Tài liệu Báo cáo khoa học:Insulin-like growth factor 1 signaling regulates cytosolic sialidase Neu2 expression during myoblast differentiation and hypertrophy doc

Insulin-like growth factor signaling regulates cytosolic sialidase Neu2 expression during myoblast differentiation and hypertrophy Alessandro Fanzani, Francesca Colombo, Roberta Giuliani, Augusto Preti and Sergio Marchesini Department of Biomedical Sciences and Biotechnology, Unit of Biochemistry, University of Brescia, Italy Keywords AKT; IGF-1; myoblast; Neu2 sialidase; gangliosides Correspondence A Fanzani, University of Brescia, Department of Biomedical Sciences and Biotechnology, viale Europa 11, 25123 Brescia, Italy Fax: +39 030 3701157 Tel: +39 030 3717568 E-mail: fanzani@med.unibs.it (Received May 2006, revised 12 June 2006, accepted 13 June 2006) doi:10.1111/j.1742-4658.2006.05380.x Cytosolic sialidase (neuraminidase 2; Neu2) is an enzyme whose expression increases during myoblast differentiation Here we show that insulin-like growth factor (IGF1)-induced hypertrophy of myoblasts notably increases Neu2 synthesis by activation of the phosphatidylinositol 3-kinase/AKT/ mammalian target of rapamycin (P13K/AKT/mTOR) pathway, whereas the proliferative effect mediated by activation of the extracellular regulated kinase ⁄ (ERK1 ⁄ 2) pathway negatively contributed to Neu2 activity Accordingly, the differentiation L6MLC ⁄ IGF-1 cell line, in which the forced postmitotic expression of insulin-like growth factor stimulates a dramatic hypertrophy, was accompanied by a stronger Neu2 increase Indeed, the hypertrophy induced by transfection of a constitutively activated form of AKT was able to induce high Neu2 activity in C2C12 cells, whereas the transfection of a kinase-inactive form of AKT prevented myotube formation, triggering Neu2 downregulation Neu2 expression was strictly correlated with IGF-1 signaling also in C2 myoblasts overexpressing the insulin-like growth factor binding protein and therefore not responding to endogenously produced insulin-like growth factor Although Neu2-transfected myoblasts exhibited stronger differentiation, we demonstrated that Neu2 overexpression does not override the block of differentiation mediated by PI3 kinase and mTOR inhibitors Finally, Neu2 overexpression did not modify the ganglioside pattern of C2C12 cells, suggesting that glycoproteins might be the target of Neu2 activity Taken together, our data demonstrate that IGF-1-induced differentiation and hypertrophy are driven, at least in part, by Neu2 upregulation and further support the significant role of cytosolic sialidase in myoblasts Skeletal muscle hypertrophy plays an important role during postnatal development and occurs in response to physical exercise [1], resulting in an increase in fiber size accompanied by the increased expression of insulin-like growth factor (IGF-1) [2,3] Since IGF-1 overexpression in the skeletal muscle of transgenic mice triggers an increase in muscle size [4–6], the emerging idea is that IGF-1 is sufficient to induce muscle hypertrophy Administration of IGF-1 to cultured muscle cells elicits a biphasic response, first promoting cell proliferation and then enhancing myogenic differentiation [7,8], reproducing the events occurring during Abbreviations AKT or PKB, protein kinase B; caAKT, constitutively active form of AKT; DM, differentiating medium; GM, growth medium; IGF-1, insulin-like growth factor 1; IGFBP5, insulin-like growth factor 1-binding protein; IRS-1, insulin receptor substrate 1; kiAKT, kinase-inactive form of AKT; LY, LY294002; Neu2, neuraminidase 2; HS, horse serum; MAP kinase, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PD, PD098059 FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3709 Insulin-like growth factor signaling A Fanzani et al the repair of damaged tissue In particular, myoblast proliferation is triggered by activation of the extracellular regulated kinase (ERK) pathway, whereas myoblast hypertrophy occurs after activation of the phosphatidylinositol 3-kinase (PI3K)–AKT pathway [9,10] Another critical regulator of myoblast hypertrophy is mammalian target of rapamycin (mTOR) [11,12], whose activation by AKT elicits the phosphorylation of two known regulators of protein synthesis, P70S6K and the eukaryotic initiation factor 4E-binding protein PHAS-1 (PHAS/4EBP1) [13,14], thereby promoting increased protein translation Among the four forms of mammalian sialidases, neuraminidase (Neu2) (EC 3.2.1.18) is unique with regard to cellular localization and tissue expression Whereas the lysosomal form Neu1 [15–17], the ganglioside sialidase Neu3 [18] and the recently cloned Neu4 [19] are membrane-bound enzymes with broad tissue expression, Neu2 has a cytosolic localization and its expression is relatively high only in the skeletal muscle [20] The involvement of Neu2 in myoblast differentiation has been proposed for the first time using L6 rat myoblasts [21]; in addition, we recently suggested a crucial role for Neu2 in C2C12 myoblasts, demonstrating its increase during myoblast differentiation and that its overexpression enhances myotube formation [22] The purpose of this work was to establish whether IGF-1 is critical in myoblasts for Neu2 expression, using pharmacologic inhibitors of the PI3K–AKT– mTOR and ERK pathways and obtaining cells either expressing the constitutively activated AKT or its dominant negative form Neu2 expression was further investigated using L6E9 myoblasts, in which forced postmitotic expression of IGF-1 stimulates a dramatic hypertrophy [23], and using C2 myoblasts overexpressing IGF-binding protein-5 (IGFBP5), in which IGF-1 signaling is selectively repressed [24] Neu2 transfectants, characterized by enhanced differentiation, were treated with inhibitors of the PI3 kinase–AKT–mTOR pathway to evaluate the effects on myotube formation; finally, parental and Neu2-transfected cells were subjected to ganglioside analysis to determine whether the endogenous sialidase activity was able to modify the cellular content of sialolipids Results Neu2 expression increases during IGF-1-induced hypertrophy of myoblasts It is well known that IGF-1 is able to induce both myoblast proliferation through the activation of the Ras–Raf–Mek–Erk pathway and cell differentiation ⁄ 3710 hypertrophy through activation of the PI3 kinase– AKT–mTOR pathway [9] Accordingly, IGF-1 treatment (5 ngỈmL)1) of C2C12 myoblasts triggered the simultaneous phosphorylation of ERK1 ⁄ and AKT proteins (Fig 1A) Subsequently, AKT phosphorylation led to activation of mTOR, detected as phosphorylation of the downstream target p70S6K (Fig 1A) When we used pharmacologic inhibitors to block selectively these pathways (Fig 1A), ERK1 ⁄ phosphorylation was prevented in the presence of 30 lm PD098059 (PD), whereas AKT phosphorylation was blocked in the presence of 20 lm LY294002 (LY), a known inhibitor of PI3 kinase activity In addition, the inhibition of mTOR activity was achieved in the presence of ngỈmL)1 rapamycin, as revealed by the absence of the phosphorylated form of p70S6K As shown in Fig 1B, C2C12 cells grown in differentiating medium (DM) until day fused into multinucleated myotubes, whereas the cells grown in DM supplemented with IGF-1 (5 ngỈmL)1) developed a marked cell hypertrophy While simultaneous treatment with IGF-1 and PD did not change the rate of cell hypertrophy, myotube formation was completely prevented by treatment with LY The block of differentiation was obtained even in the presence of rapamycin (data not shown), an inhibitor of mTOR activity, confirming the relevance of the PI3 kinase–AKT–mTOR pathway in this process The rate of cell hypertrophy was quantified by myotube diameter analysis (Fig 1B, right panel): in the presence of IGF-1 or IGF-1 supplemented with PD, the average myotube diameter was about two-fold compared to parental cells differentiated in DM alone, whereas in the presence of LY no myotubes were observed Under these experimental conditions, Neu2 expression was investigated by RT-PCR analysis (Fig 1C) and enzymatic assay (Fig 1D) Neu2 transcript upregulation was observed in myoblasts grown in the presence of IGF-1 compared to DM alone, with the upregulation being reinforced in the presence of IGF-1 supplemented with PD In addition, treatment with LY or rapamycin strongly repressed Neu2 transcription Accordingly, the enzymatic assay showed very low Neu2 activity in proliferating cells cultured in growth medium (GM), whereas myoblasts grown in DM exhibited high Neu2 activity Interestingly, a further increase of Neu2 activity was observed during myotube hypertrophy obtained in the presence of IGF-1; moreover, the effects of IGF-1 were considerably reinforced in the presence of IGF-1 supplemented with PD, suggesting that ERK1 ⁄ phosphorylation negatively contributes to Neu2 expression Finally, the treatments either with LY or rapamycin completely prevented FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS A Fanzani et al Insulin-like growth factor signaling A C B D E F G H Fig Insulin-like growth factor (IGF-1)-induced hypertrophy enhances Neu2 expression in murine myoblasts (A) ngỈmL)1 IGF-1 induces phosphorylation of ERK1 ⁄ 2, AKT and p70S6K proteins ERK1 ⁄ phosphorylation was prevented in the presence of 30 lM PD098059 (PD), AKT phosphorylation in the presence of 20 lM LY294002 (LY), and p70S6K phosphorylation in the presence of ngỈmL)1 rapamycin Western blots against total ERK1 ⁄ and tubulin were performed to verify equal loading of protein samples (B) C2C12 myoblasts were grown for days in the presence of the indicated treatments and subjected to Giemsa staining Mean myotube diameters are shown in a graph on the right and expressed in arbitrary units (n ¼ 10, *P < 0.05) (C) Neu2 transcript expression obtained by RT-PCR analysis in the presence of the indicated treatments until day The data were normalized by loading the total RNA as control (D) Neu2 enzymatic assay performed using C2C12 cells cultured for days in the presence of the indicated treatments (n ¼ 3, *P < 0.05) (E) Neu2 activity was evaluated in C2C12 cells cultured in differentiating medium (DM) until day in the presence of two different concentrations of PD (10 and 30 lM) alone or supplemented with ngặmL)1 IGF-1 (n ẳ 3, *P < 0.05) (F, G, H) Morphology (F), time-course of Neu2 enzymatic activity (G) and RT-PCR analysis of Neu2 transcript expression (H) obtained for L6MLC ⁄ IGF-1 cells compared to untreated and IGF-1-treated L6E9 cells (n ¼ 3, *P < 0.05) FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3711 Insulin-like growth factor signaling A Fanzani et al Neu2 activity, suggesting that the PI3 kinase–AKT– mTOR pathway is crucial for Neu2 expression The effects of PD treatment on Neu2 activity were further examined (Fig 1E); in particular, C2C12 cells cultured in DM for days in the presence of different concentrations of PD (10–30 lm) showed an increase of Neu2 activity compared to cells grown in DM alone, suggesting that the lower PD concentration is sufficient to inhibit the ERK1 ⁄ phosphorylation induced by endogenously secreted IGF-1 As expected, Neu2 activity further increased in the presence of PD supplemented with exogenous IGF-1, with the major effect obtained in the presence of 30 lm PD, confirming that the higher Neu2 activity is achieved only when the proliferative effect of IGF-1 is neutralized To uncouple the proliferative effects of IGF-1 on muscle cells, we used L6MLC ⁄ IGF-1 cells [23], a myogenic cell line in which IGF-1 expression is forced only after myoblast withdrawal from the cell cycle and commitment to differentiation As shown in Fig 1F, at day L6MLC ⁄ IGF-1 cells exhibited pronounced myotube hypertrophy compared to L6E9 cells treated with IGF-1 (5 ngỈmL)1), whereas untreated L6E9 cells exhibited a low rate of differentiation Accordingly, hypertrophied L6MLC ⁄ IGF-1 cells showed significantly higher Neu2 activity compared to IGF-1-treated cells (Fig 1G) In particular, L6MLC ⁄ IGF-1 cells exhibited a peak of Neu2 enzymatic activity at day that correlated with a very high degree of hypertrophy Indeed, the transcriptional profile revealed a high level of Neu2 induction in L6MLC ⁄ IGF-1 cells compared to IGF-1-treated or untreated cells (Fig 1H) These data strongly suggest that the highest Neu2 expression is obtained when IGF-1 fully exerts its myogenic effect without stimulating cell proliferation Constitutive AKT activation is per se sufficient to drive Neu2 expression It is well known that the expression of an AKT activated form is able to induce myoblast differentiation and hypertrophy, mainly through the activation of mTOR protein [11,12] To better characterize the signaling pathway triggering Neu2 upregulation, C2C12 cells were transfected using either the constitutively activated form of AKT (caAKT) or its kinase-inactive form (kiAKT) [13] After transfection, caAKT cells differentiated faster than parental cells, developing hypertrophy as revealed by morphology (Fig 2A) On the contrary, kiAKT cells did not differentiate at all, as evidenced by the weak positivity to myotube staining Myotube dia3712 meter analysis (Fig 2A, right panel) confirmed the increase in fiber size of caAKT cells of about threefold compared to parental cells, whereas kiAKT cells formed few myotubes with a reduced diameter As a consequence, stronger phosphorylation of AKT was observed in caAKT cells compared to parental cells, thus leading to enhanced phosphorylation of p70S6K (Fig 2B), whereas in kiAKT cells, activation of AKT and p70S6K was undetectable (Fig 2B) As shown in Fig 2C, a remarkable increase of Neu2 transcript was observed by RT-PCR analysis in caAKT cells compared to parental cells, whereas kiAKT cells exhibited reduced Neu2 expression In addition, caAKT myoblasts revealed about a three-fold induction of Neu2 enzymatic activity compared to parental cells, whereas kiAKT cells exhibited very low Neu2 activity (Fig 2D) As caAKT cells treated with rapamycin did not exhibit Neu2 activity (data not shown), sustained activation of the AKT–mTOR pathway seems to be crucial for Neu2 expression To determine whether IGF-1 was able to drive Neu2 expression through AKT-independent pathways, kiAKT cells were treated with IGF-1 and subjected to Neu2 activity analysis As shown in Fig 2E, IGF-1 treatment induced AKT phosphorylation in parental cells, whereas IGF-1-treated kiAKT cells showed a very low level of AKT phosphorylation; indeed, as shown in Fig 2F, Neu2 enzymatic activity was undetectable in kiAKT cells stimulated with IGF-1, whereas parental cells grown either in DM or in DM supplemented with IGF-1 exhibited Neu2 activity These data confirmed that Neu2 expression is dependent on AKT activation during IGF-1-induced differentiation of myoblasts Neu2 expression is strictly dependent on IGF-1 signaling To establish whether Neu2 expression was strictly dependent on IGF-1 signaling, we used C2BP5 cells, a well-characterized cell model consisting of C2 myoblasts overexpressing IGFBP5 and therefore not responding to endogenously produced IGF-1 [24] C2BP5 myoblasts failed to differentiate properly when grown in DM (Fig 3A), even in the presence of strong upregulation of endogenous IGF-1 transcript (Fig 3B) On the contrary, treatment of the cells with R3-IGF-1 (a mutated form of IGF-1 tat does not bind IGFBP5) was able to restore myotube formation (Fig 3A), as confirmed by strong expression of myogenin (Fig 3C) In addition, as seen in C2C12 cells, simultaneous treatment with R3-IGF-1 and PD did not interfere with myoblast differentiation, whereas FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS A Fanzani et al Insulin-like growth factor signaling A D C B F E Fig Neu2 upregulation is dependent on AKT activation (A) Parental C2C12 myoblasts, constitutively active AKT (caAKT) cells and kinaseinactive (kiAKT) cells were grown in differentiating medium (DM) for 48 h, and the myotubes were visualized by Giemsa staining Mean myotube diameters are represented in the graph on the right and expressed in arbitrary units (n ¼ 10, *P < 0.05) (B) caAKT cells grown in DM for 48 h showed stronger phosphorylation of AKT and p70S6K compared to parental C2C12 cells, whereas phosphorylation was undetectable in kiAKT cells Immunoblot analysis was performed using anti-phospho-AKT (Ser473) and anti-phospho-P70S6K (Thr389) The data were normalized using tubulin as control (C) Neu2 transcript upregulation is dependent on AKT activity Neu2 transcript analysis was performed by semiquantitative RT-PCR, using cells grown for 48 h in DM, and the data were normalized by loading the total RNA as control (D) Neu2 enzymatic assay performed on parental, caAKT and kiAKT cells grown for 48 h in DM (n ¼ 3, *P < 0.05) (E) Immunoblot analysis against the phospho-AKT form (Ser473) was performed on untreated C2C12 cells and on parental and kiAKT cells treated with insulin-like growth factor (IGF-1) for 15 The data were normalized using tubulin as control (F) Neu2 enzymatic activity of kiAKT cells treated until day with IGF-1 and compared to C2C12 cells grown either in DM or in DM supplemented with IGF-1 (n ¼ 3, *P < 0.05) the treatments with LY completely prevented myotube formation (Fig 3A) Thus, strong Neu2 transcript upregulation was found to be strictly dependent on the restoration of IGF-1 signaling (Fig 3D); in fact, C2BP5 cells treated with increasing doses of R3-IGF-1 (15 and 30 ngỈmL)1) exhibited a proportional increase of Neu2 expression, as revealed by RT-PCR analysis Indeed, during the differentiation induced by R3-IGF-1 (30 ngỈmL)1), the Neu2 enzymatic activity increased approximately four-fold compared to cells FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3713 Insulin-like growth factor signaling A Fanzani et al A B Fig Neu2 expression is strictly dependent on insulin-like growth factor (IGF-1) signaling (A) C2BP5 cells were grown after confluence until day in differentiating medium (DM) or DM supplemented with 15 ngỈmL)1 R3-IGF-1 with or without 10 lM PD098059 (PD) or 20 lM LY294002 (LY) The cells were visualized by Giemsa staining (B) RT-PCR analysis of endogenous IGF-1 transcript in C2BP5 cells grown in DM for 48 h (C) Western blot analysis of myogenin in C2BP5 cells cultured for 48 h in growth medium (GM), DM or DM supplemented with 15 ngỈmL)1 R3-IGF-1 (D) RTPCR analysis of Neu2 transcript in C2BP5 cells grown for 72 h in DM or DM supplemented with two different concentrations of R3-IGF-1 The data were normalized by loading the total RNA as control (E) Neu2 enzymatic activity was detected in C2BP5 cells only when R3-IGF-1 was added to restore myotube formation The enzymatic assays were performed using cells grown for 96 h in DM with R3-IGF-1 (30 ngỈmL)1) alone or supplemented with 30 lM PD, 20 lM LY, or ngặmL)1 rapamycin (n ẳ 3, *P < 0.05) C E D grown in DM (Fig 3E) As seen in C2C12 cells, treatment with R3-IGF-1 and PD significantly enhanced Neu2 activity compared to IGF-1 treatment alone, whereas in the presence of R3-IGF-1, supplemented with either LY or rapamycin, Neu2 expression was completely prevented morphology, except that there were many proliferating cells The treatment of Neu2-transfected cells with either LY or rapamycin prevented myotube formation also in presence of IGF-1, suggesting that Neu2 overexpression cannot override LY ⁄ rapamycin inhibition of differentiation PI3 kinase and mTOR inhibitors block the enhanced differentiation of Neu2-overexpressing cells Neu2 overexpression does not affect the ganglioside pattern in C2C12 myoblasts We previously reported that Neu2 overexpression enhances myoblast differentiation of C2C12 cells, triggering marked cell hypertrophy [22] In order to show whether C2C12 myoblasts transfected with rat Neu2 cDNA (Fig 4A) could override the pharmacologic inhibition of differentiation, untreated or IGF-1-treated Neu2 clones were added with either LY or rapamycin (Fig 4B) After 48 h in DM, Neu2 clones developed an elongated shape and a pronounced tendency to form myotubes; in the same manner, the clones that were treated with IGF-1 showed a similar 3714 Although the ability of Neu2 to hydrolyze gangliosides in vitro has been reported [25], the target of Neu2 activity during myoblast differentiation is still unknown To investigate this, we used Neu2 clones to evaluate possible modifications of the ganglioside pattern (Fig 4C) Surprisingly, both transfected and parental cell lines exhibited a similar pattern, with GM3 ganglioside as a major component, and GM2 and GD1a gangliosides present in lower amounts In addition, C2C12 myoblasts were grown in the presence of a selective inhibitor of ganglioside biosynthesis, P4 [26], and characterized for their proliferation and FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS A Fanzani et al Insulin-like growth factor signaling A B C D E Fig Neu2-induced differentiation is blocked by PI3 kinase and mammalian target of rapamycin (mTOR) inhibitors, and Neu2 overexpression does not affect the ganglioside pattern of C2C12 myoblasts (A) C2C12 cells were stably transfected with a vector harboring the rat Neu2 cDNA and tested for Neu2 transcript expression and for the increase of sialidase activity compared to untransfected cells (B) Neu2overexpressing clones grown for 48 h in differentiating medium (DM) or DM supplemented with insulin-like growth factor (IGF-1) were treated with either LY294002 (LY) or rapamycin and analyzed by Giemsa staining for their morphology (C) Ganglioside pattern obtained by TLC analysis Gangliosides were visualized in parental C2C12 cells and in C2C12 cells overexpressing Neu2 sialidase In addition, the gangliosides were undetectable in myoblasts after treatment with P4, a synthetic inhibitor of glycosphingolipid biosynthesis (D) C2C12 cells were treated with P4 and then subjected to [3H]thymidine incorporation to quantify the rate of proliferation (n ¼ 3, *P < 0.05) (E) morphology of C2C12 cells and Neu2-overexpressing clones, differentiated in either DM or DM supplemented with P4 until day Mean myotube diameters are represented in a graph on the right and expressed in arbitrary units (n ¼ 10, *P < 0.05) FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3715 Insulin-like growth factor signaling A Fanzani et al differentiation rate As shown in Fig 4C, gangliosides were undetectable in myoblasts incubated for 72 h in the presence of P4 Under these conditions, proliferation rate was decreased about two-fold compared to untreated myoblasts, as revealed by thymidine incorporation (Fig 4D) However, myoblasts grown in the presence of P4 retained the capacity to differentiate, and Neu2-overexpressing cells exhibited stronger myotube formation compared to parental cells, even when treated with P4 (Fig 4E), as confirmed by myotube diameter analysis (right panel) These data suggest that different substrates, such as sialoglycoproteins, could be the target of Neu2 enzymatic activity in myoblasts Discussion The current study establishes for the first time that Neu2 expression is strictly dependent on IGF-1 signaling in myoblasts IGFs (IGF-1 and IGF-2) are synthesized primarily in the liver, but are also produced locally in tissues, including skeletal muscle, where they can exert autocrine or paracrine effects [27] In particular, whereas IGF-2 appears to play a mitogenic role primarily during embryogenesis and regeneration [28,29], IGF-1 has been reported to be essential for muscle differentiation and hypertrophy [1–3] Unlike other growth factors, IGF-1 is able to exert pleiotropic effects on muscle cells, first supporting myoblast replication through mitogen-activated protein (MAP) kinase activation, and subsequently promoting myogenic differentiation through the PI3 kinase–AKT pathway IGF-1 receptor myoblast plasma membrane Proliferation Ras IRS-1 P13-k AKT Raf Mek Erk1/2 Neu2 down-regulation mTOR Differentiation Hypertrophy p70s6k Neu2 up-regulation myotube formation Fig Intracellular signaling pathways regulating Neu2 expression in myoblasts Insulin-like growth factor (IGF-1) receptor autophosphorylation activates, through insulin receptor substrate (IRS-1) recruitment, different downstream signals, triggering both myoblast proliferation and differentiation ⁄ hypertrophy In particular, activation of the Ras–Raf–Mek–Erk pathway stimulates proliferation, contributing to Neu2 downregulation On the contrary, activation of the PI3K–AKT–mTOR–P70S6K pathway leads to myoblast differentiation and hypertrophy, inducing strong Neu2 expression, which could play a crucial role during myotube formation 3716 The data presented here demonstrate that the signaling triggered by IGF-1 modulates Neu2 expression in myoblasts We were able to dissect the contribution of the IGF-1-induced pathways to Neu2 expression (Fig 5); in particular, the highest Neu2 activity was obtained by IGF-1 treatment with concomitant inhibition of ERK1 ⁄ phosphorylation, suggesting that this pathway contributes to Neu2 downregulation, whereas activation of the PI3 kinase–AKT–mTOR pathway stimulated strong Neu2 upregulation When we measured Neu2 activity in L6MLC ⁄ IGF-1 cells, we found a stronger increase of Neu2 enzymatic activity compared to parental L6E9 cells treated with exogenous IGF-1 Thus, Neu2 is highly expressed only when IGF-1 exerts its myogenic effect after myoblast withdrawal from the cell cycle and commitment to differentiation These observations suggest that during IGF-1-induced regeneration of muscle cells following myofiber injury, the largest contribution of Neu2 activity might be related to the postmitotic effects of IGF-1 after cell migration, presumably during the formation of new fibers We next examined the contribution of AKT to Neu2 expression The activation of AKT has been extensively suggested as a key event in myoblast differentiation and hypertrophy [14,30,31] For example, AKT is able to promote increased protein synthesis by direct activation of p70S6K and PHAS-1 ⁄ 4E-BP1 through mTOR [13,14,32,33] or through inhibition of mTOR-independent targets such as glycogen synthase kinase 3b [13,34] Here we show a dramatic increase of Neu2 activity during C2C12 cell hypertrophy induced by transfection of a constitutively active form of AKT On the contrary, the transfection of its kinase-inactive form almost completely prevented Neu2 activity, also after treatment with IGF-1, suggesting that AKT is a key regulator of Neu2 expression Interestingly, when we used rapamycin to block mTOR activity in myoblasts overexpressing the active form of AKT, complete suppression of Neu2 synthesis was observed, suggesting that Neu2 expression is completely dependent on mTOR activity To determine whether Neu2 regulation was strictly dependent on IGF-1 signaling, we used C2 myoblasts stably transfected with IGFBP5 [35]; these cells, unable to differentiate properly in response to endogenous secreted IGF-1 [23], exhibited Neu2 enzymatic activity only when myotube formation was restored by addition of the analogous form R3-IGF-1, thus confirming that Neu2 expression is dependent on IGF-1 signaling Although Neu2 overexpression was able to enhance myotube formation in C2C12 cells, treatment of Neu2 transfectants with inhibitors of PI3 kinase and mTOR proteins prevented myotube formation, suggesting that FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS A Fanzani et al Neu2 overexpression cannot override LY ⁄ rapamycin inhibition of differentiation Taken together, our data suggest that IGF-1induced differentiation and hypertrophy are associated with Neu2 upregulation, supporting the idea that the presence of a cytosolic sialidase is significant during myotube formation In accord with this hypothesis, it has been reported that the inhibition of Neu2 translation by addition of antisense oligonucleotides strongly decreases myotube formation in L6 rat myoblasts [21] The ability of sialidases to work on glycoconjugates has been long known, suggesting that modulation of these substrates is a crucial step in physiologic and pathologic states [36,37] Despite the reported Neu2 ability to hydrolyze gangliosides and glycoproteins in vitro [25], the target of this enzyme in myoblasts is still unknown It has been previously reported that Neu2 transfection decreases GM3 ganglioside in B16 melanoma cells, diminishing invasiveness and cell motility [38], whereas transfection in human carcinoma epidermoid A431 cells led to increased epidermal growth factor receptor autophosphorylation and cell proliferation caused by the decrease in GM3 [39] According to these observations, we sought to determine whether Neu2 overexpression could modify the ganglioside pattern in C2C12 cells Surprisingly, no differences were found in ganglioside pattern between parental and transfected cells, in particular with regard to GM3 ganglioside A decrease in GM3 was observed during C2C12 differentiation (data not shown), but even in this case we did not detect differences compared to Neu2 transfectants As the inhibition of ganglioside biosynthesis significantly decreased the proliferation rate of myoblasts, thus leading to differentiation, the reduction of ganglioside content could play a prodifferentiating role in myoblasts It is possible that local modulation of some gangliosides could be important for myoblast differentiation, but in our experimental conditions we were unable to detect eventual differences due to Neu2 activity However, we cannot rule out the possibility that Neu2 activity could be restricted to glycosphingolipids associated with the cytoskeleton [40,41] or soluble and organelle membrane-bound gangliosides [42,43], thus rendering the analysis quite difficult Interestingly, Neu2 trasfectants maintained the ability to enhance myotube formation even during the inhibition of ganglioside biosynthesis, suggesting that different substrates, such as glycoproteins, could be a potential target for Neu2 in myoblasts The ability of the Neu2 enzyme to hydrolyze a2,3-sialylglycoproteins has been reported, suggesting a potential role in the turnover of glycoproteins resident in the cytosolic compartment Interestingly, a cytosolic Insulin-like growth factor signaling N-glycanase has been found to release free glycans from asparagine-linked glycopeptides exported out of the endoplasmic reticulum to the cytosol [44,45] In this context, cytosolic glycans may be substrates for Neu2 activity In addition, there is a recent report of a dramatic increase of recombinant Neu2 enzymatic activity in the presence of Ca2+ [46] As Ca2+ has a crucial role in correct myoblast differentiation, it is likely that local variations in Ca2+ concentration enhance Neu2 enzymatic activity in the cytosolic compartment Finally, since IGF signaling plays a crucial role in the physiologic and pathologic states of the muscle [47], it is of interest to establish whether Neu2 impairment occurs during atrophy caused by muscle diseases A recent paper, in fact, describes the downregulation of sialidase Neu2 in a mouse model of human dysferlinopathy [48,49], indicating that altered Neu2 expression may impair muscle regeneration In conclusion, our data shed new light on the mechanisms triggering the increase of cytosolic sialidase expression during myoblast differentiation and hypertrophy, and suggest that further investigations would be useful to elucidate the target of Neu2 activity in muscle cells Experimental procedures Cell lines The mouse C2C12 myoblasts maintained at subconfluent density at 37 °C in 5% CO2 were cultured in DMEM high glucose (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 100 lgỈmL)1 penicillin ⁄ streptomycin antibiotic (SigmaAldrich), defined as growth medium (GM) Confluent cells were transferred to DM containing DMEM supplemented with 2% horse serum (HS) and the medium was changed every day To induce myoblast hypertrophy, C2C12 cells were grown in DM, and 72 h postconfluence ngỈmL)1 IGF-1 (Sigma-Aldrich) was added in order to stimulate myotube formation The L6E9 line is a subclone of the parental rat neonatal myogenic line that does not express IGF-1 but that has IGF-1 receptors L6E9 cells were maintained in GM consisting of DMEM supplemented with 20% fetal bovine serum, and differentiated at 80% confluence either in DM consisting of DMEM supplemented with 1% fetal bovine serum or in DM supplemented with ngỈmL)1 IGF-1 L6MLC ⁄ IGF-1 cells are L6E9 cells stably transfected with a vector harboring a muscle-specific IGF-1 [23], whose expression is activated by myosin light chain promoter only after myoblasts have withdrawn from the cell cycle and have committed to differentiation Hypertrophy of L6MLC ⁄ IGF-1 cells was achieved by growing the cells in DM alone FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3717 Insulin-like growth factor signaling A Fanzani et al C2BP5 cells were cultured according to previously described conditions [24] in the presence of G418 (400 lgỈmL)1) C2BP5 cells are C2 myoblasts stably transfected with mouse insulin-like growth factor binding protein-5 cDNA, which renders the cells unresponsive to endogenous IGF-1 These cells undergo minimal differentiation without the inclusion of exogenous R3-IGF-1, an IGF-1 analog lacking the IGFBP-binding region and therefore able to induce differentiation Pharmacologic treatments of myoblasts were performed using 10–30 lm PD098059 (Sigma) to inhibit ERK1 ⁄ phosphorylation, 20 lm LY294002 (Sigma) to inhibit PI3 kinase activity, and ngỈmL)1 rapamycin (Sigma) to inhibit mTOR activity To visualize myotubular structures, cells were washed three times in NaCl ⁄ Pi before fixing for 10 in 100% methanol at ) 20 °C Cells were stained with Giemsa reactive (Sigma-Aldrich) for 2–3 h and again washed in NaCl ⁄ Pi To quantify the myotube diameter in differentiating myoblasts, 10 fields were chosen randomly and 10 myotubes were measured per field The average diameter per myotube was the mean of 10 mesurements taken along the length of the myotube Stable transfections To obtain Neu2 transfectants, C2C12 myoblasts were transfected with a pCDNA expression vector harboring the rat Neu2 cDNA using Lipofectamine 2000 reagent (Invitrogen, Milan, Italy) according to the manufacturer’s instructions; the cells were cloned after 10–15 days of selection in G418 antibiotic (0.5 mgỈmL)1, Promega, Milan, Italy) and used for a few passages To obtain C2C12 myoblasts expressing either a constitutive active form of AKT or its dominant negative form, the cells were transfected using a pBABE vector in which a myristoylated AKT or a kinase-inactive AKT mutated at the ATP-binding site (K179M) were cloned [13] After transfection by Lipofectamine 2000 reagent, the mix of stable transfectants was obtained after 10 days of selection in puromycin antibiotic (2 lgỈmL)1, Sigma) and used for a few passages RNA extraction and RT-PCR analysis Total RNA was obtained by Tri-reagent extraction (Sigma) The pellet of RNA was resuspended in RNase-free water, and digested with unit of DNAase (DNA-free; Ambion, Huntingdon, UK) for h at 37 °C, according to the manufacturer’s instructions Two micrograms of total RNA was retrotranscribed with 400 units of MMLV-RT (Promega) for h at 37 °C, and the RT template was used for PCR amplification For RT-PCR analysis of murine cytosolic Neu2 sialidase expression, primers 5¢-CGAGCCAGCAAGACGGATGA G-3¢ (sense) and 5¢-GGCTCTACAAGCTTACTCACTAC CCGG-3¢ (antisense) were used, and the amplified products 3718 were normalized by loading an equal amount of extracted RNA for each sample For the screening of Neu2 transfectants, PCR analysis was performed using the primers for the rat Neu2 cDNA in order to avoid amplification of the endogenous murine Neu2 mRNA, as previously described [22] For RT-PCR analysis of rat cytosolic Neu2, primers 5¢-CCGTCCAGGACCTCACAGAG-3¢ (sense) and 5¢-TC ACTGAGCACCATGTACTG-3¢ (antisense) were used Sialidase assay A confluent 100 mm plate was washed with NaCl ⁄ Pi, and the cells harvested in 350 lL of 0.25 m sucrose ⁄ mm EDTA containing a mix of protease inhibitors (Complete Mini Protease Inhibitors; Roche Molecular Biochemicals, Monza, Italy) were then sonicated at °C for 10 s The mixture was centrifuged at 600 g (Heraeus Megafuge 1.0R, DJB Labcare Ltd., Newport Pagnell, UK) for 10 at °C, and the supernatant was ultracentrifuged at 105 000 g (Beckmann Coulter L80 S.p.A., Milan, Italy) for 60 at °C The supernatant was used as the cytosolic fraction and assayed for sialidase activity The assay mixture contained 60 nmol of the substrate 4-methylumbelliferyl N-acetylneuraminic acid (Sigma-Aldrich), 100 lg of BSA and aliquots of cytosolic fractions (50–100 lg of proteins) in a final volume of 0.2 mL of 50 mm sodium acetate buffer (pH 5.8) After incubation at 37 °C for h, the reaction was terminated by addition of 0.8 mL of 0.25 m glycine buffer (pH 10.4), and the amount of 4-methylumbelliferone released was determined fluorometrically with an excitation wavelength of 365 nm and an emission wavelength of 450 nm Western blot analysis Myoblast cells were harvested at °C in RIPA lysis buffer (1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS in 50 mm NaCl, 20 mm Tris ⁄ HCl, pH 7.6) containing a mix of protease inhibitors Lysates were cleared by centrifugation at 12 000 g (Heraeus Megafuge 1.0R) for 15 at °C before determination of protein concentration by bicinchoninic acid assay (Pierce, Celbio SRL, Milan, Italy) SDS ⁄ PAGE was performed on 10% acrylamide gel Western blots were visualized by enhanced chemiluminescence (Chemicon Ltd., Chandlers Ford, UK) For the detection of phosphorylated ERK1 ⁄ 2, a mouse monoclonal antibody was used (clone E-4; Santa Cruz Biotechnology, Santa Cruz, CA, USA) A polyclonal antibody against ERK1 ⁄ was used for the detection of total ERK1 ⁄ (Santa Cruz Biotechnology) The phosphorylated forms of AKT (Ser473) and P70S6K (Thr389) were detected using rabbit polyclonal antibodies (Cell Signalling Ltd., Hitchin, UK) The detection of myogenin was performed using a mouse monoclonal antibody (clone F5-D; Santa Cruz Biotechnology) An antibody against a-tubulin (Sigma) was used to normalize the loading in the different western blots FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS A Fanzani et al Insulin-like growth factor signaling [3H]Thymidine incorporation Cells were seeded in 24-well plates at · 10 cell ⁄ mL in DMEM containing 10% fetal bovine serum and incubated at 37 °C for 24 h in the presence or absence of ganglioside biosynthesis inhibitor P4 (1 lm) [26] After 24 h of serum starvation in DMEM, either 10% fetal bovine serum or 10% fetal bovine serum plus P4 were added to the wells Twenty hours later, cells were incubated with [3H]thymidine (1 lCiỈmL)1), and after an additional period of h, samples were directly precipitated in 5% trichloroacetic acid and incubated on ice for 30 The cells were lysed in 0.5 m sodium hydroxide, and after neutralization with 0.5 m HCl, liquid scintillator was added and the amount of [3H]thymidine incorporated was determined in a counting device Ganglioside analysis Cells were grown at confluence and harvested after three washes in NaCl ⁄ Pi Gangliosides were extracted in chloroform ⁄ methanol (C ⁄ M : 1, v ⁄ v) according to the Folch–Pi method [50] Extracts were separated on a TLC plate with a chloroform ⁄ methanol ⁄ 0.3% CaCl2 (C ⁄ M ⁄ W, 60 : 35 : v ⁄ v) solvent system, and spots were visualized with p-dimethylaminobenzaldehyde reagent, with the plate being heating at 120 °C for 10 Statistics All of the data are expressed as the means ± SE Statistical significance was determined using Student’s t-test A P-value of < 0.05 was considered 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factor signaling 14 15 16 17 18 19 20 21 22 23 24 25 A... that Neu2 expression is dependent on AKT activation during IGF -1- induced differentiation of myoblasts Neu2 expression is strictly dependent on IGF -1 signaling To establish whether Neu2 expression. .. (19 95) Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice J Biol Chem 270, 12 109? ?12 116 ` Musaro A, McCullagh