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: [email protected]
(Received 5 May 2006, revised 12 June
2006, accepted 13 June 2006)
doi:10.1111/j.1742-4658.2006.05380.x

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–

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

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-

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

(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-

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

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

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

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

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

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%

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

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-

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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