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The International Journal of Biochemistry & Cell Biology 41 (2009) 934–944

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

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ncreased spermine oxidase (SMO) activity as a novel differentiation marker ofyogenic C2C12 cells�

anuela Cervelli a, Emiliano Fratinia,b, Roberto Amendolab, Marzia Bianchia, Emanuela Signori c,lisabetta Ferrarod, Antonella Lisi c, Rodolfo Federicoa, Lucia Marcoccie, Paolo Mariottinia,∗

Dipartimento di Biologia, Università “Roma Tre”, Rome, ItalyDipartimento BAS-BiotecMed, ENEA, CR Casaccia, Rome, ItalyIstituto di Neurobiologia e Medicina Molecolare, CNR, Rome, ItalyLaboratory of Molecular Neuro-embryology, IRCCS Fondazione Santa Lucia, 00143 Rome, ItalyDipartimento di Scienze Biochimiche “A. Rossi Fanelli”, Università “La Sapienza”, Rome, Italy

r t i c l e i n f o

rticle history:eceived 9 June 2008eceived in revised form1 September 2008

a b s t r a c t

Spermine oxidase (SMO) is a FAD-containing enzyme involved in animal cell polyamines (PA) homeostasis,selectively active on spermine and producing H2O2, spermidine, and the 3-aminopropanal. In the presentstudy, we have examined the SMO gene expression during the mouse myoblast C2C12 cell differentiationinduced with two different stimuli by RT-PCR analysis, polysome-mRNP distribution and enzyme activity.

ccepted 12 September 2008vailable online 21 September 2008

eywords:olyaminespermine oxidase

SMO transcript accumulation and enzymatic activity increases during C2C12 cell differentiation and cor-relates with the decrease of spermine content. Many proteins are highly regulated during the phenotypicconversion of rapidly dividing C2C12 myoblasts into fully differentiated post-mitotic myotubes. The SMOgene induction represents a novel and additional marker of C2C12 cell differentiation. The sub-cellularlocalization of the SMO� and SMO� splice variants is not involved in the differentiation processes. Nuclear

O� p

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2C12 muscle cellsell differentiation

localization of only the SM

. Introduction

The flavin-containing spermine oxidase (SMO) plays an impor-ant role in the animal cell homeostasis of polyamines (PA), beingapable of directly oxidizing spermine (Spm) and producing sper-idine, 3-aminopropanal and H2O2 (Cervelli et al., 2003; Vujcic et

l., 2002; Wang et al., 2001). A previous study on the relative abun-ance of SMO transcript in various mouse organs showed that mus-le cells highly express two distinct splice variants of spermine oxi-ase (SMO� and �) (Cervelli et al., 2004). In the present study, we

Abbreviations: AcSpm, N1Ac-spermine; ASMA, alpha skeletal muscle actin;2C12, mouse skeletal myoblast cell line; FCM, flow cytometric; DM, differen-iation medium; GM, growth medium; IGF-1, insulin-like growth factor; APAO,cetylpolyamine oxidase; MCK, muscle creatine kinase; ODC, ornithine decarboxy-ase; SMO, spermine oxidase; SSAT, spermidine/spermine N1-acetyl-transferase; PA,olyamines; PI, propidium iodide; Put, putrescine; ROS, reactive oxygen species;pd, spermidine; Spm, spermine; TDM, transfection with differentiation mediumDM; TGM, transfection with growth medium; WS, withdrawal of serum.� Enzyme: vertebrate spermine oxidase.∗ Corresponding author at: Dipartimento di Biologia, Università di “Roma Tre”,iale G. Marconi 446, 00146 Roma, Italy. Tel.: +39 06 55176359;

ax: +39 06 5517 6321.E-mail address: mariotpa@uniroma3.it (P. Mariottini).

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357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.biocel.2008.09.009

rotein was confirmed.© 2008 Elsevier Ltd. All rights reserved.

ave examined the expression of these two isoforms in the mouse2C12 myogenic cells that represent an early stage of muscle differ-ntiation (Yaffe and Saxel, 1977). Depending on the culture condi-ions, these cells can either proliferate as myoblasts or differentiatento myotubes (Linkhart et al., 1981). When cultured in low serum-ontaining growth medium or in the presence of IGF-1 (insulin-likerowth factor 1) as a myogenic cell differentiating agent (Coolicant al., 1997; Florini et al., 1991; Milasincic et al., 1996; Tureckovat al., 2001), C2C12 cells exit the cell cycle and undergo a well-efined program of differentiation that culminates in the formationf myosin heavy chain-positive bona fide multinucleated muscleells (Coolican et al., 1997; Florini et al., 1991; Milasincic et al., 1996;ureckova et al., 2001). In order to highlight the role of polyamineetabolism during muscle differentiation, we analysed the

olyamine content and the relative transcription levels by RT-PCRnalysis of the key metabolic enzymes such as ornithinedecarboxy-ase (ODC), polyamine oxidase (APAO), N1-acetyl-transferase (SSAT)nd spermine oxidase (SMO) � and � isoforms.

To analyse the translational utilization of the SMO transcripts

uring both proliferating and differentiating conditions, the frac-ion of SMO� and SMO� mRNAs associated to polysomes wasetermined together with the fraction of the ODC, APAO and SSATranscripts, as well as the control ASMA (alpha skeletal musclectin) and MCK (muscle creatine kinase) mRNAs. C2C12 myoblast

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ells transfected with both SMO� and SMO� V5-tagged proteinsere induced to differentiate into myotubes and analysed to assess

he sub-cellular localization of the two isoforms. The nuclear local-zation of the SMO� protein was confirmed during the C2C12ell myogenic differentiation. Transcript accumulation of the PAetabolic enzymes was differentially regulated during differenti-

tion, showing, among other things, a higher expression for bothMO isoforms. Interestingly, augmented transcript level was con-istent with SMO enzymatic activity increase and Spm depletion.he importance of SMO induction during muscle differentiationould be related to hydrogen peroxide production, H2O2 being aommon signalling molecule that plays an important role in reg-lating the fate of mononuclear muscle cells (Orzechowski et al.,002). More and more data indicate that, in muscle cells, reac-ive oxygen species (ROS) and nitric oxide (NO) are continuallyenerated and that these molecules have a well-established roles physiological modulators of skeletal muscle functions, rangingrom development to metabolism and from blood flow to con-ractile functions (Clanton et al., 1999). Studies in the past twoecades suggest that strenuous muscle activity, associated withome pathological conditions and/or aging, increases the gener-tion of ROS in the skeletal muscle to a level that overwhelmshe antioxidant defence systems (Ji, 1995, 2001), thus provokingatigue, inflammation, and tissue degeneration. Thus, to betterefine the actors that play a role in oxidative stress remains a very

mportant challenge (Caporossi et al., 2003).

. Materials and methods

.1. Cell culture, differentiation and transfection

C2C12 cells were maintained in a growth medium (GM) consist-ng of Dulbecco’s modified minimum essential medium (DMEM)upplemented with 20% foetal bovine serum (Gibco BRL, Gaithers-urg, MD), penicillin (50 U/ml), and streptomycin (50 mg/ml)Sigma, Milan, Italy). An adequate number of C2C12 cells (5 × 104

ells in a 60 mm Petri dish) to maintain a non-confluent conditionuring the time course were incubated at 37◦ C and exposed to aumidified atmosphere of 5% CO2. Control GM cells were aliquotedt various time following 24 h seeding time (GM4-96h cells). Tonduce differentiation, GM cells were shifted 24 h after seeding to aifferentiation medium (DM) consisting of DMEM supplementedith 2% foetal bovine serum, penicillin (50 U/ml), and strepto-ycin (50 mg/ml) or alternatively supplemented with IGF-1 (ITS

00×, insulin transferrin) (Sigma, Milan, Italy) (DM4-96h cells).ifferentiation was followed by cell morphology under a phase-ontrast microscopy observation (Axioskop, 20× objective, Zeiss,berkochen, GE).

Ectopically expression of each vector (1 �g DNA/0.5 × 105 cells)as obtained with LipofectAMINE Plus reagent (Invitrogen) accord-

ng to the manufacturer’s instructions. After transfection andxposure to glycerol shock, the cells were washed with phosphate-uffered saline. Aliquots were cultured under normal conditionsor 24 and 48 h (TGM24-48h cells) or induced to differenti-te (TDM4-96h) to localize SMO isoforms according to the timeourses.

.2. Flow cytometric (FCM) determination of DNA content of GMnd DM C2C12 cells

To study DNA content, C2C12 cells were treated with Propidiumodide (PI), as described (Nicoletti et al., 1991). At least 105 cellsere analysed by a FACSCalibur flow cytometer (Becton Dickinson,

an Josè, CA), previously calibrated by CaliBRITE 3 beads (Bec-

eto13

hemistry & Cell Biology 41 (2009) 934–944 935

on Dickinson), with laser excitation set at 488 nm, and a 630 nmmission filter to detect red fluorescence. FCM histograms werenalysed by the Windows Multiple Document Interface (WinMDIer. 2.8, The Scripps Research Institute, La Jolla, CA) dedicatedoftware.

.3. RT-PCR analysis of SMO˛ and SMO� transcripts in DM2C12 cells

The relative levels of SMO�, SMO�, APAO, ODC, SSAT, �-actinnd rpS7 transcripts during C2C12 cell differentiation inducedy SW or IGF-1were measured by RT-PCR with specific primersescribed in Cervelli et al. (2004) and Amendola et al. (2005).

n particular, SMO� forward 5′-GTACCTGAAGGTGGAGAGC-3′ andMO� reverse 5′-TGCATGGGCGCTGTCTTGG-3′; SMO� forward 5′-AGAGCAGCAGCCTGGTCACC-3′ and SMO� reverse 5′-GGGCCC-TGCTGGAAGAGGTCTCG-3′; APAO-forward 5′-GAGCCACCACTGC-TGCC-3′ and APAO-reverse 5′-CCATGTGTGGCTTCCCC-3′; ODC-

orward 5′-TCCAGGTTCCCTGTAAGCAC-3′ and ODC-reverse 5′-CC-ACTTTGCCTTTGGATGT-3′; SSAT-forward 5′-CGTCCAGCCACTG-CTCTG-3′ and SSAT-reverse 5′-GCAAGTACTCTTTGTCAATCTTG-3′;-actin-forward 5′-TGTTACCAACTGGGACGACA-3′ and �-actin-

everse 5′-AAGGAAGGCTGGAAAAGAGC-3′; rpS7-forward 5′-CGA-GTTGGTCGG-3′ and rpS7-reverse 5′-GGGAATTCAAAATTAACA-CC-3′. The MCK (creatine kinase, GenBank accession numberM133921) specific primers were: MCK1-for 5′-GTCATCCAGA-TGGGGTGG-3′, MCK2-for 5′-GTGGCCGGCGATGAGGAG-3′, MCK3-ev 5′-AGCAGCAGAGGTGACACGG-3′, and MCK4-rev 5′-TGGAGA-CACGCGGAGGTG-3′; the ASMA (alpha skeletal muscle actin,enBank accession number XM133921) specific primers were:SMA1-for 5′-ATTGAACATGGCATCATCACC-3′, ASMA2-for 5′-AC-ACATGGAGAAGATCTGG-3′, ASMA2-rev 5′-GCAGCTCATAGCTCT-CTCC-3′ and ASMA2-rev 5′-TCCTGATGTCGATGTCGCAC-3′. TotalNA was isolated from different samples (GM and DM cells) byRIZOL reagent (Gibco BRL), according to the manufacturer’snstructions. A synthesis of the cDNAs from the RNAs of different

ouse organs was performed by primer random examers in 20 �leaction volume containing 1 �g of total RNA, according to theanufacturer’s instructions (SuperScript First-Strand Synthesis

ystem for RT-PCR, Invitrogen). Aliquots of reverse-transcribed-NA were amplified within with Taq DNA polymerase (Pharmacia)

n the linear range and in saturating experimental conditions by 20,5, 30 or 35 PCR cycles: denaturation at 94◦ C for 1 min, annealingt 60◦ C for 1 min and extension at 72◦ C for 1 min. The RT-PCRsere normalized by a comparison with the �-actin control. Further

ontrol reaction mixtures, either without template (not shown)r RT enzyme (not shown), were uniformly negative. Resultsere quantified by densitometry, using the BioRad Multianalyst

oftware (BioRad, Hercules, CA). An estimation of the relativeT-PCR amplified product amounts was obtained by dividing therea of gel bands by the area of the relative control �-actin geland. Data points are the means ± SE of three to five separatexperiments, each performed in duplicate.

.4. Polysome-mRNP distribution of SMO˛ and SMO� transcriptsn GM and DM C2C12 cells

For preparing polysomes, the procedure for cell lyses, sucroseradient sedimentation, and analysis of the polysome/subpolysomeistribution of mRNAs was essentially that described by Meyuhas

t al. (1996). Proliferating (cultured in GM) and post-mitotic (cul-ured in DM, 48 h after SW) C212 cells (1 × 106) were directly lysedn the plate with 300 ml of lyses buffer [10 mM NaCl, 10 mM MgCl2,0 mM Tris–HCl pH 7.5, 1%Triton X-100, 1% sodium deoxycholate,6 units/ml RNase inhibitor (Amersham Pharmacia Biotech), 1 mM

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ithiothreitol] and transferred to an Eppendorf tube. After 5 minncubation on ice with occasional vortexing, the lysate was cen-rifuged at 6000 × gmax for 8 min at 4◦ C. The supernatant wasrozen in liquid nitrogen and stored at −70◦ C for later analysisr immediately loaded on 15–50% (w/v) sucrose gradient con-

tc1o−

ig. 1. (A) Cellular morphology analysis during C2C12 cell differentiation (Axioskop, phaments. (B) DM C2C12 cell cycle check-points analysis. Cells were analysed at 4, 48, 72 a0/G1 cell fraction, number of events per relative amount of Propidium Iodide (PI).

hemistry & Cell Biology 41 (2009) 934–944

aining 100 mM NaCl, 10 mM MgCl2, 30 mM Tris–HCl, pH 7.5 andentrifuged in a Beckman ultracentrifuge with an SW41 rotor for00 min at 37,000 rpm. Fractions collected while monitoring theptical density at 260 nm, were ethanol-precipitated overnight at20◦ C. The distribution of SMO� SMO�, APAO, ODC, SSAT, �-actin

se-contrast 20× objective, Zeiss). The micrographs represent three separate exper-nd 96 h after WS. FCM representative histograms out of three experiments of the

M. Cervelli et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 934–944 937

Fig. 2. Sub-cellular localization of SMO� and SMO� isoforms in transiently transfected TGM and TDM C2C12 cells after SW. Transiently transfected cells are indicated asSMO�, C2C12/pcDNA3/V5-His/SMO�, and SMO�, C2C12/pcDNA3/V5-His/SMO�. Time course of 4, 48, 72 and 96 h after SW in TDM cells; TGM cells analysed at 24 and48 h after transfection. Cells were immunostained with anti-V5 (green) and nuclei were stained with 4′ ,6′-diamino-2-phenylindole (DAPI, blue). Merge row is the result ofoverlapping images. The micrographs represent three separate experiments.

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38 M. Cervelli et al. / The International Journal o

nd rpS7 transcripts were analysed by RT-PCR with the specific

rimers described above. To perform quantitative RT-PCR analysisn RNA extracted from polysome gradient fractions, each sampleas reverse-transcribed into cDNA together 15 pg of an in vitro

ranscribed RNA for ZmPAO cDNA (Zea mais polyamine oxidase,enBank accession number AJ002204), included as an internal con-

ecdt3

ig. 3. SMO� and SMO� transcript determination during the C2C12 cell myogenic differM cells were analysed at 4 h (see Section 2). The PCR products were fractionated by

ndependent replicas are shown. Densitometric analysis of gel bands represents measuremach experimental time point) is represented as an arbitrary densitometric units bar grap

hemistry & Cell Biology 41 (2009) 934–944

rol to confirm that the amount of product was not influenced by

xperimental variations in the reactions. For each PCR, differentycles and template amounts were tested, in order to avoid con-itions of saturation. ZmPAO cDNA (378 bp) was amplified withhe following primers: ZmPAO1-for 5′-ATGAACCCCATCTGGCCCAT-′ and ZmPAO2-rev 5′-GTCGCCGAAGTCGCTGAAGG-3′.

entiation. Samples of DM C2C12 cells taken after SW at 4, 48, 72 and 96 h; control1.2% agarose gel electrophoresis. Representative RT-PCR experiments from threeents of three separate experiments. Gene of interest/�-actin ratio (normalized for

h (±SE) below PCR band gel.

M. Cervelli et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 934–944 939

Fig. 4. SMO� and SMO� transcript determination during the C2C12 cell myogenic differentiation. Samples of DM C2C12 cells taken after IGF-1 treatment at 36 and 72 h;control GM cells were analysed at 4 h (see Section 2). The PCR products were fractionated by 1.2% agarose gel electrophoresis. Representative RT-PCR experiments from threeindependent replicas are shown. Densitometric analysis of gel bands represents measurements of three separate experiments. Gene of interest/�-actin ratio (normalized foreach experimental time point) is represented as an arbitrary densitometric units bar graph (±SE) below PCR bands gel.

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.5. Analysis of enzymatic activities and polyamine content

Polyamine oxidase activities on cell homogenates were deter-ined fluorometrically according to Cervelli et al. (2004). Spm and

1Ac-spermine (AcSpm) were used as the substrates of SMO andPAO enzymes, respectively. The enzyme activity was calculated asmol H2O2 produced/mg protein/min for SMO enzyme and pmol2O2 produced/mg protein/hour for APAO enzyme. Protein con-

ent was estimated by the method of Markwell et al. (1978) withovine albumin as a standard. ODC and SSAT enzyme activities,alculated as pmol CO2 produced/mg protein/hour, were deter-ined by using 14C-labeled substrate and scintillation counting

f end metabolized products, as described elsewhere (Chen et al.,003). Polyamine concentration was determined as described else-here (Mates et al., 1992) and expressed as nmoles/mg protein

or each sample, in respect to standard concentration of bovinelbumin.

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ig. 5. RT-PCR analysis of SMO� and SMO� mRNAs in polysome gradient fractions fromn sucrose gradients and total RNA extracted from gradient fractions. (B) RT-PCR reactionuantification control (cDNA for ZmPAO, see Section 2). The agarose gel photograph stripT-PCR experiment from three independent replicas is shown.

hemistry & Cell Biology 41 (2009) 934–944

.6. Intracellular localization of SMO˛ and SMO� in transfectedM C2C12 cells

C2C12 cells (0.5 × 105 cells in 35 mm Petri dishes) were tran-iently transfected in triplicate with the plasmids pcDNA3/SMO�-5 and pcDNA3/SMO�-V5 (Cervelli et al., 2004). Twenty-four hoursfter transfection cells were shifted by SW or IGF-1 treatmentnto DM medium for 4, 48, 72 and 96 h and then fixed with 3.7%araformaldheyde in PBS (15′ at 4 ◦C). After permeabilization with.2% Triton X-100 in PBS for 5 min, cells were blocked in 2% horseerum in PBS and incubated for 1 h at 37 ◦C with primary mousenti-V5 monoclonal antibody (Sigma) followed by a secondary

eaction with the goat polyclonal anti-Mouse IgG conjugated withITC (Sigma). The evaluation of the sub-cellular localization ofMO� and SMO� V5-tagged proteins was carried out as describedn Cervelli et al. (2004). Nuclei were counterstained with 1 �g/mlAPI (4′,6′-diamidino-2-phenylindole). Cells were examined under

GM and DM (48 h after SW) C2C12 cells. (A) Cytoplasmic extract was fractionateds using specific primer pairs and different PCR cycles are indicated. Also shown is aes are aligned with the A254 traces of the optical density profiles. A representative

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Leica TCS SP5 Confocal Microscopy equipped with a 40× (NA 1.25)r 63× (NA 1.4) oil-immersion objective.

. Results

.1. Cellular localization of SMO˛ and SMO� splice variantroteins during C2C12 cell myogenic differentiation

The conversion of C2C12 myoblasts into myotubes was moni-ored during the time course by cell morphology and FCM analysisFig. 1A and B). SW or IGF-1 treatment (not shown) induced alearly visible change in cell shape, while FCM determinationhowed an increased in the G0/G1 cell fraction, both analysesndicating that C2C12 cells switched from a proliferating to a post-

itotic differentiated condition. To reveal intracellular SMO� andMO� protein localization during myogenic differentiation, bothMO splice variants were microscopically localized in GM and DM2C12 cells transiently overexpressing SMO proteins (TGM andDM cells). Augmented transcript levels for each transfected iso-orms were detected in transiently transfected cells by RT-PCRnot shown). Sub-cellular localization was performed using the V5-AG as the epitope to direct the primary monoclonal antibodies.MO isoform subcellular localization in transfected TDM C2C12ells was analysed throughout differentiation (4, 48, 72 and 96 hfter SW), while aliquots of TGM C2C12 cells, cultured in a non-onfluent condition, were taken at 24 and 48 h after transfectionsee Materials and methods, Section 2.1). As depicted in Fig. 2,n both C2C12/pcDNA3/SMO�-V5 and C2C12/pcDNA3/SMO�-V5ransiently transfected TGM and TDM C2C12 cells, the sub-cellularocalization of the SMO�-V5tagged isoform is localized exclusivelyn the cytoplasm, the SMO�-V5tagged protein clearly shows auclear localization.

These results substantiate the same cell localization of SMO�nd SMO� proteins previously observed in neuroblastoma cellsCervelli et al., 2004; Amendola et al., 2005; Bianchi et al., 2005).

.2. Accumulation of SMO˛ and SMO� mRNAs during C2C12 cellyogenic differentiation

Levels of SMO�, SMO�, APAO, ODC and SSAT mRNAs were exam-ned during C2C12 myogenic differentiation by RT-PCR analysisFigs. 3 and 4). To assess differentiated status, the PCR-amplified

RNAs coding for the MCK and ASMA proteins were probed. Theousekeeping control �-actin protein and ribosomal protein S7 (rp-7), was also probed to quantify the amplified samples (see Section; Cervelli et al., 2004). Fig. 3 shows a typical time course anal-

sis of the transcripts accumulation during C2C12 differentiationobtained with SW) of DM cells at 4, 48, 72 and 96 h after induc-ion. Control GM cells were aliquoted at 4 h following 24 h seedingime (GM4h). In order to amplify each transcript within a linearange different PCR cycle conditions were used (see Section 2).

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able 1MO and APAO activities of GM and DM C2C12 cells.

ample SMO activity (pmolH2O2 produced/mgprotein/min)

SMO activity (pmolH2O2 produced/mg pro-tein/min) + MDL72,527

M4h 543.42 ± 45.9 33.3 ± 13.4M4h 288.78 ± 51.7 20.8 ± 11.2M48h 324.39 ± 36.9 30.6 ± 8.2M72h 519.90 ± 91.1 17.9 ± 12.4M96h 858.25 ± 43.4 28.4 ± 10.6MT 31.03 ± 14.4 3.9 ± 1.4

M and DM samples as defined in the text. SMT, skeletal muscle tissue. Values are the men Student’s t test.

hemistry & Cell Biology 41 (2009) 934–944 941

t can be observed that both SMO� and SMO� isoforms and alsoPAO and SSAT mRNAs were dramatically increased during differ-ntiation. By contrast, transcript coding for ODC decreased its levelrogressively during the time course monitored. The �-actin andp-S7 control transcripts showed no changes in their accumula-ion patterns during the time course (Fig. 3). Identical results werebtained using the IGF-1 as a myogenic cell differentiating agentn GM cells and DM36 and DM72 samples (Fig. 4), confirmingclear relationship between mRNA expression and cell differen-

iation patterns, independently from the differentiation stimulusdopted.

.3. Polysome-mRNP distribution of SMO˛ and SMO� mRNAs

In order to determine the amount of SMO� and SMO� mRNAsranslated during C2C12 cell myogenic differentiation, we anal-sed their distribution between polysomes and mRNPs in GMnd DM C2C12 cells induced by SW (DM48 sample, Fig. 5). Aemi-quantitative RT-PCR approach was adopted to evaluate theercentage of mRNA loading onto polysomes. Different PCR cycleumbers for each specific primer pair were chosen in order to fithe linear range of amplification. Fig. 5 shows the results of a typi-al experiment. The polysome distributions of APAO, ODC, SSAT andhe controls MCK, ASMA, �-actin and rp-S7 transcripts have beenxamined in parallel. As an additional control, to monitor any lossf material during the RNA extraction, the ZmPAO cDNA was ampli-ed and the corresponding RT-PCR product was equally present inll fractions. It appears that in both GM and DM C2C12 cells theMO� and SMO� mRNAs are mainly translated, as well as all thether analysed mRNAs that were found to be almost entirely loadednto polysomes (Fig. 5). The same experimental protocol was car-ied out on DM C2C12 cells induced by IGF-1 obtaining identicalesults (data not shown).

.4. Enzyme activities and polyamine content during C2C12 cellyogenic differentiation

During C2C12 myogenic differentiation, SMO enzymatic activ-ty at first dropped to about half the value measured in GM4hells (GM4h and DM4h samples), to linearly increase up to abouthree times (DM4-96h samples). Similarly, APAO enzymatic activ-ty decreased dramatically to one/fifteenth of the level measuredn GM4h cells (GM4h and DM4h samples), before rising to a valuelmost comparable to that of GM cells (GM4h and DM96h sam-les) (Table 1). SSAT enzymatic activity remains steady during the2C12 myogenic differentiation (DM4-96h samples). On the otherand, it increases up to four fold in GM proliferating cells that enter

nto a confluent culture condition (GM4-72h samples) (Table 2).DC enzymatic activity showed a high level in the early stagesf differentiation, to dramatically decrease to about one/thirtiethf the initial value (DM4-96h samples). ODC activity measured inroliferating C2C12 cells shows a linear decrease after seeding as

APAO activity (pmolH2O2 produced/mgprotein/h)

APAO activity (pmolH2O2 produced/mgprotein/h) + MDL72,527

4962.86 ± 59.3 20.43 ± 13.8336.48 ± 28.8 18.65 ± 8.5383.50 ± 35.1 22.78 ± 4.2

1975.23 ± 95.7 19.95 ± 5.53784.02 ± 74.7 15.05 ± 9.91264.18 ± 44.7 32.66 ± 6.8

ans ± SD of three independent experiments performed in duplicate. p < 0.01 based

942 M. Cervelli et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 934–944

Table 2ODC and SSAT activity of GM and DM C2C12 cells.

Sample ODC activity (pmol CO2

produced/mg protein/h)ODC activity (pmol CO2

produced/mg protein/h) + DFMOSSAT activity (pmolN-AcSpd/mg protein/h)

GM4h 4088.76 ± 227.6 103.21 ± 11.3 285.96 ± 76.8GM48h 2899.55 ± 119.2 142.56 ± 7.6 399.57 ± 46.0GM72h 1864.39 ± 153.7 99.72 ± 9.3 1172.04 ± 126.8DM4h 2432.81 ± 132.3 101.55 ± 5.4 1065.61 ± 152.7DM48h 102.37 ± 7.0 18.3 ± 2.4 955.58 ± 73.8DM72h 122.11 ± 15.3 12.3 ± 5.4 987.35 ± 97.3DM96h 89.72 ± 3.9 2SMT 44.77 ± 3.9 1

GM and DM samples as defined in the text. SMT, skeletal muscle tissue. Values are the me

Table 3Polyamines content of GM and DM C2C12 cells.

Sample Polyamines content (nmol/mg protein) and relative pool percentages

Put Spd Spm

GM4h 12.51 ± 0.17 (10.4%) 77.73 ± 0.83 (64.8%) 29.70 ± 0.23 (24.8%)DM4h 2.52 ± 0.12 (2.7%) 62.08 ± 0.69 (62.8%) 34.07 ± 0.19 (34.5%)DM48h 4.65 ± 0.10 (5.7%) 52.33 ± 0.76 (64.5%) 24.14 ± 0.20 (29.8%)DM72h 7.49 ± 0.11 (9.7%) 48.92 ± 0.70 (63.1%) 21.14 ± 0.22 (27.2%)D

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M96h 9.14 ± 0.12 (12.3%) 45.11 ± 0.69 (61.6%) 19.23 ± 0.20 (26.1%)

M and DM samples as defined in the text. Values are the means ± SD of threendependent experiments. p < 0.01 based on Student’s t test.

he culture condition approaches confluence (GM4-72h samples)Table 2).

The PA content of the GM and DM cells was assayed in parallel.he Spm level of proliferating and early stage differentiated C2C12ells (GM4h and DM4-48h samples) proved similar, then decreaseduring cell differentiation (DM72-96h samples). The level of sper-idine (Spd) linearly decreased during the time course analysed asell. By contrast, the putrescine (Put) content increased up to about

our fold during differentiation (DM4-96h samples), to reach a finalalue comparable to the one measured in the proliferating C2C12ells (GM4h and DM96h samples) (Table 3). It is worthy to men-ioning that the relative PA pool percentages of Spm, Spd and Putiffered in the GM4 and DM4 samples, mainly as regards Spm andut contents. After C2C12 cell differentiation the PA pools provedo be the ones observed in proliferating cells, the relative PAs per-entages being reasonably similar in the GM4 and DM96 samplesTable 3). The PA content measured with IGF-1 differentiation stim-lus was consistent with the above-mentioned results (data nothown).

. Discussion

Polyamine metabolism reflects a concerted action of enzymes,egulated at several transcriptional and post-transcriptional lev-ls, but it still is unclear how this metabolism interacts with cellifferentiation and in particular with muscle differentiation. A pre-ious report showed that SMO enzyme is highly expressed in mouseuscle tissue (Cervelli et al., 2004). This has therefore prompted

s to analyse the SMO gene expression during the conversion of2C12 myoblasts into myotubes, this conversion corresponding ton early stage of muscle differentiation (Yaffe and Saxel, 1977). Asn initial step, we monitored the conversion of C2C12 myoblastsnto myotubes by cell morphology and FCM analysis (Fig. 1A and

). Then we analysed the cell localization of the two splice vari-nts SMO� and SMO� transiently expressed in both TGM and TDM2C12 cells (Fig. 2). Identical results were obtained using TDM cells

nduced by IGF-1 (not shown). According to previous experimentsarried out on the neuroblastoma cell line N18TG2, the expected

deOdr

2.4 ± 7.7 1041.13 ± 173.71.7 ± 3.3 2137.74 ± 193.1

ans ± SD of three independent experiments. p < 0.01 based on Student’s t test.

ytoplasmic localization of the SMO� protein was confirmed, asell as the nuclear localization of the SMO� protein (Amendola et

l., 2005; Bianchi et al., 2005; Cervelli et al., 2004).These results consistently substantiate the cell localization of

hese two SMO isoforms, and in both proliferating and differ-ntiating cells. The role played by the nuclear SMO� isoformemains unclear; probably, the very low level of free Spm into theuclear moiety suggests a SMO� function not totally linked to thepm metabolism. To this end, a 60% homology has been recentlyescribed between the SMO enzyme and the FAD-dependent aminoxidase lysine-specific demethylase 1 (LSD1) (Casero & Marton).SD1 is associated with a transcriptional repressor complex, thusypothetically suggesting novel roles for SMO� and its substratepm in chromatin remodelling. Very recently, Murray-Stewart etl. (2008) have demonstrated that the human SMO/PAOh1 (homo-ogue to SMO�) and SMO5 (homologue to SMO�) isoforms possessoth cytoplasmic and nuclear localization in NCI-H157 humanon-small cell lung carcinoma cells. The finding that the predom-

nant splice variant SMO/PAOh1 is also present in the nucleusas rather unexpected. Further research is needed to establishhether this dissimilarity in cellular localization between theuman SMO/PAOh1 and the murine counterpart SMO� is due to theifferent cell lines analysed or whether reflects a different species-pecific feature.

We also examined the gene expression patterns of these spliceariants during the C2C12 myogenic differentiation induced byifferent stimuli (SW and IGF-1 treatment) by RT-PCR analysis.

nterestingly, we found that the accumulation pattern of SMO�,MO�, APAO and SSAT transcripts is quite similar to those of MCKnd ASMA mRNAs, typical myogenic markers. On the other hand,he ODC mRNA accumulation pattern shows an opposite behaviour,he transcript level being high at early stage of differentiation, laterecreasing during the time course analysed (Figs. 3 and 4). It turnedut that the accumulation of the transcripts of the murine genesoding for the PA metabolic enzymes were deeply altered and fol-owed the two mRNA accumulation opposite profiles during C2C12ell myogenic differentiation. We also analysed the translationaltilization of the SMO�, SMO�, ODC, APAO, SSAT, ASMA and MCKRNAs in GM and DM C2C12 cells, and we found that all theseRNAs are translated with high efficiency, being almost completely

oaded onto polysomes, irrespectively of the cellular status (Fig. 5).aking these results as a whole, we can depict a muscle cell differ-ntiating scenario where SMO, APAO and SSAT genes are inducedt the level of transcription, at variance with the mRNA coding forDC, whose level is reduced.

During cell differentiation, it is generally accepted that ODC is

own-regulated (Capell et al., 2000; Nitta et al., 2001; Rosandert al., 1995), and C2C12 cells represent one more proof that bothDC transcript level and enzymatic activity are reduced duringifferentiation. It must be also taken into account that ODC down-egulation is a well-known marker of non-proliferating cells. The

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resent work indicates is a further proof that ODC expressionncreases in proliferating cells as shown by FCM experimentsFig. 1B). The enzyme activities of SMO, APAO, ODC and SSAT

atch the accumulation profiles of the corresponding transcriptsTables 1 and 2), suggesting the lack of a translational control as con-rmed by the polysome loading analysis. To this end, all polyamineetabolic genes analysed in this work can be considered additionalarkers of the myogenic differentiation. In line with the observa-

ion that SMO enzyme activity is induced up to three times duringell differentiation, the intracellular polyamine pools were affectedhowing a significant decrease both of Spm and Spd, and an increasef Put (Table 3). Therefore, in C2C12 cells the induction to differen-iation provoked a dramatic effect on the PA pools. In effect, webserved a markedly different PA content between GM and DM42C12 cells, while this difference is almost absent between GM andM96 samples. This indicates that after the initial perturbation,

he polyamine homeostasis drives back the PA content to a cellularalanced equilibrium (as the relative Put, Spd and Spm content per-entages of GM and DM96 cells demonstrate). This is in line withhe well-known notion that intracellular polyamines are manda-ory for the expression of several growth-regulatory genes, such asYC, FOS, JUN and ER� (Casero and Marton, 2007 and references

herein). Accordingly, we hypothesize that during the dramaticellular stress occurring during myogenic differentiation the PAynthesis and catabolism are affected mainly by transcriptionalvents. In particular, during muscle differentiation the increase ofMO activity following transcript up-regulation overwhelms theoncerted action of the other enzymes involved in PA metabolism,eading to a considerable lowering of Spm content and a concomi-ant Put increase. This PAs variation due to an increase of SMOxpression is in accordance with previous reports of SMO ectopicalver-expression in human HEK293 (Vujcic et al., 2002) and murineeuroblastoma N18TG2 (Amendola et al., 2005; Cervelli et al., 2004)ell lines. More and more data indicate that SMO gene can be rapidlynduced in response to stress and is responsible for H2O2 produc-ion in different cell lines and malignant tissues (Babbar and Casero,006; Chaturvedi et al., 2004; Goodwin et al., 2008; Xu et al., 2004).nterestingly, the resulting H2O2 produced by SMO enzyme activityas been demonstrated to be implicated as a signalling molecule forpoptosis (Chaturvedi et al., 2004; Xu et al., 2004), inflammation-nduced carcinogenesis (Babbar and Casero, 2006), and inducedensitivity to ionizing radiation (Amendola et al., 2005; Bianchi etl., 2007).

The molecular sources of ROS in skeletal muscle are poorlynderstood, and probably differ depending on muscle fibber typer muscle region, on age, on exercise intensity or duration and onhysiologic state of the tissue. One likely candidate is mitochondriallectron transport. It also been suggested that xanthine oxidase,ADH oxidoreductase, phospholipase A2 and NADPH oxidase are

nvolved in ROS formation in skeletal muscle (Clanton et al., 1999;iao et al., 2005). Spermine oxidase is undoubtedly part of the intra-ellular redox scenario, albeit its contribution remains still unclear,ue to the compensatory and redundant mechanisms that carry outhe H2O2 production.

Muscle differentiation is a highly ordered multi-step processhat is regulated by two groups of myogenic transcription fac-ors, namely the basic helix–loop–helix muscle regulatory factorsMyoD, Myf5, myogenin, and MRF4) and the myocyte enhancer-inding factors 2 proteins (Molkentin and Olson, 1996). Insulin-likerowth factors (IGF-1 and 2) stimulate muscle differentiation by

ctivating several signalling pathways (Naya and Olson, 1999). Dur-ng the last few years, it has been shown that IGFs stimulate the

yogenic process via phosphatedylinositol 3-kinase (PI 3-kinase),38 mitogen-activated protein kinase (p38 MAPK) (Piao et al.,005). ROS can be cytotoxic when present at high and/or sustained

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evels. However, low ROS concentration has been shown to functions physiological intracellular signalling mediators. H2O2 representsn ideal signalling molecule due to its rapid synthesis and diffusion.edox-sensitive proteins are targets of specific oxidation by variousxidants. There is abundant evidence that nuclear factor-kB (NF-kB)ctivity is extremely sensitive to cellular redox status. In addition, itas been suggested that NF-kB activation by PI 3-kinase stimulateshe myogenic process by increasing the expression of inducibleitric oxide synthase (Piao et al., 2005). A possibility is that SMOxpression could be linked to IGFs pathway and that the produced2O2 could interact with NO, acting as physiological modulators of

keletal muscle functions. Different pathways are involved in ROSeneration, typical of important metabolic systems where a manda-ory interplay between compensatory and redundant mechanismo exist. In this regard the present work indicates that SMO activ-

ty could provide another piece in the puzzle of hydrogen peroxideroduction during muscle differentiation.

cknowledgements

The authors are indebted to Dr. G. Bellavia (Department of Biol-gy, Università Roma Tre, Rome, Italy), Dr. F. De Carlo (Institute ofeurobiology and Molecular Medicine, CNR, Rome, Italy) for tech-ical support and Dr Martin Bennett for the text editing. We thankhe reviewers for their suggestions, and the resulting improvementsn the paper.

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