LECTURE
Protein tyrosine phosphorylation signaling in the
differentiation of human endometrial stromal cells
Tetsuo Maruyama, Yurie Yamamoto, Nozomi Sakai, Aki Shimizu, Asuka Shimoki, Takahiro Masuda andYasunori Yoshimura
Department of Obstetrics and Gynecology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on February 27, 2002)

Abstract. Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine
kinases (PTKs) and protein tyrosine phosphatases (PTPs), is a critical element in signal transduction
pathways regulating cell growth, differentiation, apoptosis, and tumorigenesis. The differentiation ofhuman endometrial stromal cells (decidualization) is crucial for successful embryo implantation andmaintenance of pregnancy; however, little is known about the molecular events involving tyrosine
phosphorylation, PTKs, and PTPs in the process of decidualization. We have previously reported thatthe tyrosine kinase activity of c-Src belonging to the Src family kinase is increased together with alteredtyrosine phosphorylation of several cellular proteins in the in vitro model of decidualization. Focal
adhesion kinase (FAK) and paxillin are known to form a complex with c-Src at the focal contacts andto participate in the integrin-mediated signal transduction as c-Src substrates. Those focal adhesion
proteins, however, are not hyperphosphorylated on tyrosine during decidualization. Moreover, the lossof focal adhesions and the disorganization of the actin-based cytoskeleton were observed in decidualized stromal cells, suggesting that the escape from regulation by c-Src may be in part due to thedecidualization-induced disruption of the interaction between the focal adhesion proteins and c-Src.
These ﬁndings collectively indicate that decidual c-Src may activate signaling pathway(s) different fromthe integrin-mediated signaling cascade involving FAK and paxillin. This review summarizes our recent
studies on the tyrosine phosphorylation signaling pathway(s) in decidualization. (Keio J Med 51 (2): 93–
99, June 2002)
Key words: tyrosine phosphorylation, c-Src, endometrium, decidualization, focal adhesionof the NRPTKs is far less understood.1 The NRPTKs
can currently be further subdivided into 10 families,
based on structural similarities and sequence homology.1 Among them, the Src family tyrosine kinases
including c-Src and Fyn have been well characterized as
key molecules controlling the dynamic process of tyrosine phosphorylation in various types of cells.2 However, very little is known about NRPTK- and RPTKmediated signaling(s) in a variety of human female
reproductive processes.

Tyrosine Phosphorylation and Protein
Tyrosine Kinases
Protein phosphorylation on serine, threonine, andtyrosine residues is deeply involved in intracellular signal transduction. In particular, reversible protein phosphorylation on tyrosine residues, controlled coordinately by protein tyrosine kinases (PTKs) and protein
tyrosine phosphatases (PTPs), plays a pivotal role in awide variety of biological processes including cell
growth, differentiation, apoptosis, and tumorigenesis.1
The PTKs can be divided into two main categories:
the receptor-type transmembrane PTKs (RPTKs) andthe non-receptor PTKs (NRPTKs).1 Most members of
the former class function as receptors for growth- or
differentiation factors, while the physiological function

Decidualization
Decidualization is the progesterone-induced differentiation of ﬁbroblast-like stromal cells of the proliferative estrogen-primed endometrium into decidual cells,

Reprint requests to: Dr. Tetsuo Maruyama, Department of Obstetrics and Gynecology, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan, e-mail: tetsuo@sc.itc.keio.ac.jp93

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Maruyama T, et al: Tyrosine phosphorylation in decidualization

which are easily distinguishable histologically as thelarger and rounder cells appearing around the spiralarteries and eventually spreading through the most partof the endometrium in the luteal phase of the menstrual
cycle.3 Following embryo implantation, decidualization persists and extends throughout the endometrium,
leading to the formation of the pregnancy decidua. Thismorphological change is accompanied by the biochemical expression of a number of biological substances.4
These decidualization-associated factors, in turn, act as
local regulators of both decidual and trophoblast functions.5,6 Thus, decidualization is crucial for successful
embryo implantation and maintenance of pregnancy.
In the presence of estrogen and progesterone, endometrial stromal cells (ESC) isolated from human endometrium can exhibit morphological and functionalchanges in vitro that mimic in vivo decidual transformation.7,8 With the development of those in vitro models
of decidualization of ESC, many studies have addressed
the molecular mechanisms underlying decidualization.4
Intracellular signaling molecules and pathways responsible for decidualization, however, have not been fully
elucidated. Increasing bodies of evidence have implicated the activation of cAMP/protein kinase Amediated signaling cascade(s) in decidualization,9–11
which alone cannot substantially account for the multiple aspects of functional and morphological decidualization.
Alterations in the Proﬁle of Phosphotyrosinyl Proteins
and Kinase Activation of c-Src Tyrosine Kinase
Concomitant with its Decreased Dephosphorylation
upon In Vitro Decidualization of ESC
To explore possible decidual PTK-mediated signaling pathway(s), we ﬁrst examined the proﬁle ofphosphotyrosinyl proteins in in vitro decidualized ESC
using immunoblotting with the anti-phosphotyrosine(anti-PY) antibody 4G10. As shown in Fig. 1A, there
were differences in the intensities of several phosphotyrosinyl proteins between the untreated and 17-b
estradiol (E2) þ progesterone (P4)-treated ESC (upper
panel).12 A thick band of @60 kDa was clearly observed in ESC treated with control vehicles for 14 days
(Fig. 1A, upper panel, lane 1, white arrowhead), while
the @60-kDa band disappeared in ESC treated with
E2 þ P4 for the same period (Fig. 1A, upper panel, lane2).12 In contrast, the tyrosine-phosphorylated @56-kDa
and @64-kDa bands were detected in ESC treated for
14 days with E2 þ P4, but not control vehicles (Fig. 1A,
upper panel, lane 2, gray arrowheads).12 Decidualization of the E2 þ P4-treated ESC was judged by morphological changes and the production of prolactin, atypical decidualization marker (data not shown).12
The molecular weights of phosphotyrosinyl proteinsFig. 1 Alterations in the proﬁle of phosphotyrosinyl proteins andkinase activation of c-Src tyrosine kinase concomitant with its
decreased dephosphorylation upon in vitro decidualization of ESC. A.
Immunoblot analysis with anti-PY and anti-Src mAb 327 on the wholecell lysates, immunoprecipitates with mAb327, their ﬂow-throughderived from ESC treated with either control vehicles (Cx) or with
E2 þ P4 (E2 þ P4) for 14 days as indicated. Closed arrowheads, IgG
heavy chains. White and gray arrowheads, see the text. IP, immunoprecipitation. Lower panel, the same ﬁlter stripped and reprobed withmAb 327. B, In vitro kinase assay of c-Src immunoprecipitates derived
from the same samples as above. MK, [ 14 C]-labeled rainbow marker.
(From Maruyama T, Yoshimura Y, Yodoi J, Sabe H. Endocrinology
1999; 140: 2634. With permission, from Endocrinology, Volume 140,
6 1999, by the Endocrine Society, www.endo-society.org)

affected by decidualization as shown in Figure 1A weresimilar to those of Src family tyrosine kinases whose
activities are regulated by their own tyrosine phosphorylation2 Src family tyrosine kinases including c-Src
and Fyn are known to play a pivotal role in the differentiation of various types of cells.2 In addition, E2 andP4 have been reported to activate the c-Src/p21ras/Erk
pathway in human breast cancer cells.13,14 These ﬁndings collectively led us to determine whether the Src
family kinases were involved in the decidualization of
ESC in vitro.
Stripping and reprobing of the same membrane with

Keio J Med 2002; 51 (2): 93–99

95

Fig. 2 Mechanisms involved in activation of Src family kinases. The left panel shows a model of the structure of inactivated Src PTKs that arephosphorylated on the C-terminal tyrosine (Y527 in this model of Src). The middle panel shows possible mechanisms involved in activation of
Src PTKs. Y416 represents the autophosphorylation site in the activation loop of Src. The right panel represents a model for the activated stateof Src in which the intramolecular interactions of the Src homology (SH) 3 and SH2 domains are disrupted. (From Thomas SM and Brugge JS.
Annu. Rev. Cell Dev. Biol. 1997; 13: 519. With permission, from the Annual Review of Cell and Developmental Biology, Volume 13, 6 1997, by
Annual Reviews www.AnnualReviews.org)

anti-c-Src monoclonal antibody mAb327 revealed thatc-Src was expressed at almost the same level in both
untreated and treated ESC (Fig. 1A, lower panels,
lanes 1 and 2).12 We next examined employing an
in vitro kinase assay if the kinase activity of c-Src isincreased upon decidualization. As shown in Figure 1B,
c-Src immunoprecipitates with mAb327 from decidualized ESC could prominently phosphorylate enolase,a substrate for c-Src, (lane 2) when compared to thosefrom non-decidualized ESC (lane 1).12 Intriguingly, thetyrosine phosphorylation level of the decidual c-Src was
lower than that of non-decidual c-Src (Fig. 1A, upper
panel, lanes 3 and 4, white arrowheads), while the sameor greater amount of c-Src was immunoprecipitatedfrom the lysates of decidualized ESC than those of nondecidualized ESC (Fig. 1A, lower panel, lanes 3 and
4).12 Although Fyn, another Src family kinase, is also
expressed in ESC, it was not activated upon decidualization (data not shown).12 Taken together, these results
suggest that c-Src became activated and dephosphorylated during in vitro decidualization, accompanied byalterations in tyrosine phosphorylation levels of several
cellular proteins.
Possible Regulatory Mechanism(s) Underlying
Decidual c-Src Activation
The kinase activity of c-Src is known to be upregulated by dephosphorylation of its negative regulatory tyrosine residue, tyrosine 527 (Y527) (corresponding to Y530 in human), located at the C-terminus (Fig.
2).2 To date, the plausible candidate molecules regu-

lating the phosphorylation status of the C-terminal
tail are C-terminal Src kinase (Csk) and receptor-likeprotein-tyrosine phosphatase-a (RPTP-a).2 In general,Csk represses the kinase activity of c-Src throughphosphorylation of Y530, while RPTP-a activates c-Src
through dephosphorylation of the same residue.2 We
have here shown using immunoblot analysis with 4G10
that the kinase activation of decidual c-Src coincides
with its own dephosphorylation. Osusky et al. havedemonstrated that 4G10 can recognize a c-Src mutant
whose Tyr-416 is substituted to phenylalanine (cSrc[Y416F]), but cannot fully react with either a c-Src
double mutant (Y416F/Y527F) or a dephosphorylated
c-Src (Y416F).15 It has been recently reported that cSrc is dephosphorylated, as determined by immunoblotting using 4G10, with the concomitant elevation ofits kinase activity in A418 cells overexpressing RPTPa.16 Zheng et al. have reported that overexpressionof RPTP-a results in the activation of c-Src kinase
together with its dephosphorylation of Ty-527 in rat
embryo ﬁbroblasts, as assessed by cyanogen bromide
phosphopeptide mapping.17 Taken together, our results
suggest that the kinase activation of decidual c-Src may
be, at least in part, due to the dephosphorylation of thenegative regulatory site. This seems to be consistent
with our recent data using a speciﬁc and selective monoclonal antibody against the active form of human c-Src
whose Y530 is dephosphorylated (unpublished data).
Alternatively, c-Src is known to be able to couple
with a number of receptor protein tyrosine kinases suchas platelet-derived growth factor (PDGF), epidermal
growth factor (EGF), colony-stimulating factor 1 (CSF-96

Maruyama T, et al: Tyrosine phosphorylation in decidualization

Fig. 4 Src interaction with components of focal adhesion complexes.
This ﬁgure was designed to indicate that Src associates with focal
adhesions following engagement of integrins by the extracellularmatrix. The intermolecular interactions displayed are hypothetical,
based on known interactions in vitro or co-immunoprecipitation fromcell lysates and represent only a small number of proteins within thesecomplexes. (Adapted from Thomas SM and Brugge JS. Annu. Rev.
Cell Dev. Biol. 1997; 13: 531. With permission, from the Annual
Review of Cell and Developmental Biology, Volume 13, 6 1997, by
Annual Reviews www.AnnualReviews.org)

Fig. 3 Interaction of RPTKs with Src and several representative
SH2-containing signaling proteins. Src interacts with tyrosine phosphorylated motifs in RPTKs through its SH2 domain. Other proteins,
such as Shc and PLC shown here, also bind to RPTKs through related
SH2 domains. Src can also phosphorylate tyrosine residues on the receptor. The position of the Src SH2 binding motif at the juxtamembrane location represents the location of the Src binding sites on the
PDGF receptor. (From Thomas SM and Brugge JS. Annu. Rev. Cell
Dev. Biol. 1997; 13: 542. With permission, from the Annual Review ofCell and Developmental Biology, Volume 13, 6 1997, by Annual
Reviews www.AnnualReviews.org)

derived ROS that in turn mediate the signal transduction downstream of the corresponding receptors.19
Given that the expression of several redox-regulatory
proteins including superoxide dismutase and thioredoxin is induced during decidualization,20–22 it is conceivable that those redox-active proteins together withgrowth factors and cytokines might coordinately regulate the kinase activity of decidual c-Src.
Downstream Target Proteins of Decidual c-Src

1), and insulin-like growth factor (IGF) receptors upon
their binding to the relevant ligands (Fig. 3).2 In addition, many G-protein coupled receptors and cytokine
receptors including angiotensin II, bombesin, bradykinin, vasopressin, platelet activating factor (PAF), interleukin-11 (IL-11), prolactin, and oncostatin M can also
associate with c-Src.2 These transmembrane receptors
recruit and activate c-Src as well as the other Src family
kinases upon ligand binding, thereby transmitting theextracellular stimuli to the intracellular signal.2 Importantly, those c-Src activating factors are also locally
produced by the endometrium and decidua.4–6 Thus,
this knowledge collectively substantiates an idea thatseveral decidual paracrine/autocrine factors might activate signaling pathways involving c-Src.
Reactive oxygen species (ROS), generated by reduction-oxidation (redox) reactions, are also known toactivate c-Src.18 Stimulation by some growth factors
such as EGF and PDGF generates intracellular H2 O2 -To gain a clue for the c-Src-mediated signaling pathway in decidualization, we then examined the tyrosine
phosphorylation of paxillin and focal adhesion kinase(FAK). They are deeply involved in the integrinmediated signal transduction through focal adhesion
assembly, serving as substrates for the Src family tyrosine kinases (Fig. 4).2,23 As shown in Fig. 5, the whole
pattern of tyrosine phosphorylation of cellular proteins
was changed during E2 þ P4-induced decidualization(lanes 1 and 2) in a similar way as demonstrated in Fig.
1A (upper panel, lanes 1 and 2).24 Despite the alterations in the entire pattern of tyrosine phosphorylation,
there was little difference in the tyrosine phosphorylation levels of both the paxillin (Fig. 5, lanes 3 and 4,
asterisks) and 125-kDa FAK (lanes 5 and 6, sharps)
immunoprecipitates between non-decidualized anddecidualized ESC.24
The formation of focal adhesions are prerequisite
for the integrin-mediated signaling pathways respon-

Keio J Med 2002; 51 (2): 93–99

97

Fig. 6 Disappearance of the focal contacts in the decidualizationinduced multicellular nodules Immunoﬂuorescence micrographs using
anti-paxillin antibody of ESC treated with either control vehicles(Control) or with E2 þ P4 (E2 þ P) for seven days. Bars, 25 mm.
(Adapted from Maruyama T, Yoshimura Y, Sabe H. Endocrinology
1999; 140: 5984. With permission, from Endocrinology, Volume 140,
6 1999, by the Endocrine Society, www.endo-society.org)

Fig. 5 No marked changes in tyrosine phosphorylation of FAK andpaxillin upon decidualization. Immunoblot data using anti-PY antibody on whole cell lysates derived from ESC treated with either control vehicles (Cx) or with E2 þ P4 (E2 þ P4) for 14 days and immunoprecipitates with anti-FAK or anti-paxillin antibody as indicated.
Closed arrowheads, IgG heavy chains. *, tyrosine-phosphorylated
paxillin. ], tyrosine-phosphorylated FAK. (Adapted from Maruyama
T, Yoshimura Y, Sabe H. Endocrinology 1999; 140: 5984. With permission, from Endocrinology, Volume 140, 6 1999, by the Endocrine
Society, www.endo-society.org)

sible for cell growth, differentiation, migration, transformation, and contractility.23 To address the question
why tyrosine phosphorylation of FAK and paxillin was
not markedly changed despite decidual c-Src activation,
we next analyzed the subcellular localization of the
focal adhesion proteins focusing on the characteristic
decidualization-induced multicellular nodules. ESC in
the control cultures clearly possessed focal contacts, asassessed by the immunostaining of paxillin (Fig. 6, left
panel).24 In contrast, in the decidualization-induced
multicellular nodules, no discrete accumulations of paxillin staining could be detected at borders of adjoining
cells or at distinct focal adhesion plaques; but rather
paxillin was diffusely localized in the cytosol (Fig. 6,
right panel).24
The orderly recruitment and complex formation of
focal adhesion proteins are crucial for the actin-based
cytoskeletal organization.23 We further investigated thesubcellular distribution of actin stress ﬁbers in the multicellular nodules where the formation of the focal adhesion assembly was impaired. Double-labeled immunostaining of ESC using FITC-conjugated phalloidin
and anti-vinculin antibody, visualized by rhodamineconjugated secondary antibody, showed that the undifferentiated and ﬂattened ESC in the control cultures
clearly possessed focal adhesion contacts containing

vinculin, one of the focal adhesion proteins (Fig. 7, left
upper panel).24 Moreover, there were well-stretchedactin stress ﬁbers (Fig. 7, left middle panel), linking
paired focal adhesion contacts (left lower panel).24
However, well-organized F-actin could not be detected
but was rather located at the cell periphery within
decidualization-induced multicellular nodules (Fig. 7,
right panel).24 We also obtained similar ﬁndings withpaxillin (data not shown).24 Together with the elevation of the c-Src kinase activity during in vitro decidualization, our present data suggest that c-Src may activate a different signaling pathway(s), which does not
involve FAK and paxillin, in decidualized ESC. Furthermore, dissociation of the focal adhesion complex
together with the breakdown of the actin-based cytoskeleton might contribute to both functional and morphological decidualization.
Role of c-Src in Decidual Transformationc-Src primarily co-localizes with markers of endosomal membranes and associates with specialized secretory vesicles in some cell types,25,26 implicatingc-Src as a possible regulator of exocytosis.27,28 Considering the high secretory potential of decidual cells,4
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.2,29 Also, thekinase activation of c-Src has been implicated in theregulation of the actin-based cytoskeletal organizationand focal adhesion assembly.2,29 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.
Elucidation of the relevance of c-Src activation to thedecidual function awaits further studies.

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Maruyama T, et al: Tyrosine phosphorylation in decidualization

Fig. 7 Disorganization of the actin-based cytoskeleton in decidualization-induced multicellular nodules. ESC treated with either control
vehicles (Control) or with E2 þ P4 (E2 þ P) were double-stained for F-actin using FITC-conjugated-phalloidin and for vinculin using antivinculin antibody that was subsequently visualized by rhodamine-conjugated secondary antibody. Bars, 25 mm. (Adapted from Maruyama T,
Yoshimura Y, Sabe H. Endocrinology 1999; 140: 5984. With permission, from Endocrinology, Volume 140, 6 1999, by the Endocrine Society,www.endo-society.org)

Analogy to the Differentiation of
Other Cells Types
Lastly, since c-Src is a proto-oncogene product, its
dysregulated overexpression and activation are thought
to be closely associated with cell growth and tumorigenesis rather than differentiation.2,29 However, quite
a few studies have implicated c-Src and its activationin the differentiation of several types of cells such as
osteoclasts,30 keratinocytes,31 and neuronal cells.32,33
Intriguingly, 1,25-dihydroxyvitamin D3, which is required for commitment of macrophage progenitors
to osteoclast differentiation, has been shown to induce
the kinase activation of c-Src, but not the expression,
together with dephosphorylation of c-Src.29 Thus, it islikely that there may be a common mechanism involving c-Src activation for some types of differentiation
including decidualization.
Concluding Remarks
As mentioned previously, many decidualizationinduced bioactive substances such as PDGF, EGF,
CSF-1, IGF, IL-11, angiotensin II, bradykinin, PAF,
prolactin, and oncostatin M behave as ligands for thetransmembrane receptors that can couple with and activate c-Src, raising the possibility that decidual c-Src
might be a point of convergence in the action of thosefactors. In this regard, it is tempting to speculate that cSrc may act as the master regulator of decidual function
through selectively and timely transducing signals fromthe extracellular stimuli. Our current data will not only

broaden our understanding of endometrial physiology
but also contribute to the development of new drugs
and treatments for female infertility, recurrent pregnancy loss, and contraception to target the deciduaspeciﬁc tyrosine phosphorylation signaling cascade(s).
Acknowledgments: We thank Drs. H. Sabe, J. Yodoi, T. Takamatsu,
and H. Kanzaki for their valuable comments and technical advice. We
acknowledge the technical and secretarial assistance of Ms. R. Sakurai
and S. Kuwabara. This work has been 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.

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