help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-1217
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nagashima, T.
Right arrow Articles by Yoshimura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nagashima, T.
Right arrow Articles by Yoshimura, Y.
Endocrinology Vol. 149, No. 3 1227-1234
Copyright © 2008 by The Endocrine Society

Activation of SRC Kinase and Phosphorylation of Signal Transducer and Activator of Transcription-5 Are Required for Decidual Transformation of Human Endometrial Stromal Cells

Takashi Nagashima, Tetsuo Maruyama, Hiroshi Uchida, Takashi Kajitani, Toru Arase, Masanori Ono, Hideyuki Oda, Maki Kagami, Hirotaka Masuda, Sayaka Nishikawa, Hironori Asada and Yasunori Yoshimura

Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo 160-8582, Japan

Address all correspondence and requests for reprints to: Tetsuo Maruyama, M.D., Ph.D., Department of Obstetrics and Gynecology, Keio University School of Medicine, Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan. E-mail: tetsuo{at}sc.itc.keio.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone induces decidual transformation of estrogen-primed human endometrial stromal cells (hESCs), critical for implantation and maintenance of pregnancy, through activation of many signaling pathways involving protein kinase A and signal transducer and activator of transcription (STAT)-5. We have previously shown that kinase activation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) kinase is closely associated with decidualization and that SRC is indispensable for maximal decidualization in mice. To address whether SRC kinase activity is essential for decidualization in humans, hESCs were infected with adenoviruses carrying enhanced green fluorescent protein alone (Ad-EGFP), a kinase-inactive dominant-negative mutant (Ad-SRC/K295R), or an inactive autophosphorylation site mutant (Ad-SRC/Y416F). The cells were cultured in the presence of estradiol and progesterone (EP) to induce decidualization and subjected to RT-PCR, immunoblot, and ELISA analyses. Ad-EGFP-infected hESCs exhibited decidual transformation and up-regulation of decidualization markers including IGF binding protein 1 and prolactin in response to 12-d treatment with EP. In contrast, hESCs infected with Ad-SRC/K295R remained morphologically fibroblastoid without production of IGF binding protein 1 and prolactin even after EP treatment. Ad-SRC/Y416F displayed similar but less inhibitory effects on decidualization, compared with Ad-SRC/K295R. During decidualization, STAT5 was phosphorylated on tyrosine 694, a well-known SRC phosphorylation site. Phosphorylation was markedly attenuated by Ad-SRC/K295R but not Ad-EGFP. These results indicate that the SRC-STAT5 pathway is essential for decidualization of hESCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DECIDUALIZATION IS THE process by which progestin-induced fibroblastoid stromal cells of estrogen-primed endometrium differentiate into decidual cells. This process is crucial for embryo implantation and maintenance of pregnancy. In the presence of estrogen and progestin, human endometrial stromal cells (hESCs) isolated from human cycling endometrium exhibit morphological and functional changes in vitro that mimic in vivo decidual transformation (1, 2). This in vitro model has enabled a variety of studies on molecular mechanisms underlying decidualization.

To date, cAMP/protein kinase A (PKA) and progestin-mediated signaling pathways have emerged as key cellular events to drive decidual transformation (3). These two signaling pathways cooperatively regulate the activity of decidua-selective transcription factors through their cross talk and convergence, thereby up-regulating decidua-specific genes, eventually leading to terminal differentiation (3). Also, decidualized hESCs produce many bioactive substances, including growth factors and cytokines, whose downstream signaling pathways contribute to decidual transformation in a paracrine/autocrine manner (4, 5, 6). For instance, signal transducer and activator of transcription (STAT)-5, a latent transcription factor activated by numerous cytokines and peptide growth factors, is a candidate signaling molecule thought to regulate decidualization (7, 8, 9).

We previously reported that kinase activation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (SRC) is closely associated with in vitro and in vivo decidualization of hESCs (10, 11). SRC is a nonreceptor tyrosine kinase that associates with many surface receptors including growth factors and cytokine receptors, becomes activated upon ligand binding, and converts the extracellular stimuli to intracellular signals (12). The kinase activity of SRC is up-regulated by dephosphorylation of its negative regulatory tyrosine residue, tyrosine 527 (corresponding to tyrosine 530 in humans), located at the carboxyl terminus and further enhanced by autophosphorylation of tyrosine 416 (12). Given that many growth factors and cytokines are locally produced from decidual cells (4, 5, 6), it seems reasonable that SRC activation is accompanied by decidualization. However, it remains uncertain whether it is absolutely essential for the process of decidual changes. To address the essential role of SRC in decidualization of hESCs, we previously performed knockdown experiments to abrogate decidual SRC activity using specific inhibitors of SRC family kinases, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) (13); however, these inhibitors unexpectedly promoted decidualization together with paradoxical SRC activation (13). Although we demonstrated that SRC is an indispensable signaling component for maximal decidualization in mice (14), it remains unclear whether SRC and its kinase activity are essential for decidualization in humans.

To clarify this point, we conducted experiments in which adenovirus was used to introduce dominant-negative mutants of SRC into hESCs. We subsequently asked whether the elimination of SRC kinase activity by overexpression of these mutants influenced decidualization of hESCs in vitro. We now provide direct evidence that SRC kinase activation together with STAT5 phosphorylation is required for decidualization of hESCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies
The mouse monoclonal antibody clone 327, which reacts with both active and inactive SRC, was obtained from Calbiochem (San Diego, CA). BD Living Colors A.v. peptide antibody, which recognizes enhanced green fluorescent protein (EGFP), was purchased from CLONTECH Laboratories (Palo Alto, CA). Phospho-p44/42 MAPK (Thr202/Tyr204) E10 monoclonal antibody, which recognizes the phosphorylated forms of MAPK3/MAPK1, was purchased from New England Biolabs (Beverly, MA). Anti-MAPK3/MAPK1 (total MAPK) antibody was purchased from Upstate Biotechnology (Lake Placid, NY). A monoclonal antibody against β-actin (ACTB) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-STAT5{alpha}/STAT5β (total STAT5) polyclonal antibody and anti-phospho-STAT5{alpha}/STAT5β (tyrosine 694) polyclonal antibody recognizing the phosphorylated forms of STAT5{alpha}/STAT5β were purchased from Cell Signaling Technology (Beverly, MA).

Plasmids and recombinant adenovirus construction
Two pBabe vectors, one encoding a chicken c-SRC kinase-inactive dominant-negative mutant (SRC/K295R) and a second chicken c-SRC inactive autophosphorylation site mutant (SRC/Y416F) were kindly provided by Hidesaburo Hanafusa and Tsuyoshi Akagi (Molecular Oncology, Osaka Bioscience Institute). The recombinant adenovirus vectors carrying SRC/K295R or SRC/Y416F genes were constructed by using the Adeno-X expression system (CLONTECH). Briefly, for the construction of pShuttle SRC/K295R and SRC/Y416F expression vectors, the pBabe-based vectors were digested with BamHI and EcoRI. Then the fragments containing the full-length SRC/K295R or SRC/Y416F cDNAs were blunted with T4 DNA polymerase and ligated with pShuttle vectors that had been digested with NheI and blunt ended. In addition, to construct adenovirus shuttle plasmid pAdeno vectors carrying SRC/K295R or SRC/Y416F, the recombined pShuttle vectors were digested with PI-SceI and I-CeuI, and ligated with pAdeno vectors. The mutated sites and junctions were verified by sequencing and restriction enzyme mapping. The newly recombined pAdeno vectors carrying SRC/K295R or SRC/Y416F were digested with PacI and transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions for the construction of adenovirus vectors. Adenovirus vectors encoding EGFP (Ad-EGFP), SRC/K295R (Ad-SRC/K295R), or SRC/Y416F (Ad-SRC/Y416F) were packaged and propagated in HEK 293 cells. After being purified by cesium chloride gradient centrifugation at 300,000 x g for 3.5 h at 4 C, the adenovirus vectors were dialyzed in PBS overnight at 4 C and stored at –80 C before cell infection in vitro. The viral titer of purified adenovirus was 2.5 x 1010 pfu/ml. About 80% NIH-3T3 cells and 70% hESCs were infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F when the multiplicity of infection reached 50.

Tissue specimens
Endometrial specimens from the proliferative or early secretory phases of the regular menstrual cycle were obtained from consenting patients undergoing endometrial biopsies or total abdominal hysterectomy for benign gynecological diseases. The use of these human specimens was approved by the Human Ethics Committee at Keio University. No abnormalities or malignancies in these specimens were detected by histological examination.

Isolation of hESCs
hESCs were isolated from human cycling endometria as previously described (15, 16). Briefly, tissue specimens were washed in DMEM (Sigma-Aldrich, St. Louis, MO) and minced with scissors into small pieces less than 1 mm3 in size. The tissues were then gently agitated in tubes for 2 h at 37 C in DMEM with 0.2% (wt/vol) collagenase (Wako Bio-chemicals, Osaka, Japan), 0.05% DNase I (Life Technologies, Gaithersburg, MD), 10% fetal bovine serum (FBS), and 1% antibiotic-antimycotic mixture (Life Technologies, Inc., Grand Island, NY). After enzymatic digestion, cell clumps were dispersed by pipetting. Most of the digested hESCs presented as single cells or small aggregates were filtered sequentially through a 70-µm cell strainer nylon filter (Falcon 2350; BD Biosciences, Franklin Lakes, NJ) to remove gland cells, layered onto Ficoll-Paque (Pharmacia, Uppsala, Sweden), and centrifuged at 500 x g for 15 min at 4 C to remove erythrocytes. The isolated hESCs were collected from the Ficoll interface, resuspended in DMEM with 10% FBS and 1% antibiotic-antimycotic mixture, and transferred to individual wells of 24-well plates, 6-cm dishes, or eight-glass chamber microscope as described below.

Cell culture, hormonal treatment, and adenovirus transfection
Murine fibroblast NIH-3T3 cells were inoculated into 6-cm dishes (1.0 x 105 cells/dish) and maintained in DMEM with 10% FBS and 1% antibiotic-antimycotic mixture at 37 C under 5% CO2 in humidified air. Once 80% confluent, the cells were washed twice in PBS and cultured under serum starvation in DMEM with 0.1% FBS. Then, NIH-3T3 cells were infected with Ad-EGFP or Ad-SRC/K295R, or Ad-SRC/Y416F or treated with control medium for 48 h (uninfected control). These cells were then treated with 100 ng/ml recombinant epidermal growth factor (EGF; Sigma-Aldrich) for 4 min and harvested before the collection of total cell lysates.

Isolated hESCs were transferred to 24-well plates (5.0 x 105 cells/well) or 6-cm dishes, and grown at 37 C under 5% CO2 in humidified air. Once 70% confluent, the cells were washed twice in PBS and infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F. After 48 h infection, the cells were maintained in DMEM with 10% FBS and 1% antibiotic-antimycotic mixture. Subsequently hESCs were stimulated without or with 10 nM 17β-estradiol (Sigma-Aldrich) plus 1 µM progesterone (Sigma-Aldrich) (EP) for 12 d to induce decidualization. Supernatants were harvested every 2 d, centrifuged at 2000 x g to remove any nonadherent cells, and stored at –80 C. On d 12, total RNA was isolated from cultured hESCs. The morphological changes of hESCs treated with or without EP were viewed with a Leica DM IRE2 inverted fluorescent microscope using a x20 objective (Leica Microsystems, Heidelberger GmbH, Germany).

Immunoblot analysis
Noninfected or infected NIH-3T3 cells in 6-cm dishes were washed twice in cold PBS containing 1 mM Na3VO4 and lysed with 200 µl cold lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM Na3VO4, 50 mM NaF, and 1 mM Na2MoO4] containing protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). After stirring three times for 15 sec and chilling for 5 min at 4 C, the cell lysates were centrifuged at 17,000 x g for 10 min at 4 C and stored immediately at –80 C until electrophoresis.

Cell extracts from hESCs were prepared by the method of Schreiber et al. (17). hESCs in 6-cm dishes were washed twice in cold PBS containing 1 mM Na3VO4. We then added 200 µl cold buffer A [20 mM HEPES (pH 7.6), 0.2 mM EDTA (pH 8.0), 1 mM dithiothreitol, 10 mM NaCl, 1.5 mM MgCl2, 50 mM Na3VO4, 20% glycerol, 0.1% Nonidet P-40] containing protease inhibitor cocktail. The cells were subjected to hypotonic lysis on ice for 5 min and then collected by means of a scraper. After chilling for 10 min, the cell lysates were pelleted by centrifugation at 500 x g for 5 min at 4 C. The supernatants were stored immediately at –80 C as cytoplasmic fractions for protein analysis by electrophoresis. The nuclear pellets were resuspended in 100 µl cold buffer B [20 mM HEPES (pH 7.6), 0.2 mM EDTA (pH 8.0), 1 mM dithiothreitol, 500 mM NaCl, 1.5 mM MgCl2, 50 mM Na3VO4, 20% glycerol, 0.1% Nonidet P-40] containing protease inhibitor cocktail and vigorously vortexed for 10 sec. After chilling for 30 min, the mixtures were pelleted by centrifugation at 17,000 x g for 15 min at 4 C. The supernatants were stored immediately at –80 C as nuclear fractions for protein electrophoresis. The protein concentrations were measured using the DC protein assay kit (Bio-Rad, Hercules, CA).

In typical experiments, 10 µg lysates of NIH3T3 cells were mixed with lysis buffer and 20 µg cytoplasmic or nuclear extracts of hESCs were mixed with buffer A or buffer B, respectively. After addition of 2x SDS sample buffer, samples were heated at 95 C for 5 min. The heated samples were separated on 8% SDS-PAGE for 2 h at 125 V and subsequently transferred onto a polyvinylidene fluoride membrane (Immobilon P; Millipore, Bedford, MA) using transfer buffer (50 mM Tris aminomethane, 40 mM glycine, 0.04% SDS, and 20% methanol) for 2 h at 52 V. The membrane was incubated in TBS-T [20 mM Tris-HCl, 100 mM NaCl (pH 7.6), and 0.1% Tween 20] containing 5% BSA (Sigma-Aldrich) for 1 h at room temperature to block nonspecific binding sites. After three 10-min washes in TBS-T, the membranes were incubated overnight with primary antibodies against SRC (1:1000 dilution), EGFP (1:1000 dilution), ACTB (1:1000 dilution), phospho-MAPK (1:5000 dilution), phospho-STAT5 (1:1000 dilution), and total-STAT5 (1:1000 dilution) in TBS-T containing 1% BSA. After overnight incubation, membranes were washed three times in TBS-T for 10 min per wash and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Bar Harbor, ME) diluted 1:10,000 in TBS-T containing 1% BSA. After washing three times for 10 min in TBS-T, bound antibodies were detected using enhanced chemiluminescence plus detection kit (Amersham Biosciences Co., Piscataway, NJ) according to the manufacturer’s instructions and exposed to x-ray films (Eastman Kodak, Rochester, NY). Immunoblots were stripped in the buffer [62.5 mM Tris (pH 6.8), 2% SDS, and 100 mM β-mercaptoethanol] at 50 C for 30 min and reprobed with antibody against total MAPK (1:5000) in TBS-T containing 1% BSA.

Immunofluorescent analysis
For visualization of filamentous actin (F-actin), hESCs grown on 8-glass chamber microscope slides were fixed with 4% paraformaldehyde/PBS for 20 min at 37 C and washed three times for 5 min each time with PBS. Then cells were permeabilized with 0.2% Triton X-100 (Wako Chemical)/PBS for 15 min at 37 C. After washing three times for 5 min each time with PBS, cells were blocked for nonspecific staining with 5% fetal calf serum/PBS for 1 h at room temperature (RT). Subsequently cells were incubated with 1: 500 Texas Red-X conjugated phalloidin (Molecular Probes, Eugene, OR) for 1 h at RT or overnight at 4 C and washed three times for 10 min each time with PBS. Counterstaining was conducted with 2 mg/ml Hoechst 33342 (Sigma-Aldrich) to visualize DNA by incubation for 20 min at RT. The morphological changes were viewed with a DMIRE2 inverted fluorescent microscope (Leica Microsystems) using a x40 oil immersion objective. For digitally enhanced images, on-chip integration, background subtraction of white noise, and frame averaging, image processing was performed using Adobe Photoshop CS2 software (Adobe Systems, San Jose, CA).

Extraction of total RNA and semiquantitative RT-PCR analysis
The expression levels of transcripts in hESCs grown on 24-well plates were determined by RT-PCR. Total RNA was isolated from cultured hESCs using Trizol (Invitrogen) according to the manufacturer’s instructions, treated with deoxyribonuclease, purified by RNeasy Spin columns (QIAGEN, Valencia, CA), and resuspended in 20 µl of diethylpyrocarbonate-treated water. Semiquantitative RT-PCR of human IGF binding protein 1 (IGFBP1), prolactin (PRL), ACTB, matrix metalloproteinase-2 (MMP2), and SRC were carried out with 500 ng of total cellular RNA, on which RT was performed, using One-Step RT-PCR kit (QIAGEN) according to the manufacturer’s recommendations. Primers used to amplify IGFBP1, PRL, ACTB, MMP2, and SRC were designed using known sequences and shown in Table 1Go. The conditions used for PCR were as follows: 50 C for 30 min, 95 C for 15 min, x-cycles (94 C for 60 sec, y C for 60 sec, and 72 C for 90 sec), and then 72 C for 10 min. The extension cycle and annealing temperature, shown as x and y, respectively, are given in Table 1Go. After PCR amplification, 10 µl of the PCR products were electrophoresed in 3% agarose gels, visualized by ethidium bromide staining, and photographed under UV illumination.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Oligonucleotide primers

 
Measurement of IGFBP1 and PRL in culture media by ELISA
Concentrations of IGFBP and PRL in culture supernatants of hESCs grown on 24-well plates were measured quantitatively using specific ELISA kits (DSL-10–7800; Diagnostic Systems Laboratories, Webster, TX) and chemiluminescence ELISA kits (Architect Prolactin; Abbott Laboratories, North Chicago, IL), respectively, according to the manufacturers’ instructions. The conditioned culture supernatants were thawed and concentrated 20-fold using Microcon centrifugal filter units (Millipore) before ELISAs. The lower detection limits of these assays were 0.25 ng/ml for IGFBP1 and 0.6 ng/ml for PRL. Measurements were performed in triplicate, and each assay was repeated twice. The results were normalized to the number of cells present in each well at each time point.

Statistical analysis
Data are mean ± SD of at least three independent experiments, except for results of RT-PCR analysis, in which case a representative experiment is depicted in the figure. ANOVA was performed and significant differences from control were determined using the Dunnett’s test. All data were analyzed with the JMP 6.0.3 statistical analysis program (SAS Institute Inc., Cary, NC). Differences were considered significant when P<0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Attenuation of EGF-induced MAPK activation by adenovirus-mediated overexpression of SRC/K295R and SRC/Y416F in NIH-3T3 cells
SRC/K295R cannot bind ATP and has neither basal nor agonist activity; therefore, it behaves as a dominant-negative mutant when overexpressed. SRC/Y416F is a point mutation in SRC at the activation loop that is predicted to eliminate regulated SRC activity and results in a molecule with only basal activity. Thus, SRC/Y416F cannot be activated by agonists. When SRC/Y416F is overexpressed, it moderately suppresses the agonist-induced activation of endogenous SRC (18, 19).

To verify our adenovirus constructs, we first examined whether Ad-SRC/K295R and Ad-SRC/Y416F function as predicted. It is known that EGF stimulation leads to phosphorylation of MAPK through kinase activation of SRC in NIH-3T3 cells (20). We therefore introduced these SRC mutants into NIH-3T3 cells via adenovirus, cultured the infected cells under serum starvation, and then stimulated them without or with EGF. After 4 min stimulation, the cells were harvested for protein extraction and subjected to immunoblot analysis.

As shown in Fig. 1AGo, NIH-3T3 cells infected with Ad-EGFP and Ad-SRC/K295R strongly expressed EGFP and SRC mutant protein, respectively (Fig. 1Go, top two panels). EGF stimulation induced expression of the phosphorylated form of MAPK in noninfected or Ad-EGFP-infected NIH-3T3 cells (Fig. 1AGo, third panel from top). As expected, EGF-induced phosphorylation of MAPK was dramatically suppressed in Ad-SRC/K295R-infected NIH-3T3 cells. Neither adenovirus infection nor EGF treatment affected the levels of total MAPK, i.e. both phosphorylated and unphosphorylated forms (Fig. 1AGo, bottom panel). In contrast, although Ad-SRC/Y416F-infected NIH-3T3 cells prominently expressed SRC mutant protein, EGF-induced phosphorylation of MAPK was mildly suppressed, compared with Ad-SRC/K295R. Thus, Ad-SRC/K295R and Ad-SRC/Y416F displayed high transduction efficiency and exhibited dominant-negative effects on the SRC kinase-mediated signaling pathway as expected.


Figure 1
View larger version (60K):
[in this window]
[in a new window]

  		FIG. 1. Attenuation of EGF-induced MAPK activation by adenovirus-mediated overexpression of SRC/K295R and SRC/Y416F in NIH-3T3 cells. NIH-3T3 cells were cultured with control medium (noninfected) or infected with Ad-EGFP, Ad-SRC/K295R (A), or Ad-SRC/Y416F (B) and cultured under serum starvation in DMEM with 0.1% FBS for 48 h. The cells were then treated without (EGF–) or with EGF (EGF+) at 100 ng/ml for 4 min, harvested for protein extraction, and then subjected to immunoblot analysis to determine the expression levels of SRC, EGFP, phosphorylated MAPK (pMAPK), and total MAPK (tMAPK).

 
Dominant-negative mutants of SRC inhibited EP-induced morphological changes in hESCs
To address whether SRC kinase activity is required for decidualization, we first tested the effects of overexpression of the two mutant constructs on EP-induced morphological changes of hESCs. hESCs were isolated from cycling human endometria, infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F or treated with control medium (noninfected). After 48 h, the cells were subsequently treated without or with EP for 12 d and then fixed, doubly stained with Texas Red-X-conjugated phalloidin to visualize F-actin and Hoechst 33342 for DNA, and subjected to fluorescent microscopy to show cell morphology. Figure 2Go shows that EP treatment induced morphological changes, including enlargement and adoption of polygonal shapes in noninfected (Fig. 2AGo) or Ad-EGFP-infected hESCs (Fig. 2Go, B and C). Notably, approximately 70% of hESCs expressed EGFP, even approximately 2 wk after infection (Fig. 2CGo), indicating the high transduction efficiency and stable transgene expression in our experimental system. In contrast to Ad-EGFP, hESCs infected with Ad-SRC/K295R remained morphologically fibroblastoid even after approximately 2wk treatment with EP (Fig. 2DGo). Ad-SRC/Y416F displayed similar but less inhibitory effects, i.e. fibroblastoid hESCs were dominant, but some hESCs became polygonal and enlarged (Fig. 2EGo).


Figure 2
View larger version (39K):
[in this window]
[in a new window]

  		FIG. 2. Inhibition of EP-induced morphological changes by dominant-negative mutants of SRC in hESCs. hESCs were isolated from human cycling endometrium, cultured in control medium (noninfected, A) or infected with Ad-EGFP (B and C), Ad-SRC/K295R (D), or Ad-SRC/Y416F (E) and treated without (EP–) or with EP (EP+) for 12 d. hESCs were then fixed, doubly stained with Texas Red-X-conjugated phalloidin to visualize F-actin and Hoechst 33342 for DNA, and subjected to immunofluorescence microscopy. Bar, 30 µm.

 
Dominant-negative SRC mutants attenuate EP-induced expression of decidualization-associated genes in hESCs
Inhibition of morphological changes by the SRC mutants prompted us to examine whether they also attenuated functional decidualization, i.e. up-regulation of two representative decidual markers, IGFBP1 and PRL. hESCs were isolated from human cycling endometrium, infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F or treated with control medium (noninfected) for 48 h and then treated without or with EP for 12 d. Cells were harvested for RNA extraction and then subjected to semiquantitative RT-PCR as previously described (16). As shown in Fig. 3Go (top panels), treatment with EP induced the expression of IGFBP mRNA in noninfected or Ad-EGFP-infected hESCs. Adenovirus infection and EP treatment did not influence the mRNA levels of ACTB (Fig. 3Go, top three panels). Two different SRC mutants were successfully overexpressed in hESCs (Fig. 3Go, third panels from top). Consistent with the morphological data, Ad-SRC/K295R completely attenuated the basal and inducible levels of IGFBP1 mRNA expression (Fig. 3Go, top panel). In contrast, Ad-SRC/Y416F did not affect the basal level of IGFBP1 mRNA expression, but it did reduce the inducible level, although to a smaller extent than K295R (Fig. 3Go, top panel).


Figure 3
View larger version (101K):
[in this window]
[in a new window]

  		FIG. 3. Attenuation of EP-induced expression of decidualization-associated genes by dominant-negative mutants of SRC in hESCs. Isolated hESCs were cultured in media (noninfected control) or infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F; treated without (EP–) or with EP (EP+) for 12 d; harvested for RNA extraction; and then subjected to simplex or duplex semiquantitative RT-PCR analysis of IGFBP1, PRL, SRC, ACTB, and MMP2 mRNA.

 
Similarly, PRL mRNA was up-regulated in noninfected or Ad-EGFP-infected hESCs treated with EP for approximately 2 wk (Fig. 3Go, second panels from top). Both Ad-SRC/K295R and Ad-SRC/Y416F reduced the basal and inducible levels of PRL; however, the inhibitory effect of Ad-SRC/K295R on PRL expression was less apparent than on IGFBP1 expression. Neither Ad-SRC/K295R nor Ad-SRC/Y416F influenced the expression levels of MMP2, a gene unrelated to decidualization (21) (Fig. 3Go, bottom panels), suggesting that the inhibitory effects of these two mutants were specific for decidualization-associated signaling pathway(s).

Dominant-negative SRC mutants inhibit EP-induced production of IGFBP1 and PRL by hESCs
To examine the effects of overexpression of these two SRC mutants on the production of IGFBP1 and PRL, we measured (by ELISA) the concentrations of IGFBP1 and PRL proteins in the media of noninfected or adenovirally infected EP-treated hESCs. As shown in Fig. 4Go, noninfected or Ad-EGFP-infected hESCs secreted IGFBP1 and PRL in a time-dependent manner in response to treatment with EP. In contrast, Ad-SRC/K295R-infected hESCs produced very little IGFBP1 and PRL throughout the treatment with EP. Infection of Ad-SRC/Y416F also inhibited the secretion of IGFBP1 and PRL, however, less effectively than Ad-SRC/K295R infection. Thus, these SRC mutants attenuated the EP-induced production of IGFBP1 and PRL, indicating that kinase activation of SRC and its downstream signaling pathway(s) is involved in IGFBP1 and PRL production.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. Inhibition of EP-induced IGFBP1 and PRL production by dominant-negative mutants of SRC in hESCs. Isolated hESCs were cultured (noninfected, red square) or infected with Ad-EGFP (green circle), Ad-SRC/K295R (dark blue triangle), or Ad-SRC/Y416F (violet diamond) and treated with EP for 12 d. Culture media were replaced every 2 d. Supernatants collected at d 0, 4, 8, and 12 d were analyzed for IGFBP1 (A) and PRL (B) concentrations using ELISA and RIA, respectively. The results were normalized to the number of cells present in each well at each time point. Each value represents mean ± SD of three separate cultures of hESCs. A, Time course of IGFBP1 concentrations in culture media of EP-treated hESCs. a, P < 0.0001 vs. noninfected and EGFP; b, P < 0.01 vs. noninfected and EGFP; c, P < 0.0001 vs. noninfected and EGFP; d, P < 0.05 vs. noninfected and EGFP; e, P < 0.001 vs. noninfected and EGFP. B, Time course of PRL concentrations in culture media of EP-treated hESCs. a, P = 0.065 vs. noninfected; b, P = 0.053 vs. EGFP; c, P < 0.05 vs. noninfected and EGFP; d, P < 0.05 vs. EGFP; e, P < 0.0001 vs. noninfected and EGFP; f, P < 0.05 vs. EGFP; g, P < 0.0001 vs. noninfected and EGFP.

 
Dominant-negative mutants of SRC inhibited decidualization-induced phosphorylation of STAT5
To elucidate signaling molecule(s) downstream of SRC, we initially hypothesized that STAT5 might be the target of SRC based on the following: 1) STAT5 is phosphorylated, dimerized, and translocated into the nucleus during decidualization, which results in activation of the decidual PRL promoter (9); 2) the decidualization-induced nuclear accumulation of phosphorylated STAT5 in hESCs is independent of Janus kinase (JAK) activity (9); 3) STAT5 is phosphorylated and thereby activated by not only JAK but also SRC (22, 23); 4) tyrosine residue 694 (Y694) in STAT5A is a SRC phosphorylation site (23); and 5) Y694 phosphorylation on STAT5 is a prerequisite for DNA binding and transcription induction (24). To test our hypothesis, we performed immunoblot analysis using an antibody that specifically recognized the Y694-phosphorylated form of STAT5, asking whether Y694 phosphorylation of STAT5 took place during normal decidualization but was abrogated by knockdown of SRC-mediated signaling pathway(s).

As shown in Fig. 5Go, hESCs infected with Ad-EGFP, Ad-SRC/K295R, and Ad-SRC/Y416F prominently expressed EGFP and SRC mutant proteins, respectively (Fig. 5Go, top two panels). Despite the reported involvement of MAPK, a downstream signaling molecule of SRC, in the course of decidualization (25), an increase in the phosphorylation level of MAPK was marginal in decidualized hESCs (Fig. 5Go, third panel from top). Notably, neither of the SRC mutants affected the phosphorylation levels of MAPK. In contrast, the Y694-phosphorylated form of STAT5 was markedly up-regulated in decidualized hESCs but dramatically inhibited by the introduction of Ad-SRC/K295R (Fig. 5Go, second and forth panels from bottom). The overall effect of Ad-SRC/K416F on STAT5 phosphorylation was less inhibitory than that of Ad-SRC/K295R (Fig. 5Go, second and forth panels from bottom).


Figure 5
View larger version (83K):
[in this window]
[in a new window]

  		FIG. 5. Inhibition of decidualization-induced phosphorylation of STAT5 by dominant-negative mutants of SRC. Isolated hESCs were cultured (noninfected) or infected with Ad-EGFP, Ad-SRC/K295R, or Ad-SRC/Y416F; treated without (EP–) or with EP (EP+) for 12 d; harvested for protein extraction; and then subjected to immunoblot analysis to determine the expression levels of SRC, EGFP, phosphorylated MAPK (pMAPK), total MAPK (tMAPK), ACTB, phosphorylated STAT5 (pSTAT5), and total STAT5 (tSTAT5). Two independent immunoblot analyses of pSTAT5 and tSTAT5 were conducted using two different endometrial samples: experiments 1 (Exp. 1) and 2 (Exp. 2).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are a number of approaches to knockdown gene/protein activity, including small interfering RNA, antisense oligo DNA, inhibitors, and dominant-negative mutants. We previously used specific inhibitors of SRC family kinases to eliminate the kinase activity of SRC; however, those inhibitors paradoxically activated SRC and enhanced in vitro decidualization (13). We therefore used an alternative approach using adenovirus-encoded dominant-negative mutants of SRC. Because EP-induced decidualization takes approximately 2 wk, high transduction efficiency and long-term stable transgene expression are required for inhibition of decidualization. This requirement was met by the use of adenovirus. The use of dominant-negative mutants of SRC had an additional advantage. Whereas the catalytic function of SRC is crucial for signaling through a variety of cell surface receptors (12), SRC also functions as a scaffolding molecule through its SRC homology 2 and SRC homology 3 domains without any requirement for kinase activity (26, 27, 28). Simple knockdown experiments using small interfering RNA or antisense oligo DNA cannot distinguish which function, kinase, or scaffolding is essential for decidualization. Inhibition of decidualization by the kinase-inactive dominant-negative SRC mutant as presented here indicates that SRC kinase activity is, at least, indispensable, substantiating the importance of SRC-mediated signaling pathways in decidualization.

Regulatory mechanisms of SRC and its downstream signaling pathways have been well elucidated in a variety of cells (12); however, these remain elusive in hESCs. Because SRC couples with many cell surface receptors for cytokines and growth factors, these locally produced factors may activate SRC in an autocrine/paracrine manner. Indeed, IGF-I, but not platelet-derived growth factor, can activate SRC in mouse endometrial cells (14). Alternatively, progestins are also one of the candidates that positively regulate SRC activity (29). Progestins can stimulate the SRC/MAPK pathway through indirect or direct interaction of ligand-bound progesterone receptors with SRC (30, 31). This interaction may be facilitated when SRC becomes conformationally open upon dephosphorylation of tyrosine 527 (530 in human). In agreement, we reported previously that decidual SRC becomes activated together with its dephosphorylation on tyrosine 530 (11, 13). We also found that, despite the activation of decidual SRC, focal adhesion kinase and paxillin, well-established substrates of SRC and components of the focal adhesion complex (12), remain hypophosphorylated in decidualized hESCs (32). Those results indicate that focal adhesion kinase and paxillin may not be substrates of SRC in decidualizing hESCs. In this study, we have demonstrated that SRC is responsible for Y694 phosphorylation of STAT5 in decidualizing hESCs, implicating STAT5 as a signaling molecule downstream of decidual SRC.

Members of the STAT family are activated by phosphorylation within the cytoplasm by diverse cell signaling pathways, including receptor-associated JAK (33, 34). Phosphorylation of a conserved tyrosine residue in all STAT family members induces their dimerization and translocation to the nucleus. Within the nucleus, they regulate genes involved in growth and differentiation of many tissues including adipocytes, hepatocytes, and mammary epithelial cells (34). In human endometrium, STAT5 is selectively expressed in glandular epithelium and a subset of stromal cells that also express PRL receptor during the secretory phase, suggesting a potential role in differentiation (7). Indeed, it has been demonstrated that treatment of primary hESC cultures with cAMP (with or without progestin) leads to induction, phosphorylation, dimerization, and nuclear translocation of STAT5, eventually enhancing the activity of the -332/-270 decidual PRL promoter region (3, 9). Furthermore, Mak et al. (9) reported that the nuclear accumulation of phosphorylated STAT5 in hESCs is independent of JAK activity, suggesting that other activating kinase(s) may regulate decidual STAT5. Importantly, SRC is capable of directly phosphorylating STAT5 on Y694 (23). Both our previous and present studies showed that in vitro decidualization of hESCs was accompanied by SRC activation (11, 13) and Y694 phosphorylation of STAT5. The latter process was abrogated by a kinase-inactive dominant-negative SRC mutant. Taken together, these findings and our data indicate that activation of the SRC-STAT5 pathway is required for decidualization of hESCs.

MAPK is phosphorylated and thereby activated by various growth factors including EGF and platelet-derived growth factor through diverse signaling pathways involving RAF, MAPK kinase, and SRC (35). Decidualization of hESCs is accompanied by SRC activation (10, 11) and production of many growth factors and cytokines (4, 5, 6). However, we demonstrated here that, in contrast to STAT5, MAPK phosphorylation was not evident in decidualized hESCs, which is consistent with a previous report (36). Although MAPK activation has been reported to be involved in decidualization (25, 37, 38, 39), it may be temporally required for the initiation of decidualization of hESCs rather than for the establishment and maintenance of decidual transformation. Because a dominant-negative SRC mutant inhibits the phosphorylation of decidual STAT5, but not MAPK, the data suggest that STAT5, but not MAPK, is a downstream signaling molecule in the major SRC-mediated signaling pathway in decidualizing hESCs. SRC has the potential to directly phosphorylate STAT5 on Y694 (23) and indirectly activate the MAPK kinase/MAPK pathway through tyrosine phosphorylation of RAF (35). It is therefore conceivable that, in decidualizing hESCs, MAPK phosphorylation may be intricately modulated through convergence of different signaling pathways, including the cAMP-PKA pathway as suggested elsewhere (40). Thus, in this context, SRC may not be the sole and major determinant of the MAPK activity.

Inhibition of IGFBP1 and PRL mRNA expression by dominant-negative SRC mutants indicates that SRC is integrated into the upstream signaling pathway(s) responsible for induction of IGFBP1 and PRL. It has been proposed that elevation of cAMP enhances expression and nuclear accumulation of FoxO1A and CCAAT/enhancer-binding protein (C/EBP)-β in hESCs, both of which, in turn, interact with ligand-activated progesterone receptor and thereby initiate transcription of IGFBP1 and PRL genes (3). Given the reported cross talk between SRC and cAMP-PKA signaling pathways (38, 40, 41, 42), it is conceivable that SRC may affect the transcriptional activity of the decidua-specific complex containing FoxO1A and C/EBPβ. Y694 phosphorylation activates STAT5 transcriptional activity (24). Thus, in addition to the upstream cross talk between SRC and cAMP-PKA signaling pathways, STAT5 (activated by SRC through Y694 phosphorylation) may translocate into the nucleus and thereby directly regulate the promoters of decidua-specific genes including IGFBP1 and PRL through its interaction with FoxO1A, C/EBPβ, and/or progesterone receptor as proposed elsewhere (3). Intriguingly, STAT5 positively regulates the decidual PRL promoter (3, 9), whereas it inhibits the GH-induced activation of the IGFBP1 promoter (43). Thus, not only upstream but also downstream cross talk between SRC and cAMP-PKA pathways may play crucial roles in the differential regulation of decidualization marker expression. In support of this idea, different regulatory mechanisms underlying the transcriptional activation of IGFBP1 and PRL in hESCs have been suggested (44, 45).

In conclusion, SRC kinase activation and subsequent STAT5 phosphorylation are essential for in vitro decidualization of hESCs. This suggests that the SRC-STAT5 signaling pathway is critical to the molecular mechanism(s) underlying decidualization. Our findings also suggest possible therapeutic strategies to modulate endometrial function by targeting SRC-STAT5 signaling pathways.


   Acknowledgments
 
We thank Hidesaburo Hanafusa and Tsuyoshi Akagi for plasmids; Hirotaka James Okano and Naoto Yano for advice on adenovirus construction; Rei Sakurai for technical assistance; and Rika Shibata for secretarial assistance.


   Footnotes
 
This work was supported in part by Grants-in-Aid from the Japan Society for the Promotion of Science (to T.M. and to Y.Y.).

Disclosure Statement: All authors have nothing to declare.

First Published Online December 6, 2007

Abbreviations: ACTB, β-Actin; Ad-EGFP, adenoviruses carrying EGFP alone; C/EBP, CCAAT/enhancer-binding protein; EGFP, enhanced green fluorescent protein; EP, estradiol and progesterone; FBS, fetal bovine serum; hESC, human endometrial stromal cell; IGFBP1, IGF binding protein 1; JAK, Janus kinase; MMP2, matrix metalloproteinase-2; PKA, protein kinase A; PRL, prolactin; RT, room temperature; SDS, sodium dodecyl sulfate; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog; STAT, signal transducer and activator of transcription; TBS-T, Tris-HCl, NaCl, and Tween 20.

Received September 4, 2007.

Accepted for publication November 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Huang JR, Tseng L, Bischof P, Janne OA 1987 Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 121:2011–2017[Abstract/Free Full Text]
  2. Irwin JC, Kirk D, King RJ, Quigley MM, Gwatkin RB 1989 Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril 52:761–768[Medline]
  3. Gellersen B, Brosens J 2003 Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair. J Endocrinol 178:357–372[Abstract]
  4. Gurpide E, Tabanelli S, Tang B 1992 Human endometrial stromal cells. In: Genazzani AR, Petraglia F, eds. Hormones in gynecological endocrinology. Carnforth, Lancastershire, UK: Partenon Press, Casterton Hall; 717–724
  5. Giudice LC 1994 Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. Fertil Steril 61:1–17[Medline]
  6. Tabibzadeh S 1991 Human endometrium: an active site of cytokine production and action. Endocr Rev 12:272–290[Abstract/Free Full Text]
  7. Jabbour HN, Critchley HO, Boddy SC 1998 Expression of functional prolactin receptors in nonpregnant human endometrium: janus kinase-2, signal transducer and activator of transcription-1 (STAT1), and STAT5 proteins are phosphorylated after stimulation with prolactin. J Clin Endocrinol Metab 83:2545–2553[Abstract/Free Full Text]
  8. Prigent-Tessier A, Barkai U, Tessier C, Cohen H, Gibori G 2001 Characterization of a rat uterine cell line, U(III) cells: prolactin (PRL) expression and endogenous regulation of PRL-dependent genes; estrogen receptor β, {alpha}(2)-macroglobulin, and decidual PRL involving the Jak2 and Stat5 pathway. Endocrinology 142:1242–1250[Abstract/Free Full Text]
  9. Mak IY, Brosens JJ, Christian M, Hills FA, Chamley L, Regan L, White JO 2002 Regulated expression of signal transducer and activator of transcription, Stat5, and its enhancement of PRL expression in human endometrial stromal cells in vitro. J Clin Endocrinol Metab 87:2581–2588[Abstract/Free Full Text]
  10. Maruyama T, Yoshimura Y, Yodoi J, Sabe H 1999 Activation of c-Src kinase is associated with in vitro decidualization of human endometrial stromal cells. Endocrinology 140:2632–2636[Abstract/Free Full Text]
  11. Yamamoto Y, Maruyama T, Sakai N, Sakurai R, Shimizu A, Hamatani T, Masuda H, Uchida H, Sabe H, Yoshimura Y 2002 Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells. Mol Hum Reprod 8:1117–1124[Abstract/Free Full Text]
  12. Thomas SM, Brugge JS 1997 Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13:513–609[CrossRef][Medline]
  13. Maruyama T, Yamamoto Y, Shimizu A, Masuda H, Sakai N, Sakurai R, Asada H, Yoshimura Y 2004 Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro. Biol Reprod 70:214–221[Abstract/Free Full Text]
  14. Shimizu A, Maruyama T, Tamaki K, Uchida H, Asada H, Yoshimura Y 2005 Impairment of decidualization in SRC-deficient mice. Biol Reprod 73:1219–1227[Abstract/Free Full Text]
  15. Maruyama T, Sachi Y, Furuke K, Kitaoka Y, Kanzaki H, Yoshimura Y, Yodoi J 1999 Induction of thioredoxin, a redox-active protein, by ovarian steroid hormones during growth and differentiation of endometrial stromal cells in vitro. Endocrinology 140:365–372[Abstract/Free Full Text]
  16. Sakai N, Maruyama T, Sakurai R, Masuda H, Yamamoto Y, Shimizu A, Kishi I, Asada H, Yamagoe S, Yoshimura Y 2003 Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells. J Biol Chem 278:16675–16682[Abstract/Free Full Text]
  17. Schreiber E, Matthias P, Muller MM, Schaffner W 1989 Rapid detection of octamer binding proteins with ‘mini-extracts,’ prepared from a small number of cells. Nucleic Acids Res 17:6419[Free Full Text]
  18. Kim HP, Lee MS, Yu J, Park JA, Jong HS, Kim TY, Lee JW, Bang YJ 2004 TGF-β1 (transforming growth factor-β1)-mediated adhesion of gastric carcinoma cells involves a decrease in Ras/ERKs (extracellular-signal-regulated kinases) cascade activity dependent on c-Src activity. Biochem J 379:141–150[CrossRef][Medline]
  19. Lee MS, Kim TY, Kim YB, Lee SY, Ko SG, Jong HS, Kim TY, Bang YJ, Lee JW 2005 The signaling network of transforming growth factor β1, protein kinase C{Delta}, and integrin underlies the spreading and invasiveness of gastric carcinoma cells. Mol Cell Biol 25:6921–6936[Abstract/Free Full Text]
  20. Belsches AP, Haskell MD, Parsons SJ 1997 Role of c-Src tyrosine kinase in EGF-induced mitogenesis. Front Biosci 2:d501–d518
  21. Lockwood CJ, Krikun G, Hausknecht VA, Papp C, Schatz F 1998 Matrix metalloproteinase and matrix metalloproteinase inhibitor expression in endometrial stromal cells during progestin-initiated decidualization and menstruation-related progestin withdrawal. Endocrinology 139:4607–4613[Abstract/Free Full Text]
  22. Olayioye MA, Beuvink I, Horsch K, Daly JM, Hynes NE 1999 ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases. J Biol Chem 274:17209–17218[Abstract/Free Full Text]
  23. Okutani Y, Kitanaka A, Tanaka T, Kamano H, Ohnishi H, Kubota Y, Ishida T, Takahara J 2001 Src directly tyrosine-phosphorylates STAT5 on its activation site and is involved in erythropoietin-induced signaling pathway. Oncogene 20:6643–6650[CrossRef][Medline]
  24. Gouilleux F, Wakao H, Mundt M, Groner B 1994 Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361–4369[Medline]
  25. Strakova Z, Srisuparp S, Fazleabas AT 2002 IL-1β during in vitro decidualization in primate. J Reprod Immunol 55:35–47[CrossRef][Medline]
  26. Kaplan KB, Swedlow JR, Morgan DO, Varmus HE 1995 c-Src enhances the spreading of src–/– fibroblasts on fibronectin by a kinase-independent mechanism. Genes Dev 9:1505–1517[Abstract/Free Full Text]
  27. Schwartzberg PL, Xing L, Hoffmann O, Lowell CA, Garrett L, Boyce BF, Varmus HE 1997 Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src–/– mutant mice. Genes Dev 11:2835–2844[Abstract/Free Full Text]
  28. Schlaepfer DD, Broome MA, Hunter T 1997 Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol Cell Biol 17:1702–1713[Abstract]
  29. Shupnik MA 2004 Crosstalk between steroid receptors and the c-Src-receptor tyrosine kinase pathways: implications for cell proliferation. Oncogene 23:7979–7989[CrossRef][Medline]
  30. Migliaccio A, Piccolo D, Castoria G, Di Domenico M, Bilancio A, Lombardi M, Gong W, Beato M, Auricchio F 1998 Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J 17:2008–2018[CrossRef][Medline]
  31. Boonyaratanakornkit V, Scott MP, Ribon V, Sherman L, Anderson SM, Maller JL, Miller WT, Edwards DP 2001 Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases. Mol Cell 8:269–280[CrossRef][Medline]
  32. Maruyama T, Yoshimura Y, Sabe H 1999 Tyrosine phosphorylation and subcellular localization of focal adhesion proteins during in vitro decidualization of human endometrial stromal cells. Endocrinology 140:5982–5990[Abstract/Free Full Text]
  33. Schindler C, Darnell Jr JE 1995 Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem 64:621–651[Medline]
  34. Darnell Jr JE 1997 STATs and gene regulation. Science 277:1630–1635[Abstract/Free Full Text]
  35. Kyriakis JM, Avruch J 2001 Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 81:807–869[Abstract/Free Full Text]
  36. Lathi RB, Hess AP, Tulac S, Nayak NR, Conti M, Giudice LC 2005 Dose-dependent insulin regulation of insulin-like growth factor binding protein-1 in human endometrial stromal cells is mediated by distinct signaling pathways. J Clin Endocrinol Metab 90:1599–1606[Abstract/Free Full Text]
  37. Thienel T, Chwalisz K, Winterhager E 2002 Expression of MAP kinases (Erk1/2) during decidualization in the rat: regulation by progesterone and nitric oxide. Mol Hum Reprod 8:465–474[Abstract/Free Full Text]
  38. Yoon MS, Koo JB, Hwang JH, Lee KS, Han JS 2005 Activation of phospholipase D by 8-Br-cAMP occurs through novel pathway involving Src, Ras, and ERK in human endometrial stromal cells. FEBS Lett 579:5635–5642[CrossRef][Medline]
  39. Ihnatovych I, Hu W, Martin JL, Fazleabas AT, de Lanerolle P, Strakova Z 2007 Increased phosphorylation of myosin light chain prevents in vitro decidualization. Endocrinology 148:3176–3184[Abstract/Free Full Text]
  40. Stork PJ, Schmitt JM 2002 Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12:258–266[CrossRef][Medline]
  41. Klinger M, Kudlacek O, Seidel MG, Freissmuth M, Sexl V 2002 MAP kinase stimulation by cAMP does not require RAP1 but SRC family kinases. J Biol Chem 277:32490–32497[Abstract/Free Full Text]
  42. Baker MA, Hetherington L, Aitken RJ 2006 Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa. J Cell Sci 119:3182–3192[Abstract/Free Full Text]
  43. Ono M, Chia DJ, Merino-Martinez R, Flores-Morales A, Unterman TG, Rotwein P 2007 Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone. Mol Endocrinol 21:1443–1457[Abstract/Free Full Text]
  44. Tang M, Mazella J, Zhu HH, Tseng L 2005 Ligand activated relaxin receptor increases the transcription of IGFBP-1 and prolactin in human decidual and endometrial stromal cells. Mol Hum Reprod 11:237–243[Abstract/Free Full Text]
  45. Buzzio OL, Lu Z, Miller CD, Unterman TG, Kim JJ 2006 FOXO1A differentially regulates genes of decidualization. Endocrinology 147:3870–3876[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nagashima, T.
Right arrow Articles by Yoshimura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nagashima, T.
Right arrow Articles by Yoshimura, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals