Direct Regulation of ß3-Integrin Subunit Gene Expression by HOXA10 in Endometrial Cells

Gaurang S. Daftary, Patrick J. Troy, Catherine N. Bagot, Steven L. Young and Hugh S. Taylor

Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520-8063

Address all correspondence and requests for reprints to: Hugh S. Taylor, 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
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen and progesterone regulate HOXA10 expression in the endometrium, where HOXA10 is necessary for implantation. The integrins are also involved in early embryo-endometrial interactions. Here we show that HOXA10 directly regulates ß3-integrin subunit expression in the endometrium, likely mediating the effect of sex steroids on ß3-integrin expression. ß3-Integrin expression was decreased in endometrium shown to have low HOXA10 expression. ß3-Integrin mRNA levels were increased in endometrial adenocarcinoma cells (Ishikawa) transfected with pcDNA3.1/HOXA10, and decreased in cells treated with HOXA10 antisense. Seven consensus HOXA10 binding sites were identified 5' of the ß3-integrin gene. Direct binding of HOXA10 protein to four sites was demonstrated by EMSA. Reporter gene expression increased in BT-20 cells cotransfected with pcDNA3.1/ HOXA10 and pGL3-promoter vector containing region F (encompassing all seven HOXA10 consensus sites). A 41-bp segment (Region A) showed highest affinity binding to HOXA10 protein. Increased reporter expression, equal in magnitude to that obtained with Region F, was obtained with Region A. HOXA10 protein binding within Region A was localized by deoxyribonuclease I footprinting. ß3-Integrin expression was directly up-regulated by HOXA10 through a 41-bp 5'-regulatory element. Sex steroids regulate the expression of endometrial ß3-integrin through a pathway involving HOXA10.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE HOMEOBOX GENE Hoxa10 is expressed in the murine endometrium and is essential for fertility in mice. Mice with a targeted disruption of the Hoxa10 gene are infertile due to defective embryo implantation (1). Embryos from these mice implant normally when transferred to the uteri of wild-type mice. Embryos from wild-type mice fail to implant in Hoxa10(-/-) mice (2). We have previously shown that altering Hoxa10 expression in the adult murine endometrium affects the number of implantation sites, demonstrating the necessity of maternal Hoxa10 (3). Murine uteri transfected with Hoxa10 antisense demonstrate decreased Hoxa10 expression, number of implantation sites, and litter size. Conversely, overexpression of Hoxa10 increases litter size. In the human, HOXA10 is expressed in the endometrial glands and stroma throughout the menstrual cycle (4). Estrogen and progesterone regulate HOXA10 expression in the endometrium (4). Expression rises dramatically at the time of implantation, suggesting a role for HOXA10 in this process in humans.

Estrogen and progesterone are necessary for endometrial development during the menstrual cycle. However, the molecular pathways and target genes through which the sex steroids regulate such diverse processes as growth, differentiation, and development of receptivity to embryo implantation have still not been characterized. HOXA10 is a transcription factor, known to be regulated by estrogen and progesterone. In turn, HOXA10 is likely to mediate a subset of sex steroid effects by activation or repression of downstream genes.

Although HOXA10 is essential to implantation, few direct targets of HOXA10 transcriptional regulation have been described. Embryo implantation involves interactions of the blastocyst with the endometrium. These interactions are likely mediated by cell adhesion molecules such as the integrins (5, 6). The integrins have well characterized expression patterns in the endometrium (7). They participate in cell-to-cell and cell-to-substratum interactions by anchoring extracellular proteins to intracellular cytoskeletal components. They are heterodimeric glycoproteins comprised of distinct {alpha}- and ß-subunits. The family of integrins includes the cell surface receptors for fibronectin, laminin, collagen, and vitronectin (5).

Here we investigated the possibility that a cell adhesion molecule, ß3-integrin, is a target of HOXA10 regulation. HOXA10 and ß3-integrin subunit are coexpressed in the endometrium at the time of embryonic implantation. Like other integrins, ß3-integrin subunit exhibits a cell-specific and menstrual cycle-specific pattern of expression in the endometrium (7). It appears on endometrial epithelial cells at the time of blastocyst implantation (7). The trophoblast also expresses the {alpha}vß3-integrin receptor at this time (8). These receptors contain the Arg-Gly-Asp (RGD) tripeptide as a ligand recognition sequence (9, 10). Osteopontin is a ligand of this receptor and has been found in abundance in the endometrium (11, 12). It has an RGD-dependent binding site and binds integrin receptors through RGD-dependent and -independent mechanisms. It has been proposed as a bridging molecule between the endometrium and trophoblast (11, 13). Estrogen and progesterone have not been shown to directly regulate ß3-integrin subunit expression in the endometrium. Here we identify and characterize a signal transduction pathway involving HOXA10 by which the sex steroids may mediate ß3-integrin subunit expression in periimplantation endometrium.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human Endometrium with Low HOXA10 mRNA Abundance Demonstrates Decreased ß3-Integrin Subunit Expression as Compared with Normal Endometrium
We have previously reported decreased HOXA10 mRNA levels in endometrium obtained from women with endometriosis as compared with HOXA10 mRNA levels in the endometrium of fertile controls (14). ß3-Integrin levels have also been shown to be decreased in the endometrium of a subset of patients with implantation defects (15). We investigated whether ß3-integrin subunit expression is decreased in endometrium with diminished HOXA10 expression. RNA isolated from periimplantation endometrium from women with endometriosis (E) or normal fertile controls (C) was analyzed by Northern blot using HOXA10 and ß3-integrin subunit riboprobes. Representative autoradiograms of Northern analyses (Fig. 1AGo) demonstrate the levels of ß3-integrin subunit mRNA from either HOXA10-deficient endometrium obtained from patients with endometriosis (E) or from fertile controls (C). Figure 1BGo shows HOXA10 and ß3-integrin expression normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH). A statistically significant 10-fold decrease (P < 0.001) in ß3-integrin subunit expression compared with controls was noted in HOXA10-deficient endometrium. The results are an average of 10 samples ± SEM.



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Figure 1. ß3-Integrin Subunit Expression Is Decreased in HOXA10-Deficient Human Endometrium

A, ß3-Integrin subunit mRNA levels are decreased in endometrium associated with implantation defects and low HOXA10 expression. Northern analysis was performed on endometrium from 10 subjects with endometriosis (E), previously shown to be associated with low HOXA10 expression and in 10 controls (C). The blots were probed with HOXA10 and ß3-integrin riboprobes. Representative samples are shown. G3PDH was used to control for loading. B, Northern blots were analyzed by densitometry and normalized to G3PDH expression. ß3-Integrin mRNA levels are decreased 10-fold in endometriosis (E) as compared with normal endometrium (C). (* Equals statistically significant difference from controls at P < 0.001 using t test.) The results are an average of 10 samples ± SEM.

 
HOXA10 Regulates ß3-Integrin Subunit Expression in Endometrial Cells
To determine whether ß3-integrin subunit expression is regulated by HOXA10, Ishikawa cells were treated with constructs designed to alter HOXA10 expression. Ishikawa cells are a well differentiated endometrial adenocarcinoma cell line that has been shown to express ERs and PRs (16, 17, 18, 19). HOXA10 and ß3-integrin subunit expression in this cell line has been well characterized (4, 7, 20, 21, 22). A 30-bp antisense phosphothiorate-modified oligodeoxyribonucleotide was designed complementary to the translation start site of the HOXA10 gene. A 25-cm2 cellular monolayer was transfected with either this antisense construct or with a control missense construct of the same length and nucleotide composition but in random order. As demonstrated in Fig. 2AGo (rows 1 and 2), when Northern analysis was performed with a ß3-integrin subunit probe, ß3-integrin expression was greatly reduced or eliminated in the HOXA10 antisense-treated cells (-) as compared with control missense-treated cells (C). Densitometric analysis of the Northern blot revealed that ß3-integrin subunit mRNA expression was decreased 3-fold in HOXA10 antisense-treated cells (-) as compared with controls (C) (Fig. 2BGo). The results are an average of four experiments.



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Figure 2. HOXA10 Regulates ß3-Integrin Subunit Expression in Endometrial Cells

A, Altering the expression of HOXA10 in Ishikawa cells causes corresponding alterations in ß3-integrin subunit mRNA expression. Ishikawa cells were transfected with a 30-bp antisense phosphothiorate-modified oligodeoxyribonucleotide complementary to the translation start site of the HOXA10 gene (lane -). Transfection with a missense construct of the same length and nucleotide composition but in random order was used as a control (lane C). Ishikawa Cells were also transfected with pCDNA3.1/HOXA10, which constitutively expresses HOXA10 cDNA (lane +). The total cellular RNA was extracted 48 h later and analyzed by Northern blot using HOXA10 and ß3-integrin subunit probes. The blots were probed with G3PDH to control for loading. ß3-Integrin subunit mRNA is decreased in cells with decreased HOXA10 expression and increased in cells with increased HOXA10 expression (rows 1 and 2). We have previously shown and confirmed here by Western blot that treatment with this HOXA10 antisense construct decreases HOXA10 protein levels by approximately 50%, and treatment with pcDNA3.1/HOXA10 increases HOXA10 protein levels (row 3) (3 ). B, Densitometric analysis of the northern Blot revealed that ß3-integrin subunit mRNA expression normalized to G3PDH is decreased 3-fold in HOXA10 antisense-treated cells (-) as compared with controls (C). Likewise ß3-integrin mRNA levels are increased 8-fold in cells where HOXA10 is overexpressed (+). (* Equals statistically significant difference from control at P < 0.001 using t test.) The results are an average of four experiments ± SEM.

 
Ishikawa cells were then transfected with pCDNA3.1/ HOXA10, which constitutively expresses HOXA10 cDNA. Increased HOXA10 levels led to increased ß3-integrin subunit mRNA levels (+) as compared with control missense-treated cells (C) (Fig. 2AGo, rows 1 and 2). Figure 2BGo reveals densitometric analysis of four independent experiments. ß3-Integrin mRNA levels were increased 8-fold in cells where HOXA10 is overexpressed (+).

We have previously shown that the HOXA10 antisense construct decreases HOXA10 protein levels by approximately 50% (3). In addition, transfection with pcDNA3.1/HOXA10, which constitutively expresses HOXA10, led to dramatically increased HOXA10 protein levels (3). Here we confirmed the effects of these constructs on HOXA10 protein expression by Western analysis (Fig. 2AGo, rows 3 and 4).

The region 5' of the ß3-Integrin Gene Contains Seven Putative HOXA10 Binding Sites
To determine the molecular mechanism by which HOXA10 regulates ß3-integrin subunit expression, the region -2,038 to -147, 5' of the ß3-integrin subunit gene transcription initiation site, was assessed for the presence of potential HOXA10 binding sites. Seven consensus Abd-B type HOXA10 binding sites (TTAT) were identified (Fig. 3Go) (23). These seven consensus sites were localized to five regions termed A to E. Region F encompasses regions A–E. Oligonucleotides (A–F) corresponding to regions A–F along with flanking base pairs were selected. The sites were evaluated for HOXA10 protein binding and ability to drive reporter gene expression.



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Figure 3. The Region 5' of the ß3-Integrin Gene Contains Seven Putative HOXA10 Binding Sites

A schematic representation of the region -2,038 to -147 of the transcription start site of the ß3-integrin subunit gene is shown. Regions A–F represent oligonucleotide sequences containing consensus HOXA10 binding sites. These oligonucleotides were used in EMSA and reporter constructs. Oligonucleotides A–F corresponding to Regions A–F are displayed. These oligonucleotides contain one (B, C, and E), two (A and D), or seven (F) putative HOXA10 binding sites as underlined.

 
HOXA10 Directly Binds to a 41-bp Region That Contains Two Consensus Binding Sites
To evaluate HOXA10 binding to the consensus HOXA10 binding sites identified above, EMSAs were performed. A Flag-HOXA10 construct was designed that substituted the Flag tag for five N-terminal amino acids of HOXA10. Flag-HOXA10 was cloned into pcDNA 3.1 and transfected into Ishikawa cells. Flag-HOXA10 protein was isolated and used for EMSA. Each oligonucleotide containing a putative binding site was tested for ability to bind Flag-HOXA10 protein. No shift was obtained when 32P-labeled oligonucleotide A was loaded in the absence of HOXA10 protein (Fig. 4Go, lane 1). When Flag-HOXA10 protein was loaded along with 32P-labeled oligonucleotides from the ß3-integrin subunit regulatory region, a shift was obtained with oligonucleotides A (lanes 2, 3, and 4) and D (lane 5) as demonstrated in Fig. 4Go. The relative binding affinity of Flag-HOXA10 protein is greater to Region A (lanes 2, 3, and 4) than to Region D (lane 5). Region A demonstrated a dose-responsive increase in the intensity of the shifted band with serially increasing concentrations of Flag-HOXA10 protein (lanes 2, 3, and 4). Binding to labeled oligonucleotide A was inhibited by the addition of excess (160 molar fold) unlabeled oligonucleotide A (lane 6). Minimal or no binding was demonstrated to the other putative HOXA10 binding sites (Regions B, C, and E; data not shown). A supershift was obtained when polyclonal HOXA10 antibody was used along with Flag-HOXA10 and labeled oligonucleotide A (lane 7). Binding of Flag-HOXA10 to region A was not diminished when excess unlabeled oligonucleotide without a HOXA10 binding site was added (data not shown). The binding of Flag-HOXA10 protein to either one or both binding sites within the oligonucleotides A and D may result in multiple shifts, as seen in Fig. 4Go (lanes 2–5). It is likely that there are differences in the binding affinities of the two HOXA10 binding sites, such that at lower concentrations of added protein, preferential binding occurs to the higher affinity binding site with minimal or no binding to the other site. At high concentration, both consensus HOXA10 sites are bound with protein, resulting in a prominent lower mobility shift.



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Figure 4. HOXA10 Directly Binds to a 41-bp Region That Contains Two Consensus Binding Sites

EMSAs were performed to evaluate HOXA10 protein binding to the consensus HOXA10 binding sites located in the region -2,038 to -147 of the ß3-integrin subunit gene. Ishikawa cells were transfected with a Flag-pcDNA3.1/HOXA10 construct. Flag-HOXA10 protein was isolated and used for EMSA. For EMSA, Flag-HOXA10 was added to 32P-labeled oligonucleotides from the ß3-integrin subunit regulatory element containing putative HOXA10 binding sites. Lane 1 contains 32P-labeled oligonucleotide with no Flag-HOXA10 protein. Lanes 2, 3, and 4 contain end-labeled oligonucleotide A with serially increasing concentrations of Flag-HOXA10 protein and demonstrate two specific shifted bands. Lane 5 contains end-labeled oligonucleotide D with Flag-HOXA10 protein at maximal concentration as used with oligonucleotide A. Two specific shifts of lower intensity are obtained. Lane 6 demonstrates competitive assay with 160 molar-fold excess unlabeled oligonucleotide A. In lane 7 a supershift was obtained when polyclonal HOXA10 antibody was added to Flag-HOXA10 protein and labeled oligonucleotide A. Minimal or no binding was demonstrated to the other putative HOXA10 binding sites (Regions B, C, and E; data not shown). The arrows indicate specific shifts, NS indicates nonspecific binding, and * indicates the supershift.

 
HOXA10 Activates Reporter Gene Expression via a 41-bp ß3-Integrin Subunit Element
To determine whether HOXA10 directly activates ß3-integrin subunit gene expression, BT-20 cells were transfected with artificial promoter constructs containing Regions A and F identified above. BT-20 is a breast adenocarcinoma cell line that has been demonstrated not to express HOXA10 by RT-PCR (24). The cells were cotransfected with either pcDNA3.1 or pcDNA3.1/HOXA10 (which constituitively expresses HOXA10 cDNA). Additionally the cells were cotransfected with pcDNA3.1/Lac-Z as a control for transfection efficiency. Luciferase and ß-galactosidase activity was measured in the cellular lysate. Luciferase activity was normalized to ß-galactosidase activity. Empty pGL3-promoter plasmid showed no change in luciferase activity in the presence of HOXA10 (Fig. 5Go). Luciferase expression was increased 5-fold when Region F (ß3F) containing all seven consensus HOXA10 binding sites was cotransfected with HOXA10. An equivalent 5-fold increase in expression was obtained when Region A (ß3A) was used. The results of reporter gene activation were consistent with our finding of low HOXA10 binding to Region D (Figs. 3Go and 4Go) relative to Region A, and minimal to no binding to Regions B, C, and E (Fig. 3Go and data not shown) as described above.



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Figure 5. HOXA10 Activates Reporter Gene Expression via a 41-bp ß3-Integrin Subunit Element

Expression of HOXA10 in BT-20 cell is used to drive reporter gene expression. BT-20 is a breast adenocarcinoma cell line that has been demonstrated not to express HOXA10 by RT-PCR. BT-20 cells were cotransfected with artificial promoter constructs containing Regions A and F identified above along with either pcDNA3.1 or the HOXA10 expression vector pcDNA3.1/HOXA10. Additionally, the cells were cotransfected with pcDNA3.1/Lac-Z as a control for transfection efficiency. Luciferase and ß-galactosidase activity was measured in the cellular lysate 48 h after transfection. Luciferase activity was normalized to ß-galactosidase activity. pGL3 plasmid without the ß3-integrin subunit gene inserts showed no increased expression in the presence of HOXA10. Luciferase expression was increased 5-fold when the reporter construct containing Region F was cotransfected with HOXA10 expression vector as compared with the empty pcDNA3.1 expression vector. An equivalent 5-fold increase in expression was obtained when Region A was used. The results of luciferase assays are the average of eight experiments ± SEM. *, Statistically significant difference from control at P < 0.001 for Region A and P = 0.002 for Region F using Mann-Whitney Rank Sum Test. Luciferase expression was not significantly different for Regions A and F, when cotransfected with HOXA10 expression vector.

 
HOXA10 Binding Is Localized to One Consensus HOXA10 Binding Sites Located 5' of the ß3-Integrin Subunit Gene
To localize HOXA10 transcription factor binding within the 41-bp element, region A, deoxyribonuclease I (DNase I) footprinting was performed. This 41-bp region of the ß3-integrin gene was footprinted using FLAG-HOXA10 protein. HOXA10 binding was confined to a 7-bp region that contained one of the HOXA10 consensus sites. Figure 6Go demonstrates the results of the footprinting analysis and oligonucleotide A along with the 7-bp protected fragment.



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Figure 6. HOXA10 Binding Is Localized to One Consensus HOXA10 Binding Site Located 5' of the ß3-Integrin Subunit Gene

Using DNase I footprinting analysis, HOXA10 transcription factor binding within Region A was localized to a 7-bp region that contained one of the HOXA10 consensus sites. The solid bar on the right indicates the extent of the footprint. No Flag-HOXA10 protein was added to the lanes labeled 0. The triangle above indicates serially increasing Flag-HOXA10 protein concentrations. The oligonucleotide sequence containing the nucleotides protected by DNase I footprinting is displayed. The footprinted sequence is underlined.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The role of Hoxa10 has been demonstrated in the developing paramesonephric duct, where it is necessary for proper uterine development (21, 25). In the adult, Hoxa10 continues to be expressed in the uterus (25). The necessary role of Hoxa10 in reproduction has been demonstrated by defective implantation of embryos in Hoxa10(-/-) mice (1, 2). In the adult human, HOXA10 expression is regulated by sex steroids and demonstrates a dynamic expression pattern in the uterine endometrium (4). HOXA10 is expressed at low levels in the proliferative phase of the menstrual cycle but rapidly increases to high levels in the midsecretory phase. In primary endometrial cell cultures, estrogen increases expression of HOXA10 mRNA 2-fold. Progesterone increases HOXA10 mRNA expression in a dose-dependent manner inducing a 25-fold elevation when the progesterone concentration is increased from 10- 9 to 10-6 M. When treated with both estrogen and progesterone, HOXA10 mRNA expression in endometrial cells is increased further over treatment with either estrogen or progesterone alone. This pattern of HOXA10 mRNA expression in response to sex steroids is consistent with the finding that levels of HOXA10 in the endometrium increase during the secretory phase of the menstrual cycle and reach high levels in the midsecretory phase in association with peak progesterone levels. The up-regulation of HOXA10 expression by progesterone was blocked with RU486, further demonstrating the role of progesterone and its receptor in regulating HOXA10 expression (4). Although HOXA10 encodes a transcription factor, the target genes activated by HOXA10 are still poorly characterized, and none have been identified in the endometrium. Here we identify ß3-integrin subunit as the first known target of HOX gene regulation in the endometrium and describe its regulation by HOXA10.

ß3-Integrin is a subunit of the vitronectin receptor {alpha}vß3. These receptors contain the Arg-Gly-Asp (RGD) tripeptide as a ligand recognition sequence (9, 10). Osteopontin is a ligand of this receptor and has been found in abundance in the endometrium (11). It has an RGD-dependent binding site and binds integrin receptors through RGD-dependent and independent mechanisms. It has been proposed as a bridging molecule between the endometrium and trophoblast (11, 12, 13). Immunohistochemical analysis of the endometrium revealed that {alpha}v-subunit expression increased throughout the menstrual cycle, whereas ß3-integrin subunit appeared on endometrial epithelial cells only after cycle day 20 (7). Estrogen does not regulate ß3-integrin subunit expression as ß3-integrin subunit is absent in proliferative phase endometrium. In the mid-late secretory phase, the number of PRs in the endometrial epithelium decline. ß3-Integrin subunit expression is therefore not regulated by progesterone and indeed has been demonstrated not to be directly regulated by sex steroids (7).

Given the importance of both HOXA10 and the ß3-integrin subunit in implantation and their spatial and temporal coexpression pattern, we investigated the possibility that ß3 integrin subunit is a target gene of HOXA10. We determined that ß3-integrin subunit expression was decreased in HOXA10-deficient endometrium associated with implantation defects. ß3- Integrin subunit mRNA was increased when HOXA10 was overexpressed in endometrial cells and was decreased when HOXA10 expression was diminished using HOXA10 antisense. Analysis of the region 5' of the transcription start site of the ß3-integrin gene revealed seven consensus Abd-B type HOX binding sites (TTAT) (23). HOXA10 protein directly bound sites located within this region. We identified a 41-bp element that drove reporter gene expression in response to HOXA10. This element contained two putative HOXA10 binding sites. DNase I footprinting localized the binding of HOXA10 protein within this region.

The traditional model for steroid-mediated gene transactivation is through the combination of a ligand-steroid receptor complex with a steroid response element. The binding of this transcription complex to DNA leads to activation or repression of target genes. Although ß3-integrin exhibits a sex steroid-responsive expression pattern in the endometrial epithelium in vivo, it is not a direct target of sex steroid-mediated transactivation (7). Here we describe a novel pathway of sex-steroid signal transduction in the endometrium that involves an intermediary transcription factor, HOXA10. We postulate that in the absence of direct regulation, sex steroids may mediate endometrial ß3-integrin subunit expression via HOXA10.

Although sex steroids up-regulate epithelial HOXA10 expression, in the mid-late secretory phase when HOXA10 expression is at high levels, there is a concomitant decline in epithelial cell PR numbers. It is likely that one of the mechanisms by which sex steroids contribute to HOXA10 expression in epithelial cells is via stromal-epithelial interactions. Inductive interactions between apposed cell layers have been demonstrated in morphogenetic experiments (26). In studies using heterotypic recombinants, epithelial cell patterning has been demonstrated to be dependent on mesenchymal factors secreted by adjacent stromal cells (27). Likewise, graded Hox gene expression in response to a morphogen-secreting adjacent cell type has been demonstrated in transplantation experiments (28). Stromal-epithelial interactions have been demonstrated to mediate steroid action in the endometrium (29, 30, 31). It is likely that paracrine regulation is responsible for maintenance of epithelial HOXA10 expression in the late secretory phase. Persistent high levels of expression of HOXA10 in the epithelium in turn directly up-regulate ß3-integrin expression on the epithelial cell surface despite diminished PR concentration.

Defective expression of either HOXA10 or ß3-integrin subunit have been previously shown to diminish implantation in both murine and human models. Decreased HOXA10 levels in murine endometrium treated with HOXA10 antisense leads to diminished litter size (3). Implantation in the mouse has also been successfully prevented with the use of a ß3-integrin subunit antibody (32). In women with infertility due to implantation defects, such as in endometriosis, both HOXA10 and ß3-integrin subunit expression are decreased (14, 15). Both these molecules are temporally coexpressed in the endometrial epithelium. Their similar expression pattern and participation in the same functional process may be explained by the molecular relationship demonstrated here.

The precise mechanism by which HOX genes activate or repress target genes is not known. Despite the diverse phenotypic effects encoded by individual HOX genes, their DNA binding specificities are similar (33). Subtle differences in DNA binding affinity between Hox genes have been observed (34, 35). Binding differences may confer functional specificity to certain HOX-DNA interactions. Consistent with this observation, we found selective HOXA10 protein binding to, at most, four of seven consensus binding sites in the region -2,038 to -147, 5' of the transcription start site of the ß3-integrin subunit gene. A 41-bp region containing two of the four HOXA10 binding sites was found to drive reporter gene expression and likely mediates the regulation of the ß3-integrin subunit gene by HOXA10. Functional activity, as measured by cellular reporter gene expression, may result from differences in binding specificity or from binding of additional cofactors. However, in this system, relative binding affinity predicted functionality. Taken together, these data support differences in DNA binding specificity as determinants of homeotic specificity.

In summary, we have identified the first known target gene of HOXA10 in the uterus. We have proposed a molecular mechanism by which sex steroid signaling leads to regulation of ß3-integrin expression, through HOXA10 as an intermediary. We have described the characteristics of the regulatory element by which HOXA10 regulates ß3-integrin. It will be interesting to determine whether other molecules involved in the implantation process are regulated by HOXA10 and to further characterize their uterine-specific HOXA10 response elements.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Ishikawa cells were a generous gift of R. Hochberg. BT-20 cells were a generous gift of B. Kaczinski. Ishikawa cells were maintained in MEM (Life Technologies, Inc., Gaithersburg, MD) with 2.0 mM L-glutamine and Earl’s salts supplemented with 10% FBS, 1% sodium pyruvate, and 1% penicillin/streptomycin. BT-20 cells were maintained in RPMI 1640 (Life Technologies, Inc.) with 2.0 mM L-glutamine and Earl’s salts supplemented with 10% FBS, 1% sodium pyruvate, and 1% penicillin/streptomycin.

Tissue Collection
Endometrium was collected on menstrual cycle day 23–24 from 10 women with a histological diagnosis of endometriosis and from 10 fertile controls at the time of tubal ligation under an institutionally approved Human Investigations Committee protocol. All women were in the reproductive age group (age 25–35 yr) and none were receiving hormonal medications. In the study group, surgical staging had revealed early-stage endometriosis (stages I or II) according to the revised American Society of Reproductive Medicine criteria (36). We have previously demonstrated diminished HOXA10 mRNA expression in the endometrium of patients with endometriosis (14). The tissue was immediately placed in liquid nitrogen and stored at -72 C. Menstrual cycle dating was determined from menstrual cycle history and confirmed histologically using established criteria (37).

Northern Blot Analysis
RNA from tissue or cells was isolated using Trizol (Life Technologies, Inc.) pursuant to the manufacturers guidelines. Total RNA (40–50 µg) was size fractionated on 1% agarose/0.66 M formaldehyde gel and transferred to nylon membranes. The membrane was hybridized to a riboprobe complementary to the 3'-untranslated region of the ß3-integrin subunit gene. A 101-bp region of the ß3 integrin subunit gene was amplified by PCR. The product was phenol/chloroform purified, ethanol precipitated, and cloned into the SrfI site in PCR Script-SK (+) plasmid (Stratagene, La Jolla, CA). The vector was linearized with EcoRI, ethanol precipitated, and used as a template for riboprobe synthesis. RNA probes were generated by in vitro transcription using T3 polymerase (Promega Corp., Madison, WI).

HOXA10 Fusion Protein and in Vitro Translation
HOXA10 cDNA was a gift of C. Largman. The fusion protein was constructed by cloning HOXA10 cDNA into the EcoRI site of pcDNA3.1 (+) plasmid (Invitrogen). Subsequently, oligodeoxynucleotides encoding the FLAG tag were synthesized, annealed, and cloned in frame into an NheI site in the polylinker immediately 5' to the pcDNA3.1-HOXA10.FLAG Tag sense sequence: 5'-TCTGCTAGCCCCATGGACTACGAAGGACGACG ATGACAACGATCTCCCGCCCGCGCTAGCCTCCTCT-3'. Ishikawa cells were grown to 70% confluence in 25-cm2 flasks and transfected for 5 h with 6 µg of HOXA10FLAG-pcDNA3.1 plasmid using Lipofectamine (Life Technologies, Inc.). After transfection, cells were washed with PBS and allowed to grow for an additional 72 h. HOXA10-FLAG protein was isolated using an Anti-Flag M1 Affinity Gel (Sigma, St. Louis, MO).

EMSAs
Complementary single-stranded oligodeoxynucleotides were synthesized and annealed to incorporate putative HOXA10 binding sites and flanking sequences located in the region -2,038 to -147, 5' of the transcription start site of the ß3-integrin subunit gene (Fig. 3Go), GenBank accession no. AF020552. Sequences of oligonucleotides A–E are as follows: Oligonucleotide A (-1,973 to -1,933), 5'-GGGGGGGCTTATAATGTTATTTTTAGTTTACAG GTTCTTAC-3'; Oligonucleotide B (-1,774 to -1,739), 5'-AAAATTATCCCAAAAGAAAGCA GAAAAAGAGAACA-3'; Oligonucleotide C (-1,569 to -1,608), 5'-GCACAATTATGTCATGTTTGCTAGGGCTT GGGCTAGGGTT-3'; Oligonucleotide D (-1,439 to -1,478), 5'-TTGGTTTTATATAT ATGGGAATGGAGCAGATAACTAAATA-3'; Oligonucleotide E (-859 to -900), 5'-CCTTG TTTTTATCACCATCAGGACTACCCATTGAGGCAGG-3'. Complementary oligonucleotides were annealed and end labeled with 32P-dATP (Amersham Pharmacia Biotech, Arlington Heights, IL) using T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA) and purified with Quick Spin columns (Roche Molecular Biochemicals, Indianapolis, IN). Binding reactions were performed as previously described (38). Briefly, 0.05(+), 0.1(++), or 1.0(+++) µg of HOXA10FLAG purified recombinant HOXA10 protein 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 were incubated on ice for 30 min. Samples were fractionated for 3 h at 200 V in a 4% nondenaturing polyacrylamide gel containing 1x Tris-borate-EDTA at 4 C. The gel was dried under vacuum at 80 C for 45 min and exposed overnight on X-OMAT film (Eastman Kodak Co., Rochester, NY) and subsequently developed.

Reporter Constructs and Expression Plasmids
Oligonucleotide A described above and oligonucleotide F, inclusive of all seven consensus HOXA10 binding sites (-1,973 to -859, GenBank accession no. AF020552), 5' of the transcription start site of ß3-integrin subunit gene, were annealed and cloned in pGL3 reporter constructs (Promega Corp.). The identity of inserts was confirmed by sequencing.

Transfection and Luciferase Assays
BT-20 cells were grown to 75–80% confluence in 25-cm2 flasks and transiently transfected with the appropriate plasmid using Lipofectamine (Life Technologies, Inc.), a mixture of liposomes consisting of a 3:1 (wt/wt) formulation of 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N-N-dimethyl-1-propanium trifluoroacetate and dioleoylphosphatidyl ethanolamine. The cells were transfected with 4 µg of either pcDNA3.1-HOXA10 or pcDNA3.1 plasmid. The cells were also cotransfected with 4 µg of a pGL3-ß3-integrin subunit reporter construct, or empty pGL3 plasmid. Additionally, 4 µg of pcDNA3.1/LacZ were used as an internal control for transfection efficiency. After 5 h, the cells were washed and allowed to grow for an additional 48 h. The cells were washed with cold PBS and lysed with 1x Reporter Lysis Buffer (Promega Corp.), and the lysates was 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 determined using the Luciferase Assay Kit (Promega Corp.) and luminometer. ß-Galactosidase activity was determined using the ß-Galactosidase kit (Tropix, Inc., Bedford, MA) and luminometer. ß-Galactosidase was used to normalize luciferase values.

DNase I Footprinting
DNase I footprinting experiments were carried out as described previously except that 7 U DNase were used per reaction (39). FLAG-HOXA10 protein was produced in Ishikawa cells and purified as described above.

Western Blot Analysis
Ishikawa cells, transfected with a DNA/liposome complex containing either HOXA10 antisense oligonucleotide or pcDNA3.1(+)/HOXA10, were lysed in single detergent lysis buffer and centrifuged at 12,000 x g at 4 C for 2 min. The supernatant was loaded on to a 6% SDS polyacrylamide gel, size fractioned, and transferred to a nitrocellulose membrane. The membrane was immersed in a 3% gelatin-Tris buffered saline (TBS) (20 mM Tris, 500 mM NaCl) blocking solution for 30 min at room temperature, washed for 10 min in TBST (20 mM Tris, 500 mM NaCl, 0.05% Tween-20, pH 7.5) and incubated for 1 h with a 1:1,000 dilution of HOXA10 polyclonal antibody (BabCo, Richmond, CA). The membrane was washed with TBS for 5 min at room temperature and incubated for 1 h with a 1:200 dilution of goat antimouse IgG-horseradish peroxidase (Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was then washed in TBS (2x) for 5 min at room temperature and immersed in a horseradish peroxidase color developer buffer (Bio-Rad Laboratories, Inc.) for 30 min. Photographs were taken immediately.


    ACKNOWLEDGMENTS
 


    FOOTNOTES
 
Abbreviations: DNase, Deoxyribonuclease; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; TBS, Tris-buffered saline.

Received for publication May 17, 2001. Accepted for publication November 27, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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