14, 19) demonstrated the presence of
a protein/DNA complex in ECC-1 NE (Fig. 6B). The electrophoretic
mobility of this complex was retarded by the addition of rabbit antirat
BTEB antiserum, but not by preimmune rabbit serum. Nuclear extracts
prepared from Hec-1-A cells exhibited the formation of a similar,
albeit weaker intensity, protein/DNA complex that was also supershifted
by anti-BTEB antiserum, but not by preimmune serum (Fig. 6B).
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and 8).
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). Cotransfection of BTEB and PR (plus P) had additive effects
on the activity of both promoter constructs (Fig. 7). However, although
the effect of BTEB on UF promoter activity was more pronounced for the
longer construct (P < 0.05), that of PR/P did not
differ between constructs (P = 0.107). In ECC-1 cells,
cotransfection of BTEB and PR (in the presence of P) resulted in a
significant trans-activation of the -1935UF-Luc promoter
construct at a level that was higher (P < 0.05) than
the additive effects of the two expression vectors when tested
separately (Fig. 8). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary cultures of GE and ST cells from early pregnancy pig endometrium and the human endometrial transformed (carcinoma) cell lines that exhibit the epithelial phenotype were used to examine the cell context dependence of BTEB action for a number of reasons. First, isolated ST cells do not manifest BTEB protein, either endogenously or upon transient transfection with the BTEB expression construct, despite the presence of the BTEB transcript. Second, ECC-1 and Hec-1-A cells have diminished BTEB protein levels relative to primary cultures of endometrial GE cells; hence, a temporal association between increased expression of this protein and induction of UF promoter activity could be readily established. Finally, isolated GE cells represent a more physiologically relevant environment for UF gene expression. Using these models, we obtained several lines of evidence to support the involvement of BTEB in the transcriptional regulation of the UF gene: 1) BTEB, in the absence or presence of PR, increased UF promoter activity in endometrial epithelial cells; 2) UF mRNA levels in pig endometrial GE cells were induced by increased BTEB levels; 3) endometrial carcinoma cells exhibiting diminished BTEB protein content, relative to endometrial epithelial cells of pregnancy, do not express the endogenous UF gene; and 4) ST cells, which express PR at a level comparable to that of GE cells, but not BTEB, have undetectable expression of UF. Collectively, these results indicate that BTEB, similar to its family member Sp1 (2, 9, 10, 12), mediates the trans-activation of an endometrial epithelial gene.
In support of previous studies (13), the data presented here demonstrate the cell context-dependent translational control of BTEB expression in two cell systems, namely the porcine endometrial stromal fibroblastic cells and human endometrial carcinoma cells exhibiting the epithelial phenotype. Thus, although BTEB transcripts in these cells are readily detectable, expression of the corresponding protein is minimal (ECC-1, Hec-1-A) or undetectable (ST cells). Interestingly, transient transfection of a human BTEB expression construct that lacked specific 5'-untranslated sequences previously identified to mediate translational repression of BTEB mRNA in HeLa cells (13) increased the expression of corresponding BTEB protein in ECC-1 and Hec-1-A cells, as measured by increased transcription from the UF gene promoter. By contrast, similarly transfected ST cells did not exhibit the anticipated increase in UF promoter activity. These results suggest that additional factors, unrelated to those involved in translational repression, might modulate the synthesis and/or functional activity of BTEB protein in ST cells.
The apparent requirement for coexpression of BTEB and P-dependent PR for the high level transcriptional activity of UF, a gene preferentially expressed by uterine epithelial cells of pregnancy (Ref. 20 and this study), raises the interesting possibility that uterine differentiation associated with pregnancy events may involve a threshold concentration of each of these factors, below which the functional signaling mechanism(s) responsible for activation of specific gene targets is not achieved. Indeed, GE cells isolated from early pregnancy pig endometrium showed a modest, but consistently higher, level of UF mRNA upon transfection with BTEB expression construct, relative to control transfected cells. As GE cells express functional PR (20), this finding suggests that increasing the ratio of BTEB to PR might favor UF gene expression. However, day 60 pregnancy endometrium, which manifests a similar BTEB to PR ratio as day 12 pregnancy endometrium in vivo, exhibits higher expression of the UF gene (Ref. 33 and this study). This lack of correlation between relative expression of UF and the BTEB/PR ratio could be due to the presence of other factors or hormonal influences that act in synergy with or modulate the effects of BTEB and PR/P. In this regard, we have previously shown that an 80-kDa nuclear protein that binds a negatively acting cis element upstream of the UF gene promoter is expressed at higher levels in ECC-1 cells and in the early pregnancy pig endometrium than in the midpregnancy pig endometrium (19, 23). A similar pattern of expression was observed for the nuclear protein Sp1 in ECC-1 cells and in early vs. midpregnancy pig endometrium (unpublished data from this laboratory). As Sp1 binds to GC-rich sequences with the same affinity as BTEB (6) and, hence, can compete for BTEB-binding sites associated with the UF gene promoter, Sp1 may function as a trans-repressor of BTEB action. Taken together, these results are consistent with the idea that the magnitude of BTEB and PR/P activities, as assessed by their effects on UF gene expression, is dependent on endometrial cell context and may constitute a critical determinant of epithelial-associated gene transcription.
The localization of binding sites for PR and BTEB in close proximity to each other within the UF gene promoter region (18, 19) suggested the possibility that a functional PR/BTEB complex might mediate the induction of UF gene transcription in endometrial epithelial cells. Indeed, the formation of transcriptionally active ER or PR complexes with Sp1 that then bind to GC-rich enhancer elements has been implicated in the steroid hormone induction of gene transcription (3, 9, 35). The data presented here cannot directly address this possibility; however, our observations that BTEB increased UF promoter activity in the absence of PR or any added cofactor and that PR/P enhanced UF promoter activity in the absence of BTEB suggest that trans-activation by BTEB or PR occurs mainly via their direct binding to respective recognition sequences on the UF promoter. The functional relevance of the additive vs. synergistic effects of BTEB and activated PR shown here on the activation of endometrial epithelial-associated genes remains to be ascertained. It is interesting to note, however, that ECC-1 cells exhibit higher levels of endogenous BTEB protein than Hec-1-A cells, suggesting that, at least in vitro, a higher BTEB/PR ratio might favor enhanced UF promoter activity. Alternatively, the extent of the interactions between BTEB and the PR/P complex might require the participation of cell-specific cofactors.
In a previous study (18), three P-responsive sequences within the 2-kb 5'-flanking region of the UF gene were identified by their ability to bind the PR protein and to exhibit P-dependent enhancer activity within the context of homologous and heterologous promoters in a rabbit endometrial cell line, HRE-H9, that endogenously expresses functional PR. The present study shows that in Hec-1-A cells cotransfected with a PR expression vector, the effects of PR/P were largely mediated by sequences within the -831 construct that contained two of the three identified P-responsive sequences. In contrast, additional BTEB-responsive sequences appear to be located more upstream of the initially reported BTEB-binding sites within -768 to -749 nucleotides (19), as the effect of BTEB on the short construct was increased with additional flanking sequence. A closer examination of sequences between -831 to -1935, however, did not reveal the presence of consensus elements known to bind Sp-related family members (23). Thus, BTEB effects may also be mediated by noncanonical sequences or by interaction with other as yet unknown nuclear proteins.
In conclusion, the present study demonstrates a functional role for coexpression of P-dependent PR and BTEB in mediating the high level promoter activity of an epithelial-specific, pregnancy-associated gene. The cell context dependence of BTEB expression as well as of its trans-activation potential predict a unique role for BTEB in the regulation of endometrial epithelial gene transcription during pregnancy.
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Acknowledgments |
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Footnotes |
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Received August 20, 1998.
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