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Originally published In Press as doi:10.1074/jbc.M310443200 on November 3, 2003

J. Biol. Chem., Vol. 279, Issue 5, 3852-3861, January 30, 2004
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C/EBP-{delta} Induction by gp130 Signaling

ROLE IN TRANSITION TO MYELIN GENE EXPRESSING PHENOTYPE IN A MELANOMA CELL LINE MODEL*

Anil K. Kamaraju, Sophie Adjalley, Peilin Zhang, Judith Chebath, and Michel Revel{ddagger}

From the Department of Molecular Genetics, Weizmann Institute of Science, Rehov Herzl, Rehovot 76100, Israel

Received for publication, September 22, 2003 , and in revised form, October 30, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{delta} in DRG cultures and in the F10.9 cell line. As shown here, silencing of C/EBP-{delta} 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-{delta} was sufficient to induce accumulation of P0 and MBP mRNAs, the effect being selective, because C/EBP-{delta} did not affect several other genes strongly regulated by IL6RIL6. Interestingly, GFAP was inhibited by C/EBP-{delta} overexpression, leading to a modulation of the ratio between myelin gene products versus GFAP and suggesting that C/EBP-{delta} 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-{delta} 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-{delta} mRNA, we found an inhibition of P0 mRNA induction by IL6RIL6, showing that the role of C/EBP-{delta} on this myelin gene is not unique to the melanoma system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Embryonic neural crest-derived precursors develop into postnatal mature myelinating Schwann cells (SC)1 through a series of stages defined by the expression of specific gene markers (see Refs. 1-3 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 (4, 5).

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 (6). Triggering of the gp130 receptor by IL6RIL6, a fusion protein comprising IL-6 and soluble IL-6 receptor (7), 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 (8). 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 (9). 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 (10-12). 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 (13). Ectopic Pax3c expression counteracts the effects of IL6RIL6, restoring MITF (13), and inhibiting myelin P0 and MBP promoter activities (9). 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 (9).

An early and sustained induction of CCAAT/enhancer-binding protein delta (C/EBP-{delta}, CRP3, NF-IL6{beta}, and CELF) was noted in the F10.9 cells during analysis of the IL6RIL6-dependent gene expression changes by DNA microarrays.2 Induction of C/EBP-{delta} was also observed in dissociated DRGs cells.3 C/EBP-{delta} belongs to a family of transcriptional regulators dimerizing through long leucine zipper domains (14) and known to couple growth factor signal transduction to cellular differentiation in adipocytes (15, 16), mammary cells (17, 18), and neural cells (19, 20). Proteins of the C/EBP family (C/EBP-{alpha}, -{beta}, -{gamma}, -{delta}, -{epsilon}, and -{zeta}) 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. 21-23 for reviews). Their activities result not only from specific DNA binding sites but also from various protein-protein interactions (24) (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 (25), interference with caspases (26), or interactions with retinoblastoma pRb (27). C/EBP-{beta} and {delta} (NF-IL6 and NF-IL6{beta}) are involved in the induction and function of IL-6 (28-32). Both are induced by cytokines of the IL-6 family via Stat3-dependent and independent mechanisms (33, 34).

In view of the strong induction of C/EBP-{delta}, much over that of C/EBP-{beta}, in the IL6RIL6-treated transdifferentiating F10.9 cells, we examined in the present study whether C/EBP-{delta} is necessary for the induction of myelin gene products in F10,9 and in DRG cells. We also investigated the role of C/EBP-{delta} overexpression for non-myelinating glial cell markers, for the turn-off of the melanogenic pathway and for cell growth.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures and Cytokines—Murine B16 melanoma metastatic clone F10.9 cells (35) 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) (7, 13) 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 (36, 37). 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 (7, 13).

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 [GenBank] ), 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-{delta}. Amplification with F1/R1 is shown in all results. Myelin basic protein (MBP) (M15060 [GenBank] ), F: 136-156 and R: 466-485. Two fragments of 473 and 360 bp are amplified. Peripheral myelin protein-22 (PMP-22) (Z38110 [GenBank] ) F: 1-19; R: 466-483. Microphtalmia (MITF) (NM_008601 [GenBank] ), F: 82-101; R: 1382-1401. Tyrosinase (D00131 [GenBank] ), F: 804-827; R: 1398-1421. Suppressor of cytokine signaling-3 (SOCS3) (U88328 [GenBank] ), F: 248-301; R: 673-690. Proteinase inhibitor Spi2 (eb4) (M64086 [GenBank] ), F: 902-922; R: 1287-1308. CCAAT/enhancer-binding protein delta (C/EBP-{delta}) (X61800 [GenBank] ), F: 772-795; R: 1091-1113. C/EBP-{beta} (NM_009883 [GenBank] ), 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 x 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 [GenBank] ), F: 262-286; R: 667-697. C/EBP-{delta} rat (M65149 [GenBank] ), F: 492-614; R: 1030-1052. Annealing temperatures: 58 °C (P0), 52 °C (C/EBP-{delta}), and 30 cycles.

RNA Silencing Experiments—The p-SUPER and p-Retro-SUPER (pRS) plasmids were a gift of Dr. Agami (38, 39). The following oligonucleotides were cloned in p-SUPER cut with BamH1 and HindIII. C/EBP-{delta}1: Sense and antisense are given 5'-3' and contain nucleotides 981-999 of sequence X61800 [GenBank] , as verified by sequencing. gatccccGCTGGTGGAGTTGTCGGCCttcaagagaGGCCGACAACTCCACCAGCtttttggaaaagcttttccaaaaaGCTGGTGGAGTTGTCGGCCtctcttgaaGGCCGACAACTCCACCAGCggg. C/EBP-{delta} 2: contains the nucleotides 921-939 of sequence X61800 [GenBank] . gatccccCATCGCTGTGCGCAAGAGCttcaagagaGCTCTTGCGCACAGCGATGtttttggaaaagcttttccaaaaaCATCGCTGTGCGCAAGAGCtctcttgaaGCTCTTGCGCACAGCGATGggg p-RS-C/EBP-{delta} 2 was obtained by cloning the EcoRI/XhoI fragment of p-SUPER-C/EBP-{delta} 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 (40), 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-{delta} and p-BABE C/EBP-{beta} (gifts of Dr. R. Schwartz) with BamHI and containing, respectively, C/EBP-{delta} and C/EBP-{beta} coding sequences, were cloned in the EcoRV unique site of pBI-EGFP to create pBI-EGFP C/EBP-{delta} and C/EBP-{beta}. Deletion of 5' cDNAs sequences, removing amino acids 1-165 of C/EBP-{delta} (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-{delta} and C/EBP-{beta}, 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 (9). Oligonucleotides representing the consensus target sequence of C/EBP proteins 5'-TGCAGATTGCGCAATCTGCA were 5'-labeled with [{alpha}-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-{delta} (M-17) or C/EBP-{beta} (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 (13). 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 (13). 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-{delta} (M-17) or C/EBP-{beta} (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 x 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 x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
C/EBP-{delta} 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 (9, 13). In a microarray analysis of gene expression profiles after IL6RIL6 addition to the F10.9 cells we noticed that C/EBB{delta} is one of the most rapidly and intensely induced genes.2 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-{beta} (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 (23, 28, 41). The same pattern was observed at the protein level (Fig. 1, C and D). Whereas the C/EBP-{delta} protein was strongly induced from very low levels in the untreated cells, the C/EBP-{beta} 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-{beta} (42) was reduced in the IL6RIL6-treated cells (Fig. 1C).



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  		FIG. 1.
Preferential induction of C/EBP-{delta} expression in F10.9 cells treated with IL6RIL6. Total RNA extracted from F10.9 cells at different times of incubation with or without IL6RIL6 was converted to biotin-labeled cRNA, for hybridization on mouse cDNA microarrays MG-U74A (Affymetrix). Among IL6RIL6-regulated genes, C/EBP-{delta} was the most induced transcription factor. The graph shows expression levels of three members of the C/EBP factors family in non-treated (NT) cells (part A) and IL6RIL6-treated cells (part B). Vertical bars indicate standard deviation for the two repeats. Detergent radioimmune precipitation assay buffer extracts of F10.9 cells proteins were resolved on SDS-10% PAGE (30 µg of proteins/lane) and blotted on nitrocellulose. Blots were sequentially reacted with specific rabbit anti-C/EBP-{beta} (C) or anti-C/EBP-{delta} (D) antibodies, and goat anti-rabbit Ig coupled with horseradish peroxidase, followed by ECL reaction. After signal stripping, the same blots were reacted with anti-ERK1/2 antibodies to verify equal loading of proteins.

 
Silencing of C/EBP-{delta} Expression Reduces Induction of Myelin Genes by IL6RIL6—The role of C/EBP-{delta} was first investigated by inhibiting its mRNA expression with small double-stranded interfering siRNAs (43, 44). Several oligonucleotides containing an inverted repeat of 19 bp specific to the murine C/EBP-{delta} sequence were cloned under the RNA polymerase III-dependent H1 RNA promoter in the p-SUPER vector (38). The IL6RIL6-dependent induction of C/EBP-{delta} mRNA could be inhibited by transient transfection of the F10.9 cells with p-SUPER C/EBP-{delta}-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-{beta} was not reduced (and even somewhat increased) by the C/EBP-{delta}-specific siRNAs, and the same differential inhibition of C/EBP-{delta} 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-{delta} 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-{delta} appears to be necessary for the effect of IL6RIL6 on myelin gene expression in the transdifferentiating cells.



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  		FIG. 2.
C/EBP-{delta}-specific siRNAs inhibit C/EBP-{delta} and myelin gene mRNA induction. The empty p-SUPER plasmid (ctrl) or two p-SUPER plasmids expressing short double-stranded RNAs containing C/EBP-{delta} sequences (pSUPER-C/EBP-{delta}-1 and 2) were transiently transfected in F10.9 cells. Cells were left untreated or treated with IL6RIL6 for 48 h. Total RNA (A and B), or in another experiment, proteins (C), were extracted from transfected cells, about 60 h after transfection start. In part A, RT-PCR was used to amplify C/EBP-{delta}, C/EBP-{beta}, or myelin genes P0 and MBP cDNAs. In part B, mRNA concentrations normalized on glyceraldehyde-3-phosphate dehydrogenase content were evaluated by using Image Gauge and reported on the graph. RT-PCR results from pSUPER (Ctrl) or pSUPER-C/EBP-{delta}-2 (RNAi II)-transfected cells are recorded. In part C, a protein blot was successively reacted with anti-C/EBP-{delta}, anti-C/EBP-{beta}, and anti-ERK1/2 antibodies. The C/EBP-{beta} 37-kDa protein is shown.

 
Ectopic Expression of C/EBP-{delta} Is Sufficient to Induce Myelin Gene Transcripts—F10.9 stably transformed clones were obtained, which contain the C/EBP-{delta} 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-{delta} 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-{delta} 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-{delta}-EGFP pool showed after doxycycline a strong induction of C/EBP-{delta}-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-{beta}. Notably, in the C/EBP-{delta} pool after doxycycline, the C/EBP-{delta} DNA binding activity became higher than that of C/EBP-{beta}.



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  		FIG. 3.
Doxycycline-regulated C/EBP-{delta} expression, and C/EBP-{delta} DNA-binding activity. A, Western blot analysis of radioimmune precipitation assay buffer extracts from individual lines or pool of lines (P) permanently transfected with the pBI-EGFP-C/EBP-{delta} plasmid and reacted with anti-C/EBP-{delta} and ERK1/2 antibodies. Doxycycline treatment was for 60 h. B, nuclear extracts of control and pBI-EGFP-C/EBP-{delta}-transfected pools, non-treated or treated with IL6RIL6, doxycycline, or both for 48 h, were mixed with 32P-labeled double-stranded oligonucleotide representing the C/EBP consensus binding site, and DNA protein complexes were analyzed by electrophoresis on a native polyacrylamide gel. Antibodies against C/EBP-{delta} (d), C/EBP-{beta} (b), or the tumor suppressor gene pRb were added to some samples.

 
Activation of the C/EBP-{delta} 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-{delta}; no P0 mRNA induction was seen in clones that did not express C/EBP-{delta} after doxycycline. In the positive pool treated by doxycycline for 60 h, the intensity of the P0 RNA band was comparable or even higher than with IL6RIL6 (Fig. 4B). As an equivalent to the LIP transcriptional repressor of C/EBP-{beta} (42), we deleted the N-terminal 165 residues of C/EBP-{delta}, preserving the basic region and the leucine zipper, and isolated tetracycline-regulated clones expressing this Dip mutant. No induction of P0/MZP RNA was observed in the Dip pool of clones expressing the 19-kDa Dip polypeptide after doxycycline (Fig. 4, B and D). Moreover, when the cells were treated by IL6RIL6 thereby increasing the endogenous 35-kDa C/EBP-{delta} (Fig. 4D), the expression of Dip reduced the induction of the P0 mRNA (Fig. 4C). In analogy to other C/EBP factors (23) the ability of C/EBP-{delta} to interact with other proteins through its N-terminal transactivation domain may be part of its overall transcriptional activation functions, and this domain appears essential for the C/EBP-{delta}-mediated accumulation of the myelin gene transcripts.



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  		FIG. 4.
Ectopic expression of C/EBP-{delta} is sufficient to induce myelin gene transcripts. RNA extracted from individual pBI-EGFP-C/EBP-{delta}-transfected lines (A), or from the pools of C/EBP-{delta}- and Dip-transfected lines (B and C) were analyzed by RT-PCR for P0 mRNA expression, or for expression of C/EBP-{delta} mRNA from the transgene (C/EBP-{delta} ext). The pool of C/EBP-{delta}-transfected cells was treated with 140 ng/ml IL6RIL6. In B or C the pool of Dip-transfected lines was treated, respectively, with 70 or 140 ng/ml IL6RIL6. In C, the graph represents P0 band intensities scanned from the Dip pool assays (B). Part D presents a Western blot analysis of Dip pool radioimmune precipitation assay buffer extracts after 48 h of treatment with IL6RIL6 with or without doxycycline (Doxy). The doxycycline-induced band of 19 kDa represents the Dip protein, and the 35-kDa IL6RIL6-induced band is the C/EBP-{delta} protein.

 
Comparative Effects of C/EBP-{delta} on Myelin and Other IL6RIL6-induced Genes—In addition to myelin P0 mRNA, the ectopic expression of C/EBP-{delta} also led to the accumulation of MBP mRNA in a manner similar to that produced by IL6RIL6 (Fig. 5A). In contrast, the glial fibrillary acidic protein GFAP mRNA was not increased by C/EBP-{delta}, and moreover the increase in GFAP mRNA produced by IL6RIL6 was markedly reduced when the cells were also treated with doxycycline (Fig. 5A; see below). That C/EBP-{delta} expression in the F10.9 cells does not result in a general effect similar to that of IL6RIL6 was further demonstrated by examining genes such as the serine protease inhibitor Spi-2 and the suppressor of cytokine signaling Socs3, that are regulated at the transcription level by gp130 and Stat3 activation (45, 46). The doxycycline-dependent expression of C/EBP-{delta} neither increased Spi-2 and Socs3 transcripts nor interfered with their induction by IL6RIL6 (Fig. 5B). This would rule out that C/EBP-{delta} acts by causing the cells to produce IL-6 and/or sIL-6R. Furthermore, attempts to detect in conditioned medium from C/EBP-{delta} pool cells treated with doxycycline, a soluble factor that would increase P0 or MBP mRNA in naïve F10.9 cells remained negative (not shown).



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  		FIG. 5.
C/EBP-{delta} does not reproduce all the effects of IL6RIL6 on F10.9 cell gene expression. Total RNA was extracted from a pool of F10.9 clones permanently transfected with pBI-EGFP plasmid (Ctrl pool) or a pool of clones transfected with pBI-EGFP-C/EBP-{delta}, non-treated or treated with IL6RIL6 (48 h), doxycycline (60 h), or both (respectively, 48 and 60 h). PCR experiments shown in A and B involved two reverse transcription reactions with the same RNAs.

 
An intermediate result was obtained for another glial protein, peripheral myelin protein PMP-22: although its mRNA was not induced by the expression of C/EBP-{delta} alone, there was an increase in the level of PMP-22 mRNA produced by IL6RIL6 treatment when ectopic C/EBP-{delta} was simultaneously expressed by doxycycline (Fig. 5B). Moderate but reproducible additive effects of IL6RIL6 and C/EBP-{delta} expression were also observed on P0 and MBP, contrasting with the negative effect observed on the GFAP gene (Fig. 5A).

A C/EBP-{delta}-regulated Switch between P0 and GFAP—During the last steps of SC differentiation, the expression of myelin protein genes is strongly up-regulated in SC destined to become myelinating cells, whereas at the same time GFAP expression, which is high in immature SC cells is down-regulated, remaining high only in the non-myelinating SC (1, 2) (for review). In the F10.9 cell model, IL6RIL6 leads to an increase in myelin gene expression as well as in GFAP mRNA. IL6RIL6 leads to activation of P0 and MBP promoter transcriptional activity (9), and we have similarly found that a reporter gene linked to 2 kb of the GFAP gene 5'-flanking sequence (47, 48) is activated over 10 times by IL6RIL6 in F10.9 cells, an activation abrogated if the palindromic Stat sites are mutated in the GFAP upstream promoter (not shown). It is therefore of interest that the rise in P0 or MBP mRNA can be dissociated from that in GFAP RNA in the C/EBP-{delta}-transformed cells. Time-course experiments confirmed that at no time does doxycycline increase GFAP mRNA, whereas it causes an upsurge of P0 RNA at 48-72 h (Fig. 6A). Actually, IL6RIL6 itself induces GFAP mRNA early and with a peak between 12 to 24 h followed by a decrease at later times, whereas P0 mRNA is significantly increased only after 24 h and continues to rise up to 72 h (Fig. 6A).



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  		FIG. 6.
C/EBP-{delta} overexpression inhibits the induction by IL6RIL6 of the glial gene GFAP at the mRNA and protein level. In part A, total RNA was extracted from a pool of F10.9 clones permanently transfected with pBI-EGFP-C/EBP-{delta} and left non-treated or treated for the indicated periods with IL6RIL6 or doxycycline, added at the same time. The levels P0, GFAP, and C/EBP-{delta} mRNAs were compared using RT-PCR. In part B, the pools of pBI-EGFP-transfected cells (ctrl), or pBI-GFAP-C/EBP-{delta}-transformed cells were left non-treated, or treated with IL6RIL6 at the concentrations of 140, 70, and 35 ng/ml, or were treated with doxycycline, without or with the same concentrations of IL6RIL6. After RNA extraction, P0 and GFAP mRNA levels were compared with RT-PCR. In part C, proteins were extracted from wild-type F10.9 cells, from control pool, from the pool of pBI-EGFP-C/EBP-{delta}-transformed cells, and from pool of Dip-transformed cells. After blotting onto nitrocellulose, blots were reacted with anti-GFAP and anti-ERK1/2 antibodies.

 
The dissociation between P0 and GFAP regulation is further made evident upon overexpression of C/EBP-{delta} in the IL6RIL6-treated cells. At suboptimal concentrations of IL6RIL6 (35-70 ng/ml), the overexpression of C/EBP-{delta} markedly reduces GFAP RNA, whereas it increases P0 mRNA (Fig. 6B). Under these conditions, the P0/GFAP ratio increases from 0.6 in the control pool to about 2.5 in the C/EBP-{delta}-expressing pool. The decrease in GFAP when C/EBP-{delta} is overexpressed in IL6RIL6-treated cells was confirmed at the protein level (Fig. 6C). Like the induction of P0, the decrease of GFAP requires the N-terminal domain of C/EBP-{delta} and is not observed in the Dip pool (Fig. 6C).

As noted above, GFAP is increased early after IL6RIL6 addition and decreases again from 24 to 72 h (Fig. 6A). This decrease of GFAP at late time may be attributed to the negative effect of C/EBP-{delta} accumulation in the IL6RIL6-treated cells. In contrast, C/EBP-{delta} may contribute to the maintenance of a high level of P0 mRNA at late times. Interestingly, our experiments provide support for a late action of C/EBP-{delta}, because doxycycline acts more slowly than IL6RIL6 to induce P0 mRNA despite the fact that the C/EBP-{delta} RNA is observed from 6 to 72 h (Fig. 6A) and the C/EBP-{delta} protein level is already maximal at 12 h (not shown). The MBP mRNA followed the same pattern as P0 (not shown).

C/EBP-{delta} Down-regulates Pax3 and MITF—The transdifferentiation of the F10.9 cells from melanocytic to glial phenotype in response to gp130 activation is characterized by a decrease in Pax3 mRNA and protein, and a subsequent down-regulation of MITF and the melanogenesis pathway (13). Pax3 is a repressor of both MBP and P0 promoter activity (9) and is down-regulated during the differentiation of SC to the myelinating phenotype (4, 8). Analysis of the F10.9 C/EBP-{delta} pool showed that doxycycline caused a decrease in Pax3 protein at 48 h after treatment, whereas in the control pool doxycycline had no such effect (Fig. 7A). In both pools, as in the untransformed F10.9 cells, IL6RIL6 down-regulated Pax3. Screening the response of a number of individual C/EBP-{delta}-transformed clones to doxycycline indicated a good correlation between C/EBP-{delta} induction and Pax3 decrease. In addition, in these doxycycline-treated clones, there was a good correlation also between the Pax3 decrease and the induction of myelin P0 mRNA (Fig. 7B).



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  		FIG. 7.
Doxycycline-induced overexpression of C/EBP-{delta} inhibits Pax3c expression, regulating P0 and MITF mRNAs. A, Western blot analysis of radioimmune precipitation assay buffer extracts of F10.9, control, and C/EBP-{delta} pools, treated for 60 h with doxycycline and 48 h with IL6RIL6. Blot was reacted with anti-Pax3 and anti-ERK1/2 antibodies. Extracts of individual clones of C/EBP-{delta} as shown in Fig. 3 were also tested for Pax3 expression, and in parallel, RNA of the same clones were tested for P0mRNA levels. The recorded intensities of the bands were reported in the graph in B. The reverse-transcribed RNAs used in Fig. 5 were used to amplify the MITF mRNA (C).

 
The level of MITF mRNA was partially reduced by doxycycline treatment of the C/EBP-{delta} pool but not of the control pool (Fig. 7C). The decrease in MITF related to C/EBP-{delta} induction was, however, lower than that produced by IL6RIL6. As detailed elsewhere (13), Pax3 is only one of the transcription factors that regulate the MITF promoter and in addition to Pax3 decrease there is also a loss of the Pax3-Sox10 cooperative effect after IL6RIL6 treatment of the F10.9 cells. We examined also the level of tyrosinase, a key gene in melanogenesis, transactivated by MITF. Interestingly, we found that the basal level of tyrosinase mRNA was lower in the C/EBP-{delta}-transformed cells than in the control clones (not shown). The tyrosinase enzymatic activity present in the C/EBP-{delta} pool was also very low (Table I). Nevertheless, the induction of C/EBP-{delta} by doxycycline produced a further 10-fold decrease in tyrosinase activity, whereas in the control pool the drug did not significantly reduce the tyrosinase (Table I). Cells of the untreated C/EBP-{delta} pool showed also less black pigmentation granules than the control cells. This might result from leaky regulation of C/EBP-{delta} (Fig. 3A), or from a low survival rate of tyrosinase- and melanin-expressing cells during the selection with hygromycin. Whatever the reason, the leakiness or selective pressure did not seem to affect the other gene products examined in this study.


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  		TABLE I
Low constitutive levels of tyrosinase and inhibition by doxycycline in Tet-R-C/EBP-{delta}-transformed F10.9 cells

Cells seeded on 9-cm plates were incubated for 72 h, and four replicas of each sample extract were tested for tyrosinase enzymatic activity, using L-Dopa conversion to Dopa-quinone. Optical density at 570 nm against blank is shown.

 
The overall growth of the melanoma cells was also reduced as a result of C/EBP-{delta} induction by doxycycline (Table II). The reduction was smaller than the one caused by IL6RIL6 treatment and was not observed in the F10.9 cells or control transformed pool exposed to doxycycline. A much stronger growth inhibition was observed when we expressed C/EBP-{beta} under doxycycline control in F10.9 cells.2 Moreover, with C/EBP-{delta}, the epithelioid morphology of the melanoma cells was conserved after doxycycline (not shown), unlike the IL6RIL6-treated cells that undergo elongation and form extended cell arrays (13).


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  		TABLE II
Doxycycline inhibits the growth of Tet-R-C/EBP-{delta}-transformed F10/9 cells

Cells seeded in 96-well plates were incubated for 72 h, fixed, and stained with crystal violet. Ten replicas were analyzed for each condition. Optical density of the eluted dye at 540/630 nm and S.D. (between parentheses) are shown. The percentage of growth inhibition is calculated relatively to non-treated samples values.

 
C/EBP-{delta} mRNA Knockdown in Dissociated Embryonic Rat DRG Cells Reduces P0 mRNA Induction by IL6RIL6—Embryonic E14 DRG cells cultured in defined medium supplemented with fibroblast growth factor-2 contain a mixture of mature neurons and precursor cells common to neuron/glial cell lineages that can multiply and differentiate in the presence of various inducers (36). Treatment of such cells with IL6RIL6 increases P0 and C/EBP-{delta} mRNA levels, as verified by RT-PCR and analysis on DNA-arrays.3 In this system, the basal level of P0, and C/EBP-{delta} mRNAs in the absence of IL6RIL6 is not negligible. To evaluate the role of C/EBP-{delta} in P0 mRNA regulation in this system containing precursor glial cells, we infected the cells with viral particles containing pRS-C/EBP-{delta}2, and control pRS. RNAs were extracted after 40 h of infection with viruses and an additional 36 h of treatment with IL6RIL6. The percentage of cells infected with a virus of the same family expressing the fluorescent EGFP protein was about 70%. In Fig. 8 (A and B), two RT-PCR experiments, using RNAs coming from two different infections, are shown. We can see that, in cells infected with the virus pRS-C/EBP-{delta}2, not only C/EBP-{delta} mRNA but also P0 mRNA induction by IL6RIL6 is inhibited (Fig. 8), demonstrating that C/EBP-{delta} plays a role in the induction of P0 expression by IL6RIL6. RT-PCR reactions were performed with two different cDNA preparations of each RNA sample, and average values of PCR band intensities are reported on the graph (Fig. 8C). This graph shows that C/EBP-{delta} expression was very efficiently inhibited (about 3-fold), and P0 mRNA inhibited by 50-60%, in basal as well as IL6RIL6-induced levels.



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  		FIG. 8.
C/EBP-{delta} siRNA inhibit P0 mRNA expression in dissociated cells of embryonic DRGs. A and B, RT-PCR analysis of C/EBP-{delta} and P0 in RNAs coming from two experiments (A and B) of infection of DRG cells with pRS control (ctrl) and pRS-C/EBP-{delta}2 retroviruses. Cells were treated or not with IL6RIL6 for the last 36 h of the culture. In part C, RT-PCR images were scanned, the intensities of PCR products were evaluated by Image Gauge (intensities related to glyceraldehyde-3-phosphate dehydrogenase), and the average values were related to values obtained with the control samples in the presence of IL6RIL6 as 100%.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The preferential induction of C/EBP-{delta} by the gp130 activator was observed at all time points from 3 to 48 h in the F10.9 cells. In comparison, C/EBP-{beta} (NF-IL6) was much less enhanced. None of the other C/EBP family members scored significant changes in the gene microarray analysis. A similar pattern was seen in cultures of rat embryo E14 DRG cultures treated 48 h with IL6RIL6 and exhibiting differentiation of SC to the myelinating phenotype (8): the mean activation of C/EBP-{delta} was about 15-fold, versus about 3.5-fold for C/EBP-{beta}.2 Silencing of C/EBP-{delta} gene expression indicates that this transcriptional regulator is necessary for the induction and/or accumulation of myelin P0 and MBP gene transcripts in response to the gp130 triggering by IL6RIL6 in F10.9 cells. Moreover, overexpression of C/EBP-{delta} in these cells without IL6RIL6 treatment leads to increase of P0 and MBP mRNAs comparable to those produced by IL6RIL6. This effect of C/EBP-{delta} is selective and not seen for several other IL6RIL6-dependent gene products, indicating that C/EBP-{delta} does not mediate all the effects of the cytokine. The slower kinetic observed for the rise of P0 and MBP RNA in cells expressing C/EBP-{delta} as compared with cells treated by IL6RIL6 would further argue for a different mode of action. We previously demonstrated that reporter genes under the control of the P0 and MBP promoter sequences are strongly activated by IL6RIL6 and identified P0 promoter elements involved on the response as well as positively acting transcription factors, including Sox10, ZBP99, and Krox20/Egr2 (9). Attempts to demonstrate similar promoter activation following induction of C/EBP-{delta}, or inhibition of the effect of IL6RIL6 on the reporter genes by silencing C/EBP-{delta} expression, have, however, remained negative. Thus, after C/EBP-{delta} induction by doxycycline for 48-72 h there was no significant effect on a reporter gene containing 1.2 kb of 5'-flanking sequences of the rat P0 promoter (data not shown). More investigations will be needed to determine whether C/EBP-{delta} regulates P0 gene transcription or regulates the post-transcriptional accumulation of P0 transcripts. The fact that cells transformed with C/EBP-{delta} deleted of its N-terminal activation domain (Dip) do not produce cellular P0 transcripts and partially decrease the level of transcripts observed after IL6RIL6 treatment, does confirm that C/EBP-{delta} through its regulatory domain plays some important role in the accumulation of the cellular myelin P0 mRNA. The gp130-mediated signaling by IL6RIL6 may trigger several mechanisms inducing myelin gene transcripts: some of these promote transcription from proximal promoter sequences, whereas others could be later post transcriptional regulatory effects or may act on other gene or chromatin regulatory elements. In this respect the recruitment of p300 by C/EBP factors may be of interest (49). The present results would indicate that only some of these multiple effects are mediated by C/EBP-{delta}.

Another major outcome of IL6RIL6 is the down-regulation of Pax3, which is observed in the F10.9 cells (13) as well as in embryonic DRG cells (8), and is part of the differentiation of SC to the myelinating stage (2). Overexpression of C/EBP-{delta} resulted in a decrease of the Pax3 protein level in the F10.9 cells. However, silencing of the C/EBP-{delta} by siRNA did not abolish the Pax3 decrease caused by IL6RIL6, suggesting again that C/EBP-{delta} is not the sole mediator involved in IL6RIL6 action. In addition to its role as a repressor of the MBP and P0 promoters (4, 9), Pax3 has (in synergism with Sox10) a positive transcriptional effect on the MITF promoter and we have shown that Pax3 decrease by IL6RIL6 is involved in the loss of MITF, tyrosinase, and melanogenesis (13). Decrease in MITF and low tyrosinase were observed here in the C/EBP-{delta} transformed F10.9 cells. Pax3 and MITF also promote growth and survival of melanoma cells (10, 50, 51) and C/EBP-{delta}, like IL6RIL6, reduced F10.9 cell growth. An involvement of C/EBP-{delta} in GADD45-{gamma} induction and growth arrest was reported (52). Because other factors such as IL-1, transforming growth factor-{beta}, tumor necrosis factor-{alpha}, and hepatocyte growth factor/scatter factor can reduce melanogenesis in melanoma cells (53-55), it may be of interest to investigate the role of C/EBP-{delta} in the context of these cytokines as well.

A major finding in our study is that C/EBP-{delta} differentially affects the expression of GFAP versus that of myelin P0 and MBP mRNAs. This may be relevant for the switch from embryonic and adult non-myelinating SC, expressing GFAP (56), to the myelinating phenotype expressing the myelin gene products; this switch being operative not only in the embryo development but recapitulated after axonal injury during nerve regeneration (2). Gp130 signaling by cytokines of the IL-6 family, in particular ciliary neurotrophic factor, is known to be involved in the induction of GFAP expression and astrocyte differentiation (47, 48). In embryonic E14 DRG cell cultures, addition of IL6RIL6 nevertheless increased myelin gene expression much more than GFAP, which was even sometimes decreased (8). The rise in C/EBP-{delta} following IL6RIL6 may be one of the mechanisms reducing GFAP mRNA, as seen here by overexpression of C/EBP-{delta} in IL6RIL6-treated F10.9 cells. Under these conditions, the ratio of myelin P0 to GFAP mRNAs was much higher when C/EBP-{delta} was elevated. Conversely, we found that silencing of C/EBP-{delta} in the IL6RIL6-treated cells, using either specific C/EBP-{delta} siRNA (this study), or overexpression of the C/EBP antagonist A-C/EBP2 (19), led to a low ratio of P0 to GFAP (not shown). In embryonic brain cortical cells, the inhibition of C/EBP activity by A-C/EBP was found to enhance the ciliary neurotrophic factor-mediated generation of astrocytes, which are characterized by expression of GFAP (19). In the same system, the A-C/EBP was found to inhibit neuron differentiation, although it could not be determined which member of the C/EBP family is involved in these two effects. Our results indicate that C/EBP-{delta} has the capacity to down-regulate GFAP expression, and it is possible that its inhibition was responsible for the enhanced GFAP in the study of Menard et al. (19). In this hypothesis, the marked elevation of C/EBP-{delta} caused by gp130 activation may contribute to driving glial cells toward myelinating functions in the brain as well as in the peripheral nervous system.

To confirm the role of C/EBP-{delta} in non-transformed cells, we used a system of dissociated embryonic DRG cells containing Schwann cell precursors, cultured in defined medium allowing cell multiplication and maturation (36). The response to IL6RIL6 of this cell population has been analyzed at the RNA level by RT-PCR and on gene arrays after 24 and 48 h of treatment.3 We used viral particles containing the vector for C/EBP-{delta}-specific siRNAs to knockdown C/EBP-{delta} mRNA. We used cells shortly after infection and found that knocking down C/EBP-{delta} inhibited P0 mRNA expression. The phenomenon observed in F10.9 cells can thus be observed also in non-transformed cells, suggesting that C/EBP-{delta} is one of the components of the mechanisms up-regulating P0 mRNA during Schwann cell maturation.

C/EBP factors were not implicated before in the induction of myelin genes or in the myelinating process. C/EBP factors, and particularly C/EBP-{delta} and C/EBP-{beta}, are redundant in their activities. C/EBP-{delta}/C/EBP-{beta} double knockouts are deficient in brown lipid tissue and die at early neonatal stage (16). Mice with only C/EBP-{delta} gene inactivation are viable, but were reported to exhibit neural/behavioral alterations (57). That C/EBP-{delta} has the capacity to up-regulate expression of myelin genes and to down-regulate GFAP may have significant developmental implications.


   FOOTNOTES
 
* This work was supported by InterPharm (Weizmann Industrial Park, Israel) and Serono Group (Geneva, Switzerland). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 972-8-934-2103 (or -2101); Fax: 972-8-9343174; E-mail: michel.revel{at}weizmann.ac.il.

1 The abbreviations used are: SC, Schwann cell(s); P0, myelin protein zero; MBP, myelin basic protein; GFAP, filament glial fibrillary acid protein; DRG, dorsal root ganglia; IL-6, interleukin-6; MITF, microphtalmia; Stat3, signal transducer and activator of transcription 3; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcription; EGFP, enhanced green fluorescent protein; siRNA, small interference RNA; rtTA, reverse tetracycline-controlled transcriptional activator; ERK, extracellular signal-regulated kinase. Back

2 A. K. Kamaraju, L. Ben Simchon, S. Saban, J. Chebath, and M. Revel, manuscript in preparation. Back

3 P. Zhang, L. Ben Simchon, S. Saban, J. Chebath, and M. Revel, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Reuven Agami for the gift of the p-SUPER plasmid and to Dr. Richard Schwartz for the gift of pBabe C/EBP-{delta} and C/EBP-{beta} plasmids. The assistance of Raya Zwang, Rosalie Kaufmann, Zipora Marks, and Lia Chazin is gratefully acknowledged. We are especially grateful to Dr. Dalia Gurari for developing the Tet-on system and supervising its analysis. We thank Dr Levana Ben-Simchon for bioinformatics expertise.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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