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Endocrinology, doi:10.1210/en.2006-1673
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Endocrinology Vol. 148, No. 7 3176-3184
Copyright © 2007 by The Endocrine Society

Increased Phosphorylation of Myosin Light Chain Prevents in Vitro Decidualization

Ivanna Ihnatovych, WenYang Hu, Jody L. Martin, Asgerally T. Fazleabas, Primal de Lanerolle and Zuzana Strakova

Departments of Obstetrics and Gynecology (I.I., A.T.F., Z.S.) and Physiology and Biophysics (W.H., P.d.L.), University of Illinois at Chicago, Chicago, Illinois 60612; and Loyola University Medical Center (J.L.M.), Maywood, Illinois 60153

Address all correspondence and requests for reprints to: Zuzana Strakova, Ph.D., Department of Obstetrics and Gynecology, The University of Illinois at Chicago, 820 South Wood Street (M/C 808), Chicago, Illinois 60612-7313. E-mail: zstrakov{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Differentiation of stromal cells into decidual cells, which is critical to successful pregnancy, represents a complex transformation requiring changes in cytoskeletal architecture. We demonstrate that in vitro differentiation of human uterine fibroblasts into decidual cells includes down-regulation of {alpha}-smooth muscle actin and ß-tubulin, phosphorylation of focal adhesion kinase, and redistribution of vinculin. This is accompanied by varied adhesion to fibronectin and a modified ability to migrate. Cytoskeletal organization is determined primarily by actin-myosin II interactions governed by the phosphorylation of myosin light chain (MLC20). Decidualization induced by cAMP [with estradiol-17ß (E) and medroxyprogesterone acetate (P)] results in a 40% decrease in MLC20 phosphorylation and a 55% decline in the long (214 kDa) form of myosin light-chain kinase (MLCK). Destabilization of the cytoskeleton by inhibitors of MLCK (ML-7) or myosin II ATPase (blebbistatin) accelerates decidualization induced by cAMP (with E and P) but inhibits decidualization induced by IL-1ß (with E and P). Adenoviral infection of human uterine fibroblast cells with a constitutively active form of MLCK followed by decidualization stimuli leads to a 30% increase in MLC20 phosphorylation and prevents decidualization. These data provide evidence that the regulation of cytoskeletal dynamics by MLC20 phosphorylation is critical for decidualization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DIFFERENTIATION OF uterine stromal cells into decidual cells (decidualization) is critical for human implantation and establishment of pregnancy. Decidual cells control trophoblast invasion and protect the embryo from maternal immune rejection. During decidualization, fibroblast-like stromal cells change morphologically and functionally into epithelial-like secretory cells. Decidual cells generate local secretion of different regulators of trophoblast invasion (reviewed in Refs. 1 and 2). The major secretory products of the human decidualized endometrium include IGF binding protein (IGFBP)-1 (3) and prolactin (4).

Despite the importance of decidualization, little is understood about the factors and pathways involved in this process. A previous in vivo study in the baboon (5) and in vitro studies in baboon and human stromal fibroblasts established the cytokine IL-1ß as one of the mediators of decidualization (6, 7). This was based on previous identification of IL-1 as a paracrine factor that modulates the communication between the human maternal endometrium and the embryo (8, 9). Because cAMP leads to in vitro decidualization of baboon and human stromal fibroblasts, it is widely used as an exogenous decidualization stimulus (10, 11). Microarray analysis of human uterine fibroblasts treated with cAMP to induce decidualization demonstrated a striking reprogramming of genes associated with extracellular matrix, tissue strength and tissue integrity, suggesting that these changes are a prominent feature of decidualization (12, 13, 14).

The cytoskeleton plays a crucial role in cellular processes such as smooth muscle contraction, mitosis, cell growth, cell motility, aging, and apoptosis (see review in Ref. 15). The organization and the plasticity of the cytoskeleton are determined primarily by the forces generated within it by actin-myosin II interactions (16) that are governed by the phosphorylation and dephosphorylation of myosin light chain (MLC20) (17, 18). The phosphorylation status of MLC20 is mainly regulated by myosin light-chain kinase (MLCK), which phosphorylates MLC20 of myosin II on Thr18 and Ser19 residues (17, 19, 20).

Because the cytoskeleton provides the necessary intracellular scaffolding to support and organize the cellular components, its ability to deform and reform is critical for differentiation. Even though decidualization has been extensively studied, many questions remain regarding the molecular mechanisms that regulate cytoskeletal reorganization during this process. Previously we demonstrated that the differentiation of stromal fibroblasts into the secretory decidual phenotype is initially associated with changes in the cytoskeleton, particularly with the down-regulation of {alpha}-smooth muscle actin (SMA) (5, 6, 10). We also showed that the disruption of the actin filaments by cytochalasin D promotes decidualization (21).

The aim of this study was to investigate the molecular mechanism of cytoskeleton reorganization underlying decidualization induced by an exogenous stimulus (cAMP) and a stimulus of embryonic origin (IL-1ß). Our goal was to test the hypothesis that MLC20 phosphorylation, by affecting cytoskeletal dynamics, plays a central role in the ability of stromal cells to decidualize.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IL-1ß was obtained from R&D Systems, Inc. (Minneapolis, MN). ML-7 and blebbistatin were purchased from EMD Biosciences (La Jolla, CA). Mouse monoclonal antihuman {alpha}-SMA antibody clone 1A4 was from Dako (Glastrup, Denmark). Monoclonal ß-actin antibody clone AC 15, monoclonal antibody to vinculin, and N6, 2'-O-dibutyryladenosine cAMP were purchased from Sigma Chemical Co. (St. Louis, MO). Mouse monoclonal antibody to ß-tubulin was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies to focal adhesion kinase (FAK) and FAK phosphorylated on Tyr925 (FAK925) were from Cell Signaling Technology, Inc. (Beverly, MA), and mouse p27 was from BD Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibody to MLCK and rabbit polyclonal antibody to MLC20 were prepared and characterized previously (22, 23). Rhodamine-conjugated phalloidin and Prolong antifade mounting medium containing 4',6'-diamino-2-phenylindole were purchased from Molecular Probes (Eugene, OR). All cell culture supplies were obtained from Invitrogen (Grand Island, NY). Other reagents of cell culture grade were purchased from Fisher Scientific (Itasca, IL) or Sigma.

Cell culture
Human uterine fibroblasts (HuF) were isolated from the decidua parietalis dissected from the placental membranes after normal vaginal delivery at term, as previously described (6). These cells represent a proliferating population of nondifferentiated fibroblastic cells, which are maintained in the decidualized uterine endometrium and closely resemble endometrial stromal cells (24, 25). Although HuF cells are not identical with stromal cells isolated from human endometrium, an advantage lies in their easy availability from term placenta, robust proliferative abilities, and their confirmed use as a model for in vitro decidualization in several studies (6, 13, 24, 25). All studies were approved by the Institutional Review Board of the University of Illinois.

Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated and charcoal-stripped fetal bovine serum (SFBS), 0.1 mM sodium pyruvate, and 1% penicillin/streptomycin. At confluence cells were trypsinized, propagated, and used for experiments in passage numbers three to five. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (Dako) and vimentin (Zymed Laboratories, Inc., San Francisco, CA). The purity of fibroblast cells used in studies was greater than 95%.

Treatment of cells
Treatment with decidualization stimuli.
Decidualization studies were carried out in RPMI 1640 with 2% SFBS. Confluent HuF cells were subjected to no treatment (control) or treated for 6 or 12 d with the decidualization stimuli: 10 ng/ml IL-1ß or 0.1 mM dibutyryl cAMP [both in the presence of 36 nM estradiol-17ß (E) and 1 µM medroxyprogesterone acetate (P), sometimes referred to in the manuscript as steroid hormones (H)]. Cell culture medium was changed every 2 d.

Treatment with inhibitors.
All drug experiments were carried out at 80–90% cell confluence in six-well plates in serum-free RPMI 1640 supplemented with sodium pyruvate and penicillin/streptomycin. HuF cells were pretreated for 4 h with 20 µM ML-7 or 50 µM blebbistatin. After the pretreatment with inhibitors, medium was exchanged for RPMI 1640 with 2% SFBS, and the cells were treated with decidualization stimuli as described above. The medium was collected on 3 and 6 d of treatment and frozen at –70 C until further analysis. All experiments were repeated a minimum of three times and each experiment was done in triplicate.

Immunodetection analysis
After each treatment, HuF cells were rinsed twice with PBS and lysed on ice with lysis buffer as previously described (6). The protein concentration was estimated by the Bradford assay (Bio-Rad Laboratories, Inc., Webster, TX). Equal amounts of total protein (10 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After the transfer, the blots were probed with specific antibodies against {alpha}-SMA, ß-actin, ß-tubulin, FAK, FAK925, and p27 according to the protocols provided by the manufacturers. The immunoreactive bands were detected by enhanced chemiluminescence.

Preparation of cells with a truncated catalytic domain of MLCK (HuF-tMK)
An adenovirus that contains an active form of MLCK (A-tMK) was made by homologous recombination in an adenovirus packaging cell line (AD-293 cell) to transfer the tMK gene (26) into the adenovirus genome. Briefly, the tMK DNA (1.2 kb) was inserted into a shuttle vector, pACCMVpLpASR (8.8 kb), at EcoRI/HindIII sites. This construct was cotransfected with pJM17 (40.3 kb), which contains the human adenovirus type 5 genome, into AD-293 cells using SuperFect transfection reagent kit (QIAGEN, Valencia, CA). Recombinant adenoviruses were amplified and cesium chloride gradient purified, dialyzed, and titered by standard methods. A-tMK viruses were identified by DNA sequencing. Confluent HuF cells grown in six-well plates were infected with an empty adenovirus (EA) or increasing concentrations (1 x 107, 2 x 107, 4 x 107, and 8 x 107 plaque-forming units of the A-tMK for 2 h in serum-free RPMI 1640 medium. RPMI 1640 medium with 5% SFBS was added for further incubation for 48 h. Preliminary experiments using a green fluorescent protein vector construct resulted in nearly 100% infection efficiency. The expression of tMK protein was verified in cell lysates by immunoblotting with specific antibodies against MLCK (22). Overall toxicity of constructs was determined by a cell viability assay using trypan blue staining of HuF-EA and HuF-tMK after 6 d of infection. There was no significant difference observed in the total number of cells in HuF-EA and HuF-tMK in comparison with noninfected controls. The percentage of dead cells in noninfected controls, HuF-EA, and HuF-tMK was 13.0 ± 6.2, 8.0 ± 2.7, and 14.6 ± 8.7%, respectively.

Induction of decidualization in HuF-tMK and HuF-EA cells
HuF cells grown in six-well plates were infected with 3 x 107 plaque-forming units of the EA or A-tMK in serum-free RPMI 1640 medium. After 2.5 h of infection, culture medium was changed into 2% SFBS, and the cells were subjected to no treatment (control) or treated for 6 d with the decidualization stimuli. Cell culture medium was changed every 2 d. On 6 d of treatment, the medium was collected and the IGFBP-1 and prolactin concentrations were measured using an ELISA kit (Diagnostic Systems Laboratories, Webster, TX). The cells were rinsed twice with ice-cold PBS, and the cellular proteins were precipitated with ice-cold 10% trichloroacetic acid (TCA) and 10 mM dithiothreitol and used for further analysis as described below.

Measurement of MLC20 phosphorylation
Changes in MLC20 phosphorylation in HuF were quantified according to Chew et al. (27). Briefly, the TCA precipitates were washed with ethyl ether; dissolved in 9 M urea, 10 mM dithiothreitol, and 20 mM Tris (pH 7.5); and separated using urea-glycerol PAGE. The proteins were transferred to polyvinylidene difluoride membrane, and the un-, mono-, and diphosphorylated forms of MLC20 were identified using an affinity-purified antibody to MLC20 (23) and horseradish peroxidase-conjugated secondary antibody (Bio-Rad Laboratories). Protein bands were visualized using an enhanced chemiluminescence kit. The stoichiometry of phosphorylation (mol PO4/mol MLC20) was calculated as a ratio of phosphorylated forms to total MLC20 as previously described (23).

Immunofluorescence staining
HuF cells grown on glass coverslips and exposed to decidualization treatments for 6 d were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and then blocked with 5% BSA at room temperature. Incubations with primary antibodies against {alpha}-SMA (1:500), vinculin (1:100), and FAK925 (1:100) were conducted at room temperature for 2 h, followed by incubation for 1 h with fluorescein isothiocyanate-conjugated secondary antibodies (1:100). For double staining of filamentous actin (F-actin) and vinculin, rhodamine-conjugated phalloidin was included with a secondary fluorescein isothiocyanate-conjugated antibody. Coverslips were mounted with Prolong antifade containing 4',6'-diamino-2-phenylindole and examined using an LSM 510 laser confocal microscope (Carl Zeiss Microimaging, Thornwood, NY).

Wound-healing assay
HuF cells grown on glass coverslips were treated with cAMP in the presence of E and P for 6 d. Then wounds were made (350–400 µm) across the monolayer of stimulated HuF and untreated controls using 10 µl micropipette tips. The coverslips were rinsed twice with PBS to remove wound-derived cell aggregates, and the medium was replaced very carefully. The coverslips were incubated in complete media for 16 h at 37 C, 5% CO2, and subsequently fixed and processed for immunofluorescence as described above. Quantitative measurements of the distances between the wound edges (by averaging the width of four randomly chosen regions of single wound) were made at 0 and 16 h, and the percentage of the closure were calculated as described by Nobes and Hall (28).

Adhesion assay
HuF cells grown in six-well plates were subjected to decidualization stimuli for 6 d. The ability of control and treated HuF cells to adhere to fibronectin was detected using Innocyte extracellular matrix (ECM) cell adhesion microplate assay (EMD Biosciences) according to the manufacturer’s protocol.

Statistical analysis
Statistical analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL). Results are expressed as mean ± SD. One-way ANOVA was used to test the null hypothesis of group differences, followed by a two-tailed t test for pairwise comparison or post hoc tests using Tukey’s and Bonferroni correction for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Changes in the cytoskeleton organization during in vitro decidualization
To understand cytoskeletal remodeling during decidualization, HuF cells were treated for 6 and 12 d with two decidualization stimuli: IL-1ß and cAMP (both in the presence of steroid hormones E and P). Western blot analyses demonstrated a significant decline in the {alpha}-SMA protein on d 6 of treatment with cAMP and on d 12 of treatment with IL-1ß (Fig. 1AGo). Stimulation of HuF for 6 d with cAMP or IL-1ß resulted in a decreased level of ß-tubulin. Further treatment for 12 d enhanced the down-regulation of ß-tubulin. No changes were detected in ß-actin during decidualization. In contrast to {alpha}-SMA and ß-tubulin, the expression of the cell cycle inhibitor p27 slightly increased during treatment with cAMP. We also used immunofluorescence to detect changes in cytoskeleton proteins and focal adhesion complex (FAC) proteins (Fig. 1BGo). Immunofluorescence with {alpha}-SMA antibody confirmed a decrease in {alpha}-SMA in HuF undergoing decidualization induced by cAMP, compared with untreated controls (Fig. 1BGo, a and c). Treatment for 6 d with IL-1ß led to significant changes in overall cell shape and organization (Fig. 1BGo, b, e, h, and k). HuF cells became prominently elongated and formed net-like structures with empty spaces between the cell aggregates. The most intense {alpha}-SMA staining was detected on the cell edges (Fig. 1BGo, b). Immunodetection of activated FAK, the key protein of the FAC, and vinculin, one of the FAC proteins, further confirmed the differences in the cytoskeleton architecture in HuF undergoing decidualization induced by different stimuli. Whereas treatment with IL-1ß increased FAK925 staining (Fig. 1BGo, e), treatment with cAMP did not change it at all (Fig. 1BGo, f), compared with untreated controls (Fig. 1BGo, d). Immunodetection of vinculin and filamentous actin revealed: 1) a similar pattern of overall parallel stress fiber organization in HuF cells treated with both decidualization stimuli and in untreated controls (Fig. 1BGo, j, k, and l); 2) vinculin staining follows F-actin fibers in controls (Fig. 1BGo, g and j) and HuF treated for 6 d with IL-1ß (Fig. 1BGo, h and k); 3) notable consolidation of vinculin in HuF treated for 6 d with cAMP (Fig. 1BGo, i and l). Changes in FAK phosphorylation during decidualization were further confirmed by Western blot analysis (Fig. 1CGo). Whereas IL-1ß caused an evident increase in FAK phosphorylation, no changes in the level of FAK925 protein were detected in other treatment groups. The total FAK detection was used as the loading control. These results showed that decidualization in vitro is associated with prominent changes in cytoskeletal organization.


Figure 1
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  		FIG. 1. Changes in the cytoskeleton organization during decidualization. A, Distribution of {alpha}-SMA, ß-tubulin, ß-actin, and p27 in total cell lysates of HuF undergoing decidualization for 6 and 12 d immunodetected with appropriate antibodies. B, Confocal microscopy of immunofluorescent staining of {alpha}-SMA (a–c), FAK925 (d–f), vinculin (g–i), and double F-actin (visualized with rhodamine-conjugated phalloidin)-vinculin staining (j–l) in untreated cells (a, d, g, and j) and HuF treated for 6 d with IL-1ß and H (b, e, h, and k) or cAMP and H (c, f, i, and l). Scale bar, 50 µm. C, The densitometric evaluation of the ratio of FAK925 to total FAK in HuF treated for 6 d with steroid hormones E and P (H), IL-1ß, and E and P (IL-1ß+H) and cAMP and E and P (cAMP+H). Inset, Representative immunoblot of FAK925 during decidualization. The blots were reprobed with antibody to total FAK as loading control (CTR).

 
Cell adhesion and migration during in vitro decidualization
We next investigated how changes in cytoskeletal architecture during decidualization would affect cell adhesion and migration. We determined the ability of HuF treated for 6 d with decidualization stimuli to attach to the ECM protein, fibronectin. Treatment with IL-1ß resulted in a 30% increase in cell adhesion (Fig. 2AGo), whereas treatment with cAMP slightly decreased it. These differences in adhesion to fibronectin in HuF treated for 6 d with two different stimuli correlated with the changes in the cytoskeletal and FAC proteins.


Figure 2
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  		FIG. 2. Changes in adhesion and migration during decidualization. A, HuF cells grown in six-well plates were subjected to decidualization treatment for 6 d and adhesion assays were performed. Note the significant difference (*, P < 0.05) in the ability to adhere to fibronectin between HuF cells treated for 6 d with cAMP and steroid hormones E and P (cAMP+H), compared with those treated with IL-1ß and E and P (IL-1ß+H). There was no significant difference between IL-1ß+H and untreated controls (P = 0.06). B, Confocal images of control HuF (a) and HuF treated for 6 d with steroid hormones E and P, H (b), or cAMP and H (c) after the initial wounding (t = 0); and migration of the cells after 16 h (d–f, untreated or treated with H or cAMP and H, respectively). Wounded cells were fixed at t = 0 and t = 16 h, and actin filaments were visualized with rhodamine-conjugated phalloidin. Scale bar, 200 µm. C, Percent wound closure calculated for wounds in untreated HuF cells (CTR), treated with H, and cAMP with H. Values represent means ± SD for at least three different experiments. Note a significant difference (*, P < 0.05) in wound closure between H, cAMP+H-treated cells, and untreated controls.

 
The wound-healing assay was performed to characterize the ability of cells to migrate after exposure for 6 d to decidualization inducing treatments. Unfortunately, because treatment by IL-1ß results in the net-like cell organization, it made the wounding of these cells impossible. HuF treated for 6 d with steroid hormones E and P (E+P) and cAMP (with E+P) as well as untreated cells were wounded and fixed at t = 0 and t = 16 h, and actin filaments were visualized with phalloidin (Fig. 2BGo). In control cells, there was almost complete coverage of the wound with cells. In contrast, the wound-healing assay demonstrated a significant decrease in migration in HuF treated with E+P and cAMP (with E+P). The calculated percent wound closure was significantly lower, compared with untreated controls (Fig. 2CGo).

Effect of the cytoskeleton destabilization on decidualization
Cytoskeleton organization and dynamics depend on actin-myosin interactions. To investigate the importance of these interactions to the decidualization process, we destabilized the cytoskeleton by inhibiting of either myosin II ATPase activity or MLCK. HuF cells were pretreated for 4 h with a specific myosin II ATPase inhibitor, blebbistatin (50 µM), or a MLCK inhibitor, ML-7 (20 µM) and exposed to decidualization stimuli for 6 d. Results revealed opposite effects of these drugs on decidualization induced by IL-1ß and cAMP as estimated by measurement of the synthesis of markers of decidualization: IGFBP-1 (Fig. 3Go) and prolactin (data not shown). Both blebbistatin and ML-7 significantly increased synthesis of IGFBP-1 in HuF treated by cAMP with E+P (Fig. 3Go, upper panel). In contrast, the drugs significantly inhibited the level of IGFBP-1 in HuF stimulated by IL-1ß with E+P (Fig. 3Go, lower panel). Thus, these results provided further evidence that although different molecular mechanisms underlie decidualization induced by exogenous (cAMP) and embryonic (IL-1ß) stimuli, the cytoskeletal organization is critical for both of them.


Figure 3
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  		FIG. 3. Destabilizing the cytoskeleton has opposite effects on decidualization induced by IL-1ß and cAMP. HuF cells were pretreated with ML-7 (20 µM) or blebbistatin (50 µM) followed by treatment with decidualization stimuli as described in Materials and Methods. The level of IGFBP-1 synthesis was measured in conditioned medium on d 3 and 6 of treatment with cAMP and steroid hormones E and P (cAMP+H; upper panel) and on 6 d of treatment with IL-1ß and steroid hormones (IL-1ß+H; lower panel). Note that whereas pretreatment with both inhibitors caused significant increase in synthesis of IGFBP-1 in HuF undergoing decidualization induced by cAMP+H, it significantly decreased the level of IGFBP-1 in HuF cells treated induced by IL-1ß+H, compared with HuF without pretreatment (–) (*, P < 0.05).

 
MLC20 phosphorylation in HuF cells undergoing decidualization
Because actin-myosin interactions are mainly regulated by the phosphorylation status of MLC20, our next set of experiments aimed to determine the role of MLC20 phosphorylation in cytoskeletal remodeling during decidualization. The un-, mono-, and diphosphorylated forms of MLC20 were identified in TCA precipitates using an affinity purified antibody to MLC20 (23). In the cells treated with cAMP, the band representing diphosphorylated MLC20 almost completely disappeared, which resulted in a 40% decrease in total MLC20 phosphorylation (Fig. 4AGo). Treatment with IL-1ß did not have any effect on MLC20 phosphorylation. Because the level of MLC20 phosphorylation depends on the balance of three enzymes, MLCK, MLC phosphatase and Rho-associated kinase, the amounts of these proteins were detected in the same samples. The changes in MLC20 phosphorylation were associated with a 55% decline in the nonmuscle (long, 214 kDa) form of MLCK (Fig. 4BGo). There were no changes in the amounts of Rho-associated kinase and MLC phosphatase target subunit (data not shown). The results emphasized the importance of MLCK for MLC20 phosphorylation during decidualization induced by cAMP (with E+P).


Figure 4
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  		FIG. 4. Decreased MLC20 phosphorylation and down-regulation of nonmuscle MLCK during decidualization. A, The stoichiometry of MLC20 phosphorylation quantified in control (CTR, no treatment) and HuF treated for 6 d with steroid hormones E and P (H), IL-1ß and steroid hormones (IL-1ß+H), and cAMP and steroid hormones (cAMP+H). The inset shows the migration of the MLC20 species (un-, mono-, and diphosphorylated MLC20 from top to bottom). B, The levels of nonmuscle (long form, black bars) and muscle (short form, white bars) isoforms of MLCK were expressed as percent (±SD) of those determined in untreated (CTR) HuF cells (100%). Inset, Representative Western blot showing changes in protein levels of MLCK isotypes during decidualization. Cell extracts from untreated (CTR) cells and HuF treated with steroid hormones E and P (H), IL-1ß and steroid hormones (IL-1ß+H), and cAMP and steroid hormones (cAMP+H) were resolved by SDS-PAGE and immunoblotted with specific antibody (22 ). Note significant decrease (*, P < 0.05) in nonmuscle isotype of MLCK in HuF treated with cAMP+H in comparison with CTR.

 
Effect of the increased levels of phosphorylated MLC20 on decidualization
Finally, to demonstrate the importance of MLCK in cytoskeletal remodeling underlying decidualization, we manipulated the cytoskeleton using an adenovirus construct containing a truncated, constitutively active form of MLCK. The dose-dependent expression of tMK in HuF was verified by the immunodetection with antibody against MLCK (22). The appearance of a 55-kDa band corresponded to tMK (Fig. 5AGo). Then HuF cells were infected with either an EA or A-tMK construct and subjected to decidualization inducing treatments. The phosphorylation status of MLC20 was detected in TCA precipitates on d 6 of treatment. Western blot analysis revealed that infection of HuF cells with A-tMK caused a 30% increase in total MLC20 phosphorylation (Fig. 5BGo, filled bars), compared with cells infected with EA (Fig. 5BGo, open bars) or noninfected HuF cells (Fig. 4AGo), or treatment with cAMP did not induce a decrease in MLC20 phosphorylation.


Figure 5
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  		FIG. 5. Increasing MLC20 phosphorylation by expressing active MLCK prevents decidualization. A, HuF cells were infected with an EA or increasing concentrations of the A-tMK adenovirus as described in Materials and Methods. The expression of tMK was detected in total cell lysates by immunoblotting with specific antibody to MLCK (22 ). B, The stoichiometry of MLC20 phosphorylation was quantified in HuF transfected with EA (open bars) or A-tMK (filled bars) and consequently treated for 6 d with steroid hormones E and P (H), IL-1ß and steroid hormones (IL-1ß+H), and cAMP and steroid hormones (cAMP+H) or left untreated (CTR). The insets show the migration of the MLC20 species (un-, mono-, and diphosphorylated MLC20 from top to bottom). C, HuF cells were infected with EA (white bars) or A-tMK (black bars) followed by the treatment for 6 d with IL-1ß and steroid hormones (IL-1ß+H) and cAMP and steroid hormones (cAMP+H). IGFBP-1 and prolactin protein released into the medium was measured using an ELISA. Note the significant decrease in IGFBP-1 and prolactin synthesis in HuF treated with A-tMK in comparison with HuF treated with EA (*, P < 0.05).

 
The ability of HuF transfected with EA or A-tMK to transform into decidual cells was verified by measurement of the levels of IGFBP-1 and prolactin (Fig. 5CGo) in conditioned media on d 6 of treatment by specific ELISAs. The results clearly demonstrated that HuF cells infected with EA construct were able to differentiate as indicated by their ability to induce synthesis of both markers (Fig. 5CGo). The levels of IGFBP-1 and prolactin synthesis were comparable with levels of decidualization in control cells (no virus introduced; see Fig. 3Go). However, introduction of the A-tMK construct resulted in a 70% inhibition of decidualization in both decidualization-inducing treatments (Fig. 5CGo). These results confirmed that changes in MLC20 phosphorylation precede decidualization and that up-regulation of MLC20 phosphorylation prevents decidualization.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study analyzes molecular events involved in cytoskeletal reorganization during in vitro decidualization of human stromal fibroblasts. We show that decidualization induced by the exogenous stimulus cAMP or the embryonic stimulus IL-1ß (both in the presence of E and P) is accompanied by changes in the cytoskeletal architecture. We also provide evidence of changes in actin-myosin interactions during HuF differentiation. We show, for the first time, that the phosphorylation status of MLC20 significantly affects decidualization. Our data also demonstrate substantial complexity in the involvement of the cytoskeleton during decidualization.

Cytoskeletal dynamics have been implicated in decidualization previously. We demonstrated a down-regulation of {alpha}-SMA in the stromal cells undergoing decidualization at the early implantation site in the baboon (d 15 of pregnancy), which is in close contact with the conceptus (5). A similar decrease in {alpha}-SMA staining in the cells close to the apical surface and underneath the epithelial plaques was attributed directly to the action of IL-1ß in our in vivo baboon model of simulated pregnancy (5). IL-1ß had a specific effect on epithelial cells (decrease in cyclooxygenase-1 protein) and induced decidualization in stromal cells as evidenced by their IGFBP-1 expression. This effect of IL-1ß on in vivo decidualization is in agreement with our previous in vitro studies in both baboon and human stromal fibroblasts (6, 7) and further validates the use of IL-1ß as the mediator of decidualization.

The mechanism of IL-1ß (with E and P)-induced decidualization is complex due to the fact that IL-1ß without support of steroid hormones is not able to induce IGFBP-1 expression and in vitro decidualization (6). Regulation by steroid hormones, particularly progesterone, is also absolutely critical for in vivo decidualization process (29, 30). Our previous in vivo studies have shown that gene and protein expression during the menstrual cycle or the window of implantation in the baboon model of simulated pregnancy are significantly compromised if the action of progesterone is antagonized with progesterone receptor antagonists (29, 30). Thus, the presence of steroid hormones and cross-talk with their pathways is crucial for IL-1ß-induced decidualization.

In this study, both the stimulus of embryonic origin (IL-1ß) and the exogenous stimulus (cAMP) were used to induce the decidualization process in vitro. However, these pathways do not work in synergy. In contrast to IL-1ß, cAMP is able to temporarily induce prolactin expression and in vitro decidualization in human stromal fibroblasts in the absence of steroid hormones (11). Moreover, we previously reported that the addition of IL-1ß antagonizes the ability of cAMP to induce decidualization in vitro (6). We also described the IL-1ß activation of MAPK, matrix-metalloproteinase-3, and cyclooxygenase-2/prostaglandin E2 pathways and reviewed their possible contribution to cytoskeleton reorganization in baboon and human stromal fibroblasts (see reviews in Refs. 31 and 32).

Focal adhesions on decidual cells may be key interacting regions between maternal and embryonic cells because decidual ß1 integrin and FAK participate in extensive outgrowth of blastocysts during implantation (33, 34). In addition, focal adhesions are thought to function as connections between the cytoskeleton and the ECM to confer structural integrity. FAK serves as an adaptor or scaffold for a number of signaling molecules that participate in cell migration, survival, multiplication, adhesion, and cell shape (35, 36). In agreement with these findings, we show that decidualization in vitro is accompanied by notable changes in the actin cytoskeleton (down-regulation of {alpha}-SMA and MLCK) and microtubule organization (decrease in ß-tubulin) as well as focal adhesion sites (increase in FAK phosphorylation and reorganization of vinculin). These changes in cytoskeletal and FAC proteins during decidualization result in changes in adhesion and migration.

FAK phosphorylation appears to be an integral part of decidualization induced by IL-1ß. After initial autophosphorylation, FAK can be further phosphorylated on several residues, including Tyr925, as detected in our study. Only HuF cells treated for 6 d with IL-1ß demonstrate increased phosphorylation of FAK. This observation is consistent with the demonstration that IL-1ß increases tyrosine phosphorylation of FAK in human gingival fibroblasts (37). The Tyr925 residue of FAK is the site of phosphorylation by tyrosine kinase Src (35, 36), and rapid Src phosphorylation was observed after IL-1ß stimulation (38). Src has been shown to be indispensable component of signaling pathway leading to in vivo decidualization in mice (39). Src transduces signals through the activation of ERK and the phosphorylation of MLCK (40). Because IL-1ß stimulates ERK in human fibroblasts (Ref. 41 and our unpublished results) and IL-1ß-induced ERK activity requires the stability of the actin cytoskeleton (42), we speculate that the ERK cascade is involved in IL-1ß-triggered decidualization. Importantly, neither E nor P alone or cAMP in combination with both steroid hormones increases phosphorylation of FAK. This finding is in agreement with a previous study involving decidualization induced by hormonal treatment for 12 d (43).

In contrast to IL-1ß, cAMP is known to decrease FAK phosphorylation via protein kinase A signaling (44) and cause the loss of focal adhesions, and fragmentation of stress fibers (45). It also activates tyrosine phosphatase involved in the dephosphorylation of tyrosine residues on FAK (46).

Our data presented above demonstrate the complexity of, and difference between, the mechanisms underlying cAMP- and IL-1ß-induced decidualization. Proper cytoskeletal organization and function depend on interactions between actin and myosin II, which in turn are regulated by actin dynamics and MLC20 phosphorylation. MLC20 phosphorylation and dephosphorylation are required for smooth muscle contraction and relaxation (17, 20). It also plays a central role in cell motility (47), endothelial (48) and epithelial (49) barrier function, cell division (50), cytoskeletal dynamics (51), and apoptosis (52). The role of MLC20 phosphorylation during decidualization, however, has never been studied. Intuitively, one would predict that stabilizing the cytoskeleton by increasing MLC20 phosphorylation would promote decidualization and strengthen the barrier to trophoblast invasion. However, it has been shown that disrupting actin filaments with cytochalasin D leads to a significant enhancement of in vitro decidualization induced by cAMP (21). In agreement with this work, we demonstrate that initial destabilization of the cytoskeleton, by inhibiting MLCK or myosin II ATPase activity, also significantly enhances decidualization by cAMP.

The importance of decreasing cytoskeletal stiffness on decidualization induced by cAMP is underscored by the unique decrease in the expression of the long form of MLCK. MLCK is expressed in HuF cells as long (214 kDa protein) and short (130 kDa protein) isoforms. Both isoforms originate from unique transcriptional sites within a single gene (53, 54), and they may have specific and unique functions that regulate contractile processes in cells and tissues (55). It has also been shown that factors responsible for the induction of the long form of MLCK (by TNF from exon 1A) are dependent on the cellular differentiation state of intestinal epithelial cells (56). Our results demonstrate down-regulation of the long isoform of MLCK after cAMP treatment, which subsequently results in a decrease in MLC20 phosphorylation. Previous studies have shown that increasing MLC20 phosphorylation increases cytoskeletal stiffness (57), whereas ML-7 decreases cytoskeletal stiffness (58). Thus, the decrease in MLC20 phosphorylation presumably leads to an inhibition of actin-myosin II interactions and destabilization of the cytoskeleton and promotes decidualization.

In contrast, IL-1ß-induced decidualization is almost completely blocked by ML-7 as well as by blebbistatin. This suggests that the decidualization pathway activated by IL-1ß requires a certain level of cytoskeletal organization. The level of cytoskeletal organization must be very precisely regulated because the introduction of constitutively active MLCK increased MLC20 phosphorylation and prevented both cAMP- and IL-1ß-induced differentiation of HuF into decidual cells. Our interpretation of these data are that unstimulated HuF cells have a certain level of cytoskeletal organization and stiffness that has to be maintained for IL-1ß to differentiate HuF into decidual cells. Reducing cytoskeletal organization or stiffness promotes cAMP-induced differentiation but inhibits IL-1ß-induced differentiation. However, stabilizing or increasing cytoskeletal stiffness by increasing MLC20 phosphorylation inhibits both processes. We propose the hypothetical integrated model to explain the role of the cytoskeleton reorganization in vitro decidualization of human stromal fibroblasts (Fig. 6Go).


Figure 6
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  		FIG. 6. Hypothetical model of involvement of the cytoskeleton in differentiation of human uterine stromal cells into decidual cells. Different molecular mechanisms underlie in vitro decidualization of HuF induced by cAMP and IL-1ß. IL-1ß induces multiple signaling pathways resulting in regulation of cytoskeletal organization: including FAK and MAPK activation and gene induction. Inhibition of MLCK (by ML-7) and myosin II ATPase (by blebbistatin) completely blocks IL-1ß-induced decidualization. On the other hand, treatment with cAMP leads to down-regulation of MLCK and subsequent decrease in MLC20 phosphorylation. This, in turn, negatively affects actin-myosin interactions and allows changes in cytoskeletal dynamics, leading to decidualization. Treatment with ML-7 and blebbistatin enhances MLC20 dephosphorylation and destabilization of actin-myosin interactions that further accelerates cAMP-induced decidualization. The artificial manipulation with cytoskeleton properties by increasing MLC20 phosphorylation prevents decidualization induced by both stimuli. COX, Cyclooxygenase; MMP, matrix metalloproteinase; MLCP, MLC phosphatase; ROCK, Rho-associated kinase.

 
Taken together, the data presented here suggest that cytoskeletal dynamics, rather than the absolute level of cytoskeletal stiffness or the precise organization of the cytoskeleton, are the more important element in the differentiation of HuF into decidual cells. This evidence ultimately provides new cytoskeleton targets for possible treatment of diseases connected with infertility caused by decidualization defects.


   Footnotes
 
This work was supported by National Institutes of Health Grants HD-44713 (to Z.S.) and HD-36759 and HD-42280 (to A.T.F.) and The Lalor Foundation grant (to I.I.).

Disclosure Statement: The authors have nothing to declare.

First Published Online April 5, 2007

Abbreviations: A-tMK, Adenovirus that contains truncated catalytic domain of MLCK; E, estradiol-17ß; EA, empty adenovirus; ECM, extracellular matrix; FAC, focal adhesion complex; F-actin, filamentous actin; FAK, focal adhesion kinase; FAK925, FAK phosphorylated on Tyr925; H, steroid hormones; HuF, human uterine fibroblast; HuF-tMK, HUF cells infected with truncated catalytic domain of MLCK; IGFBP, IGF binding protein; MLC, myosin light chain; MLCK, myosin light-chain kinase; P, medroxyprogesterone acetate; SFBS, charcoal-stripped fetal bovine serum; SMA, smooth muscle actin; TCA, trichloroacetic acid.

Received December 13, 2006.

Accepted for publication March 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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