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Endocrinology, doi:10.1210/en.2007-0849
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Endocrinology Vol. 149, No. 3 1015-1025
Copyright © 2008 by The Endocrine Society

Opposite Regulation of Transforming Growth Factors-β2 and -β3 Expression in the Human Endometrium

Héloïse P. Gaide Chevronnay, Patricia B. Cornet, Denis Delvaux, Pascale Lemoine, Pierre J. Courtoy, Patrick Henriet1 and Etienne Marbaix1

Cell Biology Unit (H.P.G.C., P.B.C., D.D., P.L., P.J.C., P.H., E.M.), de Duve Institute, and Department of Pathology (E.M.), Medical School, Université Catholique de Louvain, B-1200 Bruxelles, Belgium

Address all correspondence and requests for reprints to: Etienne Marbaix, Cell Biology Unit, UCL-7541, Avenue Hippocrate, 75, B-1200 Bruxelles, Belgium. E-mail: etienne.marbaix{at}uclouvain.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TGF-βs have been reported to mediate the repression by progesterone of several matrix metalloproteinases in the human endometrium, thereby preventing menstrual breakdown. Because of conflicting reports on the expression profiles, source, and regulation of the TGF-β system in this tissue, we investigated by real-time RT-PCR and ELISA the expression of the three TGF-βs (total and mature forms) and their two receptors throughout the menstrual cycle, and their regulation by ovarian steroids in cultured explants including their microdissected epithelial and stromal compartments. Regulation by cAMP and MAPK was further investigated. This comprehensive study on a large collection of endometrial samples evidenced a differential regulation of TGF-β isoforms expression, both in vivo and in explant culture. In vivo, TGF-β2 increased by about 5-fold at the mid-late secretory phase then declined after menstruation; TGF-β3 increased at menstruation and remained high during the proliferative phase; TGF-β1 was maximal at menstruation. In explants cultured without ovarian steroids both TGF-β2 and -β3 were preferentially expressed in the stroma. Ovarian steroids strongly repressed both TGF-β2 and -β3 in stroma but only TGF-β2 in glands. cAMP prevented inhibition by ovarian steroids of TGF-β2 but not -β3. In presence of ovarian steroids, MAPK inhibitors (p38 and ERK pathways) stimulated TGF-β3 but inhibited TGF-β2 expression. In conclusion, TGF-β2 and -β3 are differentially expressed during the menstrual cycle and regulated by progesterone in epithelial vs stromal cells. The opposite regulation of TGF-β2 and -β3 by cAMP and MAPK could account for their distinct expression in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE THREE TGF-βs (TGF-β1, -β2, and -β3), encoded by different genes, regulate cell growth and differentiation and play a central role during implantation and pregnancy in the human (1). Their effects are mediated by type I and type II TGF-β receptors (TGFβ-Rs) (2, 3). TGF-βs are not redundant because invalidation of each isoform induces a different phenotype in mouse (4). Indeed, TGF-β1 knockout (KO) mice either die at midgestation due to defective yolk-sac vasculogenesis or survive with altered immune response, decreased bone mass, and propensity to colon cancer. TGF-β2 KO mice present multiple malformations defects, leading to perinatal death. TGF-β3 KO mice die shortly after birth due to defective palate growth and delayed lung development.

A crucial role of TGF-βs is to stimulate extracellular matrix (ECM) synthesis and induce wound healing (5). The functional layer of the endometrium in cycling women is a spectacular paradigm of sequential ECM remodeling, including stromal decidualization at the midsecretory phase, menstrual breakdown (if no pregnancy is initiated), and full tissue repair during the proliferative phase. TGF-βs are thought to be instrumental in these processes (1). Menstruation is induced by a fall of estrogen and progesterone production in the late secretory phase, leading to proteolytic ECM breakdown and shedding of the functionalis. Matrix metalloproteinases (MMPs) are key enzymes for this process (6): the expression of most MMPs is repressed during the secretory phase and increases dramatically at or slightly before menstruation (7); conversely, MMP inhibitors prevent menstrual-like ECM breakdown in cultured explants (8). Progesterone and cytokines coordinately regulate MMP expression in time and space (6). Among cytokines, TGF-βs have been shown to mediate the inhibition by progesterone of MMP-3 secretion by stromal cells and MMP-7 by epithelial cells (9, 10). Progesterone was reported to promote TGF-β1 and -β2 expression in endometrial explants (9, 11) and stimulate TGF-β1 but decrease TGF-β3 expression in purified stromal cells (12).

However, the expression profile of the various actors of the TGF-β system during the menstrual cycle as well as the source, regulation, and activation of TGF-βs in the human endometrium are unclear in the literature. Contradictory reports indicated that TGF-β1 mRNA level was stable throughout the menstrual cycle (13, 14) or increased during the secretory phase (15, 16) and at menstruation (11). TGF-β2 mRNA expression was only estimated by in situ hybridization (16, 17). Opposite reports indicated that TGF-β3 mRNA selectively increased during the secretory phase (13) or during the proliferative phase (12). At the protein level, the various TGF-β isoforms were compared only by immunolocalization and reported to either increase during the secretory phase (9) or peak during the late proliferative and midsecretory phases (17). TGF-β1 protein was reported to be stably expressed during the menstrual cycle and TGF-β3 protein to increase during the secretory phase (13, 18). The cellular origin of TGF-βs is also disputed, with opposite claims on their preferential localization in epithelial (17) or stromal cells (18), with variations throughout the cycle (9, 13, 17, 18).

In an attempt to clarify the expression profiles and regulation of the various actors of the TGF-β system, we obtained normal endometrium from 137 spontaneously cycling women and precisely measured their mRNA level by real-time RT-PCR and protein level by ELISA in fresh tissue and in large numbers of paired groups of explants cultured with or without ovarian steroids. The compartment of production was clarified by laser capture microdissection combined with real-time RT-PCR. These techniques yielded more sensitive assays and provided the comprehensive quantitative analyses of the TGF-β system in the human endometrium. We further investigated in explant cultures the molecular mechanisms controlling the expression of the three TGF-β isoforms, by looking at the cAMP and MAPK signaling pathways and at a possible role of retinoic acid. Regulation of ERK and p38 activity was also analyzed during the menstrual cycle and on explant treatment with ovarian steroids. Major differences in expression profiles and opposite regulation were observed between TGF-β2 and -β3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection and culture
Normal endometrium was provided by 137 spontaneously cycling women (26–57 yr old), after informed consent. Part of this collection was already analyzed in previous reports addressing different questions (7, 19). The Ethical Committee of the Université Catholique de Louvain approved the study. A fraction of the endometrium was fixed in formalin for histological analysis. Independent dating by an expert pathologist (E.M.) following microscopic criteria was finely adjusted according to the last menstrual period recorded in the clinical files. The rest of the tissue was either frozen at –80 C for protein analysis and/or RNA extraction (SV total RNA isolation system; Promega, Madison, WI) or used for organ culture.

Explants of about 1 mm side were cultured as described (8) on Millicell inserts (Millipore, Bedford, MA; 24 explants per 30-mm insert) in six-well Cellstar culture plates (Greiner Bio-One, Kremsmuenster, Austria) with daily-renewed DMEM (Invitrogen-Life Technologies, Merelbeke, Belgium; 1200 µl per 24 explants), devoid of phenol red and serum. This medium was supplemented with 1 nM water-soluble 17β-estradiol and/or 100 nM progesterone, or 300 nM 2-hydroxypropyl-β-cyclodextrin as vehicle. For specific experiments, 0.5 mM 8-bromoadenosine-cAMP, 1 µM all-trans-retinoic acid (all above reagents from Sigma-Aldrich, St. Louis, MO), 50 µM U0126 (Promega), 10 µM SB-203580 (Calbiochem, Darmstadt, Germany), or 0.5% dimethylsulfoxide as vehicle were further added. After 1 or 2 d of culture, explants were either frozen at –80 C for protein analysis or RNA extraction, or embedded in Tissue-Tek O.C.T. compound (Sakura, Zoeterwoude, The Netherlands) and frozen in liquid nitrogen-cooled isopentane for laser capture microdissection. For immunohistochemical analysis, some explants were fixed in formalin and embedded in paraffin.

Real-time RT-PCR
Extracted RNA was quantified by spectrophotometry at 260 nm. Its integrity was assessed by electrophoresis or the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). Aliquots of 200 ng RNA were reverse transcribed using the oligo(dT) protocol of Thermoscript RT-PCR system (Invitrogen-Life Technologies).

The oligonucleotide primers and TaqMan probes (Table 1Go; Invitrogen-Life Technologies) were designed as reported for TGF-β1, TGF-β2, prolactin, and β-actin (19, 20, 21) or derived from their cDNA sequences for TGF-β3, TGFβ-RI (ALK-5) and TGFβ-RII, using the Primer3 software (http://frodo.wi.mit.edu). A second set of 3'-located β-actin primers (β-actin-2 in Table 1Go) was specifically used with the microdissected materials because of preferential linear amplification of the mRNA 3'-end (see below).


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  		TABLE 1. Oligonucleotide primer and probe sequences for real-time RT-PCR amplification of TGF-βs, TGFβ-RI, TGFβ-RII, prolactin, and β-actin mRNA

 
Standards for real-time PCR were prepared as described (19). Quantitative PCR amplification was performed on the MyIQ single-color real-time PCR detection system (Bio-Rad, Hercules, CA) with the LightCycler FastStart DNA master HybProbes kit (Roche/Boehringer, Mannheim, Germany) for TGF-β1, TGF-β2, and β-actin-1, the qPCRCore kit (Eurogentec, Seraing, Belgium) for TGFβ-RI, the LightCycler FastStart DNA MasterPLUS SYBRGreen I kit (Roche/Boehringer) for TGFβ-RII and the Platinum SYBR Green qPCR Supermix-UDG kit (Invitrogen-Life Technologies) for TGF-β3, prolactin, and β-actin-2. The reaction was performed in a final volume of 20 µl with 500 nM specific primers and 200 nM TaqMan probe for TGF-β1, -β2, TGFβ-RI, and β-actin-1. The optimal final concentration of MgCl2 was 3 mM for prolactin, β-actin-1 and -2, 4 mM for TGF-β3, 5 mM for TGF-β1 and TGF-β2, and 7.5 mM for TGFβ-RI. MgCl2 was not added to the reaction mixture for TGFβ-RII amplification. After denaturation at 95 C for 10 min, the cDNAs were subjected to 45 amplification cycles, each one including 20 sec of cDNA denaturation at 95 C and 30 sec of annealing and elongation at 60 C for TGF-β1, -β2, -β3, TGFβ-RI, TGFβ-RII, prolactin, and β-actin-2 or 45 sec at 67 C for β-actin-1. The fluorescence signal was measured after each cycle. A standard curve and a control reaction without template were performed in each assay. A single specific melting temperature was consistently obtained at the end of each run for TGF-β3, TGFβ-RII, prolactin, and β-actin-2, confirming the validity of SYBR Green-based assays. All PCRs were performed in independent duplicates. The level of β-actin mRNA normalized to total RNA did not change throughout the cycle, including the menstrual phase (not shown).

Laser capture microdissection
Laser capture microdissection was performed on PALM MicroBeam system (PALM Microlaser Technologies, Bernried, Germany). Briefly, 7-µm-thick cryosections were fixed in 75% ethanol for 1 min. Embedding medium was removed in water and sections were dehydrated in 75, 95, and 100% ethanol for 30 sec each and then xylene for 5 min. Microdissection was performed using the PALM Robot software. Typically, 20 slides were prepared per sample and about 50 microdissected glands or stromal foci were pooled separately. RNA was extracted by the phenol/chloroform procedure using Tripure (Roche/Boehringer) in the presence of 40 µg glycogen (Roche/Boehringer) to visualize the pellet in the final steps of purification. A linear amplification of 10 ng RNA was performed with the two-cycle amplification kit (Affymetrix, Santa Clara, CA) and 400 ng of the obtained cRNA was reverse transcribed with the Thermoscript RT-PCR system using 3 µM forward primers for β-actin-2 and TGF-βs.

Protein assay
Tissue was homogenized in PBS (Sigma-Aldrich) containing 1% Tween 20 (Merck, Darmstadt, Germany) supplemented with Complete protease inhibitor cocktail (1 tablet for 50 ml; Roche/Boehringer). Protein concentration in the lysates was measured by the bicinchoninic acid method (Sigma-Aldrich). Conditioned media were concentrated about 2-fold with Microcon centrifugal filter devices (Amicon; Millipore, Bedford, MA), according to the manufacturer’s recommendations, and protein concentration was measured using the Bradford protein assay (Bio-Rad). Concentration of TGF-β1, -β2, or -β3 was measured using DuoSet ELISA development kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s recommendations. Mature TGF-β was directly assayed in the samples, whereas total TGF-β (latent and mature forms) was assayed after treatment with 0.1 M HCl at 20 C for 10 min.

Western blotting
Tissue was homogenized in 150 mM NaCl, 2 mM CaCl2, and 20 mM HEPES (pH 7.4), supplemented with 10 mM 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, Complete mini EDTA-free protease inhibitor cocktail (1 tablet for 10 ml; Roche/Boehringer), and 2 mM sodium orthovanadate 2 mM sodium pyrophosphate, and 2 mM sodium fluoride (all from Sigma-Aldrich) to inhibit phosphatases. Loading was normalized based on DNA content measured by spectrophotometry at 460 nm. Proteins were resolved by SDS-PAGE (10% polyacrylamide) and transferred at 90 V for 1.5 h to a nitrocellulose membrane (Hybond-Cextra; Amersham Biosciences, Roosendaal, The Netherlands). Blots were blocked for 2 h at room temperature in Tris-buffered saline [TBS; 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl], 5% nonfat milk, and 0.05% Tween 20 and incubated overnight at 4 C in TBS, 5% BSA, and 0.05% Tween 20 with rabbit antibodies to p44/42 (ERK) MAPK (1:1000; no. 9102), phospho-p44/42 ERK (Thr202/Tyr204; 1:1000, clone 197G2), p38 MAPK (1:1000, no. 9212), or phospho-p38 (Thr180/Tyr182; 1:500, clone 3D7) (all from Cell Signaling Technology, Beverly, MA). After three washes in TBS and 0.05% Tween 20, blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat antirabbit IgG (1:10,000 or 1:20,000; Invitrogen-Life Technologies). Membranes were washed once with TBS and 0.05% Tween 20 and twice with TBS. Immunoreactive bands were visualized using chemiluminescence (ECL Advance Western blotting detection kit; Amersham Bioscience) and quantified using Kodak 1D Image Analysis (Eastman Kodak Co., Rochester, NY). Data were normalized by reference to glyceraldehyde phosphate dehydrogenase (GAPDH) after membrane stripping for 30 min with Re-blot Plus strong antibody stripping solution (Chemicon International, Temecula, CA), incubation with anti-GAPDH mouse monoclonal antibody (0.5 µg/ml; clone 6C5; Ambion, Austin, TX) overnight at 4 C, and blot development as above.

Immunohistochemistry
Prolactin was immunolabeled on paraffin sections of explants as described (19) except that no antigen retrieval was performed. Briefly, sections were incubated overnight at 4 C with rabbit antiprolactin polyclonal antibodies (1:500; DakoCytomation, Glostrup, Denmark), washed, and revealed with peroxidase-conjugated dextran molecules carrying antirabbit secondary antibodies (Envision; DakoCytomation) followed by incubation with H2O2 and diaminobenzidine. Negative results were consistently obtained when replacing the primary antibody by a similar dilution of rabbit antifluorescein polyclonal antibodies (DakoCytomation).

Data presentation and statistical analysis
Most data are presented on logarithmic scales, using geometric means (obtained from arithmetic mean of logarithmic values) and 95% confidence intervals (CIs). Logarithmic transformation was applied to give equal weight to decreases and increases on graphic representations, in contrast to arithmetic means, which emphasize increases but hide decreases. Significance was tested using the two-tailed Wilcoxon two-sample (Figs. 1Go and 2Go) or signed rank tests (see Figs. 3Go, 4Go, 6Go, and 7Go). Differences were considered significant for P < 0.05.


Figure 1
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  		FIG. 1. Expression of TGF-β and TGFβ-R mRNA in the cycling human endometrium. The amount of mRNA molecules of TGF-β1, TGF-β2, TGF-β3 (63 samples), TGFβ-RI (46 samples), and TGFβ-RII (53 samples) were quantified by real-time RT-PCR in endometria collected throughout the menstrual cycle. Target mRNA amounts were normalized to β-actin mRNA (quantified with the β-actin-1 primer) and are presented on a logarithmic scale. The menstrual cycle is divided into early secretory (d 15–19; white circles at left), mid- and late-secretory (d 20–27; gray circles), menstrual (d 28 to d 5; black circles), and proliferative phases (d 6–14; white circles at right). Statistically significant differences are indicated in the text.

 

Figure 2
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  		FIG. 2. Expression of total and mature TGF-β proteins in the cycling human endometrium. The amount of TGF-β1, TGF-β2, and TGF-β3 proteins was measured by ELISA in 64 endometria collected throughout the menstrual cycle, 18 of which were also used in Fig. 1Go. Samples were treated with 0.1 M HCl for 10 min to activate the latent forms to measure the total amount of TGF-βs. Target protein amounts were normalized to total protein concentration and are presented on a logarithmic scale. Broken lines indicate the threshold of detection. Phases of the menstrual cycle are presented as at Fig. 1Go. Statistically significant differences are reported in the text.

 

Figure 3
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  		FIG. 3. Time course of expression of TGF-β and TGFβ-R mRNA in cultured explants and effects of estradiol and progesterone. A and B, Explants were cultured in the absence (–H) or presence (+EP) of 1 nM 17β-estradiol (E) and 100 nM progesterone (P) for 24 h (for TGF-βs, n = 21 proliferative and n = 18 secretory endometria; for TGFβ-RI, n = 11 proliferative and n = 11 secretory endometria; for TGFβ-RII, n = 15 proliferative and n = 14 secretory endometria) or 48 h (for TGF-βs, n = 11 proliferative and n = 11 secretory endometria; for TGFβ-RI, n = 6 proliferative and n = 11 secretory endometria; for TGFβ-RII, n = 11 proliferative and n = 9 secretory endometria). Normalized target mRNA level after culture was quantified as in Fig. 1Go and expressed as relative values by reference to the noncultured tissue (NC, 0 h) or explants cultured without hormone (–H), as indicated at right of the panels. These values after 0, 24, and 48 h of culture are presented separately for proliferative (A) and secretory (B) endometria. C, Normalized mRNA levels in explants from seven endometria cultured +E, +P, or +EP are compared with controls without hormone (–H). Results are presented as geometric means with 95% CI. *, P < 0.05; **, P < 0.005.

 

Figure 4
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  		FIG. 4. Time course of expression of TGF-β proteins in cultured explants and effects of estradiol and progesterone. Explants were cultured in the absence (–H) or presence (+EP) of 1 nM 17β-estradiol (E) and 100 nM progesterone (P) for 24 h (n = 6 proliferative and n = 4 secretory endometria) or 48 h (n = 9 proliferative and n = 5 secretory endometria). TGF-β proteins measured in the conditioned medium were added to those measured in the explants lysates to yield total TGF-β production, normalized to the total protein amount in explants and presented as relative values to reference tissues as in Fig. 3Go. Total TGF-βs were measured after treatment with HCl as described at Fig. 2Go. Results are presented as geometric means with 95% CI. *, P < 0.05; **, P < 0.005.

 

Figure 6
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  		FIG. 6. Effect of cAMP on prolactin and TGF-β expression in cultured explants. Explants were cultured for 24 or 48 h in the absence (–cAMP) or presence of 0.5 mM 8-bromoadenosine-cAMP (+cAMP), either alone (–H) or combined with ovarian steroids (+EP). A, Prolactin mRNA expression was quantified by real-time RT-PCR in five proliferative and 4 secretory endometria and normalized to β-actin mRNA. This figure shows the effect of cAMP addition in explants cultured without (–H) or with ovarian steroids (+EP) for 24 or 48 h, as ratio of cAMP-treated over untreated samples. B, Prolactin protein immunostaining in a representative explant cultured for 48 h with EP and cAMP. Arrowheads point to immunolabeled stromal cells. C, TGF-β mRNA expression was quantified as in Fig. 3Go in explants from five proliferative and three secretory endometria. D, Total TGF-β protein amount was measured as in Fig. 4Go in six proliferative endometria. The data are presented as the effects of EP addition (EP/–H) as in the lower panels of Figs. 3Go, A and B, and 4AGo. Results are presented as geometric means with 95% CI. NS, Not significant. *, P < 0.05; **, P < 0.005.

 

Figure 7
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  		FIG. 7. Contribution of ERK and p38 MAPK pathways to the regulation of TGF-βs in the human endometrium. A and B, Effect of MAPK inhibitors on TGF-β mRNA and protein expression. Explants were cultured for 24 h (mRNA) or 48 h (proteins) in the absence (–H) or presence of ovarian steroids (+EP). The culture medium was supplemented or not with 50 µM U0126 or 10 µM SB-203580, as indicated. Normalized target mRNA (A) and total protein (B) levels in explants from six proliferative and four secretory endometria (A) and from six proliferative and one secretory endometria (B) are expressed as relative values by reference to explants cultured without MAPK inhibitor. Total protein amount was quantified as at Fig. 4Go. Results are presented as geometric mean with 95% CI. *, P < 0.05; **, P < 0.005. C, Expression and phosphorylation of ERK and p38 on estradiol and progesterone treatment in explants cultured for 24 h (four proliferative and one secretory endometria) and 48 h (three proliferative and one secretory endometria) in the absence (–) or presence (+) of estradiol and progesterone (EP). D, Expression and phosphorylation of ERK during the menstrual cycle in lysates from five secretory (S), four menstrual (M), and six proliferative (P) endometria. Lysates were analyzed by Western blotting for total and phosphorylated forms of ERK and p38. Data were normalized to GAPDH after blot stripping. Activity corresponding to the ratio between normalized phospho-protein (pERK and pp38) and total protein (ERK and p38) is presented as mean with 95% CI. NS, Not significant.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-β and TGFβ-R expression profile in the cycling human endometrium
The mRNA levels of TGF-β1, -β2, -β3, TGFβ-RI, and -RII were quantified by real-time PCR and normalized to β-actin mRNA in 83 samples collected throughout the menstrual cycle (Fig. 1Go). Strikingly, TGFβ-RII mRNA level was about 1000-fold higher than TGFβ-RI throughout the cycle, whereas TGF-β1 was the most abundant TGF-β mRNA, with an average 6- and 3-fold higher level than TGF-β2 and -β3, respectively. The range of interpatient variation within a given phase of the cycle was generally low for TGFβ-RI; intermediate for TGFβ-RII, TGF-β3, and TGF-β1; and high for TGF-β2. Along the menstrual cycle, three distinct expression profiles could be identified: 1) TGF-β1, TGFβ-RI, and TGFβ-RII mRNA levels were rather constant, with a modest increase during the menstrual phase for TGF-β1 and TGFβ-RI (P < 0.01 and P < 0.02 vs. all other phases); 2) the average TGF-β2 mRNA level abruptly increased by 5-fold from the early secretory to the mid-late secretory (P < 0.01) and the menstrual phase and then briskly decreased by about 5-fold from the menstrual to the proliferative phase (P < 0.001), and its level was significantly lower in the proliferative and early secretory than in the mid-late secretory and menstrual phases (P < 0.0001); and 3) the average TGF-β3 mRNA level increased by 3-fold from the secretory to the menstrual phase (P < 0.001) and remained elevated throughout the proliferative phase (P < 0.001, compared with the secretory phase).

The level of total (latent and mature) and mature forms of all three TGF-β proteins was measured by ELISA in 64 endometria collected throughout the menstrual cycle (Fig. 2Go). As predicted from mRNA studies, TGF-β1 was the most abundant protein, with an average total level 2.5-fold higher than TGF-β2 and 20-fold higher than TGF-β3. The individual TGF-βs also showed the same expression profiles along the menstrual cycle as their mRNA. First, total and mature TGF-β1 protein levels were more abundant at the menstrual phase (P < 0.002 vs. the rest of the cycle, for both forms). Total TGF-β1 protein slightly decreased from menstrual to proliferative (P < 0.05) and further to secretory phase (P < 0.02 vs. proliferative; P < 0.002 vs. menstrual). Second, total and mature TGF-β2 level strongly increased by 5- and 2.5-fold during the mid-late secretory phase (P < 0.001 and P < 0.01 for total and mature forms respectively; d 23–27 vs. d 15–22) and remained high at the menstrual phase [P < 0.0001 and P < 0.02; late secretory (d 23–27) combined with menstrual phase vs. rest of the cycle]. Whereas total TGF-β2 level then decreased by about 4-fold from the menstrual to the proliferative phase (P < 0.002), mature TGF-β2 did not significantly decrease. And third, total and mature TGF-β3 protein levels gradually increased, by 3-fold from the secretory to the menstrual phase (P < 0.001 and P < 0.02). In contrast to the mature form, the total TGF-β3 level continued to increase about 2-fold during the proliferative phase (P < 0.005 vs. menstrual phase). The level of total and mature TGF-β3 then sharply dropped about 7-fold after ovulation (P < 0.001; proliferative vs. secretory phase, for both forms).

On average, the amount of mature TGF-β1 and TGF-β2 was 8- and 4-fold higher than that of TGF-β3 (P < 0.001 for both comparisons), although the proportion of mature TGF-β3 (26% of total) was 3-fold higher than that of TGF-β1 (9% of total; P < 0.01) but not significantly different from that of TGF-β2 (14% of total). The mature form proportion did not appreciably change throughout the menstrual cycle for TGF-β1 and -β3, but that of TGF-β2 was 3- to 4-fold lower at the secretory than at the menstrual (P < 0.02) and proliferative phase (P < 0.001).

Altogether the different expression profiles throughout the menstrual cycle suggested a differential regulation of TGF-β2 and -β3 expression by ovarian steroids.

Effect of ovarian steroids on TGF-β and TGFβ-R expression in explants
To directly test for differential effects of ovarian steroids, explants from proliferative or secretory endometria were cultured for up to 48 h in the presence or absence of estradiol and progesterone (Fig. 3Go for mRNAs; Fig. 4Go for proteins). In the absence of ovarian steroids, TGF-β2 mRNA was strikingly induced during the first day of culture [by 28-fold in proliferative explants (Fig. 3AGo) and 4-fold in secretory explants (Fig. 3BGo), compared with noncultured tissues], an increase that was strongly but not completely prevented by progesterone, be it alone or combined with estradiol (Fig. 3CGo). In the absence of ovarian steroids, TGF-β3 mRNA behaved differently from TGF-β2 mRNA: it did not change in proliferative explants and decreased moderately (by 2-fold) and transiently (after 24 h of culture) in secretory explants. However, TGF-β3 mRNA was also clearly repressed by progesterone, either alone or combined with estradiol (Fig. 3CGo). In contrast, the relative levels of TGF-β1, TGFβ-RI, and TGFβ-RII mRNA showed only modest changes during culture. The modest increase of TGFβ-RI and -RII was insensitive to ovarian steroids, but that of TGF-β1 was prevented by progesterone (Fig. 3CGo). Estradiol antagonized the effect of progesterone on TGF-β1 expression and had no appreciable effect on the other targets (Fig. 3CGo).

To further examine whether differential regulation of transcription was also reflected at the protein level, total and mature TGF-β amounts were measured in explants and conditioned culture media (Fig. 4Go). Surprisingly, the mature form of TGF-β1 was detected in only 43% of cultured explants and 7% of conditioned media, and, when detected, its level was much lower than in vivo (P < 0.001). In contrast, the proportion of mature TGF-β2 and -β3 was similar in culture and in vivo. TGF-β2 was mostly retained inside the explants because conditioned media contained only 24 ± 4% (mean ± 95% CI) of the produced protein; TGF-β3 was found in similar amounts in the conditioned media and inside the explants.

Profiles of protein expression for TGF-β1 and -β2 were similar to those of corresponding mRNA. Indeed, total TGF-β1 level rose (4-fold) after 1 d of culture, and then leveled off. The level of total and mature TGF-β2 showed a stronger increase after 1 d of culture without ovarian steroids (total 7-fold; mature 4-fold) and further increased at the second day of culture (total 20-fold; mature 10-fold). In contrast to mRNA, total and mature TGF-β3 protein expression increased by 5-fold after 1 d of culture and then leveled off at the second day of culture. Combined estradiol and progesterone inhibited the expression of the three TGF-β isoforms, after 1 d for TGF-β2 and after 2 d for TGF-β1 and -β3.

These experiments showed that TGF-β2 and -β3 were differentially expressed in cultured endometrial explants, although both were repressed by ovarian steroids.

Effect of estradiol and progesterone on TGF-β mRNA level in microdissected glands and stroma
Because the identity of TGF-β-producing cells in the human endometrium is still unclear, we speculated that the differential regulation observed in vivo (Figs. 1Go and 2Go) and on explant culture (Figs. 3Go and 4Go) could reflect a distinct cellular origin. The level of TGF-β mRNA was therefore analyzed separately in epithelial glands and stroma after isolation by laser capture microdissection from explants cultured for 24 h in the absence or presence of combined estradiol and progesterone. The procedure is illustrated in Fig. 5AGo. The three TGF-βs showed distinct cellular expression profiles (Fig. 5BGo): 1) TGF-β1 mRNA level was similar between stroma and glands and was not consistently affected by ovarian steroids, as expected; 2) TGF-β2 mRNA was much more abundant in stroma than in glands, both when explants were cultured in the absence (35-fold) or presence of ovarian steroids (26-fold); furthermore, TGF-β2 expression was strongly inhibited by estradiol and progesterone, both in glands (6-fold) and stroma (10-fold); and 3) TGF-β3 mRNA level was 14-fold higher in stroma than glands in the absence of ovarian steroids but identical in their presence because ovarian steroids decreased by 30-fold the TGF-β3 expression in stromal cells, down to the level of the hormone-insensitive epithelial cells. Thus, TGF-β2 and -β3 expression further showed a distinct regulation by ovarian steroids between stroma and glands.


Figure 5
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  		FIG. 5. Effect of estradiol and progesterone on TGF-β mRNA levels in microdissected glands and stroma. A, Selectivity of laser capture microdissection of stromal compartments (a–c) and epithelial glandular cells (d–f) from explants. a and d, Broken lines outline selected compartments; b and e, fields after microdissection and expulsion of selected cells; c and f, recovery of microdissected tissues in the tube caps. B, TGF-β mRNA expression in stromal (open circles) and glandular compartments (closed circles) isolated by laser capture microdissection. mRNA of glandular or stromal compartments isolated from three proliferative endometria cultured for 24 h in the absence (–H) or presence of combined estradiol and progesterone (+EP) was linearly amplified, reverse-transcribed with specific primers for TGF-βs and β-actin-2, and quantified by real-time PCR. Normalized mRNA levels were expressed as relative values by reference to the stromal compartment microdissected from explants cultured without ovarian steroids and are represented as geometric means with SEM.

 
Effects of cAMP and retinoic acid on TGF-β mRNA expression
We were puzzled by the apparent contradiction between two observations: ex vivo, the surge of TGF-β2 mRNA expression in explant culture was inhibited by progesterone, whereas in vivo, TGF-β2 mRNA level abruptly increased at the midsecretory phase, i.e. when progesterone concentration is maximal and stromal decidualization is initiated. Because decidualization is induced by both progesterone and cAMP (22), we looked for an additional regulation by cAMP. To this aim, explants were cultured for up to 2 d with 8-bromoadenosine-cAMP (Fig. 6Go). This treatment was effective because exposure to cAMP increased by about 10-fold the expression of prolactin mRNA already after the first day of culture (Fig. 6AGo). Prolactin expression was apparently induced in both decidualizing stromal cells and epithelial glandular cells (Fig. 6BGo), as previously reported (23). cAMP reversed the inhibition of TGF-β2 mRNA by combined estradiol and progesterone partially after 24 h of culture and totally after 48 h (Fig. 6CGo). At the protein level, reversion was moderate (Fig. 6DGo). cAMP had no effect on TGF-β1 and -β3 expression, neither at the mRNA nor at the protein level. Again, TGF-β3 was poorly affected by estradiol and progesterone at the protein level, despite a strong decrease of the mRNA level.

Retinoic acid was reported to stimulate the expression of TGF-β2 in mouse keratinocytes (24) and regulate the expression of TGF-βs in the endometrium (25). At 1 µM, we found no appreciable effect on the expression of the TGF-β mRNA in human endometrial explants (data not shown).

Involvement of MAPK in the regulation of TGF-β mRNA expression
As a further attempt to unravel the molecular mechanisms responsible for the divergent regulation of TGF-β isoforms in vivo and ex vivo, the effect of MAPK signaling cascades was explored because of the presence of different binding sites for MAPK effectors on the promoter of each TGF-β isoform. Specific inhibitors of ERK (U0126) or p38 (SB-203580) pathways were tested in explants cultured for 24 h (mRNA) or 48 h (proteins), in combination or not with ovarian steroids (Fig. 7Go). Whereas U0126 specifically and totally inhibited ERK phosphorylation after 48 h of culture, SB-203580 specifically inhibited p38 phosphorylation by 70% (data not shown). Both inhibitors had strikingly opposite effects on TGF-β2 and TGF-β3 expression (Fig. 7Go, A and B). Indeed, TGF-β2 mRNA and protein levels were decreased by the addition of SB-203580 in the absence of ovarian steroids and by both inhibitors in their presence. Conversely, both inhibitors increased TGF-β3 mRNA and protein levels in explants cultured with ovarian steroids but increased only TGF-β3 mRNA level in their absence. TGF-β1 mRNA level was affected by neither SB-203580 nor U0126, whereas TGF-β1 protein level was slightly decreased by both inhibitors in the absence of hormone. This provided an additional evidence of the opposite regulation between TGF-β2 and TGF-β3.

Finally, to test the relevance of MAPK pathways in the cycling human endometrium and the effect of ovarian steroids on their activity in cultured explants to ovarian steroids, the expression and phosphorylation of ERK and p38 were analyzed by Western blotting. ERK and its phosphorylated form were detected in all samples throughout the menstrual cycle without obvious difference between phases (Fig. 7DGo). ERK was also detected in explants in which its phosphorylation increased after 24 h and 48 h of culture without hormone (Fig. 7CGo). Whereas p38 showed no variation throughout the menstrual cycle, its phosphorylated form was not detected except in one menstrual sample of four tested (data not shown). In contrast, p38 phosphorylation was readily detected in explant culture and increased after 24 and 48 h of culture without hormones (Fig. 7CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates three different global expression profiles in the human endometrium, throughout the menstrual cycle and in cultured explants, for the five actors of the TGF-β system, namely the three TGF-β isoforms and the two receptors TGFβ-RI and -RII. It further clarifies the cell-specific expression of the three TGF-β isoforms and their distinct response to ovarian steroids. An opposite regulation of TGF-β2 and TGF-β3 was also singled out, for the first time, by experimental manipulation of cAMP and MAPK pathways.

TGF-βs are general regulators of cell proliferation, differentiation, and ECM turnover and are proposed as major actors of endometrium differentiation for implantation and pregnancy (1). In particular, TGF-βs have been reported to mediate the repression by progesterone of several MMPs, thereby preventing ECM menstrual breakdown (9, 10). Accordingly, expression of TGF-βs was predicted to predominate during the secretory phase of the menstrual cycle, in particular at decidualization, when ECM synthesis is reprogrammed and most MMPs are repressed by high progesterone concentrations. At the opposite, we found that the three TGF-βs showed a high global expression at the menstrual phase when the endometrium breaks down and is shed but distinct temporal expression profiles, suggesting different roles in the cycling endometrium. In contrast, TGFβ-R mRNA level was rather stable throughout the cycle. TGF-β1 expression was remarkably high throughout the cycle and further increased at menstruation. TGF-β2 level was much lower than TGF-β1 around ovulation and then increased at the midsecretory phase, with a 2- to 3-d lag between the mRNA and the protein level and remained high during the menstrual phase. Although occurring late during the secretory phase (around d 21), this rise could contribute to mediate the inhibition of MMP expression. Using part of the same collection of endometrial samples as further exploited in the present study, we previously reported that mRNA of lefty-A/endometrial bleeding-associated factor (EBAF), an inhibitor of TGF-β signaling, increases during the late secretory phase of the cycle to peak at menstruation (19). The protein lefty-A/EBAF is also more abundant at the end of the secretory phase and during menstruation (26). Lefty-A/EBAF increases the expression of MMP-3, -7, and -9 in human endometrium (19, 27). A balance between lefty-A/EBAF and TGF-β2 expression could thus modulate MMP expression at the late secretory and menstrual phases of the cycle. TGF-β3 expression level was also low after ovulation but, in contrast to TGF-β2, steadily increased throughout the rest of the cycle to culminate at the end of the proliferative phase. Because TGF-βs are involved in wound healing (5), TGF-β3 could participate in the tissue repair, which is already initiated at menstruation and proceeds during the proliferative phase.

Altogether these results agree with some previous studies but are at variance with other reports (12, 13, 14, 15, 16, 17, 18). The considerable interpatient variation observed at each phase of the cycle likely explains discrepancies when comparing small series. We suggest that the large number of patients we could collect brings more confidence in the distinct expression profiles we identified among the three TGF-βs throughout the cycle. Furthermore, this is, to the best of our knowledge, the first quantitative analysis of the expression profile of the three TGF-β isoforms at the protein level, including their mature form, which is relevant for their biological effects. The profile of each mature form evolved in parallel to the total level (mature and latent forms) throughout the cycle.

To investigate the regulation underlining these distinct profiles, especially between TGF-β2 and -β3, whereas avoiding interpatient variation, the effects of physiological concentrations of ovarian steroids were tested on paired explants. Endometrium explants showed strikingly different effects between TGF-β2 and TGF-β3 expression: in the absence of ovarian steroids, TGF-β2 mRNA strongly increased in both proliferative and secretory explants, whereas TGF-β3 mRNA did not change in proliferative explants and even transiently decreased in secretory explants. An opposite regulation of TGF-β2 and TGF-β3 expression has been previously reported in keratinocytes (28). Whereas TGF-β2 protein showed a strikingly similar pattern to its mRNA, TGF-β3 protein increased during the first day of culture in contrast to the mRNA. This may reflect either a rapid but transient increase of mRNA, a specific posttranscriptional regulation of TGF-β3 mRNA (29), or an inhibition of protein degradation. To further investigate whether differences in global mRNA regulation could result from distinct cellular responses, we looked for TGF-β mRNA in glands and stroma isolated by laser capture microdissection. This approach showed that stroma contributes the bulk of TGF-β2 and -β3 mRNAs, at least in cultured explants. Our rigorous measurements by real-time RT-PCR after laser capture microdissection partially confirm a previous in situ hybridization analysis (18). In addition, ovarian steroids strongly inhibited TGF-β2 mRNA in both stroma and glands and to the same extent, whereas it inhibited TGF-β3 expression only in stroma, in agreement with a previous report on purified stromal cells (12). The lack of effect of progesterone on TGF-β3 expression by epithelial cells, which express the progesterone receptor during the proliferative phase of the cycle, was already reported for MMP-7 in purified epithelial cells (9). Because progesterone inhibits both TGF-β2 and -β3 in the stroma but not the glands, we may conclude that progesterone regulates TGF-β2 and TGF-β3 expression by different mechanisms.

TGF-β1 mRNA and protein and TGFβ-R mRNA levels were not appreciably affected during culture. Progesterone inhibited the expression of all three TGF-βs and estradiol antagonized this effect for TGF-β1 mRNA. Estradiol was shown to increase TGF-β1 mRNA expression in purified endometrial stromal cells, whereas medroxyprogesterone acetate had a slight stimulatory effect (12). The inhibition by progesterone of TGF-β expression in endometrial explants we consistently observed after 2 d of culture contrasts with previous studies, which reported that progesterone increases the mRNA level of TGF-β1 and TGF-β2 in explants cultured for 4 d (9, 11). To reconcile these two lines of evidence, one could speculate on a biphasic effect of progesterone: first inhibition and then stimulation, as already observed for cyclin D1 in human breast cancer cells (30).

Surprisingly, mature TGF-β1 was found in small amount in only a limited number of cultured explants, whereas it was abundant in vivo throughout the menstrual cycle. There was no such difference between in vitro and in vivo conditions for the other TGF-βs. This suggests either that TGF-β1 mature isoform is much more sensitive to proteolytic degradation in vitro than in vivo, or that TGF-β1 is activated by different mechanisms than TGF-β2 and TGF-β3. This alternative activation would require molecular partners absent in the culture system, such as those supplied through blood. Furthermore, the decrease of the proportion of mature TGF-β2 at the secretory phase also suggests a distinct mechanism of activation regulated throughout the cycle. TGF-βs are secreted as latent complexes; bind to various ECM and pericellular molecules; and can be activated by plasmin, several MMPs, thrombospondin, and integrins (31, 32, 33). Plasmin is thought to play a minor role in TGF-β1 activation because plasminogen KO mice do not reproduce the phenotype of TGF-β1 KO mice (34). In contrast, thrombospondin-1 KO mice exhibit a similar pattern of inflammation to TGF-β1 KO mice, suggesting a major role of thrombospondin-1 in TGF-β1 activation (35). Platelet-derived thrombospondin-1 is thus a potential activator of TGF-β1 in the endometrium.

The distinct regulation of TGF-β genes likely results from differences in their promoter (36). TGF-β2 and TGF-β3 promoters contain the classical TATA box elements; there is neither a TATA nor a CAAT box in TGF-β1 promoter. In addition, no estrogen- or progesterone-receptor binding element has been identified in the promoter of any TGF-β, suggesting that ovarian steroids receptors do not interact directly with them. Promoters of TGF-β1 and TGF-β3, respectively, contain seven and three putative binding sites for specificity protein-1, but none is present in TGF-β2 promoter. In contrast, activator protein-1 sites have been identified in TGF-β1 and TGF-β2 promoters but not in that of TGF-β3. Interestingly, both TGF-β2 and TGF-β3 promoters contain a cAMP response element (CRE)/activating transcription factor (ATF), suggesting that cAMP may regulate their expression. Indeed, overexpression of both CRE binding protein and ATF increased TGF-β2 promoter activity in embryonal carcinoma cells (37), and we here report that cAMP prevented the inhibition by progesterone of TGF-β2 expression in endometrial explants, suggesting that cAMP contributes to the midsecretory increase of TGF-β2 mRNA level in vivo. Prostaglandins E2 and F2{alpha}, which are produced during the mid-late secretory phase (38), could stimulate cAMP synthesis at that phase of the cycle.

Because the promoters of TGF-β isoforms have differences in binding sites for MAPK effectors, i.e. activator protein-1, specificity protein-1, and CRE/ATF sites, we further investigated the effect of MAPK inhibitors on TGF-β expression. MAPKs are activated by a variety of stimuli, including by growth factors via the ERK pathway and by stress via the p38 pathway. Both pathways were reported to stimulate expression of the three TGF-βs in cultured cells (39, 40, 41). In human endometrial explants, TGF-β2 and TGF-β3 were oppositely regulated by MAPK: both ERK (in combination with ovarian steroids) and p38 pathways stimulated the expression of TGF-β2 but inhibited that of TGF-β3 without appreciable effect on TGF-β1. Inhibition of TGF-β3 at the protein level was observed only in combination with estradiol and progesterone.

ERK was reported to be phosphorylated in response to progesterone, prolactin, and relaxin (42, 43, 44), whereas estradiol stimulated p38 phosphorylation (45). In the explant culture system, both ERK and p38 pathways were activated, and combined estradiol and progesterone partially inhibited this activation. However, the opposite effects of MAPK on TGF-β2 and TGF-β3 were maintained despite the inhibition. Furthermore, the effect of ERK on TGF-β2 and both pathways on TGF-β3 protein was not observed in the absence of estradiol and progesterone, suggesting that ovarian steroids cooperate with MAPK pathways to regulate the expression of TGF-βs. In vivo, we found that ERK was phosphorylated throughout the cycle without variation between phases of the cycle in contrast to a previous report of intense ERK2 expression and activity during the secretory phase (46). We could not detect the phosphorylated form of p38 except in one menstrual endometrium of the four tested, although phosphorylated p38 was intensely immunolocalized in the functionalis of proliferative and secretory endometria in another study (45). The ERK pathway could thus be a main regulator of TGF-β expression in vivo, in cooperation with ovarian steroids as suggested in cultured explants, even though activation of this pathway is not increased at the secretory phase.

Although they share the same TGFβ-RI and -RII receptors, TGF-β isoforms also interact at different affinities with the coreceptors endoglin and betaglycan. For instance, endoglin binds TGF-β1 and TGF-β3 but not TGF-β2 (47), and ligand interaction with endoglin may differentially modulate signaling through TGFβ-RI (48). Together with differences in sequestration, activation, and coreceptor binding affinities, the differential temporal expression of TGF-βs throughout the menstrual cycle and their opposite regulation by MAPK points to a distinct role of TGF-β isoforms in the cycling human endometrium, as observed during embryogenesis (49). This concept could also be relevant for other tissues undergoing extensive remodeling, such as wound healing and carcinomas.


   Acknowledgments
 
We are grateful to the patients as well as Professor J. Donnez, the staff of the Gynaecologic Department of the Cliniques Universitaires Saint-Luc, Drs. B. Van Der Meersch and J.-C. Verougstraete for providing the endometrial tissues. We also thank Professor Y. Guiot for help in the laser capture microdissection technology.


   Footnotes
 
This work was supported by the Fonds de la Recherche Scientifique (FRS), the Interuniversity Attraction Poles and the Concerted Research Actions of the Communauté Française de Belgique, and the Fonds Spécial de Recherche of the Université Catholique de Louvain. P.H. is research associate, and P.B.C. was research fellow at the FRS. H.P.G.C. is recipient of a fellowship from the Fonds pour la Recherche en Industrie et en Agronomie.

Disclosure Statement: H.P.G.C., P.B.C., D.D., P.L., P.J.C., P.H., and E.M. have nothing to declare.

First Published Online November 26, 2007

1 P.H. and E.M. were equal senior authors. Back

Abbreviations: ATF, Activating transcription factor; CI, confidence interval; CRE, cAMP response element; EBAF, endometrial bleeding-associated factor; ECM, extracellular matrix; GAPDH, glyceraldehyde phosphate dehydrogenase; KO, knockout; MMP, matrix metalloproteinase; TBS, Tris-buffered saline; TGFβ-R, TGF-β receptor.

Received June 25, 2007.

Accepted for publication November 14, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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