Molecular Human Reproduction, Vol. 8, No. 12, 1117-1124,
December 2002
© 2002 European Society of Human Reproduction and Embryology
Molecular events in the uterus |
Expression and subcellular distribution of the active form of c-Src tyrosine kinase in differentiating human endometrial stromal cells
1 Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 and 2 Department of Molecular Biology, Osaka Bioscience Institute, 6-2-4, Furuedai, Suita City, Osaka 565-0874, Japan
Abstract
Decidual growth factors and locally produced cytokines are thought to activate specific phosphorylation signalling pathway(s), thereby eliciting a variety of decidual functions. We have previously reported the activation of c-Src tyrosine kinase during ovarian steroid-induced decidualization of cultured human endometrial stromal cells. As chicken c-Src is known to be activated upon dephosphorylation of tyrosine 527 (Y527, corresponding to Y530 in human), we here employed a monoclonal antibody, clone 28, directed against the active form of human c-Src whose Y530 is dephosphorylated, and investigated whether c-Src became dephosphorylated at Y530 and thereby activated during decidualization. We found that the active form of c-Src was up-regulated and demonstrated increased kinase activity during in-vitro decidualization. Immunohistochemistry revealed that decidual cells in early pregnancy decidua were intensely stained with clone 28 when compared with the stromal cells in the non-pregnant endometrium. Moreover, the active form of c-Src translocated from a perinuclear region to the cytoplasm upon decidualization. Thus, the Y530 dephosphorylation, kinase activation, and subcellular translocation of c-Src may be intracellular signalling events associated with decidualization in vivo as well as in vitro.
c-Src/decidualization/progesterone/tyrosine kinase/tyrosine phosphorylation
Introduction
Human cycling endometrium and pregnancy decidua produce a large number of bioactive substances under the influence of ovarian steroid hormones (Tabibzadeh, 1991
; Giudice, 1994). These factors act as local regulators of endometrial function in an autocrine/paracrine manner, thereby controlling a wide variety of endometrial processes including cell growth, differentiation, and tissue breakdown (Tabibzadeh, 1991; Giudice, 1994). In particular, during progesterone-induced differentiation of estrogen-primed endometrial stromal cells (i.e. decidualization), a number of biological factors locally produced play a pivotal role in the initiation and maintenance of pregnancy by controlling uterine receptivity, trophoblast invasion, endometrial and placental vasculogenesis, and vascular integrity (Lockwood et al., 1993; Tabibzadeh and Babaknia, 1995; Cheung, 1997; Smith, 1998). However, little is known about signal transduction pathway(s) and signalling molecule(s) responsible for decidualization. Elucidation of the intracellular signalling events is important in that it will contribute not only to a further understanding of endometrial physiology, but also to the development of new drugs to regulate possible endometrium/decidua-specific signalling pathways for contraception or treatment of infertility.
It is well known that reversible protein phosphorylation on tyrosine residues, coordinately controlled by protein tyrosine kinases and phosphatases, plays an important role in a variety of biological processes including cell growth, differentiation, apoptosis, and tumorogenesis (Hunter, 1998). We have previously demonstrated that decidualization of cultured endometrial stromal cells is accompanied by reduced c-Src phosphorylation and increased c-Src kinase activity (Maruyama et al., 1999b). Our results seem to fit with the generally accepted model that c-Src becomes activated upon dephosphorylation at its negative regulatory tyrosine residue, Y527 (corresponding to Y530 in human) (Thomas and Brugge, 1997).
To further examine whether the low phosphorylation level of decidual c-Src is due to dephosphorylation of Y530, we have employed a murine monoclonal antibody, termed clone 28, which was raised against the C-terminal peptides of c-Src containing dephosphorylated Y530 and therefore selectively recognizes the active form of c-Src (Kawakatsu et al., 1996). This antibody has been successfully demonstrated to specifically react with the activated c-Src (Kawakatsu et al., 1996; Sakai et al., 1998; Jiang et al., 1999; Tominaga et al., 2000; Wu et al., 2000). Here we show using clone 28 that c-Src becomes dephosphorylated on Y530 and activated during in-vivo as well as in-vitro decidualization. We also found that the active form of c-Src translocated from a perinuclear region to the cytoplasm upon decidualization. This is the first report to provide evidence suggesting that signalling pathway(s) involving Y530 dephosphorylation, kinase activation, and subcellular translocation of c-Src may participate in the process of differentiation of human endometrial stromal cells.
Materials and methods
Reagents
Clone 28 (Kawakatsu et al., 1996) was kindly provided by Dr Koji Owada (Kyoto Pharmaceutical University, Kyoto, Japan). The mouse monoclonal antibody clone 327, which reacts with both active and inactive c-Src, was obtained from Calbiochem (San Diego, CA, USA). Anti-ß-actin mouse monoclonal, anti-Fyn mouse monoclonal, anti-IGFBP-1 (insulin-like growth factor binding protein-1) goat polyclonal, and donkey horse-radish peroxidase (HRP)-conjugated pre-adsorbed anti-goat antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat Cy3-conjugated anti-mouse IgG and HRP-conjugated anti-mouse IgG antibodies were purchased from Amersham International (Buckinghamshire, UK). Purified epidermal growth factor (EGF) was obtained from Wako (Osaka, Japan). Progesterone and 17ß-estradiol (E2) were purchased from Sigma (St Louis, Missouri, MO, USA). A peptide corresponding to the C-terminal region of human c-Src (QYQPGENL, residues 529–536) was synthesized (TaKaRa Biomedicals, Tokyo, Japan).
Plasmid construction
To construct a pShuttle chicken c-Src expression vector, the pcDNA3-based vector for wild-type chicken c-src cDNA was digested with BamHI and EcoRI. The fragment containing the full length of c-src cDNA was then blunt-ended with T4 DNA polymerase and ligated with pShuttle vector (Clontech, Palo Alto, CA, USA) that had been digested with NotI and blunt-ended. The orientation and junctions were verified by sequencing and restriction enzyme mapping.
Tissue specimens
Endometrial specimens from the proliferative (day 7–14; n = 26), early secretory (day 15–21; n = 19), and mid- to late secretory (day 22–28; n = 6) phases of the menstrual cycle were obtained from consenting patients undergoing endometrial biopsies or total abdominal hysterectomy for myomas. The use of the specimens was approved by Keio University Ethics Committee. In addition, early pregnancy decidua was obtained from the first trimester pregnancy termination (7–9 gestational weeks; n = 4). There was no abnormality or malignancy in these specimens as diagnosed by histological examination. Dating was confirmed according to published criteria (Noyes et al., 1950). The specimens diagnosed as late proliferative phase or early secretory phase were also used for the cell cultures.
Isolation of endometrial stromal cells
Endometrial stromal cells were isolated from human cycling endometria as previously described (Maruyama et al., 1999a). In brief, tissue samples were washed with Dulbecco's modified Eagle's medium (DMEM) and minced into small pieces of <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, USA), 1% antibiotic– antimycotic mixture (Life Technologies), and 10% fetal bovine serum (FBS). After enzymatic digestion, cell clumps were dispersed by pipetting. Most of the stromal cells that were present as single cells or small aggregates were strained through a 70 µm cell strainer (Falcon 2350; Becton Dickinson, Franklin Lakes, NJ, USA). The filtrates were washed twice, and the number of viable cells was counted by Trypan Blue dye exclusion.
Cell culture, hormonal treatment and transfections
Two million viable endometrial stromal cells were inoculated into 6 cm dishes (Falcon). Alternatively, they were seeded into each well of 6-well plates (Falcon). The cells were pre-cultured for ~2 days to be grown to subconfluence in DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic mixture. The cells were then cultured in the absence or presence of 10 nmol/l E2 plus 1 µmol/l progesterone to induce differentiation. The cells were incubated for different periods with renewal of the medium every 2 days, 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 EGF on c-Src activation, the cells were pre-cultured for 48 h in DMEM supplemented with 0.1% calf serum, then treated with EGF (100 ng/ml) for 5, 10 and 20 min, and harvested for various types of analyses as indicated. NIH-3T3 cells were also transfected with pShuttle chicken c-Src expression vector using Lipofectamine (Life Technologies) according to the manufacturer's instructions and then harvested for immunoblot analyses 48 h after transfection.
Immunoprecipitation and immunoblotting
Total cell lysates were prepared with RIPA buffer (20 mmol/l Tris–HCl pH7.5, 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Na-deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mmol/l Na3VO4, 50 mmol/l NaF, 1 mmol/l Na2MoO4) (Maruyama et al., 1999b,c) containing protease inhibitor cocktail (Roche Molecular Biochemicals). The protein concentration was measured using DC protein assay kit (BioRad, Hercules, CA, USA). In typical experiments, 100–200 µg of cell lysates were immunoprecipitated by incubation with clone 327 (0.2 µg) or anti-Fyn antibody (0.2 µg) for 4 h at 4°C, followed by incubation with protein G-sepharose beads (Amersham Biosciences). The immune complexes were washed three times with RIPA buffer and then resuspended in the 2xSDS sample buffer.
The washed immunoprecipitates or 30 µg of the lysates derived from the cultured stromal cells, NIH-3T3 cells, or the endometrial/decidual tissues were separated by 8% SDS–polyacrylamide gel electrophoresis (PAGE), and transferred onto a PVDF membrane (Immobilon P; Millipore, Bedford, MA, USA). Non-specific binding sites were blocked in 5% bovine serum albumin (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 ECL plus detection kit (Amersham Biosciences). When indicated, immunoblots were stripped in the buffer (62.5 mmol/l Tris pH 6.8, 2% SDS, 100 mmol/l ß-mercaptoethanol) at 50°C for 30 min, and reprobed with another specific antibody. The intensity of the signals on the immunoblot was quantified using NIH Image program version 1.62. The value of intensity of the active c-Src band from non-decidualized cells was adjusted to 1.0.
In-vitro kinase assay
In-vitro kinase assay was performed as previously described (Maruyama et al., 1999b). Briefly, immunoprecipitates of c-Src and Fyn from 100–200 µg of the cell lysates were washed three times with RIPA buffer, twice with kinase buffer (50 mmol/l Tris–HCl pH 7.4, 0.1% NP-40, 0.1 mmol/l Na3VO4, 3 mmol/l MgCl2, 3 mmol/l MnCl2), and then incubated with 20 µl kinase buffer containing 10 µCi [
-32P]ATP and 2 µg of acid-treated enolase (Sigma) for 10 min at 30°C. After termination of the reactions by adding 2xSDS sample buffer with 1 mmol/l EDTA, the samples were boiled and separated by 8% SDS–PAGE. The gels were dried and subjected to autoradiography.
Immunohistochemistry
The deparaffinized sections from formaldehyde-fixed specimens were washed with 50 mmol/l Tris–HCl (pH 7.6), containing 150 mmol/l NaCl, microwaved for 10 min, and washed again. The internal peroxidase activity and non-specific binding sites were blocked by 0.3% hydrogen peroxide–methanol for 20 min and blocking buffer (Dako Co., Capinteria, CA, USA) for 10 min. The slides were then incubated with clone 28 (3 µg/ml) for 1 h. As a negative control, mouse control IgG (Dako Japan, Kyoto, Japan) was used at the same concentration as clone 28. After being washed, bound antibody was visualized using biotinylated anti-mouse IgG (Vector, Burlingame, CA, USA) and Vectastain Elite ABC Kit (Vector) according to the manufacturer's instructions. The samples were then incubated with 0.02% 3,3'-diaminobenzidine for 5 min. Nuclei were lightly stained by haematoxylin or methyl green. Each step was performed at room temperature.
Immunofluorescence microscopy
The isolated endometrial stromal cells, prepared as mentioned above, were seeded and grown on plastic chamber slides (Permanox, Lab-Tek Chamber Slide; Nalgen Nunc International, Naperville, IL, USA) to subconfluence. Subsequently, the cells were treated with or without 10 nmol/l E2 plus 1 µmol/l progesterone for 10 days. Cells were then fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS at room temperature. After being blocked by 10% BSA in PBS for 30 min at room temperature, slides were incubated with either clone 28 (3 µg/ml), clone 28 (3 µg/ml) that had been preincubated with the competitive peptide (100 µg/ml), or clone 327 (1 µg/ml) for 90 min at 37°C. After being washed three times in PBS, bound antibody was visualized using Cy3-conjugated antibody for 30 min at 37°C in a moist chamber. The slides were washed extensively in PBS and mounted. Cells were simultaneously stained with the nuclear dye, bisbenzimide (Hoechst dye 33258; Sigma).
Statistical analysis
Differences in the quantified relative intensity of the immunoblot signals between the non-decidualized and decidualized cells were statistically assessed using Wilcoxon rank-sum test. Differences were considered significant if P < 0.05.
Results
Specificity of clone 28 for the active form of c-Src
To verify the specificity of clone 28, we examined whether, in NIH 3T3 cells, clone 28 could react with the active form of c-Src induced by EGF stimulation, a known c-Src activator (Belsches et al., 1997). We found that the intensity of immunoblot staining with clone 28 was increased in a time-dependent manner upon EGF treatment (Figure 1A, arrow). To investigate the association between immunoreactivity with clone 28 and kinase activity, c-Src immunoprecipitates derived from the EGF-treated cells were subjected to both immunoblot staining with clone 28 and in-vitro kinase assay using enolase as a c-Src substrate (Figure 1B). As shown in Figure 1B, the c-Src immunoprecipitates exhibited an EGF-induced increase in the immunoreactivity with clone 28 (top panel), together with an elevation of the kinase activity for phosphorlyation of enolase (bottom panel). Both the immunoreactivity with clone 28 and the phosphorylation activity displayed similar kinetics upon EGF treatment. Stripping and reprobing the immunoblot with clone 327 showed almost equal loading of these immunoprecipitates for each sample (Figure 1B, middle panel).
|
We also found, using immunoblot analysis with clone 28, that the active form of c-Src was up-regulated by the other known c-Src activators, platelet-derived growth factor-BB (DeMali et al., 1999
) and hydrogen peroxide (Abe et al., 1997), in NIH 3T3 cells (data not shown). Thus, clone 28 can selectively recognize the active form of c-Src as reported previously (Kawakatsu et al., 1996).
Kinase activation and Y530 dephosphorylation of c-Src during in-vitro decidualization
We have previously shown that decidualization of cultured endometrial stromal cells is accompanied by reduced c-Src phosphorylation and increased c-Src kinase activity (Maruyama et al., 1999b). These findings prompted us to determine whether reduced phosphorylation of decidual c-Src is due to dephosphorylation of specific tyrosine residues including Y530.
Immunoblot analyses with clone 28 revealed that cultured decidualized cells exhibited a greater abundance of active c-Src than did non-decidualized cells (Figure 2A, top panel, arrow). In contrast, both non-decidualized and decidualized cells displayed almost the same level of ß-actin expression, indicating that the amount of the cell lysates was equally loaded in each sample (Figure 2A, middle panel). In addition, decidualization of E2 + progesterone-treated cells was verified by the prominent expression of IGFBP-1, a typical decidualization marker (Figure 2A, bottom panel).
|
As observed in Figures 1 and 2A
, two bands were occasionally detected by immunoblot staining of total cell lysates with clone 28. Since the molecular weight of c-Src is known to be 60 kDa, the upper band appeared to correspond to the active form of c-Src. To determine which band represents the active form of c-Src, we compared the c-Src immunoprecipitate derived from non-decidualized and decidualized cells with the corresponding input (Figure 2B, upper panel). Immunoblot analysis of these samples with clone 28 revealed that the single band in the c-Src immunoprecipitate exhibited the same mobility as the upper band of the input (Figure 2B, upper panel, arrow). In agreement with immunoblot analysis on the whole cell lysates (Figure 2A), the c-Src immunoprecipitate from decidualized cells displayed an increase in the immunoreactivity with clone 28 compared with that from non-decidualized cells (Figure 2B, upper panel). Although the band of IgG heavy chain was expected to migrate very closely to that of c-Src, we confirmed that the single band reactive with clone 28 as well as with clone 327, as observed in the clone 327 immunoprecipitates, was not the heavy chain of mouse IgG (data not shown).
We also compared the SDS-based molecular weight of endogenous stromal c-Src with that of exogenously overexpressed c-Src derived from NIH 3T3 cells transfected with the pShuttle c-Src expression vector (Figure 2B, two lower panels). Immunoblot analysis with clone 28 revealed that exogenous c-Src was so prominently overexpressed that the two bands could not be differentiated (Figure 2B, lower left panel). To distinguish the two bands, the same membrane was exposed to the film for a shorter time (lower right panel), enabling us to see that clone 28 preferentially recognized the upper band corresponding to the exogenously overexpressed c-Src (lower right panel, arrow). These results collectively indicate that the upper band represented the active form of c-Src, consistent with the previous report (Kawakatsu et al., 1996). Although the nature of the lower band remained unclear, it did not correspond to the inactive form of c-Src, because its molecular weight was obviously <60 kDa and it did not react with clone 327 (Figure 2B).
We then tested whether the c-Src immunoprecipitate derived from decidualized cells not only possessed the enhanced immunoreactivity with clone 28 but also displayed the elevated kinase activity. As shown in Figure 2C (left panels), immunoprecipitates with clone 327 derived from decidualized cells exhibited a prominent immunoblot staining with clone 28 (left top panel), coinciding with an enhanced kinase activity for phosphorylation of enolase (left bottom panel) when compared to the non-decidualized cells. Stripping and reprobing the immunoblot with clone 327 showed almost equal loading of these c-Src immunoprecipitates in each sample (left bottom panel). In contrast, the immunoprecipitates of Fyn, another Src family kinase, did not show an enhanced kinase activity upon decidualization (Figure 2C, two right panels), consistent with a previous report (Maruyama et al., 1999b).
The series of the independent experiments using five different cell cultures revealed that the immunostaining intensity of the activated c-Src in stromal cells treated with E2 + progesterone for 10–14 days was ~5-fold higher than that in the non-treated cells (mean ± SE: 4.78 ± 2.78, P < 0.05, Wilcoxon rank-sum test).
Expression of the active form of c-Src in the endometrium during the menstrual cycle and early pregnancy
Since our study was focused on in-vitro decidualization, we then determined if there might be a similar mechanism in vivo by which decidual c-Src is activated through dephosphorylation of Y530. Immunoblot analyses of cycling endometria and early pregnancy decidua revealed that the decidua displayed increased levels of active c-Src expression when compared to the non-pregnant endometria (Figure 3, top panel). The decidual tissues showed a prominent expression of IGFBP-1 (Figure 3, bottom panel), whereas the levels of ß-actin were not dramatically changed upon decidualization (Figure 3, middle panel). There was little difference in the expression of active c-Src between proliferative and secretory endometria (Figure 3, top panel).
|
We noted, however, that the active form of c-Src in the non-pregnant endometrium appeared to be more abundant than expected from our in-vitro data. To address this discrepancy, we examined the immunohistochemical localization of active c-Src in cycling endometria and early pregnancy decidua. Consistent with our in-vitro data, we found that decidual cells in the pregnancy decidua were more intensely stained with clone 28 when compared with the stromal cells in the non-pregnant endometrium (Figure 4A–D
). Moreover, decidualizing stromal cells adjacent to spiral arteries (predecidualized cells) in the secretory endometrium also exhibited more prominent staining for active c-Src than stromal cells in the proliferative endometrium (Figure 4A and B). In contrast, the level of the active form of c-Src present in the glandular and luminal epithelium was relatively high and constant throughout the menstrual cycle (Figure 4A and B). These findings may account for the unexpectedly high levels of active c-Src expression even in the non-pregnant endometrium (Figure 3). In addition, as predecidualization occurs focally in the secretory endometrium, high and constant levels of glandular active c-Src expression may undermine an increase in the level of stromal active c-Src expression in the secretory endometrium, possibly explaining why there was little difference in the expression of activated c-Src between proliferative and secretory endometria (Figure 3).
|
Diffuse cytoplasmic distribution of the active form of stromal c-Src upon decidualization
Besides an increase in the levels of active c-Src expression during decidualization, immunohistochemistry also revealed that the active form of stromal c-Src appeared to translocate from a perinuclear region to the cytoplasm during the process of decidualization (Figure 4E and F
). In contrast, the localization of the active form of glandular c-Src remained unchanged.
To further examine whether the active form of c-Src behaves similarly in vitro, we performed immunofluorescence of cultured stromal cells with clone 28 or clone 327, together with Hoechst dye 33258 for nuclear staining. As shown in Figure 5A, the active form of c-Src was highly concentrated in a specific region in the non-decidualized cells (arrowheads). Nuclear staining with Hoechst dye 33258 revealed that the active form of c-Src was confined to the perinuclear region in non-decidualized cells (Figure 5B, arrowheads), while it became more diffusely distributed in the cytoplasm of decidualized cells (Figure 5C). This characteristic staining pattern of decidualized cells disappeared when clone 28 was preincubated with the peptide corresponding to the C-terminal region of human c-Src (QYQPGENL, residues 529–536) (Figure 5D), indicating that immunoreactivity of clone 28 represents the active form of c-Src. Total c-Src (both inactive and active c-Src) reactive with clone 327 was less obviously concentrated around the nucleus than the active form of c-Src alone (Figure 5A, B and E). Upon decidualization, the total c-Src became distributed more evenly throughout the cytoplasm in a similar way to the active form of c-Src (Figure 5F).
|
Discussion
To date, the plausible candidate molecules regulating the phosphorylation status of the C-terminal tail are C-terminal Src kinase (CSK) and several protein tyrosine phosphatases (PTPases) including receptor-like protein-tyrosine phosphatase-
(Brown and Cooper, 1996; Thomas and Brugge, 1997). In general, CSK represses the kinase activity of c-Src through phosphorylation of Y530, while PTPases activate c-Src through dephosphorylation of the same residue (Brown and Cooper, 1996; Thomas and Brugge, 1997). CSK and several PTPases play a pivotal role, through regulation of c-Src, in the differentiation of a variety of cell types such as osteoclasts, keratinocytes, and neurons (Zhao et al., 1992; den Hertog et al., 1993; Chappel et al., 1997; Takayama et al., 1997). It is, therefore, conceivable that CSK and/or PTPases may regulate the phosphorylation status of Y530 and thereby activate c-Src upon decidualization, though so far these molecules have not been identified in human endometrium and decidua.
c-Src is known to associate with a number of receptor protein tyrosine kinases such as PDGF, EGF, colony stimulating factor-1, and insulin-like growth factor receptors (Thomas and Brugge, 1997) and thereby to be activated upon binding of the receptors to the corresponding ligands. 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 receptors can also associate with c-Src and activate it (Thomas and Brugge, 1997). Importantly, those Src-activating factors are thought to be locally produced in the decidua, acting as paracrine/autocrine regulators of decidual function (Tabibzadeh, 1991; Gurpide et al., 1992; Schrey et al., 1992; Giudice, 1994; Jokhi et al., 1997; Morgan et al., 1998; Ogata et al., 2000; Popovici et al., 2000). Thus, they may have a potential to behave as ligands for their relevant transmembrane receptors that can exert the recruitment and activation of decidual c-Src. Although we here demonstrated a PDGF- or EGF-induced dephosphorylation of Y530 in NIH 3T3 cells, it remains to be elucidated whether decidual EGF and PDGF are involved in the Y530 dephosphorylation and kinase activation of c-Src.
In addition to local growth factors, estrogen and progesterone have been reported to activate c-Src/p21ras/Erk pathway in human breast cancer cells (Migliaccio et al., 1996, 1998). Very recently, it has been shown that progesterone receptors directly interact with the SH3 domain of c-Src and activate it upon ligand binding (Boonyaratanakornkit et al., 2001). Thus, although we here showed that kinase activation of decidual c-Src by Y530 dephosphorylation was an event tightly associated with decidualization, further studies are required to elucidate the upstream and downstream signalling pathways of decidual c-Src.
All vertebrate Src family kinases possess C-terminal extensions of 15–17 residues following the end of the kinase domain, and these tails contain tyrosine (Y527 in chicken Src) in a constant position and conserved sequence (Thomas and Brugge, 1997). Therefore, it is possible that clone 28 may cross-react with other src family tyrosine kinases such as Fyn and Yes because this antibody was raised against the c-Src C-terminal peptides. Indeed, it has been recently suggested that clone 28 cannot distinguish the active form of c-Src from the active Fyn (Wu et al., 2000). However, based on our previous (Maruyama et al., 1999b) and present results demonstrating no remarkable activation of Fyn upon decidualization, we assume that the active form of stromal c-Src was the main component of the ~60 kDa band recognized by clone 28.
We here found that the total c-Src (both inactive and active c-Src) reactive with clone 327 located at the perinuclear area, consistent with many previous reports on the subcellular localization of c-Src as well as v-Src, the oncogenic viral src gene product (Resh and Erikson, 1985; David-Pfeuty and Nouvian-Dooghe, 1990; Kaplan et al., 1992; Redmond et al., 1992; Fincham et al., 1996). In addition, we here showed that its active form reactive with clone 28 exhibited more evident perinuclear localization than the total c-Src reactive with clone 327. These findings are in agreement with the previous biochemical fractionation studies showing that a large portion of endogenous c-Src kinase activity in chicken embryo fibroblasts is associated with the perinuclear membrane system (Resh and Erikson, 1985). In this study, clone 28 localized decidual c-Src dephosphorylated at Y530 to the cytosol but failed to localize it to either the cell periphery or focal adhesions, apparently contradicting the findings of one study (Kaplan et al., 1994). Intriguingly, the redistribution of c-Src into the cytosol and cell peripheries requires the proper organization of actin cytoskeleton (Fincham et al., 1996). Given the loss of focal adhesions and the disorganization of actin-based cytoskeleton in decidualized cells as previously reported (Maruyama et al., 1999c), it is possible that activated decidual c-Src dephosphorylated at Y530 may not be able to translocate from the perinuclear area to the cell peripheries and focal adhesion plaques.
c-Src primarily co-localizes with markers of endosomal membranes and associates with specialized secretory vesicles in some cell types (Grandori and Hanafusa, 1988; Johnston et al., 1989; Kaplan et al., 1992; Linstedt et al., 1992), implicating c-Src as a possible regulator of exocytosis (Oddie et al., 1989; Barnekow et al., 1990; Ely et al., 1994). Considering a high secretory potential of decidual cells (Gurpide et al., 1992), it is possible that c-Src may regulate secretion of many decidualization-associated bioactive substances. Alternatively, v-Src, a constitutively kinase-active variant of c-Src, possesses an oncogenic transformation activity including cell rounding and detachment, and additionally kinase activation of c-Src has been implicated in the regulation of the actin-based cytoskeletal organization and focal adhesion assembly (Brown and Cooper, 1996; Thomas and Brugge, 1997; Abram and Courtneidge, 2000). Therefore, it is conceivable that c-Src might play a role in a unique decidual transformation from fibroblastic stromal cells into metabolically active, round and enlarged decidual cells.
In summary, we here verified dephosphorylation at the C-terminus as a component of decidual c-Src regulation and identified differences in subcellular localization of the active form of c-Src. Alterations in the subcellular distribution of c-Src from its site of synthesis to its site of action, together with its increased kinase activity, must be required for c-Src to work as an effective signalling molecule (Abram and Courtneidge, 2000). Taken together, our present results suggest that c-Src may actively participate in the process of decidualization in vivo as well as in vitro, further prompting us to elucidate the decidual c-Src function with knock-out studies.
Acknowledgements
We thank Dr Koji Owada (Kyoto Pharmaceutical University, Kyoto, Japan) for his generous gift of clone 28, Dr Hidesaburo Hanafusa (Osaka Bioscience Institute, Osaka, Japan) for pcDNA3-based expression vector for wild-type chicken c-src cDNA, Mr Ryuich Taki (Mitsubishi Chemical BCL, Tokyo, Japan) for his technical advice and assistance on immunohistochemistry, and Ms Shino Kuwabara for her secretarial assistance. This study was supported, in part, by grants from the Ministry of Education, Science, and Culture of Japan (B:12470348), Keio Gijuku Academic Development Funds, and grants from the Keio Health Counseling Center. Part of this work was presented at the 2000 International Symposium on Cell and Molecular Biology of Endometrium in Health and Disease, Kobe, Japan and at the 35th Annual Meeting of the Society for the Study of Reproduction, Baltimore, USA.
Notes
3 To whom correspondence should be addressed. E-mail: tetsuo{at}sc.itc.keio.ac.jp 
References
Abe, J., Takahashi, M., Ishida, M., Lee, J.D. and Berk, B.C. (1997) c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1 (BMK1). J. Biol. Chem., 272, 20389–20394.
Abram, C.L. and Courtneidge, S.A. (2000) Src family tyrosine kinases and growth factor signaling. Exp. Cell. Res., 254, 1–13.[ISI][Medline]
Barnekow, A., Jahn, R. and Schartl, M. (1990) Synaptophysin: a substrate for the protein tyrosine kinase pp60c–src in intact synaptic vesicles. Oncogene, 5, 1019–1024.[ISI][Medline]
Belsches, A.P., Haskell, M.D. and Parsons, S.J. (1997) Role of c-Src tyrosine kinase in EGF-induced mitogenesis. Front. Biosci., 2, d501–518.[Medline]
Brown, M.T. and Cooper, J.A. (1996) Regulation, substrates and functions of src. Biochim. Biophys. Acta, 1287, 121–149.[Medline]
Boonyaratanakornkit, V., Scott, M.P., Ribon, V., Sherman, L., Anderson, S.M., Maller, J.L., Miller, W.T. and Edwards, D.P. (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.[ISI][Medline]
Chappel, J., Ross, F.P., Abu-Amer, Y., Shaw, A. and Teitelbaum, S.L. (1997) 1,25-dihydroxyvitamin D3 regulates pp60c–src activity and expression of a pp60c–src activating phosphatase. J. Cell. Biochem., 67, 432–438.[ISI][Medline]
Cheung, C.Y. (1997) Vascular endothelial growth factor: possible role in fetal development and placental function. J. Soc. Gynecol. Invest., 4, 169–177.[ISI][Medline]
David-Pfeuty, T. and Nouvian-Dooghe, Y. (1990) Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells. J. Cell Biol., 111, 3097–3116.
DeMali, K.A., Godwin, S.L., Soltoff, S.P. and Kazlauskas, A. (1999) Multiple roles for Src in a PDGF-stimulated cell. Exp. Cell Res., 253, 271–279.[ISI][Medline]
den Hertog, J., Pals, C.E.G.M., Peppelenbosch, M.P., Tertoolen, L.G.J., de Laat, S.W. and Kruijer, W. (1993) Receptor protein tyrosine phosphatase activates pp60c–src and is involved in neuronal differentiation. EMBO J., 12, 3789–3798.[ISI][Medline]
Ely, C.M., Tomiak, W.M., Allen, C.M., Thomas, L., Thomas, G. and Parsons, S.J. (1994) pp60c–src enhances the acetylcholine receptor-dependent catecholamine release in vaccinia virus-infected bovine adrenal chromaffin cells. J. Neurochem., 62, 923–933.[ISI][Medline]
Fincham, V.J., Unlu, M., Brunton, V.G., Pitts, J.D., Wyke, J.A. and Frame, M.C. (1996) Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins. J. Cell Biol., 135, 1551–1564.
Giudice, L.C. (1994) Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. Fertil. Steril., 61, 1–17.[ISI][Medline]
Grandori, C. and Hanafusa, H. (1988) pp60c–src is complexed with a cellular protein in subcellular compartments involved in exocytosis. J. Cell Biol., 107, 2125–2135.
Gurpide, E., Tabanelli, S. and Tang, B. (1992) Human endometrial stromal cells. In Genazzani, A.R. and Petraglia F. (eds), Hormones in Gynecological Endocrinology. Parthenon Press, Casterton Hall, Carnforth, Lancs, UK, pp. 717–724.
Hunter, T. (1998) The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease. Phil. Trans. R. Soc. Lond. B. Biol. Sci., 353, 583–605.[ISI][Medline]
Jiang, G., den Hertog, J., Su, J., Noel, J., Sap, J. and Hunter, T. (1999) Dimerization inhibits the activity of receptor-like protein-tyrosine phosphatase-. Nature, 401, 606–610.[Medline]
Johnston, P.A., Cameron, P.L., Stukenbrok, H., Jahn, R., De Camilli, P. and Sudhof, T.C. (1989) Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells. EMBO J., 8, 2863–2872.[ISI][Medline]
Jokhi, P.P., King, A. and Loke, Y.W. (1997) Cytokine production and cytokine receptor expression by cells of the human first trimester placental–uterine interface. Cytokine, 9, 126–137.[ISI][Medline]
Kaplan, K.B., Swedlow, J.R., Varmus, H.E. and Morgan, D.O. (1992) Association of pp60c–src with endosomal membranes in mammalian fibroblasts. J. Cell Biol., 118, 321–333.
Kaplan, K.B., Bibbins, K.B., Swedlow, J.R., Arnaud, M., Morgan, D.O. and Varmus, H.E. (1994) Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527. EMBO J., 13, 4745–4756.[ISI][Medline]
Kawakatsu, H., Sakai, T., Takagaki, Y., Shinoda, Y., Saito, M., Owada, M.K. and Yano, J. (1996) A new monoclonal antibody which selectively recognizes the active form of Src tyrosine kinase. J. Biol. Chem., 271, 5680–5685.
Linstedt, A.D., Vetter, M.L., Bishop, J.M. and Kelly, R.B. (1992) Specific association of the proto-oncogene product pp60c–src with an intracellular organelle, the PC12 synaptic vesicle. J. Cell Biol., 117, 1077–1084.
Lockwood, C.J., Nemerson, Y., Krikun, G., Hausknecht, V., Markiewicz, L., Alvarez, M., Guller, S. and Schatz, F. (1993) Steroid-modulated stromal cell tissue factor expression: a model for the regulation of endometrial hemostasis and menstruation. J. Clin. Endocrinol. Metab., 77, 1014–1019.[Abstract]
Maruyama, T., Sachi, Y., Furuke, K., Kitaoka, Y., Kanzaki, H., Yoshimura, Y. and Yodoi, J. (1999a) 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.
Maruyama, T., Yoshimura, Y., Yodoi, J. and Sabe, H. (1999b) Activation of c-Src kinase is associated with in vitro decidualization of human endometrial stromal cells. Endocrinology, 140, 2632–2636.
Maruyama, T., Yoshimura, Y. and Sabe, H. (1999c) Tyrosine phosphorylation and subcellular localization of focal adhesion proteins during in vitro decidualization of human endometrial stromal cells. Endocrinology, 140, 5982–5990.
Migliaccio, A., di Domenico, M., Castoria, G., de Falco, A., Bontempo, P., Nola, E. and Auricchio, F. (1996) Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol–receptor complex in MCF-7 cells. EMBO J., 15, 1292–1300.[ISI][Medline]
Migliaccio, A., Piccolo, D., Castoria G., di Domenico, M., Bilancio, A., Lombardi, M., Gong, W., Beato, M. and Auricchio, F. (1998) Activation of the Src/p21ras/Erk pathway by progesterone receptor via cross-talk with estrogen receptor. EMBO J., 17, 2008–2018.[ISI][Medline]
Morgan, T., Craven, C. and Ward, K. (1998) Human spiral artery renin–angiotensin system. Hypertension, 32, 683–687.
Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.[Medline]
Oddie, K.M., Litz, J.S., Balserak, J.C., Payne, D.M., Creutz, C.E. and Parsons, S.J. (1989) Modulation of pp60c–src tyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells. J. Neurosci. Res., 24, 38–48.[ISI][Medline]
Ogata, I., Shimoya, K., Moriyama, A., Shiki, Y., Matsumura, Y., Yamanaka, K., Nobunaga, T., Tokugawa, Y., Kimura, T., Koyama, M. et al. (2000) Oncostatin M is produced during pregnancy by decidual cells and stimulates the release of HCG. Mol. Hum. Reprod., 6, 750–757.
Popovici, R.M., Kao, L.-C. and Giudice, L.C. (2000) Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology. Endocrinology, 141, 3510–3513.
Redmond, T., Brott, B.K., Jove, R. and Welsh, M.J. (1992) Localization of the viral and cellular Src kinases to perinuclear vesicles in fibroblasts. Cell Growth Differ., 3, 567–576.[Abstract]
Resh, M.D. and Erikson, R.L. (1985) Highly specific antibody to Rous sarcoma virus src gene product recognizes a novel population of pp60v–src and pp60c–src molecules. J. Cell Biol., 100, 409–417.
Sakai, T., Kawakatsu, H., Fujita, M., Yano, J. and Owada, M.K. (1998) An epitope localized in c-Src negative regulatory domain is a potential marker in early stage of colonic neoplasms. Lab. Invest., 78, 219–225.[ISI][Medline]
Schrey, M.P., Holt, J.R., Cornford, P.A., Monaghan, H. and al-Ubaidi, F. (1992) Human decidua is a target tissue for bradykinin and kallikrein: phosphoinositide hydrolysis accompanies arachidonic acid release in uterine decidua cells in vitro. J. Clin. Endocrinol. Metab., 74, 426–435.[Abstract]
Smith, S.K. (1998) Angiogenesis, vascular endothelial growth factor and the endometrium. Hum. Reprod. Update, 4, 509–519.
Tabibzadeh, S. (1991) Human endometrium: an active site of cytokine production and action. Endocr. Rev., 12, 272–290.[ISI][Medline]
Tabibzadeh, S. and Babaknia, A. (1995) The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Hum. Reprod., 10, 1579–1602.
Takayama, Y., Nada, S., Nagai, K., and Okada, M. (1997) Role of Csk in neural differentiation of the embryonic carcinoma cell line P19. FEBS Lett., 406, 11–16.[ISI][Medline]
Thomas, S.M. and Brugge, J.S. (1997) Cellular functions regulated by Src family kinases. Annu. Rev. Cell Dev. Biol., 13, 513–609.[ISI][Medline]
Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S.A. and Alberts, A.S. (2000) Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling. Mol. Cell, 5, 13–25.[ISI][Medline]
Wu, Y., Ozaki, Y., Inoue, K., Satoh, K., Ohmori, T., Yatomi, Y. and Owada, K. (2000) 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, 1497, 27–36.[Medline]
Zhao, Y., Sudol, M., Hanafusa, H. and Krueger, J. (1992) Increased tyrosine kinase activity of c-Src during calcium-induced keratinocyte differentiation. Proc. Natl. Acad. Sci. USA, 89, 8298–8302.
Submitted on April 18, 2002; resubmitted on July 17, 2002; accepted on September 20, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 M. M. Desouki and B. G. Rowan Src Kinase and Mitogen-Activated Protein Kinases in the Progression from Normal to Malignant Endometrium Clin. Cancer Res., January 15, 2004; 10(2): 546 - 555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Maruyama, Y. Yamamoto, A. Shimizu, H. Masuda, N. Sakai, R. Sakurai, H. Asada, and Y. Yoshimura Pyrazolo Pyrimidine-Type Inhibitors of Src Family Tyrosine Kinases Promote Ovarian Steroid-Induced Differentiation of Human Endometrial Stromal Cells In Vitro Biol Reprod, January 1, 2004; 70(1): 214 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sakai, T. Maruyama, R. Sakurai, H. Masuda, Y. Yamamoto, A. Shimizu, I. Kishi, H. Asada, S. Yamagoe, and Y. Yoshimura Involvement of Histone Acetylation in Ovarian Steroid-induced Decidualization of Human Endometrial Stromal Cells J. Biol. Chem., May 2, 2003; 278(19): 16675 - 16682. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||