Institut de Génétique et de Biologie Moléculaire et ... · 10/1/2001 · Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/ INSERM/ ULP, BP 163, 67404 ILLKIRCH

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The RSKs, CREB and STAT3 proteins are regulated by different LIF signaling

pathways in mouse Embryonic Stem cells

Boeuf*, H., Merienne, K., Jacquot, S., Duval.D., Zeniou, M., Hauss, C., Reinhardt, B.,

Huss-Garcia, Y., Dierich A., Frank, D.A.1, Hanauer, A. and C. Kedinger2

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/ INSERM/ ULP, BP 163,

67404 ILLKIRCH Cedex, C.U. de Strasbourg, France. 1Dana-Farber Cancer Institute,

Department of Adult Oncology, 44 Binney St., Boston, MA 02115, USA. 2ESBS - FRE 2370 -

Pôle API, Boulevard Sébastien Brant, 67400 Strasbourg-Illkirch

Corresponding author : H. Boeuf

I.G.B.M.C.,

BP 163,

67404 ILLKIRCH Cedex, C.U. de Strasbourg, France.

Tel : 03 88 65 34 53

Fax : 03 88 65 32 01

e-mail : [email protected]

Running title: LIF signaling in ES cells

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 1, 2001 as Manuscript M106718200 by guest on O

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SUMMARY

Mouse embryonic stem (ES) cells remain pluripotent in vitro in the

continuous presence of Leukaemia Inhibitory Factor (LIF). In the absence of LIF, ES

cells are irreversibly committed to differentiate into various lineages. In this study we

have set up an in vitro assay, based on the anti-apoptotic activity of LIF, to

distinguish «pluripotent» from «differentiation-committed» ES cells. We have

examined the phosphorylation profiles of known (STAT3 and ERKs) and identified

new (RSKs and CREB) LIF-regulated targets in ES and in ES-derived neuronal

cells. We have demonstrated that while STAT3, a crucial player in the maintenance

of ES cell pluripotency, is induced by LIF in all cell-types tested, the LIF-dependent

activation of RSKs is restricted to ES cells. We have shown that LIF-induced

phosphorylation of RSKs, in ES cells, is dependent on ERKs while STAT3

phosphorylation is not mediated by any known MAPK activities. Our results also

demonstrate that the LIF-dependent phosphorylation of CREB is partially under the

control of the RSK2 kinase.

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INTRODUCTION

Leukaemia Inhibitory Factor (LIF) is a pleiotropic cytokine which belongs to the

Interleukin 6 (IL6) cytokine family including Ciliary Neurotrophic Factor (CNTF),

Oncostatin M (Onco M) and Cardiotrophin-1 (CT-1). It is secreted by various cell

types and mediates opposite effects (either proliferative or differentiative)

depending on the cell lineages and stage of differentiation (1-3). LIF also influences

the survival, differentiation and response to injury of neuronal cell lineages and

synergizes with CNTF for moto-neuron cell survival (4-7). The pleiotropic effects of

LIF signaling are transduced by the heterodimeric gp130/gp190 (LIFRβ) receptor

which becomes phosphorylated on tyrosine residues by the constitutively

associated, LIF-activated JAK tyrosine kinases. Different parts of the gp130 subunits

serve as docking sites for cell-type specific complexes, leading to the activation of

ras/Mitogen Activated Protein Kinase (MAPK) and Signal Transducer and Activator

of Transcription (STAT) pathways (8-12). However the identity of LIF-induced

proteins in different cell contexts has not been precisely characterised.

One major goal in this field is to identify LIF-dependent pathways in LIF-

sensitive cell lines derived from the same founder cells in which LIF may trigger

various effects. The most appropriate cells to study LIF signaling, which may satisfy

these criteria, are the mouse Embryonic Stem (ES) cells. These cells are derived

from the inner cell mass of blastocysts and they remain pluripotent in vitro when

maintained in the presence of LIF (13,14). Upon LIF withdrawal ES cells

differentiate heterogeneously into various cell types and part of the cells die by

apoptosis during the differentiation process (15). ES cells can also be induced to

differentiate homogeneously into determined cell types (16-20).

MAPKs are Ser/Thr kinases that are activated by several growth factors,

cytokines and stress signals. They are classified into 3 families [Extracellular-signal-

Regulated Kinases (ERKs), c-Jun N-terminal Kinases (JNKs) and p38s] involved in

cell proliferation, differentiation and apoptotic processes (21-23). Among the direct

targets of ERKs (ERK1, ERK2 and ERK5), are kinases such as the Ser/Thr kinases

of the families of Mitogen/Stress Kinases (MSKs) and Ribosomal S6 Kinases

(RSKs) (24-27). The RSKs (or Mitogen Activated Protein Kinase Activated Protein-

Kinase 1/ MAPKAP-K1) family includes three members in mouse cells: RSK1/

MAPKAP-K1a; RSK2/ MAPKAP-K1b and RSK3/ MAPKAP-K1c. These kinases are

regulated by growth factors, cytokine and stress, and are involved in several

biological processes including cell survival and proliferation (25,28,29). The cAMP


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Responsive Element Binding (CREB) transcription factor is activated by

phosphorylation on Ser 133 by different pathways like the cAMP-dependent Protein

Kinase A (PKA), the Nerve Growth Factor (NGF)-dependent p38 and ERK pathways

and the Epidermal Growth Factor (EGF)-dependent RSK2 and MSK1 pathways (30-

34).

STAT3 is at the heart of opposite effects mediated by LIF and it is involved in

many cell processes (apoptosis, anti-apoptosis, cell differentiation and cell

proliferation) depending upon the cell types (35). For example, inactivation of

STAT3 leads to differentiation of ES cells and in contrast blocks the LIF-dependent

differentiation of the myeloid M1 cell line (11,36-40). Activation of STAT3 by

phosphorylation on Tyr 705 and Ser 727 residues has been documented in cells

treated with various growth factors and cytokines (41-46). Phosphorylation on Tyr

705 is essential for STAT3 DNA-binding activity, while phosphorylation on Ser 727,

a residue within the transactivation domain (TAD), is rather involved in

transactivation processes (42,47). Several observations suggest that MAP kinases

are involved in STAT3 phosphorylation: (i) Ser 727 lies within a consensus MAPK

recognition sequence (48) and (ii) ERK2 is activated upon LIF treatment in ES cells

(38). In addition, activation of the ras/ERK pathway diminishes the requirement of

LIF in ES cells (49). Phosphorylation of STAT3 on Ser 727 leads to positive and

negative effects on STAT3 functions, depending on the cell type and the nature of

inducers (45,50-53).

To get insight into LIF signaling in mouse ES cells, we have followed the LIF-

dependent phosphorylation profiles and DNA binding activities of the STAT3

transcription factor under different ES cell growth conditions : « pluripotent », in

the continuous presence of LIF ; « reversibly differentiation-committed », without

LIF for 20h and « irreversibly differentiation-committed », 48h without LIF. We have

also characterised the activation profile of MAPKs and identified new LIF targets

(RSKs and CREB) regulated by ERKs, whose role in the maintenance of ES cell

pluripotency is discussed. Also, by using Ser/Thr kinase inhibitors we have

distinguished LIF-induced-ERK and PKC-dependent pathways. In addition,

activation profiles of some of these LIF-induced proteins have also been studied in

an ES-derived neuronal cell line that we have characterized as being a LIF-

sensitive cell line.


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EXPERIMENTAL PROCEDURES

Cells and reagents

ES cells were derived from the inner cell mass of mouse blastocysts as

described (54). The ES S1 cell line, grown in LIF-containing medium without feeder

cells was used in these experiments unless indicated. The ES H1 wild-type (WT)

and the derived ES H1RSK2– (X/Y cells in which the X-linked RSK2 gene has been

deleted by homologous recombination) were grown on feeder cells, in the presence

of LIF (55). In this RSK2– cell line, shortened mRNA corresponding to the in-frame

skipping of exon 2 (in which was inserted the neomycine resistance gene and the

stop codons) has been detected by RT-PCR analysis, indicating that the RSK2– cell

line used in this study may express an hypomorph allele of RSK2. However, no

RSK2 protein was detected in this cell line, under classical western blot conditions.

H1-derived cell lines were passaged twice without feeder cells in the continuous

presence of LIF, prior to LIF withdrawal and reinduction.

The polyclonal anti-phospho Ser727-STAT3 (56), anti-phospho Tyr705-

STAT3 (QCB), anti-STAT3, ERK2 (C-14), ERK1 (C-16) and RSK2 (E-1) (Santa

Cruz), anti-phospho JNK1/JNK2, anti-phospho p38 (Cell Signalling), the

monoclonal anti-phospho ERK1/ERK2 (Biolabs), and anti-JNK1 (Pharmingen)

antibodies, the U0126 (Promega) and H7 (Biomol) compound were used as

recommended by the manufacturers. The monoclonal anti-phospho Thr577-RSK

antibody has been described (29).

When indicated, quantification of the signals have been performed with the

Biorad, GS700, imaging densitometer by using Molecular analyst, version 2.1,

software.

In vitro pluri. test

ES cells were grown in medium without LIF (DMEM 4.5g/l glucose ; 10%

FCS ; glutamax ; β2 mercaptoethanol 0.1 mM) for various time periods extending

from 6 to 72h and reinduced with LIF (1000 units/ml) up to cell lysis which was

performed 72h after the beginning of the experiment. Apoptosis is scored by the

appearance of a DNA ladder following DNA extraction by the Hirt procedure as

described previously (15).


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Pluripotent and differentiation-committed cell growth conditions

S1 cells were grown in the continuous presence of LIF, and medium was

changed every other day. Cell lysates were prepared without medium change or 15

min after LIF-containing medium addition (« pluripotent » conditions). ES cells

grown without LIF for 20h [differentiation-commited (Dif. com.), reversible] or 48h

[differentiation-commited (Dif. com.), irreversible] were refed with LIF-containing

medium 15 min before harvesting. Cell lysates were prepared from cells grown

under these different conditions.

Neuronal differentiation

Neuronal differentiation of ES cells was mainly induced as described, (16),

with minor modifications: the S1 ES cell line were grown as embryoid bodies

(300,000 cells/ml) on bacterial Petri dishes in Dulbecco's Modified Minimal

Essential Medium (DMEM Gibco/BRL) supplemented with 15% fetal calf serum

(Hyclone), 0.1 mM β mercaptoethanol, 0.1% non-essential aminoacids (Gibco

/BRL), 50 units/ml penicillin and 50 µg/ml streptomycin (Gibco /BRL), in presence of

10-6 M all-trans retinoic acid (RA, Sigma). After four days of RA treatment the

embryoid bodies were trypsinized and the cells were resuspended in the same

medium. 40 000 cells/ml were then plated in DMEM on coated culture dishes (for

biochemical studies) or glass coverslips (for immunocytochemistry). Coating was

performed successively with 0.1% gelatine, 10 µg/ml poly-D-lysine and 1 µg/ml

laminine. After two hours, the culture medium was replaced by a defined medium

(16).

The cell medium was changed every other day. LIF induction [30 min with

250 or 500 units of LIF/ml] was performed four days after plating, on homogeneous

neuronal cell populations.

Immunocytochemistry

Cells (plated on glass coated coverslips, coated as described above), fixed

with 4% paraformaldehyde (15 min) were treated with 3 % H2O2 and

permeabilized with 0.1% Triton X100 (10 min). After preincubation with 10% normal

goat serum (NGS) for 30 min cells were treated with rabbit antiserum directed

against Neurofilament 200 (Sigma) diluted 1/100 or mouse antiserum directed


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against MAP1 (Sigma) diluted 1/300. Peroxidase-conjugated anti-rabbit and anti-

mouse antibodies were used as secondary antibody respectively.

Kinase inhibitor treatments

ES cells grown without LIF for 20h were pretreated for 1 h with 50 µM H7 or

various concentration of U0126, before reinduction with LIF in the presence of the

inhibitors, at the same concentrations.

Cell lysates and bandshift experiments

Cytosolic and nuclear cell lysates were prepared as described (57). Whole cell

lysates were a mixture of cytosolic and nuclear lysates at a 2:1 ratio. Bandshift

experiments were performed with the nuclear lysates (30 µg) on the high-affinity

STAT3 DNA binding site of the c-fos promoter [c-Sis-Induced Element (SIE)], as

described (38).

Western blots

Nuclear or whole cell lysates were resolved by SDS-PAGE and electro-

transferred onto nitrocellulose membranes in the presence of 0.07% SDS. Proteins

were reacted with the different antibodies, as recommanded by the manufacturers.


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RESULTS

ES cells are irreversibly committed to cell differentiation and apoptosis when grown

in the absence of LIF for at least 36 h

Mouse ES cells were grown in the continuous presence of LIF and were fed

with fresh medium or passaged every other day. In the absence of LIF, ES cells are

committed to differentiation. Analysis of the expression profile of genes know as

« pluripotent » (rex-1, FGF4, ESP) or « differentiated » cell markers (FGF-5)

indicates that by 24h of LIF withdrawal, cells are committed to differentiate [(58-60)

and Duval et al., submitted]. In addition, ES cells grown without LIF for 48 h have

lost their pluripotency, as suggested by their inability to colonise embryos (61). We

have developped a quicker test to determine the point of irreversible commitment of

cell differentiation, when cells are grown in the absence of LIF. Based on our

previous observation that, during the differentiation process, 30% of the cells are

dying by apoptosis, [(15) and Duval et al, submitted], we have set up an in vitro

pluripotency test (in vitro pluri. test): cells were grown in the absence of LIF for

different time periods (between 6 and 72 h) and then refed with LIF-containing

medium, up to 72h, before harvesting. Cells were scored for apoptosis by the

appearance of a DNA ladder, a qualitative test which allows rapid detection of

apoptotic cells (Fig. 1). The morphology of the cells and integrity of their DNA were

unaltered when LIF was withdrawn for 24 h, compared to cells maintained in the

continuous presence of LIF. By contrast, from 36 h onward after LIF withdrawal, ES

cell clumps start to dissociate and cells begin to spread reflecting the differentiation

process. Meanwhile dying cells were detected and increasing proportions of DNA

were degraded, indicating that part of the cells were dying by apoptosis. Therefore,

we conclude that ES cells are irreversibly committed to differentiation and/or

apoptosis, starting at 36 h after LIF withdrawal. Based on this in vitro test, as well as

on in vivo data (14, 61), we will refer in the following to « pluripotent »,

« reversibly differentiation-committed » or « irreversibly differentiation-committed »

cells.

LIF-dependent phosphorylation of STAT3 in ES cells

STAT3 is critical for the maintenance of pluripotent ES cells as well as in

various LIF-sensitive differentiated cells (38,62-64). Phosphorylation of STAT3 at

the Tyr 705 residue is crucial for its activity in ES cells (11,38). It is also


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phosphorylated at the Ser 727 MAPK consensus site in response to various stimuli

like EGF or IL6. We were interested to follow the STAT3 activation profile under

experimental conditions as defined above. In these experiments LIF was withdrawn

for 20 h or 48 h as a way to determine LIF-dependent STAT3 activation at a very

early stage of differentiation commitment, while the process is still reversible (20 h

after LIF withdrawal), as well as in irreversibly differentiation-committed cells (48 h

after LIF withdrawal).

Western blot analyses of nuclear extracts from ES cells grown under these

conditions were performed using specific antibodies which recognize the activated

STAT3 proteins phosphorylated on the Tyr705 (P-Y705-STAT3) or Ser727 (P-

S727-STAT3) residues (Fig. 2). Phosphorylated STAT3 is detected in the

continuous presence of LIF with a clear enhancement of the level of

phosphorylation when fresh medium with LIF is added (Fig. 2 :« Pluripotent » cell

growth conditions). By contrast, no phosphorylation on Tyr 705 and Ser 727

residues is detected in ES cells grown in the absence of LIF for 20 h or 48 h.

However, a rapid phosphorylation at both sites is induced upon LIF addition

indicating that these cells are still LIF responsive (Fig. 2: «Dif. com. at 20h» and

«Dif. com. at 48h» cell growth conditions). STAT3 phosphorylation is correlated

with its specific DNA binding activity as detected on the SIE probe, in the

«Pluripotent» and «Dif. com.» cells (Fig. 2). Protein-DNA complex formation on a

LIF-unresponsive site was constitutive and unchanged in both cell types, indicating

that cell extracts were equally functional [(38) and data not shown]. However, we

noticed a reduction in the amount of STAT3-dependent DNA-binding complexes

formed in «Dif. com.» cells at 48 h, upon LIF induction. Also, we have found that the

cytosolic extracts, in which phosphorylated STAT3 could be detected (44), did not

exibit specific DNA binding activity indicating that nuclear partners may stabilize the

DNA-phospho-STAT3 complexes (data not shown). These results indicate that LIF-

induced STAT3-dependent complexes, which include phosphorylated STAT3

proteins, are present in ES cells grown under the pluripotent and differentiation-

committed cells.

None of the MAPKs are required for STAT3 phosphorylation at the Ser 727 site in

ES cells

We have analysed the activation profile of members of the three MAPK

families (ERK, JNK and p38) in ES cells. Western blot analysis of total cell lysates


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from ES cells maintained under various conditions were performed with antibodies

against MAPKs which specifically recognize the activated dually phosphorylated

forms of ERK1/ERK2 (P-ERK1/P-ERK2, P-ERKs), JNK1/JNK2 (P-JNK1/P-JNK2) and

p38 (P-p38). As shown in Fig. 3A , the ERK1 and ERK2 proteins are induced by LIF

in cells maintained in the presence of low or high serum concentrations, in good

agreement with previous studies (38,49). However, MAPKs as well as their targets,

like the RSKs proteins, are serum-induced proteins (25). Therefore, it was of interest

to test the direct effect of serum and LIF independently since both are required for

the proper growth of ES cells (our unpublished observation). We were also

interested to determine the sensitivity of the various LIF-dependent targets in the

presence of the U0126 compound, a specific MAP ERK Kinase (MEK) inhibitor

which acts downstream of MEK1 and MEK2, impairing phosphorylation of ERK1

and ERK2 at the specific TEY site (65,66).

ES cells, grown 20 h without LIF, were induced in the basic medium (10%

FCS) or in LIF-containing medium (10% FCS + LIF) for 15 min in the absence or

presence of various concentrations of U0126 (see experimental procedures). As

shown in Fig. 3B, we observed an additive effect of LIF and serum on ERK1/2

phosphorlation while phosphorylation of JNK1 and p38 were strictly serum-

dependent, with a strong and a mild activation for JNK1 and p38, respectively. In

vitro kinase assays, performed on exogenous substrates after ERK1, ERK2, JNK1

and p38 immunoprecipitation, confirmed that the phosphorylated forms of these

proteins corresponded to the activated enzymes [(38) and data not shown]. We

observed that ERK1 was less activated than ERK2, in agreement with the low

amount of P-ERK1 reproducibly observed with the P-ERKs antibody (Fig. 3A and B

and data not shown). Similar results were obtained in ES cells deprived of LIF for

48h (not shown). From these experiments, we conclude, also, that phosphorylation

of STAT3 is strictly LIF-dependent and, based on its insensitivity to the U0126

inhibitor, that phosphorylation of STAT3 at Ser 727 is ERKs-independent.

The H7-sensitive phosphorylation of STAT3 on Ser 727 is not required for LIF-

dependent STAT3 DNA binding activity

PKC proteins are LIF-regulated kinases which may phosphorylate STAT3

upon cytokine induction (41,67). Experiments with the PKC inhibitor H7, and with

U0126, have been performed in cells deprived of LIF for 20 h and reinduced for 15

min with LIF, in the presence of the inhibitors (Fig. 4A). H7 blocked STAT3


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phosphorylation at the Ser 727 residue, without affecting phosphorylation at the Tyr

705 residue. U0126, which, as expected, impairs phosphorylation of ERK1 and

ERK2, did not abolish STAT3 phosphorylation on Ser 727 as previously shown

(see Fig 3B). STAT3 DNA binding activity was not affected in the presence of either

H7 or U0126, indicating that phosphorylation of Tyr 705 was sufficient to mediate

the DNA binding activity of STAT3 (Fig. 4B).

Phosphorylation of RSKs and CREB proteins is LIF-dependent in ES cells

To characterize LIF-regulated effectors of ERKs, we have examined the

activation of members of the RSK family by using a monoclonal antibody raised

against a phosphorylated peptidic sequence well conserved among the RSK

proteins (anti-P-RSKs), thus recognizing activated RSK1, 2 and 3. The peptide

includes a unique threonine residue in the C-terminal domain of RSKs (Thr 577 in

the case of RSK2) which is phosphorylated by ERK1/ERK2 in response to mitogen

and UV stimulation (29). Constant amounts of RSK1, 2 and 3 were present in ES

cells, whether treated or not with serum and LIF (Fig. 3B and data not shown). As

detected with the phosphospecific antibodies (P-RSKs), it was clear that, in the

presence of serum, the induced ERK1 and ERK2 kinases did not phosphorylate the

RSKs which were phosphorylated only in the presence of LIF. However,

phosphorylation was abolished in the presence of U0126, indicating that LIF-

dependent RSKs phosphorylation at this site may be regulated by the LIF-induced

ERK proteins.

We have also analysed the activation profile of CREB transcription factor, a

known EGF induced-dependent RSK target and have shown that it was

phosphorylated at the Ser 133 residue upon LIF treatment. The U0126 compound

repressed CREB phosphorylation indicating that the RSKs may contribute to this

phosphorylation in ES cells.

Altogether, our results suggest that at least two pathways, distinguishable by

their sensitivity to U0126, lead to LIF-dependent activation of proteins : the U0126-

sensitive (ERKs/RSKs/CREB) and the U0126-insensitive (STAT3) pathways.

The overall phosphorylation of RSKs by LIF is diminished in the RSK2 – cells

RSK2 was the most efficient LIF-dependent RSK, as deduced from

comparative in vitro kinase assays performed after selective immunoprecipitation of

RSK1, 2 or 3 (not shown). To directly investigate the effect of RSK2 on ES cell


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pluripotency, an hemizygote (X/Y) RSK2– ES cell line has been derived, in which

the X-linked RSK2 locus was targetted by homologous recombination (55). Western

blot analyses with the specific antibodies revealed LIF-dependent phosphorylation

of the RSK proteins in the wild-type cell line. In the derived RSK2– line, in which we

did not detect RSK2 proteins, while RSK1 and RSK3 were present, LIF-dependent

phosphorylation was clearly reduced, with a residual signal likely reflecting

phosphorylation of RSK1 and RSK3 proteins (Fig. 5 and data not shown).

LIF-dependent CREB phosphorylation at Ser 133 residue is half-reduced in the

RSK2 – cell line

The phosphorylation status of CREB and STAT3 has been investigated in the

RSK2– cells. As shown in Fig. 5, LIF-dependent CREB phosphorylation at Ser 133

was diminished in the RSK2– cells: densitometry scanning revealed that the P-

CREB signal was reduced by half in RSK2- compared to the WT cell line. This

indicates that RSK2 partly contributes to the LIF-dependent Ser 133

phosphorylation of CREB in ES cells. However, the LIF-dependent phosphorylation

of STAT3 at Ser 727 was not altered in the RSK2– cell line, as expected from our

results which show that phosphorylation of STAT3 at the Ser727 residue is not

sensitive to U0126 (Fig. 3B).

LIF-dependent phosphorylation of STAT3 but not of RSKs in ES-derived neuronal

cells

STAT3 proteins have pleiotropic effects in different cell lineages in embryos as

well as in adult tissues. Interestingly, this transcription factor is induced by LIF under

the different ES cell growth conditions that we have tested (pluripotent and

differentiation-commited). We were wondering if the LIF-dependent induction of

STAT3 observed in ES cells and early heterogeneous ES cell derivatives was also

observed in homogeneously differentiated derivatives and if we can use STAT3 as

a paradigm to test the LIF sensitivity of ES-derived differentiated cell lineages. LIF is

a survival factor in many neuronal cell types and can also trigger differentiation of

cortical precursors into astrocytes (7,63,68). ES cells were differentiated into a

neuronal cell line which expresses specific neuronal markers, such as the

neurofilament and MAP1 proteins (Fig. 6A and experimental procedures).

Homogeneously differentiated neuronal cells were treated with LIF for 30 min and

the phosphorylation status of STAT3 and RSKs were determined with the


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corresponding phospho-specific antibodies. As shown in Fig. 6B, STAT3

phosphorylation at both sites was induced in this ES-derived neuronal cell line

indicating that these neurons expressed the LIF receptor and were LIF- sensitive.

However, we observed a basal constitutive phosphorylation of ERKs and RSKs and

the level of expression of CREB proteins is barely detectable in this particular

neuronal cell type (Fig. 6B and data not shown).

In conclusion, we have isolated a LIF-sensitive neuronal cell line which will be

usefull to study LIF and STAT3 functions in neurons.


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DISCUSSION

In this study we have characterized the point of no return (36 h) at which ES

cells are irreversibly committed to differentiation when grown in the absence of LIF,

by an in vitro assay based on the anti-apoptotic effect of LIF. We have also

analysed the LIF-dependent activation profile of the STAT3 transcription factor

under various cell growth conditions and in an ES-derived neuronal cell line. In

addition, we have identified new LIF-responsive targets and shown that, at least,

two distinct pathways are activated by LIF in ES cells. Finally, we have

demonstrated that the LIF-dependent CREB phosphorylation is under the control of

RSKs in ES cells.

Concomitant LIF-dependent STAT3 phosphorylation at Tyr 705 and Ser 727 in ES

cells

This study reveals that LIF induces concomitant STAT3 phosphorylation at the

level of two residues, Tyr 705 and Ser 727 irrespective of the cell growth conditions,

in ES cells. STAT3 phosphorylation at both regulatory sites is transient reaching a

maximum after 30 min of induction and decreasing after two hours to the level

detected in ES cells continuously maintained with LIF (data not shown). Because

an irreversible process of differentiation is engaged in ES cells, when STAT3

phosphorylation is impaired (11,38,40), we postulate that the transient STAT3

phosphorylation observed when fresh medium containing LIF is added, is critical for

the maintenance of ES cell pluripotency. These results are in good agreement with

previous work showing that stable expression of a tamoxifene-activated STAT3-ER

fusion protein, phosphorylated at both canonical Ser and Tyr residues, allows the

maintenance of ES cell pluripotency in the absence of LIF, upon tamoxifen

treatment (40). By contrast, continuous activation of STAT3 leads to the

differentiation of the myeloid M1 cell line, indicating that sustained versus transient

LIF-dependent STAT3 activation may be at the origin of opposite effects of LIF

triggered in ES and M1 cell lines (36). Our findings are divergent with studies

suggesting that Ser 727 phosphorylation of STAT3 negatively regulates Tyr 705

phosphorylation, as observed in EGF-stimulated cells (50). This observation

suggests that the antagonistic effect of STAT3 phosphorylation at Tyr 705 and Ser

727 might be cell-type and inducer specific.


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MAPKs are not involved in STAT3 Ser 727 phosphorylation

MAPKs like ERKs and JNKs are induced by serum in many cell types and

experiments performed in serum-deprived cells indicate that LIF activates the ras

signaling pathway in ES cells to the same extent as achieved by serum (49).

However, since ES cells could not be propagated properly in the absence of serum

(our unpublished observation) we have conducted our experiments in media

supplemented with 10% FCS or 10% FCS and 1000u/ml LIF. Under these

conditions, the three classes of MAPKs are activated in ES cells, by serum only

(p38 and JNK1) or, additively, by serum and LIF (ERKs). Members of the three

MAPK families have been proposed as potential candidates for STAT3

phosphorylation, among which ERKs would be more specifically involved in growth-

factor-dependent phosphorylation (50,69-71). Based on the effects of U0126, we

rule out the involvement of ERKs in LIF-dependent Ser 727 phosphorylation. In

addition, staurosporine (a more general kinase inhibitor), which inactivates ERK1

and ERK2, did not affect Ser 727 phosphorylation (not shown). Inhibition of p38

activity (by PD169316) or JNK1 (by high concentration of U0126) did not affect

STAT3 Ser 727 phosphorylation (not shown). Finally, we show that STAT3

phosphorylation is not under the direct control of RSK2 but that a PKC isoform, that

would be activated by LIF and inhibited by H7 in ES cells, may phosphorylate

STAT3 at Ser 727, in agreement with earlier findings (41,72). Recent studies

indicate that PKCδ is involved in IL6-dependent phosphorylation of STAT3 leading

to repression of STAT3-dependent transcription (53). Our observation that a specific

inhibitor of PKCδ (Rottlerin) did not decrease the level of STAT3 phosphorylation at

the Ser 727 residue (unpublished data) suggested that the LIF and IL6-dependent

STAT3 phosphorylations at Ser 727 are mediated by distinct H7-sensitive

pathways. Also, our experiments indicate that LIF-dependent STAT3 DNA-binding

activity relies only upon Tyr 705 phosphorylation. This is in good agreement with

previous reports showing that stimulation by IFNγ (for STAT1) and EGF (for STAT3),

which induced phosphorylation at Tyr 705 on STAT1 and STAT3, triggers specific

DNA binding activity of these factors (73,74).


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RSKs and CREB are LIF-dependent targets in ES cells

We show that phosphorylation of members of the RSK family and of the

CREB transcription factor is induced by LIF but not by serum in ES cells. Based on

their sensitivity to U0126, RSKs and CREB behave as dowstream ERK targets.

However, since CREB phosphorylation is not completly abolished in the presence

of U0126, it will be of interest to examine the LIF-dependency of the MSK-1

(Mitogen-Serum kinase-1), a recently identified CREB kinase (24,34). Our results

show also that serum-mediated induction of ERK1 and ERK2 does not result in

RSKs phosphorylation, suggesting that in addition to ERK1 and ERK2, another

U0126-sensitive LIF-induced ERK protein may phosphorylate the RSK proteins.

The ERK5 protein, a U0126-sensitive but serum-independent MAPK, represents a

potential candidate whose involvement in LIF signaling in ES cells is presently

under investigation (27,75-77).

LIF-dependent CREB phosphorylation is partly under RSK2 control

As concluded from an in vitro kinase assay, RSK2 was the most active LIF-

dependent kinase when compared with RSK1 and RSK3 (data not shown). By

using RSK2- ES cells, in which the RSK2 gene had been deleted by homologous

recombination, we observed a residual LIF-dependent phosphorylation of the

remaining RSKs (most likely RSK1 and RSK3). This indicates not only that other

members of the RSK family are LIF-responsive, but also that there is no

compensatory stimulation of these kinases, in the absence of RSK2. Similar results

have been obtained with cells derived from human fibroblasts of a Coffin Lowry

patient, in which the mutated RSK2 gene encodes an unstable RSK2 protein

lacking kinase activity [(28) and data not shown]. Together, these observations

suggest a potential redundancy of RSKs in LIF signaling, both in mouse and human

cell systems. Our results also revealed that LIF-dependent phosphorylation of the

CREB transcription factor is diminished in the RSK2- cell line, pointing to the

contribution of RSK2 in LIF-dependent CREB phosphorylation. However, RSK2-

cells stayed undifferentiated in vitro in the presence of LIF (our unpublished

observations) and CREB was not involved at early stages of embryogenesis (78),

indicating that RSK2 and CREB are LIF targets that are not involved in the

maintenance of ES cell pluripotency.


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Activation of ERKs, JNK1, p38, RSKs and CREB in ES cells: for which purpose?

The concomitant strong activation of JNK1, ERK1/2, RSKs and CREB and

mild activation of p38 by serum and/or LIF may contribute to ES cell survival, since

apoptosis is often correlated with the simultaneous activation and repression of

different members of the MAPK family, depending on the cell system (79,80). As a

matter of fact, we have recently shown that LIF prevents apoptosis in ES cells and

that the p38 MAP kinase is strongly activated, while JNKs and ERK1/2 are

repressed, during the apoptotic process triggered upon LIF withdrawal [(15) and

Duval et al, submitted]. In addition, several studies are consistent with the idea that

activated ERKs are involved in a negative regulatory pathway controlling cell

proliferation in ES cells (18,40,61). The demonstration that LIF-dependent activated

ERKs leads to the degradation of the specific GP190 LIF receptor subunit in various

cell lines, strongly indicates that activated ERKs contribute to LIF receptor recycling

(81). In addition, RSK2 blocks Bad-mediated cell death and inhibition of CREB Ser

133 phosphorylation triggers apoptosis in particular subtypes of neurons, indicating

that RSK2 and CREB may also function as survival factors (82,83). It is likely

therefore that a fine balance between activated ERKs, JNK1, p38, RSKs and CREB

is required for proper ES cell survival in the presence of LIF (Fig.7).

STAT3, a paradigm to study LIF signaling in various ES cell derivatives

The STAT3 transcription factor is an effector of LIF signaling in various cell

types in which LIF may trigger opposite effects (62,63). We have characterized an

ES-derived neuronal cell line in which phosphorylation of STAT3, but neither ERKs

nor RSKs, was induced by LIF. This indicates the presence of LIF receptors in this

cell line and raises the possibility to study LIF and STAT3 signaling in a neuronal

cell type. Indeed, while activation of STAT3 in ES cells is critical for the

maintenance of ES cell pluripotency (11,38,40), the precise role of LIF and

activated STAT3 has not yet been further investigated in this LIF-sensitive neuronal

cell model.


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In conclusion, this report, in which we identify two LIF-dependent pathways by

following LIF-induced proteins in pluripotent and in various ES cell derivatives,

emphasizes the interest of the versatile ES cell system to study alternative LIF

signaling under various cell growth conditions leading to cell pluripotency or to

differentiation-committed cell lineages.

In addition, it is intriguing to note that human ES cells, which may be used in

the future for cellular therapy, can maintain pluripotency in the absence of LIF but in

the presence of still unknown factors produced by mouse feeder layer cells (84,85).

It will be of interest to examine how pluripotency pathways have evolved in mouse

compared to human ES cells. Understanding the molecular basis of pluripotency in

the mouse model may be very useful in characterizing key human pluripotency

factors.


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ACKNOWLEDGMENTS

We thank D. Queuche and E. Blondelle for ES cells and materials, B. Chatton

and M. Vigneron for helpful discussions and N. Ghyselinck for the critical reading of

the manuscript. We are grateful to the staffs of the cell culture, biocomputing and

artwork facilities for providing help and material. This work was supported by funds

from the Centre National de la Recherche Scientifique, the Institut National de la

Santé et de la Recherche Médicale, the Centre Hospitalier Universitaire Régional,

the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le

Cancer and the Université Louis Pasteur of Strasbourg.


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FIGURE LEGENDS

Figure 1

In vitro pluri. test

A - Phase contrast pictures: ES cells grown in the absence of LIF for 6, 12,

24, 36, 48 or 72h and reinduced with LIF up to 72h, after the beginning of the

experiment, as indicated. Note that cells corresponding to the time point 72h without

LIF were not reinduced. Bars,100 µm.

B - 20 µg of DNA from each plate grown under the indicated conditions, were

loaded on a 2% agarose gel and stained with ethydium bromide after

electrophoresis. The reverse photograph of stained DNA is presented.

Figure 2

LIF-dependent activation profile of STAT3 in ES cells grown under

pluripotent or differentiation-committed conditions

Western blot analysis of STAT3 was performed with nuclear lysates from ES

cells cultivated in the continuous presence of LIF (Pluripotent), without LIF for 20 h

(Dif. com. at 20h) or without LIF for 48 h (Dif. com. at 48h) and reinduced with LIF for

15 min (+). The blots were probed with antibodies directed against phospho-

Tyr705-STAT3 (P-Y705-STAT3), phospho-Ser727-STAT3 (P-S727-STAT3), and

STAT3. Bandshift analysis were performed with the corresponding nuclear cell

lysates, with the SIE67 probe. The asterisk indicates a non-specific complex.

Figure 3

Differential activation of MAPKs, of MAPKAPKs and transcription

factors by serum and LIF

A - Western blot analyses were performed with total lysates from ES cells

grown in serum-containing medium (10 % FCS) or starved for 20 h (0.5% FCS), in

medium without LIF for 20 h (-) or reinduced with LIF for 15 min, with the anti-

phospho-ERK1/2 (P-ERKs), anti-ERK1 and anti-ERK2 antibodies as indicated.

B – Western blot analysis were performed with total lysates from ES cells

grown in serum-containing medium without LIF for 20 h (-) and reinduced in the

serum-containing medium without LIF (+ 10% FCS) or in the serum-containing

medium with LIF (+ LIF) in the absence (-) or presence of various concentrations of


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U0126 as indicated, with phospho-specific antibodies directed against activated

p38 (P-p38), JNK1/JNK2 (P-JNK1), P-ERKs, phospho-T577-RSK (P-RSKs),

phospho-S133-CREB (P-CREB), P-S727-STAT3 and P-Y705-STAT3 and with

antibodies directed against p38, JNK1, ERK1, ERK2, RSK2, CREB and STAT3.

Exposure time for P-ERK1 was 30 min while exposure time for P-ERK2 was 1 min

(panel B).

Figure 4

ERKs are not responsible for phosphorylation of STAT3 at Ser 727.

Western blot (A) and bandshift (B) analyses, were performed with whole cell

(A) or nuclear lysates (B) from ES cells cultivated without LIF for 20 h (-) and

reinduced with LIF for 15 min (+), in the absence or presence of 50 µM H7 or 10 µM

U0126, as indicated. A 100 molar excess of wild type (wt) or mutated (m)

oligonucleotides were used as competitors in B. Other symbols and annotations

were as in Fig. 2 and 3.

Figure 5

LIF-dependent phosphorylation of RSKs, CREB and STAT3 proteins in

WT and in RSK2- ES cell lines

Western blot analyses with anti-P-RSKs, RSK2, P-CREB, CREB, P-S727-

STAT3 and STAT3 antibodies were performed with whole cell lysates from the wild-

type (WT) or RSK2- ES cells grown without LIF for 20 h (-) or reinduced with LIF for

15 min (+).

Figure 6

LIF-dependent phosphorylation of STAT3 but not of RSKs in ES-

derived neurons

A – Pictures of ES cell-derived neurons taken four days after plating of the RA-

treated embryoid bodies in defined medium : phase contrast ; staining with anti-

neurofilament and MAP1 antibodies (bars, 100 µm).

B - Western blot analyses of total cell lysates from ES-derived neuronal cells

induced in the basic medium (-) or in basic medium with 250u/ml (+) or 500u/ml (++)

of LIF for 30 min.


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Figure 7

Recapitulative scheme of the serum and LIF pathways operating in

mouse ES cells

A drawing of the alternative roads, induced by serum and/or LIF, including

PKC, MAPKs (p38, JNKs and ERKs), MAPKAPKs (RSKs) and two transcription

factors (CREB and STAT3), is presented. The thickness of the arrows refers to minor

(thin line) moderate or strong (heavy line) effects. The position of the blocking effect

of MEK inhibitor (U0126) and PKC inhibitor (H7) is indicated. The potential

physiological effects of these activated pathways, Apop. (apoptose), Dif.

(differentiation), Prolif. (proliferation) as well as the postulated mechanisms of action

of activated ERKs on feedback control regulation and differentiation are deduced

from different studies [(18,23,30,78,81), Duval et al, submitted]. Question marks

indicate yet unknown intermediates.


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FOOTNOTES

Key words : ES cells; ES-derived neuronal cells; LIF; MAPKs; RSKs; STAT3; CREB

The abbreviations used are :

CREB: cAMP Responsive Element Binding; ERK: Extracellular signal Regulated

Kinase; ES: Embryonic Stem; JNK: c-Jun N-terminal Kinase; LIF: Leukaemia

Inhibitory Factor; MAPK : Mitogen Activated Protein Kinase ; MAPKAPK : Mitogen

Activated Protein Kinase Activated Protein Kinase; MSK: Mitogen Stress Kinase;

PKC: Protein Kinase C; RSK: Ribosomal S6 Kinase; SIE: c-Sis Inducible Element;

STAT: Signal Transducer and Activator of Transcription


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Andre Hanauer and Claude KedingerHauss, Beatrice Reinhardt, Yolande Huss-Garcia, Andree Dierich, David A. Frank,

Helene Boeuf, Karine Merienne, Sylvie Jacquot, David Duval, Maria Zeniou, Charlottepathways in mouse embryonic stem cells

The RSKs, CREB and STAT3 proteins are regulated by different LIF signaling

published online October 1, 2001J. Biol. Chem.

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Institut de Génétique et de Biologie Moléculaire et ... · 10/1/2001 · Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/ INSERM/ ULP, BP 163, 67404 ILLKIRCH