Increased spermine oxidase (SMO) activity as a novel differentiation marker of myogenic C2C12 cells

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

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Increased spermine oxidase (SMO) activity as a novel differentiation marker of myogenic C2C12 cells夽 Manuela Cervelli a , Emiliano Fratini a,b , Roberto Amendola b , Marzia Bianchi a , Emanuela Signori c , Elisabetta Ferraro d , Antonella Lisi c , Rodolfo Federico a , Lucia Marcocci e , Paolo Mariottini a,∗ a

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

a r t i c l e

i n f o

Article history: Received 9 June 2008 Received in revised form 11 September 2008 Accepted 12 September 2008 Available online 21 September 2008 Keywords: Polyamines Spermine oxidase C2C12 muscle cells Cell differentiation

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 H2 O2 , spermidine, and the 3-aminopropanal. In the present study, we have examined the SMO gene expression during the mouse myoblast C2C12 cell differentiation induced with two different stimuli by RT-PCR analysis, polysome-mRNP distribution and enzyme activity. SMO transcript accumulation and enzymatic activity increases during C2C12 cell differentiation and correlates with the decrease of spermine content. Many proteins are highly regulated during the phenotypic conversion of rapidly dividing C2C12 myoblasts into fully differentiated post-mitotic myotubes. The SMO gene induction represents a novel and additional marker of C2C12 cell differentiation. The sub-cellular localization of the SMO␣ and SMO␮ splice variants is not involved in the differentiation processes. Nuclear localization of only the SMO␮ protein was confirmed. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction The flavin-containing spermine oxidase (SMO) plays an important role in the animal cell homeostasis of polyamines (PA), being capable of directly oxidizing spermine (Spm) and producing spermidine, 3-aminopropanal and H2 O2 (Cervelli et al., 2003; Vujcic et al., 2002; Wang et al., 2001). A previous study on the relative abundance of SMO transcript in various mouse organs showed that muscle cells highly express two distinct splice variants of spermine oxidase (SMO␣ and ␮) (Cervelli et al., 2004). In the present study, we

Abbreviations: AcSpm, N1 Ac-spermine; ASMA, alpha skeletal muscle actin; C2C12, mouse skeletal myoblast cell line; FCM, flow cytometric; DM, differentiation medium; GM, growth medium; IGF-1, insulin-like growth factor; APAO, acetylpolyamine oxidase; MCK, muscle creatine kinase; ODC, ornithine decarboxylase; SMO, spermine oxidase; SSAT, spermidine/spermine N1 -acetyl-transferase; PA, polyamines; PI, propidium iodide; Put, putrescine; ROS, reactive oxygen species; Spd, spermidine; Spm, spermine; TDM, transfection with differentiation medium TDM; TGM, transfection with growth medium; WS, withdrawal of serum. 夽 Enzyme: vertebrate spermine oxidase. ∗ Corresponding author at: Dipartimento di Biologia, Università di “Roma Tre”, Viale G. Marconi 446, 00146 Roma, Italy. Tel.: +39 06 55176359; fax: +39 06 5517 6321. E-mail address: [email protected] (P. Mariottini). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.09.009

have examined the expression of these two isoforms in the mouse C2C12 myogenic cells that represent an early stage of muscle differentiation (Yaffe and Saxel, 1977). Depending on the culture conditions, these cells can either proliferate as myoblasts or differentiate into myotubes (Linkhart et al., 1981). When cultured in low serumcontaining growth medium or in the presence of IGF-1 (insulin-like growth factor 1) as a myogenic cell differentiating agent (Coolican et al., 1997; Florini et al., 1991; Milasincic et al., 1996; Tureckova et al., 2001), C2C12 cells exit the cell cycle and undergo a welldefined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells (Coolican et al., 1997; Florini et al., 1991; Milasincic et al., 1996; Tureckova et al., 2001). In order to highlight the role of polyamine metabolism during muscle differentiation, we analysed the polyamine content and the relative transcription levels by RT-PCR analysis of the key metabolic enzymes such as ornithinedecarboxylase (ODC), polyamine oxidase (APAO), N1 -acetyl-transferase (SSAT) and spermine oxidase (SMO) ␣ and ␮ isoforms. To analyse the translational utilization of the SMO transcripts during both proliferating and differentiating conditions, the fraction of SMO␣ and SMO␮ mRNAs associated to polysomes was determined together with the fraction of the ODC, APAO and SSAT transcripts, as well as the control ASMA (alpha skeletal muscle actin) and MCK (muscle creatine kinase) mRNAs. C2C12 myoblast

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cells transfected with both SMO␣ and SMO␮ V5-tagged proteins were induced to differentiate into myotubes and analysed to assess the sub-cellular localization of the two isoforms. The nuclear localization of the SMO␮ protein was confirmed during the C2C12 cell myogenic differentiation. Transcript accumulation of the PA metabolic enzymes was differentially regulated during differentiation, showing, among other things, a higher expression for both SMO isoforms. Interestingly, augmented transcript level was consistent with SMO enzymatic activity increase and Spm depletion. The importance of SMO induction during muscle differentiation could be related to hydrogen peroxide production, H2 O2 being a common signalling molecule that plays an important role in regulating the fate of mononuclear muscle cells (Orzechowski et al., 2002). More and more data indicate that, in muscle cells, reactive oxygen species (ROS) and nitric oxide (NO) are continually generated and that these molecules have a well-established role as physiological modulators of skeletal muscle functions, ranging from development to metabolism and from blood flow to contractile functions (Clanton et al., 1999). Studies in the past two decades suggest that strenuous muscle activity, associated with some pathological conditions and/or aging, increases the generation of ROS in the skeletal muscle to a level that overwhelms the antioxidant defence systems (Ji, 1995, 2001), thus provoking fatigue, inflammation, and tissue degeneration. Thus, to better define the actors that play a role in oxidative stress remains a very important challenge (Caporossi et al., 2003). 2. Materials and methods 2.1. Cell culture, differentiation and transfection C2C12 cells were maintained in a growth medium (GM) consisting of Dulbecco’s modified minimum essential medium (DMEM) supplemented with 20% foetal bovine serum (Gibco BRL, Gaithersburg, MD), penicillin (50 U/ml), and streptomycin (50 mg/ml) (Sigma, Milan, Italy). An adequate number of C2C12 cells (5 × 104 cells in a 60 mm Petri dish) to maintain a non-confluent condition during the time course were incubated at 37◦ C and exposed to a humidified atmosphere of 5% CO2 . Control GM cells were aliquoted at various time following 24 h seeding time (GM4-96h cells). To induce differentiation, GM cells were shifted 24 h after seeding to a differentiation medium (DM) consisting of DMEM supplemented with 2% foetal bovine serum, penicillin (50 U/ml), and streptomycin (50 mg/ml) or alternatively supplemented with IGF-1 (ITS 100×, insulin transferrin) (Sigma, Milan, Italy) (DM4-96h cells). Differentiation was followed by cell morphology under a phasecontrast microscopy observation (Axioskop, 20× objective, Zeiss, Oberkochen, GE). Ectopically expression of each vector (1 ␮g DNA/0.5 × 105 cells) was obtained with LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer’s instructions. After transfection and exposure to glycerol shock, the cells were washed with phosphatebuffered saline. Aliquots were cultured under normal conditions for 24 and 48 h (TGM24-48h cells) or induced to differentiate (TDM4-96h) to localize SMO isoforms according to the time courses. 2.2. Flow cytometric (FCM) determination of DNA content of GM and DM C2C12 cells To study DNA content, C2C12 cells were treated with Propidium Iodide (PI), as described (Nicoletti et al., 1991). At least 105 cells were analysed by a FACSCalibur flow cytometer (Becton Dickinson, San Josè, CA), previously calibrated by CaliBRITE 3 beads (Bec-

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ton Dickinson), with laser excitation set at 488 nm, and a 630 nm emission filter to detect red fluorescence. FCM histograms were analysed by the Windows Multiple Document Interface (WinMDI ver. 2.8, The Scripps Research Institute, La Jolla, CA) dedicated software. 2.3. RT-PCR analysis of SMO˛ and SMO transcripts in DM C2C12 cells The relative levels of SMO␣, SMO␮, APAO, ODC, SSAT, ␤-actin and rpS7 transcripts during C2C12 cell differentiation induced by SW or IGF-1were measured by RT-PCR with specific primers described in Cervelli et al. (2004) and Amendola et al. (2005). In particular, SMO␣ forward 5 -GTACCTGAAGGTGGAGAGC-3 and SMO␣ reverse 5 -TGCATGGGCGCTGTCTTGG-3 ; SMO␮ forward 5 CAGAGCAGCAGCCTGGTCACC-3 and SMO␮ reverse 5 -GGGCCCCTGCTGGAAGAGGTCTCG-3 ; APAO-forward 5 -GAGCCACCACTGCCTGCC-3 and APAO-reverse 5 -CCATGTGTGGCTTCCCC-3 ; ODCforward 5 -TCCAGGTTCCCTGTAAGCAC-3 and ODC-reverse 5 -CCAACTTTGCCTTTGGATGT-3 ; SSAT-forward 5 -CGTCCAGCCACTGCCTCTG-3 and SSAT-reverse 5 -GCAAGTACTCTTTGTCAATCTTG-3 ; ␤-actin-forward 5 -TGTTACCAACTGGGACGACA-3 and ␤-actinreverse 5 -AAGGAAGGCTGGAAAAGAGC-3 ; rpS7-forward 5 -CGAAGTTGGTCGG-3 and rpS7-reverse 5 -GGGAATTCAAAATTAACATCC-3 . The MCK (creatine kinase, GenBank accession number XM133921) specific primers were: MCK1-for 5 -GTCATCCAGACTGGGGTGG-3 , MCK2-for 5 -GTGGCCGGCGATGAGGAG-3 , MCK3rev 5 -AGCAGCAGAGGTGACACGG-3 , and MCK4-rev 5 -TGGAGATCACGCGGAGGTG-3 ; the ASMA (alpha skeletal muscle actin, GenBank accession number XM133921) specific primers were: ASMA1-for 5 -ATTGAACATGGCATCATCACC-3 , ASMA2-for 5 -ACGACATGGAGAAGATCTGG-3 , ASMA2-rev 5 -GCAGCTCATAGCTCTTCTCC-3 and ASMA2-rev 5 -TCCTGATGTCGATGTCGCAC-3 . Total RNA was isolated from different samples (GM and DM cells) by TRIZOL reagent (Gibco BRL), according to the manufacturer’s instructions. A synthesis of the cDNAs from the RNAs of different mouse organs was performed by primer random examers in 20 ␮l reaction volume containing 1 ␮g of total RNA, according to the manufacturer’s instructions (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen). Aliquots of reverse-transcribedRNA were amplified within with Taq DNA polymerase (Pharmacia) in the linear range and in saturating experimental conditions by 20, 25, 30 or 35 PCR cycles: denaturation at 94◦ C for 1 min, annealing at 60◦ C for 1 min and extension at 72◦ C for 1 min. The RT-PCRs were normalized by a comparison with the ␤-actin control. Further control reaction mixtures, either without template (not shown) or RT enzyme (not shown), were uniformly negative. Results were quantified by densitometry, using the BioRad Multianalyst software (BioRad, Hercules, CA). An estimation of the relative RT-PCR amplified product amounts was obtained by dividing the area of gel bands by the area of the relative control ␤-actin gel band. Data points are the means ± SE of three to five separate experiments, each performed in duplicate. 2.4. Polysome-mRNP distribution of SMO˛ and SMO transcripts in GM and DM C2C12 cells For preparing polysomes, the procedure for cell lyses, sucrose gradient sedimentation, and analysis of the polysome/subpolysome distribution of mRNAs was essentially that described by Meyuhas et al. (1996). Proliferating (cultured in GM) and post-mitotic (cultured in DM, 48 h after SW) C212 cells (1 × 106 ) were directly lysed on the plate with 300 ml of lyses buffer [10 mM NaCl, 10 mM MgCl2 , 10 mM Tris–HCl pH 7.5, 1%Triton X-100, 1% sodium deoxycholate, 36 units/ml RNase inhibitor (Amersham Pharmacia Biotech), 1 mM

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

taining 100 mM NaCl, 10 mM MgCl2 , 30 mM Tris–HCl, pH 7.5 and centrifuged in a Beckman ultracentrifuge with an SW41 rotor for 100 min at 37,000 rpm. Fractions collected while monitoring the optical density at 260 nm, were ethanol-precipitated overnight at −20◦ C. The distribution of SMO␣ SMO␮, APAO, ODC, SSAT, ␤-actin

Fig. 1. (A) Cellular morphology analysis during C2C12 cell differentiation (Axioskop, phase-contrast 20× objective, Zeiss). The micrographs represent three separate experiments. (B) DM C2C12 cell cycle check-points analysis. Cells were analysed at 4, 48, 72 and 96 h after WS. FCM representative histograms out of three experiments of the G0/G1 cell fraction, number of events per relative amount of Propidium Iodide (PI).

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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 as SMO␣, 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 and 48 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 of overlapping images. The micrographs represent three separate experiments.

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and rpS7 transcripts were analysed by RT-PCR with the specific primers described above. To perform quantitative RT-PCR analysis on RNA extracted from polysome gradient fractions, each sample was reverse-transcribed into cDNA together 15 pg of an in vitro transcribed RNA for ZmPAO cDNA (Zea mais polyamine oxidase, GenBank accession number AJ002204), included as an internal con-

trol to confirm that the amount of product was not influenced by experimental variations in the reactions. For each PCR, different cycles and template amounts were tested, in order to avoid conditions of saturation. ZmPAO cDNA (378 bp) was amplified with the following primers: ZmPAO1-for 5 -ATGAACCCCATCTGGCCCAT3 and ZmPAO2-rev 5 -GTCGCCGAAGTCGCTGAAGG-3 .

Fig. 3. SMO␣ and SMO␮ transcript determination during the C2C12 cell myogenic differentiation. Samples of DM C2C12 cells taken after SW at 4, 48, 72 and 96 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 three independent replicas are shown. Densitometric analysis of gel bands represents measurements of three separate experiments. Gene of interest/␤-actin ratio (normalized for each experimental time point) is represented as an arbitrary densitometric units bar graph (±SE) below PCR band gel.

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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 three independent replicas are shown. Densitometric analysis of gel bands represents measurements of three separate experiments. Gene of interest/␤-actin ratio (normalized for each experimental time point) is represented as an arbitrary densitometric units bar graph (±SE) below PCR bands gel.

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2.5. Analysis of enzymatic activities and polyamine content Polyamine oxidase activities on cell homogenates were determined fluorometrically according to Cervelli et al. (2004). Spm and N1 Ac-spermine (AcSpm) were used as the substrates of SMO and APAO enzymes, respectively. The enzyme activity was calculated as pmol H2 O2 produced/mg protein/min for SMO enzyme and pmol H2 O2 produced/mg protein/hour for APAO enzyme. Protein content was estimated by the method of Markwell et al. (1978) with bovine albumin as a standard. ODC and SSAT enzyme activities, calculated as pmol CO2 produced/mg protein/hour, were determined by using 14 C-labeled substrate and scintillation counting of end metabolized products, as described elsewhere (Chen et al., 2003). Polyamine concentration was determined as described elsewhere (Mates et al., 1992) and expressed as nmoles/mg protein for each sample, in respect to standard concentration of bovine albumin.

2.6. Intracellular localization of SMO˛ and SMO in transfected DM C2C12 cells C2C12 cells (0.5 × 105 cells in 35 mm Petri dishes) were transiently transfected in triplicate with the plasmids pcDNA3/SMO␣V5 and pcDNA3/SMO␮-V5 (Cervelli et al., 2004). Twenty-four hours after transfection cells were shifted by SW or IGF-1 treatment into DM medium for 4, 48, 72 and 96 h and then fixed with 3.7% paraformaldheyde in PBS (15 at 4 ◦ C). After permeabilization with 0.2% Triton X-100 in PBS for 5 min, cells were blocked in 2% horse serum in PBS and incubated for 1 h at 37 ◦ C with primary mouse anti-V5 monoclonal antibody (Sigma) followed by a secondary reaction with the goat polyclonal anti-Mouse IgG conjugated with FITC (Sigma). The evaluation of the sub-cellular localization of SMO␣ and SMO␮ V5-tagged proteins was carried out as described in Cervelli et al. (2004). Nuclei were counterstained with 1 ␮g/ml DAPI (4 ,6 -diamidino-2-phenylindole). Cells were examined under

Fig. 5. RT-PCR analysis of SMO␣ and SMO␮ mRNAs in polysome gradient fractions from GM and DM (48 h after SW) C2C12 cells. (A) Cytoplasmic extract was fractionated on sucrose gradients and total RNA extracted from gradient fractions. (B) RT-PCR reactions using specific primer pairs and different PCR cycles are indicated. Also shown is a quantification control (cDNA for ZmPAO, see Section 2). The agarose gel photograph stripes are aligned with the A254 traces of the optical density profiles. A representative RT-PCR experiment from three independent replicas is shown.

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a Leica TCS SP5 Confocal Microscopy equipped with a 40× (NA 1.25) or 63× (NA 1.4) oil-immersion objective. 3. Results 3.1. Cellular localization of SMO˛ and SMO splice variant proteins during C2C12 cell myogenic differentiation The conversion of C2C12 myoblasts into myotubes was monitored during the time course by cell morphology and FCM analysis (Fig. 1A and B). SW or IGF-1 treatment (not shown) induced a clearly visible change in cell shape, while FCM determination showed an increased in the G0/G1 cell fraction, both analyses indicating that C2C12 cells switched from a proliferating to a postmitotic differentiated condition. To reveal intracellular SMO␣ and SMO␮ protein localization during myogenic differentiation, both SMO splice variants were microscopically localized in GM and DM C2C12 cells transiently overexpressing SMO proteins (TGM and TDM cells). Augmented transcript levels for each transfected isoforms were detected in transiently transfected cells by RT-PCR (not shown). Sub-cellular localization was performed using the V5TAG as the epitope to direct the primary monoclonal antibodies. SMO isoform subcellular localization in transfected TDM C2C12 cells was analysed throughout differentiation (4, 48, 72 and 96 h after SW), while aliquots of TGM C2C12 cells, cultured in a nonconfluent condition, were taken at 24 and 48 h after transfection (see Materials and methods, Section 2.1). As depicted in Fig. 2, in both C2C12/pcDNA3/SMO␣-V5 and C2C12/pcDNA3/SMO␮-V5 transiently transfected TGM and TDM C2C12 cells, the sub-cellular localization of the SMO␣-V5tagged isoform is localized exclusively in the cytoplasm, the SMO␮-V5tagged protein clearly shows a nuclear localization. These results substantiate the same cell localization of SMO␣ and SMO␮ proteins previously observed in neuroblastoma cells (Cervelli et al., 2004; Amendola et al., 2005; Bianchi et al., 2005). 3.2. Accumulation of SMO˛ and SMO mRNAs during C2C12 cell myogenic differentiation Levels of SMO␣, SMO␮, APAO, ODC and SSAT mRNAs were examined during C2C12 myogenic differentiation by RT-PCR analysis (Figs. 3 and 4). To assess differentiated status, the PCR-amplified mRNAs coding for the MCK and ASMA proteins were probed. The housekeeping control ␤-actin protein and ribosomal protein S7 (rpS7), was also probed to quantify the amplified samples (see Section 2; Cervelli et al., 2004). Fig. 3 shows a typical time course analysis of the transcripts accumulation during C2C12 differentiation (obtained with SW) of DM cells at 4, 48, 72 and 96 h after induction. Control GM cells were aliquoted at 4 h following 24 h seeding time (GM4h). In order to amplify each transcript within a linear range different PCR cycle conditions were used (see Section 2).

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It can be observed that both SMO␣ and SMO␮ isoforms and also APAO and SSAT mRNAs were dramatically increased during differentiation. By contrast, transcript coding for ODC decreased its level progressively during the time course monitored. The ␤-actin and rp-S7 control transcripts showed no changes in their accumulation patterns during the time course (Fig. 3). Identical results were obtained using the IGF-1 as a myogenic cell differentiating agent on GM cells and DM36 and DM72 samples (Fig. 4), confirming a clear relationship between mRNA expression and cell differentiation patterns, independently from the differentiation stimulus adopted. 3.3. Polysome-mRNP distribution of SMO˛ and SMO mRNAs In order to determine the amount of SMO␣ and SMO␮ mRNAs translated during C2C12 cell myogenic differentiation, we analysed their distribution between polysomes and mRNPs in GM and DM C2C12 cells induced by SW (DM48 sample, Fig. 5). A semi-quantitative RT-PCR approach was adopted to evaluate the percentage of mRNA loading onto polysomes. Different PCR cycle numbers for each specific primer pair were chosen in order to fit the linear range of amplification. Fig. 5 shows the results of a typical experiment. The polysome distributions of APAO, ODC, SSAT and the controls MCK, ASMA, ␤-actin and rp-S7 transcripts have been examined in parallel. As an additional control, to monitor any loss of material during the RNA extraction, the ZmPAO cDNA was amplified and the corresponding RT-PCR product was equally present in all fractions. It appears that in both GM and DM C2C12 cells the SMO␣ and SMO␮ mRNAs are mainly translated, as well as all the other analysed mRNAs that were found to be almost entirely loaded onto polysomes (Fig. 5). The same experimental protocol was carried out on DM C2C12 cells induced by IGF-1 obtaining identical results (data not shown). 3.4. Enzyme activities and polyamine content during C2C12 cell myogenic differentiation During C2C12 myogenic differentiation, SMO enzymatic activity at first dropped to about half the value measured in GM4h cells (GM4h and DM4h samples), to linearly increase up to about three times (DM4-96h samples). Similarly, APAO enzymatic activity decreased dramatically to one/fifteenth of the level measured in GM4h cells (GM4h and DM4h samples), before rising to a value almost comparable to that of GM cells (GM4h and DM96h samples) (Table 1). SSAT enzymatic activity remains steady during the C2C12 myogenic differentiation (DM4-96h samples). On the other hand, it increases up to four fold in GM proliferating cells that enter into a confluent culture condition (GM4-72h samples) (Table 2). ODC enzymatic activity showed a high level in the early stages of differentiation, to dramatically decrease to about one/thirtieth of the initial value (DM4-96h samples). ODC activity measured in proliferating C2C12 cells shows a linear decrease after seeding as

Table 1 SMO and APAO activities of GM and DM C2C12 cells. Sample

SMO activity (pmol H2 O2 produced/mg protein/min)

GM4h DM4h DM48h DM72h DM96h SMT

543.42 288.78 324.39 519.90 858.25 31.03

± ± ± ± ± ±

45.9 51.7 36.9 91.1 43.4 14.4

SMO activity (pmol H2 O2 produced/mg protein/min) + MDL72,527 33.3 20.8 30.6 17.9 28.4 3.9

± ± ± ± ± ±

13.4 11.2 8.2 12.4 10.6 1.4

APAO activity (pmol H2 O2 produced/mg protein/h) 4962.86 336.48 383.50 1975.23 3784.02 1264.18

± ± ± ± ± ±

59.3 28.8 35.1 95.7 74.7 44.7

APAO activity (pmol H2 O2 produced/mg protein/h) + MDL72,527 20.43 18.65 22.78 19.95 15.05 32.66

± ± ± ± ± ±

13.8 8.5 4.2 5.5 9.9 6.8

GM and DM samples as defined in the text. SMT, skeletal muscle tissue. Values are the means ± SD of three independent experiments performed in duplicate. p < 0.01 based on Student’s t test.

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

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

GM4h GM48h GM72h DM4h DM48h DM72h DM96h SMT

4088.76 2899.55 1864.39 2432.81 102.37 122.11 89.72 44.77

± ± ± ± ± ± ± ±

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

227.6 119.2 153.7 132.3 7.0 15.3 3.9 3.9

103.21 142.56 99.72 101.55 18.3 12.3 22.4 11.7

± ± ± ± ± ± ± ±

11.3 7.6 9.3 5.4 2.4 5.4 7.7 3.3

SSAT activity (pmol N-AcSpd/mg protein/h) 285.96 399.57 1172.04 1065.61 955.58 987.35 1041.13 2137.74

± ± ± ± ± ± ± ±

76.8 46.0 126.8 152.7 73.8 97.3 173.7 193.1

GM and DM samples as defined in the text. SMT, skeletal muscle tissue. Values are the means ± SD of three independent experiments. p < 0.01 based on Student’s t test.

Table 3 Polyamines content of GM and DM C2C12 cells. Sample

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

GM4h DM4h DM48h DM72h DM96h

12.51 2.52 4.65 7.49 9.14

Spd ± ± ± ± ±

0.17 (10.4%) 0.12 (2.7%) 0.10 (5.7%) 0.11 (9.7%) 0.12 (12.3%)

77.73 62.08 52.33 48.92 45.11

Spm ± ± ± ± ±

0.83 (64.8%) 0.69 (62.8%) 0.76 (64.5%) 0.70 (63.1%) 0.69 (61.6%)

29.70 34.07 24.14 21.14 19.23

± ± ± ± ±

0.23 (24.8%) 0.19 (34.5%) 0.20 (29.8%) 0.22 (27.2%) 0.20 (26.1%)

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

the culture condition approaches confluence (GM4-72h samples) (Table 2). The PA content of the GM and DM cells was assayed in parallel. The Spm level of proliferating and early stage differentiated C2C12 cells (GM4h and DM4-48h samples) proved similar, then decreased during cell differentiation (DM72-96h samples). The level of spermidine (Spd) linearly decreased during the time course analysed as well. By contrast, the putrescine (Put) content increased up to about four fold during differentiation (DM4-96h samples), to reach a final value comparable to the one measured in the proliferating C2C12 cells (GM4h and DM96h samples) (Table 3). It is worthy to mentioning that the relative PA pool percentages of Spm, Spd and Put differed in the GM4 and DM4 samples, mainly as regards Spm and Put contents. After C2C12 cell differentiation the PA pools proved to be the ones observed in proliferating cells, the relative PAs percentages being reasonably similar in the GM4 and DM96 samples (Table 3). The PA content measured with IGF-1 differentiation stimulus was consistent with the above-mentioned results (data not shown). 4. Discussion Polyamine metabolism reflects a concerted action of enzymes, regulated at several transcriptional and post-transcriptional levels, but it still is unclear how this metabolism interacts with cell differentiation and in particular with muscle differentiation. A previous report showed that SMO enzyme is highly expressed in mouse muscle tissue (Cervelli et al., 2004). This has therefore prompted us to analyse the SMO gene expression during the conversion of C2C12 myoblasts into myotubes, this conversion corresponding to an early stage of muscle differentiation (Yaffe and Saxel, 1977). As an initial step, we monitored the conversion of C2C12 myoblasts into myotubes by cell morphology and FCM analysis (Fig. 1A and B). Then we analysed the cell localization of the two splice variants SMO␣ and SMO␮ transiently expressed in both TGM and TDM C2C12 cells (Fig. 2). Identical results were obtained using TDM cells induced by IGF-1 (not shown). According to previous experiments carried out on the neuroblastoma cell line N18TG2, the expected

cytoplasmic localization of the SMO␣ protein was confirmed, as well as the nuclear localization of the SMO␮ protein (Amendola et al., 2005; Bianchi et al., 2005; Cervelli et al., 2004). These results consistently substantiate the cell localization of these two SMO isoforms, and in both proliferating and differentiating cells. The role played by the nuclear SMO␮ isoform remains unclear; probably, the very low level of free Spm into the nuclear moiety suggests a SMO␮ function not totally linked to the Spm metabolism. To this end, a 60% homology has been recently described between the SMO enzyme and the FAD-dependent amino oxidase lysine-specific demethylase 1 (LSD1) (Casero & Marton). LSD1 is associated with a transcriptional repressor complex, thus hypothetically suggesting novel roles for SMO␮ and its substrate Spm in chromatin remodelling. Very recently, Murray-Stewart et al. (2008) have demonstrated that the human SMO/PAOh1 (homologue to SMO␣) and SMO5 (homologue to SMO␮) isoforms possess both cytoplasmic and nuclear localization in NCI-H157 human non-small cell lung carcinoma cells. The finding that the predominant splice variant SMO/PAOh1 is also present in the nucleus was rather unexpected. Further research is needed to establish whether this dissimilarity in cellular localization between the human SMO/PAOh1 and the murine counterpart SMO␣ is due to the different cell lines analysed or whether reflects a different speciesspecific feature. We also examined the gene expression patterns of these splice variants during the C2C12 myogenic differentiation induced by different stimuli (SW and IGF-1 treatment) by RT-PCR analysis. Interestingly, we found that the accumulation pattern of SMO␣, SMO␮, APAO and SSAT transcripts is quite similar to those of MCK and ASMA mRNAs, typical myogenic markers. On the other hand, the ODC mRNA accumulation pattern shows an opposite behaviour, the transcript level being high at early stage of differentiation, later decreasing during the time course analysed (Figs. 3 and 4). It turned out that the accumulation of the transcripts of the murine genes coding for the PA metabolic enzymes were deeply altered and followed the two mRNA accumulation opposite profiles during C2C12 cell myogenic differentiation. We also analysed the translational utilization of the SMO␣, SMO␮, ODC, APAO, SSAT, ASMA and MCK mRNAs in GM and DM C2C12 cells, and we found that all these mRNAs are translated with high efficiency, being almost completely loaded onto polysomes, irrespectively of the cellular status (Fig. 5). Taking these results as a whole, we can depict a muscle cell differentiating scenario where SMO, APAO and SSAT genes are induced at the level of transcription, at variance with the mRNA coding for ODC, whose level is reduced. During cell differentiation, it is generally accepted that ODC is down-regulated (Capell et al., 2000; Nitta et al., 2001; Rosander et al., 1995), and C2C12 cells represent one more proof that both ODC transcript level and enzymatic activity are reduced during differentiation. It must be also taken into account that ODC downregulation is a well-known marker of non-proliferating cells. The

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present work indicates is a further proof that ODC expression increases in proliferating cells as shown by FCM experiments (Fig. 1B). The enzyme activities of SMO, APAO, ODC and SSAT match the accumulation profiles of the corresponding transcripts (Tables 1 and 2), suggesting the lack of a translational control as confirmed by the polysome loading analysis. To this end, all polyamine metabolic genes analysed in this work can be considered additional markers of the myogenic differentiation. In line with the observation that SMO enzyme activity is induced up to three times during cell differentiation, the intracellular polyamine pools were affected showing a significant decrease both of Spm and Spd, and an increase of Put (Table 3). Therefore, in C2C12 cells the induction to differentiation provoked a dramatic effect on the PA pools. In effect, we observed a markedly different PA content between GM and DM4 C2C12 cells, while this difference is almost absent between GM and DM96 samples. This indicates that after the initial perturbation, the polyamine homeostasis drives back the PA content to a cellular balanced equilibrium (as the relative Put, Spd and Spm content percentages of GM and DM96 cells demonstrate). This is in line with the well-known notion that intracellular polyamines are mandatory for the expression of several growth-regulatory genes, such as MYC, FOS, JUN and ER␣ (Casero and Marton, 2007 and references therein). Accordingly, we hypothesize that during the dramatic cellular stress occurring during myogenic differentiation the PA synthesis and catabolism are affected mainly by transcriptional events. In particular, during muscle differentiation the increase of SMO activity following transcript up-regulation overwhelms the concerted action of the other enzymes involved in PA metabolism, leading to a considerable lowering of Spm content and a concomitant Put increase. This PAs variation due to an increase of SMO expression is in accordance with previous reports of SMO ectopical over-expression in human HEK293 (Vujcic et al., 2002) and murine neuroblastoma N18TG2 (Amendola et al., 2005; Cervelli et al., 2004) cell lines. More and more data indicate that SMO gene can be rapidly induced in response to stress and is responsible for H2 O2 production in different cell lines and malignant tissues (Babbar and Casero, 2006; Chaturvedi et al., 2004; Goodwin et al., 2008; Xu et al., 2004). Interestingly, the resulting H2 O2 produced by SMO enzyme activity has been demonstrated to be implicated as a signalling molecule for apoptosis (Chaturvedi et al., 2004; Xu et al., 2004), inflammationinduced carcinogenesis (Babbar and Casero, 2006), and induced sensitivity to ionizing radiation (Amendola et al., 2005; Bianchi et al., 2007). The molecular sources of ROS in skeletal muscle are poorly understood, and probably differ depending on muscle fibber type or muscle region, on age, on exercise intensity or duration and on physiologic state of the tissue. One likely candidate is mitochondrial electron transport. It also been suggested that xanthine oxidase, NADH oxidoreductase, phospholipase A2 and NADPH oxidase are involved in ROS formation in skeletal muscle (Clanton et al., 1999; Piao et al., 2005). Spermine oxidase is undoubtedly part of the intracellular redox scenario, albeit its contribution remains still unclear, due to the compensatory and redundant mechanisms that carry out the H2 O2 production. Muscle differentiation is a highly ordered multi-step process that is regulated by two groups of myogenic transcription factors, namely the basic helix–loop–helix muscle regulatory factors (MyoD, Myf5, myogenin, and MRF4) and the myocyte enhancerbinding factors 2 proteins (Molkentin and Olson, 1996). Insulin-like growth factors (IGF-1 and 2) stimulate muscle differentiation by activating several signalling pathways (Naya and Olson, 1999). During the last few years, it has been shown that IGFs stimulate the myogenic process via phosphatedylinositol 3-kinase (PI 3-kinase), p38 mitogen-activated protein kinase (p38 MAPK) (Piao et al., 2005). ROS can be cytotoxic when present at high and/or sustained

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levels. However, low ROS concentration has been shown to function as physiological intracellular signalling mediators. H2 O2 represents an ideal signalling molecule due to its rapid synthesis and diffusion. Redox-sensitive proteins are targets of specific oxidation by various oxidants. There is abundant evidence that nuclear factor-kB (NF-kB) activity is extremely sensitive to cellular redox status. In addition, it has been suggested that NF-kB activation by PI 3-kinase stimulates the myogenic process by increasing the expression of inducible nitric oxide synthase (Piao et al., 2005). A possibility is that SMO expression could be linked to IGFs pathway and that the produced H2 O2 could interact with NO, acting as physiological modulators of skeletal muscle functions. Different pathways are involved in ROS generation, typical of important metabolic systems where a mandatory interplay between compensatory and redundant mechanism do exist. In this regard the present work indicates that SMO activity could provide another piece in the puzzle of hydrogen peroxide production during muscle differentiation. Acknowledgements The authors are indebted to Dr. G. Bellavia (Department of Biology, Università Roma Tre, Rome, Italy), Dr. F. De Carlo (Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy) for technical support and Dr Martin Bennett for the text editing. We thank the reviewers for their suggestions, and the resulting improvements in the paper. References Amendola R, Bellini A, Cervelli M, Degan P, Marcocci L, Martini F, et al. Direct oxidative DNA damage, apoptosis and radio sensitivity by spermine oxidase activities in mouse neuroblastoma cells. Biochim Biophys Acta—Rev Cancer 2005;1755:15–24. Babbar N, Casero Jr RA. Tumor necrosis factor-alpha increases reactive oxygen species by inducing spermine oxidase in human lung epithelial cells: a potential mechanism for inflammation-induced carcinogenesis. Cancer Res 2006;64:8521–5. Bianchi M, Amendola R, Federico R, Polticelli F, Mariottini P. Two short protein domains are responsible for the nuclear localization of mouse spermine oxidase (mSMO)␮ isoform. FEBS J 2005;272:3052–9. Bianchi M, Bellini A, Cervelli M, Degan P, Marcocci L, Martini F, et al. Chronic sublethal oxidative stress by spermine oxidase over activity induces continuous DNA repair and hypersensitivity to radiation exposure. Biochim Biophys Acta—Mol Cell Res 2007;1773:774–83. Capell T, Bassie L, Topsom L, Hitchin E, Christou P. Simultaneous reduction of the activity of two related enzymes, involved in early steps of the polyamine biosynthetic pathway, by a single antisense cDNA in transgenic rice. Mol Gen Genet 2000;264:470–6. Caporossi D, Ciafrè SA, Pittaluga M, Savini I, Farace MG. Cellular responses to H(2)O(2) and bleomycin-induced oxidative stress in L6C5 rat myoblasts. Free Radic Biol Med 2003;35(11):1355–64. Casero Jr RA, Marton LJ. Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov 2007;6:373–90. Cervelli M, Bellini A, Bianchi M, Marcocci L, Nocera S, Polticelli F, et al. Mouse spermine oxidase gene splice variants: nuclear sub-cellular localization of a novel active isoform. Eur J Biochem 2004;271:760–70. Cervelli M, Polticelli F, Federico R, Mariottini P. Heterologous expression and characterization of mouse spermine oxidase. J Biol Chem 2003;278:5271–6. Chaturvedi R, Cheng Y, Asim M, Bussiere FI, Xu H, Gobert AP, et al. Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization. J Biol Chem 2004;279:40161–73. Chen Y, Vujcic S, Liang P, Diegelman P, Kramer DL, Porter CW. Genomic identification and biochemical characterization of a second spermidine/spermine N1-acetyltransferase. Biochem J 2003;373:661–7. Clanton TL, Zuo L, Klawitter P. Oxidants and skeletal muscle function: physiologic and pathophysiologic implications. Proc Soc Exp Biol Med 1999;222:253–62. Coolican SA, Samuel DS, Ewton DZ, McWade FJ, Florini JR. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J Biol Chem 1997;272:6653–62. Florini JR, Ewton DZ, Roof SL. Insulin-like growth factor-I stimulates terminal myogenic differentiation by induction of myogenin gene expression. Mol Endocrinol 1991;5:718–24. Goodwin AC, Jadallah S, Toubaji A, Lecksell K, Hicks JL, Kowalski J, et al. Increased spermine oxidase expression in human prostate cancer and prostatic intraepithelial neoplasia tissues. The Prostate 2008;68:766–72.

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