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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 2 720-729
Copyright © 2003 by The Endocrine Society

Differential Expression of ß1c Integrin Messenger Ribonucleic Acid and Protein Levels in Human Endometrium and Decidua during the Menstrual Cycle and Pregnancy

Rosa A. Vacca, Ersilia Marra, Giuseppe Loverro, Eugenio Maiorano, Anna Napoli, Mariarosaria Lovecchio, Luigi Selvaggi and Elda Perlino

Center of Study on Mitochondria and Energy Metabolism (R.A.V., E.Mar., M.L., E.P.), Consiglio Nazionale delle Ricerche Bari, and Departments of Obstetrics and Gynecology (G.L., L.S.) and Pathological Anatomy and Genetics (E.Mai., A.N.), University of Bari School of Medicine, I-70126 Bari, Italy

Address all correspondence and requests for reprints to: Dr. Rosa Anna Vacca, Center of Study on Mitochondria and Energy Metabolism, CNR Bari, Via Amendola 165/A, I-70126 Bari, Italy. E-mail: csmmrv09{at}area.ba.cnr.it.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
ß1C and ß1A integrins are alternatively spliced variants of the human ß1-subunit; the former has been shown to inhibit cell proliferation, and the latter to promote it. Although some components of the ß1 integrin subfamily are expressed in human endometrial and decidual cells during the menstrual cycle and early pregnancy, to date no information is available about the expression of ß1C integrin in endometrial and decidual tissues and its possible roles during implantation and pregnancy. To gain further insight on this subject, we have explored ß1C integrin expression in endometrial (proliferative, secretory, and atrophic) and decidual (from the first and third trimesters of pregnancy) tissue samples at both gene and protein levels by Northern and Western blotting analyses and by immunohistochemistry. ß1A protein levels were also measured in the same tissues as a control. The results of this study demonstrate that both ß1C- and ß1A-subunits are expressed in the endometrium and decidua. In the former, maximal ß1C expression was detected in atrophic endometria, whereas ß1A expression levels were increased in secretive and decreased in atrophic endometrial tissues compared with proliferative endometria. In addition, whereas ß1A levels were significantly increased in decidual tissues, compared with proliferative endometria, ß1C expression was dramatically reduced in the same tissues, thus pointing to selective down-regulation of ß1C expression in the decidua.

These data suggest that the expression of ß1C integrin, a very efficient inhibitor of cell proliferation, may be modulated by the maternal microenvironment and may play a fundamental role in mediating trophoblast outgrowth and migration during pregnancy.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THE HUMAN ENDOMETRIUM undergoes remarkable changes throughout the menstrual cycle in response to circulating sex steroids. The estrogen-dominant proliferative phase is characterized by intense mitotic activity of both epithelial and stromal cells, whereas during the progesterone-dominant secretory phase functional differentiation takes place that prepares the endometrial compartment for embryonic implantation; this persists and becomes much more extensive if pregnancy occurs. The endometrial changes at the time of implantation, leading to decidualization, mainly consist of modifications of both epithelial and stromal cells, which became large and polygonal and provide a hospitable environment for subsequent implantation of the blastocyst (1, 2). During human implantation, a large number of trophoblastic cells infiltrate through the decidua, and the maternal microenvironment is believed to regulate trophoblastic migration, thus ensuring adequate migration but preventing overinvasion of the decidua (3).

Extracellular matrix (ECM) proteins such as fibronectin, vitronectin, collagen, and laminin, produced by endometrial stromal or decidual cells, are important for endometrial structure and integrity and for trophoblastic attachment (4, 5, 6). Therefore, endometrial and decidual cells should be able to interact with ECM proteins by means of adequate cellular receptors.

The integrin superfamily represents the major class of receptors that mediates cell-cell and cell-ECM interactions and plays a fundamental role in the regulation of gene expression, cell proliferation, and differentiation. These receptors link to the ECM proteins and transduce mechanical and informational signals from the complex extracellular environment into the cell, incrementing the activation of cellular transduction pathways after binding with soluble mediators, cytokines, and growth factors (7, 8). Integrins are heterodimeric transmembrane glycoproteins, consisting of noncovalently associated {alpha}- and ß-subunits. Both subunits have a large extracellular domain, a transmembrane segment, and a short cytoplasmic domain. The role of the integrin cytoplasmic domain in modulating integrin functions and signaling events is well established (9). The ligand specificity of any heterodimer is determined by the specific combination of different {alpha}- and ß-subunits. Diversity within the family of integrins is generated not only by the large number of {alpha}- and ß-subunits, but also by the existence of alternatively spliced variants of the cytoplasmic domain for some {alpha}- and ßsubunits (10, 11).

An alternatively spliced variant of the ß1 integrin subunit is named ß1C. ß1C integrin is generated by the presence of an unspliced sequence that causes a frameshift in the wild-type 3'-end of the ß1 integrin subunit and codes for a unique 48-amino acid carboxyl-terminal sequence in the cytoplasmic domain (10, 11, 12). In contrast with its wild-type counterpart, ß1A, ß1C expression inhibits cell proliferation and causes growth arrest at the late G1 phase of the cell cycle (13, 14, 15). Furthermore, ß1C integrin expression is selectively reduced in prostate (16, 17), breast (18), and pulmonary (19) cancer, and its down-regulation correlates with acquisition of the neoplastic phenotype.

During implantation and pregnancy, trophoblastic cells invade the decidua, thus simulating, to some extent, the process of stromal invasion by malignant cells (20, 21, 22). This is a complexly regulated process, and to date no data are available on the expression of ß1C integrin in human endometrial and decidual tissues or on its possible roles during implantation and pregnancy. It has been demonstrated that human endometrial and decidual cells do express ß1 integrin on their surfaces and that this expression is a dynamic process throughout the menstrual cycle (23, 24, 25). In addition, ß1 integrin expression in the human endometrium increases after implantation and remains high in the decidua during early pregnancy (26). However, the role of ß1 integrin variants in human decidua during early and late pregnancy remains to be clarified.

The current study was aimed at investigating the expression of ß1 integrin variants at both gene and protein levels by Northern and immunoblotting analyses and by immunohistochemistry in both endometrial (proliferative, secretory, and atrophic) tissues and decidua from first and third trimesters of pregnancy, as well as describing their cellular localization and postulating their possible roles in the proliferation and differentiation of both endometrial and decidual tissues.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients

This study was performed on 43 tissue samples obtained from women hospitalized at the Department of Obstetrics and Gynecology of the University of Bari School of Medicine during the years 1998–2000. Informed consent was obtained from all patients for inclusion in this study, and all materials were handled in agreement with the international regulations for the treatment of human subjects.

The patients were divided into three groups. Group 1 included 18 normally menstruating women (mean age, 46 yr; range, 38–55 yr) with large subserous or infraligmentary leiomyomas abutting toward the peritoneal cavity. These women underwent hysteroscopy with microbiopsy to exclude the presence of relevant endometrial lesions and, subsequently, simple hysterectomy. None had received hormonal therapy before surgery. These tissues, which have been considered normal in our previous studies (27, 28, 29), are far less prone to interfere with endometrial functions. Among these samples, 11 were proliferative endometria, and 7 were secretory endometria.

Group 2 included six postmenopausal women (mean age, 52 yr; range, 48–56 yr; median postmenopausal age, 4 yr) who underwent hysterectomy with bilateral salpingo-oophorectomy for uterine prolapse. In these women previous hysteroscopy with microbiopsy had excluded relevant endometrial lesions. All samples from this group showed histological features of endometrial atrophy.

Group 3 included 7 women who underwent pregnancy termination during the first trimester of pregnancy (6–12 wk gestation) and 12 women at the third trimester of pregnancy (37–40 wk gestation) who underwent cesarean section. The mean age of these women was 31 yr (range, 20–39 yr). The specimens of decidua basalis were obtained at the time of pregnancy termination by Novak curette; cesarean section biopsy was performed on decidua basalis using small scissors.

Tissue processing

Soon after surgical removal, a representative sample of endometrial and decidual tissue was taken from the specimens, snap-frozen, and cryopreserved in liquid nitrogen for RNA and protein extraction. The remaining samples were fixed in 10% neutral buffered formalin for 12–24 h, embedded in paraffin, cut, and stained with hematoxylin-eosin.

One sample of histologically proven normal human liver, obtained during cholecystectomy, was also included in this study and submitted to mRNA extraction to serve as a negative control, whereas the HL60 promyelocytic cell line served as a positive control (10).

RNA extraction and Northern analysis

The frozen tissue samples were pulverized to fine powder, and cellular RNA was extracted using the guanidinium isothiocyanate-cesium chloride procedure (30). Total RNA (25 µg) isolated from the tissues was electrophoresed through 1% denaturing agarose gel containing 660 mmol/liter formaldehyde and transferred (31) to nylon membrane (Hybond N+, Amersham Pharmacia Biotech Italy Srl, Milan, Italy). The RNA-containing filters were subsequently prehybridized overnight at 42 C with a buffer consisting of 50% formamide, 5x Denhardt’s solution (1% Ficoll 400, 1% polyvinylpyrrolidone, and 1% BSA), 5x SSPE [3 mol/liter sodium chloride, 200 mmol/liter sodium phosphate (pH 7.0), and 19 mmol/liter EDTA], 0.5% sodium dodecyl sulfate, and 100 µg/ml sonicated salmon sperm DNA. The RNA blots were then hybridized for 20 h at 42 C in the same mixture with 3 x 106 cpm labeled probe/ml hybridization solution.

The radiolabeled probe was generated using the Megaprime DNA labeling kit (Amersham Pharmacia Biotech Italy Srl), 5 µl [{alpha}-32P]deoxy-CTP (3000 Ci/mmol; Amersham Pharmacia Biotech Italy Srl) (32) and 25 ng double-stranded 116-bp fragment specific for the ß1C integrin from the cDNA clone of human ß1C (17) or a full-length human ß1 cDNA that recognizes all ß1 variants (10). The specific ß1C cytoplasmic cDNA fragment representing the 116-bp ß1C insertion (nucleotides 2435–2551) was generated by PCR from the pBS-ß1C plasmid and cloned in the pBluescript vector (17).

The blots were washed once with 2x SSPE/0.1% sodium dodecyl sulfate for 10 min at room temperature and then with 1x SSPE, 0.1% sodium dodecyl sulfate at 42 C and up to 0.1x SSPE, 0.1% sodium dodecyl sulfate at 65 C, and finally exposed at -80 C overnight or longer on Kodak X-OMAT AR 5 film (Eastman Kodak Co., Rochester, NY).

mRNA levels were normalized with a constitutively expressed gene such as ribosomal 28S RNA (33). For this purpose the blots were stripped in 0.1% boiling sodium dodecyl sulfate and reprobed with the radiolabeled 28S cDNA probe. Quantitative analysis was performed by densitometric scanning of the autoradiographs using a GS-700 densitometer (Bio-Rad Laboratories, Inc., Richmond, CA); multiple expressions of the same Northern blots in a linear range were performed. The ratio of either the ß1C mRNA or the ß1 mRNA band intensity over the 28S band intensity was calculated for each sample to take into account differences in RNA loading. ß1C and ß1 RNA levels were calculated as a percentage of the control (proliferative endometrium), taken as 100 in arbitrary units. The mean value (±SEM) of the results obtained in at least three experiments was calculated for each specimen.

Immunoblotting

Both endometrial and decidual frozen tissue specimens were homogenized in lysis buffer containing 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 50 mmol/liter Tris-HCl (pH 7.5), 150 mmol/liter NaCl, 200 mmol/liter LiCl, 5 mmol/liter EDTA, 10% glycerol, 10 µg/ml aprotinin, 120 µg/ml leupeptin, and 170 µg/ml phenylmethylsulfonylfluoride. The homogenate was sonicated for 20 sec, then centrifuged for 30 min at 14,000 x g at 4 C. 2-Mercaptoethanol (1%) was added to each lysate for 30 min at 4 C to further solubilize potentially cross-linked molecules; it was then centrifuged, and supernatants were collected. One hundred and fifty micrograms of tissue extracts were electrophoresed under reducing conditions on 7.5% sodium dodecyl sulfate-polyacrylamide gel for ß1C and ß1 protein detection. Immunoblotting was carried out as previously described (16) using one of the following primary antibodies, 5 µg/ml rabbit polyclonal affinity-purified antibody to ß1C integrin (provided by Dr. M. Fornaro) or 1 µg/ml mouse monoclonal antibody to ß1 integrin extracellular domain (Transduction Laboratories, Inc.-BD Biosciences, Franklin Lakes, NJ), or using a 1:500 dilution of polyclonal antibody to ß1A integrin cytoplasmic domain (Chemicon International, Temecula, CA) or 10 µg/ml mouse monoclonal antibody to ß-tubulin (Sigma-Aldrich Italia, Milan, Italy). The blots were incubated with the antibody for 16 h at 4 C in TBS-T [20 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, and 0.2% Tween 20] containing 5% nonfat dry milk. The membrane was then washed three times in TBS-T and incubated with horseradish peroxidase-conjugated goat affinity-purified antibody to rabbit and mouse IgG (Amersham Pharmacia Biotech Italy Srl) for ß1C integrin and ß1 or ß1A integrins, respectively, diluted 1:20,000 in TBS-T for 1 h at room temperature. After three washes in TBS-T, the proteins were visualized using the enhanced chemiluminescent system according to the manufacturer’s instructions (Amersham Pharmacia Biotech Italy Srl). Protein levels were normalized using the constitutively expressed ß-tubulin, protein. For this purpose, the blots were stripped at 55 C for 20 min with stripping buffer [2% sodium dodecyl sulfate, 10 mM ß-mercaptoethanol, and 6 mM Tris-HCl (pH 6.8)] and hybridized, as described above, with 10 µg/ml antibody to ß-tubulin. Densitometric values for immunoreactive bands were quantified using a GS-700 imaging densitometer (Bio-Rad Laboratories, Inc.); multiple expressions of the same immunoblots in a linear range were performed. ß1C, ß1, and ß1A protein levels were calculated as a percentage of the control (proliferative endometrium), taken as 100 in arbitrary units, after normalization using ß-tubulin as a control for protein loading. The mean value (±SEM) of the results obtained in at least three experiments was calculated for each specimen.

Immunohistochemistry

A single paraffin block per case was selected for immunostaining based on good morphological preservation. Five-micron-thick sections were cut, collected on positively charged slides, dewaxed, and rehydrated. After quenching of endogenous peroxidase with 3% H2O2 for 15 min at room temperature, the sections were immunostained for ß1 integrin and its spliced variant, ß1C, using an avidin-biotin peroxidase (ABC) technique with an automated immunostainer (TecMate 500, DAKO Corp., Glostrup, Denmark). Before the staining procedure, the sections to be incubated with anti-ß1 integrin antibodies were immersed in 0.1% sodium dodecyl sulfate for 10 min at room temperature. A mouse monoclonal antibody against ß1 integrin (BD Biosciences, Franklin Lakes, NJ; clone 18; dilution, 1:20) and a rabbit antiserum against ß1C integrin (provided by Dr. M. Fornaro; 1.7 µg/ml dilution) as previously reported (16) were used as primary antibodies with overnight incubations at 4 C.

Control sections for specificity included the staining of positive controls (normal and carcinomatous breast) and of negative control sections, which were incubated with the Ig fraction of normal mouse or rabbit serum in place of the specific immunoreagent.

Evaluation of immunoreactivity

In all cases ß1 integrin and ß1C integrin immunoreactivities were independently evaluated by two pathologists (E.Mai. and A.N.) by separately counting the relative number of immunoreactive endometrial/decidual stromal or epithelial cells in 10 different microscopic fields, observed at x400 magnification; the extent of the immunoreactivity within each cell component (stromal vs. epithelial), meant as the percentage of immunoreactive cells, was semiquantitatively scored as follows: 0, absence of immunoreactive stromal/epithelial cells; 1, 1–10% immunoreactive stromal/epithelial cells; 2, 11–25% immunoreactive stromal/epithelial cells; 3, 26–50% immunoreactive stromal/epithelial cells; 4, more than 50% immunoreactive stromal/epithelial cells.

HL60 cells

Human promyelocytic leukemia (HL60) cells were grown in RPMI 1640 (Life Technologies, Inc., Milan, Italy) with 50 µg/ml gentamicin, 2 mmol/liter glutamine, and 15% inactivated fetal calf serum at 37 C in the presence of 5% CO2. Total RNA from differentiated cells was prepared 24 h after incubation with 160 nmol/liter phorbol 12-myristate 13-acetate (Sigma-Aldrich Italia) as previously described (34).

Statistical analysis

The data are reported as the mean ± SEM for the indicated experiments. Statistical analysis was performed using t test. All experiments were repeated at least three times.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Gene expression of ß1C and ß1 integrin subunits in endometrium and decidua

The expression of ß1C and ß1 integrins was examined at the RNA level using either a ß1C-specific probe (17) or a ß1 full-length probe that hybridizes with all the ß1 variants (10). Because of the low amounts of RNA obtained from the tissue samples, total RNA rather than polyadenylated RNA was analyzed. Steady state levels of ß1C and ß1 mRNA were evaluated by Northern blotting analysis (Fig. 1Go) of the total RNA extracted from endometrial tissues in the proliferative phase (lanes 3–5) and the secretory phase (lanes 6–8) of the menstrual cycle and from postmenopausal endometria (lanes 9–11). The expression of ß1C and ß1 was also examined in decidual tissues from patients in the first trimester of pregnancy (lanes 12–14) and in the third trimester of pregnancy (lanes 15–17).



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  		Figure 1. Northern blotting analysis of ß1C and ß1 mRNA in endometrium and decidua. Twenty-five micrograms of total RNA were used for each sample and hybridized with either the 116-bp ß1C specific or full-length ß1 or 28S rRNA [{alpha}-32P]deoxy-CTP-labeled cDNA probes, as described. Lane 1, HL60 cell line; lane 2, human liver; lanes 3–5, proliferative endometria; lanes 6–8, secretory endometria; lanes 9–11, atrophic endometria; lanes 12–14, first trimester decidua; lanes 15–17, third trimester decidua.

 
A 4.3-kb transcript was found in all samples. This band corresponds to ß1C mRNA, as the probe used represents the specific 116-bp ß1C insertion that is found only in ß1C (17). Other ß1 variants do not hybridize to this specific probe, as the ß1C-specific 116-bp insertion is not present in other ß1-spliced mRNA variants (12). Total RNA extracted from HL60 cells (Fig. 1Go, lane 1) and human liver RNA (lane 2) were used as positive and negative controls of ß1C gene expression, respectively, because ß1C is expressed by HL 60 cells and is barely detectable in normal human liver (10, 14). ß1C mRNA expression in proliferative endometrial tissues (lanes 3–5) was lower (37%) than the expression of ß1C mRNA in HL60 cells.

To normalize the differences due to mRNA loading and transfer, the same blots were dehybridized and rehybridized again with human 28S rRNA cDNA probe. The ß1C expression in all tissues was related to the 28S RNA expression accounting for the RNA loading in each sample and was calculated as a percentage of the control value (proliferative endometrium). Finally, the mean values of at least three separate measurements for each patient are reported in Fig. 2Go. As far as endometrial tissues are concerned, similar (P > 0.05) expression of ß1C mRNA levels was detected in the specimens of secretive endometria compared with proliferative endometrium, which was used as a control. In fact, the ß1C mRNA levels ranged from 56–150%, with an average value of 116 ± 16%. A statistically significant (P < 0.005) slight increase was found in atrophic endometrial specimens; ß1C mRNA ranged from 105–127%, with an average value of 117 ± 6% compared with the control.



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  		Figure 2. Statistical analysis of ß1C and ß1 mRNA expression in endometrium and decidua. The average of either ß1C or ß1 mRNA levels in 11 samples of proliferative endometrium (P) was set at 100, and the mRNA levels in the other tissues were calculated as a percentage of P. S, Secretory endometrium from seven patients; A, atrophic endometrium from three patients; FD, first trimester decidua from three patients; TD, third trimester decidua from nine patients. The mean values (±SEM) from at least three different experiments for each specimens are reported.

 
In decidual tissues, the densitometric analysis (Fig. 2Go) revealed a statistically significant (P < 0.005) decrease in ß1C mRNA levels in both samples obtained at the first (mean, 92 ± 13%) and third trimesters of pregnancy (range of expression, 50–96%; mean, 76 ± 6%) compared with the expression in proliferative endometrium.

The same tissue specimens were analyzed using the full-length ß1 cDNA probe that hybridizes with all ß1 variants. In Fig. 1Go the 4.2-kb transcript corresponding to the full-length ß1 mRNA is shown, and the statistical analysis is reported in Fig. 2Go.

In endometrial tissues, increased levels of ß1 mRNA were found in both secretive and atrophic endometria compared with proliferative endometrium; ß1 mRNA expression ranged from 73–198%, with an average value of 138 ± 21%, in secretory endometrium and from 110–131%, with an average value of 121 ± 10%, in atrophic endometrium. The differences from proliferative endometria were statistically significant (P < 0.005).

ß1 mRNA levels were higher in decidual tissues than in proliferative endometrium; in the former, ß1 mRNA expression ranged from 90–184%, with an average value of 139 ± 17% in the first trimester of pregnancy and 129 ± 14% in the third trimester.

Protein expression of ß1C, ß1A, and ß1 in endometrium and decidua

The expressions of ß1C and ß1A integrin subunits were examined at the protein level using a specific affinity-purified antibody to ß1C protein (12) or a specific antibody to the cytoplasmic domain of ß1A. An antibody to the ß1 extracellular domain that recognizes all ß1 variants was also used as a control. ß1C, ß1A, and ß1 protein levels were evaluated by immunoblotting analysis using both the same endometrial and decidual tissues specimens in which mRNA expression was analyzed and an additional two atrophic endometrial, four first trimester decidual, and two third trimester decidual specimens.

The results of a typical immunoblotting experiment are illustrated in Fig. 3Go. A 130,000–140,000 molecular weight integrin protein was found in all samples, showing that both ß1C and ß1A proteins are expressed in proliferative (lanes 1–3), secretory (lanes 4–6), and atrophic (lanes 7–9) endometria as well as in first trimester (lanes 10–12) and third trimester (lanes 13–15) decidua.



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  		Figure 3. Immunoblotting analysis of ß1C, ß1A, and ß1 protein expression in endometrium and decidua. One hundred micrograms of tissue detergent extracts were electrophoresed on 7.5% SDS-PAGE under reducing conditions, transferred to nitrocellulose membrane, and immunostained using antibodies to ß1C, ß1A, or ß1 integrin or to ß-tubulin as detailed in Patients and Methods. Lanes 1–3, Proliferative endometria; lanes 4–6, secretory endometria; lanes 7–9, atrophic endometria; lanes 10–12, first trimester decidua; lanes 13–15, third trimester decidua.

 
The values of ß1C, ß1A, and ß1 protein expression in endometrial and decidual tissues were calculated as a percentage of those in proliferative endometrium, which was used as a control, after normalization with ß-tubulin protein expression.

In endometrial tissues, as already observed for mRNA expression, the densitometric analysis (Fig. 4Go) showed that the levels of ß1C protein were not statistically different (P > 0.05) in secretive endometria (range of expression, 80–130%; average, 105 ± 6%) with respect to proliferative endometria, but these were significantly (P < 0.005) higher (range, 128–140%; average, 134 ± 3%) in atrophic endometria compared with proliferative endometria. In the same endometrial tissues, increased ß1A protein levels were found in secretory specimens (range of expression, 102–150%; average, 130 ± 5%), and decreased levels were found in atrophic endometria (range, 77–100%; mean, 87% ± 4) compared with the levels detected in proliferative endometrial tissues.



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  		Figure 4. Statistical analysis of ß1C, ß1A, and ß1 protein expression in endometrium and decidua. The average of either ß1C, ß1A, or ß1 protein levels in 11 samples of proliferative endometrium (P) was set at 100, and the protein levels in the other tissues were calculated as a percentage of P. S, Secretory endometrium from 7 patients; A, atrophic endometrium from 5 patients; FD, first trimester decidua from 7 patients; TD, third trimester decidua from 11 patients. The mean values (±SEM) from at least 3 different experiments for each specimen are reported.

 
As far as decidual samples are concerned, drastic down-regulation of ß1C protein levels was detected in all samples compared with that in proliferative endometria. ß1C protein levels ranged from 12–40%, with an average of 27 ± 8% in the first trimester and 28 ± 4% in the third trimester of pregnancy compared with the levels found in control tissues (P < 0.005). In contrast to ß1C expression, ß1A expression was increased in both the first trimester (range of expression, 150–280%; mean, 215 ± 17%) and the third trimester (range of expression, 164–255%; mean, 191 ± 11%) of pregnancy compared with the levels in proliferative endometria (P < 0.005).

Using the antibody to the ß1 extracellular domain that recognizes all ß1 variants, a statistically significant (P < 0.005) increase in ß1 protein levels in both secretive (range, 110–160%; average, 126 ± 7%) and atrophic (range, 128–138%; average, 133 ± 2%) endometria was found. In decidual specimens, as illustrated for ß1A expression, a marked increase in ß1 protein levels was detected compared with proliferative endometrial samples. In fact, ß1 protein levels ranged from 180–300%, with an average value of 227 ± 20% in the first trimester of pregnancy and 210 ± 12% in the third trimester compared with the levels found in proliferative endometria.

ß1C and ß1 integrin immunohistochemistry

The results of the semiquantitative evaluation of ß1 and ß1C immunoreactivities are schematically illustrated in Fig. 5Go, and the corresponding mean values, calculated on the scores of individual cases, are reported in Table 1Go.



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  		Figure 5. ß1C and ß1 protein immunoreactivity in endometrial and decidual tissues. The mean value of both ß1C and ß1 percentile immunoreactive cells are reported for the epithelial and stromal cells of endometrial tissues from 11 patients in the proliferative phase (P), 7 in the secretory phase (S) of the menstrual cycle, 6 postmenopausal patients (A), and decidual tissues from 6 patients (D).

 

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  		Table 1. ß1C and ß1 immunoreactivity in endometrial and decidual tissues

 
Overall, the percentages of both ß1C- and ß1-immunoreactive cells were comparatively higher in the epithelial than in the stromal cell compartment of both endometrial and decidual tissues, and the staining was more intense (moderate to strong in an arbitrary scale) in the former than in the latter (weak to moderate) cell types. Furthermore, ß1C immunoreactivity in epithelial cells appeared as a fine granularity, mainly localized at the basal cytoplasmic pole, whereas ß1 immunoreactivity in the same cells was homogeneously distributed throughout the cytoplasm, with a tendency to apical reinforcement. This different staining pattern was not apparent in the stromal cell compartment, which showed uniform cytoplasmic positivity of variable intensity.

In the tissue samples from the proliferative phase, the immunolocalization of both ß1C and ß1 integrin was almost exclusively restricted to the glandular epithelium, and the percentages of ß1C-positive epithelial cells (mean score, 4.0 ± 0) were comparable (P > 0.05) to those in ß1-reactive epithelial cells (mean score, 3.9 ± 0.1). The samples obtained during the secretory phase exhibited lower percentages of ß1C-reactive epithelial cells (mean score, 3.1 ± 0.1; P < 0.005) and ß1 immunoreactivity (mean score, 3.9 ± 0.1), comparable (P > 0.05) to those observed during the proliferative phase. The tissue samples from postmenopausal women showed results similar to those in proliferative endometria (mean score, 4 ± 0 for both ß1C and ß1 immunoreactivities), but the staining intensity was much weaker (Fig. 6Go).



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  		Figure 6. ß1C immunoreactivity in endometrial cells. Almost all epithelial cells display ß1C immunoreactivity that is mainly localized at the basal pole of the cytoplasm. A much lower number of stromal cells, if any, demonstrate faint ß1C immunoreactivity. A, Proliferative endometrium; B, secretory endometrium; C, atrophic endometrium. The ABC technique was used. Magnification for anti-ß1C: x160 for A and B, and x100 for C.

 
For ß1C and ß1 immunoreactivity in the endometrial stromal compartment, the highest percentages were detected in secretory endometria (mean score, 1.7 ± 0.2 and 2 ± 0, respectively), lower percentages were demonstrated in proliferative tissue samples (mean score, 0.5 ± 0.1 and 0.7 ± 0.2, respectively), and no immunoreactivity for either antigen could be detected in atrophic endometria.

In all decidual samples, statistically significant (P < 0.005) decreased percentages of ß1C-immunoreactive epithelial cells were found (mean score, 1.2 ± 0.2) compared with proliferative endometria, and no ß1C positivity was found in the stromal compartment. In contrast, ß1 integrin immunoreactivity was detected in both glandular epithelial cells (mean score, 3.5 ± 0.5) and stromal cells (mean score, 3 ± 0) at levels comparable to those in proliferative endometria (Fig. 7Go).



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  		Figure 7. ß1C and ß1 immunoreactivity in epithelial and stromal decidual cells. Several epithelial cells and very rare stromal cells demonstrate ß1C immunoreactivity. In the former, the positivity is mainly localized at the basal pole of the cytoplasm. A, ß1C Immunoreactivity in epithelial decidual cells; B, ß1C immunoreactivity in stromal decidual cells. Almost all epithelial cells and numerous stromal cells demonstrate ß1 immunoreactivity. In the former, the positivity is homogeneously distributed in the cytoplasm, with apical reinforcement. C, ß1 Immunoreactivity in epithelial decidual cells; D, ß1 immunoreactivity in stromal decidual cells. The ABC technique was used. Magnification for anti-ß1C and anti-ß1, x250.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Both the endometrium and the decidua represent an effective physiological model to study the modulation of integrin subunit expression in correlation with the variable cell proliferation rates during the menstrual cycle and pregnancy. This paper was aimed at evaluating the expression of ß1C and ß1A integrins, two variants that differentially affect cell proliferation (11), during the menstrual cycle and pregnancy, and at exploring their effects in modulating cell proliferation and differentiation in physiological conditions. In this light, ß1C expression was studied at both the mRNA and protein levels using a 116-bp fragment-specific probe (17) and a specific antibody against the ß1C cytoplasmic domain (16). ß1A expression was studied at the protein level using a commercial specific antibody against the ß1A cytoplasmic domain. Therefore, there is documented evidence that the ß1C and ß1A expression values detected in this study do not reflect the expression of other ß1 integrin isoforms.

This study shows that ß1C and ß1A integrin subunits are expressed in both the endometrium and decidua and that ß1C expression is down-regulated in decidual tissue compared with endometrium at both the mRNA and protein levels, whereas ß1A expression is up-regulated.

The current study reports for the first time that ß1C integrin expression is maximal in atrophic endometrium, a quiescent and nonproliferative tissue. This is in agreement with previous studies performed on prostate and breast cells, which demonstrated that ß1C integrin is mainly expressed in differentiated quiescent cells and is down-regulated in proliferating cells (16, 18). It should be emphasized that the protein expression of the overall ß1 integrin subunits in atrophic endometrium is comparable to that of ß1C integrin, but the protein levels of the ß1A variant were lower in the same samples. The two ß1 integrin subunits, ß1C and ß1A, that differ from each other just for the carboxyl-terminal cytodomain, play an important role in cell growth as they differentially influence cell proliferation, with ß1C inhibiting and ß1A promoting this process. Therefore, the contribution of ß1A in purportedly nonproliferative tissues, such as atrophic endometrium, should be minimal, if any. Consequently, in atrophic endometria we can suppose that the contribution of the ß1C variant is a determinant in the evaluation of the overall ß1 integrin expression, considering that the other two spliced variants, ß1B and ß1D, are unlikely to be detected in the endometrial and decidual tissues because other researchers have previously shown that the former is restricted to skin and liver tissues (35, 36), whereas ß1D is found in skeletal and cardiac muscle fibers only (37, 38).

Differently, in the current study both mRNA and protein levels of ß1 integrins in toto were higher than ß1C in secretive endometrium; ß1C expression was comparable to that in proliferative endometrium. As we demonstrated increased expression of the specific ß1A variant in secretive endometria, its contribution in the evaluation of the overall ß1 expression levels should be more relevant than that of ß1C. In consideration of the variable expression of ß1A throughout the menstrual cycle and of increased expression occurring predominantly during the secretory phase, it can be speculated that among the ß1-subunit variants, ß1A integrin expression is progesterone dependent, whereas ß1C is not.

As for ß1C and ß1 immunoreactivity in endometrial samples, a comparable percentage of ß1C and ß1-positive epithelial cells was found throughout the different phases of the menstrual cycle and in atrophic endometria. Differently, both ß1C- and ß1-positive stromal cells were more numerous in the secretive phase than in the proliferative phase and were completely absent in atrophic endometria. These data indicate that stromal cells actively contribute to regulation of the ß1 integrin system in a variable fashion during the different phases of the menstrual cycle and that they may be part of a complex paracrine loop in which both steroid hormones and integrins regulate the fine balance of proliferation and differentiation of the stromal and epithelial compartments.

Taking into account that no previous data are available on ß1C immunoreactivity in endometrial tissues, the results of this study are in general agreement with the results reported by others (23, 24, 25, 26) and point to a relevant role of the integrin superfamily in endometrial physiology.

Increased expression of ß1-subunits, at both the mRNA and protein levels, was detected in decidual tissues compared with that in proliferative endometrium. As very low levels of ß1C protein and much higher levels of ß1A protein were detected in decidual samples compared with endometrial tissues, the increase in ß1 expression in toto is likely to reflect ß1A expression, thus indicating selective down-regulation of the unique ß1C sequence.

The results of the immunohistochemical studies apparently confirm the occurrence of selective down-regulation of ß1C in decidual tissues, as lower percentages of epithelial cells and no stromal cells displayed ß1C immunoreactivity compared with endometrial tissues. The detection of decreased ß1C mRNA levels in the decidua suggests that either transcriptional and/or post-transcriptional regulation of ß1C expression is conceivable, whereas the strongly decreased ß1C protein levels detected in the same tissues might be ascribed to translational activity or protein degradation.

As ß1C is one of the spliced variants of the ß1 integrin superfamily, down-regulated ß1C expression could derive from an altered splicing mechanism occurring in the decidua, which results in abrogation of inhibition of trophoblastic cell proliferation. Tamura et al. (39) and Belkin et al. (40) have demonstrated that splicing mechanisms control the expression of specific integrins at different stages of differentiation. Therefore, further investigations of the real occurrence of alterations in factors controlling the integrin splicing machinery are worth performing.

Down-regulation of ß1C integrin expression during human embryo implantation and pregnancy stresses the role of this integrin in the negative regulation of cell cycle progression and proliferation, which has been shown in both nontumorigenic and tumorigenic cells (13, 16). Integrins may have several active roles in modulating the interactions between trophoblasts and uterus. They may facilitate, via intermediary molecules, the attachment of the embryo and the invasion of the extracellular matrix after hatching. Additional evidence of the role of integrins and their ligands in cell proliferation and differentiation has been obtained from studies of cancer cells showing that alterations of integrin expression in tumor cells correlate with increased invasiveness, malignant progression, and metastatic potential in vivo and in vitro (41, 42, 43). Some of the key mechanisms guiding different steps of trophoblast invasion through the basement membrane into the decidua are somewhat analogous to those involved in tumor cell invasion. In fact, during the first trimester of pregnancy, fetal trophoblast actively invades the maternal decidua, causing extracellular matrix degradation like that during invasion and metastasis of neoplastic cells (20, 21, 22). The strong down-regulation of ß1C integrin expression in decidual tissues from both the first and third trimesters strongly suggests that ß1C may play a crucial role in the modulation of trophoblastic invasion, the latter being regulated by the maternal microenvironment.

Experimental evidence also suggests that the uterine microenvironment may exert an effective control over trophoblastic invasion through the production of negative regulators of cell growth. The main inhibitory factor, detectable in the conditioned medium from the first trimester decidual cells and capable of suppressing trophoblastic invasion, has been identified as being TGFß1 (44, 45). TGFß cytokine 1 is produced by both fetal trophoblast and decidual stromal cells and may induce the endogenous or exogenous synthesis of tissue inhibitors of matrix metalloproteinase, thus indirectly influencing trophoblastic migration by controlling its proteolytic activity (45). In this regard, we found increased TGFß1 expression in both first and third trimester decidua compared with proliferative endometrium (data not shown), as previously reported (44, 45, 46). Therefore, one may speculate that ß1C integrin is important in mediating trophoblastic outgrowth and migration, as in the case of neoplastic cells, and TGFß1 might play a key role in regulating an adequate, but not excessive, invasion of the maternal decidua. Such a mechanism, involving both down-regulation of ß1C integrin expression (17, 18, 19) and drastically reduced TGFß1 expression (27, 47), has been demonstrated in neoplastic tissues.

The detection of increased ß1 integrin expression in the decidua is consistent with the results of recent studies showing that the expression of ß1 integrin in the human endometrium increases at the time of implantation and remains high in the decidua during early pregnancy. Such data highlight the important role of ß1 integrin in mediating organization of the ECM proteins derived from embryos during implantation (23, 24, 25, 26). Furthermore, the high expression of ß1 integrins and, in particular, of its ß1A variant not only during early pregnancy, but also during late pregnancy, suggests that ß1A integrin expression on decidual cells may be important for the attachment and subsequent development and differentiation of the embryo.

The data reported in this study suggest a fundamental role for the ß1C and ß1A variants in human endometrium and decidua in mediating cell proliferation. Further studies of pregnancy disorders associated with inadequate or overinvasion of the decidua by trophoblast will amplify our understanding of the role of ß1C integrin in implantation and of the regulatory events in trophoblast-decidual interactions. Moreover, studies of ß1C and ß1A integrin expression in benign and malignant hyperproliferative lesions of the uterus (currently ongoing in our laboratory) will lead to an advance in the understanding of molecular control mechanisms of cellular growth.


   Acknowledgments
 
We thank Dr. Richard Lusardi for linguistic consultation, and Mr. V. Cataldo for photographic assistance.


   Footnotes
 
This work was supported in part by a grant from ASI (ARS-99-77, to E.P.) and by a grant from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (Piani di potenziamento della rete scientifica e tecnologica–Cluster 03 to E.Mar.).

Abbreviations: ABC, Avidin-biotin peroxidase; ECM, extracellular matrix.

Received July 15, 2002.

Accepted October 28, 2002.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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