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 1 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
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:
(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.

FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3709
the 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-binding 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
Insulin-like growth factor 1 signaling A. Fanzani et al.
3710 FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS
AB
DE
GF
H
C
Fig. 1. Insulin-like growth factor 1 (IGF-1)-induced hypertrophy enhances Neu2 expression in murine myoblasts. (A) 5 ngÆmL
)1
IGF-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
)1
rapamycin. 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
)1
IGF-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).
A. Fanzani et al. Insulin-like growth factor 1 signaling
FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3711
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 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
Insulin-like growth factor 1 signaling A. Fanzani et al.
3712 FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 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
B
C
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).
A. Fanzani et al. Insulin-like growth factor 1 signaling
FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3713
grown 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

)1
R3-IGF-1 with or without 10 lM
PD098059 (PD) or 20 lM 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
)1
R3-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 lM LY, or
5ngÆmL
)1
rapamycin (n ¼ 3, *P<0.05).
Insulin-like growth factor 1 signaling A. Fanzani et al.

3714 FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS
AB
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).
A. Fanzani et al. Insulin-like growth factor 1 signaling
FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3715
differentiation 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.
Insulin-like growth factor 1 signaling A. Fanzani et al.
3716 FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS
Neu2 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 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 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 Ca
2+
[46]. As Ca
2+
has a
crucial role in correct myoblast differentiation, it is
likely that local variations in Ca
2+
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 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 °Cin5%CO
2
were cultured in DMEM
high glucose (Sigma-Aldrich, Milan, Italy) supplemented
with 10% fetal bovine serum (Sigma-Aldrich) and
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 ng ÆmL
)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.
A. Fanzani et al. Insulin-like growth factor 1 signaling
FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS 3717
C2BP5 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
5ngÆmL
)1
rapamycin (Sigma) to inhibit mTOR activity.
To visualize myotubular structures, cells were washed
three times in NaCl ⁄ P
i
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 ⁄ P
i
. 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 37 °C, 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 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
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 ⁄ P
i
, and
the cells harvested in 350 lL of 0.25 m sucrose ⁄ 1mm EDTA
containing a mix of protease inhibitors (Complete Mini Pro-
tease Inhibitors; Roche Molecular Biochemicals, Monza,
Italy) were then sonicated at 4 °C for 10 s. The mixture was
centrifuged at 600 g (Heraeus Megafuge 1.0R, DJB Labcare
Ltd., Newport Pagnell, UK) for 10 min at 4 °C, and the

supernatant was ultracentrifuged at 105 000 g (Beckmann
Coulter L80 S.p.A., Milan, Italy) for 60 min at 4 °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 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 37 °C 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 4 °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 centrifu-
gation at 12 000 g (Heraeus Megafuge 1.0R) for 15 min at
4 °C 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
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 Biotechno-
logy). An antibody against a-tubulin (Sigma) was used to
normalize the loading in the different western blots.
Insulin-like growth factor 1 signaling A. Fanzani et al.
3718 FEBS Journal 273 (2006) 3709–3721 ª 2006 The Authors Journal compilation ª 2006 FEBS
[
3
H]Thymidine incorporation
Cells were seeded in 24-well plates at 2 · 10
4
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 [
3
H]thymidine
(1 lCiÆmL
)1
), and after an additional period of 6 h, samples
were directly precipitated in 5% trichloroacetic acid and
incubated on ice for 30 min. 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 [
3
H]thymi-

dine incorporated was determined in a counting device.
Ganglioside analysis
Cells were grown at confluence and harvested after three
washes in NaCl ⁄ P
i
. Gangliosides were extracted in chloro-
form ⁄ methanol (C ⁄ M2:1,v⁄ v) according to the Folch–Pi
method [50]. Extracts were separated on a TLC plate with
a chloroform ⁄ methanol ⁄ 0.3% CaCl
2
(C ⁄ M ⁄ W, 60 : 35 : 8
v ⁄ v) solvent system, and spots were visualized with p-di-
methylaminobenzaldehyde reagent, with the plate being
heating at 120 °C for 10 min.
Statistics
All of the data are expressed as the means ± SE. Statisti-
cal significance was determined using Student’s t-test. A
P-value of < 0.05 was considered significant.
Acknowledgements
We are grateful to Antonio Musaro
`
(Department of
Histology and Medical Embryology, University of
Rome, Italy) for kindly providing L6MLC ⁄ IGF-1
cells, Peter Rotwein (Department of Biochemistry and
Molecular Biology, Oregon Health & Science Univer-
sity, Portland, USA) for the C2BP5 cell line, and Clau-
dio Basilico (Microbiology Department, New York
University, USA) for the vectors harboring the consti-
tutive activated form of AKT and its kinase-inactive

form. This work was partially supported by grants
from 60% MIUR, from MIUR (FIRB, 2001) to AP
and from CIB (Consorzio Italiano Biotecnologie,
2004-05) to SM.
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