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Department of Obstetrics, Gynecology, and Reproductive Sciences (Y.S., H.C., U.A.K., A.A.), Yale University School of Medicine, New Haven, Connecticut 06520; and Department of Histology and Embryology (Y.S., U.A.K.), Akdeniz University School of Medicine, Antalya 07070, Turkey
Address all correspondence and requests for reprints to: Aydin Arici, M.D., Division of Reproductive Endocrinology and Infertility, Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, Connecticut 06520-8063. E-mail: aydin.arici{at}yale.edu.
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Abstract |
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Objective: We hypothesized that estrogen might activate the p38 MAPK pathway in endometrial cells and exert some of its actions through this pathway in the endometrium.
Interventions: p38 MAPK phosphorylation was analyzed using in vivo and in vitro techniques.
Results: Total and phosphorylated p38 MAPK immunostainings were more intense in epithelial cells compared with stromal cells, and the phosphorylated/total p38 MAPK ratio was significantly higher in the functional endometrial layer compared with the basal layer (P < 0.05). Estradiol significantly increased p38 MAPK phosphorylation in endometrial stromal cells in culture within 2 min (P < 0.05), and this phosphorylation was blocked by a specific p38 MAPK inhibitor. Moreover, tamoxifen and raloxifene also increased phosphorylation of p38 MAPK. The estrogen receptor antagonist ICI 182,780 reversed the estrogen-induced p38 MAPK phosphorylation in endometrial stromal and epithelial cells, suggesting involvement of the estrogen receptor.
Conclusion: Our results indicate the involvement of estrogen in regulating p38 MAPK activity in endometrial cells, suggesting a nongenomic action of estrogen through this MAPK in the endometrium.
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Introduction |
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and ERß) and interacting with the estrogen response element on the promoter region of a target gene (2). However, in addition to this well-established genomic pathway, it has also been shown that E2 can exert early physiological effects that are extremely rapid. The time course of these effects is similar to those elicited by growth factors and peptide hormones, suggesting the presence of a "nongenomic" pathway for estrogen action (3, 4). Physiologically important nongenomic estrogen signaling pathways occur in human vascular endothelial cells, human breast cancer cells (MCF-7), and rat primary cortical neurons (5, 6, 7). There is compelling evidence showing that E2 activates distinct signal transduction pathways, such as protein kinase A, phosphotidylinositol-3 kinase, and MAPK signaling pathways (7, 8). These rapid nongenomic effects of estrogen on multiple signaling pathways have been recently attributed to cell membrane-localized ER (5, 9).
MAPKs compose a family of protein kinases, whose function and regulation have been conserved during evolution (10). MAPKs phosphorylate specific serines and threonines of target protein substrates and regulate cellular activities ranging from gene expression, mitosis, movement, metabolism, survival, and programmed cell death. The MAPK superfamily consists of three well-characterized subfamilies (11). ERKs respond to growth factors or other external mitogenic signals and are involved in promoting cell proliferation. The p38 MAPK and c-Jun N-terminal kinase pathways are distinguished by generally being activated in response to stress and therefore are called the "stress-activated kinases" that promote inflammation and programmed cell death (12, 13).
The p38 MAPK family consists of four isoforms, , ß, 
, and 
, which are activated by several stimuli, including hormones, ligands for G protein-coupled receptors, inflammatory cytokines such as IL-1 and TNF-, and stresses such as UV radiation and osmotic and heat shock (14). The substrates of p38 MAPKs are usually either other protein kinases or transcription factors. It has also been shown that p38 MAPK phosphorylates and activates the molecular chaperone protein, heat shock protein 27 (15), which has been implicated in cellular movement. The downstream activities of p38 MAPK include cytokine production, apoptosis, cell-cycle arrest, regulation of RNA splicing or stabilization, and cell differentiation (16).
The human endometrium is a dynamic structure that undergoes cyclical changes that include proliferation and differentiation in response to ovarian steroid hormones. Given the role of p38 MAPK in a variety of cellular processes such as cytokine expression and proliferation, which occur in the endometrium under the influence of E2, we hypothesized that E2 may exert some of its actions by activating the p38 MAPK through the nongenomic pathway in endometrial cells. To determine the effects of E2 on p38 MAPK phosphorylation, we analyzed in vivo p38 MAPK phosphorylation in the endometrium by immunohistochemistry and in vitro in endometrial stromal cells (ESCs) and epithelial cells by Western blot analysis and ELISA.
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Subjects and Methods |
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For immunohistochemical studies, endometrial tissues were obtained from 27 fertile women (mean age, 35.7 yr; range, 28–44 yr) with regular menstrual cycles undergoing laparoscopy or hysterectomy for benign gynecological conditions other than endometrial disease. The indications for the surgeries were leiomyomata (n = 17), elective sterilization (n = 6), benign adnexial masses (n = 3), and myometrial hypertrophy (n = 1). The day of the menstrual cycle was established from the women’s menstrual history and was confirmed by endometrial histology using the criteria of Noyes et al. (17). All endometrial samples were grouped according to the menstrual cycle phase: proliferative (n = 13) and secretory (n = 14) phases.
For ESC cultures, endometrial samples (n = 24, 15 from the proliferative and 9 from the secretory phase of the cycle) were obtained at the time of hysterectomy or laparoscopy from fertile women (mean age, 38.3 yr; range, 29–48 yr), who did not receive any hormonal medications in the preceding 3 months. The indications for the surgeries were leiomyomata (n = 15), elective sterilization (n = 5), and benign adnexial masses (n = 4). The endometrial samples were placed in Hank’s balanced salt solution and transported to the laboratory for ESC isolation and long-term culture. Written informed consents were obtained from each patient using consent forms and protocols approved by the Human Investigation Committee of Yale University.
Immunohistochemistry
Formalin-fixed paraffin-embedded samples were cut into 5-µm sections. After deparaffinization, slides were boiled in 10 mM citrate buffer (pH 6.0) for 15 min for antigen retrieval. Then, sections were immersed in 3% hydrogen peroxide (in 50% methanol/50% distilled water) for 15 min to block endogenous peroxidase. Slides were then incubated in a humidified chamber with 5% blocking horse serum (LabVision, Fremont, CA) in Tris-buffered saline (TBS) for 30 min at room temperature. Afterward, excess serum was drained and sections were incubated with primary antibodies [rabbit polyclonal antihuman p38 MAPK antibody, 1:200 dilution in TBS (Cell Signaling Technology, Beverly, MA) and rabbit monoclonal antihuman phospho-specific p38 MAPK (pTpY180/182) antibody, 1:200 dilution in TBS (Cell Signaling Technology)] overnight at 4 C in a humidified chamber. For negative controls, normal rabbit IgG isotypes were used at the same concentrations. Term placenta sections from uncomplicated pregnancies were used as positive controls (18). The sections were washed three times for 5 min with TBS, and then biotinylated goat antirabbit antibody (1.5 mg/ml; Vector Laboratories, Burlingame, CA) was added at 1:400 dilution for 30 min at room temperature. The antigen-antibody complex was detected by using an avidin-biotin-peroxidase kit (LabVision). Diaminobenzidine (3,3-diaminobenzidine tetrahydrochloride dihydrate; LabVision) was used as the chromogen, and sections were counterstained with hematoxylin.
The intensity for total and phosphorylated p38 MAPK immunoreactivity was semiquantitatively evaluated using the following intensity categories: 0, no staining; 1+, weak but detectable staining; 2+, moderate or distinct staining; 3+, intense staining. For each tissue, a histological score (HSCORE) value was derived by summing the percentages of cells that stained at each intensity category and multiplying that value by the weighted intensity of the staining, using the formula HSCORE = 
Pi (i + l), where i represents the intensity scores, and Pi is the corresponding percentage of the cells. In each slide, five randomly selected areas were evaluated under a light microscope (x40 magnification), and the percentage of the cells for each intensity within these areas was determined at different times by two investigators blinded to the type and source of the tissues. The intraindividual and interindividual coefficients of variation were 10 and 12%, respectively, for the HSCORE evaluation. The average score of two was used.
Chemicals and cell line
E2, progesterone (P), and tamoxifen were purchased from Sigma-Aldrich (St. Louis, MO). The ER antagonist ICI 182,780 (ICI) was obtained from Tocris Cookson (Ballwin, MO). The p38 MAPK pathway inhibitor SB203580 was obtained from Calbiochem (San Diego, CA). Estrone, 17-ethinyl estradiol, raloxifene, and Ishikawa cells (a well-differentiated endometrial adenocarcinoma cell line) were a kind gift from Dr. R. Hochberg (Yale University School of Medicine, New Haven, CT).
Isolation and culture of human ESC
ESCs were separated and maintained in monolayer culture as described previously (19). Briefly, endometrial tissue was minced with a sterile surgical blade and digested in Hank’s balanced salt solution (Sigma-Aldrich) containing collagenase B (1 mg/ml, 15 U/mg; Roche, Indianapolis, IN), deoxyribonuclease I (0.1 mg/ml, 1500 U/mg; Roche), penicillin (200 U/ml), and streptomycin (200 mg/ml) for 60 min at 37 C with agitation. The dispersed endometrial cells were separated by filtration through a wire sieve (73-µm-diameter pore; Sigma-Aldrich) and were cultured in DMEM Ham’s F-12 (1:1 vol/vol; Sigma-Aldrich) containing fetal bovine serum (10% vol/vol; Invitrogen, Carlsbad, CA). The cultures were maintained in a standard 95% air/5% CO2 incubator at 37 C.
Experimental setup
After one passage, ESCs were plated in 100-mm culture plates and grown to confluence. ESCs after first passage were assayed immunocytochemically using specific cell-surface markers and were found previously to contain 0–7% epithelial cells, no detectable endothelial cells, and 0.2% macrophages (20, 21). The confluent ESCs and epithelial (Ishikawa) cells were treated with serum-free, phenol red-free media (Sigma-Aldrich) for 24 h before treatment with steroids. Each experiment with ESCs was repeated at least three times using cells prepared from endometrial tissue specimens obtained from at least three different patients.
For Western blot analysis, cell cultures were treated with vehicle (control), E2 (10–6 to 10–12 M), P (10–7 M), or the specific ER antagonist ICI (10–6 M) alone or with E2. To evaluate the effect of a p38 MAPK inhibitor, the cultures were preincubated with SB203580 (1–20 µM) for 30 min before the treatment with E2 (10–8 M) for 10 min. In another experimental setup, cell cultures were treated with vehicle, E2 (10–8 M), estrone (10–7 M), 17-ethinyl estradiol (10–7 M), raloxifene (10–6 M), or tamoxifen (10–7 M) for 5, 10, and 20 min.
Western blot analysis
Total protein from treated ESCs and Ishikawa cells was extracted with cell extraction buffer (BioSource International, Camarillo, CA) containing 3 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma-Aldrich). The protein concentration was determined by a detergent-compatible protein assay (Bio-Rad, Hercules, CA). Samples (40 µg) were loaded on 10% Tris-HCl Ready Gels (Bio-Rad), electrophoretically separated, and electroblotted onto nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 h to reduce nonspecific binding. Subsequently, the membrane was incubated for 2 h with primary antibodies against total and phosphorylated p38 MAPK [polyclonal rabbit antihuman p38 MAPK and monoclonal rabbit antihuman phospho-specific p38 MAPK (pTpY180/182), both at 1:1000 dilution, in 2% nonfat dry milk in TBS-T (Cell Signaling Technology)]. The membrane was washed with TBS-T for 1 h and incubated with horseradish peroxidase-conjugated antirabbit secondary antibody (Vector Laboratories) diluted at 1:10000 in TBS-T. The protein was visualized by light emission on film (Amersham Biosciences, Buckinghamshire, UK) with enhanced chemiluminescence substrate (Amersham Biosciences). Immunoblot bands for total and phosphorylated p38 MAPK were quantified using a laser densitometer.
Immunoassay for p38 MAPK
The cells were preincubated in a specific p38 MAPK inhibitor, SB203580 (1–20 µM) or with vehicle for 30 min. Afterward, cell cultures were treated with E2 (10–8 M) for 10 min. Protein was isolated as described above. Total and phospho-p38 MAPK proteins were quantified using immunoassay kits according to instructions provided by the manufacturer (BioSource International). The sensitivities of these kits were 16 pg/ml for human total p38 MAPK and 0.8 U/ml for human phospho-p38 MAPK (pTpY180/182). The intraassay and interassay coefficients of variation were 5.1 and 7.3% for total p38 MAPK assay and 4.3 and 8.4% for phospho-p38 MAPK assay, respectively. The quantified phospho-p38 MAPK content was normalized to the total p38 MAPK content.
Statistical analysis
Because the data from the immunoassay and Western blot were not normally distributed (as determined by Kolmogorov-Smirnov test), pairwise multiple comparisons were analyzed with nonparametric ANOVA on ranks (Kruskal-Wallis test), followed by post hoc Student-Newman-Keuls test. In contrast, the data from immunohistochemistry were normally distributed and, therefore, were analyzed with Student’s t test or one-way ANOVA, followed by post hoc Holm-Sidak test when appropriate. Statistical calculations were performed using SigmaStat for Windows, version 3.0 (Jandel Scientific, San Rafael, CA). Statistical significance was defined as P < 0.05.
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Results |
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Total p38 MAPK immunoreactivity was both cytoplasmic and nuclear in the endometrial epithelial cells and ESCs (Fig. 1
). However, phospho-p38 MAPK staining was mostly nuclear. Both total and phospho-p38 MAPK stainings were more intense in epithelial cells compared with ESCs (P < 0.05). Their expression did not change throughout the menstrual cycle. Epithelial and ESCs in the functional endometrial layer showed the highest total and phospho-p38 MAPK immunoreactivity, and their expression declined precipitously in the basal layer (P < 0.05). However, phosphorylated/total p38 MAPK ratio was significantly higher in the functional endometrial layer compared with the basal layer (P < 0.05) (Fig. 2). Moreover, phosphorylated/total p38 MAPK ratio was also higher in epithelial cells compared with ESCs in the functional layer (P < 0.05) (Fig. 2).
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To investigate the effects of sex steroids on p38 MAPK activation, cultured ESCs were treated with vehicle, E2 (10–8 M), or P (10–7 M) for 5 and 10 min. ESCs in culture expressed both p38 MAPK and phospho-p38 MAPK. The total p38 MAPK levels did not show any change with time or any of the treatments.
According to Western blot analysis, E2 induced a rapid increase in the phosphorylation of p38 MAPK. In contrast, P decreased p38 MAPK phosphorylation at both time points investigated (Fig. 3A).
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To determine the time course of this E2 action, cells were treated with E2 (10–8 M) for 0, 2, 5, 10, 20, 30, 60, and 180 min. Western blot analysis revealed that E2 was able to increase phospho-p38 MAPK levels within 2 min (P < 0.05). The E2-induced phosphorylation of p38 MAPK was transient, and the levels of phospho-p38 MAPK started to decline at 20 min and reached the baseline levels within 1 h (Fig. 3C).
E2-induced activation of p38 MAPK is inhibited by a specific p38 MAPK inhibitor
To assess the effect of a p38 MAPK inhibitor on the induction of p38 MAPK phosphorylation by E2, we pretreated the cells for 30 min with either vehicle or SB203580 (1 to 20 µM) and afterward added E2 (10–8 M) to the cultures for 10 min. The immunoassay confirmed the Western blot analysis results in which E2 increased p38 MAPK phosphorylation. SB203580 significantly inhibited E2-induced phosphorylation of p38 MAPK at all concentrations used (P < 0.05). Pretreatment with SB203580 (10 µM) induced a 60 ± 3% inhibition of E2-induced phosphorylation (P < 0.05) (Fig. 4). SB203580 alone also induced a significant decrease in basal phospho-p38 MAPK levels (P < 0.05). Interestingly, E2 was able to induce p38 MAPK phosphorylation even in the presence of SB203580 (P < 0.05).
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To investigate the ER-mediated p38 MAPK activation, cells were treated with vehicle (control), E2 (10–8 M), the specific ER antagonist ICI (10–6 M), or E2 plus ICI for 5, 10, and 20 min. E2 rapidly and significantly increased p38 MAPK phosphorylation within 5 min (P < 0.05), with the maximum effect observed at 10 min (41 ± 2% above control levels; P < 0.05). Then at 20 min, the amount of phospho-p38 MAPK was similar to the control levels. When used alone, ICI induced a slight nonsignificant increase in p38 MAPK phosphorylation when compared with untreated cells. Conversely, the administration of ICI with E2 totally reversed the E2-induced activation of p38 MAPK (57 ± 3% decrease compared with E2 alone; P < 0.05 at 10 min) at all of time points investigated (Fig. 5A). Likewise, E2 increased the phosphorylation of p38 MAPK in endometrial epithelial (Ishikawa) cells, and addition of ICI blunted the effect of E2 on p38 MAPK phosphorylation at 10 min (Fig. 5B).
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To determine whether p38 MAPK activation is also regulated by selective ER modulators (SERMs) and other estrogen derivatives, ESCs were treated with vehicle, E2 (10–8 M), estrone, (10–7 M), 17-ethinyl estradiol, a synthetic ester of estrogen (10–7 M), and two SERMs, raloxifene (10–6 M) and tamoxifen (10–7 M), for 5, 10, and 20 min.
Western blot revealed that all of the treatments induced a minimal but nonsignificant increase in phospho-p38 MAPK levels at 5 min. At 10 min, only E2 and tamoxifen induced significantly higher levels of phospho-p38 MAPK compared with control (P < 0.05) (Fig. 6).
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Discussion |
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In addition to its ability to promote ER-dependent gene transcription, estrogen rapidly triggers a variety of second-messenger signaling events, including the stimulation of cAMP (23), calcium mobilization (3), generation of inositol phosphate (24), and activation of the MAPKs (25). Although it is not clear by which mechanisms these rapid signaling events occur, because of the quickness (within 5 min) by which they are activated, it is presumed that they are initiated at the plasma membrane or cytoplasm and do not involve ER-mediated gene transcription.
Knowledge of the ability of E2 to activate rapid nongenomic mechanisms has evolved during the past two decades. The ability of E2 to induce these effects has been recently attributed to the cell-surface ERs. Morey et al. (26) described the presence of membrane receptors in primary cultures of human vascular smooth muscle cells, which appear to be ERs, and also have shown that, when transfected into Chinese hamster ovary cells, the ER is membrane bound and can activate several signaling pathways, such as the ERK and c-Jun N-terminal kinase pathways (27). Previously, Guzeloglu-Kayisli et al. (28) have shown that E2 rapidly induces the phosphorylation of Akt/protein kinase B (PKB), which is a downstream regulator of phosphotidylinositol-3 kinase in ESCs. Recently, Acconcia et al. (29) demonstrated the stimulation of Akt/PKB, ERK, and p38 MAPK by E2 in ER- and/or ERß-containing cell lines. They showed that, although E2 stimulated p38 MAPK in both ER- and ERß-expressing cell lines, it stimulated the phosphorylation of Akt/PKB and ERK in only ER-expressing cell lines. Although many studies have so far addressed some of the nongenomic actions in various tissues and cells, to our knowledge, our study is the first to determine the regulation of p38 MAPK by E2 in detail in human endometrial cells.
In the present study, we demonstrated the in vivo expression of total and phosphorylated p38 MAPK in human endometrium. We did not find any cyclical changes in the expression of both total and phosphorylated p38 MAPK throughout the menstrual cycle. We may speculate that presence of various growth factors or cytokines capable of activating p38 MAPK such as IL-11 and leukemia inhibitory factor in the secretory phase is likely to mask the increase in phosphorylated p38 MAPK in proliferative phase (29, 30, 31, 32).
In the present study, we also demonstrated that E2 rapidly induces the phosphorylation of p38 MAPK in ESCs. We observed that E2-induced phosphorylation of p38 MAPK in ESCs is rapid and transient, which occurs within 2 min and starts to return to baseline levels after 20 min.
There are contradictory results about the effects of SB203580 on p38 MAPK phosphorylation. Although some studies indicate that SB203580 does not inhibit the phosphorylation but rather inhibits the downstream activities of p38 MAPK (33, 34), there are some other studies showing the inhibitory effect of this agent on p38 MAPK phosphorylation (35, 36). The present study revealed that SB203580 decreased both E2-induced and basal levels of phospho-p38 MAPK in ESCs. Strikingly, E2 stimulated the p38 MAPK phosphorylation even in the presence of SB203580 to a degree suggesting the involvement of another pathway in the phosphorylation of p38 MAPK by E2.
In our study, similar to that of Acconcia et al. (29), the E2-induced p38 MAPK phosphorylation was prevented by the pure ER antagonist ICI, whereas ICI alone induced a slight but nonsignificant increase in the phosphorylation of p38 MAPK. There are many contradictory results of the ICI effect on rapid estrogen signaling. For instance, it has been shown previously that E2-induced phosphorylation of Akt/PKB was not inhibited by ICI in ESCs (28). It has also been reported that ICI prevents estrogen-induced activation of ERK-1 and ERK-2 (25). In our study, the inhibition of the stimulatory effect of E2 on p38 MAPK phosphorylation by ICI implies the involvement of ERs in the achievement of this rapid and specific effect. These observations correlate with other studies that demonstrate that several steroid hormones and their antihormones may act through membrane receptors to facilitate rapid nongenomic signaling.
Furthermore, we observed that estrone, one of the three naturally occurring estrogens, as well as the SERMs raloxifene and tamoxifen, and 17-ethinyl estradiol, a synthetic estrogen component of many oral contraceptives, also rapidly phosphorylate p38 MAPK, suggesting the involvement of these agents in the regulation of this pathway in ESCs, which may have additional implications that need to be investigated.
Phosphorylated p38 MAPK contributes to the activation of nuclear transcription factors, including nuclear factor 
B, which regulates the gene expression of various cytokines and adhesion molecules (37). Recently, Mori-Abe et al. (38) have shown that E2 induces apoptosis in vascular smooth muscle cells by activating the p38 MAPK pathway. Arici et al. (39, 40, 41) has shown previously that the endometrium is capable of producing local cytokines such as IL-8 and monocyte chemotactic protein-1 in a cycle-dependent manner, suggesting the involvement of sex steroids in the expression of these cytokines. Other studies have shown that IL-6 and IL-8 mRNA stability is regulated by p38 MAPK (42) and that monocyte chemotactic protein-1 (43), IL-12p40 (44), and granulocyte-macrophage colony-stimulating factor expressions (45) are all p38 MAPK dependent. Based on these studies, the finding that E2 rapidly induces the phosphorylation of p38 MAPK in ESCs has implications in the regulation of the timely expression of several cytokines in the endometrium under the influence of sex steroids.
In conclusion, we have shown that E2 stimulates p38 MAPK in ESCs, and this effect is too rapid to be mediated through the genomic pathway. Moreover, E2-induced phosphorylation of p38 MAPK is inhibited by a specific ER antagonist, which altogether implies a membranous or cytosolic ER in the rapid and specific E2-induced activation of p38 MAPK signaling. Our results indicate the involvement of estrogen in regulating p38 MAPK, suggesting a nongenomic action of estrogen through this MAPK pathway in the endometrium. Therefore, estrogen may exert part of its effects in the endometrium through the p38 MAPK pathway.
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Acknowledgments |
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Footnotes |
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First Published Online March 7, 2006
Abbreviations: E2, Estradiol; ER, estrogen receptor; ESC, endometrial stromal cell; HSCORE, histological score; ICI, ICI 182,780; P, progesterone; PKB, protein kinase B; SERM, selective estrogen receptor modulator; TBS, Tris-buffered saline; TBS-T, TBS containing 0.1% Tween 20.
Received September 26, 2005.
Accepted March 1, 2006.
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and ERß expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319 and ERß activated non-genomic signaling. J Cell Physiol 203:193–201[CrossRef][Medline], and transforming growth factor-ß expression in endometrium, placenta, and placental membranes. Am J Obstet Gynecol 163:1430–1437[Medline]- or IL-1ß-stimulated rheumatoid synovial fibroblasts. FEBS Lett 465:23–27[CrossRef][Medline]-induced expression of monocyte-chemoattractant protein-1 in endothelial cells. Blood 93:857–865This article has been cited by other articles:
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, P < 0.01 vs. stromal cells in secretory phase.
