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• W2145144258 abstract "Expression of genes encoding structural myelin proteins marks the inception of the myelinating Schwann cell (SC) phenotype. Earlier embryonic SC as well as adult non-myelinating SC produce the intermediate filament glial fibrillary acid protein (GFAP), which disappears from the myelinating SC. We previously observed that triggering of the gp130 receptor system by the IL6RIL6 ligand, comprising interleukin-6 (IL-6) fused to the soluble IL-6 receptor, induces myelin gene expression in rat embryonic dorsal root ganglia (DRG) cultures as well as in the murine melanoma cell line B16/F10.9. Study of target genes regulated by IL6RIL6 indicates a strong and selective induction of the transcriptional regulator C/EBP-δ in DRG cultures and in the F10.9 cell line. As shown here, silencing of C/EBP-δ mRNA and protein expression by introduction of small interference RNA-producing plasmids in the F10.9 cells prevented the induction of myelin protein zero (P0) and myelin basic protein (MBP) mRNAs by IL6RIL6. Doxycycline-regulated overexpression of C/EBP-δ was sufficient to induce accumulation of P0 and MBP mRNAs, the effect being selective, because C/EBP-δ did not affect several other genes strongly regulated by IL6RIL6. Interestingly, GFAP was inhibited by C/EBP-δ overexpression, leading to a modulation of the ratio between myelin gene products versus GFAP and suggesting that C/EBP-δ plays a role in the switch to a myelinating phenotype. The down-regulation of Pax3, also typical of the transition to myelinating cells, was observed after C/EBP-δ expression in correlation to P0 induction and to decrease of melanogenesis and cell growth. In cultures of dissociated cells of embryonic rat DRG, where we knocked-down the C/EBP-δ mRNA, we found an inhibition of P0 mRNA induction by IL6RIL6, showing that the role of C/EBP-δ on this myelin gene is not unique to the melanoma system. Expression of genes encoding structural myelin proteins marks the inception of the myelinating Schwann cell (SC) phenotype. Earlier embryonic SC as well as adult non-myelinating SC produce the intermediate filament glial fibrillary acid protein (GFAP), which disappears from the myelinating SC. We previously observed that triggering of the gp130 receptor system by the IL6RIL6 ligand, comprising interleukin-6 (IL-6) fused to the soluble IL-6 receptor, induces myelin gene expression in rat embryonic dorsal root ganglia (DRG) cultures as well as in the murine melanoma cell line B16/F10.9. Study of target genes regulated by IL6RIL6 indicates a strong and selective induction of the transcriptional regulator C/EBP-δ in DRG cultures and in the F10.9 cell line. As shown here, silencing of C/EBP-δ mRNA and protein expression by introduction of small interference RNA-producing plasmids in the F10.9 cells prevented the induction of myelin protein zero (P0) and myelin basic protein (MBP) mRNAs by IL6RIL6. Doxycycline-regulated overexpression of C/EBP-δ was sufficient to induce accumulation of P0 and MBP mRNAs, the effect being selective, because C/EBP-δ did not affect several other genes strongly regulated by IL6RIL6. Interestingly, GFAP was inhibited by C/EBP-δ overexpression, leading to a modulation of the ratio between myelin gene products versus GFAP and suggesting that C/EBP-δ plays a role in the switch to a myelinating phenotype. The down-regulation of Pax3, also typical of the transition to myelinating cells, was observed after C/EBP-δ expression in correlation to P0 induction and to decrease of melanogenesis and cell growth. In cultures of dissociated cells of embryonic rat DRG, where we knocked-down the C/EBP-δ mRNA, we found an inhibition of P0 mRNA induction by IL6RIL6, showing that the role of C/EBP-δ on this myelin gene is not unique to the melanoma system. Embryonic neural crest-derived precursors develop into postnatal mature myelinating Schwann cells (SC) 1The abbreviations used are: SCSchwann cell(s)P0myelin protein zeroMBPmyelin basic proteinGFAPfilament glial fibrillary acid proteinDRGdorsal root gangliaIL-6interleukin-6MITFmicrophtalmiaStat3signal transducer and activator of transcription 3DMEMDulbecco's modified Eagle's mediumRTreverse transcriptionEGFPenhanced green fluorescent proteinsiRNAsmall interference RNArtTAreverse tetracycline-controlled transcriptional activatorERKextracellular signal-regulated kinase. through a series of stages defined by the expression of specific gene markers (see Refs. 1Zorick T.S. Lemke G. Curr. Opin. Cell Biol. 1996; 8: 870-876Google Scholar, 2Kioussi C. Gruss P. Trends Genet. 1996; 12: 84-86Google Scholar, 3Jessen K.R. Mirsky R. Microsc. Res. Tech. 1998; 41: 393-402Google Scholar for reviews). The genes encoding structural protein components of the nerve myelin sheaths, such as myelin protein zero (P0) or myelin basic protein (MBP) are strongly activated only at the latest stages of SC differentiation. The non-myelinating compartment of the SC expresses the glial fibrillary acidic protein (GFAP), which is present already in the embryonic progenitors and is not turned-off. The non-myelinating SC continue to express several other early markers that disappear in the myelinating SC, in particular transcription regulators such as POU domain Octamer-6 (Oct-6/SCIP) and paired homeodomain Pax3, which repress P0 or MBP gene expression (4Kioussi C. Gross M.K. Gruss P. Neuron. 1995; 15: 553-562Google Scholar, 5Monuki E.S. Kuhn R. Lemke G. Mech. Dev. 1993; 42: 15-32Google Scholar). Schwann cell(s) myelin protein zero myelin basic protein filament glial fibrillary acid protein dorsal root ganglia interleukin-6 microphtalmia signal transducer and activator of transcription 3 Dulbecco's modified Eagle's medium reverse transcription enhanced green fluorescent protein small interference RNA reverse tetracycline-controlled transcriptional activator extracellular signal-regulated kinase. In cultures of rat embryo dorsal root ganglia (DRG), we previously observed that expression of myelin genes P0 and MBP can be activated at the premyelinating stage by interleukin-6 (IL-6) type signaling (6Haggiag S. Chebath J. Revel M. FEBS Lett. 1999; 457: 200-204Google Scholar). Triggering of the gp130 receptor by IL6RIL6, a fusion protein comprising IL-6 and soluble IL-6 receptor (7Chebath J. Fischer D. Kumar A. Oh J.W. Kolett O. Lapidot T. Fischer M. Rose-John S. Nagler A. Slavin S. Revel M. Eur. Cytokine. Netw. 1997; 8: 359-365Google Scholar), led to a rapid induction of the myelin genes in embryonic day E14 DRG cells, whereas GFAP was not or less increased, and Pax3 was profoundly down-regulated, indicating a transition toward the myelinating SC phenotype (8Haggiag S. Zhang P.L. Slutzky G. Shinder V. Kumar A. Chebath J. Revel M. J. Neurosci. Res. 2001; 64: 564-574Google Scholar). Transcriptional activation of the P0 and MBP promoters by the IL6RIL6 stimulus was demonstrated in the melanoma cell line B16/F10.9 that undergoes morphological transdifferentiation from a melanocytic to a glial phenotype (9Slutsky S.G. Kamaraju A.K. Levy A.M. Chebath J. Revel M. J. Biol. Chem. 2003; 278: 8960-8968Google Scholar). Development of melanocytic lineage from the neural crest is controlled to a large extent by transcription factors Pax3, SRY-box Sox10, and their target microphtalmia-associated MITF (10Goding C.R. Genes Dev. 2000; 14: 1712-1728Google Scholar, 11Bondurand N. Pingault V. Goerich D.E. Lemort N. Sock E. Caignec C.L. Wegner M. Goossens M. Hum. Mol. Genet. 2000; 9: 1907-1917Google Scholar, 12Watanabe A. Takeda K. Ploplis B. Tachibana M. Nat. Genet. 1998; 18: 283-286Google Scholar). The F10.9 cells respond to IL6RIL6 by a Stat3-mediated down-regulation of Pax3c, a loss of Pax3-Sox10 synergism, leading to a reduction of transcription factor MITF and thereby of tyrosinase and melanogenic activity (13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar). Ectopic Pax3c expression counteracts the effects of IL6RIL6, restoring MITF (13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar), and inhibiting myelin P0 and MBP promoter activities (9Slutsky S.G. Kamaraju A.K. Levy A.M. Chebath J. Revel M. J. Biol. Chem. 2003; 278: 8960-8968Google Scholar). Sox10 is increased by IL6RIL6, and the modulation of Sox10 and Pax3 by tetracycline-dependent regulation, respectively, increased and decreased the cellular level of myelin P0 mRNA in F10.9 cells. IL6RIL6 also induces zinc finger protein ZBP99, which acts in synergism with Sox10 to activate the P0 promoter (9Slutsky S.G. Kamaraju A.K. Levy A.M. Chebath J. Revel M. J. Biol. Chem. 2003; 278: 8960-8968Google Scholar). An early and sustained induction of CCAAT/enhancer-binding protein delta (C/EBP-δ, CRP3, NF-IL6β, and CELF) was noted in the F10.9 cells during analysis of the IL6RIL6-dependent gene expression changes by DNA microarrays. 2A. K. Kamaraju, L. Ben Simchon, S. Saban, J. Chebath, and M. Revel, manuscript in preparation. Induction of C/EBP-δ was also observed in dissociated DRGs cells. 3P. Zhang, L. Ben Simchon, S. Saban, J. Chebath, and M. Revel, manuscript in preparation. C/EBP-δ belongs to a family of transcriptional regulators dimerizing through long leucine zipper domains (14Williams S.C. Cantwell C.A. Johnson P.F. Genes Dev. 1991; 5: 1553-1567Google Scholar) and known to couple growth factor signal transduction to cellular differentiation in adipocytes (15Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Google Scholar, 16Tanaka T. Yoshida N. Kishimoto T. Akira S. EMBO J. 1997; 16: 7432-7443Google Scholar), mammary cells (17Dearth L.R. DeWille J. J. Biol. Chem. 2003; 278: 11246-11255Google Scholar, 18Gigliotti A.P. Johnson P.F. Sterneck E. DeWille J.W. Exp. Biol. Med. (Maywood). 2003; 228: 278-285Google Scholar), and neural cells (19Menard C. Hein P. Paquin A. Savelson A. Yang X.M. Lederfein D. Barnabe-Heider F. Mir A.A. Sterneck E. Peterson A.C. Johnson P.F. Vinson C. Miller F.D. Neuron. 2002; 36: 597-610Google Scholar, 20Sterneck E. Johnson P.F. J. Neurochem. 1998; 70: 2424-2433Google Scholar). Proteins of the C/EBP family (C/EBP-α, -β, -γ, -δ, -ϵ, and -ζ) have a basic DNA binding domain, share 90% homology in the C-terminal end (leucine zipper) and diverge in the N-terminal region (see Refs. 21Vinson C.R. Sigler P.B. McKnight S.L. Science. 1989; 246: 911-916Google Scholar, 22Vinson C. Myakishev M. Acharya A. Mir A.A. Moll J.R. Bonovich M. Mol. Cell Biol. 2002; 22: 6321-6335Google Scholar, 23Ramji D.P. Foka P. Biochem. J. 2002; 365: 561-575Google Scholar for reviews). Their activities result not only from specific DNA binding sites but also from various protein-protein interactions (24McKnight S.L. Cell. 2001; 107: 259-261Google Scholar) (for review). C/EBPs can regulate growth and differentiation either by direct transactivation/repression effects or, indirectly, for example by repression of E2F-dependent transcription (25Porse B.T. Pedersen T.A. Xu X. Lindberg B. Wewer U.M. Friis-Hansen L. Nerlov C. Cell. 2001; 107: 247-258Google Scholar), interference with caspases (26Buck M. Poli V. Hunter T. Chojkier M. Mol. Cell. 2001; 8: 807-816Google Scholar), or interactions with retinoblastoma pRb (27Charles A. Tang X. Crouch E. Brody J.S. Xiao Z.X. J. Cell Biochem. 2001; 83: 414-425Google Scholar). C/EBP-β and δ (NF-IL6 and NF-IL6β) are involved in the induction and function of IL-6 (28Kinoshita S. Akira S. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1473-1476Google Scholar, 29Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Google Scholar, 30Akira S. Isshiki H. Sugita T. Tanabe O. Kinoshita S. Nishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Google Scholar, 31Ramji D.P. Vitelli A. Tronche F. Cortese R. Ciliberto G. Nucleic Acids Res. 1993; 21: 289-294Google Scholar, 32Alonzi T. Gorgoni B. Screpanti I. Gulino A. Poli V. Immunobiology. 1997; 198: 144-156Google Scholar). Both are induced by cytokines of the IL-6 family via Stat3-dependent and independent mechanisms (33Cantwell C.A. Sterneck E. Johnson P.F. Mol. Cell Biol. 1998; 18: 2108-2117Google Scholar, 34Alonzi T. Maritano D. Gorgoni B. Rizzuto G. Libert C. Poli V. Mol. Cell Biol. 2001; 21: 1621-1632Google Scholar). In view of the strong induction of C/EBP-δ, much over that of C/EBP-β, in the IL6RIL6-treated transdifferentiating F10.9 cells, we examined in the present study whether C/EBP-δ is necessary for the induction of myelin gene products in F10,9 and in DRG cells. We also investigated the role of C/EBP-δ overexpression for non-myelinating glial cell markers, for the turn-off of the melanogenic pathway and for cell growth. Cell Cultures and Cytokines—Murine B16 melanoma metastatic clone F10.9 cells (35Porgador A. Feldman M. Eisenbach L. J. Immunogenet. 1989; 16: 291-303Google Scholar) were cultured as a monolayer at 37 °C, 5% CO2,in Dulbecco's modified Eagle's medium with 8% fetal calf serum (Biolabs, Bet Ha-Emek, Ness Ziona, Israel), supplemented with glutamine, penicillin, and streptomycin. Cells were subcultured every 3 days at 10-30% confluence. Fused IL6RIL6 chimera was produced as described using mammalian Chinese hamster ovary cells and immunoaffinity purification of the secreted 85-kDa protein (Interpharm, Israel) (7Chebath J. Fischer D. Kumar A. Oh J.W. Kolett O. Lapidot T. Fischer M. Rose-John S. Nagler A. Slavin S. Revel M. Eur. Cytokine. Netw. 1997; 8: 359-365Google Scholar, 13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar) and used routinely at 140 ng/ml. Dorsal root ganglia (DRG) were excised from Wistar rats day 14 embryos. About 250 DRGs were treated with 10 μl of collagenase-dispase (Gibco-BRL), in 0.2 ml Dulbecco's modified Eagle's medium/F-12, for 30 min at 37 °C. Dissociated cells were seeded at 106 cells per 9-cm tissue culture plate coated with poly-d-lysine and fibronectin. Cells were grown at 37 °C, 5% CO2 in defined medium (Dulbecco's modified Eagle's medium/F-12) containing 20 ng/ml fibroblast growth factor-2 with 1% N2 and 2% B27 supplements (Invitrogen), containing 15% chicken embryo extract as described previously (36Morrison S.J. White P.M. Zock C. Anderson D.J. Cell. 1999; 96: 737-749Google Scholar, 37Stemple D.L. Anderson D.J. Cell. 1992; 71: 973-985Google Scholar). After 3 days, cells were frozen at -180 °C, and after thawing contained 80% live cells. For experiments reported here, cells were used at passage one. F10.9 cell growth and tyrosinase assays were performed as described before (7Chebath J. Fischer D. Kumar A. Oh J.W. Kolett O. Lapidot T. Fischer M. Rose-John S. Nagler A. Slavin S. Revel M. Eur. Cytokine. Netw. 1997; 8: 359-365Google Scholar, 13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar). RT-PCR—Total RNA was extracted with Tri reagent (Molecular Research Center), as recommended by the manufacturer. For RT-PCR, RNA samples (2 μg/assay) were reverse-transcribed with SuperscriptII (Invitrogen Molecular Biology) in the presence of oligo(dT) in 20 μl, and 2 μl of the RT reaction was used for amplification with Taq polymerase. The primers used to amplify specific mouse cDNAs were as follows: glial fibrillary acidic protein (GFAP) (accession number L27219), forward (F): 1301-1605 and reverse (R): 2175-2194; myelin protein zero (P0) (XM_110344), F1: 113-133 and R1: 745-766; F2: 150-174; R2: 854-878; F3: 1-26 with R3: 221-246. Several couples of primers were used to verify identity of P0 mRNA induced in melanoma treated with IL6RIL6 or in cells expressing C/EBP-δ. Amplification with F1/R1 is shown in all results. Myelin basic protein (MBP) (M15060), F: 136-156 and R: 466-485. Two fragments of 473 and 360 bp are amplified. Peripheral myelin protein-22 (PMP-22) (Z38110) F: 1-19; R: 466-483. Microphtalmia (MITF) (NM_008601), F: 82-101; R: 1382-1401. Tyrosinase (D00131), F: 804-827; R: 1398-1421. Suppressor of cytokine signaling-3 (SOCS3) (U88328), F: 248-301; R: 673-690. Proteinase inhibitor Spi2 (eb4) (M64086), F: 902-922; R: 1287-1308. CCAAT/enhancer-binding protein delta (C/EBP-δ) (X61800), F: 772-795; R: 1091-1113. C/EBP-β (NM_009883), F: 561-580; R: 942-981. Glyceraldehyde-3-phosphate dehydrogenase primers (Clontech) were used to verify RNA loading. Amplification conditions were 94 °C (1 min) 52-58 °C (30 s), 72 °C (1 min) for 29 cycles (GFAP, P0, and MBP), or for 20 cycles (glyceraldehyde-3-phosphate dehydrogenase). PCR fragment sequences were verified on DNA analyzer 3700 (PE Applied Biosystems, Hitachi). Gel photographs or films were scanned and processed with Adobe Photoshop. DRG Cell Infection with p-SUPER Retroviruses and RT-PCR—After thawing, DRG cells were seeded on poly-d-lysine- and fibronectin-coated wells of 6-well plates (7 × 104 cells/well) and cultured for 3 days in growth medium. Each well received 0.8 ml of HEK-293/NFK-conditioned medium containing p-SUPER retroviruses (see below), and 0.5 ml of growth medium supplemented with Polybrene (8 μg/ml). Cells were incubated with viruses for 6 h, and after complementing the medium volume to 2.5 ml with growth medium, left for 36 h with virus. Medium was changed and IL6RIL6 added at 140 ng/ml. RNA extraction was done on two wells/assay, 36 h after IL6RIL6 addition. For RT-PCR, the following primers were used. P0 rat (K03242), F: 262-286; R: 667-697. C/EBP-δ rat (M65149), F: 492-614; R: 1030-1052. Annealing temperatures: 58 °C (P0), 52 °C (C/EBP-δ), and 30 cycles. RNA Silencing Experiments—The p-SUPER and p-Retro-SUPER (pRS) plasmids were a gift of Dr. Agami (38Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Google Scholar, 39Brummelkamp T.R. Bernards R. Agami R. Cancer Cell. 2002; 2: 243-247Google Scholar). The following oligonucleotides were cloned in p-SUPER cut with BamH1 and HindIII. C/EBP-δ1: Sense and antisense are given 5′-3′ and contain nucleotides 981-999 of sequence X61800, as verified by sequencing. gatccccGCTGGTGGAGTTGTCGGCCttcaagagaGGCCGACAACTCCACCAGCtttttggaaaagcttttccaaaaaGCTGGTGGAGTTGTCGGCCtctcttgaaGGCCGACAACTCCACCAGCggg. C/EBP-δ 2: contains the nucleotides 921-939 of sequence X61800. gatccccCATCGCTGTGCGCAAGAGCttcaagagaGCTCTTGCGCACAGCGATGtttttggaaaagcttttccaaaaaCATCGCTGTGCGCAAGAGCtctcttgaaGCTCTTGCGCACAGCGATGggg p-RS-C/EBP-δ 2 was obtained by cloning the EcoRI/XhoI fragment of p-SUPER-C/EBP-δ 2 into the p-Retro-SUPER plasmid. Tetracycline-dependent Expression System—We first selected F10.9 cell clones expressing reverse tetracycline-controlled transcriptional activator (rtTA) isolated from the pTet-On regulator plasmid (Clontech) cloned in the bicistronic vector pEF-IRES puro (40Hobbs S. Jitrapakdee S. Wallace J.C. Biochem. Biophys. Res. Commun. 1998; 252: 368-372Google Scholar), using puromycin. The plasmid pBI-EGFP was made by inserting the coding sequence of enhanced green fluorescent protein from EGFP-N1 (Clontech) in the unique NotI site of the multiple cloning site II of the pBI vector bearing a bi-directional Tet-regulated element (Clontech). This plasmid was transfected into several rtTA-transformed F10.9 clones, and we chose the clone where the differential fluorescence signal, without and with doxycycline, was highest. This clone has the same phenotype as F10.9 wild-type cells concerning shape, melanogenesis, growth rate, and response to IL6RIL6. We co-transfected the rtTA-transformed clone with pBI-EGFP and pSV2-Hygro and a pool of about 200 hygromycin- and puromycin-resistant clones was amplified to constitute our control pool cells. The fragments excised from plasmids p-BABE C/EBP-δ and p-BABE C/EBP-β (gifts of Dr. R. Schwartz) with BamHI and containing, respectively, C/EBP-δ and C/EBP-β coding sequences, were cloned in the EcoRV unique site of pBI-EGFP to create pBI-EGFP C/EBP-δ and C/EBP-β. Deletion of 5′ cDNAs sequences, removing amino acids 1-165 of C/EBP-δ (replaced by Met-Ala), in the same plasmid created pBI-EGF Dip. These plasmids were transfected in the selected rtTA clone with pSV2-Hygro, as above. Only clones fluorescent after treatment with doxycycline were retained. Expression from transgenes was verified by RT-PCR, using the pBI vector reverse primer 5′-ACTCACCCTGAAGTTCTCAG and forward primers specific of C/EBP-δ and C/EBP-β, and by Western blots with specific antibodies. The doxycycline dose used routinely (200 ng/ml) does not change F10.9 cell growth or differentiation. Cloned cells were passed in the presence of puromycin (0.5 μg/ml) or hygromycin alternatively, except for preparation of assay samples. Electrophoretic Mobility Shift Assays—F10.9 cells were grown in 9-cm dishes for 48 h without or with IL6RIL6, doxycycline, or both, and nuclear extracts prepared as described (9Slutsky S.G. Kamaraju A.K. Levy A.M. Chebath J. Revel M. J. Biol. Chem. 2003; 278: 8960-8968Google Scholar). Oligonucleotides representing the consensus target sequence of C/EBP proteins 5′-TGCAGATTGCGCAATCTGCA were 5′-labeled with [α-32P]ATP (104 cpm/fmol) and polynucleotide kinase, denatured by heat, annealed, and isolated on a non-denaturing 8% polyacrylamide gel. About 20,000 cpm of the oligonucleotide probe (20 fmol) was incubated with 2 μl of nuclear extracts for 20 min on ice in a final volume of 20 μl. The incubation buffer final composition was 20 mm Hepes, pH 7.9, 60 mm NaCl, 1 mm dithiothreitol, 5% glycerol, 5 mm MgCl2, 3 μg/ml bovine serum albumin, and 2 μg of poly(dI)-poly(dC) alternate copolymers per assay (Roche Applied Science). Assays were loaded at 50 V on 15-cm long 5% native polyacrylamide gel, and separation made at 170 V. Gels were fixed, dried, and exposed to film. When 2 pmol of wild-type cold probe were used as competitor, all the complexes were competed out. To check the specificity of the bands, in some assays, 4 μl of specific anti-C/EBP-δ (M-17) or C/EBP-β (C19) antibodies (Santa Cruz Biotechnology) were added together with the probe. Western Blots—F10.9 cells or transformed clones were seeded at less than 50% confluence. At the end of the treatment period, cells washes and extraction with radioimmune precipitation buffer, containing protease inhibitors (Calbiochem), were as described (13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar). Cell extracts were analyzed on 10% or 12% SDS-PAGE, and proteins were transferred to nitrocellulose membranes Protran Ba85 (Schleicher & Schuell). For immunodetection, the blots were first blocked and incubated with primary antibodies and with secondary antibodies goat anti-rabbit (or anti-mouse) horseradish peroxidase-conjugated immunopurified IgGs (Jackson Immunoresearch Laboratories) as described (13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar). Antibody binding was revealed with Pierce ECL reagents, and exposure to films. Rabbit anti-Pax3 antibody (Geneka Biotechnology Inc.) were used at 1:2000 dilution, and rabbit IgG against C/EBP-δ (M-17) or C/EBP-β (C-19) (Santa Cruz Biotechnology) were used at 1:2000 dilution. Monoclonal mouse antibody against glial fibrillary acidic protein (GFAP) (Sigma Israel Ltd.) was diluted 1:1000. The secondary antibody was diluted 1:10,000. Anti-extracellular signal-regulated kinase (ERK) 1/2 antibodies (Sigma Israel) were used as control. Cell Transfections—For transient transfections with p-SUPER plasmids, F10.9 cells in the log phase of growth were seeded in 6-well Costar plates (2 × 105 cells/well). After 16 h, each well received 1.2 ml of mixture containing 2 μg of plasmid DNA, 10 μl of LipofectAMINE (Invitrogen) in F-12 medium without antibiotics or serum. After 10 h at 37 °C in a CO2 incubator, the mixture was replaced by normal growth medium, and treatment with IL6RIL6 (140 ng/ml) was started. For permanent transfections, cells were plated as before and transfected with 2 μg of pEF-IRES puro/rtTA and LipofectAMINE for 10 h. After medium change, cells were left in complete growth medium for 24 h, trypsinized, and plated at 200,000 cells per 9-cm tissue culture plate in growth medium containing 500 ng/ml puromycin. The same procedure was applied for co-transfection with pBI (1.5 μg) and pSV2hygro (0.5 μg), except that selection was with hygromycin (180 μg/ml). To obtain p-RS self-inactivating viruses, p-RS plasmids (5 μg) were transfected in HEK 293 NFK-packaging cells (1.6 × 106 cells/9-cm plate, seeded 24 h before transfection), using DNA/CaPO4 co-precipitation. After 14 h, the medium was replaced by fresh growth medium, and supernatants were collected for 48 h, every day. C/EBP-δ Up-regulation in F10.9 Cells Treated by IL6RIL6—Exposure of melanoma F10.9 cells to the gp130 activator, IL6RIL6, leads to down-regulation of genes involved in melanogenesis followed after 24-48 h by the transcriptional induction of genes producing myelin proteins such as P0, MBP, and CNP (9Slutsky S.G. Kamaraju A.K. Levy A.M. Chebath J. Revel M. J. Biol. Chem. 2003; 278: 8960-8968Google Scholar, 13Kamaraju A.K. Bertolotto C. Chebath J. Revel M. J. Biol. Chem. 2002; 277: 15132-15141Google Scholar). In a microarray analysis of gene expression profiles after IL6RIL6 addition to the F10.9 cells we noticed that C/EBBδ is one of the most rapidly and intensely induced genes. 2A. K. Kamaraju, L. Ben Simchon, S. Saban, J. Chebath, and M. Revel, manuscript in preparation. Starting from low levels in the untreated cells, increases of 5- to 100-fold were recorded from the 3-h time point and up to 48 h after IL6RIL6 treatment (Fig. 1, A and B). Interestingly, C/EBP-β (NF-IL6) was much less increased during the culture of F10.9 cells with IL6RIL6 (Fig. 1, A and B), despite the usual co-regulation of the two C/EBP genes after gp130 signaling (23Ramji D.P. Foka P. Biochem. J. 2002; 365: 561-575Google Scholar, 28Kinoshita S. Akira S. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1473-1476Google Scholar, 41Ramji D.P. Hughes T.R. Sabatakos G. Biochem. Soc. Trans. 1994; 22: 358SGoogle Scholar). The same pattern was observed at the protein level (Fig. 1, C and D). Whereas the C/EBP-δ protein was strongly induced from very low levels in the untreated cells, the C/EBP-β activating isoform (LAP 37 kDa) was only moderately affected after 48 h. The 22-kDa LIP isoform that contains only the bZIP domain and not the activating domain of C/EBP-β (42Descombes P. Schibler U. Cell. 1991; 67: 569-579Google Scholar) was reduced in the IL6RIL6-treated cells (Fig. 1C). Silencing of C/EBP-δ Expression Reduces Induction of Myelin Genes by IL6RIL6—The role of C/EBP-δ was first investigated by inhibiting its mRNA expression with small double-stranded interfering siRNAs (43Dykxhoorn D.M. Novina C.D. Sharp P.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 457-467Google Scholar, 44Fire A. Trends Genet. 1999; 15: 358-363Google Scholar). Several oligonucleotides containing an inverted repeat of 19 bp specific to the murine C/EBP-δ sequence were cloned under the RNA polymerase III-dependent H1 RNA promoter in the p-SUPER vector (38Brummelkamp T.R. Bernards R. Agami R. Science. 2002; 296: 550-553Google Scholar). The IL6RIL6-dependent induction of C/EBP-δ mRNA could be inhibited by transient transfection of the F10.9 cells with p-SUPER C/EBP-δ-1 and -2, the inhibition reaching 90% with the latter siRNA vector as compared with cells transfected by the empty p-SUPER vector (Fig. 2, A and B). The expression of C/EBP-β was not reduced (and even somewhat increased) by the C/EBP-δ-specific siRNAs, and the same differential inhibition of C/EBP-δ was observed at the protein level (Fig. 2C). The induction of myelin gene transcripts was measured 48 h after treatment with IL6RIL6 (Fig. 2, A and B). As compared with F10.9 cells transfected by the control vector, the C/EBP-δ siRNA resulted in a reduction of up to 80% of the P0 mRNA and over 60% inhibition of MBP mRNA. Three independent transfections with different siRNA plasmids gave the same results. Thus, the induction of C/EBP-δ appears to be necessary for the effect of IL6RIL6 on myelin gene expression in the transdifferentiating cells. Ectopic Expression of C/EBP-δ Is Sufficient to Induce Myelin Gene Transcripts—F10.9 stably transformed clones were obtained, which contain the C/EBP-δ cDNA and a green fluorescent protein (EGFP) cDNA on both sides of a bidirectional doxycycline-regulated promoter (pBI plasmid), as well as rtTA. Clones of cells fluorescent in the presence of doxycycline were selected and examined as a pool (P) of eight clones showing a strong drug-induced expression of the C/EBP-δ protein (Fig. 3A). Individual clones showing different levels of tetracycline regulated or of basal expression were also used in the analysis. The doxycycline-induced C/EBP-δ was functionally active as seen from electrophoresis mobility shift assays with a consensus sequence target of all C/EBP factors (Fig. 3B). Nuclear extracts of the pBI-C/EBP-δ-EGFP pool showed after doxycycline a strong induction of C/EBP-δ-specific DNA complexes, forming slower migrating complexes (supershift) with specific antibodies (Fig. 3B). These complexes were completely absent in extracts from a control pool of clones with only pBI-EGFP, unless the cells were IL6RIL6-treated (Fig. 3B). With both pools, there were complexes supershifted by antibodies to C/EBP-β. Notably, in the C/EBP-δ pool after doxycycline, the C/EBP-δ DNA binding activity became higher than that of C/EBP-β. Activation of the C/EBP-δ ectopic transgene by doxycycline produced a strong induction of the myelin P0 mRNA (Fig. 4A). The P0 mRNA structure, verified by RT-PCR with various couples of primers (see “Experimental Procedures”), followed by sequencing, is the one of mature mRNA found in myelinating SCs. In individual clones, the accumulation of P0 mRNA was always correlated with the induction of C/EBP-δ; no P0 mRNA induction was seen in clones that did not express C/EBP-δ after doxycycline. In the positive pool treated by doxycycline for 60 h" @default.
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• W2145144258 title "C/EBP-δ Induction by gp130 Signaling" @default.
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