Mol. Hum. Reprod. Advance Access originally published online on March 9, 2007
Molecular Human Reproduction 2007 13(5):317-322; doi:10.1093/molehr/gam001
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PI3K/Akt And ERK1/2 signalling pathways are involved in endometrial cell migration induced by 17ß-estradiol and growth factors
1 Ospedale Maggiore Policlinico-Mangiagalli-Regina Elena, University of Milano, Milano 2 Department of Obstetrics and Gynaecology, Clinica ‘Macedonio-Melloni’, University of Milano, Milano 3 Istituto Auxologico Italiano, Via Zucchi 18, Cusano Milanino, Milano
4 To whom correspondence should be addressed at: Molecular Biology Laboratory, Istituto Auxologico Italiano Via Zucchi 18, Cusano Milanino, Mi, Italy. E-mail: a.diblasio{at}auxologico.it
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Abstract |
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Cell motility and invasion are crucial events for endometrial cells, not only for the establishment of pathological states but also during the physiological tissue remodelling that occurs during the menstrual cycle and embryo implantation. We have characterized these phenomena in endometrial stromal cells evaluating cell migration-specific stimuli and the biochemical pathways involved. Ability of endometrial cells to migrate on collagen type IV substrate was evaluated by means of chemotaxis experiments. Modulation of this phenomenon by different growth factors and steroid hormones and their ability to activate extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/Akt signalling in this context were examined. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor and fibroblast growth factor-2 as chemoattractant agents stimulated basal migration of endometrial stromal cells through the rapid activation of both ERK1/2 and PI3K/Akt signalling pathways. Experiments using wortmannin and PD98059, specific inhibitors of the PI3K/Akt and ERK1/2 activity, respectively, showed that the activation of both pathways is required for growth-factor-induced cell motility responses. Similarly, 17ß-estradiol (10–6–10–8 M) could enhance both constitutive and PDGF-induced migration of the cells and their rapid treatment with the hormone significantly increased phosphorylation of ERK1/2 and Akt. Conversely, progesterone did not interfere with the basal migration but inhibits the PDGF-induced motility of this cell type. Rapid activation of intracellular signalling cascades ERK1/2 and PI3K/Akt by growth factors and estrogens is involved in the migration of normal endometrial stromal cells.
Key words: endometrium/ERK1/2/migration/PI3K/Akt
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Introduction |
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Cell migration is a coordinated physiological process used by normal cells during several processes such as embryonic morphogenesis, wound healing and cell trafficking and by neoplastic cells to spread within tissues (Kassis et al., 2001; Alessandro and Khon, 2002; Friedl and Wolf, 2003; Mareel and Leroy, 2003). The protein–protein interactions and signalling events that regulate cell migration involve (i) an initial propulsion due to the elongation of leading pseudopodes driven by actin polymerization and assembly to filaments; (ii) binding of the growing cell protrusions to the adjacent extracellular matrix (ECM) via adhesion molecules, most notably receptors of the integrin family; (iii) the development of focal contacts from focal complexes derived from integrin clustering; (iv) recruitment of surface proteases towards attachment sites, which in turn degrade ECM components; (v) cell contraction by actin stress fibres. Eventually, these events lead to the generation of membrane protrusions and traction forces, which allow the cell to change shape and establish interactions with ECM substrates (Friedl and Wolf, 2003).
Human uterine endometrium is a plastic tissue in which cells undergo a variety of adaptation reactions in response to the physiological changes that occur in the different phases of the cycle and during embryo implantation (Salamonsen, 2003; Matsumoto et al., 2005). Unlike most normal adult tissues, the functional layer of the uterine endometrium undergoes cyclic growth and tissue remodelling throughout the reproductive years. The remodelling of endometrial tissue, which is regulated by ovarian steroids as well as by various cytokines and growth factors, shares features with repair of mucosal injury characterized by a migratory phenotype with specialized cytoskeletal and matrix-receptor reorganizations and specialized matrix-dependent signalling patterns (Salamonsen, 2003; Matsumoto et al., 2005). In various species, during implantation, endometrial focal adhesions develop as aggregates composed of ECM proteins, integrins and cytoskeletal proteins, which promote and stabilize the attachment of trophectoderm (Johnson et al., 2003). Moreover, in pathological conditions such as endometriosis, endometrial cell spreading and invasion imply migration as a critical process in terms of changes in adhesion molecule expression profiles for the binding to a different ECM environment and gain of a proteolytic activity for the implantation into the peritoneal surface (Viganò et al., 2004; Yoshida et al., 2004).
Migration is regulated by many gene products and complicated signalling integrated in the concept of focalized adhesion. Main protagonists are protein kinases such as extracellular signal-regulated protein kinase (ERK)1/2, which are the most widely expressed members of the mitogen-activated protein (MAP) kinase family, the phosphatidylinositol 3 kinase (PI3K), the focal adhesion kinase (FAK) and others that can be activated by growth factors, cytokines and ECM (Friedl and Wolf, 2003). Interestingly, the importance of estrogens in modulating rapid signalling effects that act on these targets has been recently highlighted (Acconcia and Kumar, 2005; Acconcia et al., 2006).
Notwithstanding the importance of these events, to date, process of migration of normal endometrial cells has not been characterized in detail. The phenomenon has been studied in endometrial cancer cell lines (Acconcia et al., 2006), without sufficient information in the physiological condition. Cell migration-specific stimuli, the potential involvement of steroid hormones and the biochemical pathways involved remain to be established. To address these questions, we have analysed the regulation of endometrial cell migration by growth factors and steroid hormones. A second goal was to determine the possible mechanism(s) and pathway(s) underlying such regulation.
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Reagents and antibodies
Platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), fibroblast growth factor (FGF)-2, 17ß-estradiol (E2), progesterone, PD98059 and wortmannin were from Sigma as well as collagen type IV as a solubilized basement membrane preparation extracted from Engelbreth–Holm–Swarm mouse sarcoma. Antibodies used were: rabbit polyclonal anti-ERK (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and anti-phospho-ERK (Thr-202/Tyr-204) (New England Biolabs, Beverly, MA, USA), rabbit polyclonal anti-phospho-Akt (Ser-473) and Pan-Akt (Biosource International, Nivelles, Belgium). Secondary antibody conjugated to peroxidase, ECL and Hybond ECL nitrocellulose membranes was from Amersham Biosciences, Cologno Monzese, MI, Italy. BCA Protein Assay Kit was obtained from Pierce Biotechnology, Rockford, IL, USA.
Sample collection
Human samples were obtained from women who attended the endoscopic surgical service of the Department of Obstetrics and Gynecology of the University of Milan at ‘Macedonio-Melloni’ clinic to undergo gynaecological laparoscopy for pelvic pain, benign ovarian pathology or leiomyomas. Women with previous autoimmune, neoplastic, hepatic or thyroid disorders were excluded from the study. All subjects were younger than 40, had regular menstrual cycles and none had received hormones for at least 3 months.
Samples of uterine endometrium were obtained from n = 32 women. Laparoscopic examination demonstrated normal pelvic organs in n = 7 cases, benign ovarian pathology in n = 13 cases and benign uterine pathology in n = 12 cases. Fourteen samples were in proliferative and n = 18 in the secretory phase of the cycle. Dating was based on the last menstrual period and histological examination of the samples.
Patients were informed in detail that tissue samples would be used for research purposes and they gave written consent. Approval for this study was granted by the local Human Institutional Investigation Committee.
Cell preparation and culture
We established stromal cell monolayers from eutopic endometrial samples (Viganò et al., 2002; Mihalich et al., 2003). Briefly, the tissues were gently minced into small pieces (1–2 mm3) and washed in fresh medium to remove mucus or debris. Thereafter, they were incubated for 2 h at 37°C in a shaking water bath in 10 ml Dulbecco's modified Eagle's medium (DMEM) containing 0.1% collagenase. At the end of the incubation, cell clumps were mechanically dispersed by aspiration through a Pasteur pipette. Single stromal cells were separated from large clumps of epithelium during a 10 min period of differential sedimentation at single gravity. The top 8 ml of medium, containing predominantly stromal cells, were then slowly removed and the cells were collected by centrifugation (200g). The stromal-enriched fraction was washed twice in DMEM supplemented with 10% FBS and antibiotics and allowed to adhere selectively to 25 cm2 tissue culture dishes for 15 min at 37°C in a 95% air and 5% CO2 incubator. Thereafter, non-attached epithelial cells still present were removed and a purified stromal preparation was obtained. Endometrial cells were cultured to subconfluence in DMEM with 10% FBS and antibiotics in a humidified atmosphere of 95% air and 5% CO2 at 37°C. Subconfluence was reached after a minimum of 8 days, during which the culture medium was changed every other day. Before each experiment, cells were serum-deprived for 24 h. To evaluate the effects of steroid hormones on endometrial migration, before the assay, cells were cultured in DMEM for 24 h with and without two different doses of E2 (10–6 and 10–8 M) or with progesterone at 10–7 M or with a combination of the two steroids.
Cell migration assay
Endometrial stromal cell migration was evaluated by means of chemotaxis experiments in a 48-well-modified Boyden chamber. The Boyden chamber system uses two hollow plastic chambers, separated by a porous membrane. During the migration assay, cells can migrate towards a chemoattractant added in a lower part of the chamber through the pores of the membrane which can be coated with components of the ECM (in our case, collagen type IV) able to stimulate cell adhesion without occluding the micropores. Briefly, the chemotaxis experiments were performed using 8 µm nucleopore polyvinylpyrrolidone-free polycarbonate filters coated with 10 µg ml–1 of type IV collagen and placed over a bottom chamber containing various amounts of PDGF or EGF or FGF-2 or E2 (10–6 and 10–8 M) as the chemoattractant factors. Serum-free medium was used as a negative control. In some experiments, cells were pretreated for 30 min with PD98059 (10 µM) or wortmannin (100 nM). Cells were added to the upper chamber at a density of 4 x 104 cells/well. After 6 h of incubation at 37°C, the non-migrated cells on the upper surface of the filter were removed by scraping. The cells that had migrated to the lower side of the filter were stained with Diff-Quick stain (VWR Scientific Products, Bridgeport, NJ, USA), and 5–8 unit fields per filter were counted at 200x magnification using a Zeiss microscope. The assays were run in triplicate.
Western blot analysis
Samples of endometrial stromal cell cultures were treated with a lysis buffer containing 150 mM NaCl, 10 mM Tris–HCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulphonyl fluoride, 10 µg ml–1 leupeptin and 10 µg ml–1 aprotinin. Lysates were subsequently centrifuged at 13 000g for 15 min and the supernatant was collected for protein analysis. Sample protein concentration was determined using a commercial BCA protein assay kit. Equal amounts of protein from cell lysates were resuspended in sample buffer containing 62 mM Tris–HCl (pH 6.8), 2% sodium dodecylsulphate (SDS), 10% glycerol, 5% 2-mercaptoethanol and 0.04% bromphenol blue, resolved by SDS-polyacrylamide gel electrophoresis and transferred to Hybond ECL nitrocellulose membranes. For Akt and ERK1/2 detection, after brief washing in tris-buffered saline Tween-20 (TBST) [25 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.1% Tween-20], the membrane was blocked with 5% skim milk/TBST overnight at 4°C. All subsequent steps were performed at room temperature. The membrane was incubated for 3 h with the indicated primary antibody diluted 1:1000 in 25 ml of 5% skim milk. After incubation with the appropriate secondary peroxidase-conjugated antibody, the membrane was washed in TBST for 30 min and the immunoreactive bands were visualized with chemoluminescence.
Statistical analysis
Difference between groups was compared, as appropriate, by analysis of variance and the Fisher PLSD test as post-test. Probability < 0.05 was considered statistically significant.
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Migration of endometrial stromal cells is growth-factor-mediated and involves both Akt and ERK1/2 activation
PDGF-BB, EGF and FGF-2 were tested as chemoattractants for endometrial stromal cells using collagen-coated Boyden chamber. Serum-starved cells showed a basal migration behaviour in the Boyden chamber assay. No difference has been observed for the basal migration in relation to the phase of the cycle (data not shown). The chemotaxis assays demonstrated the ability of the cells to increase their migration towards all the three agents, PDGF-BB, EGF and FGF-2 (Figure 1). PDGF-BB significantly stimulated the chemotactic migration of endometrial stromal cells in a dose-dependent manner. The peak stimulatory effect was reached at a concentration of 25 ng ml–1, corresponding to an increase in chemotaxis of 3-fold. Similarly, EGF and FGF-2 exerted a dose-dependent stimulation of endometrial cell migration (data not shown). The peak stimulatory effect of
2- and 1-fold was obtained at doses of 50 and 20 ng ml–1 for EGF and FGF-2, respectively.
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It has been recently shown that the protein kinase Akt and MAPK pathways are involved in PDGF-induced motility in various cell types (Pola et al., 2003). Thus, to elucidate the pathways responsible for the pro-migratory effect of PDGF-BB, EGF and FGF-2 on endometrial cells, cells were pretreated with wortmannin to block the PI3K/Akt pathway or PD98059 to block the ERK1/2 pathway. The results shown in Figure 1 demonstrate that both drugs could reduce growth-factor-stimulated migration, thus suggesting that both pathways are required for the phenomenon. The inhibitory effect of PD98059 was 25, 52 and 37% for PDGF-BB, EGF and FGF-2, respectively. Wortmannin induced an inhibition of 43, 55 and 37% for PDGF-BB, EGF and FGF-2, respectively. In order to exclude that these inhibitory effects were dependent upon the inhibition of the basal migration of endometrial cells, in some experiments, the action of wortmannin and PD98059 alone was tested on the basal migratory response. Both drugs could affect the basal migration of endometrial cells only to a limited extent (percentage of inhibition of control migration was 10% for wortmannin and 9% for PD98059). Assays of cell death did not reveal a loss of cell viability due to pretreatment with inhibitors (data not shown).
To confirm that these intracellular pathways are indeed responsible for the migratory activity of endometrial cells, we evaluated phosphorylation levels of Akt and ERK1/2 in the presence and absence of the different compounds. Figure 2 shows total and phospho-Akt and total and phospho-ERK1/2 in endometrial stromal cells in the presence or absence of different growth factors. Total Akt and ERK1/2 protein levels were similar in cells cultured in the presence or absence of growth factors. The amount of phospho-Akt and phospho-ERK was strongly induced by a short (15 min) incubation with PDGF-BB, EGF and FGF-2 (Figure 2).
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Migration of endometrial stromal cells is hormone responsive
We next investigated whether steroid hormones could impact basal or growth-factor-mediated cellular migration of endometrial cells. The effect that exogenously added E2 and progesterone had upon cell migration was assessed by pretreating cells for 24 h with the compounds before the Boyden chamber assay. Treatment with E2 at different concentrations (10–6 and 10–8 M) resulted in a significant 2-fold increase of migrating cell numbers (Figure 3A). Moreover, pre-incubation of the cells with E2 stimulated the migration induced by PDGF-BB in an additive manner (Figure 3A). In contrast, although pretreatment of cells with progesterone (10–7 M) could minimally and not significantly affect basal migration of endometrial cells, the hormone was able to significantly inhibit the migration induced by PDGF-BB of
35% (Figure 3B). Progesterone could also inhibit, although not significantly, E2-induced migration (by 13%) when the two hormones were added simultaneously (Figure 3A).
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Effects of E2 upon cell migration was also tested after a shorter treatment (6 h) performed by adding the steroid at different concentrations in the lower part of the Boyden chamber system. Results obtained indicate that E2, tested as chemoattractants, can stimulate the migratory behaviour of endometrial stromal cells at both 10–6 and 10–8 M doses (Figure 3C).
Induction of Akt and ERK1/2 phosphorylation by E2 in endometrial stromal cells
As mentioned above, activation of both ERK1/2 and PI3K/Akt pathways was required for the migratory effects of growth factors on endometrial stromal cells. We therefore evaluated the possibility that the stimulatory effect of E2 and the inhibitory effect of progesterone might be mediated by their ability to interfere with the activation of these intracellular pathways. First, we incubated hormone pretreated endometrial cells with specific PI3K/Akt and ERK1/2 pathway inhibitors for 30 min before the chemotaxis assay. The inhibitors could block the E2-mediated induction of migration thus suggesting that the two signalling pathways are indeed involved in the phenomenon (Figure 3). To better clarify this mechanism, the effect of the hormones on Akt and ERK1/2 phosphorylation was studied. Cells were treated with two different concentrations of E2 (10–6 and 10–8 M) for short (5–30 min) and long exposure (24 h) (Figure 4A). E2 treatment for short exposure induced a significant increase in the phosphorylation of Akt and ERK1/2 at both concentrations tested, although the effect was more evident for the lower dose. For both doses, the increase in Akt phosphorylation was evident at 5 min and was sustained at 30 min; a similar pattern of phosphorylation was present for ERK1/2. In contrast, in cells treated with E2 for 24 h, phospho-Akt and phospho-ERK1/2 levels were not different from baseline.
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The ligand-induced ERK and Akt activation was not due to a direct effect of E2 on total protein content, since no changes in the total ERK and Akt expression levels were detected after reprobing the membranes with total Akt and ERK1/2 antibodies. These results indicate that E2 can activate non-genomic signalling pathways in endometrial stromal cells.
Incubation of cells with progesterone at 10–7 M for 24 h did not interfere with basal or PDGF-induced activation of ERK or PI3K/Akt pathways (Figure 4B).
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Discussion |
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The human endometrium undergoes vast architectural modifications during each menstrual cycle (Johnson et al., 2003). At the end of the cycle, with withdrawal of the steroid hormone support, the activity of matrix-degrading enzymes induces endometrial destruction with the resultant loss of blood vessel integrity and shedding of most of the functionalis layer (Salamonsen, 2003). When substantial endometrial tissue loss occurs during menstruation, endometrial stromal cells in-growth, ECM deposition, angiogenesis and tissue contraction act in concert to re-establish organ integrity. In this phase, regeneration begins with the outgrowth of basal glands and in-growth from the intact peripheral surface membrane bordering the denuded basalis. The stromal tissue begins to grow when the endometrial wound is completely re-epithelialized, and endometrial cell movement is necessary to repopulate the space created by tissue loss and to avoid excessive fibroplasia. These events are regulated in large measure by steroid hormones, the timing and concentrations of which dictate the balance between endometrial growth and transformation (Guzeloglu-Kayisli et al., 2004). In the present study, we have shown that growth factors are also very potent promoters of the dynamic behaviour of endometrial stromal cells and that they induce the migration by activating both PI3K/Akt and ERK1/2 pathways. Although this was already known for PDGF-BB, this study provides the first description of this phenomenon also for FGF-2 and EGF. However, the most important novelty of the present study is that E2 can stimulate both basal and growth-factor-mediated motility of these cells and that the steroid hormone can rapidly induce the same signalling routes used by growth factors. In contrast, progesterone had an inhibitory effect on PDGF-mediated migration.
Cell movement is a complex process involving a number of steps, including the disruption of cell–cell junctions, cytoskeletal rearrangements and constant remodelling of adhesive contacts with the ECM. It is this complexity of events that makes the evaluation of the entire phenomenon particularly interesting in the context of the human endometrium as a dynamic tissue (Kassis et al., 2001; Friedl and Wolf, 2003; Mareel and Leroy, 2003). We have been able to identify a novel cellular mechanism supporting the role of E2 in stimulating the remodelling of normal endometrial tissue during the menstrual cycle, which has the ability to favour motility of these cells. In the same way, we have been able to observe an opposite action of progesterone. Together with proliferation, apoptosis, adhesion and invasion, the processes described herein are certainly supportive of physiological tissue plasticity and implantation preparation.
Depending on the cell type, various signalling pathways have been shown to be involved in the growth-factor-induced motility (Anand-Apte and Zetter, 1997; Alessandro and Kohn, 2002; Geraldes et al., 2002; Pola et al., 2003). The PI3Ks are a family of lipid kinases that phosphorylate the 3'-OH group of the inositol ring of membrane-bound phospho-inositides. The main phospholipid formed (phosphatidylinositol-3,4,5-trisphosphate) and the protein kinase that is activated by it (Akt) trigger a cascade of responses from cell growth and proliferation to survival and motility (Alessandro and Kohn, 2002). On the other hand, MAPK pathways are also crucial signal transducing systems that are involved in the regulation of important cellular processes like cell proliferation, cell survival and also in some cases, cell motility (Alessandro and Kohn, 2002). The results presented herein indicate that disruption of the PI3K cascade by a specific inhibitor prevents the ability of endometrial cells to migrate in response to PDGF-BB, EGF and FGF-2. Moreover, the ERK1/2 pathway also potently interferes with the regulation of cell motility, as PD98059 (a specific blocker of this pathway activation) has an inhibitory effect on growth-factor-stimulated migratory behaviour. The role of ERK1/2 in growth-factor-induced motility is controversial and could depend on the cell type studied (Pola et al., 2003). The PDGF-induced chemotaxis of Rat1 fibroblasts was unaffected by the PD98059 treatment (Anand-Apte et al., 1997). In contrast, the chemotactic response to PDGF was dependent on ERK1/2 in human mesangial cells (Choudhury et al., 1997) and retinal pigment endothelial cells (Hinton et al., 1998). Our data support the idea that both PI3K/Akt and ERK pathways mediate PDFG-BB-, EGF- and FGF-2-stimulated motility responses in endometrial cells. These findings are totally in agreement with the rapid induction of phosphorylation of ERK1/2 and Akt upon growth factor incubation.
Contradictory information is also available on the role of E2 in regulating cell migration. Although E2 induces the event in mammary carcinoma cell lines (Marino et al., 2003), in some endometrial cancer cell lines (Acconcia et al., 2006) and in aortic endothelial cells (Geraldes et al., 2002), recent reports indicate that E2 can inhibit migration in vascular smooth muscle cells (Geraldes et al., 2002). Together, these findings suggest cell-type-specific E2-dependent mechanisms for the modulation of cytoskeletal remodelling and migration. In endometrial cancer cells in particular, these processes have been shown to be dependent upon non-genomic activation of ERK1/2 and FAK/c-Src, which are non-receptor tyrosine kinases regulating specific downstream cytoskeleton-associated signalling pathways (Acconcia et al., 2006). No data have been reported so far for a potential role exerted by E2 in non-cancer endometrial cells in this regard. On the basis of the results of the western blot experiments of the present study, E2 can mediate rapid activation of non-genomic signalling molecules in normal endometrial stromal cells as well and, at least for Akt phosphorylation, this observation is in line with previous reported data (Guzeloglu-Kayisli et al., 2004). Furthermore, the pro-migratory effect of an acute treatment (6 h) of endometrial cells with E2 and the inhibition of E2-induced motility by wortmannin and PD98052 leads to speculation that these non-genomic signalling pathways ERK1/2 and PI3K/Akt are strongly involved in mediating the effect of the steroid on the migration of this population of cells.
We cannot exclude that classical or non-classical genomic effects also induced by estrogens and the reactivation of the intracellular signalling induced by matrix protein engagement during the migration assay might have triggered endometrial cell movement. Supporting this concept, it has been shown that many intracellular signalling molecules are activated by integrin engagement, including components of the Ras/Raf/MEK/ERK pathway, the PI3K/Akt pathway, Src and Abl tyrosine kinases and FAK (Bill et al., 2004). Moreover, in various cell types, estradiol has been demonstrated to increase integrin expression and function (Cid et al., 1999). This could be particularly true for the higher dose of the hormone whose rapid effects, as shown in western blotting, were limited. Further studies are needed to definitively clarify this aspect.
Results obtained treating cells with progesterone alone tend to rule out any action of the hormone on endometrial cell migration. However, the hormone was able to inhibit the PDGF- and E2-mediated increase of cell motility, probably through a classical genomic effect. Indeed, we failed to observe any influence of progesterone on the PDGF-induced activation of ERK1/2 and PI3K/Akt pathways. In this regard, it has to be noted that there is a convergence of progesterone with growth factor signalling in different tissues (Richer et al., 1998; Lange et al., 1998). A cross talk between the two occurs at the level of STATs and progestin treatment regulates expression of type I growth factor receptors. Moreover, it has been demonstrated that progestins can decrease the secretion of PDGF isoforms in breast cancer cells. Again, other studies are needed for a proper assessment of the overall effects of progestins on endometrial cell dynamics.
In conclusion, the results of this study support the following observations: (i) human endometrial stromal cells are constitutively able to migrate on collagen type IV substrate; (ii) growth factors as chemoattractant agents stimulate basal migration of these cells through the rapid activation of ERK1/2 and PI3K/Akt signalling pathways; (iii) pretreatment with E2 for 24 h ‘prime’ these cells for both constitutive and PDGF-induced migration signals; E2 is also able to stimulate cell migration when used as chemoattractant. Non-genomic signalling cascades are likely to have an impact on these effects and (iv) pretreatment with progesterone for 24 h does not interfere with the basal migration but inhibits the PDGF-induced motility of this cell type.
The rapid stimulation of non-classical intracellular pathways by steroid hormones in relation to endometrial tissue remodelling warrants further investigations.
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Submitted on January 5, 2007; accepted on January 8, 2007.
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