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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Research Article

Zinc promotes proliferation and activation of myogenic cells via the PI3K/Akt and ERK signaling cascade Kazuya Ohashia, Yosuke Nagataa, Eiji Wadaa, Peter S. Zammitb, Masataka Shiozukaa, Ryoichi Matsudaa,n a

Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Japan Randall Division of Cell and Molecular Biophysics, King's College London, London SE1 1UL, UK

b

article information

abstract

Article Chronology:

Skeletal muscle stem cells named muscle satellite cells are normally quiescent but are activated

Received 30 July 2014

in response to various stimuli, such as injury and overload. Activated satellite cells enter the cell

Received in revised form

cycle and proliferate to produce a large number of myogenic progenitor cells, and these cells then

2 March 2015

differentiate and fuse to form myofibers.

Accepted 4 March 2015 Available online 13 March 2015 Keywords: Skeletal muscle C2C12 Reserve cells Zinc Insulin

Zinc is one of the essential elements in the human body, and has multiple roles, including cell growth and DNA synthesis. However, the role of zinc in myogenic cells is not well understood, and is the focus of this study. We first examined the effects of zinc on differentiation of murine C2C12 myoblasts and found that zinc promoted proliferation, with an increased number of cells incorporating EdU, but inhibited differentiation with reduced myogenin expression and myotube formation. Furthermore, we used the C2C12 reserve cell model of myogenic quiescence to investigate the role of zinc on activation of myogenic cells. The number of reserve cells incorporating BrdU was increased by zinc in a dose dependent manner, with the number dramatically further increased using a combination of zinc and insulin. Akt and extracellular signal-regulated kinase (ERK) are downstream of insulin signaling, and both were phosphorylated after zinc treatment. The zinc/insulin combination-induced activation involved the phosphoinositide 3-kinase (PI3K)/Akt and ERK cascade. We conclude that zinc promotes activation and proliferation of myogenic cells, and this activation requires phosphorylation of PI3K/Akt and ERK as part of the signaling cascade. & 2015 Elsevier Inc. All rights reserved.

Abbreviations: IR, insulin receptor; IGFR, insulin-like growth factor receptor; IRS-1, insulin receptor substrate-1; PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin; Grb2, growth factor receptor-bound protein 2; FGF2, fibroblast growth factor 2; DTPA, diethylenetriamine pentaacetic acid; GM, growth medium; DMEM, Dulbecco's modified Eagle medium; BSA, bovine serum albumin; PBS, phosphate buffered saline; BrdU, 5-bromo-20 -deoxyuridine; EdU, 5-ethynyl-20 -deoxyuridine; HS, horse serum; SDS, sodium dodecyl sulfate; MyHC, myosin heavy chain; ERK, extracellular signal-regulated kinase n Correspondence to: Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902, Japan. Fax: þ81 354544306. E-mail addresses: [email protected] (K. Ohashi), [email protected] (Y. Nagata), [email protected] (E. Wada), [email protected] (P.S. Zammit), [email protected] (M. Shiozuka), [email protected] (R. Matsuda).

http://dx.doi.org/10.1016/j.yexcr.2015.03.003 0014-4827/& 2015 Elsevier Inc. All rights reserved.

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Introduction Muscle satellite cells play an indispensable role in regeneration of skeletal muscle [1]. Satellite cells are located between the basal lamina and the plasma membrane, and usually exist in a mitotically quiescent state, until activated by injury or overload [2]. Once activated, satellite cells enter the cell cycle and proliferate to produce a large number of myoblast progeny expressing Myf5 and MyoD, which then commit to differentiation and express myogenin. Myoblasts then fuse to existing myofibers to provide new myonuclei, or fuse together to form myotubes de novo, which subsequently mature to muscle fibers [3]. Insulin is a ligand for the insulin receptor (IR) or the insulin-like growth factor receptor (IGFR). When insulin binds to these receptors, tyrosine kinase in the β subunit of the receptor is activated, resulting in autophosphorylation of the kinase regulatory domain. The phospho-tyrosine of IR allows activation of insulin receptor substrate-1 (IRS-1), which operates through several signaling pathways including the phosphoinositide 3-kinase (PI3K)/Akt cascade [4,5]. The PI3K/Akt cascade has various roles, such as controlling proliferation and differentiation, and being anti-apoptotic. Especially in skeletal muscle, PI3K/Akt signaling plays a role in hypertrophy [6,7], and IGF-1-induced PI3K/Akt signaling contributes to increase in size of C2C12 myotubes [8]. In satellite cells, the PI3K/Akt cascade is involved in G1/S cell cycle progression in vitro [9]. Akt induces phosphorylation of mammalian target of rapamycin (mTOR), which is a master regulator of cell growth and cell cycle progression [10,11]. In myogenic cells, mTOR is involved in muscle differentiation [12], and is known to serve as a signal for muscle hypertrophy [7,13]. IR and IGFR also activate extracellular signal-regulated kinase (ERK) through Shc and growth factor receptor-bound protein 2 (Grb2) [4]. ERK is known to regulate cell proliferation, differentiation and cell survival. We previously reported that fibroblast growth factor 2 (FGF2)-induced phosphorylation of ERK through Grb2 has a role in the activation of quiescent myogenic cells [14]. Although the physiological roles of growth factors or hormones on skeletal muscle plasticity have been extensively investigated, the effects of minerals on skeletal muscle function have not been studied in detail. Zinc is an important element for skeletal muscle biology, for example increasing isometric twitch tension and endurance [15,16]. Zinc is an essential element and mainly distributed in skeletal muscle and bone in human. The concentration of zinc in human serum is around 15 mM [17]. More than 300 enzymes require zinc for their function and so zinc is involved in many biological activities including cell growth and DNA synthesis [18]. Addition of zinc into diethylenetriamine pentaacetic acid (DTPA)-treated medium rescues the inhibitory effects of metal ion chelation on muscle differentiation [19]. Surprisingly, zinc also induces phosphorylation of IR and IRS-1 in myoblasts, even in the absence of insulin [20], and increases phosphorylation of Akt in skeletal muscle of mice fed with zinc supplemented diet [21]. Furthermore, the zinc-containing drug, Z103, improves muscle function in the mdx murine model of Duchenne muscular dystrophy [22,23]. Although zinc has been shown to be important for proliferation of several types of cells and function of skeletal muscle, the role of zinc in myoblast or muscle satellite cell biology remains largely unknown. Here, we investigated the effects of zinc on activation, proliferation and differentiation of myogenic cells. We found that zinc

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promotes activation and proliferation of myoblasts, partly via the phosphorylation of PI3K/Akt and ERK.

Materials and methods Cell culture C2C12 myogenic cells (ATCC, Manassas, VA) were cultured in growth medium (GM) (Dulbecco's Modified Eagle Medium [DMEM], Gibco, Grand Island, NY) containing 20% fetal bovine serum (JRH Bioscience, St. Lenexa, KC), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco) at 37 1C in 5% CO2. It should be noted that DMEM does not contain zinc. To induce differentiation, 1  105 cells were seeded in a 35 mm tissue culture dish with GM and were incubated for 24 h. The medium was replaced with serum-free differentiation medium containing insulin–transferrin–sodium selenite media supplement (Sigma-Aldrich, St. Louis, MO) and 1 mg/ml bovine serum albumin (BSA) (Sigma-Aldrich). To isolate reserve cells, C2C12 cells were cultured for 5 days and then treated with 0.05% trypsin (Gibco) with phosphate buffered saline (PBS) containing CaCl2 and MgCl2 [PBS(þ)], for 5 min at 37 1C as reported previously [14].

Proliferation analysis We used 10 mM 5-ethynyl-20 -deoxyuridine (EdU) or 10 mM 5bromo-20 -deoxyuridine (BrdU) to assess cell proliferation. 5  103 cells were seeded in an 8 well chamber slide (Nunc, UK) or 1  104 cells were seeded in a gelatin-coated-24 well culture plate with GM and incubated for 24 h. Then, the medium was changed to 2% horse serum (HS) or 1 mg/ml BSA/DMEM with zinc chloride (ZnCl2) or zinc sulfate (ZnSO4), and EdU or BrdU was added for the last 2 h of the 48 h incubation. Following that, cells were fixed with 10% formalin for 15 min. To visualize BrdU, cells permeabilized with 0.5% Triton X-100. After 1 N hydrochloric acid treatment for 30 min, incorporated BrdU was detected with the antiBrdU rat monoclonal antibody BU1/75 (Abcam, Cambridge, UK) and Alexa Fluor 594-conjugated anti-rat IgG antibody (Molecular Probes, Eugene, OR) and 100 ng/ml Hoechst 33258. Finally, cells were mounted in Mowiol mounting medium. Approximately 800 cells were counted in each experiment. To visualize EdU, we used the Click-it EdU imaging kit (Invitrogen, Life Technologies, Paisley, UK).

Activation analysis To induce activation of reserve cells, cells were stimulated with ZnCl2 (the concentration was from 25 to 100 mM) and 10 mg/ml insulin (Sigma-Aldrich). When the effects of the inhibitors were assessed, cells were treated with each inhibitor for 30 min prior to reserve cell stimulation. LY294002 (Cayman Chemical, Ann Arbor, MI), Triciribine (Merck, Whitehouse Station,NJ), rapamycin (Cayman Chemical) and U0126 (Merck) stock solutions were prepared in dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1% in a culture medium and the same dose of DMSO was added to control conditions. To evaluate activation of reserve cells, cells were incubated with the medium containing 10 mM BrdU for 24 h. Following incubation, cells were fixed with 10% formalin in PBS for 15 min, and then immunostained to detect BrdU.

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Western blotting

mouse monoclonal antibody (Ser473, clone 587F11), anti-Akt rabbit monoclonal antibody (pan, 11E7), anti-phospho-p42/44 rabbit monoclonal antibody (T202/Y204, clone 20G11) (all from Cell Signaling Technology, Danvers MA), and anti-ERK 1/2 rabbit polyclonal antibody (Promega, Madison, WI).

Reserve cells were isolated as described at day 4, and then cultured with 1 mg/ml BSA/DMEM for 24 h. At day 5, cells were fixed with 10% trichloroacetic acid in PBS for 30 min at 4 1C and collected. Cells were homogenized with a sonicator in Complete Lysis-M Reagent (Roche Diagnosis, Indianapolis, IN) containing 2% sodium dodecyl sulfate (SDS). Protein concentrations were measured with the bicinchoninic acid method, and adjusted to 1 mg/ml in SDSsample buffer (50 mM Tris–HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, bromophenol blue). Samples were boiled for 5 min and separated by 12.5% SDS-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with Odyssey Blocking Buffer (LI-COR Biosciences, Inc., Lincoln, NE) and incubated with primary antibodies overnight at 4 1C. The membranes were washed with Tris-buffered saline containing 0.05% Tween-20 and incubated with Alexa Fluor 680 or IR Dye-conjugated secondary antibodies. The analysis was performed with an Odyssey Infrared Imaging System (LI-COR). The primary antibodies used were as follows: anti-phospho-Akt

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Immunofluorescent staining Cells were fixed with 10% formalin in PBS for 15 min, and permeabilized with 0.5% Triton X-100. Cells were incubated with primary antibodies: anti-myogenin rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-myosin heavy chain (MyHC) mouse monoclonal antibody MF20 (Developmental Studies Hybridoma Bank, Iowa City, IA) for 1 h at room temperature. After washes, secondary antibodies Alexa Fluor 488conjugated anti-mouse IgG antibody, Alexa Fluor 594conjugated anti-rabbit IgG antibody (Molecular Probes, Eugene, OR), and 100 ng/ml Hoechst 33258 were applied for 1 h at room temperature. The samples were the washed and mounted with Mowiol mounting medium.

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Fig. 1 – The effect of zinc on the myogenic differentiation of C2C12. C2C12 differentiation without (A, C, E, G) or with 50 lM ZnCl2 (B, D, F, H) at day 3 of differentiation. Fixed C2C12 cells were immunostained with anti-MyHC antibody (C, D), anti-myogenin antibody (E, F) and Hoechst 33258 (G, H). Differentiation at day 3 was assessed by expression of myogenin (I) and fusion index (J) and ratio of myogenin with MyHC positive cells (K). Data represent the mean7SEM (n ¼3). Asterisks indicate statistically significant difference (po0.05) by Student's t-test. The scale bars of A–B and C–H are 200 and 100 lm, respectively.

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Statistical analysis The data were analyzed by Student's t-tests or one-way analysis of variance (ANOVA) with Tukey's multiple comparison test for two independent groups or multiple comparisons, respectively. The values are expressed as mean7standard error of mean (SEM). The p-valueso0.05 were considered statistically significant.

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46 h in 2% HS/DMEM supplemented with ZnCl2, and pulsed with EdU for 2 h before being fixed. Treatment with zinc increased the proportion of myoblasts that had incorporated EdU (Fig. 3A) and number of the total cells per field (Fig. 3B). Similar results were obtained in serum-free medium (Fig. 3C and D). We also used a different source of zinc, namely zinc sulfate (ZnSO4), which has different ionic strength to ZnCl2. ZnSO4 also enhanced proliferation of C2C12 cells (Fig. 3E). These results clearly demonstrate that zinc promotes proliferation of C2C12 cells.

Results Effects of zinc on reserve cell activation Zinc suppresses myogenic differentiation of C2C12 myoblasts To first examine the effect of ZnCl2 treatment on myogenic differentiation, murine C2C12 myoblasts were treated with 25 or 50 mM zinc at the time of induction of differentiation. Myotubes at day 3 with 50 mM zinc were thinner than non-treated control (Fig. 1A and B). Immunostaining for myogenin and MyHC was performed to assess differentiation of C2C12 cells (Fig. 1C–H), with the percentage of myogenin positive cells decreased significantly by zinc treatment (Fig. 1I). Furthermore, the fusion index was also reduced by zinc (Fig. 1J). However, there was no statistical difference in the ratio of MyHC positive cells/myogenin positive cells between zinc treatment and non-treated control (Fig. 1K) suggesting that zinc affected expression of myogenin or earlier genes associated with differentiation, rather than MyHC. These results indicate that zinc suppresses C2C12 differentiation. Crucially, removal of zinc at day 3 resulted in normal myotube formation at day 5 (Fig. 2), showing that the inhibitory effect of zinc is reversible.

Zinc enhances proliferation of C2C12 myoblasts Since differentiation of C2C12 was inhibited by zinc, we also assessed its effects on proliferation. C2C12 cells were cultured for

ZnCl2

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Upon induction of differentiation, C2C12 cells can undergo different fates: either form multinucleated myotubes, become mitotically quiescent mononucleated “reserve cells” or apoptosis [14,24]. Reserve cells can then again be activated to enter the cell cycle on exposure to serum containing medium. To assess the effects of zinc on activation of reserve cells, we treated reserve cells with 25, 50, or 75 mM zinc in 2% HS-containing DMEM with BrdU for 24 h. BrdU incorporation was increased by treatment with zinc in a dose dependent manner (Fig. 4A). Zinc also induced activation of reserve cells in serum free medium, as revealed by the greater proportion of BrdU positive cells after zinc exposure (Fig. 4B). We also found that BrdU incorporation was enhanced with ZnSO4 (Fig. 4C). These results suggested that zinc induces activation of quiescent myogenic reserve cells.

Zinc acts through phosphorylation of Akt and ERK To investigate intracellular pathways meditating activation of reserve cells after zinc exposure, we focused on ERK and Akt. Zinc was reported to be involved in phosphorylation of ERK [25,26], and we recently reported that ERK is phosphorylated during activation of reserve cells [14,27]. ERK is essential for cell proliferation in general [28]. Akt also plays essential roles in cell proliferation and survival, and is an insulin signaling pathway protein.

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MyHC

Hoechst Fig. 2 – The effects of zinc removal on myogenic differentiation. At day 3, differentiation medium with or without ZnCl2 was replaced with fresh differentiation medium without ZnCl2, and cells were cultured for 2 more days. At day 5, the cells were fixed and immunostained for MyHC (A, B) and with Hoechst 33258 (C, D). The scale bar is 100 lm.

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Fig. 3 – The effect of zinc on the proliferation of C2C12 cells. C2C12 cells were cultured with 2% HS-containing medium (A, B) or serum-free medium (C, D). Proliferating C2C12s were cultured for 48 h without or with ZnCl2 and pulsed for 2 h with EdU before fixation, and EdU incorporation was analyzed (A, C) and the number of cells per field counted (B, D). The effect of ZnSO4 was analyzed by BrdU incorporation (E). Data represent the mean7SEM (n¼ 3). Asterisks indicate statistically significant differences (po0.05) with Tukey's multiple comparison tests.

We examined whether zinc induced phosphorylation of Akt and ERK in quiescent myogenic cells using Western blot analysis. Akt phosphorylation was detected from 0.5 h, becoming stronger after 1 h, and was even maintained for 6 h. 100 mM zinc caused stronger phosphorylation of Akt than 50 mM zinc. ERK phosphorylation was detected in cells treated with either 50 or 100 mM zinc from 0.5 h, and was maintained for 6 h as with Akt phosphorylation (Fig. 5).

Zinc and insulin synergize to enhance reserve cell activation and phosphorylation of Akt We recently reported that activation of reserve cells is enhanced by a combination of insulin and FGF2 or EGF, to a level similar to serum-induced activation, and phosphorylation of ERK is involved [14,27]. Insulin plays roles in activation of both the PI3K/Akt and

ERK cascade [4]. In rat adipocytes, zinc inhibits dephosphorylation of IR [29]. Therefore, we hypothesized that combination of zinc and insulin could enhance activation of reserve cells. While incubation for 24 h with either insulin or zinc alone slightly increased the proportion of reserve cells incorporating BrdU compared with non-treated control, zinc and insulin together dramatically further increased BrdU incorporation in reserve cells (Fig. 6). We next evaluated whether the zinc/insulin combination treatment affected Akt or ERK phosphorylation. We harvested cells 0.5, 1, and 3 h after the addition of zinc and/or insulin (Fig. 5). Phosphorylated Akt was detected 0.5 h after adding zinc and insulin, and it was increased and sustained compared to treatment with each agent individually. On the other hand, we did not observe a synergistic effect of zinc and insulin on ERK phosphorylation (Fig. 7). To compare intracellular signaling pathways of

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Fig. 4 – The effect of zinc on the activation of reserve cells. Reserve cells were incubated in 2% HS-containing DMEM (A), or BSA containing DMEM (B, C) supplemented with ZnCl2 (A, B) or ZnSO4 (C) in the presence of BrdU for 24 h, then fixed and immunostained for BrdU. Data represent the mean7SEM (n¼3). Asterisks indicate statistically significant differences compared to 2% HS non-treated control (A) or non-treated control (B and C) (po0.05) with Tukey's multiple comparison tests.

reserve cell activation between ZnCl2/insulin and insulin/EGF, we assessed the phosphorylation of Akt and ERK induced by insulin, EGF or combination of the two. We observed that phosphorylation of Akt and ERK was induced mainly with insulin and EGF, respectively. The combination of insulin and EGF enhanced phosphorylation of Akt, but the synergic effect on phosphorylation of ERK was less obvious (Fig. 8).

cells were pretreated with the inhibitor for 30 min before zinc and insulin stimulation for 24 h in the presence of BrdU. LY294002, Triciribine, rapamycin and U0126 all significantly reduced the proportion of reserve cells incorporating BrdU. These observations demonstrate that activation of reserve cells by zinc/ insulin functions through the PI3K/Akt and ERK signaling cascade (Fig. 9C).

Inhibition of PI3K, Akt, mTOR, and ERK suppress zinc/ insulin-mediated activation of reserve cells

Discussion

It was important to determine whether zinc/insulin-mediated activation of reserve cells operated through PI3K, Akt, mTOR, and/ or ERK. To investigate the involvement of these signaling proteins, inhibitors were used: to inhibit phosphorylation of PI3K we used LY294002, for Akt we used triciribine, for mTOR we used rapamycin, and for ERK we used U0126 (Fig. 9A and B). Reserve

The activation and proliferation of satellite cells are required for muscle regeneration and growth. In this study, we examined the effects of zinc in C2C12 myogenic cells. Although zinc plays roles in the growth and proliferation of many types of cells, its effects on myogenic cells remain unclear. Here, we demonstrate that the addition of zinc to serum-free differentiation medium inhibits

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Fig. 5 – Zinc-stimulated phosphorylation of ERK and Akt in reserve cells. Reserve cells were stimulated with 50 or 100 lM ZnCl2. At 0.5–6 h after stimulation, reserve cells were harvested for the analyses of Akt and ERK phosphorylation by Western blotting.

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Fig. 7 – The combinatorial effect of zinc and insulin for phosphorylation of Akt and ERK. Reserve cells were stimulated with 50 lM ZnCl2 and 10 lg/ml insulin. At 0.5–3 h after stimulation, reserve cells were harvested for the analyses of Akt and ERK phosphorylation. Time (min)

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Fig. 6 – The combinatorial effect of zinc and insulin for activation of reserve cells. Reserve cells were stimulated with 50 lM ZnCl2 and 10 lg/ml insulin in the presence of BrdU. After 24 h, cells were fixed and immunolabeled to analyze the level of BrdU incorporation. Data represent the mean7SEM (n¼ 3). Asterisks indicate statistically significant differences compared to non-treated control (po0.05) with Tukey's multiple comparison tests.

C2C12 myoblast differentiation and promotes proliferation. Furthermore, we demonstrate that zinc induces activation of quiescent myogenic reserve cells. Finally, the combination of zinc and insulin enhances the activation of reserve cells through the PI3K/Akt cascade and ERK. Chelation of zinc from culture medium inhibits the differentiation of myogenic cells of the chick embryo, suggesting that zinc is an important element for muscle differentiation [19]. Another study proposed that zinc is required for initiation of muscle differentiation [30]. Here, we observed inhibition of myotube formation and an increase in the number of cells by zinc addition. Moreover, we noted a decreased number of myogenin-positive cells and a lower fusion index with 50 mM zinc. Interestingly, there was no difference in MyHC expression in myogenin positive cells after day 3 of differentiation in C2C12 cells. Although it is possible that zinc negatively regulates maturation of myotubes, differentiation of C2C12 cells after myogenin expression occurred normally in zinc-supplemented medium. It suggests that zinc affects expression of myogenin or earlier genes associated with

pERK1/2

ERK1/2

Fig. 8 – The phosphorylation of Akt and ERK induced by insulin and EGF. Reserve cells were stimulated with 10 lg/ml insulin and 25 ng/ml EGF for 10–60 min. The phosphorylation of Akt and ERK was analyzed.

differentiation. Our results might seem contradictory to previous studies reporting that zinc is essential for myogenic differentiation. In this study, zinc concentration was higher than serum, whereas previous studies reported the effects of depletion of zinc [19,30]. We assume that there is optimal range of zinc for differentiation of myogenic cells. Muscle differentiation is strictly regulated by several factors such as FGF, IGF [31,32,33,34]. We found that zinc promoted proliferation and suppressed differentiation of C2C12 cells. Furthermore, the proliferative effect of zinc was promoted in the presence of serum to a greater extent than observed in the absence of serum. Therefore, we speculate that some components of serum enhanced zinc-induced proliferation. In addition, we observed similar results in activation of reserve cells. Zinc is required for Ca2þ uptake of 3T3 cells stimulated with growth factors such as IGF-1, and Ca2þ influx is essential for proliferation of the cells [35,36]. Furthermore, zinc enhances EGF-stimulated DNA synthesis in mouse hepatocytes [37]. Hence, it is possible that the increased proliferation of C2C12 cells induced by zinc is related to the effects of EGF and/or IGF-1.

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Fig. 9 – The involvement of insulin pathways for the activation of reserve cells induced by combination of zinc and insulin. LY294002, triciribine, rapamycin or U0126 were pretreated for 30 min before ZnCl2 and insulin stimulation of reserve cells. ZnCl2/ insulin-induced phospho-Akt at 3 h and -ERK at 0.5 h were confirmed with 25.0 lM LY294002, 5.0 lM triciribine, 100.0 nM rapamycin or 10.0 lM U0126 (A, B). Next, cells were fixed and immunostained to analyze BrdU incorporation (C). Data represent the mean7SEM (no inhibitor control; n¼6, with inhibitor; n¼ 3). Asterisks indicate statistically significant differences compared to no inhibitor control (po0.05) using Tukey's multiple comparison tests.

Importantly, we found that ZnSO4 has similar effects as ZnCl2 on promoting proliferation and activation of reserve cells, suggesting that these effects are mediated by zinc ions. However, most zinc exists as zinc complexes which bind to some proteins in serum, so more experiments are required to determine whether these effects are by zinc ions or zinc complexes. We have recently shown that combination of insulin and EGF stimulated DNA synthesis in quiescent C2C12 cells [27]. Zinc was shown to activate IR by receptor protein tyrosine kinases [20,26,38], and zinc was also reported to affect phosphorylation of ERK pathway via EGF receptor phosphorylation in several cell types [25,26]. Previous studies demonstrate that the metal chelator DTPA canceled the effect of IGF-1 for the S-phase entry of quiescent 3T3 cells, and that the function of IGF was rescued by the addition of 400 mM zinc [35,39], suggesting that zinc is required for cell cycle progression. In this study, we demonstrated that zinc induced activation of reserve cells, and zinc induced phosphorylation of downstream proteins of IR and EGFR in quiescent myogenic cells, Akt and ERK. These results are

consistent with previous studies that showed zinc induces activation of PI3K, Akt and mTOR-dependent p70S6K [38,40,41]. It was also found that supplemental dietary zinc influenced phosphorylation of the mTOR signaling pathway in skeletal muscle in vivo [21]. We found that activation of reserve cells was dramatically enhanced by treatment with a combination of zinc and insulin. This combination also enhanced phosphorylation of Akt, but not of ERK. This enhanced activation was suppressed by inhibition of PI3K, Akt, mTOR and ERK suggesting that the PI3K/Akt cascade as well as ERK is required for activation of reserve cells. However, reserve cell activation was not suppressed completely, suggesting incomplete inhibition or that other pathways are involve. The combination-induced phosphorylation of Akt was sustained longer than adding either agent individually. Furthermore, we found that the phosphorylation of ERK induced by zinc was stronger than that induced by insulin alone. We previously reported that activation of reserve cells is induced with a combination of EGF and insulin, and that the

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activation required phosphorylation of ERK [27]. However, mechanisms other than ERK required for activation of reserve cells are unclear. In this study, we showed phosphorylation of ERK and Akt was induced by insulin and EGF. Furthermore, Akt phosphorylation was enhanced by combination treatment. Zinc exerted effects similar to EGF on Akt phosphorylation either alone or in combination with insulin. Here, we demonstrated that Akt and ERK phosphorylation is induced by ZnCl2/insulin and insulin/ EGF during activation of reserve cells. Moreover, the inhibitors of the PI3K/Akt and ERK cascades suppressed reserve cell activation. Hence, we consider that reserve cell activation requires phosphorylation of both pathways, PI3K/Akt and ERK. Furthermore, the effect of zinc is similar to the effect of EGF on Akt and ERK phosphorylation. In addition, synergic effects on activation of reserve cells between ZnCl2/insulin and insulin/EGF are similar. Therefore, zinc may function instead of EGF in the enhanced activation. Zinc was shown to be involved in EGFR-induced intracellular signaling [25,26] and phosphorylation of ERK is induced by IGFR and EGFR [26]. Zinc has the potential to induce the activation of Akt and ERK pathways in mutant Chinese hamster ovary cells deficient of IR activity [26]. In contrast, zinc failed to activate ERK1/2 and Akt in IGF-1R knockout cells [26], indicating that IGF-1R is more effective than IR for signaling pathways mediated with zinc. Because insulin binds to IGFR when insulin concentrations are high, IGFR rather than IR would be important for the activation of reserve cells stimulated with zinc. Therefore, it is possible that zinc-induced phosphorylation of ERK and Akt in quiescent C2C12 cells observed in this study occurs through both IGFR- and EGFR. Dephosphorylation of phosphotyrosyl residues is inhibited by zinc [42], and it was also proposed that zinc may indirectly activate IGF1R and EGFR by preventing dephosphorylation of receptor protein tyrosine kinases [26]. Another group reported that 1 mM zinc inhibited dephosphorylation of IR in rat adipocytes [29]. Hence, we speculate that enhanced phosphorylation of Akt, following treatment with the combination of zinc and insulin, was caused by the inhibition of protein tyrosine phosphatases. In conclusion, zinc promotes proliferation in myoblasts, but inhibits their myogenic differentiation. Quiescent myogenic cells were also activated by addition of zinc, to augment proliferative potential with serum containing medium. These effects were also observed even in serum free medium. Furthermore, the effects of zinc were enhanced when combined with insulin. These results demonstrated that zinc promotes proliferation and activation of myoblast. Because zinc is mainly present in skeletal muscle in the human body, it is possible that zinc, discharged from damaged muscle fibers, may contribute to the activation and proliferation of muscle satellite cells.

Acknowledgement This work was supported in part by a Grant-in-Aid for a Health and Labour Sciences Research Grant for Comprehensive Research on Disability Health and Welfare (H22-016); Intramural Research Grant (26-8) for Neurological and Psychiatric Disorder of National Center of Neurology and Psychiatry; a grant-in aid #25650106 from Ministry of Education, Culture, Sports, Science and Technology-Japan; the Ichiro Kanehara Foundation (25-3) and the Sasakawa Scientific Research Grant (13-209) from The Japan Science Society.

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