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Glucocorticoid-induced Leucine Zipper (GILZ) and Long GILZInhibit Myogenic Differentiation and Mediate Anti-myogenicEffects of Glucocorticoids*□S

Received for publication, September 28, 2009, and in revised form, January 8, 2010 Published, JBC Papers in Press, February 2, 2010, DOI 10.1074/jbc.M109.070136

Stefano Bruscoli‡1, Valerio Donato‡1, Enrico Velardi‡1, Moises Di Sante‡, Graziella Migliorati‡, Rosario Donato§,and Carlo Riccardi‡2

From the ‡Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, and the§Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Anatomia, Universita di Perugia, 06122 Perugia, Italy

Myogenesis is a process whereby myoblasts differentiate andfuse intomultinucleatedmyotubes, the precursors ofmyofibers.Various signals and factorsmodulate this process, and glucocor-ticoids (GCs) are important regulators of skeletalmusclemetab-olism. We show that glucocorticoid-induced leucine zipper(GILZ), a GC-induced gene, and the newly identified isoformlong GILZ (L-GILZ) are expressed in skeletal muscle tissue andin C2C12 myoblasts where GILZ/L-GILZmaximum expressionoccurs during the first few days in differentiation medium.Moreover, we observed that GC treatment of myoblasts, whichincreased GILZ/L-GILZ expression, resulted in reduced myo-tube formation, whereas GILZ and L-GILZ silencing dampenedGCeffects. Inhibition of differentiation causedbyGILZ/L-GILZoverexpression correlatedwith inhibitionofMyoD function andreduced expression of myogenin. Notably, results indicate thatGILZ and L-GILZ bind and regulate MyoD/HDAC1 transcrip-tional activity, thus mediating the anti-myogenic effect of GCs.

Glucocorticoids (GCs)3 are important agents widely em-ployed in the therapy of inflammatory, autoimmune, and neo-plastic diseases (1). They regulate cell survival, proliferation,and differentiation by modulating the expression of a variety ofmolecules and signaling cascades, in many cells and tissues. Inparticular, GCs are potent modulators of skeletal musclemetabolism, regulating the expression of contractile proteinsand promotingmuscle atrophy in vivo and in vitro (2, 3). More-over, GC receptor activation takes part in angiotensin II-relatedmuscle wasting (4). Recent reports have shown that activation

of FoxO proteins and the consequent activation of the ubiq-uitin-proteasome pathway represent the molecular mecha-nisms responsible for GC-mediatedmuscle atrophy (5, 6). Nev-ertheless, GC effects on differentiatingmyoblasts have not beenextensively investigated. In fact, despite the evidence that dexa-methasone (DEX) treatment results in reducedmyogenesis andinhibition of the activation of the adult stem cells in skeletalmuscle tissue known as satellite cells (7, 8), themolecular deter-minants of these biological effects are still poorly understood.We have previously identified aGC-induced, 15-kDa protein

that we named glucocorticoid-induced leucine zipper (GILZ),whichmediates some of the effects ofGCs, such as regulation ofthymocyte survival (9, 10), inhibition of NF-�B transcriptionalactivity (11–14), counteraction of extracellular signal-regulatedkinases (ERKs) 1/2 activation (15, 16), and inhibition of Ras-driven cell proliferation and oncogenic Ras-dependent trans-formation (17). GILZ expression is not restricted to lymphoidcells, and GILZ has been shown to play regulatory roles in adi-pocytes, osteoblasts, and tubular renal cells (18–21).Moreover,this factor is expressed in a variety of tissues, including skeletalmuscle tissue (22).Myogenesis is a multistep process by which undifferentiated

mononucleated precursors, the myoblasts, differentiate andfuse into multinucleated myotubes. This process takes placeduring skeletal muscle tissue development and during regener-ation of damaged skeletal muscle tissue; in this latter case, skel-etalmuscle adult stemcells, the satellite cells, become activated,proliferate, and differentiate into fusion-competent myoblasts(23, 24). The myogenic development program is tightly regu-lated, in which myoblasts exit the cell cycle and express themuscle-related factors, includingMyoD,which is the best char-acterized. MyoD activation represents the convergence of sev-eral signals from the plasma membrane to the nucleus, such asthe activation of the pro-myogenic kinases p38 and Akt by thereceptor for insulin and insulin-like growth factors 1 and 2 (25),the receptor of advanced glycation end products (26), and thecell-to-cell contact signaling mediators CDO (cell adhesionmolecule-related/down-regulated by oncogenes) and N-Cad-herin (27, 28). MyoD, which belongs to the basic helix-loop-helix protein superfamily, is expressed in proliferating, undif-ferentiated myoblasts, exhibiting a nuclear localization. In thenucleus MyoD is bound to Id1 and HDAC1, which renderMyoD inactive (24, 29, 30). Sustained p38 and Akt kinase activ-ities promote MyoD effects by enhancing its activation and

* This work was been supported by grants from Associazione Italiana per laRicerca sul Cancro Milan (to C. R.) and by Ministero Istruzione Universitadella Ricerca, Fondo per gli Investimenti della Ricerca di Base GrantRBPR05NWWC CHEM-PROFARMA-NET.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 These authors contributed equally to this work.2 To whom correspondence should be addressed: Via del Giochetto, 06122

Perugia, Italy. Tel.: 390755857467; Fax: 390755857405; E-mail: [email protected].

3 The abbreviations used are: GC, glucocorticoid; GILZ, glucocorticoid-in-duced leucine zipper; L-GILZ, long GILZ; DEX, dexamethasone; TGF, trans-forming growth factor; DMEM, Dulbecco’s modified Eagle’s medium; GM,growth medium; DM, differentiation medium; TSA, trichostatin A; HA,hemagglutinin; shRNA, small hairpin RNA; PBS, phosphate-buffered saline;Ab, antibody; mAb, monoclonal Ab; MyHC, myosin heavy chain; IP, immu-noprecipitation; ChIP, chromatin immunoprecipitation; ORF, open readingframe; RNAi, RNA interference.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 14, pp. 10385–10396, April 2, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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binding to co-factors such as chromatin regulators (31–33).Activation of MyoD leads to the induction of myogenin, anearly differentiationmarker that is amuscle-related factor itselfand is required for cell-to-cell fusion (24). Intriguingly, duringthe differentiation process, not all myoblasts undergo fusion; afraction of them enter quiescence after cell cycle withdrawaland constitute a pool of reserve cells that reactivate, proliferate,and fuse after muscle damage to repair damaged myofibers.The events regulating the preservation of this pool of undiffer-entiated cells are still poorly understood. It is known thatmem-bers of the TGF-� superfamily (i.e. TGF-� and myostatin),which are expressed and released by differentiating cells, act inan autocrinemanner to counteractMyoD transcriptional activ-ity and promote quiescence (34, 35).In this study, we analyzed the expression and role of GILZ in

GC-mediated inhibition of myogenic differentiation. Duringthe course of these studies, we observed that, in addition to the15-kDa GILZ, differentiating myoblasts expressed a 28-kDaGILZ isoform that we named long GILZ (L-GILZ). We foundthat both GILZ and L-GILZwere induced by DEX and contrib-uted to DEX anti-myogenic effects. Moreover, GILZ andL-GILZ expression spontaneously rose during myoblast differ-entiation. Notably, both GILZ isoforms affected HDAC1 activ-ity on myogenin promoter and thus inhibited MyoD transcrip-tional activity. Collectively, our data show that GILZ andL-GILZmediate theDEXanti-myogenic effect duringmyoblastdifferentiation.

EXPERIMENTAL PROCEDURES

Cloning of L-GILZ—We designed primers according to thesequence uc009ulb.1 (University of California Santa Cruz(UCSC) Genome Browser mouse data base), which representsthe longest transcript of GILZ gene. Total RNA isolated fromC2C12 cells was extractedwithTRIzol reagent (Invitrogen) andretro-transcribed with SuperScript III (Invitrogen). The prim-ers used were 5�-CACTCCCCTTCTCACTCTGC-3� (sense)and 5�-GAACTTTATAAGCAGTCATCCC-3� (antisense).Cell Cultures and Reagents—Primary myoblasts were iso-

lated from 7-day-old C57Bl6/J mice using a modification of theprocedure described by Rando and Blau (36). In brief, the cellswere isolated from dissected muscle by trypsin (Invitrogen)treatment and were preplated onto tissue culture plastic twicefor 1 h. The cells were cultured in DMEMwith 20% fetal bovineserum. C2C12 myoblasts were obtained by Dr. Pier LorenzoPuri (The Burnham Institute, La Jolla, CA) and were cultivatedin DMEM, 20% fetal calf serum (growth medium (GM)) or dif-ferentiation medium (DM) (DMEM, 2% horse serum) at 37 °Cwith 5%CO2. TGF-�, trichostatin A (TSA), andDEXwere pur-chased from Sigma-Aldrich.Cell Transfections and Plasmids—C2C12 were transfected

with Lipofectamine 2000 (Invitrogen). 24 h after transfection,the cells were switched to DM to induce myogenic differentia-tion. We transfected C2C12 with an enhanced green fluores-cent protein vector to evaluate transfection efficiency (rangingfrom 40 to 45%) by fluorescence-activated cell sorter analysis.GILZ-Myc and L-GILZ-FLAG were cloned in pcDNA3.1(Invitrogen), pcDNA3-MyoD-HA was a gift from Dr. MilenaGrossi (University of Rome, “La Sapienza,” Rome); pGL3-myo-

genin-luc (MyoG-luc) was a gift from Dr. Pier Lorenzo Puri(The Burnham Institute, La Jolla, CA).Retroviral Infections—shRNA for GILZ and L-GILZ knock-

down were inserted into pSUPER.retro.puro (Oligoengine).The target sequences were: GILZ, 5�-CAAUUUCUCCAUCU-CCUUC-3�; L-GILZ, 5�-CACUGACAAGCUGAACAAC-3�;GILZ/L-GILZ, 5�-ACAGCUUCACCUGACAAUG-3�; andscramble, 5�-GUACCGGACGAGUUAGAAC-3�. Retroviruseswere generated with Phoenix packaging cells following theNolan Lab protocol. 48 h after transfection, the medium wascollected, filtered, and added with polybrene to a suspension of105 C2C12 cells.Real Time PCR Analysis—Total RNA was extracted using

TRIzol (Invitrogen). Reverse transcription-PCRwas done usingQuantiTect reverse transcription (Qiagen). For real time PCR,the primers were: for GILZ, sense 5�-GGTGGCCCTAGA-CAACAAGA-3� and antisense 5�-TCTTCTCAAGCAGCTC-ACGA-3�; for L-GILZ, sense 5�-ACCGCAACATAGACCAG-ACC-3� and antisense 5�-TCTTCTCAAGCAGCTCACGA-3�;and for glyceraldehyde-3-phosphate dehydrogenase, sense 5�-GCCTTCCGTGTTCCTACCC-3� and antisense 5�-CAGTG-GGCCCTCAGAUGC-3�. PCR was done in CHROMO 4 (MJResearch Bio Rad, Milan, Italy) using a DyNAmo HS SYBRGREEN qPCR kit (Finnzymes; Celbio). Relative amounts ofGILZ, L-GILZ, and glyceraldehyde-3-phosphate dehydro-genase mRNA were calculated by the Comparative ��C(t)method. TheC(t) values were determined using OpticonMon-itor 2 software (MJ Research Bio Rad).May-Grunwald-Giemsa Staining—The differentiation rate

was determined by calculating themyogenic index as a percent-age of nuclei inmyotubes relative to total nuclei. 48 h after DM,the cells were fixed with paraformaldehyde (4% in PBS) for 10min at room temperature and stained with May-Grunwald-Giemsa (Carlo Erba, Milan) as previously described (26). Thecells were viewed in a phase contrast microscope (OlympusIX51) equipped with a digital camera (Olympus C-5050ZOOM), and the images were acquired at 20� magnification.The images were analyzed using UTHSCSA ImageTool soft-ware for calculation of the myogenic index. We consideredmyotubes only multinucleated cells containing at least threenuclei.Immunofluorescence—Cells seeded directly on glass cover-

slips were subjected to transfection after adherence. The cellswere fixed with 4% paraformaldehyde for 10 min at room tem-perature and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 30 min at room temperature. After threewashes with PBS, the cells were treated with sodium borohy-dride (Sigma-Aldrich) 1 mg/ml for 5 min at room temperature,washed again with PBS, and blocked overnight (3% bovineserum albumin and 1% glycine in PBS). Primary and secondaryAbs were incubated for 1 h at room temperature in PBS, 3%bovine serum albumin, and 0.1% Triton X-100. Primary Abswere: mouse mAb anti-Myc (Invitrogen); mouse mAb anti-FLAG (Sigma-Aldrich). The anti-mouse secondary Ab wasAlexa-Fluor 568-conjugated (Molecular Probes). Imageacquisition was performed using a Leica (Milano, Italy)microscope equipped with Diagnostic Instruments Spot RTColor camera under oil immersion (Sigma) at 100� magni-

GILZ and L-GILZ Inhibit Myogenesis

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fication; image merging was obtained using DiagnosticInstruments Spot Advance software.For GILZ protein endogenous detection, the myoblasts were

stained with GILZ monoclonal antibody (eBioscience) andmounted with Prolong antifade (Invitrogen). Confocal pho-tomicrographs of immunostained tubules were acquired with aLeica TCS SP2 (Leica) equipped with three laser lines (argon488, 543, and 633 nm). Each channel was acquired separatelyusing specific laser lines to avoid bleed-through of the fluoro-chromes. The photomicrographs were acquired with LAS AFSoftware (Leica) at 1024 � 1024 pixels.Immunohistochemistry—To detect myosin heavy chain

(MyHC) by immunocytochemistry, C2C12 myoblasts culti-vated in DM were fixed in cold methanol at �20 °C for 7 minand subjected to immunocytochemistry with a monoclonalanti-developmental MyHC antibody (Biogenesis) at a 1:1,000dilution. The immune reaction product was visualized usingthe Vectastain Elite ABC kit (Vector Laboratories Inc.). Theimages were acquired at 20� magnification.Immunoprecipitation—For endogenous co-immunoprecipi-

tation (co-IP) assays, whole cell extracts were prepared withnondenaturing lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl,1% Nonidet P-40, 5 mM EGTA). Immunoprecipitations wereperformed in co-IP buffer (50 mM Tris, pH 7.5, 150 mM NaCl,1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA);antigen-antibody complexes were precipitated with protein Abound to agarose beads (Sigma-Aldrich) prior to SDS-PAGE.For IP rabbit anti-MyoD Ab (Santa Cruz) and control isotypeAb (Sigma-Aldrich) were used. For exogenous co-IP assays,HEK293 cells were transfected with different combinations ofexpression vectors for GILZ-Myc, MyoD-HA, HDAC1-FLAG,and HDAC2-FLAG. Total proteins were immunoprecipitatedwith an anti-Myc Ab (Invitrogen), and co-IP was revealed withanti-HA (Santa Cruz) or anti-FLAG (Sigma-Aldrich) Abs.Western Blot—The proteins were separated on a SDS-PAGE

and subjected to Western blotting as previously described(37). The following primary Abs were used: anti-GILZ (SantaCruz), mAb anti-MyoD (Novocastra), mAb anti-myogenin(BD Pharmingen), mAb anti-MyHC (Novocastra), mAbanti-HDAC1 (Upstate Biotech), mAb anti-�-tubulin (Sigma-Aldrich), mAb anti-Myc and mAb anti-HA (Invitrogen), andmAb anti-FLAG (Sigma-Aldrich). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary Abs werefrom Thermo Scientific. Polyvinylidene difluoride mem-brane (Hybond plus) was from Amersham Biosciences.Western blot films were scanned, and band signal intensitieswere determined using Scion Image software (ScionCorporation).Luciferase Assay—MyoDactivity onmyogenin promoterwas

performed using a kit from Roche Applied Science, followingthe manufacturer’s instructions. Luciferase activity was mea-sured 6 h after switching transfected C2C12 cells to DM.West-ern blot analysis with anti-HA, anti-FLAG, and anti-Myc Abswas assessed as described in the previous paragraph to checkthe protein levels of all ectopically expressed proteins (data notshown).Chromatin Immunoprecipitation (ChIP)—The anti-HDAC1

and anti-acetyl-histone H3 (Upstate Biotech) ChIP was per-

formed according to the protocol of ChIP assay kit fromUpstate Biotech. The experiments were performed using 5 �106 cells for sample. 2�g of anti-HDAC1 or anti-acetyl-histoneH3 or nonimmune rabbit IGG was added to the cell lysates forimmunoprecipitations. After elution, the samples were depro-teinated, and DNAwas resuspended after precipitation with 20�l of double distilled H2O. The samples were quantified by realtime PCR using an Mx3000P real time PCR system (Strat-agene). The sequences of the primers against the mouse myo-genin promoter region used for CHIP were as follows: forward,5�-GAATCACATGTAATCCACTGGA-3�, and reverse, 5�-ACGCCAACTGCTGGGTGCCA-3�. As negative control forCHIP PCR, a nonpromoter genomic region was amplifiedwith the following primers: forward, 5�-TCAGAACCCAA-CTCCTTTGG-3�, and reverse, 5�-GCCTTCACAAGAGC-AGGAAC-3�. The relative amount of immunoprecipitatedDNA fragments were determined based on the thresholdcycle (Ct) for each PCR product (38). The data were quanti-tatively analyzed according to the formula 2��[Ct(IP)�Ct(input)] �2��[Ct(control IgG)�Ct(input)].Enzyme-linked Immunosorbent Assay—Supernatants from

differentiating C2C12 were collected, and TGF-� content wasevaluated by sandwich enzyme-linked immunosorbent assay,following the manufacturer’s recommendation. Anti-TGF-�antibody was purchased from Pharmingen.Statistical Analysis—All of the experiments were repeated at

least three times. Student’s t test was used with the STATPACcomputerized program for data analysis, and p� 0.05 was con-sidered significant.

RESULTS

GILZ Is Expressed in Muscle, and L-GILZ Is an AlternativeTranscript Encoded by GILZ Gene—It has been previouslyreported that GILZ is expressed in a variety of human tissues(22). With real time PCR, we analyzed the expression of GILZmRNA in different mouse tissues (Fig. 1A). We found thatGILZ is expressed in various tissues, including adult skeletalmuscle where its expression is comparable with that in lymph-oid tissues, such as spleen (Fig. 1A). These results were con-firmed byWestern blot analysis (Fig. 1B). Interestingly, in addi-tion to the expected 15-kDa GILZ band, a 28-kDa band wasdetected both in spleen andmuscle extracts (Fig. 1B). DifferentGILZ splice variants, arising from alternative transcript of thefirst exon in the GILZ gene have been already described (16),but no one translates a protein of a putative molecular mass of�28 kDa. To investigate the nature of this protein and its pos-sible relationship to GILZ, we initially performed a homologysearch in the UCSCGenome Browser BLAT search tool usingGILZ cDNA sequence (GenBankTM accession numberAF024519). Fig. 1C shows a schematic map of the analysis forGILZgenomic locus. Because a splice variant generated by exon1b-2 that splices on GILZ exon 3 and 4 has been previouslydescribed (16), we investigated its expression in C2C12murinemyoblast cell line. Primers corresponding to the 5�- and 3�-un-translated regions were used for reverse transcriptase PCR oftotal RNA from C2C12 myoblasts. PCR product was clonedinto pcDNA3.1 vector, and we named this sequence L-GILZ(GenBankTM accession number EU 818782). The longest open




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FIGURE 1. A, GILZ mRNA expression in murine tissues. Total mRNA was isolated from both immune system-related and not related adult mouse tissue andunderwent real time PCR analysis for GILZ mRNA semi-quantitative expression related to the housekeeping gene as described under “Experimental Proce-dures” (n � 3). GILZ mRNA from fresh thymus was used as unit. B, Western blot analysis of total protein extracted from adult mouse skeletal muscle and spleen.An anti-GILZ antibody recognizes the expected GILZ band and a higher molecular mass band that is expressed both in muscle and spleen. Western blot of�-tubulin is included to show total protein loading. C, schematic representation of GILZ locus. The conventional GILZ exons are named 1a, 3, and 4; exons 1band 2 are alternative exons, and they represent the origin of a putative alternative GILZ isoform. D, schematic representation of GILZ alternative isoforms. Thetwo predicted proteins share an amino acidic sequence that includes the TGF-�-stimulated clone box (TSC), leucine zipper domain (LZ), and proline- andglutamic acid-rich region (PER); the striped box represents region of identity. E, representation of L-GILZ ORF. Translation starts at a noncanonical CUG startcodon, which is related to the most conserved Kozak sequence. F and G, mutagenic analysis of L-GILZ ORF. In F mutational strategy is schematically repre-sented. Starting from L-GILZ ORF, a mutant carrying a point mutation on the CUG translation start site (L-GILZ-CUG�) and a mutant carrying a deletionupstream of the canonical AUG start codon (�L-GILZ) were generated. In G, WT L-GILZ and the mutants L-GILZ-CUG�and �L-GILZ were transfected in HEK293cells. Western blot analyses with anti-GILZ antibody confirmed that the noncanonical CUG start codon is the translation start site and revealed that synthesisof a shorter protein (�22 kDa) is allowed when the CUG codon is inactivated or when the ORF region upstream of the canonical AUG is deleted. WT, wild type.




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reading frame (ORF) predicted by software analysis startingfrom the first ATG, at position �202, is 22 kDa, but transienttransfection of HEK293 cells with pcDNA3.1-L-GILZ resultedin the expression of a �28-kDa protein, similar to the banddetected in muscle tissue (Fig. 1G, first lane). To explain thisdiscrepancy, we postulated that this new variant starts from anoncanonical non-AUG translation start codon. Indeed, a non-canonical non-AUG (CUG) translation start codon is present atposition �103, which is characterized by a high degree ofhomology with the Kozak consensus sequence (Fig. 1E). Thishypothetical splice variant generated a transcript with an ORFof 705 bp and encodes a protein of 234 amino acids with apredictive molecular mass of 26-kDa that differed from GILZprotein only in the N-terminal part (National Center for Bio-technology Information (NCBI) accession number ACJ09091).This transcript shared an identical conserved TGF-�-stimu-lated clone (TSC) box, leucine zipper domain, and C-terminalregion with GILZ (Fig. 1D). To confirm the involvement of thealternative translational start site, we generated a L-GILZ-CUG� mutant in which the thymidine of the starting codon ofthe cDNA was replaced by an adenosine (CUG to CAG). Thismutation impaired L-GILZ transcription, and only a shorterprotein product was detected following transfection (Fig. 1G,second lane). Thus, disruption of the starting CUG codonallowed the translation of a protein starting from a downstreamin-frame AUG codon, characterized by a molecular mass of�22 kDa (Fig. 1, E and F). Next, we generated a GILZ mutant,named �L-GILZ, in which DNA started from position �202,deleting the part including the CUG start codon (Fig. 1F).Transfection of this mutant form in HEK293 cells induced theexpression of a 22-kDa protein exhibiting an electrophoreticmigration profile identical to that of the L-GILZ-CUG�mutant(Fig. 1G, third lane). Taken together, these results demonstratethat the new GILZ transcript variant L-GILZ generates a28-kDa protein, which is characterized by a non-AUG transla-tional start codon (NCBI accession number ACJ09091).Glucocorticoids Inhibit Myoblast Differentiation in a GILZ/

L-GILZ-dependent Manner—GILZ has been previously de-scribed as a GC-induced protein (9, 11). Because GCs areinvolved in skeletal muscle metabolism both at a physiologicaland pharmacological level, we treated themwithDEXundiffer-entiated primary myoblasts derived from neonatal muscle ofC57BL/6 mice. We performed Western blot analysis in myo-blasts during differentation in vitro (DM) with or without DEX10�6 M treatment. The results indicated that GILZ isoformswere not present in undifferentiated myoblasts (maintained inGM), whereas their expression was induced in differentiationconditions.Moreover, GILZ and L-GILZwere strongly up-reg-ulated by DEX 10�6 M treatment as evidenced by proteinexpression (Fig. 2A). Real time analysis confirmed the up-reg-ulation of GILZ and L-GILZ mRNA expression induced byDEX (Fig. 2, B and C). Together these results indicated thatGILZ and L-GILZ are expressed in murine myoblast duringdifferentiation and that GCs control L-GILZ as well as GILZexpression, as previously shown in other tissues (9, 21).Next, to address the role of GILZ and L-GILZ in GC effects

during myogenic differentiation, we employed C2C12 myo-blasts, an established model of in vitro myogenesis (39). Mor-

phological analyses, evaluated by staining of MyHC, a muscledifferentiationmarker, revealed a reduction ofmyotube forma-tion inDEX-treated cells comparedwith the untreated controls(Fig. 3A). We also found that DEX treatment at 10�5 M, a con-centration that is known to be pro-atrogenic in differentiatedmyotubes (5), caused a sustained induction of GILZ andL-GILZ expression (Fig. 3B) and reduced C2C12 myogenicpotential at early differentiation stages. Indeed, in addition to areduction of the fusion index, we observed a significant reduc-tion of myogenin expression (Fig. 3B). On the contrary, MyoDprotein did not undergo significant regulation (Fig. 3B).To test the contribution of GILZ/L-GILZ to DEX-induced

regulation of myogenesis, we evaluated DEX effects on GILZand/or L-GILZ silenced cells. We performed gene knockdownexperiments by targeting GILZ and L-GILZ transcripts withspecific shRNA sequences cloned into pSUPER.retro vector.Control cells were infected with a pSUPER.retro vector con-taining a scramble sequence. Gene knockdown efficiency wasassessed in each experiment with real time PCR, with a degreeof knockdown greater than 70% (not shown). Confluent controland L-GILZ/GILZ knockdown cells were switched to DM andsubjected to MyHC staining 48 h later. We found that thesimultaneous GILZ and L-GILZ knockdown resulted in a com-

FIGURE 2. DEX up-regulates GILZ and L-GILZ expression in primary myo-blast cultures. A, Western blot analyses of GILZ/L-GILZ in primary myoblastsinduced to differentiate for a 48-h absence (CNTRL) or in the presence of 10�6

M DEX. B and C, real time analysis of GILZ (B) and L-GILZ (C) expression inprimary myoblasts induced to differentiate for 24 h absence (CNTRL) or inpresence of 10�6

M DEX (n � 3).




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plete reversal of DEX-mediated anti-myogenic effects, asrevealed by inhibition of myotube formation (Fig. 4A, comparepanels b and d). Moreover, the individual contribution of GILZand L-GILZ was assessed by shRNAs targeting specific forGILZ or L-GILZ mRNA sequences. The results showed thatknocking down individual isoforms similarly dampened DEXactivity (not shown). Furthermore, performing Western blotanalysis to evaluate the expression of myogenic markers of dif-ferentiation, we found that GILZ and L-GILZ knockdown con-ferred resistance toDEX-induced inhibition ofmyogenin but notof MyoD expression, at 24 h upon DM switch (Fig. 4, B and C,compare second and third rows). To note, after 24 h in DM, thelate myogenic marker of differentiation MyHC alreadyappeared in GILZ/L-GILZ silenced myoblasts, either with orwithoutDEX treatment (Fig. 4,B andC, first rows). Collectively,these results support the conclusion that GILZ and L-GILZ

share anti-myogenic activity andmediate DEX-induced anti-myo-genic effects.GILZ and L-GILZ Expression Is

Developmentally Regulated duringMyogenesis, and Their DeregulationAffects Myoblast Differentiation—We found that GILZ and L-GILZwere undetectable in undifferenti-ated proliferatingmyoblasts, where-as both isoforms, either at the pro-tein (Fig. 5A) or the mRNA (Fig.5B) level, were expressed duringmyoblast differentiation, at a timewhen myotube formation becameevident.To elucidate the possible partici-

pation of GILZ and L-GILZ in themyogenic differentiation program,we transfected preconfluent C2C12cells with GILZ-Myc- or L-GILZ-FLAG-expressing vectors, and 24 hlater the cells were induced to dif-ferentiate by transfer to DM, andthe extent of myogenic differentia-tion was evaluated by morphologicand biochemical analyses. C2C12myoblasts transiently transfectedwith a pcDNA3.1 empty vectorserved as a control. We found a sig-nificant inhibition of myotube for-mation 48 h after the switch ofGILZ-Myc and/or L-GILZ-FLAGtransfected myoblast cultures toDM compared with control cells, asinvestigated byMyHC staining (Fig.5C, panels b and c versus panel a).The expression levels of ectopicGILZ-Myc and L-GILZ-FLAG atdifferent time points (at 0, 6, 24, and48 h in DM) are shown in Fig. 5 (Dand E, bottom rows). In parallel, we

checked byWestern blot the expression levels of the earlymyo-genic marker myogenin and the late marker MyHC. We foundthat the expression of bothmarkers was impaired inGILZ-Mycor L-GILZ-FLAG transfected cells, compared with controls(Fig. 5, D and E). Remarkably, the expression of MyoD was notsignificantly reduced (Fig. 5,D and 5E, second rows). Notably, atlate differentiation stages, the effects of overexpressed GILZ orL-GILZonmyogenin expressionwere lost (Fig. 5,D andE, thirdrows, 24 and 48 h); this event can be regarded as a consequenceof the time-dependent reduction of the expression of theectopic proteins (Fig. 5,D andE, bottom rows, 24 and 48 h). Thisnotwithstanding, the GILZ/L-GILZ-induced delay in myoge-nin accumulation resulted in a significant inhibition of myo-blast fusion and MyHC expression. Thus, GILZ and L-GILZoverexpression appeared to inhibit myogenic differentiation bycountering pro-myogenic events that induce cell-to-cell fusion.

FIGURE 3. DEX reduces myogenic differentiation and up-regulates GILZ and L-GILZ expression in C2C12myoblast cultures. A, C2C12 myoblasts were induced to differentiate for 48 h in absence (CNTRL, panel a) or inpresence (panel b) of 10�5

M DEX. The cultures were subjected to MyHC staining. Bar, 50 �m. B, Western blotanalyses of GILZ/L-GILZ induction by DEX and of expression of biochemical markers of differentiation in CNTRLand DEX-treated cells at the indicated time points. C, densitometric analysis of the Western blots from B areshown in C (myogenin/�-tubulin ratio).




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These results are in agreement with a simultaneous decreaseof GILZ and L-GILZ expression levels, induced by RNA inter-ference (RNAi) previously shown in Fig. 4A, resulting in a sig-nificant increase in myotube formation (Fig. 4A, compare pan-els c and a). Moreover, biochemical analyses in GILZ silencedmyoblasts showed a significant increase of MyHC and myoge-nin expression, whereas MyoD expression was not affected(Fig. 4B). These experiments revealed that the physiologicalup-regulation of GILZ and L-GILZ, which takes place after the

differentiation onset, results inanti-myogenic effects that occurdownstream of MyoD activation.Taken together, these results indi-cate that GILZ and L-GILZ behaveasmodulators ofmyogenic differen-tiation, being able to inhibit myo-genesis and to dampen the expres-sion of differentiation markers suchas MyHC and myogenin.GILZ and L-GILZ Target MyoD

Transcriptional Activity—The re-sults shown in Fig. 5 suggested thatGILZ and L-GILZ inhibit myogenicdifferentiation at early stages andthat their effects might not berelated to repression of MyoD pro-tein expression levels. Given thatMyoD is indirectly activated by thesustained kinase activity of p38 andAkt (32), we looked for changes inthe phosphorylation status of thetwo kinases; no detectable differ-ences were detected when controland GILZ-Myc or L-GILZ-FLAGoverexpressing cells were inducedto differentiate (not shown). More-over, flow cytometric assays did notreveal any significant effects on cellcycle and viability that could coun-teract myogenesis (not shown).During myogenic differentiation,

MyoD is stably expressed in thenucleus (24). We thus analyzed thesubcellular localization of GILZ andL-GILZ. We analyzed the localiza-tion of ectopic GILZ and L-GILZ inC2C12 cells transiently transfectedwith GILZ-Myc- and L-GILZ-FLAG-expressing vectors. Immu-nofluorescence studies revealed adifferential subcellular localizationof GILZ-Myc and L-GILZ-FLAG(not shown) depending on culturemedium conditions; in GM bothGILZ and L-GILZ mainly localizedto the cytoplasm (Fig. 6A, top row),whereas at 6 h after the switch toDM, they were localized to the

nuclei (Fig. 6A, bottom row). Moreover, confocal images ofC2C12 myoblasts at 24 h after the switch revealed a clearnuclear localization of GILZ protein (Fig. 6B).Because immunofluorescence results suggested that differ-

entiation stimuli promoted a nuclear localization of GILZ andL-GILZ, we asked whether GILZ isoforms could directly targetMyoD transcriptional activity. As shown in Fig. 7A, when aMyoD-HA-expressing vector was co-transfected with a lucifer-ase reporter-gene containing the myogenin promoter (MyoG-

FIGURE 4. DEX effects on C2C12 differentiation are dependent on GILZ and L-GILZ expression, andknockdown of GILZ/L-GILZ results in enhanced myoblast differentiation. A, control (CNTRL) and or GILZ/L-GILZ silenced C2C12 myoblasts (shRNA) were induced to differentiate for 48 h in the presence or absence (NT)of 10�5

M DEX. The cultures were subjected to MyHC staining. Bar, 50 �m. B and C, Western blot of expressionof biochemical markers of differentiation was taken over in CNTRL versus small hairpin GILZ � small hairpinL-GILZ (shRNA) C2C12 myoblasts with (C) or without (B) DEX treatment. Western blot of �-tubulin is included toshow total protein loading. D, densitometric analysis of Western blots from B are shown in C (myogenin/�-tubulin ratio). *, p � 0.01 (n � 3). E, densitometric analysis of Western blots from C are shown in E (myogenin/�-tubulin ratio). *, p � 0.01 (n � 3).




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FIGURE 5. A and B, GILZ and L-GILZ are expressed in differentiating C2C12 myoblasts. The cells underwent lysis for protein extraction or RNA extractionafter cultivation in GM or at the indicated time points in DM. Western blot (A) and real time PCR (B) are shown (n � 3). C–E, overexpression of GILZ andL-GILZ results in reduced C2C12 myoblast differentiation. C, morphological evaluation of GILZ and L-GILZ overexpression effects on myogenesis. C2C12myoblasts were transfected with GILZ-Myc vector (GILZ, panel b) and/or L-GILZ-FLAG vector (L-GILZ, panel c; GILZ�L-GILZ , panel d) and then inducedto differentiate. The cells transfected with empty vector were used as control (CNTRL, panel a). MyHC staining of myoblast cultures after 48 h in DM. Bar,50 �m. D and E, Western blot analyses of myogenic markers (MyoD, myogenin, and MyHC) in control (CNTRL) and GILZ-Myc (D) or L-GILZ-FLAG-transfected cells (E) during cultivation in DM. Anti-Myc and anti-FLAG antibodies were employed to detect GILZ-Myc and L-GILZ-FLAG, respectively.




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luc) in C2C12 cells, a marked increase in luciferase activity wasmeasured compared with base-line luminescence after theswitch to DM.WhenGILZ and/or L-GILZwere co-transfectedwith MyoD-HA, we observed a dose-dependent reduction ofluciferase activity, suggesting that overexpression of GILZ andL-GILZ resulted in a reduction ofMyoD transcriptional activityon the myogenin promoter (Fig. 7A).Next, we asked whether GILZ and/or L-GILZ could interact

withMyoDat the protein level in differentiatingmyoblasts. Theresults showed that GILZ and L-GILZ co-immunoprecipitatedwith MyoD (Fig. 7B, fourth lane). To address the molecularmechanism by which GILZ/L-GILZ inhibited MyoD activity,we searched for a possible role for HDAC1. In fact, it has beenpreviously suggested that GILZ could act as a transcriptionalrepressor via interaction with and recruitment of HDAC1 (19).Moreover, HDAC1 is known to be aMyoD partner when myo-blasts are cultivated in GM (30) and during early stages of dif-ferentiation (40). We found that HDAC1 co-immunoprecipi-tated with MyoD (Fig. 7B, fourth lane). Intriguingly, in theabsence of GILZ/L-GILZ expression, when both isoforms wereknocked down by RNAi, HDAC1 did not bind MyoD (Fig. 7B,third lane).As further evidence of protein-to-protein interaction, we

performed a co-immunoprecipitation experiment in MyoD-HA/GILZ-Myc transfected HEK293 cells; we confirmed thatGILZ and MyoD interacted in a protein-to-protein manner(Fig. 7C). In the same way, co-immunoprecipitation assay per-formed in HEK293 cells co-transfected with GILZ-Myc andHDAC1-FLAG or HDAC2-FLAG revealed that GILZ was ableto interact directly with HDAC1 but not HDAC2 (Fig. 7D).To understand the significance of the GILZ/HDAC1 inter-

action, we investigated whether GILZ/L-GILZ could affect

HDAC1 recruitment to myogenin promoter, at the same bind-ing site where MyoD is present. In fact, it has been alreadyshown that HDAC1, depending on differentiation status, co-localizes with and binds to MyoD on myogenin promoter andthus represses MyoD transcriptional activity (41). To this end,ChIP assay was performed in differentiated C2C12 using ananti-HDAC1 antibody. The results showed thatHDAC1 boundto the region containing the MyoD-binding site in the myoge-nin promoter after 24 h in DM (Fig. 7E). When GILZ/L-GILZisoforms were both silenced by RNAi, the degree of binding ofHDAC1 in myogenin locus was noticeably lower (Fig. 7E), thusindicating that GILZ/L-GILZ contribute to HDAC1/MyoDinteraction on themyogenin promoter region.Moreover, in thesame experiment, we performed ChIP assay to assess the acety-lation status of myogenin promoter. Acetylation of histones isan essential process in transcriptional activation (42), and it hasbeen already shown that MyoD transcriptional activity medi-ates histone acetylation in myogenin promoter. Specific anti-body for the acetylated form of H3 histones (AcH3) wasemployed in ChIP assay; the results showed that acetylation ofH3 histones was specifically induced after 24 h in DM (Fig. 7F,third column). When GILZ and L-GILZ were both silenced byRNAi, acetylation of myogenin locus was significantly higher(Fig. 7F, fourth column). These results are consistent with pre-vious data showing an increase in the myotube formation inGILZ/L-GILZ silenced C2C12 cells in DM conditions (Fig. 4A)and suggest that the observed increase in myogenin expressionupon GILZ/L-GILZ silencing (Fig. 4, B–E) is in part dependenton the reduction of HDAC1 activity.Finally, to confirm the possible involvement of HDAC1 in

GILZ/L-GILZ-mediated inhibition of myogenesis, we treatedGILZ-Myc- and L-GILZ-FLAG-transfected C2C12 cells withTSA (50 nM), a pharmacological inhibitor of HDACs (43, 44),prior to switching the cells from GM to DM. Morphologicalanalysis revealed that TSA treatment reverted GILZ andL-GILZ anti-myogenic effects (Fig. 8), further suggesting thatthe GILZ/L-GILZ anti-myogenic effect was dependent onHDAC1 activity. Collectively, these data indicate thatGILZ andL-GILZ are involved in the MyoD/HDAC1 interaction and theconsequent inhibition of myogenin expression.

DISCUSSION

The results described here indicate that GILZ and L-GILZare involved in the regulation of myogenesis and mediate GC-induced anti-myogenic activity. Skeletal muscle regeneration,after direct trauma or primary and secondary myopathies, isunsatisfactory. In fact, myofibers mainly die by necrosis, withconsequent local inflammation, and this event contributes tocreate, in the regenerating muscle, a micro-environment thatdoes not favor satellite cell survival and myoblast fusion.Intriguingly, inflammation-induced monocyte recruitment tothe damaged skeletal muscle tissue contributes to satellite cellactivation (45). However, in severemyopathic diseases, such as,for example, Duchenne dystrophy, this regenerative capacity isexhausted, because of altered satellite cell regeneration, pro-longed inflammation, impaired vascular adaptation, and fibro-sis (46, 47). Given the important role of inflammation inmyopathies, GCs are employed in the pharmacological treat-

FIGURE 6. Subcellular localization analysis of overexpressed and endog-enous GILZ in myoblasts. GILZ-Myc (A) was transfected in C2C12 myoblasts,which were kept in GM (upper row) or switched to DM (lower row). Anti-Mycantibody was employed for GILZ-Myc ectopic protein localization (red color).4�,6�-Diamino-2-phenylindole (DAPI) has been employed for nuclear staining(blue color). Right panel of each row shows the image merge. Bar, 20 �m.B, confocal images of C2C12 myoblasts at 24 h after switch. Anti-GILZ anti-body was employed for GILZ protein localization (red color). 4�,6�-Diamino-2-phenylindole has been employed for nuclear staining (blue color). The rightlane shows the image merge. Bar, 10 �m.




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ment of these diseases (48–50). However, this treatment isunsatisfactory in the long term because GCs are active on vir-tually all tissues including skeletal muscle tissue, promoting theconversion of proteins into glucose during stress or pharmaco-logical treatments (51), and thus GC-related side effects onskeletal muscle tissue, such as wasting and interference withmyogenic differentiation (2), probably contribute to the failureof anti-dystrophic therapy and to other characteristic GC-me-diated side effects such as those typical of the iatrogenic Cush-ing syndrome (52). This notwithstanding, molecular determi-nants of myogenesis and antagonists of muscle inflammationare worthy of investigation.In the present study we focused our attention in GILZ, a

rapidly GC-induced protein shown to be a relevant mediator ofGC effects in immune-related cells (9, 11–13, 53) and to be

expressed in mesenchyme-derived cells (19, 21, 54). We foundthat in skeletal muscle tissue GILZ is partnered by a longeralternative isoform, L-GILZ, which shares with GILZ a largeportion of primary structure. Notably, all previously describedGILZ functional domains (14, 17) are also present in L-GILZ.GCs were able to induce both GILZ and L-GILZ in primarymyoblasts and C2C12 myoblasts, and their induction was longlasting and associated with inhibition of myogenic differentia-tion. Also, forced expression of GILZ and/or L-GILZ in C2C12cells resulted in anti-myogenic effects, thus mimicking GCeffects. Moreover, experiments with an RNAi approach re-vealed that both GILZ and L-GILZ contribute to GC anti-myo-genic effects. Notably, experiments of selective RNAi directedtoward GILZ or L-GILZ revealed that the specific knockdownof eitherGILZ isoformwas able to dampenGCeffects, although

FIGURE 7. GILZ and L-GILZ target MyoD transcriptional activity. A, MyoD transcriptional activity was measured with a luciferase reporter gene containingthe myogenin promoter (MyoG-luc). *, p � 0.01 (n � 3). B, MyoD interacts with GILZ, L-GILZ, and HDAC1 in differentiating myoblasts. C2C12 myoblasts werecultivated in DM for 24 h before lysis. The cell lysates were subjected to immunoprecipitation with an anti-MyoD polyclonal antibody, and MyoD, GILZ, L-GILZ,and HDAC1 were identified by Western blot. C, GILZ directly interacts with MyoD. HEK293 cells were transfected with different combinations of plasmidsencoding GILZ-Myc and MyoD-HA. GILZ-Myc was immunoprecipitated with an anti-Myc antibody, and co-immunoprecipitation was revealed with an anti-HAantibody. The bottom row represents MyoD-HA input. D, GILZ is able to interact with HDAC1 but not with HDAC2. HEK293 cells were co-transfected withexpression vectors encoding GILZ-Myc and HDAC1-FLAG (upper panels) or HDAC2-FLAG (lower panels); immunoprecipitation was carried out with an anti-Mycantibody, and co-immunoprecipitation was revealed with an anti-FLAG antibody. E and F, for ChIP analysis, equivalent amounts of chromatin from same lysateused in endogenous IP assay described in B, control (CNTRL), and GILZ/L-GILZ silenced C2C12 myoblasts (shRNA) were immunoprecipitated in parallel withnormal rabbit IgG and antibodies specific for HDAC1 (E) or acetylated H3 histones (F). The purified myogenin promoter then were analyzed by real timePCR. Bar graphs show the relative levels of bound DNA in HDAC1 (E) or acetylated H3 histones (F) immunoprecipitates, and results from threeindependent ChIP experiments combined are expressed as percentages of input chromatin, normalized to control IgG samples. UR, unrelated genomicregion; PR, specific promoter region. *, p � 0.05 fourth column versus third column; **, p � 0.05 third column versus first column; ***, p � 0.05, fourth columnversus third column (n � 3).




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to a smaller extent compared with the simultaneous GILZ/L-GILZ gene silencing. In fact, when both proteins were knockeddown,GCs completely lost their anti-myogenic effects, and for-mation of large caliber myotubes was allowed.Moreover, we found that GILZ and L-GILZ were spontane-

ously regulated during C2C12 myogenic differentiation, andRNAi experiments indicated that inhibition of GILZ/L-GILZexpression was sufficient to enhance C2C12myoblast differen-tiation. These results suggest that GILZ and L-GILZ might actas developmentally regulatedmodulators ofmyogenic differen-tiation. In this context, we investigated the possible function ofGILZ and L-GILZ in regulating the TGF-� superfamily signal-ing, a well described system of myogenic terminal differentia-tion regulation (35). The results showed that no reciprocal reg-ulation of expression was found between GILZ/L-GILZ andTGF-� (supplemental Figs. S1 and S2).At a molecular level, we found that overexpressed GILZ and

L-GILZ inhibited not only cell-to-cell fusion, but also theexpression of the muscle transcription factor myogenin.Because the two GILZ isoforms did not affect MyoD proteinexpression levels, nor did they interfere with the activation ofpro-myogenic kinases such as p38 and Akt, we looked for adirect action of GILZ/L-GILZ onMyoD activity.We found thatGILZ and L-GILZ counteracted MyoD transcriptional activity.This effect was due to a protein-to-protein interaction betweenGILZ and L-GILZ with MyoD. Moreover, we found that GILZwas able to interact with HDAC1, thus suggesting that GILZandL-GILZ act as transcriptional repressors by the recruitmentof HDAC1. Considering that TSA, a known HDAC inhibitor,reverted the differentiation block induced by the forced expres-sion of GILZ or L-GILZ, we speculate that those molecules actthrough regulation of HDAC1 activity. Collectively, our datasupport the idea that GILZ/L-GILZ could be targets for futuretherapies of myopathies and for control of GC-mediated anti-myogenic effects. Indeed, GC treatment has been reported toresult in reducedmyogenesis and inhibition of the activation of

muscle satellite cells (7, 8). These observations might call for areconsideration of anti-inflammatory GC therapy of myopa-thies aswell for new therapeutic strategies aimed to counterGCside effects at themuscle tissue level. In fact, our present resultsshow that GCs increase GILZ/L-GILZ in myoblasts and exertanti-myogenic effects via induction of GILZ/L-GILZ. There-fore, countering GILZ and L-GILZ expression and/or functioninmuscle tissuemight promote satellite cell activation and con-tractile protein accumulation in myofibers.Currently, efforts are dedicated to the synthesis and charac-

terization of the so-called “safe GCs” (55). Given the tissuespecificity of different GC receptor isoforms (51, 56) and thespecificity of post-transductional modifications that differen-tially affect target gene expression (57), GCs endowed with theability to selectively activate specific GC receptor in inflamma-tory cells could bemade available, thus protecting skeletalmus-cle tissue against GC-induced GILZ/L-GILZ expression andwasting. In this respect, GILZ/L-GILZ null mice could providean interesting tool in the upcoming future to verify whetherabsence of GILZ/L-GILZ in skeletal muscle tissue enhancesmuscle regeneration in vivo and whether GCs might be moreeffective in the symptomatic treatment of dystrophy whenGILZ and L-GILZ cannot be pharmacologically induced inmuscle tissue.

Acknowledgments—We are grateful to Dr. Pier Lorenzo Puri (TheBurnham Institute, La Jolla, CA) andDr.MilenaGrossi (University ofRome “La Sapienza”, Rome, Italy) for C2C12 cells and plasmids.

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Migliorati, Rosario Donato and Carlo RiccardiStefano Bruscoli, Valerio Donato, Enrico Velardi, Moises Di Sante, Graziella

Differentiation and Mediate Anti-myogenic Effects of GlucocorticoidsGlucocorticoid-induced Leucine Zipper (GILZ) and Long GILZ Inhibit Myogenic

doi: 10.1074/jbc.M109.070136 originally published online February 2, 20102010, 285:10385-10396.J. Biol. Chem.

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