Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print October 1, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.021527
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/1/214    most recent
biolreprod.103.021527v1
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 My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maruyama, T.
Right arrow Articles by Yoshimura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maruyama, T.
Right arrow Articles by Yoshimura, Y.
Agricola
Right arrow Articles by Maruyama, T.
Right arrow Articles by Yoshimura, Y.
BIOLOGY OF REPRODUCTION 70, 214–221 (2004)
DOI: 10.1095/biolreprod.103.021527
© 2004 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Pyrazolo Pyrimidine-Type Inhibitors of Src Family Tyrosine Kinases Promote Ovarian Steroid-Induced Differentiation of Human Endometrial Stromal Cells In Vitro1

Tetsuo Maruyama2, Yurie Yamamoto, Aki Shimizu, Hirotaka Masuda, Nozomi Sakai, Rei Sakurai, Hironori Asada, and Yasunori Yoshimura

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 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), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.

decidua, female reproductive tract, kinases, progesterone, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tyrosine phosphorylation plays an important role in a variety of biological processes, including cell growth, differentiation, apoptosis, and tumorigenesis [1]. Src family tyrosine kinases (SFKs) are key signaling molecules regulating tyrosine phosphorylation of a number of cellular proteins [2]. A prototype of the SFK members, c-Src is associated with the cellular membranes through its amino-terminal myristoylation [2]. Its kinase activity is known to be up-regulated by dephosphorylation of its negative regulatory tyrosine residue, tyrosine 527 (Y527) (corresponding to Y530 in human), located at the carboxyl terminus [2]. To date, the plausible candidate molecules regulating the phosphorylation status of the carboxyl-terminal tail are carboxyl-terminal Src kinase (Csk) and receptor-like protein-tyrosine phosphatase-{alpha} (RPTP-{alpha}) [2, 3]. In general, Csk represses the kinase activity through phosphorylation of Y527, while RPTP-{alpha} activates c-Src through dephosphorylation of the same residue [2, 3].

In addition, c-Src couples with various surface receptors, including platelet-derived growth factor (PDGF), epidermal growth factor, colony-stimulating factor 1, and insulin-like growth factor receptors [2]. In addition, many G-protein-coupled receptors and cytokine receptors, including angiotensin II, bombesin, bradykinin, vasopressin, platelet-activating factor, interleukin-11, prolactin, and oncostatin M, associate with c-Src [2]. These transmembrane receptors recruit and activate c-Src as well as the other SFKs on ligand binding, thereby transmitting the extracellular stimuli to the intracellular signal. Furthermore, c-Src interacts with the components of focal adhesion complexes downstream of integrins, regulating cytoskeleton, cell shape, and cell motility [2]. Thus, c-Src plays crucial roles in a variety of cell functions, such as growth, differentiation, and tumorigenesis.

Decidualization is the progestin-induced differentiation of fibroblast-like stromal cells of the estrogen-primed endometrium into decidual cells, which is crucial for embryo implantation and maintenance of pregnancy. In the presence of estrogen and progestin, endometrial stromal cells (ESCs) isolated from human cycling endometrium can exhibit morphological and functional changes in vitro that mimic in vivo decidual transformation [4, 5]. We have previously reported the alterations in the profile of several cellular phosphotyrosyl proteins on in vitro decidualization of human ESCs [6]. Moreover, we have found that c-Src kinase activation together with dephosphorylation at tyrosine 530 (Y530) is the cellular event tightly associated with in vitro as well as in vivo decidualization [7]. However, it remains to be elucidated whether c-Src functionally contributes to the process of decidualization.

In this study, to gain a clue for the role of decidual c-Src activation, we examined the effect of PP1 and PP2, both potent and selective SFK inhibitors [8–10], on the phosphorylation status of endometrial c-Src, its kinase activity, and differentiation of ESCs isolated from human cycling endometrium. We here provide evidence suggesting that kinase activation and Y530 dephosphorylation of c-Src are involved in functional and morphological decidualization and that pyrazolo pyrimidine-type inhibitors of SFKs have a potential to regulate endometrial function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies

Clone 28 [11], a mouse monoclonal antibody that specifically recognizes the active form of c-Src whose Y530 is dephosphorylated, was kindly provided by Dr. Koji Owada (Kyoto Pharmaceutical University, Kyoto, Japan). Clone 327, a mouse monoclonal antibody that reacts with both active and inactive c-Src, was obtained from Calbiochem (San Diego, CA). Phospho-p44/42 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204) E10 monoclonal antibody was purchased from New England Biolabs (Beverly, MA). Anti-insulin-like growth factor binding protein-1 (IGFBP-1) goat polyclonal and horseradish peroxidase (HRP)-conjugated preadsorbed anti-goat antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-prolactin (PRL) antibody was obtained from Upstate Biotechnology Inc. (Lake Placid, NY).

Tissue Collection

Endometrial specimens (n = 21) from the proliferative or early secretory phases of the menstrual cycle were obtained from consenting patients undergoing endometrial biopsies or total abdominal hysterectomy for benign gynecological disease. The use of these human specimens was approved by the Keio University Ethics Committee. There was no abnormality or malignancy in these specimens as diagnosed by histological examination. Dating was confirmed according to the criteria of Noyes et al. [12].

Isolation of Endometrial Stromal Cells

Endometrial stromal cells (ESCs) were isolated from human cycling endometria as previously described [13]. In brief, tissue samples were washed with Dulbecco Modified Eagle Medium (DMEM) and minced into small pieces of less than 1 mm3. The tissues were then incubated for 2 h at 37°C in DMEM containing 0.2% (w/v) collagenase (Wako, Osaka, Japan), 0.05% DNase I (Life Technologies, Gaithersburg, MD), 1% antibiotic-antimycotic mixture (Life Technologies), and 10% fetal bovine serum (FBS). After enzymatic digestion, cell clumps were dispersed by pipetting. Most of ESCs that were present as single cells or small aggregates were strained through a 70-µm cell strainer (Falcon 2350; Becton Dickinson, Franklin Lakes, NJ), which allowed the ESCs to pass through while intact glands were retained. The filtrates were washed twice and inoculated into 6-cm dishes or each well of six-well plates.

Cell Culture and Hormonal Treatment

The isolated ESCs were pre-cultured for about 2 days to be grown to subconfluence in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic mixture. The cells were cultured in the absence or the presence of 10 nM 17ß-estradiol (E2; Sigma, St. Louis, MO) plus 1 µM progesterone (P4; Sigma) for 10–14 days to induce decidualization. In addition, ESCs were cotreated with PP1 (BIOMOL Research Laboratories, Plymouth Meeting, PA), PP2 (BIOMOL), or herbimycin A (Calbiochem-Novabiochem, Darmstadt, Germany). The cells were incubated with every 2-day renewal of the medium according to the experimental protocol.

Murine fibroblast NIH-3T3 cells (clone 5611; JCRB#0615) were obtained from Human Science Research Resource Bank (Osaka, Japan). NIH-3T3 cells were maintained in DMEM supplemented with 10% calf serum. To examine the effect of PDGF-BB on c-Src activation, the cells were precultured for 48 h in DMEM supplemented with 0.1% calf serum, then treated with 50 ng/ml recombinant PDGF-BB (Sigma) for 5, 10, and 20 min and harvested for immunoblot analyses. Where indicated, NIH-3T3 cells were preincubated with PP1 or PP2 for 1 h, followed by treatment with PDGF-BB.

Immunoprecipitation and Immunoblotting

Total cell lysates were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% sodium-deoxycholate, 0.1% SDS, 1 mM Na3VO4, 50 mM NaF, 1 mM Na2MoO4) as previously described [6]. The protein concentration was measured using DC protein assay kit (Bio-Rad, Hercules, CA). One hundred micrograms of cell lysate protein were immunoprecipitated by incubation with clone 327 for 4 h at 4°C, followed by incubation with protein G-sepharose beads (Amersham Biosciences Inc., Piscataway, NJ). Half the immune complexes were washed three times with RIPA buffer and then resuspended in the 2x SDS sample buffer.

The resuspended immunoprecipitates or 30 µg of the total lysates from the cultured ESCs or NIH-3T3 cells were separated on 8% SDS-PAGE, transferred onto polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). Nonspecific binding sites were blocked in 2% BSA in Tris-buffered saline for 1 h at room temperature. The membranes were incubated with the primary antibodies for 1 h at room temperature. Blots were washed three times, incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, and then washed three times. Blots were developed using an enhanced chemiluminescence (ECL) plus detection kit (Amersham). When indicated, immunoblots were stripped in the buffer (62.5 mM Tris [pH 6.8], 2% SDS, 100 mM ß-mercaptoethanol) at 50°C for 30 min and reprobed with another specific antibody. The intensity of the signals on the immunoblot was quantitated using NIH Image program version 1.62 (Research Services Branch, National Institutes of Health, Bethesda, MD). In ESCs treated with E2+P4 alone, the value of the active c-Src or IGFBP-1 band intensity was adjusted to 1 when indicated.

In Vitro Kinase Assay

In vitro kinase assay was performed as previously described [6]. Briefly, immunoprecipitates with clone 327 from 100 µg of the cell lysates were washed three times with RIPA buffer, twice with kinase buffer (50 mM Tris-HCl pH 7.4, 0.1% NP-40, 0.1 mM Na3VO4, 3 mM MgCl2, 3 mM MnCl2), and half of them were then incubated with 20 µl kinase buffer containing 10 µCi [{gamma}-32P] ATP and 2 µg of acid-treated enolase (Sigma) for 10 min at 30°C. After termination of the reactions by adding 2x SDS sample buffer with 1 mM EDTA, the samples were boiled and separated on 8% SDS-PAGE. The gels were dried and subjected to autoradiography.

PRL Assay

PRL levels were measured in the culture media by RIA (Daiichi Radioisotope Laboratory Ltd, Tokyo, Japan) and normalized to protein content of the harvested cells. The detection limit of this assay was 0.3 ng/ml, and the intra- and interassay coefficients of variation were 1.9–7.1% and 1.6–3.6%, respectively.

Northern Blotting

Total RNA was extracted using TRIzol reagent (Life Technologies) according to the manufacturer's instruction. Ten micrograms of total RNA were electrophoresed and transferred to Maximum Strength Nytran nylon (Schleicher & Schuell, Keene, NH) by TurboBlotter system (Schleicher & Schuell) as described previously [14]. The IGFBP-1 probe was a 482-base pair (bp) reverse transcriptase-polymerase chain reaction (RT-PCR) fragment amplified from total RNA of human decidualized stromal cells. The primers for the PCR reaction were 5'-AACCTCTGCACGCCCTCACC-3' and 5'-CTGGCAG-TTGGGGTCTCCCCTG-3'. The filter was hybridized with the human IGFBP-1 probe labeled by a Gene Images random prime-labeling module and detected by a Gene Images CDP-star detection module (Amersham) according to the manufacturer's instructions. The membranes were exposed to x-ray film for 30–60 min.

Semiquantitative RT-PCR

Total RNA was extracted from cell cultures using TRIzol reagent according to the manufacturer's instruction. Semiquantitative RT-PCR was carried out with 0.3 µg of total cellular RNA, on which reverse transcription was performed, using the OneStep RT-PCR kit from Qiagen (Valencia, CA) according to the manufacturer's recommendations. After the reaction for 30 min at 50°C, the samples were heated for 15 min at 90°C as the initial PCR activation step. PCR amplifications were performed under the following conditions: 60 sec at 94°C, 60 sec at 56°C, and 90 sec at 72°C for 26 cycles, followed by 10 min at 72°C as the last primer extension step. Primers used to amplify human IGFBP-1 and ß-actin genes [15] as follows: IGFBP-1, 5'-AACCTCTGCACGCC-CTCACC-3' and 5'-AGGGATCCTCTTCCCATTCCAAGGGTAGA-3'; ß-actin, 5'-CCCAGGCACCAGGGCGTGATC-3' and 5'-TCAAACATGATCTGGGTCAT-3', respectively. Preliminary experiments determined the optimum PCR cycle number within the linear range of amplification for each gene being measured. After PCR amplification, 15-µl aliquots were electrophoresed in 3% agarose gels, followed by photographic recording of the gels stained with ethidium bromide. Gel photos were scanned, and densitometric analyses of PCR products were performed using the image analysis program NIH Image, version 1.62.

Statistical Analysis

Data were analyzed by Wilcoxon rank-sum test or Kruskal-Wallis test followed by post hoc Dunn test. A P-value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PP1 and PP2 Attenuate PDGF-Induced Expression of the Active Forms of Both c-Src and MAPK in NIH 3T3 Cells

The c-Src is known to be coupled with the PDGF-{alpha} and -ß receptors and to be activated on PDGF stimulation [16]. To validate PP1 and PP2, we first examined whether these SFK inhibitors attenuate the activation of c-Src in NIH 3T3 cells treated with PDGF-BB that activates all PDGF receptors. For the experiments, we have employed a murine monoclonal antibody clone 28 [11] whose specificity for the active form of c-Src has been successfully verified as described elsewhere [7, 11, 17–20]. Although immunoblot staining of total cell lysates with clone 28 occasionally detects two bands, we have previously identified the upper band, which corresponds to immunoprecipitated or exogenously overexpressed c-Src, as the active form of c-Src [7].

After PDGF-BB stimulation, the intensity of the immunoblot staining with clone 28 was increased in a time-dependent manner (Fig. 1A, top panel, arrow). Pretreatment with PP1 or PP2 for 1 h attenuated the PDGF-induced expression of the active form of c-Src (Fig. 1A, middle and bottom panels). PDGF-BB is also known to provoke phosphorylation and activation of p44/p42 MAPK through SFK-mediated signaling pathways [21]. In agreement, using a specific antibody against the phosphorylated p44/p42 MAPK, we demonstrated that treatment with PDGF induced phosphorylation of p44/p42 MAPK in NIH-3T3 cells, which was blocked by preincubation with PP1 or PP2 (Fig. 1B).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 1. Pyrazolo pyrimidine-type inhibitors of SFKs attenuate PDGF-induced expression of the active forms of both c-Src and MAPK in NIH 3T3 cells. A) Immunoblot analysis of the active form of c-Src derived from NIH-3T3 cells pretreated without or with 10 µM of PP1 or PP2 for 1 h before stimulation with PDGF for the indicated time. Whole cell lysates of NIH-3T3 cells treated as indicated were prepared and subjected to immunoblot (IB) with clone 28. The arrows indicate the active form of c-Src. B) Immunoblot analysis of the active form of MAPK derived from NIH-3T3 cells pretreated without or with 10 µM of PP1 or PP2 for 1 h before stimulation with PDGF for the indicated time. Whole cell lysates of NIH-3T3 cells treated as indicated were prepared and subjected to immunoblot (IB) with anti-phospho p44/p42 MAPK antibody. The single asterisk and double asterisks indicate the phosphorylated p44 and p42 MAPK, respectively

PP1 Paradoxically Promotes E2+P4-Induced Up-Regulation of the Active Form of c-Src and the Kinase Activation

Based on the kinase activation of c-Src during decidualization as previously reported [6], we initially expected that PP1 could inactivate decidual c-Src. As shown in Figure 2A, immunoblot staining with clone 28 was increased in E2+P4-treated ESCs as compared with nontreated ESCs, which is consistent with our previous results [7]. However, PP1 paradoxically enhanced the E2+P4-induced expression of the active form of c-Src in a dose-dependent manner (Fig. 2A). Densitometric analysis of the individual band intensities obtained from five independent experiments revealed that the effect of PP1 was significantly dose dependent (Fig. 2A, lower panel).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 2. PP1 paradoxically activates decidual c-Src. A) Immunoblot analysis of the active form of c-Src derived from ESCs treated for 10 days without or with E2+P4 in combination with increasing concentrations of PP1 as indicated. Whole cell lysates of ESCs treated as indicated were prepared and subjected to immunoblot (IB) with clone 28. The arrow indicates the active form of c-Src. Lower panel: graphic representation of the mean relative expression levels of active c-Src in ESCs treated for 10–14 days without or with E2+P4 in combination with increasing concentrations of PP1 as indicated. Mean (±SD, n = 5) ratios of the immunostaining intensity of active c-Src, as determined by image analysis, with E2+P4-treatment set at 1. *P < 0.05 versus control vehicles alone. **P < 0.05 versus E2+P4 alone. B) In vitro kinase assay and immunoblot analysis of c-Src derived from ESCs treated for 14 days without or with E2+P4 in combination with increasing concentrations of PP1 as indicated. One hundred micrograms of total cell lysates of ESCs treated as indicated were prepared and subjected to each immunoprecipitation (IP) with clone 327. The c-Src immunoprecipitates were divided into each two samples: one was subjected to in vitro kinase assay using enolase as a substrate (top panel), while the other was subjected to immunoblot staining with clone 327 (middle panel). Immunoblot with clone 327 was stripped and reprobed with clone 28 (bottom panel)

Recently, Wu et al. [20] reported that clone 28 recognizes not only the active form of c-Src but also active Fyn, another SFK. We have previously reported that c-Src, but not Fyn, is dominantly activated during decidualization [6, 7]. Although we have shown the specificity of clone 28 for the kinase-active c-Src in decidualized ESCs [7], we further examined whether c-Src reactive with clone 28 from PP1-treated decidualized ESCs could be a bona fide active form having an enhanced kinase activity. As expected, PP1 increased not only the immunoreactivity with clone 28 (Fig. 2B, bottom panel) of decidual c-Src immunoprecipitates (Fig. 2B, middle panel) but also the kinase activity to phosphorylate enolase (Fig. 2B, top panel) in a dose-dependent manner (0.1–1 µM). However, the kinase activity appears to increase more markedly than the active c-Src expression detected by clone 28. It is well known that Y527 dephosphorylation is required for kinase activation and that further Y416 phosphorylation elicits the maximal activation of c-Src [2]. Clone 28 can detect the former molecular event but not the latter, which may account for some discrepancies between the kinase activity and the expression of active c-Src as shown in Figure 2B.

PP1 Advances E2+P4-Induced Morphological Decidualization

During a series of these in vitro experiments, we noticed that morphological changes characteristics of decidualization were more evident in cultured ESCs treated with E2+P4 in combination with PP1 than those treated with E2+P4 alone. As shown in Figure 3, fibroblastic cells were dominant in cultures of ESCs treated with control vehicles, while polygonal enlarged cells were abundant in the presence of E2+P4, which was further augmented by cotreatment with PP1.



View larger version (86K):
[in this window]
[in a new window]
  		FIG. 3. PP1 advances E2+P4-induced morphological decidualization. Phase contrast micrographs of ESCs treated for 14 days without or with E2+P4 in combination with 1 µM PP1. Bars = 100 µm

PP1 Enhances E2+P4-Induced Production of IGFBP-1 and PRL

The increase in c-Src kinase activity and enhancement of morphological changes on PP1 treatment prompted us to determine if functional decidualization is also augmented in response to PP1. To demonstrate this, we examined whether PP1 potentiates the action of ovarian steroid hormones to induce IGFBP-1, an established decidualization marker. As shown in Figure 4A, PP1 dramatically increased the expression of IGFBP-1 (bottom panel) as well as the active form of c-Src (top panel) in E2+P4-induced decidualized cells, while the level of total c-Src was almost constant across the treatment (middle panel). Densitometric analysis of the individual band intensities obtained from four independent experiments revealed that PP1 significantly enhanced E2+P4-induced IGFBP-1 expression in a dose-dependent manner (Fig. 4B).



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 4. PP1 enhances production of decidual IGFBP-1 and PRL proteins. A) Immunoblot analysis of c-Src and IGFBP-1 derived from ESCs treated for 14 days without or with E2+P4 in combination with increasing concentrations of PP1 as indicated. Whole cell lysates of ESCs treated as indicated were prepared and subjected to immunoblot (IB) with clone 327 and anti-IGFBP-1 antibody (middle and bottom panels, respectively). Immunoblot with clone 327 was stripped and reprobed with clone 28 (top panel). B) Graphic representation of the mean relative expression levels of IGFBP-1 in ESCs treated for 10–14 days with E2+P4 in combination with increasing concentrations of PP1 as indicated. Mean (±SD, n = 5, except treatment with 10 µM PP1, n = 1) ratios of the immunostaining intensity of IGFBP-1, as determined by image analysis, with E2+P4-treatment set at 1. *P < 0.05 versus E2+P4. **P < 0.05 versus E2+P4 + 5 µM PP1. C) Graphic representation of the mean (±SD, n = 3) concentrations of PRL in the media of ESCs treated for 10–14 days without or with E2+P4 in combination with increasing concentrations of PP1 as indicated. *P < 0.05 versus control. **P < 0.05 versus E2+P4 alone. ***P < 0.05 versus E2+P4 + 5 µM PP1

To further verify the enhancing effect of PP1 on decidualization, we tested whether PP1 could promote the production of PRL, another established decidualization marker, in E2+P4-treated stromal cells. Consistently, PP1 also augmented the secretion of prolactin at a relatively low concentration (0.1–1 µM) (Fig. 4C), while the enhancing effect of PP1 was no longer observed at a higher concentration (5 µM). In addition to the protein levels, PP1 also enhanced the expression of decidual IGFBP-1 gene, as determined by Northern blot and RT-PCR analyses (Fig. 5, A and B, respectively).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 5. PP1 augments induction of decidual IGFBP-1 gene. A) Northern blot analysis of IGFBP-1 mRNA derived from ESCs treated for 14 days without or with E2+P4 in combination with PP1 as indicated. Total RNA was extracted, electrophoresed, transferred to the Nytran membrane, and then hybridized with IGFBP-1 cDNA probe. Lower panel: ethidium bromide-stained 26S and 18S ribosomal RNA subunits. B) RT-PCR analysis of IGFBP-1 mRNA derived from ESCs treated for 14 days without or with E2+P4 in combination with PP1 as indicated. Total RNA was extracted and subjected to RT-PCR analysis for IGFBP-1 and ß-actin mRNA. MK, 100-bp ladder marker

PP2, but Not Herbimycin A, Also Enhances E2+P4-Induced Production of Decidualization Marker Proteins

Since PP1 displayed an unexpected effect, we then examined whether PP2, another potent and selective inhibitor of SFKs [8], also affected decidualization in a similar way. As expected, PP2 advanced characteristic morphological changes, which is comparable with PP1 (data not shown). Likewise, PP2 dramatically potentiated the action of ovarian steroid hormones to induce IGFBP-1, while PP2 alone did not exhibit any inducing effect (Fig. 6A). Either PP2 alone or in combination with E2+P4 did not affect the expression of c-Src reactive with clone 327 (Fig. 6A). Densitometric analysis of the individual band intensities obtained from three independent experiments showed that PP2 significantly enhanced the expression of decidual IGFBP-1 in a dose-dependent manner (Fig. 6A, lower panel), which was similar to PP1 (Fig. 4B). As shown in Figure 6B, PP2 also augmented the expression of decidual PRL concomitantly with up-regulation of the active form of c-Src. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, did not dramatically affect the expression of active c-Src and IGFBP-1 at concentrations up to 1 µM (Fig. 6C). Treatment with more than 1 µM of herbimycin A induced cell death in ESCs within a couple of days (data not shown), thus not allowing for further biochemical analyses.



View larger version (39K):
[in this window]
[in a new window]
  		FIG. 6. PP2, but not herbimycin A, enhances expression of the active form of decidual c-Src as well as decidualization marker proteins. A) Immunoblot analysis of c-Src and IGFBP-1 derived from ESCs treated for 14 days without or with E2+P4 in combination with increasing concentrations of PP2 as indicated. Whole cell lysates of ESCs treated as indicated were prepared and subjected to immunoblot (IB) with clone 327 and anti-IGFBP-1 antibody. Lower panel: graphic representation of the mean relative expression levels of IGFBP-1 in ESCs treated for 10–14 days with E2+P4 in combination with increasing concentrations of PP2 as indicated. Mean (±SD, n = 3, except treatment with 5 µM PP2, n = 2) ratios of the immunostaining intensity of IGFBP-1, as determined by image analysis, with E2+P4-treatment set at 1. *P < 0.05 versus E2+P4 in combination with 1 µM PP2. B) Immunoblot analysis of c-Src and PRL derived from ESCs treated for 14 days without or with E2+P4 in combination with increasing concentrations of PP2 as indicated. Whole cell lysates of ESCs treated as indicated were prepared and subjected to immunoblot (IB) with clone 28 and anti-PRL antibody. Immunoblot with clone 28 was stripped and reprobed with clone 327. The arrow indicates the active form of c-Src. C) Immunoblot analysis of active c-Src, total c-Src, and IGFBP-1 derived from ESCs treated for 14 days without or with E2+P4 in combination with increasing concentrations of herbimycin A (Herb A) as indicated. Whole cell lysates of ESCs treated as indicated were prepared and subjected to immunoblot (IB) with clone 327 or anti-IGFBP-1 antibody. Immunoblot with clone 327 was stripped and reprobed with clone 28


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We here demonstrate the novel enhancing effect of PP1 and PP2 on ovarian steroid-induced differentiation of human ESCs concomitant with paradoxical kinase activation of c-Src. SFKs, including c-Src, are known to play a crucial role in differentiation of many types of cells [2]. Intriguingly, whether SFKs participate in the process of differentiation in a stimulatory or an inhibitory manner depends on the cell type [6, 22–24]. Hence, SFK inhibitors, including PP1 and PP2, exhibit a positive or negative effect on differentiation through inhibition of the SFK activity [22–24].

Although we found a positive correlation between the expression levels of the active form of c-Src and decidualization markers such as IGFBP-1 and PRL on treatment with PP1 or PP2, these SFK inhibitors paradoxically provoked the kinase activation of c-Src together with Y530 dephosphorylation. PP1 and PP2 are widely used as selective and potent inhibitors of c-Src [8, 25]. We here verified that PP1 and PP2 used in this study authentically behaved as inhibitors in that they attenuated PDGF-induced activation of c-Src and MAPK (Fig. 1), as has been described elsewhere [26–28]. Thus, our data on the paradoxical effects of PP1 and PP2 on decidual c-Src are novel and convincing, although molecular mechanisms underlying the paradoxical activation remain to be elucidated. Some studies have reported that PP1 does not act expectedly as a Src inhibitor when used for treatment of some cell types [29–31], and therefore there are several possibilities to explain our observation.

First, although PP1 and PP2 are thought to be selective and potent inhibitors of SFKs, these chemicals exhibit little specificity between SFKs and cannot be used to dissect the role of individual SFKs that coexist in a given cell [9]. A structure-based study reveals that threonine 338 common in the kinase domain of SFKs is a critical determinant of the selectivity and potency of PP1 and PP2 to bind to SFKs and inhibit the kinase activity [9]. Csk, also belonging to the SFKs, possesses a threonine at the same position 338 [9]. Csk is a well-known negative regulator of c-Src, which phosphorylates c-Src at Y530 and thereby inactivates it [2]. Taken together, it is conceivable that both PP1 and PP2 may repress the kinase activity of Csk, thereby leading to Y530 dephosphorylation and kinase activation of c-Src. Although whether PP1 and PP2 inhibit the kinase activity of Csk remains to be clarified, our hypothesis appears to be supported by our present findings that both PP1 and PP2 up-regulated the active form of decidual c-Src, whose Y530 was dephosphorylated.

Second, PP1 and PP2 may primarily target as-yet-unidentified tyrosine kinase(s) other than SFKs, as proposed elsewhere [29]. The assertion that PP1 and PP2 are truly SFK selective, in particular, in vivo has not been thoroughly tested. We found that, unlike coaddition of ovarian steroid hormones, PP1 or PP2 alone did not enhance either kinase activation of c-Src or induction of IGFBP-1 and PRL. These results suggest that ovarian steroids and/or decidua-specific factors may be required for the inhibitors to elicit the paradoxical effects as observed in this study. Indeed, it has been demonstrated that PP1 and PP2 appear to behave in a cell-type-specific manner [29, 30]. It is therefore tempting to speculate that the SFK inhibitors may target possible decidualization-induced tyrosine kinase(s) or signaling pathway(s) that might regulate the c-Src kinase activity.

In this context, PP1 and PP2 may modulate the function of the transcription factor FKHR (forkhead homologue in rhabdomyosarcoma), which has been reported to participate in the transcriptional regulation of PRL and IGFBP-1 during decidualization together with CEBPß (CCAAT/enhancer-binding protein ß) and Hoxa 10, respectively [32, 33]. FKHR is phosphorylated by Akt/protein kinase B (PKB) in cells, forcing FKHR to exit from the nucleus [34]. Therefore, kinase activation of Akt/PKB by phosphorylation hampers the FKHR activity [34, 35]. In agreement, it has been reported that hypophosphorylation of Akt/PKB is tightly associated with in vitro decidualization of ESCs [36]. PP1 and PP2 have been demonstrated to inhibit phosphorylation of Akt/PKB and thereby reduce the kinase activity [37–39]. Intriguingly, stem cell factor-induced phosphorylation of Akt/PKB is not inhibited by SU6656 [39], a different type of SFK inhibitor, suggesting that SFKs, including c-Src, may not be involved in activation of Akt/PKB in some types of cells. Thus, it is plausible that pyrazolo pyrimidine-type SFK inhibitors might directly inhibit the kinase activation of decidual Akt/PKB, thereby enhancing the FKHR activity.

In contrast to PP1 and PP2, herbimycin A showed little enhancing effect on both decidualization and c-Src kinase activity. Although herbimycin A has been widely used as a tyrosine kinase inhibitor with Src selectivity, several studies demonstrate that it behaves in a manner different from pyrazolo pyrimidine-type SFK inhibitors [31, 40, 41]. PP1 and PP2 act as competitive inhibitors of ATP for SFKs involving threonine 338 common in the kinase domain [9, 10], which, however, has become controversial [42]. Herbimycin A covalently interacts with sulfhydryl groups on protein tyrosine kinases [43–45], accounting for the differential selectivity and various actions of these two types of inhibitors as presented herein and also previously described [31, 40, 41]. Further studies will be required to elucidate the molecular basis for the effect of PP1 and PP2 on decidual c-Src activation as well as decidualization.

There were some differences in the effects of each of the inhibitors on IGFBP-1 expression versus PRL secretion (Fig. 4, B and C). Treatment with a high concentration (more than 5 µM) of PP1 resulted in a further increase in the intracellular levels of IGFBP-1 but a decline in the secretion of PRL; c-Src has been implicated as a positive regulator of exocytosis [46–48], and PP1 negatively interferes with secretory processes in some types of cells [49, 50]. Intriguingly, Furuyama and Fujisawa have reported that 10 µM of PP1 inhibit the secretion of cathepsins K and L and thereby increase their intracellular levels without affecting their synthesis in osteoclasts [49]. It is therefore tempting to speculate that inhibition of the secretion, but not synthesis, of PRL and presumably IGFBP-1 by a high concentration of PP1 and PP2 may lead to the intracellular accumulation of IGFBP-1 and PRL and thereby a dramatic increase in their intracellular protein levels in ESCs as presented herein.

Since we here just demonstrate that the positive modification of the c-Src kinase activity results in the enhancement of morphological and functional decidualization in vitro, we do not conclude that c-Src is essential for stromal decidualization. Rather, our results support the idea that the network of signaling pathways mediated by SFKs, including c-Src, is functionally involved in the differentiation of human ESCs. Alternative knockout studies using, for example, adenovirus encoding dominant negative c-Src may address the question whether c-Src is essential for decidualization of human ESCs. However, we emphasize that this is the first report to demonstrate that pyrazolo pyrimidine-type SFK inhibitors have a potential to accelerate terminal differentiation of ESCs in the presence of ovarian steroid hormones.

In summary, PP1 and PP2 enhanced ovarian steroid-induced differentiation of human ESCs and paradoxically promoted kinase activation of c-Src together with Y530 dephosphorylation. These results indicate that activation of c-Src may be a crucial signaling component for the functional and morphological differentiation of human ESCs, implicating the use of pyrazolo pyrimidine-type SFK inhibitors, including PP1 and PP2, in investigating intracellular signaling. Recently, the possible use of Src inhibitors as therapeutic agents for the treatment of osteoporosis and cancer has been extensively discussed [25]. Along with this idea, our present data may provide a clue for a possible therapeutic potential of specific pyrazolo pyrimidine-type of SFK inhibitors or the related compounds in the hormonal treatment of endometrium-derived diseases.


   ACKNOWLEDGMENTS
 
We thank Dr. Koji Owada (Kyoto Pharmaceutical University, Kyoto, Japan) for his generous gift of clone 28 and Ms. Shino Kuwabara for her secretarial assistance.


   FOOTNOTES
 
1 This study was supported, in part, by the Ministry of Education, Science, and Culture of Japan (grants B15390511 to T.M., B12470348 to Ya.Y., and A14207066 to Ya.Y.), by Keio Gijuku Academic Development Funds (to T.M.), by grants from the Keio Health Counseling Center (to T.M.), and by grants from Mitsukoshi Fund of Mitsukoshi Health and Welfare Foundation 2002 (to T.M.). Part of this work was presented at the 35th Annual Meeting of Society for the Study of Reproduction held in 2002, Baltimore, Maryland. Back

2 Correspondence: Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. FAX: 81 3 3226 1667; tetsuo{at}sc.itc.keio.ac.jp Back

Received: 24 July 2003.

First decision: 12 August 2003.

Accepted: 12 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hunter T. The Croonian Lecture. 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease. Philos Trans R Soc Lond B Biol Sci 1998 353:583-605[Abstract/Free Full Text]
  2. Thomas SM, Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 1997 13:513-609[CrossRef][Medline]
  3. Pallen CJ. Protein tyrosine phosphatase alpha (PTPalpha): a Src family kinase activator and mediator of multiple biological effects. Curr Top Med Chem 2003 3:821-835[CrossRef][Medline]
  4. Huang JR, Tseng L, Bischof P, Janne OA. Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells. Endocrinology 1987 121:2011-2017[Abstract/Free Full Text]
  5. Irwin JC, Kirk D, King RJ, Quigley MM, Gwatkin RB. Hormonal regulation of human endometrial stromal cells in culture: an in vitro model for decidualization. Fertil Steril 1989 52:761-768[Medline]
  6. Maruyama T, Yoshimura Y, Yodoi J, Sabe H. Activation of c-Src kinase is associated with in vitro decidualization of human endometrial stromal cells. Endocrinology 1999 140:2632-2636[Abstract/Free Full Text]
  7. Yamamoto Y, Maruyama T, Sakai N, Sakurai R, Shimizu A, Hamatani T, Masuda H, Uchida H, Sabe H, Yoshimura Y. Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells. Mol Hum Reprod 2002 8:1117-1124[Abstract/Free Full Text]
  8. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 1996 271:695-701[Abstract/Free Full Text]
  9. Liu Y, Bishop A, Witucki L, Kraybill B, Shimizu E, Tsien J, Ubersax J, Blethrow J, Morgan DO, Shokat KM. Structural basis for selective inhibition of Src family kinases by PP1. Chem Biol 1999 6:671-678[CrossRef][Medline]
  10. Schindler T, Sicheri F, Pico A, Gazit A, Levitzki A, Kuriyan J. Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. Mol Cell 1999 3:639-648[CrossRef][Medline]
  11. Kawakatsu H, Sakai T, Takagaki Y, Shinoda Y, Saito M, Owada MK, Yano J. A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase. J Biol Chem 1996 271:5680-5685.[Abstract/Free Full Text]
  12. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950 1:3-25
  13. Maruyama T, Sachi Y, Furuke K, Kitaoka Y, Kanzaki H, Yoshimura Y, Yodoi J. Induction of thioredoxin, a redox-active protein, by ovarian steroid hormones during growth and differentiation of endometrial stromal cells in vitro. Endocrinology 1999 140:365-372[Abstract/Free Full Text]
  14. Maruyama T, Kitaoka Y, Sachi Y, Nakanoin K, Hirota K, Shiozawa T, Yoshimura Y, Fujii S, Yodoi J. Thioredoxin expression in the human endometrium during the menstrual cycle. Mol Hum Reprod 1997 3:989-993[Abstract/Free Full Text]
  15. Sakai N, Maruyama T, Sakurai R, Masuda H, Yamamoto Y, Shimizu A, Kishi I, Asada H, Yamagoe S, Yoshimura Y. Involvement of histone acetylation in ovarian steroid-induced decidualization of human endometrial stromal cells. J Biol Chem 2003 278:16675-16682[Abstract/Free Full Text]
  16. DeMali KA, Godwin SL, Soltoff SP, Kazlauskas A. Multiple roles for Src in a PDGF-stimulated cell. Exp Cell Res 1999 253:271-279[CrossRef][Medline]
  17. Sakai T, Kawakatsu H, Fujita M, Yano J, Owada MK. An epitope localized in c-Src negative regulatory domain is a potential marker in early stage of colonic neoplasms. Lab Invest 1998 78:219-225[Medline]
  18. Jiang G, den Hertog J, Su J, Noel J, Sap J, Hunter T. Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase-alpha. Nature 1999 401:606-610[CrossRef][Medline]
  19. Tominaga T, Sahai E, Chardin P, McCormick F, Courtneidge SA, Alberts AS. Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling. Mol Cell 2000 5:13-25[CrossRef][Medline]
  20. Wu Y, Ozaki Y, Inoue K, Satoh K, Ohmori T, Yatomi Y, Owada K. Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn. Biochim Biophys Acta 2000 1497:27-36[Medline]
  21. Feig LA, Schaffhausen B. Signal transduction: the hunt for Ras targets. Nature 1994 370:508-509[CrossRef][Medline]
  22. Osterhout DJ, Wolven A, Wolf RM, Resh MD, Chao MV. Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase. J Cell Biol 1999 145:1209-1218[Abstract/Free Full Text]
  23. Klint P, Kanda S, Kloog Y, Claesson-Welsh L. Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation. Oncogene 1999 18:3354-3364[CrossRef][Medline]
  24. Walker JL, Zhang L, Menko AS. Transition between proliferation and differentiation for lens epithelial cells is regulated by Src family kinases. Dev Dyn 2002 224:361-372[CrossRef][Medline]
  25. Susva M, Missbach M, Green J. Src inhibitors: drugs for the treatment of osteoporosis, cancer or both?. Trends Pharmacol Sci 2000 21:489-495.[CrossRef][Medline]
  26. Conway AM, Rakhit S, Pyne S, Pyne NJ. Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase. Biochem J 1999 337:171-177
  27. Waltenberger J, Uecker A, Kroll J, Frank H, Mayr U, Bjorge JD, Fujita D, Gazit A, Hombach V, Levitzki A, Bohmer FD. A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling. Circ Res 1999 85:12-22[Abstract/Free Full Text]
  28. Oak JN, Lavine N, Van Tol HH. Dopamine D(4) and D(2L) receptor stimulation of the mitogen-activated protein kinase pathway is dependent on trans-activation of the platelet-derived growth factor receptor. Mol Pharmacol 2001 60:92-103[Abstract/Free Full Text]
  29. He H, Hirokawa Y, Levitzki A, Maruta H. An anti-Ras cancer potential of PP1, an inhibitor specific for Src family kinases: in vitro and in vivo studies. Cancer J 2000 6:243-248[Medline]
  30. Brockdorff JL, Gu H, Mustelin T, Kaltoft K, Geisler C, Ropke C, Odum N. Gab2 is phosphorylated on tyrosine upon interleukin-2/interleukin-15 stimulation in mycosis-fungoides-derived tumor T cells and associates inducibly with SHP-2 and Stat5a. Exp Clin Immunogenet 2001 18:86-95[CrossRef][Medline]
  31. Sunohara JR, Ridgway ND, Cook HW, Byers DM. Regulation of MARCKS and MARCKS-related protein expression in BV-2 microglial cells in response to lipopolysaccharide. J Neurochem 2001 78:664-672[CrossRef][Medline]
  32. Christian M, Zhang X, Schneider-Merck T, Unterman TG, Gellersen B, White JO, Brosens JJ. Cyclic AMP-induced forkhead transcription factor, FKHR, cooperates with CCAAT/enhancer-binding protein beta in differentiating human endometrial stromal cells. J Biol Chem 2002 277:20825-20832[Abstract/Free Full Text]
  33. Kim JJ, Taylor HS, Akbas GE, Foucher I, Trembleau A, Jaffe RC, Fazleabas AT, Unterman TG. Regulation of insulin-like growth factor binding protein-1 promoter activity by FKHR and HOXA10 in primate endometrial cells. Biol Reprod 2003 68:24-30[Abstract/Free Full Text]
  34. Woods YL, Rena G. Effect of multiple phosphorylation events on the transcription factors FKHR, FKHRL1 and AFX. Biochem Soc Trans 2002 30:391-397[CrossRef][Medline]
  35. Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999 68:965-1014[CrossRef][Medline]
  36. Yoshino O, Osuga Y, Hirota Y, Koga K, Yano T, Tsutsumi O, Taketani Y. Akt as a possible intracellular mediator for decidualization in human endometrial stromal cells. Mol Hum Reprod 2003 9:265-269[Abstract/Free Full Text]
  37. Chen R, Kim O, Yang J, Sato K, Eisenmann KM, McCarthy J, Chen H, Qiu Y. Regulation of Akt/PKB activation by tyrosine phosphorylation. J Biol Chem 2001 276:31858-31862[Abstract/Free Full Text]
  38. Nijhuis E, Lammers JW, Koenderman L, Coffer PJ. Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning. J Leukoc Biol 2002 71:115-124[Abstract/Free Full Text]
  39. Tatton L, Morley GM, Chopra R, Khwaja A. The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases. J Biol Chem 2003 278:4847-4853[Abstract/Free Full Text]
  40. Yan C, Takahashi M, Okuda M, Lee JD, Berk BC. Fluid shear stress stimulates big mitogen-activated protein kinase 1 (BMK1) activity in endothelial cells. Dependence on tyrosine kinases and intracellular calcium. J Biol Chem 1999 274:143-150[Abstract/Free Full Text]
  41. Franch HA, Wang X, Sooparb S, Brown NS, Du J. Phosphatidylinositol 3-kinase activity is required for epidermal growth factor to suppress proteolysis. J Am Soc Nephrol 2002 13:903-909[Abstract/Free Full Text]
  42. Karni R, Mizrachi S, Reiss-Sklan E, Gazit A, Livnah O, Levitzki A. The pp60c-Src inhibitor PP1 is non-competitive against ATP. FEBS Lett 2003 537:47-52[CrossRef][Medline]
  43. Uehara Y, Fukazawa H, Murakami Y, Mizuno S. Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds. Biochem Biophys Res Commun 1989 163:803-809[CrossRef][Medline]
  44. Fukazawa H, Uehara Y, Murakami Y, Mizuno S, Hamada M, Takeuchi T. Labeling of v-Src and BCR-ABL tyrosine kinases with [14C]herbimycin A and its use in the elucidation of the kinase inactivation mechanism. FEBS Lett 1994 340:155-158[CrossRef][Medline]
  45. Senga T, Miyazaki K, Machida K, Iwata H, Matsuda S, Nakashima I, Hamaguchi M. Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A. Oncogene 2000 19:273-279[CrossRef][Medline]
  46. Oddie KM, Litz JS, Balserak JC, Payne DM, Creutz CE, Parsons SJ. Modulation of pp60c-src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells. J Neurosci Res 1989 24:38-48.[CrossRef][Medline]
  47. Barnekow A, Jahn R, Schartl M. Synaptophysin: a substrate for the protein tyrosine kinase pp60c-src in intact synaptic vesicles. Oncogene 1990 5:1019-1024[Medline]
  48. Ely CM, Tomiak WM, Allen CM, Thomas L, Thomas G, Parsons SJ. pp60c-src enhances the acetylcholine receptor-dependent catecholamine release in vaccinia virus-infected bovine adrenal chromaffin cells. J Neurochem 1994 62:923-933[Medline]
  49. Furuyama N, Fujisawa Y. Regulation of collagenolytic protease secretion through c-Src in osteoclasts. Biochem Biophys Res Commun 2000 272:116-124[CrossRef][Medline]
  50. Mocsai A, Jakus Z, Vantus T, Berton G, Lowell CA, Ligeti E. Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases. J Immunol 2000 164:4321-4331[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
T. Nagashima, T. Maruyama, H. Uchida, T. Kajitani, T. Arase, M. Ono, H. Oda, M. Kagami, H. Masuda, S. Nishikawa, et al.
Activation of SRC Kinase and Phosphorylation of Signal Transducer and Activator of Transcription-5 Are Required for Decidual Transformation of Human Endometrial Stromal Cells
Endocrinology, March 1, 2008; 149(3): 1227 - 1234.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
G. Daoud, E. Rassart, A. Masse, and J. Lafond
Src family kinases play multiple roles in differentiation of trophoblasts from human term placenta
J. Physiol., March 15, 2006; 571(3): 537 - 553.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
A. Shimizu, T. Maruyama, K. Tamaki, H. Uchida, H. Asada, and Y. Yoshimura
Impairment of Decidualization in SRC-Deficient Mice
Biol Reprod, December 1, 2005; 73(6): 1219 - 1227.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
70/1/214    most recent
biolreprod.103.021527v1
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 My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Maruyama, T.
Right arrow Articles by Yoshimura, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Maruyama, T.
Right arrow Articles by Yoshimura, Y.
Agricola
Right arrow Articles by Maruyama, T.
Right arrow Articles by Yoshimura, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS