help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-1694
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 Martin, R.
Right arrow Articles by Taylor, H. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martin, R.
Right arrow Articles by Taylor, H. S.
Related Collections
Right arrow Pediatric Endocrinology
Right arrow Female Endocrinology
The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 5 1920-1926
Copyright © 2007 by The Endocrine Society

Differential Cell-Specific Modulation of HOXA10 by Estrogen and Specificity Protein 1 Response Elements

Ryan Martin, Melissa B. Taylor, Graciela Krikun, Charles Lockwood, G. Edda Akbas and Hugh S. Taylor

Departments of Obstetrics, Gynecology and Reproductive Sciences (R.M., M.B.T., G.K., C.L., G.E.A., H.S.T.) and Molecular, Cellular and Developmental Biology (H.S.T.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Hugh S. Taylor, Associate Professor, Yale University School of Medicine, 333 Cedar Street, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: hugh.taylor{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: HOX genes are highly evolutionarily conserved regulators of embryonic development. HOXA10 also regulates differentiation of the adult reproductive tract and mammary gland in response to sex steroids.

Objective: We recently identified two HOXA10 estrogen response elements (EREs). Here we demonstrate that estrogen-responsive HOXA10 expression is cell type specific.

Design and Setting: We conducted an in vitro study at an academic medical center.

Main Outcome Measure: Reporter assay, gel shift assays (electrophoretic mobility shift assay), and immunohistochemistry were done.

Results: The HOXA10 EREs and a specificity protein 1 (Sp1) binding site differentially drive the cell-type-specific E2 response. In electrophoretic mobility shift assays, both estrogen receptor-{alpha} and -ß bound both EREs but not the Sp1 site. In reporter assays, both EREs and the Sp1 site demonstrated estrogen responsiveness and tissue specificity; transiently transfected uterine Ishikawa cells or breast MCF-7 cells showed differential responses to E2 treatment. Each response element (Sp1, ERE1, and ERE2) drove distinct differential expression in each cell type. Sp1 protein was expressed in a menstrual-cycle stage-specific expression pattern in endometrium, first expressed in perivascular cells.

Conclusions: Tissue specificity inherent to a regulatory element as well as differential cellular expression of transcription factors imparts differential tissue-specific estrogen responsiveness.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HOX GENES ARE highly evolutionarily conserved and act as regulators of embryonic morphogenesis and differentiation (1). These transcription factors mediate embryonic development by determining regional patterning along the anterior-posterior axis, including that of the reproductive tract (2). HOXA10 is not only expressed during embryonic development but also continues to have a regulatory role in the adult female reproductive tract (3, 4, 5, 6, 7). HOXA10 is regulated by 17ß-estradiol (E2) and progesterone; the effects of these sex steroids are in part the direct result of their receptors binding to regulatory regions of HOXA10 (7).

Although it is known that E2 regulates HOXA10 gene expression, the molecular mechanisms by which this regulation is mediated are not fully characterized. Classically, E2 binds to either {alpha}- or ß-receptor; the liganded dimeric receptor complex regulates the transcription of specific genes by interacting with specific estrogen-responsive cis-acting elements (8). Tissue specificity of response was thought to be primarily regulated by differential expression of both estrogen receptor (ER){alpha} and ERß (9). These ERs bind estrogen response elements (EREs) as heterodimers or homodimers and activate gene expression in response to E2. Many estrogen-responsive genes have been found to contain EREs, and most vary from the consensus sequence by one or more nucleotides (10).

Alternatively, estrogen-responsive gene expression can also be mediated by specificity protein 1 (Sp1). Sp1 is a transcription factor that binds to GC-rich promoter sites causing gene activation (11). The DNA binding domain comprises three conserved zinc fingers (12). Its role and function as a transcription factor are diverse and dependent on promoter and cell context (13, 14). Typically, Sp1 forms multimeric complexes with other transactivating proteins. Transactivation by Sp1 can be the result of interaction with other DNA-bound transcription factors, or alternatively activation of Sp1 can occur through dimerization with proteins not directly bound to DNA (15). E2 can induce gene expression via ER-Sp1 interactions with GC-rich promoter elements in which Sp1, but not ER, binds DNA.

Two HOXA10 EREs were recently identified, ERE1 and ERE2, which drive estrogen-responsive reporter expression in the Ishikawa uterine endometrial adenocarcinoma cell line (16). The HOXA10 ERE 1 and ERE 2 vary from the consensus ERE by three and four nucleotides, respectively; these changes from the consensus ERE may be necessary to impart tissue specificity, ligand specificity, or dose responsiveness to estrogen flux during the reproductive cycle (16). The HOXA10 EREs drive differential expression based on ligand specificity, distinct from the consensus ERE.

In addition to ligand-mediated stimulation, most estrogen-responsive genes show distinct cellular specificity and are typically differentially regulated in breast and uterine tissue. This may be due in part to tissue-specific EREs. Estrogen-responsive HOXA10 expression has been demonstrated in both benign and malignant breast tissue as well as MCF-7 cells, a well differentiated ER-positive cell line (17). In addition, HOXA10 is expressed in uterine, epithelial, and stromal cells as well as the Ishikawa cell line. Here we demonstrate that HOXA10 is regulated differentially by E2 in breast and uterine cells; this regulation is mediated by distinct cellular specificity of each ERE. In addition to estrogen response imparted by ER binding sites, EREs also contain cues that impart tissue-specific regulation. Finally, we identified an Sp1 site that regulates HOXA10 expression in response to E2 in a cell-specific manner.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PCR

PCR was used to generate HOXA10 5' regulatory regions. Human genomic DNA was used as a template in PCR to generate five contiguous segments from the first 2000 nucleotides in the 5' regulatory region of HOXA10 (Eppendorf Mastercycler Gradient, NY). Five pairs of primers that specifically amplified each of these segments were designed (GenBank accession no. AC004080). Amplification was performed using an annealing temperature optimized for each segment. The PCR was conducted in a total volume of 50 µl containing 10x PCR buffer, 10 µM each of 5' and 3' primers. All the components for PCR were purchased from Promega (Madison, WI) with the exception of the primers that were synthesized by the Yale University School of Medicine, Department of Pathology. The PCR products were separated on 2.5% agarose-TAE (40 mM Tris-acetate, 1 mM EDTA) gels containing ethidium bromide (10 mg/ml) and visualized by UV light. Representative PCR products were excised from agarose gels and identity confirmed by DNA sequencing. The PCR products were phenol/chloroform purified, ethanol precipitated, and subsequently cloned into reporter constructs.

Reporter constructs and expression plasmids

The luciferase reporter plasmids contained five nested segments from the first 2000 nucleotides of the 5' regulatory region of HOXA10 and were subcloned into the KpnI and EcoRI sites of pGL3-promoter (Promega). Two putative EREs identified by sequence analysis of the above regions, a consensus ERE, and the Sp1 site were each subcloned into the KpnI and EcoRI sites of pGL3-promoter. The 40-, 41-, and 25-mer sequences used were as follows: ERE1, 5'-CCAGAGTGTCAGGAGCCCAGAGTGAACTAGAAGCTGACTT-3'; ERE2, 5'-CCCTACCCGGACGTGAGCCCCATACCGGGGTCCCTTAGAAG-3'; and Sp1, 5'-CGCGGGCGCGCGGCGGGCGGGCGCG-3' (nucleotide sequences are in italics).

All plasmids were sequenced to confirm their primary sequence and the identity of the inserts. Site-directed mutagenesis was also used to alter each ERE and the Sp1 site in the context of the whole element.

Cell culture

Ishikawa cells are a well differentiated endometrial adenocarcinoma cell line in which we have previously characterized estrogen regulation of HOX genes (4, 17, 18, 19, 20, 21, 22). These cells were cultured in phenol red-free Eagle’s MEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% (vol/vol) charcoal-stripped fetal bovine serum and supplemented with penicillin/streptomycin (100 µg/ml), L-glutamine (2 mM), and sodium pyruvate (1 mM). MCF-7 cells were also cultured in MEM containing 10% (vol/vol) charcoal-stripped fetal bovine serum and supplemented with penicillin/streptomycin (100 µg/ml), L-glutamine (2 mM), and sodium pyruvate. Expression of both ER{alpha} and -ß was verified by ELISA in both cell lines according to the manufacturer’s instruction (Abbot Laboratories, Weisbaden, Germany). Cells were grown in plastic flasks (75 cm2; Falcon, Franklin Lakes, NJ) and maintained at 37 C in a humidified atmosphere (5% CO2 in air). The 70–80% confluent monolayers were maintained in serum-free media for 24 h and subsequently treated with E2 (1 x 10–8 M; Sigma Chemical Co., St. Louis, MO).

Transfection and luciferase assays

To analyze the enhancer activity of these reporter constructs, Ishikawa cells or MCF-7 cells were grown to 50–60% confluence in 25-cm2 flasks and transiently transfected with the appropriate plasmid using Lipofectamine (Life Technologies), a mixture of liposomes consisting of a 3:1 (wt/wt) formulation of 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N-N-dimethyl-l-propanium trifluoroacetate and dioleoylphosphatidyl ethanolamine. After 5 h, the cells were washed with 1x PBS and allowed to grow for an additional 24 h. The 70–80% confluent monolayers were maintained in serum-free media for 24 h and subsequently treated with 10–8 M E2 (Sigma) for 6 h. The cells were washed with cold 1x PBS and lysed with 1x reporter lysis buffer (Promega), and the lysates were collected. The cells were then snap-frozen in dry ice/ethanol and microcentrifuged at maximum speed for 2 min, and supernatant was collected. Luciferase activity was assayed by using the luciferase assay kit (Promega) and a luminometer. A Renilla luciferase reporter construct was cotransfected to correct for variations in transfection efficiency. Reporter activity was normalized to Renilla expression.

Electrophoretic mobility shift assay (EMSA)

EMSAs were performed as previously described (20, 21). Complementary single-stranded oligodeoxynucleotides were synthesized and annealed to incorporate putative ER binding sites and flanking sequences located at the 5' end of the transcription site of the HOXA10 gene. Sequences of oligonucleotides are as follows: ERE1, 5'-CCAGAGTGTCAGGAGCCCAGAGTGAACTAGAAGCTGACTT-3'; ERE2, 5'-CCCTACCCGGACGTGAGCCCCATACCGGGGTCCCTTAGAAG-3'; consensus ERE, 5'-CAGGTCAGAGTGACCTG-3'; and Sp1, 5'-CGCGGGCGCGCGGCGGGCGGGCGCG-3'. Binding reactions were performed on ice for 30 min using 0.5–1. 0 µg recombinant ER{alpha} and ERß and 80,000 cpm of labeled DNA in a final volume of 25 µl containing 25 mM HEPES (pH 7.6), 50 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, 5 mM MgCl2, 10 µg/ml salmon sperm DNA, and 10% glycerol. All samples were fractionated for 3 h at 200 V in a 4% nondenaturing polyacrylamide gel containing 1x TBE at 4 C. The gel was dried under a vacuum at 80 C for 45 min and exposed overnight on X-OMAT film (Kodak, Rochester, NY) and subsequently developed.

Real-time PCR

Quantitative real-time RT-PCR was performed using the LightCycler SYBR Green RT-PCR kit from Roche (Stockholm, Sweden). One microgram of total RNA was reverse transcribed in 20 µl reaction mixture containing l0 mM each of dATP, dCTP, dGTP, and deoxythymidine triphosphate; 20 pmol oligo(dT); 40 U/µl ribonuclease inhibitor, l0 U/µl avian myeloblastosis virus-reverse transcriptase, and 10x AMV-RT buffer for 30 min at 61 C. PCR for HOXA10 was performed for 45 cycles of 95 C for 2 sec, 65 C for 5 sec, and 72 C for 18 sec. PCR for control ß-actin was performed for 45 cycles of 95 C for 2 sec, 61 C for 5 sec, and 72 C for 18 sec. The HOXA10 intron-spanning primers were selected using the primer selection program Primer3 developed by the Whitehead Institute for Biomedical Research (Cambridge, MA). All experiments were conducted four times. A standard curve was constructed, and HOXA10 expression was normalized to ß-actin. A Student’s t test was used to determine statistically significant differential expression.

Tissue collection

Endometrium was collected from 10 normal cycling reproductive-age (20–35 yr) women by endometrial biopsy with informed consent, under an approved Human Investigations Committee protocol. Tissue was fixed in formalin for histological examination and immunohistochemistry. Menstrual cycle dating was determined by menstrual history and confirmed by histological examination using the criteria of Noyes et al. (23).

Immunohistochemistry

Immunohistochemistry was performed in paraffin-fixed sections of endometrium. Peroxidase staining was conducted with the ABC Elite kit from Vector Laboratories, Inc. (Burlingame, CA) as per the manufacturer. Rabbit polyclonal antibodies to Sp1 (Accurate Surgical and Scientific Corp., Westbury, NY) were used at a concentration of 0.2 mg/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 regulates HOXA10 expression in both Ishikawa and MCF-7 cells

E2 is known to regulate HOXA10 gene expression in human breast and uterine tissue (4, 16, 17). To compare E2-driven cell-type-specific HOXA10 expression in uterine and breast cells, Ishikawa adenocarcinoma and MCF-7 breast cancer cell lines were grown to 50–60% confluence. Cells from both lines were cultured in steroid-free medium then treated with 10–5 to 10–9 M E2 or treated with diluent as a control for 24 h. Real-time PCR was used to quantify HOXA10 expression levels as shown in Fig. 1Go. HOXA10 expression was normalized to ß-actin. Both MCF-7 and Ishikawa cell express HOXA10 mRNA at similar levels. Ishikawa and MCF-7 cells each demonstrated increased HOXA10 expression in response to E2 when compared with controls. Ishikawa cells showed a dose-responsive increase in HOXA10 mRNA in response to increasing levels of E2. At each concentration of E2, Ishikawa cells demonstrated significantly greater HOXA10 expression than MCF-7 cells. Breast and uterine cells show differential HOXA10 expression in response to E2.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 1. Quantification of HOXA10 gene expression in response to E2 using real-time PCR in Ishikawa and MCF-7 cell lines. All results were normalized to ß-actin expression. In MCF-7 cells, there was a significant 3-fold increase in HOXA10 expression after treatment with 10–5 M E2. There was no difference from the negative controls at E2 concentrations of 10–7 and 10–9 M, respectively. In Ishikawa cells, HOXA10 expression was dose responsive to treatment with E2. *, Statistically significant difference of P < 0.05.

 
Identification of HOXA10 EREs and Sp1 site

To identify HOXA10 EREs, we cloned 2 kb 5' of the HOXA10 transcription start site by PCR and tested the ability of nested segments to drive reporter gene expression in transient transfection assays. Segments approximately corresponding to nucleotides –2000 to –1500, –1500 to –1000, –1000 to –500, –500 to –200, and –200 to +10 were individually cloned into pGL3-promoter and assayed for reporter gene activity after liposome-mediated transfection in Ishikawa cells (Fig. 2Go). Ishikawa cells are from a well-differentiated uterine endometrial carcinoma cell line in which we have previously characterized the estrogen regulation of HOX genes (4, 17, 18, 19, 20, 21, 22). Additionally, cells were transfected with a Renilla luciferase expression construct as a control for transfection efficiency. Luciferase activity was normalized to Renilla activity. Each assay was performed with and without physiological (10–8 M) E2. As demonstrated in Fig. 2Go, one construct consisting of nucleotides –1000 to –500 drove reporter gene expression in response to E2. A second segment encompassing –500 to –200 base pairs drove reporter expression in response to E2 stimulation but to a lesser extent than the previous segment. Sequence analysis revealed two previously defined EREs in each of these segments. ERE-1 (5'-GCCCAgagTGAAC-3') had 70% homology to the consensus ERE (5'-GGTCAnnnTGACC-3') (7). ERE-2 (5'-GGTCCcttAGAAG-3') had 60% sequence identity to the consensus element (Fig. 2Go). A third novel estrogen-responsive area in the promoter region of HOXA10 was identified and found to contain an Sp1-type binding site (5'-CGCGGGCGGGCGGCGGGCGGGCGCG-3').


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 2. Schematic representation of the HOXA10 gene regulatory region and E2-responsive expression. A, Five regions comprising 2000 bp of the HOXA10 5' regulatory region were individually tested for estrogen responsiveness in transient transfection assays. Putative EREs were previously identified in two regions that drove estrogen-responsive reporter gene expression. The distal and proximal regions were designated ERE1 and ERE2, respectively. A putative Sp1 site was also identified. B, The sequence of the two EREs, the consensus ERE, and Sp1 are shown. Bases identical to the consensus sequence are underlined. ERE1 and ERE2 demonstrated 70 and 60% sequence homology to the consensus ERE, respectively. An Sp1 binding site was identified, and the characteristic GC-rich promoter region sequence is shown. C, Reporter assays showing fold induction after E2 treatment are shown by the black bars using each element. In Ishikawa cells, ERE1 and ERE2 each showed a significant fold induction in response to E2 (P < 0.01). Similarly, the Sp1 element drove reporter expression in response to E2 (P < 0.01). White bars indicate reporter gene induction in response to E2 using modified elements in which each ERE or Sp1 element was mutated. The mutated elements failed to demonstrate E2 responsiveness. Each experiment was performed in duplicate and repeated a minimum of four times. D, Reporter assays were also preformed using the entire regulatory element in which each ERE or the Sp1 binding site was mutated. Mutation of each site diminished the ability to drive reporter gene expression in response to E2 treatment.

 
A second set of oligonucleotides was constructed that altered the putative EREs or Sp1 binding site. Mutations of three bases from each ERE construct were introduced, such that each no longer resembled a consensus ERE. These oligos were used to generate constructs for reporter assays. Each mutant response element was tested in luciferase reporter constructs for E2-driven reporter expression (Fig. 2CGo). The mutated EREs no longer responded to E2 treatment. These results are represented by the white bars in Fig. 2Go shown below each corresponding element stimulated with E2. Similarly, a mutated Sp1 element was constructed with five nucleotides (A or T) introduced into the GC-rich element. This reporter construct did not respond to E2 treatment as shown in Fig. 2Go. Mutation of each element resulted in loss of E2 responsiveness.

Reporter assays were also preformed using the entire regulatory element in which each ERE or the Sp1 binding site was individually mutated (Fig. 2DGo). Mutation of each site diminished the ability to drive reporter gene expression in response to E2 treatment. Each mutation resulted in a significant reduction of E2 responsiveness roughly proportional to the E2 responsiveness of each individual element.

HOXA10 ERE 1 and ERE 2, but not the Sp1 site, bound ER{alpha} and ERß

We performed EMSA using recombinant ER{alpha} and ERß to determine whether the putative HOXA10 EREs bound to ER. To assure that the Sp1 site did not contain an unrecognized ERE, the Sp1 site and surrounding sequence was also used in EMSA to determine ER binding. EMSA demonstrated that ER{alpha} bound to 32P-labeled ERE1, ERE2, and the consensus ERE but not to the Sp1 site (Fig. 3Go).


Figure 3
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 3. HOXA10 transcriptional activation by E2 is mediated by ER binding to EREs. EMSA was performed using recombinant ER{alpha} or ERß and 32P-labeled ERE1, ERE2, consensus ERE, and the Sp1 binding site. Upper panel, EMSA demonstrated that ER{alpha} bound to 32P-labeled ERE1, ERE2, and the consensus ERE but not to Sp1. ER{alpha} binds to each element with varying affinity. The lanes labeled 0 (lanes A, E, H, and K) contain labeled probe alone without ER. Subsequent lanes (lanes B, C, D, F, G, I, J, L, and M) all contain labeled probe and ER{alpha}. The last lane of each probe set contains labeled probe, ER, and 10–8 M E2 (lanes D, G, J, and M). Lane D contains labeled probe, E2, and twice the amount of ER used in the other binding reactions to investigate potential low-affinity binding. The asterisk indicates the shifted complex, whereas FP designates the free probe. Lower panel, ERß similarly binds each ERE but not the Sp1 element as demonstrated by EMSA. The lanes labeled 0 (lanes A, E, H, and K) contain labeled probe alone without ER. Subsequent lanes (lanes B, C, D, F, G, I, J, L, and M) all contain labeled probe and ERß. The last lane of each probe set contains labeled probe, ERß, and 10–8 M E2 (lanes D, G, J, and M). Lane D contains labeled probe, E2, and twice the amount of ER used in the other binding reactions to investigate potential low affinity binding. The asterisk indicates the shifted complex, whereas FP designates the free probe.

 
As expected, the consensus ERE as well as HOXA10 ERE1 and ERE2 each bound ER{alpha} and resulted in a shifted complex. The lanes labeled 0 (lanes A, E, H, and K) contain labeled probe alone without ER. Subsequent lanes (lanes B, C, D, F, G, I, J, L, and M) all contain labeled probe and ER{alpha}. The last lane of each probe set contains labeled probe, ER, and 10–8 M E2 (lanes D, G, J, and M). Lane D contains labeled probe, E2, and twice the amount of ER used in the other binding reactions to investigate potential low-affinity binding. The asterisk indicates the shifted complex, whereas FP designates the free probe. ER{alpha} binds to each element with varying affinity. ER{alpha} bound the consensus ERE with the highest affinity and bound ERE1 with higher affinity than ERE2. No ER{alpha} binding to the Sp1 site was noted.

Similarly, ERß bound each ERE but not the SP1 site. ERß bound the consensus ERE with the highest affinity. ERE1 bound ERß with higher affinity than ERE2. There was no discernable binding of ERß to the Sp1 site.

ERE1 and ERE2 each bound both ER{alpha} and ERß. The consensus ERE was used as a positive control and bound with the greatest affinity to both ER{alpha} and ERß. ERE1 bound with greater affinity than ERE2 to both ERs. As expected, the SP1 site did not bind to either ER{alpha} or ERß, demonstrating lack of an occult ERE.

Cell-type specificity of the HOXA10 ERE

To characterize E2-induced tissue-specific HOXA10 expression, Ishikawa and MCF-7 cells were transfected with artificial promoter constructs containing the consensus ERE, ERE1, ERE2, and the Sp1 binding sites. Four double-stranded 40-bp oligonucleotides encompassing the consensus ERE, ERE1, ERE2, and Sp1 were individually synthesized and cloned into pGL3-reporter and used in transient transfection assays. Both cell lines were treated with 10–8 M E2 for 6 h and then compared (Fig. 4Go). The consensus ERE demonstrated an 8-fold greater luciferase expression in MCF-7 cells compared with Ishikawa cells. ERE-1 showed an approximate 3-fold greater E2-induced reporter expression in Ishikawa cells than in MCF-7 cells. ERE-2 also drove a smaller but significant increase in luciferase activity after treatment with E2 in Ishikawa cells. There was no ERE2-driven expression in the MCF-7 cells. In response to E2, Sp1-driven reporter gene expression was evident in Ishikawa cells but not in MCF-7 cells. All increases in reporter expression were blocked by cotreatment with 10–7 M ICI 182780 (data not shown). Each experiment was conducted four times, and a t test was used to determine statistical significance.


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 4. Cell-specific reporter gene expression driven by the HOXA10 EREs or Sp1 site. Transient transfection and reporter assays were preformed in MCF-7 cells and Ishikawa cells. Expression of ER{alpha} and ERß were confirmed by ELISA. In MCF-7 cells, the consensus ERE drove reporter expression 8-fold more than in Ishikawa cells. ERE1 demonstrated three times the reporter expression in the Ishikawa cells than in MCF-7 cells. In Ishikawa cells, ERE2 showed a 50-fold increase in expression after treatment, whereas no expression was noted in MCF-7 cells. Sp1 drove greater expression in Ishikawa cells, whereas only minimal expression was noted in MCF-7 cells. *, Statistically significant difference of P < 0.05.

 
Spatial localization of Sp1 expression

Although the expression of ER{alpha} and ERß has been well characterized in endometrium, the expression of Sp1 in this tissue has not been fully characterized. Sp1 is known to be expressed in endometrium where it regulates tissue factor gene expression (24). Here we localized Sp1 expression in this tissue using immunohistochemistry. Immunohistochemical staining for Sp1 was conducted on six proliferative-phase and six secretory-phase endometria. The slides were evaluated by two independent observers. Figure 5Go reveals that Sp1 is expressed throughout the menstrual cycle in epithelial, stromal, and endothelial cells. A high level of expression was noted in nearly all epithelial cells in both the proliferative and secretory phases. An increase in Sp1 staining was observed in secretory- compared with proliferative-phase stromal cells (mean nuclear staining of 77% secretory vs. 38% proliferative; P = 0.01). The earliest and highest levels of Sp1 expression were localized to the cells undergoing decidualization surrounding the spiral arterioles (Fig. 5CGo).


Figure 5
View larger version (104K):
[in this window]
[in a new window]

  		FIG. 5. Sp1 expression in uterine endometrium. Immunohistochemical staining for Sp1 in proliferative-phase (A) and secretory-phase (B) human endometria were carried out in paraffin-fixed sections. Sp1 is identified by brown peroxidase staining. In the glandular epithelium, strong nuclear staining was noted in both the proliferative and secretory phases. Stromal expression increased in the secretory phase compared with the proliferative phase. As demonstrated in C, Sp1 expression was prominent in perivascular areas of the secretory endometrium.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In multicellular organisms, development requires specific differentiation of distinct cell types. In addition, adults demonstrate continual functional differentiation during particular stages of the life cycle. Both HOX genes and sex steroids have been linked to the process of organogenesis and functional differentiation in the reproductive tract, breast, and other tissues. The molecular mechanism by which differential gene expression is obtained in a distinct tissue-specific fashion is not fully understood. Both Ishikawa and MCF-7 cells express ER{alpha} and ERß; however, each demonstrated distinct HOXA10 regulation in response to E2. Here we demonstrate that two EREs and an Sp1 response element impart tissue-specific estrogen responsiveness to HOXA10 gene expression.

HOX genes are evolutionarily conserved and regulate embryonic morphogenesis and differentiation (1). HOX genes are also known as selector genes; expression within a certain segment of the embryo will cause those cells to select a particular path of development (2, 25). HOX genes perform this function by acting as transcription factors (26, 27). Sex steroids are also critical in the organogenesis and differentiation of the reproductive tract and continue to maintain functional differentiation throughout adult life. HOXA10 and HOXA11 are dynamically up-regulated by sex steroids; E2 and progesterone drive HOX expression, and this regulated expression is necessary for function of the female reproductive tract (4, 28).

Estrogen action has classically thought to be transduced by ER{alpha} and ERß (29, 30). ERs are structurally organized into functional domains consisting of a highly conserved DNA binding domain and activation domain (AF1) in the N-terminal region and a ligand binding domain and activation domain (AF2) in the C-terminal region. Upon ligand binding, ERs transactivate the target gene by binding to EREs that are classically composed of two AGGTCA motifs arranged in a palindrome separated by three base pairs. The activation of the ERE results in either transcriptional activation or repression in a cellular and promoter-specific manner (30, 31). EREs then interact with a complex array of coregulatory proteins that mediate the interaction between receptor and the basal transcriptional apparatus and allow the remodeling of the chromatin structure (32, 33, 34, 35, 36, 37). ERE sequences can affect ER conformation independently from a ligand and lead to specific cofactor interactions, allowing for the desired biological effect (38, 39, 40, 41, 42).

Previously, we identified two distinct HOXA10 EREs, ERE1 and ERE2, that demonstrated ligand specificity in the uterine adenocarcinoma Ishikawa cell line when exposed to either E2 or diethylstilbestrol (16). HOXA10 ERE1 varies from the ideal ERE by three nucleotides, and these changes may be necessary to impart or alter ligand specificity of the bound receptor (16). Here, when Ishikawa cells were treated with E2, ERE1 showed greater reporter gene expression compared with ERE2 and the consensus ERE. This suggests that, in the presence of liganded ER, ERE1 was primarily responsible for expression of HOXA10 in these cells.

The HOXA10 ERE is not only ligand specific but also tissue specific. The HOXA10 ERE demonstrates cell-type-specific estrogen-responsive activity. E2 is known to activate transcription of multiple genes, including HOXA10, in the uterus and the breast. The transcriptional activation of ERE containing reporter constructs by full or partial estrogen agonists varied between the MCF-7 breast carcinoma cell line and the Ishikawa adenocarcinoma cell line, both of which express ER{alpha} and ERß. The consensus ERE showed a significant estrogen-responsive increase in expression in MCF-7 cells, whereas ERE1-driven expression was low and ERE2-driven expression was negligible. In contrast, in Ishikawa cells, ERE1 and to a lesser extent ERE2 were significantly up-regulated by estrogen. Here we showed that the HOXA10 EREs demonstrated distinct functional properties in two different estrogen-sensitive cell lines in response to E2.

Sp1 is a member of a family of transcription factors that bind and act through GC boxes to regulate gene expression. Sp1-type transcription factors contain a DNA binding domain comprising three conserved Cys2His2 zinc fingers. Interactions of Sp1 with other DNA-bound transcription factors can result in transactivation, or alternatively, Sp1 can be activated through protein-protein interactions with non-DNA-bound cofactors (10). E2 induces expression of several genes via ER-Sp1 protein interactions using GC-rich promoter elements in which Sp1, but not ER, binds DNA (15, 43). ERE deletional analyses suggest that in some genes, E2-induced transactivation may require only GC-rich Sp1 binding sites (44). Here we showed that Sp1 is expressed in the uterine endometrium in a tissue- and menstrual-cycle stage-specific fashion. We also demonstrate tissue-specific reporter gene expression driven by an Sp1 site in response to E2; distinct differences in response were observed between Ishikawa cells and MCF-7 cells. Focal Sp1 expression in perivascular stromal cells may enhance estrogen response in this region to promote decidualization. The area around the spiral arterioles is the first to decidualize and likely limits trophoblast invasion into the vasculature. Because HOXA10 is involved in regulating decidualization, we speculate that it may be induced first in this area to promote decidualization; HOXA10 induction is likely in part driven by Sp1 through the HOXA10 Sp1 enhancer identified here.

HOXA10 is expressed in both breast and uterine cells and is regulated by E2 (28). Here we show that E2-mediated expression is regulated by two distinct EREs and Sp1, each of which demonstrates distinct tissue specificity. EREs may impart upon the ER the ability to bind tissue-specific factors, either directly or by altering ER conformation. Previously, we determined that these EREs are regulated in a ligand-specific manner (16). Here we elucidate in vitro the tissue and cell-type-specific function of both ERE and Sp1 response elements. Distinct EREs enable tissue-specific responses to E2, which may allow tissues with similar ER expression to regulate genes differently in response to the same hormonal stimulus.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online February 20, 2007

Abbreviations: E2, 17ß-Estradiol; EMSA, electrophoretic mobility shift assay; ER, estrogen receptor; ERE, estrogen response element; Sp1, specificity protein 1.

Received August 8, 2006.

Accepted February 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McGinnis W, Krumlauf R 1992 Homeobox genes and axial patterning. Cell 68:283–302[CrossRef][Medline]
  2. Taylor HS 2000 The role of HOX genes in the development and function of the female reproductive tract. Semin Reprod Med 18:81–89[CrossRef][Medline]
  3. Taylor HS, Vanden Heuvel GB, Igarahi P 1997 A conserved axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the HOXA cluster genes. Biol Reprod 57:1338–1345[Abstract]
  4. Benson GV, Lim H, Paria BC, Satokata I, Dey SK, Maas RL 1996 Mechanisms of reduced fertility in Hoxa-10 mutant mice: uterine homeosis and loss of maternal Hoxa-10 expression. Development 122:2687–2696[Abstract]
  5. Lim H, Ma L, Ma WG, Maas RL, Dey SK 1999 Hoxa-10 regulates uterine stromal cell responsiveness to progesterone during implantation and decidualization in the mouse. Mol Endocrinol 13:1005–1017[Abstract/Free Full Text]
  6. Ma L, Benson GV, Lim H, Dey SK, Maas RL 1998 Abdominal B (AbdB) Hoxa genes: regulation in adult uterus by estrogen and progesterone and repression in Mullerian duct by the synthetic estrogen diethylstilbestrol (DES). Dev Biol 197:141–154[CrossRef][Medline]
  7. Taylor HS, Arici A, Olive D, Igarashi P 1998 HOXA 10 is expressed in response to sex steroids at the time of implantation. J Clin Invest 101:1379–1382[Medline]
  8. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand induced homodimer. Cell 55:145–156[CrossRef][Medline]
  9. Mosselman S, Polman J, Dijkema R 1996 ER-ß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  10. Klinge CM 2001 Estrogen receptor interaction with estrogen response elements. Nucleic Acids Res 29:2905–2919[Abstract/Free Full Text]
  11. Suske G 1999 The Sp-family of transcription factors. Gene 238:291–300[CrossRef][Medline]
  12. Safe S, Kim K 2004 Nuclear receptor-mediated transactivation through interaction with Sp proteins. Prog Nucleic Acid Res Mol Biol 77:1–36[Medline]
  13. Lania L, Mejello B, DeLuca P 1997 Transcriptional regulation by the Sp family proteins. Int J Biochem Cell Biol 29:1313–1323[CrossRef][Medline]
  14. Bouwman P, Philipsen S 2002 Regulation of the Sp1-related transcription factors. Mol Cell Endocrinol 195:27–38[CrossRef][Medline]
  15. Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA, Safe S 2000 Ligand-, cell-, and estrogen receptor subtype ({alpha}/ß)-dependent activation at GC-rich (Sp1) promoter elements. J Biol Chem 275:5379–5387[Abstract/Free Full Text]
  16. Akbas GE, Song J, Taylor HS 2004 HOXA 10 estrogen response element (ERE) is differentially regulated by 17ß-estradiol and diethylstilbestrol (DES). J Mol Biol 340:1013–1023[CrossRef][Medline]
  17. Chu MC, Selam FB, Taylor HS 2004 HOXA10 regulates p53 expression and Matrigel invasion in human breast cancer cells. Cancer Biol Ther 3:568–572[Medline]
  18. Chau MY, Pando S, Taylor HS 2002 HOXA11 silencing and endogenous HOXA11 antisense ribonucleic acid in the uterine endometrium. J Clin Endocrinol Metab 87:2674–2680[Abstract/Free Full Text]
  19. Block K, Kardana A, Igarashi P, Taylor HS 2000 In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing Mullerian system. FASEB J 14:1101–1108[Abstract/Free Full Text]
  20. Daftary GS, Troy PJ, Bagot CN, Young SL, Taylor HS 2002 Direct regulation of ß3 integrin subunit gene expression by HOXA10 in endometrial cells. Mol Endocrinol 16:571–579[Abstract/Free Full Text]
  21. Troy P, Daftary G, Bagot C, Taylor HS 2003 Transcriptional repression of peri-implantation EMX2 expression in mammalian reproduction by HOXA10. Mol Cell Biol 23:1–13[Abstract/Free Full Text]
  22. Taylor HS, Irigashi P, Olive DL, Arici A 1999 Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium. J Clin Endocrinol Metab 84:1129–1135[Abstract/Free Full Text]
  23. Noyes RW, Hertig AT, Rock J 1953 Dating of the endometrial biopsy. Fertil Steril 1:3–5[Medline]
  24. Krikun G, Schatz F, Mackman N, Guller S, Demopoulos R, Lockwood CJ 2000 Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3. Mol Endocrinol 14:393–400[Abstract/Free Full Text]
  25. Akam M, Dawson I, Tear G 1988 Homeotic genes and the control of segmental diversity. Development 104:123–133
  26. Dessain S, Gross CT, Kuziora MA, McGinnis W 1992 Anti-type homodomains have distinct binding specificities that correlate with their different regulatory functions in embryos. EMBO J 11:991–1002[Medline]
  27. Ekker SC, Jackson DG, von Kessler DP, Sun BI, Young KE, Beachy PA 1994 The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins. EMBO J 13:3551–3560[Medline]
  28. Daftary G, Taylor HS 2006 Endocrine regulation of HOX genes. Endocr Rev 27:331–355[Abstract/Free Full Text]
  29. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  30. McKenna NJ, O’Malley BW 2000 An issue of tissues: divining the split personalities of selective estrogen receptors. Nat Med 6:960–962[CrossRef][Medline]
  31. Yi P, Driscoll MD, Huang J, Bhagat S, Hilf R, Bambara RA, Muyan M 2002 The effects of estrogen-responsive element- and ligand-induced structural changes on the recruitment of cofactors and transcriptional responses by ER{alpha} and ERß. Mol Endocrinol 16:674–693[Abstract/Free Full Text]
  32. Smith CL, O’Malley BW 2004 Coregulator function: a key to understanding tissue specificity of selective receptor modulators. Endocr Rev 25:45–71[Abstract/Free Full Text]
  33. Collingwood TN, Urnov FD, Wolffe AP 1999 Nuclear receptors: coactivators, corepressors and chromatin remodeling in the control of transcription. J Mol Endocrinol 23:255–275[Abstract]
  34. Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional function of nuclear receptors. Genes Dev 14:121–141[Free Full Text]
  35. McKeena NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  36. Pham TA, Hwung YP, Santiso-Mere D, McDonnell DP, O’Malley BW 1992 Ligand-dependent and independent function of the transcription regions of the human estrogen receptor in yeast. Mol Endocrinol 6:1043–1050[Abstract/Free Full Text]
  37. Metzger D, Losson R, Bornert JM, Lemoine Y, Chambon P 1992 Promoter specificity of the two transcriptional activation functions of the human oestrogen receptor in yeast. Nucleic Acids Res 20:2813–2817[Abstract/Free Full Text]
  38. Fritsch M, Welch RD, Murdoch FE, Anderson I, Gorski J 1992 DNA allosterically modulates the steroid binding domain of the estrogen receptor. J Biol Chem 267:1823–1828[Abstract/Free Full Text]
  39. Lefstin JA, Yamamoto KR 1998 Allosteric effects of DNA on transcriptional regulators. Nature 392:885–888[CrossRef][Medline]
  40. Nardulli AM, Greene GL, Shapiro DJ 1993 Human estrogen receptor bound to an estrogen response element bends DNA. Mol Endocrinol 7:331–340[Abstract/Free Full Text]
  41. Schwabe JW, Chapman L, Finch JT, Rhodes D 1993 The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567–578[CrossRef][Medline]
  42. Wood JR, Greene GL, Nardulli AM 1998 Estrogen response elements function as allosteric modulators of estrogen receptor conformation. Mol Cell Biol 18:1927–1934[Abstract/Free Full Text]
  43. Porter W, Saville D, Hoivik D, Safe S 1997 Functional synergy between the transcription factor Sp1 and the estrogen receptor. Mol Endocrinol 11:1569–1580[Abstract/Free Full Text]
  44. Wang F, Samudio I, Safe S 2001 Transcriptional activation of rat creatine kinase B by 17ß-estradiol in MCF-7 cells involves an estrogen responsive element and GC-rich sites. J Cell Biochem 84:156–172[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
J. G. Bromer, J. Wu, Y. Zhou, and H. S. Taylor
Hypermethylation of Homeobox A10 by in Utero Diethylstilbestrol Exposure: An Epigenetic Mechanism for Altered Developmental Programming
Endocrinology, July 1, 2009; 150(7): 3376 - 3382.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
S. Safe and K. Kim
Non-classical genomic estrogen receptor (ER)/specificity protein and ER/activating protein-1 signaling pathways
J. Mol. Endocrinol., November 1, 2008; 41(5): 263 - 275.
[Abstract] [Full Text] [PDF]

Home page
 Reproductive SciencesHome page
D. Vitiello, R. Pinard, and H. S. Taylor
Gene Expression Profiling Reveals Putative HOXA10 Downstream Targets in the Periimplantation Mouse Uterus
Reproductive Sciences, May 1, 2008; 15(5): 529 - 535.
[Abstract] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 Martin, R.
Right arrow Articles by Taylor, H. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martin, R.
Right arrow Articles by Taylor, H. S.
Related Collections
Right arrow Pediatric Endocrinology
Right arrow Female Endocrinology

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals