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First published online August 25, 2005
Stem Cells Vol. 24 No. 3 March 2006, pp. 604 -614
doi:10.1634/stemcells.2005-0323; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS

Absence of Suppressor of Cytokine Signalling 3 Reduces Self-Renewal and Promotes Differentiation in Murine Embryonic Stem Cells

Ariel Forraia, Kristy Boylea, Adam H. Harta, Lynne Hartleya, Steven Rakarb, Tracy A. Willsona, Ken M. Simpsona, Andrew W. Robertsa, Warren S. Alexandera, Anne K. Vossa, Lorraine Robba

a The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia;
b Zenyth Therapeutics Limited, Richmond, Australia

Key Words. SOCS-3 • Embryonic stem cells • Endoderm

Correspondence: Lorraine Robb, M.D., Ph.D., The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia. Telephone: 61-39-345-2527; Fax: 61-39-347-0852; e-mail: robb{at}wehi.edu.au

Received July 16, 2005; accepted for publication August 12, 2005.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-ß and gp130 and signals via the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3–null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3–null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK–STAT and extracellular signal–related kinase 1/2 (ERK-1/2)–mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain–containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3–null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK–STAT and SHP-2–ERK-1/2–MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Propagation of pluripotent murine embryonic stem (ES) cells is maintained by the cytokine leukemia inhibitory factor (LIF) [1, 2]. LIF induces dimerization of the LIFR-ß–gp130 receptor complex, activating multiple cytoplasmic signalling proteins [3]. The cytoplasmic Janus kinase (JAK)–signal transducer and activator of transcription 3 (STAT-3) pathway and extracellular signal–regulated kinase 1/2 (ERK-1/2)/mitogen-activated protein kinase (MAPK) pathway are two major signalling cascades activated by LIF. In the presence of LIF, JAKs phosphorylate tyrosines on the intracellular portion of gp130 and LIFR-ß, which then act as docking sites for STAT-1 and STAT-3 proteins that also become phosphorylated [4]. Dimeric phospho-STAT-3 enters the nucleus, binds to specific DNA-binding elements, and activates transcription. STAT-3 is a critical mediator of LIF-induced signalling pathways that regulate ES cell self-renewal. Mutation of STAT-3–interacting tyrosines on gp130, targeted deletion of Stat3, or overexpression of a dominant negative STAT-3 all abrogate the ability of LIF to maintain self-renewal of ES cells, and constitutive expression of activated STAT-3 prevents differentiation of ES cells after the withdrawal of LIF [5–7].

In ES cells, activation of the Ras–ERK-1/2–MAPK signalling pathway via gp130 is dependent on phosphorylation of the SH2 domain–containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2). Phosphorylation of a single tyrosine residue (Y757) in murine gp130 is necessary and sufficient for recruitment of SHP-2, leading to its tyrosine phosphorylation in a JAK-1–dependent manner [4, 8]. SHP-2 acts as an adaptor protein, associating with growth factor receptor–bound protein (GRB)-2 and thereby activating the Ras–ERK-1/2–MAPK pathway [9]. Unlike STAT-3 signalling, the SHP-2 signalling pathway does not promote ES cell self-renewal. ERK-1/2 activation has a differentiative effect on ES cells, and inhibition of the ERK-activating enzyme MAPK/ERK kinase (MEK) has been shown to enhance self-renewal of ES cells [10]. Mutation of the SHP-2–binding tyrosine on gp130 prolongs STAT-3 activation, diminishes ERK activation, and increases ES cell self-renewal [10]. ES cells homozygous for a SHP-2 mutation demonstrate LIF hypersensitivity and greater LIF-stimulated STAT-3 phosphorylation, greater self-renewal capacity, and less differentiation [11].

An increasing number of studies has shown that the suppressor of cytokine signalling 3 (SOCS-3) protein is a key regulator of signalling mediated via gp130. SOCS proteins contain a central SH2 domain and a carboxy-terminal SOCS box. Each of these domains is thought to have a separate function in regulating cytokine signalling. The SH2 domain interacts with phosphorylated tyrosine residues in tyrosine kinases and cytokine receptors and can negatively regulate their activity [12]. The current model suggests that SOCS-3 is recruited to the signalling complex by interaction with Y757 of gp130, leading to inhibition of JAK activity [13]. Y757 is also the binding site for SHP-2, although the binding affinity of SOCS-3 is higher than that of SHP-2 [12, 14]. The SOCS box forms part of the Elongin B/C-Cul5-SOCS-box (ECS) protein complex [15–17]. The ECS complex is a member of a family of ubiquitin ligases that share a Cullin-Rbx module. SOCS box–containing proteins recruit substrates to the ECS complex, targeting them for polyubiquitination and proteasomal degradation, and thereby controlling a variety of cellular processes. SOCS-3 binds specifically to Cul5–Rbx2 complexes in mammalian cells, but to date only insulin receptor substrate (IRS)-1 and IRS-2 have been shown to be substrates for degradation [17, 18].

Gene-targeting experiments have reinforced the notion that SOCS-3 is a major physiological regulator of signalling via gp130. SOCS-3–null embryos die at midgestation as a result of placental failure, and this can be rescued by a reduction of signalling via LIFR-ß [19–21]. When the Socs3 gene was ablated in a cell type–specific manner, this resulted in prolonged STAT-3 activation and consequent changes in the functional outcome of gp130 signalling in response to interleukin 6 [22–24]. In ES cells stimulated with LIF, Socs3 is a major transcriptional target of STAT-3 [25]; however, little is known about the function of SOCS-3 in ES cells. Overexpression of SOCS-1 or SOCS-3 has been reported to block self-renewal and promote differentiation [25, 26]. Recently, SOCS-3 was identified as one of a number of genes upregulated in SHP-2–null ES cells stimulated with LIF, and overexpression of SOCS-3 in wild-type (WT) ES cells was shown to promote ES cell differentiation to hemangioblasts and primitive erythroid progenitors [27]. In order to understand the role of SOCS-3 in ES cells, we derived SOCS-3–null ES cell lines. In standard culture conditions, SOCS-3–null ES cell lines show less proliferation and greater differentiation to endoderm. In steady state, STAT-3 phosphorylation is greater in SOCS-3–null ES cells and, in contrast to WT cells, SHP-2 is phosphorylated. In response to LIF stimulation, STAT-3, SHP-2, and ERK-1/2 activation are prolonged in SOCS-3–null ES cells. Overall, we demonstrate that SOCS-3 plays a crucial role in regulating LIF signalling in ES cells and that its absence results in alterations in self-renewal and differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of SOCS-3–Null Mice and ES Cell Lines
The SOCS-3 gene-targeting vector has been described previously [20]. A second SOCS-3 targeting construct was created by replacing the neomycin resistance cassette with a hygromycin resistance cassette. Generation and verification of SOCS-3 heterozygous ES cell lines were as described elsewhere, except that the W9.5 ES cell line was used [20]. Two targeted clones were injected into C57BL/6 blastocysts and chimeric offspring were bred to C57BL/6 mice to derive mice heterozygous for the SOCS-3 mutation. To obtain SOCS-3–null ES cell lines, SOCS-3 heterozygous mice were intercrossed, and blastocysts were collected and cultured as described previously [28]. The genotype of the ES cell lines was determined by Southern blotting of BamHI-digested DNA with 5' and 3' genomic DNA probes.

Cell Culture
ES cells were maintained on a layer of irradiated primary mouse embryonic fibroblasts in ES medium—Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.5 g/l glucose, 3.4 g/l NaHCO3, 15% (vol/vol) batch-tested fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 0.1 mM 2-mercaptoethanol (Sigma), and 0.1 mM nonessential amino acids (Invitrogen, Carlsbad, CA, http://www.invitrogen.com)—as described elsewhere [29]. Feeder-independent lines were maintained on gelatin-coated tissue culture dishes. To quantitate proliferation, viable single cells were plated at 100 cells per well in gelatin-coated 24-well tissue culture plates in triplicate, and the number of cells per well was counted daily. To assess self-renewal, the number of colonies present at day 3–6 after plating was ascertained by staining plates with methylene blue and scoring all colonies, in a blinded fashion, as undifferentiated, partially differentiated, or fully differentiated. Undifferentiated colonies were tight, rounded colonies, composed purely of stem cells, partially differentiated colonies contained stem cells with, flattened, differentiated cells at the periphery of the colony, and differentiated colonies were composed entirely of differentiated cells. Embryoid bodies were formed by seeding a single-cell suspension of ES cells at 2000–5000 cells/ml in a differentiation medium as described elsewhere [30]. To test the effect of MEK inhibitors, ES cells were plated at 0.5 x 106 cells per well of a six-well tissue culture plate in standard ES cell culture medium and were supplemented with either 10 µM UO126 (Promega, Madison, WI, http://www.promega.com), 50 µM PD98059 (Promega), or 1 µl/ml dimethylsulfoxide (DMSO). The medium was changed every 2 days.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from feeder-independent ES cell lines using the RNeasy Mini RNA kit (Qiagen, Valencia, CA, http://www1.qiagen.com). DNaseI-treated samples were reverse transcribed (RT) using Superscript III (Invitrogen), the resultant cDNA preparations were standardized, and polymerase chain reaction (PCR) was performed as described elsewhere [31]. References for primers are as follows: Oct4, Nanog, Hprt: [32], Mixl1: [33], Sox1, Otx2: [34], Afp Gata1, T: [35], and Zfp42. Isl1, Dab2, Lamb1–1, Ttr, COUPTF1 COUPTF2, Gata4, Gata6, Tcf2, and HNF4a: [36]. Primers for Socs3 were: 5'-AGATTTCGCTTCGGGACTAGC-3'5'-CTGGGTCTTGACG-CTCAAGCT-3'.

Indirect Immunofluorescence
SOCS-3–null and WT ES cells were plated onto gelatinized potassium hydroxide (KOH)-treated glass coverslips at a density of 1 x 105 cells per coverslip. Cells were cultured for 48 hours, washed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature. Immunofluorescent staining was performed as previously described [37] using antibodies specific for Oct4 and GATA-4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). After application of the secondary antibody (Alexa Fluor 594 goat anti-mouse IgG, Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com), the cells were washed, counterstained with 4',6-diamidino-2-phenylindole (DAPI) mounted in DAKO (Glostrup, Denmark, http://www.dako.com) mounting media and viewed with a Zeiss Axioplan 2 (Carl Zeiss, Jena, Germany, http://www.zeiss.com) microscope. Images were captured with a Zeiss Axiocam (Carl Zeiss) and processed with Axiovision software (Carl Zeiss).

Microarray Analysis
Total RNA was extracted from two independent WT and SOCS-3–null ES cell lines grown on primary mouse embryonic fibro-blasts in standard culture conditions using Qiagen RNeasy Mini RNA purification columns. The RNA from each cell line was quantitated using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, http://www.agilent.com). cDNA was synthesized from 5 µg of RNA according to Affymetrix methodology, and biotin-labeled cRNA was synthesized using the Affymetrix Enzo BioArray High Yield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, http://www.affymetrix.com). Affymetrix mouse genome (MOE 430 2.0, Affymetrix) GeneChips were hybridized with 10 µg of biotin-labeled cRNA according to the manufacturer’s protocol. Expression levels were calculated using Robust Multichip Analysis (RMA) [38]. A moderated t-statistic was computed for each gene in the two-group comparison (SOCS-3 null vs. WT). This is of the form t* = M/SE*, where M is the log2 fold change and SE* is a smoothed estimate of the standard error, calculated according to the empirical Bayes procedure as described elsewhere [39]. We selected, as differentially expressed, those genes with an absolute value of t* = 5. This threshold was derived by examining a plot of the quantiles of t* against the quantiles of the t-distribution on the appropriate degrees of freedom, and looking for departures from linearity. All calculations were done in R, using the "affy" and "limma" packages. The microarray data contained in this manuscript have been submitted to the GEO database, available at http://ncbi.nlm.nih.gov/geo.

Chimera Generation, Induction of Teratomas, and Tumor Histology
Chimeric embryos were generated by injection of ES cells into blastocysts obtained from intercrosses of mice carrying the ROSA-26 lacZ gene trap [40]. Chimeras were collected at embryonic day 9 and analyzed for ES cell contribution, as previously described [41]. To generate teratomas, 4-week-old nude mice were injected s.c. in opposing flanks with 1 x 106 WT or SOCS-3–null ES cells in 100 µl of PBS, and tumors were harvested 4–6 weeks later. Tissue was fixed in Bouin’s fixative, dehydrated, embedded in paraffin, sectioned at 0.4 µm, and stained with hematoxylin and eosin. Eight WT and eight SOCS-3–null tumors from four ES cell lines were examined. Animal studies were approved by the Melbourne Health Research Directorate Animal Ethics Committee.

Immunoprecipitation and Western Blotting
For cytokine stimulation experiments, feeder-independent ES cell lines were grown to near confluence in standard ES cell medium and then washed three times in PBS and cultured in ES cell medium with 0.5% FCS and without LIF for 4 hours prior to restimulation with LIF (1000 units/ml). For immunoprecipitation, cells were lysed in KALB lysis buffer (1% Triton X-100(vol/vol), 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) supplemented with 1 mM Na3VO4, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride (PMSF), and complete protease inhibitor mixture (Roche Applied Science, Indianapolis, https://www.roche-applied-science.com). Total cell lysates were pre-cleared with 50% slurry protein A sepharose (PAS), incubated with 2 µg SH-PTP2 antibody (c-18, Santa Cruz), and immunoprecipitated with PAS. For Western blotting, near confluent cells were lysed in RIPA lysis buffer (1% Triton X-100(vol/vol), 0.1% SDS(w/vol), 1% sodium deoxycholate, 150 mM NaCl, 20 mM Tris-HCl pH 7.5, 0.01% sodium azide) supplemented as above. Lysates or immunoprecipitates were subjected to SDS-PAGE separation and immunoblotting using antibodies specific for SOCS-3 (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan, http://www.ibl-japan.co.jp), STAT-3 (Santa Cruz), STAT-1 (BD Signal Transduction, Franklin Lakes, NJ, http://www.bdbiosciences.com), and ERK-1/2 (Cell Signalling Technology, Beverly, MA, http://www.cellsignal.com) and antibodies specific for the phosphorylated forms of STAT-3, STAT-1, ERK-1, ERK-2, and SHP-2 (Cell Signalling). The densitometry analysis was performed on scanned Hyperfilm ECL autoradiographs using a Molecular Dynamics Densitometer and ImageQuant software (GE Healthcare Life Sciences, Piscataway, NJ, http://www.amersham.com).

Online Supplemental Material
Supplemental online Figure 1 shows the generation and validation of SOCS-3–null ES cell lines. Supplemental online Figure 2 shows alterations in global gene expression in SOCS-3–null ES cells, using an MA plot. Supplemental online Tables 1 and 2 list genes with fivefold or greater altered expression in SOCS-3–null ES cell lines.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of SOCS-3–Null ES Cell Lines
To explore the function of SOCS-3 in ES cells, we initially attempted to generate SOCS-3–null ES cell lines by serially inactivating each allele using targeting constructs with selection cassettes for neomycin resistance or hygromycin resistance [20, 42]. When electroporated into W9.5 ES cells [43], the frequency of ES cell clones with a single, correctly targeted Socs3 allele (Socs3+/–) was approximately one in nine for each construct. To generate doubly targeted clones, 2748 G418- and hygromycin-resistant ES cell clones, generated from three independent, karyotypically normal Socs3+/– cell lines, were screened by Southern blotting. ES cell lines bearing targeted deletions of both Socs3 alleles were not obtained (data not shown). This suggested that ES cells were unable to survive homozygous targeting of SOCS-3. The most rigorous step during gene targeting is the requirement for a single, targeted cell to survive electroporation and antibiotic selection to proliferate into an undifferentiated ES cell colony. To circumvent this, we derived SOCS-3–null ES cells de novo from blastocysts. Socs3+/– mice, derived from Socs3+/– W9.5 ES cells, were intercrossed, blastocysts were collected and cultured and, after hatching, the inner cell mass was picked and expanded to generate ES cell lines [28]. From 96 blastocysts, 55 ES cell lines were obtained, of which 12 were homozygous for targeted deletion of Socs3. RT-PCR and immunoblotting demonstrated that SOCS-3 RNA and protein were not detectable in the SOCS-3–null ES cell lines (supplemental online Fig. 1). In all experiments reported here, WT ES cell lines outgrown from blastocysts in the same experiment as the null lines were used as controls, and all results shown are representative of 3–6 WT and SOCS-3–null ES cell lines.

SOCS-3–Null ES Cell Lines Exhibit Reduced Proliferation and Self-Renewal
SOCS-3–null ES cell lines could be continuously passaged in culture and were recoverable after cryostorage. Initially, all lines were maintained on a feeder layer of primary mouse embryonic fibroblasts. To facilitate biochemical analysis, feeder-independent lines were established. During routine culture, the feeder-dependent and feeder-independent SOCS-3–null ES cell lines were noted to grow more slowly than the WT lines, but could be serially passaged for at least 3 months. In SOCS-3–null ES cell cultures, around 50% of the colonies were observed to be partially differentiated, with cells at the periphery of the individual colonies adopting a flattened, refractile morphology. (Fig. 1A–1HGo). To quantitate the proliferative defect in the null lines, viable single cells were plated at low density (100 cells per well in 24-well tissue culture plates) and the number of cells per well was counted daily. In comparison with WT ES cells, the SOCS-3–null cells demonstrated markedly lower proliferation (Fig. 1IGo). To assess self-renewal, the number of colonies present at day 3 after plating was ascertained and the colonies were scored as undifferentiated, partially differentiated, or fully differentiated. As shown in Figure 1JGo, clonogenicity of the null lines was markedly lower and, in addition, SOCS-3–null ES cells gave rise to a higher proportion of colonies containing differentiated cells. To assess LIF responsiveness in the null lines, cells were plated in different concentrations of LIF, and colony morphology was scored daily. At 10 units of LIF, both WT and SOCS-3–null ES cell cultures contained < 5% undifferentiated colonies. In 1000–5000 units of LIF, < 5% of WT ES cell colonies were differentiated but 50% of SOCS-3–null colonies were partially differentiated (Fig. 1KGo). Culture of the SOCS-3–null ES cells in high concentrations of LIF (up to 50,000 units) did not alter the percentage of partially differentiated colonies (not shown).


Figure 1
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  		Figure 1. SOCS-3–null ES cells exhibit altered morphology, proliferation, and clonogenicity. (A–H): Morphology of two independent wild-type (WT) and SOCS-3–null ES cell lines, grown in standard ES cell culture conditions (15% fetal calf serum and 1000 units/ml LIF), on (A–D) and after weaning off (E–H) PMEFs. Note the partially differentiated appearance of the colonies arising from the null lines. Differentiated cells present in SOCS-3–null ES cell lines cultured on or off PMEFs are indicated by arrows. ES indicates undifferentiated ES cell colonies. (I): Growth of WT and SOCS-3–null ES cell lines. At each time point, the mean and standard deviation for triplicate wells is shown. Similar results were obtained with four WT and four SOCS-3–null lines. (J): Number of colonies present at day 3 after plating 100 cells in standard ES cell culture conditions. Colonies were fixed, stained, and scored as undifferentiated, mixed, or differentiated. Means and standard deviations for triplicate wells are shown. (K): WT and SOCS-3–null colonies were enumerated as for (J) after 3 days of culture in standard ES cell medium with concentrations of LIF as shown. Abbreviations: SOCS-3, suppressor of cytokine signalling 3; ES, embryonic stem cell; PMEF, primary mouse embryonic fibroblasts; W and WT, wild type; N, SOCS-3 null; LIF, leukemia inhibitory factor. Results are shown for a single representative experiment. Scale bars = 25 µm.

 
SOCS-3–Null ES Cells Differentiate into Endoderm in the Presence of LIF
When murine ES cells are propagated in standard conditions with FCS and LIF, minimal differentiation is observed. In cultures of SOCS-3–null ES cell lines, however, we observed a greater proportion of partially differentiated ES cell colonies, together with dispersed, refractile cells with stellate morphology, reminiscent of primitive endoderm cells [44]. To establish the identity of the differentiated cell types we used RT-PCR to examine the expression of genes associated with the undifferentiated state or with differentiation into different cell lineages. In keeping with the observation that SOCS-3–null ES cells could be cultured continuously and formed colonies containing cells with typical ES cell morphology, the pluripotential cell markers Nanog, Oct4, and Zfp42 were expressed. Markers of mesoderm (Brachyury, Mixl1), neuroectoderm (Isl1, Sox1, Otx2), and trophectoderm (Hand1) were not detected in the SOCS-3–null ES cell lines. In contrast, there was a striking upregulation of endoderm-specific gene expression. Endodermal transcription factors Tcf2, Gata4, Gata6, Hnf4a, and COUPTF1 were upregulated. Markers of parietal endoderm, Lamb1–1 and Dab2, were expressed, but visceral endoderm markers, Afp and Ttr, were not, suggesting that the differentiated cells arising in the SOCS-3–null ES cell cultures were of the primitive endoderm type (Fig. 2AGo). Immunohistochemical analysis detected Oct4 protein in WT and SOCS-3–null ES cells (Fig. 2B–2DGo). GATA-4 protein was not detectable in WT ES cells, but in SOCS-3–null cultures was detectable in cells at the edge of partially differentiated colonies (Fig. 2E–2GGo). GATA-4, but not Oct4, was detectable in the dispersed cells present in the SOCS-3–null ES cell cultures, thereby establishing their identity as endoderm. (Fig. 2H–2JGo).


Figure 2
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  		Figure 2. In the absence of SOCS-3, ES cells undergo differentiation to primitive endoderm. (A): Reverse transcription-polymerase chain reaction analysis demonstrates that SOCS-3–null ES cells express typical stem cell markers and show increased expression of primitive endoderm markers. (B–G): Indirect immunofluorescence was utilized to detect Oct4 (red) and GATA4 (green) in wild-type and SOCS-3–null ES cell cultures. In merged images (H–J) and (B, E, H) Oct-4 but not GATA-4, is detectable in WT ES cell colonies. (C, F, I): In a partially differentiated SOCS-3–null ES cell colony, Oct-4 is readily detectable and GATA4 is seen in cells at the periphery of the colony. (D, G, J): Undifferentiated Oct-4 positive SOCS-3–null ES cell colony and dispersed, differentiated cells with typical endodermal morphology (arrows) in which GATA-4 protein is detected. Nuclei of all cells in (B–G) are stained with 4',6-diamidino-2-phenylindole. Abbreviations: SOCS3, suppressor of cytokine signalling 3; ES, embryonic stem cell; +ve, positive control (embryoid body or embryonic day 9 cDNA); –ve, negative (no cDNA) control; WT, wild-type. Scale bars = 50 µm.

 
To extend the analysis of transcriptional differences between SOCS-3–null and WT ES cells, gene expression in WT and SOCS-3–null ES cell lines grown in standard ES cell culture medium on a feeder layer was compared. The gene expression levels of 45,000 known and predicted gene transcripts present on the Affymetrix MOE430 GeneChip were assayed, and expression of a large number of genes was significantly different in the null lines (supplemental online Fig. 2). The 161 genes upregulated and 61 genes downregulated more than fivefold in the SOCS-3–null ES cell lines are listed in supplemental online Table 1 and Table 2. As indicated by the RT-PCR results, early endoderm-specific genes were highly upregulated in the SOCS-3–null ES cell lines, but genes characteristically expressed in embryonic definitive endoderm (e.g., Afp, Serpini1, Alb1, Ipf1) were not upregulated, further demonstrating that a major consequence of the absence of SOCS-3 is the induction of a proportion of ES cells to adopt a primitive endodermal fate (supplemental online Table 1). Of the 161 upregulated genes, 25% had previously been documented to be expressed in endoderm of the preimplantation embryo, or during differentiation of embryoid bodies or embryocarcinoma cell lines to form endoderm [45–48] (supplemental online Table 1). In keeping with the role of endoderm in the production of basement membrane, there was a prominent increase in the expression of genes encoding secretory/extracellular matrix (ECM) proteins, and enzymes associated with the post-translational modification of ECM molecules [48]. The expression of genes associated with trophectoderm was similar in WT and mutant cell lines, and genes marking the neuroectodermal and mesodermal lineages were not upregulated. Expression of genes associated with pluripotency, including Nanog, Oct4, Zpf42, and Foxd3, was not significantly different in the WT and SOCS-3–null ES cell lines. None of the other seven members of the SOCS gene family showed altered gene expression in the SOCS-3–null ES cells, indicating that functional compensation by other SOCS genes was unlikely to be contributing to the observed phenotype (supplemental online Table 2; data not shown).

SOCS-3–Null ES Cell Lines Can Differentiate into Cells Derived from All Three Germ Layers
To determine whether the absence of SOCS-3 affects ES cell differentiation potential, expression of cell lineage–specific marker genes was assessed in WT and SOCS-3–null ES cell cultures after LIF withdrawal. In addition, the lines were cultured at low density in differentiation medium to allow formation of embryoid bodies (Fig. 3AGo). Embryoid bodies formed at 5- to 10-fold lower frequencies from SOCS-3–null ES cell lines (data not shown). RNA was prepared from adherent cultures 5 days after LIF withdrawal and from embryoid bodies at day 6, and RT-PCR was performed. Differentiated WT and SOCS-3–null ES cell cultures and embryoid bodies expressed markers of mesoderm, ectoderm, and endoderm (Fig. 3BGo). To evaluate the differentiation potential of the SOCS-3–null ES cell lines in vivo, we injected ES cells s.c. into nude mice to induce the formation of teratomas. These benign tumors contain well differentiated tissues of ectodermal, mesodermal, and endodermal origin [49]. Differentiation profiles of the resulting teratomas were assessed by histological examination. Both WT and SOCS-3–null teratomas had a heterogeneous differentiation profile, with cells of ectodermal lineage (neuronal structures), mesodermal lineage (smooth muscle, cartilage), and endodermal lineage (goblet cells, respiratory epithelium) (Fig. 3C–3GGo). Both WT and SOCS-3–null ES cell–derived teratomas also contained trophoblast cells (Fig. 3HGo). When injected into genetically marked blastocysts, the SOCS-3–null ES cells contributed to all tissues of chimera embryos (data not shown). Together, the results indicate that the absence of SOCS-3 does not affect the capacity of ES cells to differentiate into tissues derived from all three germ layers.


Figure 3
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  		Figure 3. SOCS-3–null ES cells retain the capacity to differentiate into cells derived from all three germ layers. (A): WT and SOCS-3–null embryoid bodies. (B): Reverse transcription-polymerase chain reaction analysis showing gene expression in WT and SOCS-3–null ES cells maintained in LIF (+LIF) and after LIF withdrawal (–LIF) and in day 6 embryoid bodies (D6 EB). (C–H): SOCS-3–null ES cells were injected s.c. into nude mice, and the resulting teratomas were fixed, sectioned, and stained with hematoxylin and eosin. Tissues derived from neuroectoderm (C), mesoderm (E, F), and endoderm (G) were present. (C): Primitive neuroepithelial tubes. (D): Cartilage. (E): Smooth muscle. (F): Ciliated epithelium. (G): Gut epithelium. (H): SOCS-3–null embryonic stem cells and WT ES cells (not shown) also gave rise to trophoblast tissue. Abbreviations: SOCS-3, suppressor of cytokine signalling 3; WT, Wild-type; LIF, Leukemia inhibitory factor; SOCS-3–null ES cells maintained in LIF; D6 EB, in day 6 embryoid bodies; ne, neuroepithelium; c, cartilage; ce, ciliated epithelium; ge, gut epithelium; tgc, trophoblast giant cells. Magnification: x 200.

 
Greater STAT-1, STAT-3, and SHP-2 Activation in the Absence of SOCS-3
Western blots were performed using lysates prepared from feeder-independent WT and null cell lines cultured in ES cell medium with LIF (steady state) and from cultures that had been cytokine and serum starved for 4 hours prior to readdition of LIF (1000 units/ml) (Fig. 4Go). SOCS-3 was present in WT ES cells in steady state and was detected at 30 minutes after LIF stimulation. Steady-state levels of phosphorylated STAT-1 and STAT-3 were higher in the SOCS-3–null ES cell lines (Fig. 4Go). After starvation and LIF addition, STAT-3 phosphorylation in WT cells was maximal between 5 and 30 minutes, but by 1 hour had returned to the level seen during routine culture. In contrast, SOCS-3–null ES cells showed a blunted response to readdition of LIF, exhibiting sustained STAT-3 phosphorylation. After LIF stimulation, ongoing STAT-1 phosphorylation was observed in the SOCS-3–null ES cells.


Figure 4
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  		Figure 4. LIF signalling is deregulated SOCS-3–null ES cells. Feeder-independent cell lines were grown to near confluence over 48 hours in standard ES cell culture medium, after which they were washed and placed into embryonic stem cell medium containing 0.5% serum without LIF for 4 hours. After starvation, 1000 units/ml LIF was added, and cell lysates were prepared at the indicated times thereafter. Lysates were separated by SDS-PAGE, blotted, and probed with antibodies specific to SOCS-3 or to pSTAT-3, pSTAT-1, or pERK-1/2. Membranes were stripped and reprobed with antibodies to total STAT-3, STAT-1, and ERK-1/2. Total SH2 domain–containing SHP-2 was immunoprecipitated from cell lysates, separated by SDS-PAGE, and probed with antibodies specific to phosphorylated SHP-2 (pSHP-2) or total SHP2. +LIF indicates cells maintained in standard culture conditions throughout, –LIF indicates cells harvested after 4 hours of starvation without restimulation. Histograms show densitometric quantitation of mean and standard deviation of the signal from three Western blots. The phosphorylation level is reported as a percentage relative to total STAT-1, STAT-3, SHP-2, or ERK-1/2 protein. Abbreviations: LIF, Leukemia inhibitory factor; SOCS-3, suppressor of cytokine signalling 3; pSTAT, phosphorylated signal transducer and activator of transcription; pERK-1/2, phosphorylated forms of extracellular signal–related kinase 1/2; ERK; extracellular signal–regulated kinase; SHP-2, cytoplasmic SH2-containing protein tyrosine phosphatase 2; WT, wild type.

 
SHP-2 was immunoprecipitated from cellular extracts of WT and SOCS-3–null ES cells, and SHP-2 phosphorylation was analyzed by Western blotting. Strikingly, in steady-state culture conditions, SHP-2 phosphorylation was detectable in SOCS-3–null ES cells but not in WT cells. LIF stimulation of WT cells induced SHP-2 phosphorylation that was rapidly attenuated, whereas in SOCS-3–null ES cells, there was a sustained induction of SHP-2 phosphorylation. The absence of SOCS-3 in ES cells also resulted in greater steady-state MAPK activation. Phosphorylated ERK-1 and ERK-2 proteins were present in greater amounts in steady-state SOCS-3–null ES cells, and in LIF readdition experiments, phosphorylation of ERK-1/2 lasted longer than with WT cells (Fig. 4Go). In keeping with previous observations, ERK-2 was the predominant phosphorylated ERK detected in ES cells.

Addition of Ras–MAPK Inhibitors Rescues the SOCS-3–Null ES Cell Differentiation Phenotype
SHP-2–Ras–ERK-1/2–MAPK signalling provides a differentiative signal in ES cells. To establish if the upregulation of this pathway in SOCS-3–null ES cells was responsible for their phenotype, WT and null ES cell lines were treated with the MEK inhibitors PD98059 or U0126 [50, 51]. Feeder-independent WT and null ES cells were cultured in the presence of inhibitor or carrier (DMSO) and assessed for the presence of differentiating colonies. The SOCS-3–null ES cell differentiation phenotype was completely reversed by the addition of either inhibitor to the culture medium (Fig. 5A–5HGo). This was confirmed by RT-PCR analysis, which showed that treatment with MEK inhibitors reduced Gata4 expression in the SOCS-3–null lines to a level similar to that observed in WT cells (Fig. 5IGo). Immunoblotting of lysates from treated cells showed less ERK-1/2 phosphorylation in the SOCS-3–null ES cells after treatment with inhibitor (Fig. 5JGo). To assess whether MEK inhibition affected proliferation and clonogenicity, PD98059- or U0126-treated WT and SOCS-3–null cells were plated at low density; the number of cells per well was counted daily and the colony number and morphology on day 3 after plating was scored (Fig. 5K, 5LGo). Addition of U0126 or PD98059 (not shown) reduced proliferation and clonogenicity in the WT cell lines. This effect was observed even at the lowest concentration of either inhibitor that was sufficient to inhibit ERK-/2 phosphorylation (not shown). In contrast to the effect on the differentiation phenotype, the lower proliferative capacity and clonogenicity of the SOCS-3–null lines was not rescued by inhibition of Ras–ERK-1/2–MAPK signalling.


Figure 5
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  		Figure 5. Treatment of SOCS-3–null embryonic stem (ES) cells with mitogen-activated protein kinase/ERK kinase (MEK) inhibitors prevents differentiation into endoderm. (A–H): Feeder-independent wild-type (WT) and SOCS-3–null ES cell lines were trypsinized and replated in ES cell medium (–) with or without the MEK inhibitors PD98059 or U0126 or the carrier DMSO. (I): Reverse transcription-polymerase chain reaction analysis of gene expression in WT and SOCS-3–null lines after treatment with U0126 shows that Gata4 expression in treated SOCS-3–null lines, but not in controls, is reduced to near WT levels. (J): Cell lysates prepared from WT and SOCS-3–null ES cell lines prior to and after treatment with U0126 were separated by SDS-PAGE, blotted, and probed with antibodies specific to phosphorylated forms of ERK-1/2 (pERK-1/2). Membranes were stripped and reprobed with antibodies to total ERK-1/2. Note the difference in the amount of total lysate loaded in the WT and SOCS-3–null lanes. (K, L): WT and SOCS-3–null ES cells were treated with MEK inhibitors and then plated at low density. W, wild-type cells; N, SOCS-3–null ES cells. Cell (K) and colony (L) counts were performed daily to assess proliferation and response to leukemia inhibitory factor (LIF) withdrawal. Data shown in (L) are the mean triplicate wells from a representative experiment. Similar results were obtained with two WT and four SOCS-3–null ES cell lines. Abbreviations: SOCS-3, suppressor of cytokine signalling 3; DMSO, dimethylsulfoxide; W and WT, wild type; ERK, extracellular signal–regulated kinase; N, SOCS-3 null. Scale bars = 25 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
To ascertain the role of SOCS-3 in ES cells we generated SOCS-3–null ES cell lines. Null cell lines could not be derived by gene targeting but were successfully isolated from preimplantation blastocysts. Our inability to derive doubly targeted clones by gene targeting likely reflects the lower proliferation capacity and clonogenicity of SOCS-3–null ES cells. SOCS-3–null ES cell lines exhibited less self-renewal and a greater propensity to differentiate into endoderm. Undifferentiated SOCS-3–null ES cells expressed stem cell markers and retained the capacity to differentiate into tissues of ectodermal, mesodermal, and endodermal origin in vitro and in vivo. Strikingly, the differentiative phenotype could be rescued by inhibition of MAPK signalling.

The ablation of SOCS-3 resulted in greater intensity and duration of activation of both the JAK–STAT and the Ras–ERK-1/2–MAPK signalling cascades both in the steady state and in response to LIF signalling. The null cells exhibited higher STAT-3 activation in steady-state cultures and sustained activation of STAT-3 after starvation and LIF readdition. Unlike WT cells, STAT-1 was constitutively phosphorylated. Phosphorylated SHP-2 was not detectable in WT ES cells maintained in LIF and serum, but in the absence of SOCS-3, phosphorylated SHP-2 was readily detectable, presumably as a result of a lack of competition from SOCS-3 for binding to Y757 of gp130. After cytokine stimulation, greater and sustained SHP-2 activation was observed in the null cells, whereas in WT cells, SHP-2 phosphorylation waned as SOCS-3 was upregulated. Downstream of SHP-2, ERK-1/2 phosphorylation was greater in the null cells. In other systems, SOCS-3 has been shown to positively regulate MAPK activation. In human T cells, SOCS-3 is tyrosine phosphorylated in response to multiple stimuli and binds to and inactivates Ras/GTPase-activating protein (GAP), leading to Ras–ERK-1/2–MAPK activation [52]. It is not known whether a similar mechanism operates in murine ES cells.

STAT and SHP-2–Ras–ERK signalling via gp130 are key regulators of cellular homeostasis. In vitro, the simultaneous activation of Ras–ERK–MAPK and STAT-1/3 has been repeatedly shown to generate opposing signals, the balance of which determines the biological outcome and, in vivo, balanced activation of the two signalling cascades is required to prevent disease [53–56]. In murine ES cells, opposing signals via the JAK–STAT and Ras–ERK–MAPK pathways contribute to the regulation of self-renewal and differentiation [57, 58]. STAT-3 is a critical mediator of LIF-induced signalling pathways that regulate ES cell self-renewal. Disruption of STAT-3 activation abrogates the ability of LIF to maintain self-renewal of ES cells while constitutive expression of activated STAT-3 prevents differentiation of ES cells after withdrawal of LIF [5–7]. Conversely, activation of Ras–ERK–MAPK signalling results in ES cell differentiation. Overexpression of a mutant Ras that stimulates the ERK-1/2–MAPK pathway activates expression of endoderm transcription factors in ES cells and embryoid bodies, and expression of dominant negative Ras suppresses expression of endoderm transcription factors [59, 60]. When Ras–ERK–MAPK signalling in ES cells is attenuated, the self-renewal response is enhanced [10]. Furthermore, genetic disruption of Grb2 or Shp2 in ES cells enhances self-renewal and impairs differentiation [11, 59]. Inhibition of ERK-1/2–MAPK signalling does not replace the requirement for STAT-3 signalling, but rather works synergistically with it.

In SOCS-3–null ES cells, constitutive upregulation of Ras–ERK-1/2–MAPK signalling provides a differentiative signal. This is opposed by augmented self-renewal signals via phosphorylated STAT-3. However, increased STAT-3 activation is not sufficient to prevent differentiation when Ras–ERK-1/2–MAPK signalling is activated. We hypothesize that this mixture of self-renewal and differentiative signals drives the phenotype observed in the SOCS-3–null ES cell cultures and that alterations in the thresholds of self-renewal and differentiative signals in individual cells result in the partial differentiation phenotype that can be reversed by pharmacological inhibition of MEK. During continuous subculturing, the undifferentiated cells in SOCS-3–null ES cell cultures would be expected to have a preferential replating advantage over the more differentiated cells, thus supporting their long-term dominance [61].

ES cell proliferation was markedly lower in the absence of SOCS-3 despite high levels of steady-state STAT-3 phosphorylation. The role of STAT-3 activation in ES cell proliferation is unclear. While it is well established that less STAT-3 activation leads to abrogation of ES cell self-renewal, it has not been shown to affect proliferation [5, 6], and greater STAT-3 activation may negatively affect proliferation [10]. The lower proliferation observed in the SOCS-3–null ES cell lines may be in part a result of dysregulated STAT-3 activation, but it is likely that alterations in other, as yet unidentified, signals also affect proliferation.

LIF is only able to sustain ES cells in the presence of serum, suggesting that additional factors are required. Recently, evidence has emerged that bone morphogenetic proteins (BMPs) may act in combination with LIF to sustain self-renewal of murine ES cells by inducing the expression of inhibitors of differentiation (Id) genes [62]. In addition, the Wnt signalling pathway appears to sustain ES cell pluripotency [63]. Intriguingly, the homeodomain protein Nanog has been shown to maintain ES cell self-renewal, independently of the LIF–STAT-3 and BMP-4 pathways, although a combination of LIF signalling and Nanog expression promotes maximal self-renewal efficiency [62, 64, 65]. The analysis of the SOCS-3–null ES cell phenotype reported here was conducted using ES cell media supplemented with FCS. In future experiments, it will be important to dissect the contribution of individual factors to the SOCS-3–null phenotype.

The in vivo relevance of the LIF signalling pathway for early embryo development is uncertain. During embryogenesis, the inner cell mass (ICM) exists transiently and does not act as a long-term stem cell compartment. It is not clear whether a population equivalent to the ES cell ever exists in vivo. Neither LIF, LIFR-ß, nor gp130 mutants show defects in the development of the ICM or early epiblast [66–69]. Similarly, SOCS-3–null blastocysts develop normally into the multilayered embryo [19, 20]. The LIF pathway is, however, required for survival of the ICM during implantation delay (diapause), and it will be interesting to ascertain whether this process is affected in SOCS-3–null blastocysts [70].

In summary, activation of cytokine signalling pathways in murine ES cells alters cell fate and potency. Results of SOCS-3 overexpression in ES cells, together with the data presented here on the effects of SOCS-3 ablation in ES cells, point to a key role for SOCS-3 in regulating signals emanating from the LIFR-ß–gp130 receptor complex in ES cells. In doing so, SOCS-3 regulates ES cell self-renewal and insulates ES cells from the functional consequence of lineage priming via the Ras–ERK–MAPK signalling pathway.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We thank Lucille Vollaire for karyotyping, Ruili Li for blastocyst injections, and Janelle Lochland for genotyping. We also thank Tim Thomas for genotyping the ES cell lines—derived de novo from SOCS-3 heterozygous intercrosses. We are grateful to Prof. Terry Speed and Dr. Gordon Smyth for discussion regarding analysis of the microarray data and Profs. Nicos Nicola and Doug Hilton for comments on the manuscript. The project was supported by the National Health and Medical Research Council of Australia program grant 257500, NIH grant CA22556 and Zenyth Therapeutics Limited. A.F. and K.B. contributed equally to this work.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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