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• W2138829341 abstract "Mouse embryonic stem (ES) cells remain “pluripotent” in vitro in the continuous presence of leukemia 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 (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although 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, whereas 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. Mouse embryonic stem (ES) cells remain “pluripotent” in vitro in the continuous presence of leukemia 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 (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although 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, whereas 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. leukemia inhibitory factor epidermal growth factor extracellular signal regulated kinase c-Jun N-terminal kinase mitogen-activated protein kinase mitogen-activated protein kinase-activated protein mitogen stress kinase protein kinase C ribosomal S6 kinase MAP/ERK kinase cAMP-responsive element-binding protein signal transducer and activator of transcription embryonic stem fetal calf serum c-Sis-induced element wild type Leukemia inhibitory factor (LIF)1 is a pleiotropic cytokine that belongs to the interleukin 6 cytokine family including ciliary neurotrophic factor, oncostatin M, and cardiotrophin-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 (1Hilton D.J. Trends Biochem. Sci. 1992; 17: 72-76Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 2Shellard J. Perreau J. Brulet P. Eur. Cytokine Netw. 1996; 7: 699-712PubMed Google Scholar, 3Taupin J.L. Pitard V. Dechanet J. Miossec V. Gualde N. Moreau J.F. Int. Rev. Immunol. 1998; 16: 397-426Crossref PubMed Scopus (62) Google Scholar). LIF also influences the survival, differentiation, and response to injury of neuronal cell lineages and synergizes with ciliary neurotrophic factor for motoneuron cell survival (4Aloisi F. Rosa S. Testa U. Bonsi P. Russo G. Peschle C. Levi G. J. Immunol. 1994; 152: 5022-5031PubMed Google Scholar, 5Sendtner M. Gotz R. Holtmann B. Escary J.L. Masu Y. Carroll P. Wolf E. Brem G. Brulet P. Thoenen H. Curr. Biol. 1996; 6: 686-694Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 6Cheng J.G. Patterson P.H. Mol. Cell. Neurosci. 1997; 9: 372-380Crossref PubMed Scopus (27) Google Scholar, 7Gadient R.A. Lein P. Higgins D. Patterson P.H. Brain Res. 1998; 798: 140-146Crossref PubMed Scopus (32) Google Scholar). 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 (8Ernst M. Gearing D.P. Dunn A.R. EMBO J. 1994; 13: 1574-1584Crossref PubMed Scopus (139) Google Scholar, 9Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1258) Google Scholar, 10Hirano T. Nakajima K. Hibi M. Cytokine Growth. Factor. Rev. 1997; 8: 241-252Crossref PubMed Scopus (327) Google Scholar, 11Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1227) Google Scholar, 12Ernst M. Novak U. Nicholson S.E. Layton J.E. Dunn A.R. J. Biol. Chem. 1999; 274: 9729-9737Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). However the identity of LIF-induced proteins in different cell contexts has not been precisely characterized. 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 (13Brook F.A. Gardner R.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5709-5712Crossref PubMed Scopus (439) Google Scholar, 14Bradley A. Zheng B. Liu P. Int. J. Dev. Biol. 1998; 42: 943-950PubMed Google Scholar). Upon LIF withdrawal, ES cells differentiate heterogeneously into various cell types, and some of the cells die by apoptosis during the differentiation process (15Duval D. Reinhardt B. Kedinger C. Boeuf H. FASEB J. 2000; 14: 1577-1584Crossref PubMed Google Scholar). ES cells can also be induced to differentiate homogeneously into determined cell types (16Dinsmore J. Ratliff J. Deacon T. Pakzaban P. Jacoby D. Galpern W. Isacson O. Cell Transplant. 1996; 5: 131-143Crossref PubMed Scopus (166) Google Scholar, 17Li M. Pevny L. Lovell-Badge R. Smith A. Curr. Biol. 1998; 8: 971-974Abstract Full Text Full Text PDF PubMed Google Scholar, 18Aubert J. Dessolin S. Belmonte N. Li M. McKenzie F.R. Staccini L. Villageois P. Barhanin B. Vernallis A. Smith A.G. Ailhaud G. Dani C. J. Biol. Chem. 1999; 274: 24965-24972Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 19Meyer N. Jaconi M. Landopoulou A. Fort P. Puceat M. FEBS Lett. 2000; 478: 151-158Crossref PubMed Scopus (109) Google Scholar, 20Westmoreland J.J. Hancock C.R. Condie B.G. Biochem. Biophys. Res. Commun. 2001; 284: 674-680Crossref PubMed Scopus (54) Google Scholar). MAPKs are Ser/Thr kinases that are activated by several growth factors, cytokines, and stress signals. They are classified into three families (extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38s) involved in cell proliferation, differentiation, and apoptotic processes (21Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4213) Google Scholar, 22Cohen P. Trends Cell Biol. 1997; 7: 353-361Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 23Tibbles L.A. Woodgett J.R. Cell Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (547) Google Scholar). The direct targets of ERKs (ERK1, ERK2, and ERK5) include kinases such as the Ser/Thr kinases of the families of mitogen/stress kinases (MSKs) and ribosomal S6 kinases (RSKs) (24Deak M. Clifton A.D. Lucocq L.M. Alessi D.R. EMBO J. 1998; 17: 4426-4441Crossref PubMed Scopus (840) Google Scholar, 25Frodin M. Gammeltoft S. Mol. Cell. Endocrinol. 1999; 151: 65-77Crossref PubMed Scopus (611) Google Scholar, 26Nicol R.L. Frey N. Pearson G. Cobb M. Richardson J. Olson E.N. EMBO J. 2001; 20: 2757-2767Crossref PubMed Scopus (237) Google Scholar, 27Pearson G. English J.M. White M.A. Cobb M.H. J. Biol. Chem. 2001; 276: 7927-7931Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The RSK (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 (25Frodin M. Gammeltoft S. Mol. Cell. Endocrinol. 1999; 151: 65-77Crossref PubMed Scopus (611) Google Scholar, 28Jacquot S. Merienne K. Trivier E. Zeniou M. Pannetier S. Hanauer A. Am. J. Med. Genet. 1999; 85: 214-215Crossref PubMed Scopus (12) Google Scholar, 29Merienne K. Jacquot S. Zeniou M. Pannetier S. Sassone-Corsi P. Hanauer A. Oncogene. 2000; 19: 4221-4229Crossref PubMed Scopus (38) Google Scholar). The cAMP-responsive element-binding (CREB) transcription factor is activated by phosphorylation on Ser-133 by different pathways such as the cAMP-dependent protein kinase A, the nerve growth factor (NGF)-dependent p38 and ERK pathways, and the epidermal growth factor (EGF)-dependent RSK2 and MSK1 pathways (30Foulkes N.S. Sassone-Corsi P. Biochim. Biophys. Acta. 1996; 1288: F101-F121PubMed Google Scholar, 31Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1079) Google Scholar, 32De Cesare D. Jacquot S. Hanauer A. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12202-12207Crossref PubMed Scopus (259) Google Scholar, 33Pierrat B. Correia J.S. Mary J.L. Tomas-Zuber M. Lesslauer W. J. Biol. Chem. 1998; 273: 29661-29671Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 34Arthur J.S. Cohen P. FEBS Lett. 2000; 482: 44-48Crossref PubMed Scopus (167) Google Scholar). 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 (35Akira S. Oncogene. 2000; 19: 2607-2611Crossref PubMed Scopus (301) Google Scholar). 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 (11Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1227) Google Scholar, 36Nakajima K. Yamanaka Y. Nakae K. Kojima H. Ichiba M. Kiuchi N. Kitaoka T. Fukada T. Hibi M. Hirano T. EMBO J. 1996; 15: 3651-3658Crossref PubMed Scopus (518) Google Scholar, 37Minami M. Inoue M. Wei S. Takeda K. Matsumoto M. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3963-3966Crossref PubMed Scopus (310) Google Scholar, 38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar, 39Raz R. Lee C.K. Cannizzaro L.A. d'Eustachio P. Levy D.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2846-2851Crossref PubMed Scopus (317) Google Scholar, 40Matsuda T. Nakamura T. Nakao K. Arai T. Katsuki M. Heike T. Yokota T. EMBO J. 1999; 18: 4261-4269Crossref PubMed Scopus (715) Google Scholar). Activation of STAT3 by phosphorylation on Tyr-705 and Ser-727 residues has been documented in cells treated with various growth factors and cytokines (41Boulton T.G. Zhong Z. Wen Z. Darnell J.E.J. Stahl N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6915-6919Crossref PubMed Scopus (192) Google Scholar, 42Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar, 43Hoey T. Grusby M.J. Adv. Immunol. 1999; 71: 145-162Crossref PubMed Google Scholar, 44Stephens J.M. Lumpkin S.J. Fishman J.B. J. Biol. Chem. 1998; 273: 31408-31416Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 45Gollob J.A. Schnipper C.P. Murphy E.A. Ritz J. Frank D.A. J. Immunol. 1999; 162: 4472-4481PubMed Google Scholar, 46Heim M.H. J. Recept. Signal Transduct. Res. 1999; 19: 75-120Crossref PubMed Scopus (232) Google Scholar). Phosphorylation on Tyr-705 is essential for STAT3 DNA binding activity, whereas phosphorylation on Ser-727, a residue within the transactivation domain, is rather involved in transactivation processes (42Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar, 47Zhang J.J. Zhao Y. Chait B.T. Lathem W.W. Ritzi M. Knippers R. Darnell Jr., J.E. EMBO J. 1998; 17: 6963-6971Crossref PubMed Scopus (188) Google Scholar). Several observations suggest that MAP kinases are involved in STAT3 phosphorylation: (i) Ser-727 lies within a consensus MAPK recognition sequence (48Ihle J.N. Bioessays. 1996; 18: 95-98Crossref PubMed Scopus (72) Google Scholar), and (ii) ERK2 is activated upon LIF treatment in ES cells (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar). In addition, activation of the Ras/ERK pathway diminishes the requirement of LIF in ES cells (49Ernst M. Oates A. Dunn A.R. J. Biol. Chem. 1996; 271: 30136-30143Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Phosphorylation of STAT3 on Ser-727 leads to positive and negative effects on STAT3 functions, depending on the cell type and the nature of the inducers (45Gollob J.A. Schnipper C.P. Murphy E.A. Ritz J. Frank D.A. J. Immunol. 1999; 162: 4472-4481PubMed Google Scholar, 50Chung J. Uchida E. Grammer T.C. Blenis J. Mol. Cell. Biol. 1997; 17: 6508-6516Crossref PubMed Scopus (546) Google Scholar, 51Sengupta T.K. Talbot E.S. Scherle P.A. Ivashkiv L.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11107-11112Crossref PubMed Scopus (201) Google Scholar, 52Woetmann A. Nielsen M. Christensen S.T. Brockdorff J. Kaltoft K. Engel A.M. Skov S. Brender C. Geisler C. Svejgaard A. Rygaard J. Leick V. Odum N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10620-10625Crossref PubMed Scopus (119) Google Scholar, 53Jain N. Zhang T. Kee W.H. Li W. Cao X. J. Biol. Chem. 1999; 274: 24392-24400Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). 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 20 h, and “irreversibly differentiation-committed,” 48 h without LIF. We have also characterized 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 under “Discussion.” 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. ES cells were derived from the inner cell mass of mouse blastocysts as described (54Hogan B.L. Beddington R. Costantini F. Lacy E. A Laboratory Manual. Second Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 255-272Google Scholar). 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-linkedrsk2 gene has been deleted by homologous recombination) were grown on feeder cells, in the presence of LIF (55Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (423) Google Scholar). In this RSK2− cell line, shortened mRNA corresponding to the in-frame skipping of exon 2 (in which the neomycine resistance gene and the stop codons were inserted) has been detected by reverse transcriptase-polymerase chain reaction analysis, indicating that the RSK2− cell line used in this study may express a 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 Ser-727-STAT3 (56Frank D.A. Mahajan S. Ritz J. J. Clin. Invest. 1997; 100: 3140-3148Crossref PubMed Scopus (216) Google Scholar), anti-phospho Tyr-705-STAT3 (QCB/BioSource International), anti-STAT3, ERK2 (C-14), ERK1 (C-16), and RSK2 (E-1) (Santa Cruz Biotechnology); the anti-phospho-JNK1/JNK2 and anti-phospho-p38 (Cell Signaling); the monoclonal anti-phospho-ERK1/ERK2 (Biolabs) and anti-JNK1 (PharMingen) antibodies; and the U0126 (Promega) and H7 (Biomol) compound were used as recommended by the manufacturers. The monoclonal anti-phospho Thr-577-RSK antibody has been described (29Merienne K. Jacquot S. Zeniou M. Pannetier S. Sassone-Corsi P. Hanauer A. Oncogene. 2000; 19: 4221-4229Crossref PubMed Scopus (38) Google Scholar). When indicated, quantification of the signals has been performed with the Bio-Rad GS700 imaging densitometer by using Molecular Analyst, version 2.1, software. ES cells were grown in medium without LIF (10 ml/10-cm diameter Petri dish of Dulbecco's modified Eagle's medium, 4,5 g/l glucose, 10% FCS, 862 mg/ml glutamax, 0.1 mm β2-mercaptoethanol) for various time periods extending from 6 to 72 h and reinduced with LIF (1000 units/ml) up to cell lysis, which was performed 72 h 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 (15Duval D. Reinhardt B. Kedinger C. Boeuf H. FASEB J. 2000; 14: 1577-1584Crossref PubMed Google Scholar). 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 20 h (differentiation-committed, reversible) or 48 h (differentiation-committed, irreversible) were re-fed with LIF-containing medium 15 min before harvesting. Cell lysates were prepared from cells grown under these different conditions. Neuronal differentiation of ES cells was mainly induced as described (16Dinsmore J. Ratliff J. Deacon T. Pakzaban P. Jacoby D. Galpern W. Isacson O. Cell Transplant. 1996; 5: 131-143Crossref PubMed Scopus (166) Google Scholar) with minor modifications. The S1 ES cell line was grown as embryoid bodies (300,000 cells/ml) on bacterial Petri dishes in Dulbecco's modified minimal essential medium (Life Technologies, Inc.) supplemented with 15% fetal calf serum (Hyclone), 0.1 mm β2 mercaptoethanol, 0.1% non-essential amino acids (Life Technologies, Inc.), 50 units/ml penicillin, and 50 μg/ml streptomycin (Life Technologies, Inc.) in the presence of 10−6mall-trans-retinoic acid (Sigma). After 4 days of retinoic acid treatment, the embryoid bodies were trypsinized, and the cells were resuspended in the same medium. 40,000 cells/ml were then plated in Dulbecco's modified Eagle's medium on coated culture dishes (for biochemical studies) or glass coverslips (for immunocytochemistry). Coating was performed successively with 0.1% gelatin, 10 μg/ml poly-d-lysine, and 1 μg/ml laminine. After 2 h, the culture medium was replaced by a defined medium (16Dinsmore J. Ratliff J. Deacon T. Pakzaban P. Jacoby D. Galpern W. Isacson O. Cell Transplant. 1996; 5: 131-143Crossref PubMed Scopus (166) Google Scholar). The cell medium was changed every other day. LIF induction (30 min with 250 or 500 units of LIF/ml) was performed on homogeneous neuronal cell populations 4 days after plating. 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 X-100 (10 min). After preincubation with 10% normal goat serum for 30 min, cells were treated with rabbit antiserum directed against Neurofilament 200 (Sigma) diluted 1:100 or mouse antiserum directed against MAP1 (Sigma) diluted 1:300. Peroxidase-conjugated anti-rabbit and anti-mouse antibodies were used as secondary antibody, respectively. ES cells grown without LIF for 20 h were pretreated for 1 h with 50 μm H7 or various concentrations of U0126, before reinduction with LIF in the presence of the inhibitors, at the same concentrations. Cytosolic and nuclear cell lysates were prepared as described (57Sadowski H.B. Gilman M.Z. Nature. 1993; 362: 79-83Crossref PubMed Scopus (234) Google Scholar). Whole cell lysates were a mixture of cytosolic and nuclear lysates at a 2:1 ratio. Band shift 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 (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar). Nuclear or whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose membranes in the presence of 0.07% SDS. Proteins were reacted with the different antibodies as recommended by the manufacturers. 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 24 h of LIF withdrawal, cells are committed to differentiate (58Scherer C.A. Chen J. Nachabeh A. Hopkins N. Ruley H.E. Cell Growth Differ. 1996; 7: 1393-1401PubMed Google Scholar, 59Ben-Shushan E. Thompson J.R. Gudas L.J. Bergman Y. Mol. Cell. Biol. 1998; 18: 1866-1878Crossref PubMed Scopus (215) Google Scholar, 60Lake J. Rathjen J. Remiszewski J. Rathjen P.D. J. Cell Sci. 2000; 113: 555-566Crossref PubMed Google Scholar). 2D. Duval, B. Reinhardt, C. Kedinger, and H. Boeuf, submitted for publication. In addition, ES cells grown without LIF for 48 h have lost their pluripotency, as suggested by their inability to colonize embryos (61Burdon T. Stracey C. Chambers I. Nichols J. Smith A. Dev. Biol. 1999; 210: 30-43Crossref PubMed Scopus (463) Google Scholar). We have developed 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 (15Duval D. Reinhardt B. Kedinger C. Boeuf H. FASEB J. 2000; 14: 1577-1584Crossref PubMed Google Scholar),2 we have set up an in vitro pluripotency test: cells were grown in the absence of LIF for different time periods (between 6 and 72 h) and then re-fed with LIF-containing medium up to 72 h before harvesting. Cells were scored for apoptosis by the appearance of a DNA ladder, a qualitative test that 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 with 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 some 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 (14Bradley A. Zheng B. Liu P. Int. J. Dev. Biol. 1998; 42: 943-950PubMed Google Scholar, 61Burdon T. Stracey C. Chambers I. Nichols J. Smith A. Dev. Biol. 1999; 210: 30-43Crossref PubMed Scopus (463) Google Scholar), we will refer throughout the document to pluripotent, reversibly differentiation-committed, or irreversibly differentiation-committed cells. STAT3 is critical for the maintenance of pluripotent ES cells as well as in various LIF-sensitive differentiated cells (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar,62Megeney L.A. Perry R.L.S. Lecouter J.E. Rudnicki M.A. Dev. Genet. 1996; 19: 139-145Crossref PubMed Scopus (80) Google Scholar, 63Bonni A. Sun Y. Nadal-Vicens M. Bhatt A. Frank D.A. Rozovsky I. Stahl N. Yancopoulos G.D. Greenberg M.E. Science. 1997; 278: 477-483Crossref PubMed Scopus (847) Google Scholar, 64Kunisada K. Tone E. Fujio Y. Matsui H. Yamauchi-Takihara K. Kishimoto T. Circulation. 1998; 98: 346-352Crossref PubMed Scopus (201) Google Scholar). Phosphorylation of STAT3 at the Tyr-705 residue is crucial for its activity in ES cells (11Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1227) Google Scholar, 38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar). It is also phosphorylated at the Ser-727 MAPK consensus site in response to various stimuli such as EGF or interleukin 6. We were interested to follow the STAT3 activation profile under experimental conditions as defined above. In these experiments, LIF was withdrawn for 20 or 48 h as a way to determine LIF-dependent STAT3 activation at a very early stage of differentiation commitment, whereas 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 that recognize the activated STAT3 proteins phosphorylated on the Tyr-705 (P-Y705-STAT3) or Ser-727 (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 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 20 h and Dif. com. at 48 h cell growth conditions). STAT3 phosphorylation is correlated with its specific DNA binding activity, as detected on the SIE probe in the Pluripotentand 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 (Ref. 38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholarand 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 (44Stephens J.M. Lumpkin S.J. Fishman J.B. J. Biol. Chem. 1998; 273: 31408-31416Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar)) did not exhibit 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. We have analyzed the activation profile of members of the three MAPK families (ERK, JNK, and p38) in ES cells. Western blot analysis of total cell lysates from ES cells maintained under various conditions was performed with antibodies against MAPKs that 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.3 A, the ERK1 and ERK2 proteins are induced by LIF in cells maintained in the presence of low or high serum concentrations, which is in good agreement with previous studies (38Boeuf H. Hauss C. Graeve F.D. Baran N. Kedinger C. J. Cell Biol. 1997; 138: 1207-1217Crossref PubMed Scopus (179) Google Scholar, 49Ernst M. Oates A. Dunn A.R. J. Biol. Chem. 1996; 271: 30136-30143Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). However, MAPKs (as well as their targets, such as the RSK proteins) are serum-induced proteins (25Frodin M. Gammeltoft S. Mol. Cell. Endocrinol. 1999; 151: 65-77Crossref PubMed Scopus (611) Google Scholar). Therefore, it was of interest to test the direct effect of serum and LIF independently because both are required for the proper growth of ES cells. 3H. Boeuf, D. Duval, B. Reinhardt, and C. Kedinger, unpublished observations. We were also interested in determining the sensitivity of the various LIF-dependent targets in the presence of the U0126 compound, a specific MAP/ERK kinase (MEK) inhibitor that acts downstream of MEK1 and MEK2, impairing phosphorylation of ERK1 and ERK2 at the specific TEY site (65Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2728) Google Scholar, 66Tolwinski N.S. Shapiro P.S. Goueli S. Ahn N.G. J. Biol. Chem. 1999; 274: 6168-6174Abstract Full Text Full Text PDF PubMed Scopu" @default.
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• W2138829341 date "2001-12-01" @default.
• W2138829341 modified "2023-10-17" @default.
• W2138829341 title "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" @default.
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• W2138829341 doi "https://doi.org/10.1074/jbc.m106718200" @default.
• W2138829341 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11581263" @default.

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