From the Hubrecht Laboratory, Netherlands
Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht,
The Netherlands and the § Laboratory for Physiological
Chemistry, Utrecht University, Universiteitsweg 100, 3584 CG
Utrecht, The Netherlands
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ABSTRACT |
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The formation of parietal endoderm (PE) is one of the first differentiation processes during mouse development and can be studied in vitro using F9 embryonal carcinoma (EC) cells. Treatment of F9 EC cells with retinoic acid (RA) induces differentiation toward primitive endoderm (PrE), while differentiation toward PE is induced by subsequent addition of parathyroid hormone (PTH) or PTH-related peptide (PTHrP). The signal transduction mechanisms involved in this two-step process are largely unclear.
We show that the RA-induced differentiation toward PrE is accompanied by a sustained increase in Ras activity and that ectopic expression of oncogenic Ha-Ras is sufficient to induce PrE differentiation. Ras activity subsequently decreases upon PTH-induced differentiation toward PE. This is a necessary event, since expression of oncogenic Ha-Ras in PrE-like cells prevents PTH-induced PE differentiation. Expression of active PKA in PrE-like F9 cells mimics PTH-induced PE differentiation and is again prevented by oncogenic Ha-Ras. The effect of oncogenic Ras on both differentiation steps is abolished by the MEK inhibitor PD98059 and can be mimicked by constitutively active forms of Raf and MEK.
In conclusion, our data suggest that activation of the Ras/Erk is
sufficient to induce differentiation to PrE and to prevent subsequent
differentiation toward PE. Activation of PKA down-regulates Ras
activity, resulting in disappearance of this blockade and transmission
of signal(s) triggering PE differentiation.
Murine F9 embryonal carcinoma
(EC)1 cells are a suitable
in vitro model system to study the formation of
extraembryonic endoderm. Treatment with retinoic acid (RA) induces
differentiation toward a primitive endoderm (PrE)-like phenotype (1).
Subsequently, the cells can be differentiated toward a parietal
endoderm (PE)-like phenotype by addition of cAMP-elevating agents (2).
The first step of differentiation is accompanied by morphological
changes and disappearance of the stage-specific embryonic antigen
(SSEA-1) (3) and appearance of differentiation markers like tissue
plasminogen activator (tPA) (1), laminin (2), and TROMA-1 (4, 5). The
second step of differentiation is accompanied by morphological changes
as well and is characterized by the appearance of thrombomodulin (6,
7), while expression of proteins like tPA and laminin is strongly
elevated (2).
We and others have shown that PTH or PTHrP can substitute for
Bt2cAMP in the differentiation of PrE to PE (8, 9).
Furthermore, expression patterns of PTHrP and the PTH/PTHrP receptor
suggest that this signaling system functions in PE formation in
vivo (9-11).2 PTH and
PTHrP bind to a common receptor, which couples to at least two G
proteins, Gs and Gq, resulting in activation of
both the adenylate cyclase and the phospholipase C The mammalian ras family encodes membrane-bound
GTPases, which are essential in transduction of extracellular
signals involved in cell proliferation and differentiation. Activation
of receptors catalyzes the exchange of GDP for GTP on Ras. In the
active GTP-bound state, Ras will bind to its effectors and influence
their biological activity. Ras is inactivated by GTPase-activating
proteins (GAPs), which increase the rate of GTP hydrolysis, resulting
in an inactive GDP-bound state (16, 17). The best characterized
Ras-dependent signal transduction pathway involves the
activation of the Raf/Erk cascade. Activated Ras interacts with Raf-1
and translocates it to the plasma membrane where it is exposed to
activating kinases and/or lipids (18). Activated Raf-1 phosphorylates
and activates MEK, leading to the phosphorylation and activation of Erk
(16).
Besides its transforming potential, Ras can affect the differentiation
of several cell types. Oncogenic Ras inhibits myoblast differentiation
(19) and induces dedifferentiation of thyroid cells (20). On the other
hand, oncogenic Ras is sufficient to induce differentiation of
fibroblasts to adipocytes (21) and to induce neuronal differentiation
of PC12 cells (22).
Here we examined the role of Ras and Erk in endoderm differentiation of
F9 EC cells. We show that the Ras/Erk pathway plays an important role
in this two-step differentiation process, since sustained Ras activity
is observed during PrE differentiation and is sufficient to induce it,
while Ras activity needs to be down-regulated by PTH prior to
differentiation toward PE. Finally, we show that the effect of Ras on
both differentiation processes is mediated by Erk.
Materials--
Rat PTH(1-34) was purchased from Peninsula
Laboratories Europe (St. Helens, United Kingdom), Bt2cAMP
from Aldrich (Zwijndrecht, The Netherlands), all-trans-RA
from Sigma; PD98059 was from Calbiochem. [ Cell Culture, Induction of Differentiation, and
Transfections--
F9 EC cells were obtained from the ATCC and
cultured on gelatinized surface in medium consisting of a 1:1 mixture
of Dulbecco's modified Eagle's medium and Ham's F-12 containing
7.5% fetal calf serum. Unless denoted otherwise, differentiation to
PrE-like cells was performed by culture in monolayer in the presence of
1 µM RA for 3 days. To obtain PE-like cells, this
treatment was followed by 1 mM Bt2cAMP or 100 nM PTH-(1-34) for 2 days. Transient transfections were
performed using LipofectAMINE (Life Technologies, Inc.) according to
manufacturer's instructions.
In the case of transfections with
In the case of transfection for transient HA-Erk assays,
undifferentiated F9 cells, and F9 cells treated with RA for 3 days, were plated in concentrations of, respectively, 3 × 103 and 2 × 104 cells/cm2 in
six-well tissue culture clusters. The following day cells were
transfected for 6 h, using LipofectAMINE, with PSG5-HA-Erk (0.6 µg) and RasLeu-61 (0.6 µg), Raf-CAAX (0.6 µg) or
MEK-D218/222 (0.6 µg), unless denoted otherwise. PUC-RSV plasmid was
added to bring the total amount of DNA to 2 µg.
Activation and Phosphorylation of Erk--
To culture F9 EC
cells in such a concentration that cell densities were comparable after
5 days of culture, cells to be treated without or with RA for 1, 2, 3, 4, or 5 days were plated at concentrations of, respectively, 150, 200, 300, 500, 800, or 1000 cells/cm2 in six-well tissue culture
clusters. Erk phosphorylation was measured by Western blotting with
anti-p42 MAP kinase antibodies as described previously (30).
Phosphorylated p42 MAP kinase is detected as a band with reduced
mobility compared with unphosphorylated p42 MAP kinase (31).
Experiments were repeated at least three times and representative
results are shown.
For determination of Erk activity, epitope-tagged p42 HA-MAP kinase was
immunoprecipitated with protein A-Sepharose beads coupled to monoclonal
antibody 12CA5, as described previously (15). After the kinase reaction
with myelin basic protein as a substrate, the reaction mix was
subjected to SDS-polyacrylamide gel electrophoresis. Phosphorylation of
myelin basic protein was measured using a PhosphorImager and ImageQuant
software (Molecular Dynamics).
Activation of Ras--
F9 cells were plated in 9-cm tissue
culture dishes at similar concentrations as described above in
"activation and phosphorylation of Erk." In
vivo Ras activity was determined with the minimal Ras-binding
domain (RBD) of Raf-1 as an activation-specific probe for Ras, as
described previously (32, 33). In short, cell lysates were brought to
equal protein concentrations, and active Ras was precipitated with
glutathione-agarose beads coupled to GST-RBD. Precipitated Ras was
detected by Western blotting using the rat monoclonal antibody
Y13-259.
Up-regulation of Ras and Erk Activity during RA-induced PrE
Differentiation of F9 EC Cells--
Previous work has suggested an
involvement of the Ras/Erk pathway in PrE differentiation of F9 EC
cells. It was shown that expression of oncogenic Ras in F9 EC cells is
sufficient to induce differentiation to endoderm-like cells (34) and
furthermore that RA-generated PrE-like F9 cells have a higher level of
p42 MAP kinase activity than undifferentiated F9 cells (35). To get
more insight in the role of Ras and Erk in PrE differentiation, we
determined the activity of Ras and Erk during RA-induced
differentiation. This was performed by using the RBD of Raf-1 as a
specific probe for activated Ras, as described previously (32, 33, 36). Ras activity was measured as the amount of Ras precipitated with GST-Raf-RBD. Treatment of F9 EC cells with RA strongly induced Ras
activity, which was not due to increased Ras expression (Fig. 1A). RA activated Ras already
after 24 h, while longer treatments resulted in a further
up-regulation of Ras activity. Comparable kinetics were observed for
the RA-induced Erk phosphorylation (Fig. 1B), indicating
that Erk activation is mediated by Ras activation.
Oncogenic Ha-Ras Induces Differentiation of F9 EC to PrE--
It
has been reported that oncogenic Ras induces endoderm differentiation
of F9 EC cells (34). However, a lack of proper PrE- and/or PE-specific
markers made it difficult to distinguish between both cell types at
that time. Recently, thrombomodulin has been shown to be a suitable
marker for PE, both in vitro as well as in vivo
(7). We therefore transiently transfected F9 EC cells with constructs
encoding
As a positive marker for PrE differentiation we used TROMA-1 (4, 5).
Undifferentiated cells did not express TROMA-1 (Fig. 2, E,
F, and J), while RasLeu-61 induced
expression of TROMA-1 in approximately 70% of the transfected cells
(Fig. 2, G, H, and J), which was even higher than
in RA-treated cells (35%) (Fig. 2J). Furthermore,
RasLeu-61 expressing cells showed an enlarged and flattened
morphology, which was similar to the morphology of RA-differentiated
cells, and is characteristic for PrE cells (1). We could not detect expression of thrombomodulin in the
RasLeu-61-differentiated cells (not shown). Taken together,
these data indicate that the transient expression of oncogenic Ras
induced differentiation toward a PrE-like phenotype, without further
differentiation to PE.
Oncogenic Ras-induced PrE Differentiation Is Mediated by
Erk--
As was shown in Fig. 1B, the RA-induced PrE
differentiation is accompanied by a sustained increase in Erk activity.
The importance of Erk activity in RA- and/or Ras-induced PrE
differentiation was investigated with the specific MEK inhibitor
PD98059 (37). A continuous treatment of F9 EC cells with PD98059
completely blocked the activation of a transfected hemagglutinin-tagged
p42 MAP kinase by RA or cotransfected RasLeu-61 (Fig.
3A). Under these conditions,
RasLeu-61-induced PrE differentiation was completely
abolished, while RA-induced PrE differentiation was unaffected, as
based on the appearance of a PrE-like morphology (not shown) and
expression of the differentiation marker TROMA-1 (Fig. 3B).
Similar results were found by cotransfecting N Down-regulation of Ras Activity during PTH-induced PE
Differentiation--
When F9 EC cells are differentiated to PrE, they
can subsequently be differentiated to PE by addition of
Bt2cAMP, PTH, or PTHrP (2, 8, 9). In previous studies we
demonstrated that PTH, by elevation of cAMP levels, can either inhibit
or activate Erk activity, depending on the cell type studied (14, 15). Here, we determined the effect of PTH and Bt2cAMP on Ras
and Erk activity in PrE-like F9 cells. Endogenous Ras activity
decreased when cells were differentiated to PE upon addition of
PTH(1-34) (Fig. 4 A). The
PTH-induced inhibition of Ras activity is likely mediated by elevation
of cAMP levels, since treatment with Bt2cAMP rapidly
inhibited Ras activity (Fig. 4B). Longer treatment resulted in a further decrease in Ras activity, while Ras activity was almost
completely inhibited after treatment with Bt2AMP for
48 h, when the cells were fully differentiated toward PE.
Importantly, expression levels of Ras were not changed. The inhibition
of Ras activity was reflected in the down-regulation of Erk activity (Fig. 4C), which occurred with similar kinetics, suggesting
a causal relation between the inhibitory effect of PTH on Ras and Erk
activity.
Oncogenic Ha-Ras Inhibits Differentiation to PE--
The
down-regulation of Ras and Erk activity might play a crucial role in
the PTH-induced PE differentiation. We therefore investigated the
effects of ectopic expression of either oncogenic Ras, thereby
overruling the PTH-induced inhibition of Ras activity, or a dominant
negative mutant of Ras, RasAsn-17, thus mimicking the
inhibition of endogenous Ras activity. RA-treated F9 cells were
cotransfected with a construct encoding
Treatment with PTH-(1-34) induced differentiation toward PE, resulting
in the typical change in morphology and expression of thrombomodulin in
approximately 20% of the cells (Fig. 5, C, D, and
K). It should be noted that this low efficiency of
PTH-induced PE differentiation is probably due to the transfection
procedure, since normally PTH induces PE differentiation in
approximately 50% of the cells. Transfection with oncogenic Ras had a
dramatic effect on PTH action, the cells maintained their PrE-like
morphology and did not express thrombomodulin, resulting in the
occurrence of a PE-like phenotype in only 5% of the transfected cells
(Fig. 5, E, F, and K). Expression of
RasAsn-17 to interfere with Ras activation slightly
enhanced the PTH-induced PE differentiation (Fig. 5K). These
results show that high levels of Ras activity in PTH-treated cells
prevent differentiation toward PE, and that inhibition of Ras activity
by itself is necessary but not sufficient for PE differentiation to occur.
The inhibition of PTH-induced PE differentiation by Ras can be at
different levels, Ras could interfere at the level of the PTH/PTHrP
receptor or downstream of the receptor within the phospholipase C or
the adenylate cyclase pathway. We therefore determined the effect of
oncogenic Ras on Bt2cAMP-induced PE differentiation. Addition of Bt2cAMP to RA-generated PrE-like F9 cells
induced a PE-like phenotype in 45-50% of the cells (Fig.
5L). This was strongly inhibited by expression of oncogenic
Ras, to approximately 10%, suggesting that Ras interferes with
PTH-induced PE differentiation downstream of cAMP.
The major effector of cAMP elevation is PKA. However, cAMP has been
shown to induce PKA-independent effects, like direct regulation of ion
channels and transporter proteins (39-41). Furthermore, Bt2cAMP can also mediate cAMP-independent effects via
butyrate (42). To determine whether activation of PKA is sufficient to induce PE differentiation, we transfected RA-treated F9 cells with the
catalytic subunit of PKA. This induced a PE-like phenotype in
approximately 45% of the transfected cells (Fig. 5, G, H,
and M). Also the PKA-induced PE differentiation could be
inhibited by coexpression of oncogenic Ras, which reduced the number of PKA-induced thrombomodulin expressing cells to 5-10% (Fig. 5, I, J, and M). This suggests that Ras interferes
with PTH-induced PE differentiation at the level of or downstream of PKA.
Ras Inhibits PE Differentiation via Activation of Erk--
The
decrease in endogenous Ras activity, which was observed when PrE-like
F9 cells were differentiated to PE by addition of PTH or
Bt2cAMP, strongly correlated with the decrease in Erk
activity (Fig. 4). To determine whether the inhibitory effect of Ras on PE differentiation was depending on Erk activity, we used PD98059. Like
in F9 EC cells (Fig. 3A), a continuous treatment with
PD98059 blocked the RA- as well as the RasLeu-61-induced
Erk activity in PrE-like F9 cells (not shown). PD98059 did not induce a
PE-like morphology by itself (not shown) nor thrombomodulin expression
(Fig. 6A), suggesting that
inhibition of Erk activity is not sufficient to induce differentiation
of PrE-like F9 cells to PE. Interestingly, PD98059 did however
significantly enhance PKA-induced PE differentiation (29.6 versus 46.9%, Fig. 6A), indicating that the
basal Erk activity interferes with differentiation to PE. Furthermore,
the inhibitory effect of RasLeu-61 on PKA-induced PE
differentiation was completely abolished by PD98059 (Fig.
6A) and could be mimicked by Raf-CAAX and MEK-D218/222 (Fig.
6B). Together these data strongly suggest that the
inhibitory effect of Ras on PE differentiation is mediated by Erk and
that down-regulation of the Erk activity in PrE-like F9 cells is
necessary but not sufficient for PE differentiation to occur.
One of the first differentiation processes during mouse
embryogenesis takes place in the blastocyst around day 4.5 postcoitus. Cells of the inner cell mass differentiate into PrE, which subsequently differentiates into PE. The signals involved in this two-step process
are poorly understood. F9 EC cells are a suitable in vitro model system to study the formation of PrE and PE. However, lack of
proper PrE- and/or PE-specific markers made a good distinction between
PrE or PE difficult in the past. In this study we made use of
antibodies against thrombomodulin, a suitable marker for PE (6, 7), to
distinguish between PrE and PE. We show that both differentiation
processes are regulated by the Ras/Erk pathway, since expression of
constitutively active forms of Ras, Raf, and MAP kinase kinase in F9 EC
cells induced differentiation toward a PrE-like phenotype, while they
inhibited the subsequent differentiation toward PE. Accordingly,
RA-mediated PrE differentiation was accompanied by an increase in
endogenous Ras and Erk activity, while subsequent PTH-mediated PE
differentiation was accompanied by a decrease in endogenous Ras and Erk activity.
Involvement of Ras in PrE Differentiation--
Expression of
oncogenic Ha-Ras in F9 EC cells has been reported to induce
differentiation toward an endoderm-like phenotype (34). Our data
suggest that this Ras-induced phenotype resembles PrE and not PE.
Importantly, also when expressed in embryonic stem (E14) cells, which
can be differentiated in vitro to form derivatives of all
three germ layers, oncogenic Ras induces a PrE-like morphology and
expression of Troma-1.3 This
indicates that Ras/Erk-mediated PrE differentiation is not only
restricted to the F9 EC system, but a more general feature for
embryonic stem and embryonic carcinoma cells (Fig.
7).
Ras activity is up-regulated during RA-induced PrE differentiation,
again suggesting an important role for Ras in mediating PrE
differentiation. The mechanism of Ras activation by RA is unclear.
RA-induced PrE differentiation is accompanied by expression of growth
factors and/or their receptors, such as platelet-derived growth factor,
epidermal growth factor, fibroblast growth factor, and transforming
growth factor (43, 44), and thus RA might elevate Ras activity by
establishing autocrine stimulation of these receptors. Expression of
RasAsn-17, to interfere with RA-induced Ras activation, did
not affect RA-induced PrE differentiation. However, the inhibition of
RA-induced Ras activation might have been incomplete, as indicated by
residual RA-induced Erk activation in the presence of
RasAsn-17 (data not shown). Nevertheless, while PD98059
completely inhibited RasLeu-61- and RA-induced Erk
activation in EC cells, only the Ras-induced and not the RA-induced PrE
differentiation was blocked. This is in apparent contrast with the
findings of Gao et al. (35), who, using antisense p42 MAP
kinase oligonucleotides, found an inhibition of RA-induced expression
of the differentiation marker tPA. We observed, however, that complete
inhibition of p42 MAP kinase by PD98059 had no effect on RA-induced
PrE-like morphology, expression of Troma-1, or on subsequent
PTH-induced PE differentiation (data not shown). Thus, RA induces PrE
differentiation via a Ras/Erk-independent pathway as well. A possible
target of RA and Ras involved in PrE differentiation is AP-1, a
transcription factor family consisting of homo- or heterodimers of Jun,
Fos, or ATF proteins, which can be activated via
Ras-dependent and Ras-independent pathways (45). Both RA as
well as oncogenic Ras induce c-jun expression in EC cells
(34, 46, 47), while forced expression of c-fos or c-jun in EC cells is sufficient to induce differentiation
(34, 47, 48, 49). The role of AP-1 activation in RA- and Ras-induced PrE differentiation is currently being investigated.
It should be stressed here that the identity of the factor(s)
establishing primitive endoderm differentiation in vivo is
unclear. Although RA efficiently induces differentiation of F9 EC cells in vitro, its involvement in this differentiation process
in vivo is rather unlikely (50-53). Our observation that
the Ras/Erk pathway has the capacity to regulate PrE and PE
differentiation in vitro suggests that it could play a major
role in the regulatory mechanisms governing extraembryonic endoderm
differentiation in vivo.
Differentiation to PE Is Regulated by Ras and PKA--
Treatment
of PrE-like F9 cells with PTHrP, besides inducing activation of PKA,
also induces an elevation of intracellular calcium levels (data not
shown). Here we show for the first time that a strong increase in PKA
activity is sufficient to induce PE differentiation, suggesting that
PTHrP-induced PE differentiation can be mediated solely through PKA activation.
We recently reported that the PTH-induced PKA activation is sufficient
to inhibit both basal as well as growth factor-induced activation of
Erk (14). Here we show that PTH and Bt2cAMP also inhibit
Erk activity in RA-treated F9 cells. It was demonstrated in other cells
that activation of PKA interfered with Erk activation at the level of
Raf-1, leaving Ras activity unaffected (30). Our data however
demonstrate that in PrE-like F9 cells cAMP elevation inhibits Ras
activity. We observed a strong correlation between the reduction in Ras
and Erk activity, suggesting that the cAMP-induced Erk inhibition is a
consequence of the inhibition of Ras activity. The mechanism involved
in the inhibition of Ras activity by cAMP remains to be determined.
This could be mediated via interference with growth factor-induced Ras
activation or via inhibition of RasGAP (Ras GTPase activation protein),
for instance by PKA-induced phosphorylation (54).
In agreement with an inhibitory role for Ras in PE differentiation, it
has been reported that Ras is expressed at very low levels in PE during
early rat embryogenesis (55). Inhibition of Ras or Erk activation with
RasAsn-17 or PD98059, respectively, was by itself not
sufficient to induce PE differentiation, suggesting that other events
apart from inhibition of Ras activity are involved in cAMP-mediated PE
differentiation as well. Inhibition of Erk activity did however enhance
PKA-induced PE differentiation, suggesting that the basal Erk activity
in PrE-like cells interferes with differentiation of these cells to PE.
This is in agreement with the observation that sustained elevation of
Erk activity by expression of constitutive active Ras, Raf, or MAP
kinase kinase prevents PE differentiation, even when induced by cPKA.
In conclusion, our data suggest the following model for endoderm
differentiation of F9 cells (Fig. 7). Activation of the Ras/Erk pathway
is sufficient to trigger differentiation to PrE. RA induces activation
of the Ras/Erk pathway and accordingly differentiation to PrE. However,
RA induces PrE differentiation via an Erk-independent pathway as well.
The sustained Ras/Erk activity in PrE-like cells blocks signal(s)
inducing the subsequent differentiation toward PE. This Ras activity is
down-regulated by PTHrP via PKA, resulting in disappearance of the
blockade and transmission of the signal(s) triggering PE
differentiation. According to this model, one can speculate that
differentiation of PrE and PE in the early mouse embryo is tightly
regulated by a subtle cross-talk between (extracellular) signals
leading to Ras/Erk activation and signals such as PTHrP, leading to PKA activation.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-mediated signal transduction pathways (12, 13). We recently demonstrated that PTH, by
elevation of intracellular cAMP levels, can affect the Ras/Erk pathway
in a cell type-specific fashion (14, 15).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
32P]ATP
and ECL were obtained from Amersham Pharmacia Biotech (`s Hertogenbosch, The Netherlands). Monoclonal antibodies against SSEA-1
were a gift from Dr. D. Solter (Wistar Institute, Philadelphia, PA),
monoclonal antibodies against TROMA-1 were a gift from Dr. R. Kemler
(Max-Planck-Institute for Immunobiology, Freiburg, Germany), and
monoclonal antibodies 273-34A against thrombomodulin (23) were a gift
from Dr. S. J. Kennel (Oak Ridge National Laboratory, Oak Ridge,
TN). Raf-CAAX was a gift from Drs. S. Leevers and C. Marshall (24), and
MEK-D218/222 was a gift from Drs. Brunet and J. Pouysségur (25).
The expression plasmids pSV2LacZ (26), pMT-PKAC
(27),
RSV-RasLeu-61 and RSV-RasAsn-17 (28), have been
described previously, while pSG5-HA-p42 MAP kinase was provided by
Arjan Buist and constructed by subcloning the HA-tagged MAP kinase from
a cytomegalovirus-driven promotor (29) into pSG5.
-gal to identify transfected
cells, undifferentiated F9 cells and F9 cells treated with RA for 3 days, were plated in concentrations of, respectively, 3 × 103 and 2 × 104 cells/cm2 on
13-mm tissue coverslips (Sarstedt Inc., Newton, NC) in 25-compartment clusters (Greiner). The following day cells were transfected for 6 h, using LipofectAMINE, with pSV2-lacZ (0.3 µg) and
RasLeu-61 (0.3 µg), RasAsn-17 (0.3 µg),
Raf-CAAX (0.3 µg), MEK-D218/222 (0.3 µg), or pMT-PKAC
(0.3 µg), unless denoted otherwise. PUC-RSV plasmid was added to bring the
total amount of DNA to 1 µg. Where appropriate, RA, PTH-(1-34), and
Bt2AMP were added the day after transfection.
-Galactosidase Staining and Immunofluorescence--
Cells
were washed twice with PBS, fixed with 2% paraformaldehyde, incubated
with an 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside staining solution (PBS with 0.8 mg/ml X-gal (Life Technologies, Inc.),
1 mg/ml K3FE(CN)6, 1.3 mg/ml
K4FE(CN)6), and subsequently incubated with 20 mM NH4Cl for 30 min. For detection of SSEA-1, cells were incubated for 1 h in PBS, 0.2% bovine serum albumin and subsequent antibody incubations were done in PBS, 0.2% bovine serum albumin as well. For detection of TROMA-1 and thrombomodulin, cells were incubated for 1 h in NETGEL (50 mM Tris, pH
7.4, 150 mM NaCl, 5 mM EDTA, 0.05% (v/v)
Nonidet P-40, 0.25% (w/v) gelatin, 0.02% (w/v) sodium azide), and
subsequent antibody incubations were done in NETGEL as well.
Incubations with first antibodies were for 1 h at room
temperature, the cells were rinsed extensively in PBS and incubated
with CY3-conjugated secondary antibodies (Jackson ImmunoResearch, West
Groove, PA) for 1 h at room temperature, again extensively rinsed,
and mounted in Moviol.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Ras activity and Erk phosphorylation during
RA-induced PrE differentiation of F9 EC cells. A, F9 EC
cells were treated with 1 µM RA for the indicated
periods. Immunoblots against Ras in GST-RBD precipitates (upper
panel) or in aliquots of total cell lysates (lower
panel) are shown. Active Ras is detected as Ras precipitated by
GST-RBD. B, F9 EC cells were treated with 1 µM
RA for the indicated periods. Immunoblots against p42 MAP kinase are
shown. Phosphorylated Erk (pp42) is detected as a band with
reduced mobility compared with that of unphosphorylated Erk
(p42). The experiments were repeated twice, and
representative experiments are shown.
-galactosidase (
-gal) and oncogenic Ras,
RasLeu-61, and determined the phenotype of
-gal-expressing cells by morphology and by immunofluorescence using
antibodies against phenotype-specific markers. When F9 EC cells were
transfected with control plasmids, they remained undifferentiated, as
reflected in the expression of the stem cell marker SSEA-1 (3) (Fig.
2, A, B, and
I). However, when cells were transfected with
RasLeu-61, the number of transfected SSEA-1-positive cells
decreased with approximately 80% (Fig. 2, C, D,
and I). This was comparable with the decrease in
SSEA-1 expression induced by RA-treatment (Fig. 2I).
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Fig. 2.
Oncogenic Ras induces PrE differentiation of
F9 EC cells. -Gal staining (A, C, E, G) or
immunofluorescent staining on SSEA-1 (B, D) or TROMA-1
(F, H) of F9 EC cells transiently transfected with only
-gal (A, B, E, F) or with
-gal and RasLeu-61
(C, D, G, H), as described under "Experimental
Procedures." Diagrams depict the percentage of
-gal-expressing
cells positive for SSEA-1 (I) or TROMA-1 (J),
when cells were either left untreated, cotransfected with
RasLeu-61, or treated with 1 µM RA for 3 days. Data in diagrams represent the mean ± S.D. of at least
three independent experiments. Bar, 50 µm.
raf, a
dominant-negative mutant of Raf-1 (38) (data not shown). Thus,
Ras-induced PrE differentiation is dependent on Erk, in contrast to
RA-induced PrE differentiation. To determine whether activation of the
Raf/Erk cascade is sufficient to induce PrE differentiation, we
transiently transfected F9 EC cells with constitutive active forms of
Raf (Raf-CAAX) (24) and MAP kinase kinase (MEK-D218/222) (25). Both
Raf-CAAX as well as MEK-D218/222 strongly induce a PrE-like morphology
(not shown) and TROMA-1 expression (Fig. 3C). Taken
together, the data show that activation of the Ras/Erk cascade is
sufficient to induce PrE differentiation, but that RA can induce PrE
differentiation via Ras/Erk-independent pathway(s) as well.
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Fig. 3.
Ras-induced PrE differentiation is mediated
by Erk. A, F9 EC cells were transiently transfected
with hemagglutinin-tagged Erk with or without RasLeu-61
(0.2 µg) and treated with 1 µM RA and/or 50 µM PD98059 for 2 days as indicated. Medium including test
agents was changed every 24 h. The activity of HA-Erk was assayed
using MBP as a substrate, as described under "Experimental
Procedures." Data are shown as -fold increase of Erk activity in
unstimulated cells and represents the mean ± S.D. of three
independent experiments. B, F9 EC cells were transiently
transfected with -gal (control) or with
-gal and
RasLeu-61 (0.1 µg) and treated with 1 µM RA
and/or 50 µM PD98059 for 3 days as indicated. Medium
including test agents was changed every 24 h. C, F9 EC
cells were transiently transfected with
-gal and with or without
Raf-CAAX or MEK-D218/222. Diagrams B and C depict the percentage of
-gal-expressing
cells positive for TROMA-1, as similar to Fig. 2 and represent the
mean ± S.D. of at least three independent experiments.
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Fig. 4.
Ras activity and Erk phosphorylation during
PE differentiation of RA-treated F9 cells. A, F9 cells
were left untreated or treated with 1 µM RA for 5 days
with or without 100 nM PTH-(1-34) during the last 2 days.
B, F9 EC cells were left untreated, treated with 1 µM RA for 5 days, or treated with 1 µM RA
for 5 days while 1 mM Bt2cAMP was present
during the last 10 min, 2 h, or 1 or 2 days as indicated.
Immunoblots against Ras in GST-RBD precipitates (upper
panel) or in aliquots of total cell lysates (lower
panel) are shown. Active Ras is detected as Ras precipitated by
GST-RBD. C, F9 EC cells were left untreated, treated with 1 µM RA for 5 days, or treated with 1 µM RA
for 5 days while 1 mM Bt2cAMP or 100 nM PTH-(1-34) were present during the last 10 min, 2 h, or 1 or 2 d as indicated. Erk phosphorylation was determined by
a mobility shift (see legend to Fig. 1).
-gal to identify transfected
cells. PE differentiation of
-gal-expressing cells was measured by
morphology and thrombomodulin expression. The RA-treated control cells
had a PrE-like morphology and did not express thrombomodulin (Fig.
5, A and B).
Transfections with either RasLeu-61 or
RasAsn-17 did not change the PrE-like morphology nor did
they induce expression of thrombomodulin (Fig. 5K, no
ligand).
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Fig. 5.
Oncogenic Ras inhibits PE differentiation of
RA-treated F9 cells. Shown is -gal staining (A, C, E, G,
I) or immunofluorescent staining of thrombomodulin (B, D, F,
H, J). RA-treated F9 cells were transiently transfected with
-gal alone (A, B, C, D) or cotransfected with
RasLeu-61 (E, F), with cPKA (G, H),
or with both cPKA and RasLeu-61 (I, J). Cells
were subsequently left untreated for 3 days, or treated with 100 nM PTH (C, D, E, F) or 1 mM
Bt2cAMP during the last 2 days. K, L, and
M diagrams depict the percentage of
-gal-expressing cells
staining positive for thrombomodulin. Plasmids used are indicated in
each diagram. Data in diagrams represent the mean ± S.D. of at
least three independent experiments. Bar, 50 µm.
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Fig. 6.
Ras-induced inhibition of PE differentiation
is mediated by Erk. A, RA-treated F9 cells were
transiently transfected with -gal alone (control) or
cotransfected with RasLeu-61 (0.1 µg), with cPKA, or with
both cPKA and RasLeu-61 (0.1 µg). Cells were subsequently
left untreated or treated with 50 µM PD98059 for 3 days.
Medium including test agents was changed every 24 h. B,
RA-treated F9 cells were transiently transfected with
-gal and cPKA,
with or without Raf-CAAX or MEK-D218/222. Diagrams depict the
percentage of
-gal-expressing cells positive for thrombomodulin, as
similar to Fig. 5, and represent the mean ± S.D. of at least
three independent experiments.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 7.
A model for the regulation of PrE and PE
differentiation by Ras and PKA. (For description see
"Discussion").
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ACKNOWLEDGEMENTS |
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We thank Dr. S. J. Kennel for the generous supply of antibodies; Dr. C. Marshall for the Raf-CAAX construct; Dr. J. Pouysségur for the MEK-D218/222 construct; M. J. Goumans for experiments with E14 cells; and J. M. Veltmaat, Dr. P. Bird (Monash Medical school, Box Hill Hospital, Australia), and Dr. G. van der Pluijm (University Hospital, Leiden, The Netherlands) for helpful suggestions. We also thank J. Heinen and F. Vervooldeldonk for excellent photographic reproductions and J. den Hertog for critically reading the manuscript.
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FOOTNOTES |
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* This work was performed at the Hubrecht Laboratory (Netherlands Institute for Developmental Biology, Utrecht) and the Utrecht Graduate School for Developmental Biology and was supported by Netherlands Organization for Scientific Research Grant 903 46 102.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are:
EC, embryonal
carcinoma; -gal,
-galactosidase; Bt2cAMP, dibutyryl
cyclic AMP; GST, glutathione S-transferase; MAP, mitogen-activated protein; PE, parietal endoderm; PKA, protein kinase
A; PrE, primitive endoderm; PTH, parathyroid hormone; PTHrP, PTH-related peptide; RA, retinoic acid; RBD, Ras-binding domain; SSEA, stage-specific embryonic antigen; tPA, tissue plasminogen activator; MEK, MAP kinase kinase; PBS, phosphate-buffered saline.
2 M. H. G. Verheijen, M. Karperien, V. I. Chung, M. Vanwijnen, H. Heystek, J. A. A. Hendriks, J. M. Veltmaat, B. Lanske, E. Li, C. W. G. M. Löwik, S. W. De Laat, H. M. Kronenberg, and L. H. K. Defize, submitted for publication.
3 M. H. G. Verheijen and M. J. Goumans, unpublished results.
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REFERENCES |
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