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Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/ULP), BP 163, F-67404 ILLKIRCH Cedex, France
1Correspondence: IGBMC, 1 rue Laurent Fries, B.P. 163, 67404 ILLKIRCH, France. E-mail: kedinger{at}igbmc.u-strasbg.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: pluripotency • apoptosis • early response genes • transcription
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. ES cells can be
propagated and maintained pluripotent in vitro in the
presence of leukemia inhibitory factor (LIF). This cytokine belongs to
a family including interleukin 6 (IL-6), ciliary neurotrophic factor,
oncostatin M, and cardiotrophin 1, whose functions are partly redundant
and that transduce their signal through the common gp130 receptor
subunit in association with specific partners (3
, 4)
.
Studies performed with knockout mice pointed to the specificity of the
different member of this cytokine family of receptors
(5
6
7
8)
.
LIF activates at least two pathways: the Janus kinase/signal transducer
and activator of transcription (JAK/STAT) and the ras/mitogen
activated protein (MAP) kinases pathways, which may converge, leading
to phosphorylation/translocation to the nucleus of latent STAT family
members and to the transcriptional increase of early response genes
such as c-fos, c-jun, junB, and junD (9
10
11
12)
. Phenotypic
analysis of STAT mutant mice and in vitro biochemical
studies in different cell systems have emphasized the essential
contribution of STAT proteins to cell proliferation and differentiation
(13)
. MAP kinases are serine/threonine kinases activated
by growth factors, stress signals, and cytokines and are classified
into three families: extracellular regulated kinase (ERK), jun
N-terminal kinase (JNK), and p38. They are involved in cell
proliferation, differentiation, and apoptotic processes
(14
15
16)
.
Characterization of the LIF signaling pathways in ES cells is an
essential step toward understanding cell pluripotency. Also,
comparative studies of gene expression in cell types in which LIF
mediates opposite effects (e.g., ES and the myeloid M1 cell lines)
should help to characterize the critical components of LIF
cell-specific responsiveness. We have previously shown that the ERK2
MAP kinase and the STAT3 transcription factor are activated on LIF
treatment in undifferentiated ES cells (17)
. In addition,
it has been demonstrated that STAT3 impairs LIF-dependent
differentiation of myeloid M1 cells while it maintains LIF-dependent
pluripotency of ES cells (17
18
19
20)
. Furthermore, the early
embryonic lethal phenotype of mice lacking STAT3, which die before
gastrulation, demonstrates that STAT3 is critical for the early
development of mouse embryos (21)
.
The recent characterization of the Socs proteins (suppressors of
cytokine signaling), which repress LIF signaling, further accounts for
the fine-tuning of the pleiotropic effect of LIF. These proteins have
been isolated independently as JAK binding proteins, new Src homology-2
(SH2) -containing proteins, or repressors of cytokine signaling
(22
, 23)
. They harbor conserved features like a central
SH2 domain directly involved in the association with JAK kinases, and a
carboxyl-terminal Socs box whose function remains controversial
(24
, 25)
. The less conserved amino-terminal part of these
Socs proteins may be responsible for their specificity
(26
27
28
29)
. Forced expression of specific members of the
Socs family impaired the LIF-dependent differentiation of the M1
myeloid cell line, probably by an irreversible inactivation of STAT
proteins (27
, 30)
.
In the present study, we report on a new function of the LIF in ES cells and analyze the expression of selected genes that may be involved in the control of LIF signaling in ES cells. We also investigate the role of particular Socs genes (Socs-1 and Socs-3) in LIF-dependent processes in ES cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 31)
.
Nuclear cell lysates were prepared as described (32)
.
Western blot
Nuclear cell lysates were resolved by sodium dodecyl sulfate
(SDS)-gel electrophoresis and transferred on nitrocellulose membranes.
Proteins were reacted with the anti-PARP (rabbit polyclonal antibody, a
gift from G. De Murcia, ESBS, Strasbourg), the anti-phospho-p38
(Promega, Madison, Wis.), or the anti-STAT3 H190X (Santa Cruz
Biotechnology, Santa Cruz, Calif.) antibodies.
Reporter and expression vectors
The (SIE)3-TK-CAT and
(SIEm)3-TK-CAT reporter vectors contain the
trimerized high-affinity, wild-type (SIE67, sis-induced element) or
mutated (SIE25) STAT binding sites inserted upstream of the minimal
TK-CAT reporter gene construct (17
, 33)
. The expression
vectors S1WT, S3WT, S3
N, S3C, and S3NC were constructed as
follows. Polymerase chain reaction (PCR) -amplified fragments
corresponding to the full-length Socs-1 and Socs-3 cDNAs were cloned
into the NdeI/BamHI sites of the p513HA
expression vector, a SV40 promoter-based vector in which the sequences
inserted were in-frame with the hemagglutinin (HA) tag (a gift from B.
Chatton, IGBMC, Strasbourg). The mutants S3N (in which residues 1 to
28 are deleted), S3C (in which residues 176 to 224 are deleted) and
S3NC (in which residues 1 to 28 and 176 to 224 are missing) were
constructed by PCR amplification of the proper fragments from the S3WT
construct. Each construct was verified by sequencing on both DNA
strands.
Transfection and CAT assays
ES cells were transfected and CAT assays performed as described
(17)
. Briefly, cells grown in 9 cm Petri dishes were
transfected with 5 µg of the LIF-responsive
(SIE)3-TK CAT reporter vector or 15 µg of the
(SIEm)3-TK CAT (to clearly visualize basal
transcription and its potential repression), together with 1 µg of
the Socs expression vectors or the empty vector. The percentage of
chloramphenicol acetylation was determined from at least three
independent experiments, and quantitated with a Bioimaging analyzer
(Fuji Photo Film Co.).
DNA analysis
For DNA extraction (Hirt technique), the cells were directly
lysed in ETS buffer (10 mM EDTA, 10 mM Tris-HCl, pH 8, 1% SDS), 4 ml/9
cm petri dish. Cell lysates were supplemented with NaCl (1 M final),
left overnight at 4°C, and spun 10 min at 8000 g. The
supernatant was treated 3 h with proteinase K (100 µg/ml) and
the DNA was precipitated with ethanol after phenol/chloroform
extraction. After RNase treatment (10 µg/ml, Sigma), 30 min at
37°C, 20 µg of DNA was loaded on 2% agarose gels and stained with
ethidium bromide. Floating cells were recovered by centrifugation and
the DNA was prepared as above.
Semiquantitative RT-PCR
Total RNA from ES cells was prepared with the Trizol reagent kit
(Life Technologies, Inc./BRL), and treated with DNase, (5 U/100 µg
RNA, Sigma). Total RNA (4 µg) was reverse-transcribed (RT) with
random hexameric primers and the MLV Reverse Transcriptase (Sigma). The
RT reaction was split into 4 PCR reactions with 4 sets of specific
primers. One PCR reaction was performed with primers corresponding to
the 36B4 LIF-independent gene (34)
with each RNA
preparation. PCR products (20 cycles) were analyzed by Southern blot
with random-labeled cDNA fragments.
The primers used for the LIF receptor ß subunit, gp130 and ESP have
been described (35)
.
GenBank accession numbers and primers for the other genes are:
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Selection of stably transformed cells
To establish ES cells that produce the wild-type or mutated Socs
proteins, ES cells maintained in LIF-containing medium in 9 cm petri
dishes were cotransfected with 10 µg of the S1WT, S3WT, and S3NC
vectors, together with 4 µg of the PGK-neo selection vector. After 15
days of selection in the presence of 150 µg/ml of G418, individual
clones were counted. Cotransfection efficiency was determined with an
SV40-lac Z plasmid (10 µg) transfected together with 4 µg of PGK
neo (36)
. After 15 days in the presence of 150 µg/ml of
G418, stable clones were stained with X-gal. About 65% of the
neomycin-resistant clones were positive for ß-galactosidase
expression, indicating that cotransfection efficiency was at least
65%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 3 days after LIF withdrawal, dead cells could be observed (refringent
cells in Fig. 1A
), and a significant fraction of the DNA
showed the classical degradation ladder (Fig. 1B
) indicative
of apoptotic chromatin damaging (37
38
39)
. The presence of
apoptotic cells was confirmed by electron microscopy analysis (data not
shown). After 5–7 days of LIF deprivation, the remaining cells started
to morphologically differentiate and, concomitantly, the proportion of
damaged DNA decreased to undetectable levels. A similar apoptotic
process could also be observed on LIF withdrawal from a clonal cell
line derived from the D3 cell line (40)
(not shown). The
apoptosis mediated by LIF deprivation apparently did not involve the
specific caspases responsible for poly-ADP-ribose polymerase (PARP)
cleavage (41)
, since this particular apoptotic marker was
not cleaved in ES cells after LIF withdrawal (Fig. 2A
, lane 2). However, treatment of these cells with the
apoptogenic agent staurosporine led to PARP cleavage (Fig. 2A
, lane 3), indicating that the PARP-specific caspases are
indeed present and could be activated in ES cells. Also, we found that
p38 MAP kinase isoforms were induced after 4 days of LIF withdrawal
(Fig. 2B
). The STAT3 protein level, which does not fluctuate
in ES cells, is used here as an internal control of protein loading
(17)
.
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Expression of potential LIF-responsive genes in ES cells
By semiquantitative reverse transcriptase (RT) -PCR essay, we have
analyzed expression of several genes such as 1) genes whose
promoters contain STAT binding sites (like c-fos, junB, and socs-1;
refs 42
43
44
) 2) genes known to be regulated
during ES cell differentiation (like the ESP tyrosine phosphatase; ref
45
), 3) genes induced by LIF in other cell
systems (like c-jun and junD; ref 46
), and 4)
genes preventing apoptosis and exhibiting feedback repression activity
on cytokine signaling (like the Socs genes; ref 30
). In
addition, we have characterized the expression of genes encoding the
subunits of the LIF receptor (LIFrß and gp130). Total RNAs were
prepared from ES cells grown for 20 h after LIF withdrawal (-
LIF), those grown for 20 h after LIF withdrawal and reinduced for
30 min with LIF (+ reinduction), or those maintained continuously in
the presence of LIF (+ LIF). Transcripts corresponding to a ribosomal
phosphoprotein (36B4) were used as an invariant internal control
(34)
. As shown in Fig. 3
, a clear induction of the c-fos, c-jun, junB, and Socs-3 transcripts
was observed after LIF reinduction. High levels of Socs-3 mRNA also
accumulated in ES cells continuously maintained in the presence of LIF.
Under the same conditions, the mRNA levels of JunD, Socs-1, Socs-2,
ESP, and the two major components of the LIF receptor (gp130 and
LIFrß) remained constant.
|
Overexpression of Socs-1 and Socs-3 leads to repression of
LIF-dependent transcription and cell lethality
Overexpression of Socs proteins in different cell systems revealed
their critical effect in the control of cytokine-dependent
transcription (23
, 47)
. Using a LIF-responsive
(SIE)3-TK CAT reporter, we showed earlier that
the STAT3 transcription factor is essential for LIF-dependent
transcription (17)
. In this study, ES cells were
cotransfected with this LIF-responsive
[(SIE)3-TK CAT] or with a mutated,
LIF-unresponsive [(SIEm)3-TK CAT ] reporter
gene, together with vectors allowing overexpression of HA-tagged Socs-1
(S1WT) and Socs-3 (S3WT) and of mutated forms of Socs-3 in which the
first 28 amino acids (S3N), the last 50 amino acids (encompassing
the Socs box) (S3C), or both the amino- and carboxyl-terminal parts
of the protein (S3NC) were deleted (Fig. 4A
). LIF-induced transcription from the
(SIE)3-TK CAT reporter was impaired when Socs-1
and Socs-3, but not Socs-2, were overexpressed (Fig. 4B
and
data not shown). Repression of transcription was nearly abolished with
S3N, S3C, and S3NC, indicating that the integrity of both the
Socs box and the first 28 amino acids of Socs-3 is required to
efficiently block LIF-dependent transcription. No repression of
transcription was detected with the (SIEm)3-TK
CAT, indicating that Socs proteins abolished LIF-dependent
transcription rather than basal transcription. Immunostaining of cells
with the anti-HA antibody 48 h after transfection revealed similar
levels of expression from each of these Socs constructs (data not
shown).
|
As Socs overexpression reduces LIF-dependent transcription, we tested
whether overexpression of Socs-1 and Socs-3 affected ES cell
pluripotency and/or growth properties. For this purpose, we attempted
to derive stable ES cell clones overproducing either wild-type (S1WT,
S3WT) or altered (S3NC) Socs proteins. ES cells were cotransfected
with the different Socs constructs, together with the PGK-neo selection
vector. After 2 wk in selective medium, numerous neomycin-resistant
clones were obtained with the control (V) and S3NC vectors. By
contrast, the number of surviving clones recovered with S1WT and S3WT
plasmids was reduced by 80 to 90% (Table 1
). These results clearly indicate that an excess of Socs-1 and of Socs-3
leads to cell lethality, most likely by blocking proper LIF signaling
in ES cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 41)
. Caspase-independent programmed cell death
has also been described (49
, 50)
.
We present evidence indicating that ES cells deprived of LIF enter an
apoptotic crisis, a phase that precedes cell differentiation. A similar
effect of LIF withdrawal has been reported in primordial germ cells and
cardiac myocytes in which LIF is also essential for cell proliferation
(51
, 52)
. It is possible that in ES cells apoptotic
signals are necessary to trigger ES cell differentiation by an as yet
unknown mechanism. The threshold level of these signals might commit
some of the cells toward irreversible apoptosis. Alternatively, one may
imagine that some cells enter apoptosis and the remainder
differentiate, depending on their stage in the cell cycle. The PARP, a
survival factor essential in DNA-damaged cells, is specifically cleaved
by caspase-3 during apoptosis, whereas inhibition of PARP cleavage
delays apoptosis (41
, 53
, 54)
. The fact that PARP is not
cleaved in ES cells after LIF withdrawal may retard apoptosis, allowing
the differentiation program to take place.
Several observations suggest that STAT3 might prevent apoptosis
in ES cells deprived of LIF: 1) the STAT3 transcription
factor is rapidly inactivated after LIF withdrawal (17)
;
2) it prevents apoptosis in a proB-cell line and in T cells
(55
, 56)
; and 3) its constitutive activation
blocks apoptotic processes in myeloma cells (57)
.
Abrogating STAT3 function by different strategies induces morphological
differentiation of ES cells despite a continuous supply of LIF
(17
, 20
, 58
, 59)
; it will be of interest to determine
whether, as in the case of LIF withdrawal, part of the cells die by
apoptosis during this differentiation process.
We have shown that ERK1/ERK2 and JNK1 are activated on LIF treatment
(17
and unpublished results). By contrast, p38 is
activated after LIF withdrawal during the apoptotic crisis. Activation
of p38 also occurs during neuronal apoptosis, after NGF withdrawal, and
insulin-dependent cell survival has been shown to be linked to p38
repression (14
, 15)
. Although the physiological
significance of p38 activation remains to be established in our cell
system, the possibility exists that it may be necessary for apoptosis
and/or differentiation to take place.
LIF-dependent transcription of early response genes
Among the early response genes known to be activated
transiently by cytokine treatment are the c-fos and JunB genes, which
contain STAT binding sites in their promoters and whose expression
correlates with LIF-dependent STAT3 activation in ES cells (17
, 60)
. Expression of these genes as well as c-jun is essential in
LIF-responsive tissues (e.g., bones, liver; see ref 61
, 62
). Therefore, it is possible that expression of these genes in
ES cells might prefigure their LIF dependency in organs in which LIF is
a proliferative factor.
LIF triggers opposite effects, depending on the nature of the
target cells: it is rather proliferative in stem cell pools and induces
differentiation in more committed progenitor cells (63)
.
It is clear from previous studies with the myeloid M1 cell line and
from our results in ES cells that induction of early LIF-responsive
genes differs in cell systems in which LIF has opposite effects.
Indeed, c-jun, junB, and c-fos are stimulated by LIF in both cell types
whereas jun D expression, strongly induced during LIF-dependent
differentiation of M1 cells (46)
, remains constant in ES
cells (this work). Large-scale analysis of LIF-induced genes in
different cell systems should lead to the identification of genes
activated in differentiated compared to proliferative LIF-dependent
pathways. This approach should help to characterize genes involved in
the maintenance of cell totipotency.
Socs-1 and Socs-3 proteins modulate LIF signaling in ES cells
Socs-3 is strongly induced by short-term LIF treatment and highly
expressed in ES cells continuously maintained in the presence of LIF.
STAT3 binding sites, necessary for LIF-mediated activation of
transcription, have been characterized in socs-3 promoter
(64)
. In contrast, socs-1 gene expression is not induced
by LIF, although its promoter contains STAT binding sites
(44)
. This indicates that STAT3 is probably not involved
in socs-1 gene regulation in our cell system. The expression of socs-1
and socs-3 genes in ES cells differs with that reported in M1 cells in
which Socs-1 is induced by LIF but not socs3 (27)
.
However, forced expression of either socs-1 or socs-3 modulates
LIF-dependent processes; it 1) represses STAT3-dependent
transcription, 2) decreases ES cell growth, and
3) prevents M1 cell differentiation (27
; this
work). Socs-1 protein differs from Socs-3 in its amino-terminal
proline-rich region, which may exhibit specific LIF-dependent SH3
domain recognition functions (28
, 48)
.
The molecular mechanism of repression of LIF-dependent genes by the
Socs-1 and Socs-3 proteins is presently unknown. The Socs box has
previously been shown to interact with elongins B and C of the RNA
polymerase II elongation complex (24
, 25)
, raising the
possibility that Socs proteins may act by modulating transcription
elongation. It is striking that in ES cells, both the amino-terminal
part and the carboxyl-terminal Socs box domains, of the Socs-3 protein
are required to mediate the repressive activity, since like the double
mutation, deletion of each domain separately abolishes the repressive
effect. This observation suggests that the extremities of these
molecules are functionally linked, with the Socs box interacting with
part of the RNA polymerase II elongation machinery and the
amino-terminal portion perhaps contributing to the specific targeting
of LIF-responsive genes. The linker region located between the SH2
domain and the Socs box has recently been shown to be critical for the
repressive effect of Socs-3 on growth hormone signaling
(65)
. The possibility therefore exists that at least part
of this linker domain may also contribute to the negative effect of
Socs-3 on LIF-dependent transcription.
As shown in the present study, overexpression of Socs-1 or Socs-3 in ES
cells increases cell death. On the other hand, abolition of socs gene
expression, as in socs-1 null mice, has been reported to generate
apoptosis in liver and lymphoid organs (66
, 67)
. These
apparently divergent results might be explained by the disruption of a
finely tuned balance between the levels of early response gene products
and feedback regulators of LIF, whose relative concentrations must be
critical for cell survival and for the maintenance of cell
pluripotency.
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ACKNOWLEDGMENTS |
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Received for publication September 2, 1999.
Revision received January 3, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 C. Ambrosino, G. Mace, S. Galban, C. Fritsch, K. Vintersten, E. Black, M. Gorospe, and A. R. Nebreda Negative Feedback Regulation of MKK6 mRNA Stability by p38{alpha} Mitogen-Activated Protein Kinase Mol. Cell. Biol., January 1, 2003; 23(1): 370 - 381. [Abstract] [Full Text]
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 E. E. Spangenburg and F. W. Booth Multiple signaling pathways mediate LIF-induced skeletal muscle satellite cell proliferation Am J Physiol Cell Physiol, July 1, 2002; 283(1): C204 - C211. [Abstract] [Full Text] [PDF]
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H. Boeuf, K. Merienne, S. Jacquot, D. Duval, M. Zeniou, C. Hauss, B. Reinhardt, Y. Huss-Garcia, A. Dierich, D. A. Frank, et al. The Ribosomal S6 Kinases, cAMP-responsive Element-binding, and STAT3 Proteins Are Regulated by Different Leukemia Inhibitory Factor Signaling Pathways in Mouse Embryonic Stem Cells J. Biol. Chem., November 30, 2001; 276(49): 46204 - 46211. [Abstract] [Full Text] [PDF]
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H Paradis and R. Gendron LIF transduces contradictory signals on capillary outgrowth through induction of stat3 and (P41/43)MAP kinase J. Cell Sci., January 12, 2000; 113(23): 4331 - 4339. [Abstract] [PDF]
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