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• W1974986171 abstract "Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process. Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process. Embryonic stem cell pluripotency underpins their potential utility as a source of differentiated progeny for use in regenerative medicine. Leukemia inhibitory factor (LIF) 2The abbreviations used are: LIF, leukemia inhibitory factor; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; ES, embryonic stem; mES, murine ES; hES, human ES; GSK, glycogen synthase kinase; PI3K, phosphoinositide 3-kinase; STAT, signal transducer and activator of transcription; Tet, tetracycline; 4OHT, 4-hydroxy-tamoxifen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BIO, 5-bromoindirubin-3-oxime; RT-PCR, reverse transcription PCR; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. plays an important role in maintaining self-renewal of murine ES (mES) cells (1Smith A.G. Heath J.K. Donaldson D.D. Wong G.G. Moreau J. Stahl M. Rogers D. Nature. 1988; 336: 688-690Crossref PubMed Scopus (1483) Google Scholar, 2Smith A.G. Hooper M.L. Dev. Biol. 1987; 121: 1-9Crossref PubMed Scopus (310) Google Scholar) via activation of STAT3 (3Boeuf H. Hauss C. DeGraeve F. Baran N. Kedinger C. J. 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Chem. 1996; 271: 30136-30143Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), ribosomal S 6 kinases (10Boeuf H. Merienne K. Jacquot S. Duval D. Zeniou M. Hauss C. Reinhardt B. Huss-Garcia Y. Dierich A. Frank D.A. Hanauer A. Kedinger C. J. Biol. Chem. 2001; 276: 46204-46211Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), phosphoinositide 3-kinases (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar), and Src kinases (12Anneren C. Cowan C.A. Melton D.A. J. Biol. Chem. 2004; 279: 31590-31598Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). However, whereas LIF-induced STAT3 activation promotes self-renewal, LIF-induced ERK activation appears to promote differentiation (8Burdon T. Stracey C. Chambers I. Nichols J. Smith A. Dev. Biol. 1999; 210: 30-43Crossref PubMed Scopus (474) Google Scholar), leading to the proposal that the balance between STAT3 and ERK signals contributes to the determination of mES cell fate (13Burdon T. Smith A. Savatier P. Trends Cell Biol. 2002; 12: 432-438Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar). Other extrinsic factors that also play a role in maintenance of mES cell self-renewal include bone morphogenic protein 4 (BMP4), which acts in synergy with LIF to maintain self-renewal via Smad-mediated induction of Id transcriptional repressor expression (14Ying Q.L. Nichols J. Chambers I. Smith A. Cell. 2003; 115: 281-292Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). A further report suggests BMP4 inhibition of p38 mitogen-activated protein kinase (MAPK) may also contribute to maintenance of self-renewal (15Qi X. Li T.-G. Hao J. Hu J. Simmons H. Miura S. Mishina Y. Zhoa G.-Q. Proc. Natl. Acad. Sci., U. S. A. 2004; 101: 6027-6032Crossref PubMed Scopus (348) Google Scholar). Wnt signaling has been implicated in regulation of pluripotency of both mES and human ES (hES) cells, work stemming largely from use of the GSK-3 inhibitor 5-bromoindirubin-3-oxime (BIO) (16Meijer L. Skaltsounis A.-L. Magiatis P. Polychronopoulus P. Knockaert M. Leost M. Ryan X.P. Vonica C.A. Brivanlou A.H. Dajani R. Crovace C. Tarricone C. Musacchio A. Rose S.M. Pearl L. Greengard P. Chem. Biol. 2003; 10: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 17Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1749) Google Scholar). A number of intrinsic regulators in the form of transcription factors have been identified that play important roles in regulation of pluripotency, among them Oct4, Sox2, and Nanog (18Boiani M. Schoeler H.R. Nat. Rev. Mol. Cell. Biol. 2005; 6: 872-881Crossref PubMed Scopus (567) Google Scholar). Interestingly, expression of the homeodomain protein Nanog alone can overcome the requirement of mES cells for LIF (19Mitsui K. Tokuzawa Y. Itoh H. Segawa K. Murakami M. Takahashi K. Maruyama M. Maeda M. Yamanaka S. Cell. 2003; 113: 631-642Abstract Full Text Full Text PDF PubMed Scopus (2604) Google Scholar, 20Chambers I. Colby D. Robertson M. Nichols J. Lee S. Tweedie S. Smith A. Cell. 2003; 113: 643-655Abstract Full Text Full Text PDF PubMed Scopus (2678) Google Scholar) and delays differentiation induced by retinoic acid. Insights into the transcriptional networks regulated by these transcription factors have recently been reported for both human and murine ES cells (21Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murrary H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Cell. 2005; 122: 1-10Abstract Full Text Full Text PDF PubMed Scopus (3546) Google Scholar, 22Loh Y.-H. Wu Q. Chew J.-L. Vega V.B. Zhang W. Chen X. Bourque G. George J. Leong B. Liu J. Wong K.-Y. Sung K.W. Lee C.W. Zhao X.-D. Chiu K.-P. Lipovich L. Kuznetsov V.A. Robson P. Stanton L.W. Wei C.-L. Ruan Y. Lim B. Ng H.-H. Nat. Genet. 2006; 38: 431-440Crossref PubMed Scopus (1945) Google Scholar). Significant overlap in the target genes for Oct4, Sox2, and Nanog have been revealed in hES cells (21Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murrary H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Cell. 2005; 122: 1-10Abstract Full Text Full Text PDF PubMed Scopus (3546) Google Scholar), whereas in mES cells Oct4 and Nanog bind the promoter regions of many of the same genes (22Loh Y.-H. Wu Q. Chew J.-L. Vega V.B. Zhang W. Chen X. Bourque G. George J. Leong B. Liu J. Wong K.-Y. Sung K.W. Lee C.W. Zhao X.-D. Chiu K.-P. Lipovich L. Kuznetsov V.A. Robson P. Stanton L.W. Wei C.-L. Ruan Y. Lim B. Ng H.-H. Nat. Genet. 2006; 38: 431-440Crossref PubMed Scopus (1945) Google Scholar). Further evidence suggests that Oct4-Sox2 complexes, at least in part, are involved in regulation of Nanog expression (23Rodda D.J. Chew J.-L. Lim L.-H. Loh Y.-H. Wang B. Ng H.-H. Robson P. J. Biol. Chem. 2005; 280: 24731-24737Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar), whereas p53 has been implicated in repression of Nanog expression (24Lin T. Chao C. Saito S. Mazur S.J. Murphy M.E. Appella E. Xu Y. Nat. Cell Biol. 2005; 7: 165-171Crossref PubMed Scopus (705) Google Scholar). Clearly, revealing the mechanisms that regulate Nanog expression will further enhance our understanding of the pluripotent state. Phosphoinositide 3-kinases are a family of lipid kinases whose products PI(3,4)P2 and PI(3,4,5)P3 act as intracellular second messengers (25Vanhaesebroeck B. Waterfield M.D. Expr. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (764) Google Scholar, 26Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1375) Google Scholar). PI3K-mediated signaling has been implicated in an array of physiological processes, notably proliferation, cell survival, cell migration, and trafficking (25Vanhaesebroeck B. Waterfield M.D. Expr. Cell Res. 1999; 253: 239-254Crossref PubMed Scopus (764) Google Scholar, 26Vanhaesebroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Crossref PubMed Scopus (1375) Google Scholar). As observed in many somatic cells, PI3Ks have been reported to control proliferation of mES cells (27Jirmanova L. Afanassieff M. Gobert-Gosse S. Markossian S. Savatier P. Oncogene. 2002; 21: 5515-5528Crossref PubMed Scopus (179) Google Scholar, 28Hallmann D. Trumper K. Trusheim H. Ueki K. Kahn R. Cantley L.C. Fruman D.A. Horsch D. J. Biol. Chem. 2003; 278: 5099-5108Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 29Takahashi K. Mitsui K. Yamanaka S. Nature. 2003; 423: 541-545Crossref PubMed Scopus (292) Google Scholar, 30Sun H. Lesche R. Li D.M. Liliental J. Zhang H. Gao J. Gavrilova N. Mueller B. Liu X. Wu H. Proc. Natl. Acad. Sci., U. S. A. 1999; 96: 6199-6204Crossref PubMed Scopus (693) Google Scholar), whereas we (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) and others (31Watanabe S. Umehara H. Murayama K. Okabe M. Kimura T. Nakano K. Oncogene. 2006; 25: 2697-2707Crossref PubMed Scopus (279) Google Scholar, 32Pritsker M. Ford N.R. Jenq H.T. Lemischka I.R. Proc. Natl. Acad. Sci., U. S. A. 2006; 103: 6946-6951Crossref PubMed Scopus (84) Google Scholar) have reported that PI3K-mediated signaling is important for maintenance of mES cell pluripotency and very recently that of hES cells (33Armstrong L. Hughes O. Young S.G. Hyslop L. Stewart R. Wappler I. Peters H. Walter T. Stojkovic P. Evans J. Stojkovic M. Lako M. Hum. Mol. Genet. 2006; 15: 1894-1913Crossref PubMed Scopus (321) Google Scholar). Here we have examined the mechanisms regulated by PI3Ks that contribute to maintenance of mES cell self-renewal. Using a combination of genetic and biochemical approaches, we have identified Nanog as a critical target whose expression is dependent, at least in part, on PI3K-mediated signals. Plasmid Constructs—A modified form of the Tet-off expression system incorporating chromatin insulator sequences (34Anastassiadis K. Kim J.H. Daigle N. Sprengel R. Schoeler H.R. Stewart A.F. Gene. 2002; 298: 159-172Crossref PubMed Scopus (37) Google Scholar) was used for expression of a dominant negative form of the p85 regulatory subunit of class IA PI3Ks (Δp85). Δp85 lacks the p110 interaction site, and we have previously described its use as a competitive inhibitor (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 35Craddock B.L. Orchiston E.A. Hinton H.J. Welham M.J. J. Biol. Chem. 1999; 274: 10633-10640Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The region containing the tTA-responsive promoter and encoding Δp85 was amplified from pUHD10-3neo-Δp85 (35Craddock B.L. Orchiston E.A. Hinton H.J. Welham M.J. J. Biol. Chem. 1999; 274: 10633-10640Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) (see supplemental data, Note 1) and ligated into Asc1 cut pINSHygro (34Anastassiadis K. Kim J.H. Daigle N. Sprengel R. Schoeler H.R. Stewart A.F. Gene. 2002; 298: 159-172Crossref PubMed Scopus (37) Google Scholar) to generate pINSHygroΔp85-off. Nanog-ERT2 vector was generated by a two-step procedure. First, a 5′-EcoRV/XhoI-3′ PCR fragment containing the entire coding sequence of mouse Nanog (see supplemental data, Note 2) was amplified from CGR8 RNA and cloned between the EcoRV and XhoI sites in pBSK-STAT3-ERT2 (36Vallier L. Mancip J. Markossian S. Lukaszewicz A. Dehay C. Metzger D. Chambon P. Samarut J. Savatier P. Proc. Natl. Acad. Sci., U. S. A. 2001; 98: 2467-2472Crossref PubMed Scopus (39) Google Scholar) to generate pBSK-Nanog-ERT2. 3P.-Y. Bourillot and V. Savatier, unpublished information. Second, a blunt BamHI fragment containing Nanog-ERT2 from pBSK-Nanog-ERT2 was subcloned into the blunted EcoRI site in the pPyCAGIZ bicistronic supertransfection vector (5Niwa H. Burdon T. Chambers I. Smith A. Genes Dev. 1998; 12: 2048-2060Crossref PubMed Scopus (1256) Google Scholar) (which contains an Ori sequence allowing extrachromosomal replication upon transfection into cells expressing polyoma large T antigen) (37Aubert J. Dunstan H. Chambers I. Smith A. Nat. Biotech. 2002; 20: 1208-1210Crossref PubMed Scopus (270) Google Scholar), generating pPyCAGIZ-Nanog-ERT2. Cell Culture and Generation of Transfectants—E14tg2a (2Smith A.G. Hooper M.L. Dev. Biol. 1987; 121: 1-9Crossref PubMed Scopus (310) Google Scholar) murine ES cell lines were cultured as previously described (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). E14tg2a expressing the Tet-sensitive transactivator tTA (E14 tTA) (38Era T. Witte O.N. Proc. Natl. Acad. Sci., U. S. A. 2000; 97: 1737-1742Crossref PubMed Scopus (111) Google Scholar) were a kind gift from Dr. O. Witte (University of California, Los Angeles) and electroporated as previously described (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) with pINSHygroΔp85-off. Independent clones were selected in 1000 units/ml LIF, 500 ng/ml Tet, and 200 μg/ml hygromycin and screened for expression of Δp85 following 24 h of incubation plus or minus Tet. Independent clones (termed E14Δp85) exhibiting very low to undetectable basal expression and good inducible expression of Δp85 (–Tet) were selected for further analyses. Δp85 expression was induced by washing cells three times with phosphate-buffered saline and incubating in LIF-containing medium in the absence of Tet. 500 ng/ml Tet was added back to control samples. To generate Nanog-ERT2 cells, ES cells expressing the polyoma large T antigen (E14/T cells) (37Aubert J. Dunstan H. Chambers I. Smith A. Nat. Biotech. 2002; 20: 1208-1210Crossref PubMed Scopus (270) Google Scholar) were electroporated (200 V, 960 μF) with 20 μgof pPCAGIZ-Nanog-ERT2. Cells were plated at 5 × 105 cells/10-cm dish and cultured in the presence of LIF (1000 units/ml) plus zeocin (1 μg/ml) for 8 days and resistant colonies pooled and expanded. Transient transfections were carried out using Lipofectamine 2000 (Invitrogen) at a final ratio of 1:500 with 1.6 μg of plasmid/well of ES cells in a 6-well tray. pcDNA3.1 was used as a control alongside pcDNA3.1 encoding either GSK-3β S9A or R96E mutants (kindly provided by T. Dale, Cardiff University, Wales). Transfection efficiency was monitored using a green fluorescent protein expression plasmid and flow cytometry and routinely found to be 35%. 16 h after transfection medium was replaced and cells incubated for a total period of 72 h. Self-renewal Assays—To determine the ability of ES cells to retain an undifferentiated phenotype, self-renewal assays were performed as previously described (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). In principle, this assay is based on alkaline phosphatase expression, which is high in undifferentiated ES cells and lost upon differentiation. Where required, following a 4–6-h period of cell adherence, cultures were supplemented with 5 μm LY294002, 2 μm BIO, 10 μm U0126, or Me2SO alone (all Calbiochem). The additional GSK-3 inhibitor, termed here TD114-2 (10,11,13,14,16,17,19,20,22,23-decahydro-9,4:24, 29-dimetheno-1H-dibenzo[n,t] pyrrolo [3,4-q] [1, 4, 7, 10, 13, 22]-tetraoxadiazacyclotetracosine-31,33(32H) dione) cacidition brneret was synthesized following the procedure of Kuo et al. (39Kuo G.-H. Prouty C. DeAngelis A. Shen L. O'Neill D.J. Shah C. Connolly P.J. Murray W.V. Conway B.R. Cheung P. Westover L. Xu J.Z. Look R.A. Demarest K.T. Emanuel S. Middleton S.A. Jolliffe L. Beavers M.P. Chen X. J. Med. Chem. 2003; 46: 4021-4031Crossref PubMed Scopus (92) Google Scholar) (with minor modifications) and was spectroscopically identical to compound 12 in their series. (For structures of BIO and TD114-2 see supplemental data, Note 3). Alkaline phosphatase-positive colonies, indicative of undifferentiated, self-renewing ES cell colonies and unstained, differentiated colonies, were counted in triplicate for each treatment following 3–5 days in culture. Preparation of RNA and Quantitative PCR—RNA was extracted using RNeasy kits (Qiagen) with on-column DNase digestion or TRIzol (Invitrogen) according to the manufacturer’s recommendations. Quantitative RT-PCR was carried out as follows. 1 μg of RNA was incubated at 65 °C for 5 min in the presence of 250 ng of oligo(dT)15, RNasin Plus RNase inhibitor (Promega), and 200 μm dNTPs and then placed on ice for 1 min. Reverse transcription was carried out with Omniscript (Qiagen) in the presence of 200 μm dNTPs at 40 °C for 1 h. This cDNA was then used as the template for quantitative PCR using LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science) according to the manufacturer’s instructions. The gene-specific primers used in this study are available (supplemental data, supplemental Table S1). Reactions were carried out in a total volume of 20 μl, comprising 0.4 μm of each primer, 3.5 mm MgCl2, 2 μl of SYBR Green master mix, and 2 μlof diluted cDNA. Each template was analyzed in duplicate within the same run. Amplification and on-line monitoring were performed using the LightCycler™ 1.5 system (Roche Applied Science). Following 40 amplification cycles, melt-curve analyses were performed to verify specific amplification. PCR efficiency of both the target and reference genes was calculated from the derived slopes of standard curves by LightCycler software (v4.0). These PCR efficiency values were used to calculate the relative quantification values for calibrator-normalized target gene expression by the LightCycler relative quantification software (v4.0). In all cases transcript levels were normalized to β-actin. Data were analyzed for statistical significance using two-tailed paired Student’s t-tests. Preparation of Cell Lysates and Immunoblotting—Cells were placed on ice and washed three times with phosphate-buffered saline prior to lysis as described previously (40Welham M.J. Duronio V. Leslie K.B. Bowtell D. Schrader J.W. J. Biol. Chem. 1994; 269: 21165-21176Abstract Full Text PDF PubMed Google Scholar). Insoluble material was removed by centrifugation for 3 min at full speed in a microcentrifuge at 4 °C. Protein concentrations of clarified supernatants were determined using the Bio-Rad protein assay kit according to the manufacturer’s instructions. 20 μg of each cell lysate was fractionated by SDS-PAGE and immunoblotted onto nitrocellulose (40Welham M.J. Duronio V. Leslie K.B. Bowtell D. Schrader J.W. J. Biol. Chem. 1994; 269: 21165-21176Abstract Full Text PDF PubMed Google Scholar). The following primary antibodies were used: 1:1000 for rabbit polyclonal antibodies recognizing phosphotyrosine 705 of STAT3 (anti-pSTAT3, CST 9131), phosphoserines 235/236 S 6 (pS 6, CST 2211), phosphoS33/S37/T41 β-catenin (anti-p-β-catenin, CST 9561), anti-β-catenin (CST 9562), anti-p85 (06–195; Upstate Biotechnology), anti-Nanog (ab21603; Abcam), and 1:4000 for goat polyclonal antibody recognizing GAPDH (sc-20357; Santa Cruz Biotechnology). Goat anti-rabbit or rabbit anti-goat secondary antibodies conjugated to horseradish peroxidase (Dako) were used at 1:20,000 dilution and blots developed using ECL (Amersham Biosciences). Blots were stripped and reprobed as described previously (41Welham M.J. Dechert U. Leslie K.B. Jirik F. Schrader J.W. J. Biol. Chem. 1994; 269: 23764-23768Abstract Full Text PDF PubMed Google Scholar). Band intensities were determined using a GeneSnap instrument and levels of target protein (Nanog or phosphorylated β-catenin) normalized to levels of GAPDH or β-catenin as appropriate for each sample. Phosphoinositide 3-Kinase-mediated Signaling Regulates Expression of Nanog—We have previously demonstrated a role for PI3K signaling in the efficient maintenance of self-renewal of mES cells (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) and were interested to investigate whether PI3K signaling influenced expression of the intrinsic regulator of pluripotency Nanog. When assessed by quantitative RT-PCR, inhibition of PI3K-dependent signaling with the PI3K inhibitor LY294002 at a dose of 5 μm, close to its IC50 value (42Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar, 43Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar), led to a significant decrease in Nanog RNA levels within 48 h; see Fig. 1A. Nanog protein levels were similarly reduced (Fig. 1B, (i) and (ii)) within 8 h of PI3K inhibition. In contrast, and consistent with our previous findings (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar), levels of Oct4 RNA and protein were not altered significantly (supplemental Fig. S1), indicating that the observed decrease in Nanog expression is not simply due to induction of differentiation upon treatment with LY294002. Assessment of S 6 ribosomal protein phosphorylation at serines 235 and 236 (Fig. 1B), as an indicator of PI3K inhibition, demonstrated that 5 μm LY294002 effectively blocked S 6 phosphorylation, whereas no effect on STAT3 Y705 phosphorylation was observed (supplemental Fig. S2). LY294002 has also been reported to inhibit mTORC1 (43Knight Z.A. Gonzalez B. Feldman M.E. Zunder E.R. Goldenberg D.D. Williams O. Loewith R. Stokoe D. Balla A. Toth B. Balla T. Weiss W.A. Williams R.L. Shokat K.M. Cell. 2006; 125: 733-747Abstract Full Text Full Text PDF PubMed Scopus (976) Google Scholar), and whereas previous data have implicated mTORC1 in regulation of ES cell proliferation (44Murakami M. Ichisaka T. Maeda M. Oshiro N. Hara K. Edenhofer F. Kiyama H. Yonezawa K. Yamanaka S. Mol. Cell. Biol. 2004; 24: 6710-6718Crossref PubMed Scopus (495) Google Scholar), it was important to examine whether direct inhibition of mTOR affected Nanog expression. ES cells were incubated with 5 μm LY294002 or 20 nm rapamycin and Nanog expression examined by immunoblotting (Fig. 1C). As demonstrated, rapamycin did not cause an alteration in Nanog expression after 24 h, although down-regulation of Nanog was observed after 48 h of rapamycin treatment. These results indicate that the effects of LY294002 on down-regulation of Nanog occur independently of mTORC1 inhibition during the initial phase of the response. The down-regulation of Nanog observed upon longer treatment with rapamycin is consistent with the view that multiple mechanisms are involved in regulation of Nanog expression. To investigate whether class IA PI3Ks are involved in regulation of Nanog expression, we expressed a dominant negative form of the p85 regulatory subunit of class IA PI3Ks (Δp85) (used previously in Refs. 11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 35Craddock B.L. Orchiston E.A. Hinton H.J. Welham M.J. J. Biol. Chem. 1999; 274: 10633-10640Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 45Craddock B.L. Hobbs J. Edmead C. Welham M.J. J. Biol. Chem. 2001; 276: 24274-24283Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), which lacks the p110 interaction domain and reduces activation of all class IA PI3Ks, using a modified Tet-off expression system. In this modified Tet-off system chromatin insulators flanked the Δp85 cDNA insert to reduce the potential for gene silencing. Quantitative RT-PCR analyses, shown in Fig. 2A, revealed that expression of Δp85 reduced levels of Nanog RNA, with similar results observed in three independent clones. Δp85 expression also reduced Nanog protein levels, shown in Fig. 2B, supporting a role for class IA PI3Ks in regulation of Nanog expression. Glycogen Synthase Kinase 3 Plays a Role Downstream of PI3Ks in Mediating Regulation of Nanog—PI3Ks regulate a number of physiological responses via a complex network of downstream effector molecules (46Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4681) Google Scholar). We have shown previously that inhibition of PI3Ks in mES cells enhances activation of ERKs and this plays a functional role in the loss of pluripotency observed (11Paling N.R.D. Wheadon H. Bone H.K. Welham M.J. J. Biol. Chem. 2004; 279: 48063-48070Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). Sato et al. (17Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1749) Google Scholar) have reported that inhibition of GSK-3 activity by BIO enhances self-renewal of mES and hES cells. Of relevance here is the fact that PI3K signaling also leads to inactivation of GSK3α/β, via protein kinase B-mediated phosphorylation of Ser-21/9. Therefore, we investigated whether inhibition of either ERK or GSK-3 signaling could overcome the effects of PI3K inhibition and restore Nanog expression. ES cells were cultured with LIF in the presence of either LY294002 alone or together with the MEK inhibitor U0126 or with either of two structurally distinct GSK-3 inhibitors BIO (17Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1749) Google Scholar) or TD114-2 (compound 12 in Ref. 39Kuo G.-H. Prouty C. DeAngelis A. Shen L. O'Neill D.J. Shah C. Connolly P.J. Murray W.V. Conway B.R. Cheung P. Westover L. Xu J.Z. Look R.A. Demarest K.T. Emanuel S. Middleton S.A. Jolliffe L. Beavers M.P. Chen X. J. Med. Chem. 2003; 46: 4021-4031Crossref PubMed Scopus (92) Google Scholar). Inhibition of MEK/ERK signaling was unable to reverse the decrease in Nanog expression observed when PI3Ks were inhibited; see Fig. 3A, (i). In contrast, inhibition of GSK-3 by either BIO or TD114-2 reversed the effects of LY294002 treatment, and Nanog expression remained at a level similar or higher" @default.
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• W1974986171 title "Regulation of Nanog Expression by Phosphoinositide 3-Kinase-dependent Signaling in Murine Embryonic Stem Cells" @default.
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