sialidase Neu2 expression during myoblast differentiation and hypertrophy Alessandro Fanzani, Francesca Colombo, Roberta Giuliani, Augusto Preti and Sergio Marchesini Department of Biome
Trang 1sialidase 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
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 1 (IGF-1) [2,3] Since IGF-1
overexpression in the skeletal muscle of transgenic
mice triggers an increase in muscle size [4–6], the emer-ging 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 differ-entiation [7,8], reproducing the events occurring during
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 5 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 1 (IGF1)-induced hypertrophy of myoblasts notably increa-ses 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 1⁄ 2 (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 1 stimulates a dramatic hypertrophy, was accompanied by a stronger Neu2 increase Indeed, the hypertrophy induced by transfection of a constitutively activa-ted form of AKT was able to induce high Neu2 activity in C2C12 cells, whereas the transfection of a kinase-inactive form of AKT prevented myo-tube formation, triggering Neu2 downregulation Neu2 expression was strictly correlated with IGF-1 signaling also in C2 myoblasts overexpressing the insulin-like growth factor 1 binding protein 5 and therefore not responding to endogenously produced insulin-like growth factor 1 Although Neu2-transfected myoblasts exhibited stronger differentiation, we demonstrated that Neu2 overexpression does not override the block of dif-ferentiation mediated by PI3 kinase and mTOR inhibitors Finally, Neu2 overexpression did not modify the ganglioside pattern of C2C12 cells, sug-gesting 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
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.
Trang 2the repair of damaged tissue In particular, myoblast
proliferation is triggered by activation of the
extra-cellular 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
hypertro-phy is mammalian target of rapamycin (mTOR)
[11,12], whose activation by AKT elicits the
phos-phorylation of two known regulators of protein
syn-thesis, 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 2 (Neu2) (EC 3.2.1.18) is unique with
regard to cellular localization and tissue expression
Whereas the lysosomal form Neu1 [15–17], the
ganglio-side 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
differenti-ation has been proposed for the first time using L6 rat
myoblasts [21]; in addition, we recently suggested a
cru-cial 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
overexpress-ing IGF-bindoverexpress-ing protein-5 (IGFBP5), in which IGF-1
signaling is selectively repressed [24] Neu2
transfect-ants, 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
sub-jected 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⁄
hypertrophy through activation of the PI3 kinase– AKT–mTOR pathway [9]
Accordingly, IGF-1 treatment (5 ngÆmL)1) of C2C12 myoblasts triggered the simultaneous phosphorylation
of ERK1⁄ 2 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 inhib-itors to block selectively these pathways (Fig 1A), ERK1⁄ 2 phosphorylation was prevented in the pres-ence of 30 lm PD098059 (PD), whereas AKT phos-phorylation was blocked in the presence of 20 lm LY294002 (LY), a known inhibitor of PI3 kinase activ-ity In addition, the inhibition of mTOR activity was achieved in the presence of 5 ngÆmL)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 5 fused into multinucleated myotubes, whereas the cells grown in
DM supplemented with IGF-1 (5 ngÆmL)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 pre-vented by treatment with LY The block of differenti-ation was obtained even in the presence of rapamycin (data not shown), an inhibitor of mTOR activity, con-firming 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 supple-mented with PD, the average myotube diameter was about two-fold compared to parental cells differenti-ated in DM alone, whereas in the presence of LY no myotubes were observed
Under these experimental conditions, Neu2 expres-sion was investigated by RT-PCR analysis (Fig 1C) and enzymatic assay (Fig 1D) Neu2 transcript upreg-ulation was observed in myoblasts grown in the pres-ence 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; more-over, the effects of IGF-1 were considerably reinforced
in the presence of IGF-1 supplemented with PD, sug-gesting that ERK1⁄ 2 phosphorylation negatively con-tributes to Neu2 expression Finally, the treatments either with LY or rapamycin completely prevented
Trang 3A B
G F
H C
Fig 1 Insulin-like growth factor 1 (IGF-1)-induced hypertrophy enhances Neu2 expression in murine myoblasts (A) 5 ngÆmL)1IGF-1 induces phosphorylation of ERK1 ⁄ 2, AKT and p70S6K proteins ERK1 ⁄ 2 phosphorylation was prevented in the presence of 30 l M PD098059 (PD), AKT phosphorylation in the presence of 20 l M LY294002 (LY), and p70S6K phosphorylation in the presence of 5 ngÆmL)1rapamycin West-ern blots against total ERK1 ⁄ 2 and tubulin were performed to verify equal loading of protein samples (B) C2C12 myoblasts were grown for
6 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 5 The data were normalized by loading the total RNA as control (D) Neu2 enzymatic assay performed using C2C12 cells cultured for 6 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 4 in the presence of two different concentrations of PD (10 and 30 l M ) alone
or supplemented with 5 ngÆmL)1IGF-1 (n ¼ 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).
Trang 4Neu2 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 4 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⁄ 2 phosphorylation
induced by endogenously secreted IGF-1 As
expec-ted, 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
neutral-ized 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 6 L6MLC⁄ IGF-1 cells exhibited
pro-nounced myotube hypertrophy compared to L6E9
cells treated with IGF-1 (5 ngÆmL)1), whereas
untreated L6E9 cells exhibited a low rate of
differenti-ation Accordingly, hypertrophied L6MLC⁄ IGF-1
cells showed significantly higher Neu2 activity
com-pared to IGF-1-treated cells (Fig 1G) In particular,
L6MLC⁄ IGF-1 cells exhibited a peak of Neu2
enzy-matic activity at day 6 that correlated with a very
high degree of hypertrophy Indeed, the
transcrip-tional 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
stimula-ting cell proliferation
Constitutive AKT activation is per se sufficient
to drive Neu2 expression
It is well known that the expression of an AKT
activa-ted 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
activa-ted 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
dia-meter analysis (Fig 2A, right panel) confirmed the increase in fiber size of caAKT cells of about three-fold 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 com-pared to parental cells, whereas kiAKT cells exhibited reduced Neu2 expression In addition, caAKT myo-blasts 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 unde-tectable 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 depend-ent on AKT activation during IGF-1-induced differen-tiation 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 myo-blasts 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 myo-genin (Fig 3C) In addition, as seen in C2C12 cells, simultaneous treatment with R3-IGF-1 and PD did not interfere with myoblast differentiation, whereas
Trang 5the 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 ngÆmL)1) exhibited a proportional increase
of Neu2 expression, as revealed by RT-PCR ana-lysis Indeed, during the differentiation induced by R3-IGF-1 (30 ngÆmL)1), the Neu2 enzymatic activity increased approximately four-fold compared to cells
A
D
F
E
Fig 2 Neu2 upregulation is dependent on AKT activation (A) Parental C2C12 myoblasts, constitutively active AKT (caAKT) cells and kinase-inactive (kiAKT) cells were grown in differentiating medium (DM) for 48 h, and the myotubes were visualized by Giemsa staining Mean my-otube 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 undetecta-ble 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 1 (IGF-1) for 15 min The data were normalized using tubulin as control (F) Neu2 enzymatic activity of kiAKT cells treated until day 5 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).
Trang 6grown in DM (Fig 3E) As seen in C2C12 cells,
treat-ment 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
PI3 kinase and mTOR inhibitors block the
enhanced differentiation of Neu2-overexpressing
cells
We previously reported that Neu2 overexpression
enhances myoblast differentiation of C2C12 cells,
trig-gering 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-trea-ted Neu2 clones were added with either LY or
rapa-mycin (Fig 4B) After 48 h in DM, Neu2 clones
developed an elongated shape and a pronounced
ten-dency to form myotubes; in the same manner, the
clones that were treated with IGF-1 showed a similar
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 over-expression cannot override LY⁄ rapamycin inhibition
of differentiation
Neu2 overexpression does not affect the ganglioside pattern in C2C12 myoblasts 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 pat-tern (Fig 4C) Surprisingly, both transfected and par-ental cell lines exhibited a similar pattern, with GM3 ganglioside as a major component, and GM2 and GD1a gangliosides present in lower amounts In addi-tion, C2C12 myoblasts were grown in the presence of
a selective inhibitor of ganglioside biosynthesis, P4 [26], and characterized for their proliferation and
A
B
C
E D
Fig 3 Neu2 expression is strictly depend-ent on insulin-like growth factor 1 (IGF-1) signaling (A) C2BP5 cells were grown after confluence until day 4 in differentiating med-ium (DM) or DM supplemented with
15 ngÆmL)1R3-IGF-1 with or without 10 l M PD098059 (PD) or 20 l M LY294002 (LY) The cells were visualized by Giemsa stain-ing (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 supple-mented with 15 ngÆmL)1R3-IGF-1 (D) RT-PCR analysis of Neu2 transcript in C2BP5 cells grown for 72 h in DM or DM supple-mented with two different concentrations of R3-IGF-1 The data were normalized by load-ing the total RNA as control (E) Neu2 enzy-matic 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 ngÆmL)1) alone or supplemented with 30 l M PD, 20 l M LY, or
5 ngÆmL)1rapamycin (n ¼ 3, *P < 0.05).
Trang 7A B
C
E
D
Fig 4 Neu2-induced differentiation is blocked by PI3 kinase and mammalian target of rapamycin (mTOR) inhibitors, and Neu2 overexpres-sion 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) Neu2-overexpressing clones grown for 48 h in differentiating medium (DM) or DM supplemented with insulin-like growth factor 1 (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 ganglio-sides 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 [ 3 H]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 5 Mean myotube diame-ters are represented in a graph on the right and expressed in arbitrary units (n ¼ 10, *P < 0.05).
Trang 8differentiation rate As shown in Fig 4C, gangliosides
were undetectable in myoblasts incubated for 72 h in
the presence of P4 Under these conditions,
prolifer-ation rate was decreased about two-fold compared to
untreated myoblasts, as revealed by thymidine
incor-poration (Fig 4D) However, myoblasts grown in the
presence of P4 retained the capacity to differentiate,
and Neu2-overexpressing cells exhibited stronger
myo-tube 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
signa-ling in myoblasts IGFs (IGF-1 and IGF-2) are
syn-thesized primarily in the liver, but are also produced
locally in tissues, including skeletal muscle, where they
can exert autocrine or paracrine effects [27] In
partic-ular, 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
repli-cation through mitogen-activated protein (MAP)
kin-ase activation, and subsequently promoting myogenic
differentiation through the PI3 kinase–AKT pathway
The data presented here demonstrate that the signaling triggered by IGF-1 modulates Neu2 expression in myo-blasts 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⁄ 2 phosphorylation, suggesting that this path-way contributes to Neu2 downregulation, whereas activation of the PI3 kinase–AKT–mTOR pathway sti-mulated 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 dif-ferentiation These observations suggest that during IGF-1-induced regeneration of muscle cells following myofiber injury, the largest contribution of Neu2 activ-ity 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 exam-ple, AKT is able to promote increased protein syn-thesis by direct activation of p70S6K and PHAS-1⁄ 4E-BP1 through mTOR [13,14,32,33] or through inhi-bition of mTOR-independent targets such as glycogen synthase kinase 3b [13,34] Here we show a dramatic increase of Neu2 activity during C2C12 cell hypertro-phy 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, sug-gesting that AKT is a key regulator of Neu2 expres-sion 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 com-pletely dependent on mTOR activity To determine whether Neu2 regulation was strictly dependent on IGF-1 signaling, we used C2 myoblasts stably trans-fected with IGFBP5 [35]; these cells, unable to differ-entiate 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
IGF-1 receptor
myoblast plasma membrane
Differentiation Hypertrophy
Neu2 up-regulation Neu2 down-regulation
Proliferation
myotube formation
p70s6k mTOR AKT P13-k IRS-1 Ras Raf
Mek
Erk1/2
Fig 5 Intracellular signaling pathways regulating Neu2 expression
in myoblasts Insulin-like growth factor 1 (IGF-1) receptor
autop-hosphorylation activates, through insulin receptor substrate 1 (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,
contribu-ting 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.
Trang 9Neu2 overexpression cannot override LY⁄ rapamycin
inhibition of differentiation
Taken together, our data suggest that
IGF-1-induced 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
transla-tion by additransla-tion 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
deter-mine 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
com-pared to Neu2 transfectants As the inhibition of
gan-glioside biosynthesis significantly decreased the
proliferation rate of myoblasts, thus leading to
differ-entiation, the reduction of ganglioside content could
play a prodifferentiating role in myoblasts It is
poss-ible that local modulation of some gangliosides could
be important for myoblast differentiation, but in our
experimental conditions we were unable to detect
even-tual 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
mem-brane-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
glycopro-teins, could be a potential target for Neu2 in
myo-blasts 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
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 com-partment
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 impair-ment occurs during atrophy caused by muscle diseases
A recent paper, in fact, describes the downregulation
of sialidase Neu2 in a mouse model of human dysfer-linopathy [48,49], indicating that altered Neu2 expres-sion may impair muscle regeneration In concluexpres-sion, 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 37C in 5% CO2 were cultured in DMEM high glucose (Sigma-Aldrich, Milan, Italy) supplemented
100 lgÆmL)1 penicillin⁄ streptomycin antibiotic (Sigma-Aldrich), 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 5 ngÆmL)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 consist-ing of DMEM supplemented with 20% fetal bovine serum, and differentiated at 80% confluence either in DM consist-ing of DMEM supplemented with 1% fetal bovine serum or
in DM supplemented with 5 ngÆmL)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 myo-blasts have withdrawn from the cell cycle and have commit-ted to differentiation Hypertrophy of L6MLC⁄ IGF-1 cells was achieved by growing the cells in DM alone
Trang 10C2BP5 cells were cultured according to previously
described conditions [24] in the presence of G418
(400 lgÆmL)1) C2BP5 cells are C2 myoblasts stably
trans-fected with mouse insulin-like growth factor 1 binding
pro-tein-5 cDNA, which renders the cells unresponsive to
endogenous IGF-1 These cells undergo minimal
differenti-ation without the inclusion of exogenous R3-IGF-1, an
IGF-1 analog lacking the IGFBP-binding region and
there-fore able to induce differentiation Pharmacologic
treat-ments of myoblasts were performed using 10–30 lm
PD098059 (Sigma) to inhibit ERK1⁄ 2 phosphorylation,
20 lm LY294002 (Sigma) to inhibit PI3 kinase activity, and
5 ngÆmL)1rapamycin (Sigma) to inhibit mTOR activity
To visualize myotubular structures, cells were washed
three times in NaCl⁄ Pi before fixing for 10 min in 100%
methanol at) 20 C Cells were stained with Giemsa
react-ive (Sigma-Aldrich) for 2–3 h and again washed in
NaCl⁄ Pi To quantify the myotube diameter in
differenti-ating 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
trans-fected 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 mgÆmL)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
rea-gent, the mix of stable transfectants was obtained after
10 days of selection in puromycin antibiotic (2 lgÆmL)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 1 unit of DNAase (DNA-free; Ambion,
Huntingdon, UK) for 1 h at 37C, according to the
manu-facturer’s instructions Two micrograms of total RNA was
retrotranscribed with 400 units of MMLV-RT (Promega)
for 1 h at 37C, 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
were normalized by loading an equal amount of extracted RNA for each sample For the screening of Neu2 transfect-ants, 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⁄ 1 mm EDTA containing a mix of protease inhibitors (Complete Mini Pro-tease Inhibitors; Roche Molecular Biochemicals, Monza, Italy) were then sonicated at 4C for 10 s The mixture was centrifuged at 600 g (Heraeus Megafuge 1.0R, DJB Labcare Ltd., Newport Pagnell, UK) for 10 min at 4C, and the supernatant was ultracentrifuged at 105 000 g (Beckmann Coulter L80 S.p.A., Milan, Italy) for 60 min at 4C 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 cytoso-lic fractions (50–100 lg of proteins) in a final volume of 0.2 mL of 50 mm sodium acetate buffer (pH 5.8) After incu-bation at 37C for 3 h, the reaction was terminated by addi-tion 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 4C 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 centrifu-gation at 12 000 g (Heraeus Megafuge 1.0R) for 15 min at
4C before determination of protein concentration by bi-cinchoninic acid assay (Pierce, Celbio SRL, Milan, Italy) SDS⁄ PAGE was performed on 10% acrylamide gel West-ern 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⁄ 2 was used for the detection of total ERK1⁄ 2 (Santa Cruz
(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 Biotechno-logy) An antibody against a-tubulin (Sigma) was used to normalize the loading in the different western blots