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• W2109950167 abstract "Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr −/− ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor γ2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPβ and C/EBPδ is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPβ and C/EBPδ genes via the mitogen-activated protein kinase cascade. Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived from lifr −/− ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor γ2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPβ and C/EBPδ is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPβ and C/EBPδ genes via the mitogen-activated protein kinase cascade. CAAT/enhancer binding proteins embryonic stem cells leukemia inhibitory factor mitogen-activated protein kinase peroxisome proliferator-activated receptor fetal calf serum mouse embryonic fibroblasts interleukin-6 soluble IL-6 receptor glycerol-phosphate dehydrogenase ciliary neurotrophic factor glycoprotein signal transducers and activators of transcription cardiotrophin The adipocyte differentiation program is controlled by the sequential expression of recently identified transcription factors. Members of the CCAAT/enhancer binding protein (C/EBP)1 and peroxisome proliferator-activated receptor (PPAR) families are trans-acting nuclear factors playing a regulatory role in the differentiation of preadipocytes into adipocytes (1Hwang C.S. Loftus T.M. Mandrup S. Lane D.M. Annu. Rev. Cell Dev. Biol. 1997; 13: 231-259Crossref PubMed Scopus (211) Google Scholar, 2Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). PPARγ2 is predominantly expressed in adipose tissue and plays a critical role in the adipocyte differentiation process (3Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar, 4Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar). Expression of C/EBPβ and C/EBPδ genes is not restricted to adipose tissue but is induced early and transiently during the program of adipocyte differentiation. Expression of C/EBPβ and C/EBPδ decreases following adipocyte maturation, whereas expression of C/EBPα and PPARγ2 gene is induced (5Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar). C/EBP-binding sites have been identified in the PPARγ2 promoter, and it has been recently shown that C/EBP proteins directly control transcription from the PPARγ2 promoter (6Clarke S.L. Robinson C.E. Gimble J.M. Biochem. Biophys. Res. Commun. 1997; 240: 99-103Crossref PubMed Scopus (193) Google Scholar). The adipogenic role of C/EBPβ and C/EBPδ has been previously demonstrated. Their ectopic expression in fibroblasts leads, in the presence of adipogenic hormones, to the adipocyte phenotype (7Yeh W.-C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar, 8Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (481) Google Scholar, 9Wu Z. Bucher N.L. Farmer S.R. Mol. Cell. Biol. 1996; 16: 4128-4136Crossref PubMed Google Scholar). Finally, the generation by homologous recombination of C/EBPβ−/−·δ−/− mice has clearly established the essential role of these two C/EBPs for the acquisition of adipocytes both in vitro and in vivo (10Tanaka T. Yoshida N. Kishimoto T. Akira S. EMBO J. 1997; 16: 7432-7443Crossref PubMed Scopus (645) Google Scholar). However, the extracellular factors and the intracellular signaling pathways involved in the regulation of C/EBPβ and C/EBPδ expression in preadipose cells are poorly defined. During the course of our investigation to identify extracellular factors that regulate early events in adipocyte differentiation, secretion of leukemia inhibitory factor (LIF) by preadipocytes was observed. LIF is known to induce differentiation of the murine myeloid leukemia cell line M1, to maintain pluripotent embryonic stem (ES) cells (11Smith 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, 12Williams R.L. Hilton D.J. Pease S. Willson T.A. Stewart C.L. Gearing D.P. Wagner E.F. Metcalf D. Nicola N.A. Gouch N.M. Nature. 1988; 336: 684-687Crossref PubMed Scopus (1543) Google Scholar), and to modulate stem cell and differentiated cell type functions in vitro and in vivo (13Hilton D.J. Trends Biochem. Sci. 1992; 17: 72-76Abstract Full Text PDF PubMed Scopus (213) Google Scholar). LIF and the related cytokines cardiotrophin (CT-1) and ciliary neurotrophic factor (CNTF) act through heterodimeric receptors comprised of LIF receptor and gp130 (14Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1306) Google Scholar). We show in the current study that exogenous LIF stimulates terminal differentiation of Ob1771 and 3T3-F442A preadipose cells and induces adipocyte differentiation of multipotent mouse embryonic fibroblasts (MEF). Moreover, by using an antagonist of LIF receptor or genetically modified ES cells, we show that LIF receptor plays a key role during adipocyte differentiation. These results are at variance with previous reports showing an anti-adipogenic effect of LIF in 3T3-L1 preadipocytes (15Mori M. Yamaguchi K. Abe K. Biochem. Biophys. Res. Commun. 1989; 160: 1085-1092Crossref PubMed Scopus (140) Google Scholar, 16Greenberg A.S. Nordan R.P. McIntosh J. Calvo J.C. Scow R.O. Jablons D. Cancer Res. 1992; 52: 4113-4116PubMed Google Scholar). The differential response to LIF of 3T3-L1 cells versus other cells is discussed. The expression of C/EBPβ and C/EBPδ genes was rapidly induced in Ob1771 and 3T3-F442A preadipose cells after LIF addition. The role of the mitogen-activated protein kinase (MAPK) pathway in early events induced by LIF has been investigated. The results show that p42/p44 MAP kinase pathway mediates both C/EBP gene expression and adipocyte differentiation induced by LIF. Ob1771 preadipose cells were induced to undergo differentiation in serum-free conditions as previously reported (17Gaillard D. Négrel R. Lagarde M. Ailhaud G. Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (163) Google Scholar). For differentiation of 3T3-F442A in serum-free conditions, confluent preadipose cells were exposed to Dulbecco's modified Eagle's medium and Ham's F-12 medium (1:1, v/v) containing bovine serum albumin (0.1 mg/ml), T3 (200 pm), transferrin (10 μg/ml), fetuin (50 μg/ml), epidermal growth factor (50 ng/ml), BRL49653 (10 nm) plus sodium ascorbate (100 mm), and sodium selenite (20 nm). Mouse embryonic fibroblasts (MEF) were harvested from 14-day post-coitus embryos and maintained for 10 passages in 10% heat-inactivated FCS. MEF were a gift of D. Grall and G. Pagès (Nice, France). For studying effect of LIF, MEF were plated at 2.5 × 103cells/cm2 and maintained in serum-free medium for proliferation. At confluence cells were maintained in serum-free medium (17Gaillard D. Négrel R. Lagarde M. Ailhaud G. Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (163) Google Scholar) supplemented with 20 nm BRL49653. Embryonic stem (ES) cells were maintained pluripotent as described previously (18Dani C. Smith A.G. Dessolin S. Leroy P. Staccini L. Villageois P. Darimont C. Ailhaud G. J. Cell Sci. 1997; 110: 1279-1285Crossref PubMed Google Scholar). ES cells deficient for LIF receptor were maintained pluripotent by addition of IL-6 together with soluble IL-6 receptor. For differentiation, wild-type and mutant ES cells were cultivated in aggregates termed embryoid bodies and were committed into the adipocyte lineage as recently described (18Dani C. Smith A.G. Dessolin S. Leroy P. Staccini L. Villageois P. Darimont C. Ailhaud G. J. Cell Sci. 1997; 110: 1279-1285Crossref PubMed Google Scholar). LacZ-marked ES cells were generated by introducing the lacZ gene into Oct-4 locus by homologous recombination using an internal ribosome entry site-containing construct (19Mountford P. Zevnik B. Düwell A. Nichols J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar, 20Dani C. Chambers I. Johnstone S. Robertson M. Ebrahimi-Chahardahcherik B. Saito M. Taga T. Li M. Burdon T. Nichols J. Smith A. Dev. Biol. 1998; 203: 149-162Crossref PubMed Scopus (105) Google Scholar). Northern blotting and ribonuclease protection assays (for expression of LIF and LIF receptor transcripts) were performed as described previously (21Dani C. Doglio A. Amri E.-Z. Bardon S. Fort P. Bertrand B. Grimaldi P. Ailhaud G. J. Biol. Chem. 1989; 264: 10119-10125Abstract Full Text PDF PubMed Google Scholar, 22Leroy P. Dessolin S. Villageois P. Moon B.C. Friedman J.M. Ailhaud G. Dani C. J. Biol. Chem. 1996; 271: 2365-2368Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Quantification of the hybridization signal was performed using a PhosphorImager apparatus (Fujix Bas 1000). Probes for LIF, ob, GAPDH, and LIF receptor have been described (22Leroy P. Dessolin S. Villageois P. Moon B.C. Friedman J.M. Ailhaud G. Dani C. J. Biol. Chem. 1996; 271: 2365-2368Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 23Chambers I. Cozens A. Broadbent J. Robertson M. Lee M. Li M. Smith A. Biochem. J. 1997; 328: 879-888Crossref PubMed Scopus (21) Google Scholar). The C/EBPβ and C/EBPδ cDNAs were provided by S. L. McKnight (Tularik Inc., South San Francisco). The PPARγ cDNA was isolated from PPARγ2/SPORT plasmid and the a-FABP cDNA from pAL422 plasmid (gift of P. Tontonoz and B.M. Spiegelman, Dana-Farber Cancer Institute, Boston). Confluent cells were maintained in 0.5% FCS for 12 h before adding LIF. Whole-cell lysates were prepared and electrophoresed on 12.5% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes and incubated with specific antiserum against C/EBPδ or C/EBPβ. Anti-C/EBPβ (C–19) and anti-C/EBPδ (C-22) antibodies were purchased from Santa Cruz Biotechnology. Antibodies were used at supplier's recommended concentrations. LIF was obtained from Life Technologies, Inc. IL-6 and soluble IL-6 receptor preparation has been described (24Yoshida K. Chambers I. Nichols J. Smith A.G. Saito M. Yasukawa K. Shoyab M. Taga T. Kishimoto T. Mech. Dev. 1994; 45: 163-171Crossref PubMed Scopus (187) Google Scholar). BRL49653 was a gift of SmithKline Beecham. PD98059 was purchased from New England Biolabs, and U0126 was purchased from Promega. The expression of LIF and LIF receptor genes was investigated during three defined stages of the program of 3T3-F442A adipocyte differentiation using a sensitive ribonuclease protection assay. Total RNAs were prepared from either exponentially growing cells corresponding to the adipoblast stage, from cells that reach confluence corresponding to the preadipose stage characterized at the molecular level by the early expression of specific genes such as A2COL6 (21Dani C. Doglio A. Amri E.-Z. Bardon S. Fort P. Bertrand B. Grimaldi P. Ailhaud G. J. Biol. Chem. 1989; 264: 10119-10125Abstract Full Text PDF PubMed Google Scholar), or from cells that express ob gene and accumulate triacylglycerol corresponding to the adipose stage of differentiation (22Leroy P. Dessolin S. Villageois P. Moon B.C. Friedman J.M. Ailhaud G. Dani C. J. Biol. Chem. 1996; 271: 2365-2368Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 25Maffei M. Fei H. Lee G.H. Dani C. Leroy P. Zhang Y. Proenca R. Negrel R. Ailhaud G. Friedman J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6957-6960Crossref PubMed Scopus (420) Google Scholar). As shown in Fig.1, levels of RNAs encoding the two forms of secreted LIF, i.e. the diffusible form (LIF-D) and the form associated to the extracellular matrix (LIF-M) (26Rathjen P.D. Toth S. Willis A. Heath J.K. Smith A.G. Cell. 1990; 62: 1105-1114Abstract Full Text PDF PubMed Scopus (223) Google Scholar), were higher in adipoblasts and preadipocytes than in mature adipocytes. In contrast, the LIF receptor was not detectably expressed in adipoblasts. Expression of LIF receptor was induced in preadipocytes in parallel to that of A2COL6 gene (not shown) and was maintained in mature adipocytes. Transcripts encoding the soluble form (sLIF-R) and the transmembrane form (tmLIF-R) of LIF receptor, which could be distinguished by RNase protection (23Chambers I. Cozens A. Broadbent J. Robertson M. Lee M. Li M. Smith A. Biochem. J. 1997; 328: 879-888Crossref PubMed Scopus (21) Google Scholar), showed a similar pattern of expression. The differential developmental expression of LIF and LIF receptor was found to be similar during adipocyte differentiation of the 3T3-F442A and Ob1771 clonal lines (not shown). Expression of LIF and LIF receptor genes in mouse adipose tissue was investigated. The stromal-vascular fraction, containing adipoblasts and preadipocytes, and the adipocyte fraction, containing the bulk of mature adipocytes, were isolated from epididymal fat pads after cell dissociation by collagenase treatment. Total RNAs from each fraction were then prepared as described previously (21Dani C. Doglio A. Amri E.-Z. Bardon S. Fort P. Bertrand B. Grimaldi P. Ailhaud G. J. Biol. Chem. 1989; 264: 10119-10125Abstract Full Text PDF PubMed Google Scholar). LIF RNA was detected only in the stromal-vascular fraction, whereas LIF receptor was expressed in both fractions. These data are consistent with in vitro data and suggest that LIF and LIF receptor RNAs are expressed simultaneously in preadipocytes only. The capacity of preadipocytes to secrete bioactive LIF was investigated. For that purpose we studied the capacity of Ob1771 preadipocytes to support pluripotent ES cells in coculture assays. It has been previously described that the maintenance of ES cells required the presence of LIF or LIF-related factors (24Yoshida K. Chambers I. Nichols J. Smith A.G. Saito M. Yasukawa K. Shoyab M. Taga T. Kishimoto T. Mech. Dev. 1994; 45: 163-171Crossref PubMed Scopus (187) Google Scholar), and we have recently described the generation of convenient LacZ-marked ES cells in which β-galactosidase expression is restricted to pluripotent ES cells (19Mountford P. Zevnik B. Düwell A. Nichols J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar, 20Dani C. Chambers I. Johnstone S. Robertson M. Ebrahimi-Chahardahcherik B. Saito M. Taga T. Li M. Burdon T. Nichols J. Smith A. Dev. Biol. 1998; 203: 149-162Crossref PubMed Scopus (105) Google Scholar). The indicator ES cells were plated on layers of confluent Ob1771 preadipocytes and maintained in either serum-supplemented or serum-free medium (17Gaillard D. Négrel R. Lagarde M. Ailhaud G. Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (163) Google Scholar). Five days later, the number of β-galactosidase-positive colonies was determined. As shown in Fig. 2 A, Ob1771 preadipose cells maintained in both culture conditions could support pluripotent ES cells. Addition of an antagonist of LIF receptor, hLIF05 (27Vernallis A.B. Hudson K.R. Heath J.K. J. Biol. Chem. 1997; 272: 26947-26952Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), completely blocked ES cell maintenance supported by preadipocytes. The antagonist of LIF receptor is a mutated form of human LIF that antagonizes the action of LIF-D and LIF-M as well as that of CNTF and CT-1 by blocking the LIF receptor (27Vernallis A.B. Hudson K.R. Heath J.K. J. Biol. Chem. 1997; 272: 26947-26952Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 28Neophytou C. Vernallis A.B. Smith A.G. Raff M.C. Development. 1997; 124: 2345-2354Crossref PubMed Google Scholar). Part of the capacity of preadipocytes to maintain ES cells was due to LIF-D as the activity of Ob1771 preadipocyte-conditioned medium was reduced by 60–80% in the presence of a specific anti-LIF blocking antibody (Fig. 2 B). It is interesting to note that the activity of the conditioned medium was not abolished suggesting that a soluble LIF-related cytokine is also secreted by preadipocytes. In control experiments, the anti-LIF antibody was efficient to abolish activity of 0.4 ng/ml LIF when added to basal medium (not shown). LIF and LIF-like factors were specifically expressed at the preadipose stage as no activity was detected in medium conditioned by mature adipocytes (not shown). This result is in agreement with Fig. 1 showing that expression of LIF-D is inhibited in fully differentiated cells. Taken together these results show that LIF and LIF receptor expression are differentially regulated during adipocyte differentiation, suggesting that this pathway plays a regulatory role in adipogenesis. This hypothesis was further investigated. The effect of LIF on differentiation of Ob1771 preadipose cells was investigated in a chemically defined medium (17Gaillard D. Négrel R. Lagarde M. Ailhaud G. Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (163) Google Scholar). Glycerol-phosphate dehydrogenase (GPDH) is a late marker of adipocyte differentiation that faithfully reflects cells containing triacylglycerol (17Gaillard D. Négrel R. Lagarde M. Ailhaud G. Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (163) Google Scholar). Chronic exposure to LIF elicited a 20-fold increase in the specific activity of GPDH (Fig. 3) and the appearance of adipocyte-like cells (Fig. 4). The adipogenic effect of LIF was not unique to Ob1771 cells as a positive effect of LIF on differentiation of 3T3-F442A preadipocytes was observed (Fig. 3). LIF also induced adipocyte differentiation of BRL49653-treated multipotent MEF, indicating that the adipogenic effect of LIF is not restricted to clonal preadipose cell lines (Fig. 3). The effect of LIF was then investigated as a function of exposure time. Exposure of Ob1771 cells to LIF from day 0 to 3 induced a 6-fold increase of GPDH activity, and exposure from day 0 to 7 was sufficient to achieve maximal response at day 11. In contrast, exposure after day 7 was ineffective (Fig. 3, lower panel). This observation suggests that LIF exerts its adipogenic effects by acting at an early step in the differentiation process, i.e. at the preadipose stage, which in turn dictates terminal events. Chronic exposure of Ob1771 cells to low concentration of the thiazolidinedione BRL49653 (20 nm), a PPARγ ligand (3Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar, 29Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3469) Google Scholar), promoted adipocyte differentiation to a low extent. However, LIF addition to the thiazolidinedione-treated preadipose cells led to a dramatic increase in adipocyte differentiation (Fig. 3, lower panel and Fig. 4). Thus, LIF and PPARγ appear to stimulate differentiation in a synergistic manner. Cross-talk between LIF signaling and PPARγ activation remains to be elucidated.Figure 4Photomicrographic record of Ob1771 cells. Cells were maintained for 11 days in the indicated conditions (magnification, × 100).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The LIF receptor antagonist hLIF05 was used to determine whether LIF receptor and LIF secreted by preadipocytes play a role during adipocyte differentiation. At the concentration of 0.5 μg/ml, hLIF05 had no toxic effect but inhibited adipocyte differentiation enhanced by 10 ng/ml LIF (not shown). As shown in Fig.5, when hLIF05 was added during BRL49653-induced differentiation of Ob1771 cells in serum-free medium (no exogenous addition of LIF), the level of GPDH activity was reduced to 56 ± 7%. Another approach was used to examine the requirement of LIF receptor during the development of adipose cells as we have recently determined culture conditions that favor the commitment of ES cells into the adipocyte lineage (18Dani C. Smith A.G. Dessolin S. Leroy P. Staccini L. Villageois P. Darimont C. Ailhaud G. J. Cell Sci. 1997; 110: 1279-1285Crossref PubMed Google Scholar). This model provides a valuable in vitro system for studying the role of genes active during adipocyte differentiation. The role of LIF and LIF receptor was addressed by investigating whether embryoid bodies derived from lif−/− ES cells (20Dani C. Chambers I. Johnstone S. Robertson M. Ebrahimi-Chahardahcherik B. Saito M. Taga T. Li M. Burdon T. Nichols J. Smith A. Dev. Biol. 1998; 203: 149-162Crossref PubMed Scopus (105) Google Scholar) or lifr−/− ES cells 2M. Li and A. G. Smith, submitted for publication. were able to undergo adipocyte differentiation. These mutant cells have been generated by gene targeting via two rounds of homologous recombination. LIF-null ES cells underwent adipogenesis with comparable efficiency to wild-type ES cells, as determined by the expression of adipocyte-specific genes. This result was in agreement with studies of LIF mutant mice, which indicated also that a lack of LIF expression did not prevent the development of adipose tissue (not shown). Likely, both in vitro and in vivo, LIF-related cytokines such as IL-6, CT-1, or CNTF could compensate for the lack of LIF. Therefore, the phenotype of LIF receptor-null ES cells was investigated. As previously reported (24Yoshida K. Chambers I. Nichols J. Smith A.G. Saito M. Yasukawa K. Shoyab M. Taga T. Kishimoto T. Mech. Dev. 1994; 45: 163-171Crossref PubMed Scopus (187) Google Scholar), propagation of pluripotent ES cells can be maintained by addition of gp130-activating cytokines. Therefore, pluripotent lifr−/− ES cells were maintained by addition of IL-6 together with soluble IL-6 receptor. These cells are responsive to IL-6/sIL-6R but are unresponsive to LIF, CT-1, or CNTF, which act through the LIF receptor-gp130 complex (20Dani C. Chambers I. Johnstone S. Robertson M. Ebrahimi-Chahardahcherik B. Saito M. Taga T. Li M. Burdon T. Nichols J. Smith A. Dev. Biol. 1998; 203: 149-162Crossref PubMed Scopus (105) Google Scholar). The capacity of LIF receptor-deficient ES cells to undergo adipocyte differentiation was dramatically reduced. Only 5–7% of outgrowths derived from two independent clones of LIF receptor-deficient ES cells contained adipocyte colonies compared with 55–70% of outgrowths derived from wild-type ES cells. The lifr+/− cells displayed an intermediate phenotype (Fig.6 A). The lower levels of expression of adipocyte-specific genes such as PPARγ and a-FABP in mutant cells compared with wild-type and heterozygotes were consistent with a suppression of terminal differentiation (Fig. 6 B). Cardiomyocytes beating cells were detectable in wild-type and mutant cultures (not shown) and transcripts for myogenin, a skeletal muscle-specific marker, as well as for α chain 2 of type VI collagen (A2COL6), a gene preferentially expressed by mesenchymal cells (30Ibrahimi A. Bertrand B. Bardon S. Amri E.-Z. Grimaldi P. Ailhaud G. Dani C. Biochem. J. 1993; 289: 141-147Crossref PubMed Scopus (34) Google Scholar, 31Marvulli D. Volpin D. Bressan G. Dev. Dyn. 1996; 206: 447-454Crossref PubMed Scopus (32) Google Scholar), were present at similar levels in the three cell types (Fig. 5 B). These observations indicate that the reduction of adipogenesis of LIF receptor-deficient ES cells did not reflect a general defect of these cells to undergo differentiation. Moreover, addition of IL-6 together with the soluble IL-6 receptor from day 7 to day 20 after embryoid formation, which corresponds to the permissive period for terminal differentiation (18Dani C. Smith A.G. Dessolin S. Leroy P. Staccini L. Villageois P. Darimont C. Ailhaud G. J. Cell Sci. 1997; 110: 1279-1285Crossref PubMed Google Scholar), restored the capacity of LIF receptor-null cells to undergo adipocyte differentiation and to express adipocyte-specific genes (Fig. 6 B, lane 4). These results indicate that LIF receptor plays a critical role in the process of terminal differentiation of preadipose cells into adipose cells derived from ES cells. Thus, detailed studies of LIF effects and the transducing machinery were undertaken.Figure 6Inability of LIF receptor-deficient ES cells to undergo adipocyte differentiation in vitro. A, embryoid bodies derived from wild-type and mutant ES cells were induced to undergo adipocyte differentiation. Twenty days after embryoid body formation the percentage of outgrowths showing adipocyte colonies was scored. Number of outgrowths (no), number of independent clones (ncl) checked, and number of experiments (ne) performed are, respectively, lifr+/+, no = 334, ncl = 3, ne = 4; lifr+/−, no = 254, ncl = 1, ne = 3; lifr−/−, no = 609, ncl = 2, ne = 3. B, total RNAs prepared from 20-old-day outgrowths were analyzed by Northern blotting for the expression of the indicated genes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The C/EBβ and C/EBPδ transcription factors are expressed at the preadipose stage of the differentiation program. Although these trans-acting factors play a critical role in adipocyte differentiation, the nature of the extracellular factors that regulate their early expression is poorly known. Therefore, the regulation of C/EBP gene expression by LIF was investigated in Ob1771 and 3T3-F442A preadipose cells. As shown in Fig. 7, a dramatic increase in C/EBPδ expression in response to LIF was observed. This induction was rapid and transient as C/EBPδ transcript accumulation peaked within 1 h and decreased to basal level within 24 h. Similar kinetics were observed for C/EBPβ gene expression, although the stimulation remained lower due to a higher basal level at time 0. The accumulation of C/EBPβ and C/EBPδ RNAs by LIF was followed by an increase in C/EBP proteins. Accumulation of C/EBP proteins was detectable after 1 h, extended until 8 h, and disappeared 15 h after LIF addition (Fig. 7, lower panel). Not unexpectedly, the LIF receptor antagonist hLIF05 at 0.1 to 1 μg/ml abolished completely the stimulation of C/EBPβ and C/EBPδ by 10 ng/ml LIF but did not abolish that induced by 10 ng/ml IL-6 (not shown). This observation is consistent with the inhibition of adipogenesis by hLIF05 within the same range of concentrations (Fig.5). Addition of LIF to 3T3-F442A preadipose cells induced activation of p42 MAPK as determined by a decrease in electrophoretic mobility (Fig.8). The level of activation was maximal after 10 min and returned to basal levels after 25 min. A similar response was observed for activation of p44 MAPK, whereas no activation of p38 MAPK and Jun kinase was detectable (not shown). Preincubation of preadipose cells with the specific MAPK kinase inhibitor PD98059 (32Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar) prevented induction of C/EBPβ and C/EBPδ (Fig.9 A). This effect could not be mimicked by preincubation of cells with an inhibitor of protein synthesis such as cycloheximide (not sho" @default.
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• W2109950167 title "Leukemia Inhibitory Factor and Its Receptor Promote Adipocyte Differentiation via the Mitogen-activated Protein Kinase Cascade" @default.
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