The p75NTR-mediated effect of nerve growth factor in L6C5 myogenic cells
Perini et al. BMC Res Notes
NTR The p75 -mediated effect of nerve growth factor in L6C5 myogenic cells
Alessandra de Perini 0
Ivan Dimauro 0
Guglielmo Duranti 0
Cristina Fantini 0
Neri Mercatelli 0 1
Roberta Ceci 0
Luigi Di Luigi 0
Stefania Sabatini 0
Daniela Caporossi 0
0 Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Piazza Lauro de Bosis , 15, 00135 Rome , Italy
1 Laboratory of Cellular and Molecular Neurobiology, CERC , Fondazione Santa Lucia, Via del Fosso di Fiorano, 64, 00143 Rome , Italy
Objective: During muscle development or regeneration, myocytes produce nerve growth factor (NGF) as well as its tyrosine-kinase and p75-neurotrophin (p75NTR) receptors. It has been published that the p75NTR receptor could represent a key regulator of NGF-mediated myoprotective effect on satellite cells, but the precise function of NGF/p75 signaling pathway on myogenic cell proliferation, survival and differentiation remains fragmented and controversial. Here, we verified the role of NGF in the growth, survival and differentiation of p75NTR-expressing L6C5 myogenic cells, specifically inquiring for the putative involvement of the nuclear factor κB (NFκB) and the small heat shock proteins (sHSPs) αB-crystallin and Hsp27 in these processes. Results: Although NGF was not effective in modulating myogenic cell growth or survival in both standard or stress conditions, we demonstrated for the first time that, under serum deprivation, NGF sustained the activity of some key enzymes involved in energy metabolism. Moreover, we confirmed that NGF promotes myogenic fusion and expression of the structural protein myosin heavy chain while modulating NFκB activation and the content of sHSPs correlated with the differentiation process. We conclude that p75NTR is sufficient to mediate the modulation of L6C5 myogenic differentiation by NGF in term of structural, metabolic and functional changes.
NGF; p75NTR; Myogenic differentiation; Energy metabolism; NFκB; sHSPs
Skeletal muscle regeneration depends upon optimal
activation, proliferation and differentiation of myogenic
precursors, the satellite cells, whose behavior is controlled by
components, such as cytokines and growth factors,
contained in the satellite cell microenvironment [
myogenic differentiation, muscle cells produce NGF and
other neurotrophins as well as their receptors,
tyrosinekinase receptors (TrkA and TrkB) and p75-neurotrophin
receptor (p75NTR), able to act in autocrine manner on
cell morphology, proliferation and differentiation [
Beside some contractictory results , several evidences
demonstrate that cellular signaling pathways activated
by the neurotrophin/p75NTR axis stabilize the
cytoskeletal architecture and increase the fusogenic properties
of myotubes, thus promoting “in vitro” myogenic
differentiation, myotubes survival and muscle repair “in vivo”
]. Thus, despite the well known pro-apoptotic role of
p75NTR in neuron cells, this receptor might represent a
key mediator of survival in myoblasts and myotubes and
its activity during myogenesis seems important for
developing skeletal muscle [
We previously demonstrated that the activation of
NF-κB and the parallel modulation of αB-crystallin
(αB-Cry) and Hsp27 play a major role in the
antiapoptotic effect exerted by vascular endothelial growth
factor (VEGF) in C2C12 myoblasts exposed to oxidative or
hypoxic-like stress “in vitro” [
], as well as in the
positive effect exerted by platelet-rich plasma on rat skeletal
muscle healing “in vivo” [
]. NF-κB is a redox-sensitive
transcription factor known to regulate several cellular
processes (i.e. inflammation, cellular survival,
proliferation and differentiation) that recently emerged as a key
player in the regulation of skeletal muscle homeostasis
]. αB-Cry and Hsp27 are small heat shock proteins
(sHSPs) abundantly expressed in muscle tissue where
they stabilize cytoskeletal structures, especially under
pro-oxidant insult [
], modulate myogenic
] and interact with several growth
factors through the c-Jun N-terminal kinase (JNK)- and/or
NFκB- dependent regulatory mechanisms [
9, 11, 12
Considering these observations altogether, the main
aim of the present study was to analyze in L6C5
myogenic cells expressing exclusively the p75NTR receptor,
whether the effect of NGF on myoblast growth, survival,
fusion rate and expression of early and late markers of
myogenesis correlates with NFκB activation and/or
modulation of αB-Cry and Hsp27 expression.
Materials and methods
Cell culture, growth and viability
All experiments were carried out on L6C5 rat myogenic
cells (ICLC, AL00001). As already described [
myoblast cultures were maintained and subcultured
in growing medium (GM) (DMEM with 4.5 g/l glucose
and Corning® glutagro™, w/o sodium pyruvate,
Corning 10-102-CVR; 100 U/ml penicillin, 100 μg/ml
streptomycin, Euroclone ECB3001D; 10% FBS, Gibco 10270).
Differentiation medium (DM) containing 2% FBS was
utilized in 85%—confluent cells to induce myotubes.
The process was monitored through microscopy and
expression of myogenic markers [
]. NGF (Promega,
10–100 ng/ml) was added to the GM or DM medium at
the indicated time and replenished with media changes
every 3 days. For cell growth and viability, 5 × 105 cells/
well were seeded in a 96-well culture plate for 6, 12,
24, and 48 h with or without 10% FBS, in presence or
absence of NGF added from the seeding. Cell growth was
analysed by MTS assay (Promega) following the
manufacturer’s recommendations. The absorbance was
measured at 490 nm (Bio-Rad680). Viability was evaluated by
trypan blue exclusion assay performed at the same
L6C5 myoblasts grown in presence or in absence of NGF
(10, 100 ng/ml) for 48 h under standard (GM) or serum
starvation conditions were lysed (0.05 M Tris–Acetate,
250 mM sucrose, pH 7.5, 1 mM PMSF) with a protease
inhibitor cocktail (P8340, Sigma Aldrich, St. Louis, MO).
After gentle sonication (twice 10 s in ice with Vibra-Cell
CV 18 SONICS VX 11) and centrifugation (at 14,000 rpm
for 10 min at 4 °C), the supernatant was tested for protein
content (Bradford method, Sigma Aldrich, St Louis MO)
and then analysed spectrophotometrically (20–50 µl
sample, Perkin Elmer Lambda 25, Fremont, CA, USA)
for glyceraldehyde-phosphate-dehydrogenase (GAPDH),
lactate dehydrogenase (LDH), citrate synthase (CS),
3-OH acylCoA dehydrogenase (HAD) and alanine
transglutaminase (ALT) enzymatic activities as previously
Protein extraction and immunoblotting was performed
by standard methodology as already described [
Briefly, total cellular proteins were extracted by lysis
buffer (20 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA,
1 mM sodium orthovanadate, 100 mM PMSF, 10 mg/
ml leupeptin, 10 mg/ml aprotinin, 5 mg/ml
pepstatin, 50 mM NaF, 1 nM okadaic acid, 1% Triton X-100
and 10% glycerol), and quantified using the Bradford
assay (Sigma). From each sample, 15–20 μg of proteins
have been utilized for immunoblotting with the
following antibodies: myogenin (sc-576), Hsp27 (sc-1048),
p75NTR (sc-56448) and TrkA (sc-20539) (1:1000,
SantaCruz Biotechnology), β-actin (A1978) (1:3000, Sigma),
anti-embryonic MyHC (F1652) (1:1000, Biovalley) and
MyHC IIB (BFF3) (1:1000, Development Studies
Hybridoma Bank), αB-crystallin (SPA-222) (1:1000, Enzo Life
Sciences), SAPK/JNK (#9252), Phospho-SAPK/JNK
(Thr183/Tyr185) (#4668), and Bcl-2 (#2870) (1:1000,
Cell Signalling), Caspase 3 (#44976) (1:1000, Abcam).
All immunoblots were visualized with the
appropriated horseradish peroxidase-conjugated secondary
antibody (1:15,000, Millipore) followed by detection with
enhanced chemiluminescence (Amersham-Biosciences).
Bands were quantified by ImageJ software. The
expression of β-actin was used as a normalizing control.
Fusion rate analysis
Cells, grown 2 and 9 days in DM with or without NGF
(20 ng/ml), were fixed with ice-cold methanol (7′ at
− 20 °C), permeabilized (0.1% triton X-100 for 20′) and
then blocked at RT for 60′ (TBS, 10% FBS, 0.1% triton).
Myotubes were identified by double staining with
Hoechst 33258 (Sigma) and MyHC antibody (sc-12117, 1:50,
Santa-Cruz Biotechnology) as MyHC positive cells with
at least two nuclei. Since the number of total nuclei was
not modulated by NGF (data not shown), the fusion rate
was evaluated by the number of total myotubes in each
well and the number of nuclei/myotube [
NFκB activity was measured in nuclear protein extracts
(15 μg) by the TransAM NF-κB p65 protein assay (Active
Motif ), according to the manufacturer’s protocol [
assay was performed in presence or in absence of NGF
(20 ng/ml) on proliferating myoblasts (24 h from seeding)
or cells grown in DM for 2, 5 and 9 days. Experimental
samples and controls were run in duplicate.
Statistical comparisons between groups were
performed by Student’s t test. All values are given as the
mean ± standard deviation of the mean (SD). p < 0.05
was considered significant.
Results and discussion
Impairment of the satellite cell pool has been observed
in age-related muscle dysfunction and muscle
degenerative pathologies, while the poor rate of survival and
proliferation of myoblasts derived from satellite cell
transplantation represents an important limit for the
cell replacement therapy in muscle diseases [
Various components of the microenvironment play a
crucial role in the proliferative and differentiation potential
of satellite cells, thus ensuring an adequate regenerative
response to muscle insult [
]. This study was indeed
designed to confirm and extend the positive role of NGF/
p75NTR axis in “in vitro” differentiation of L6C5 myogenic
As already described in other murine cell lines and in
human muscle cells [
], L6C5 cell line expressed
only p75NTR mRNA that, differently from primary
myogenic cells , was not differentially expressed in
myoblasts or during myogenesis, at both mRNA and protein
levels (Additional file 1: Fig. S1a–c).
NGF did not affect significantly neither myoblast
growth nor viability under standard culture condition
(data not shown). Under serum starvation, the
MTSderived OD values of myoblasts treated with NGF 100 ng/
ml were statistically higher when compared to untreated
cells (p < 0.05) (Fig. 1a), although the Trypan blue assay
(Fig. 1b) and the analysis of the apoptotic index (data not
shown) excluded a significant NGF modulation of the
total number of viable and nonviable cells. Since MTS
quantification of viable cells depends upon the cellular
metabolic rate [
], we verified the activity of some key
enzymes. The results showed that, under serum
deprivation, the activity of CS and GAPDH, both involved in
carbohydrates metabolism, was increased by NGF
supplementation compared to control (CS: 21.7 and 55.2%
increase for NGF 10 and 100 ng/ml respectively; GAPDH:
37.7 and 95.7% increase for NGF 10 and 100 ng/ml
respectively (p < 0.05) (Fig. 1c). No NGF-dependent
differences were detected under standard conditions (data
not shown). Previous data concerning the correlation
between NGF and an increased activity in cycle Krebs
enzymes also involved the activation of the JNK pathway
]. Indeed, we demonstrated a significant enhancement
of JNK phosphorylation after 15′–30′ min from NGF
(20 ng/ml) supplementation in L6C5 myoblasts grown
under serum starvation (p < 0.05) (Fig. 1d). At present,
the biological significance of this result is unknown, but
it agrees with published data in L6 myogenic cells
showing that modification of mitochondrial homeostasis
correlates with JNK phosphorylation and insulin resistance
], opening to speculations on the role of NGF in the
metabolic homeostasis of myogenic cells and possible
ergogenic effect in muscle tissue [
In 2011, Colombo et al. [
] proved that p75NTR
modulates myogenesis and dystrophin expression, proposing
this receptor as a novel marker of human
differentiationprone muscle precursor cells. In agreement with Deponti
et al. [
], we confirmed that NGF at the concentration
of 20 ng/ml, the most effective to promote L6 myoblast
proliferation and differentiation [
], did not modulate the
expression of myogenin, an early myogenic marker [
both at protein (Fig. 2a) and transcriptional level (data
not shown). However, later during differentiation (9 days
in DM), NGF increased both the Embrional (HC Emb)
and type IIB (HC IIB) MyHC isoform contents (p < 0.05)
(Fig. 2b). Thus, we showed for the first time that NGF,
similar to growth hormone [
], exerted a positive
effect on the terminal markers of myogenesis [
also found that, at the same stage, NGF induced a
significant increase in the number of fibers (p < 0.05) (Fig. 2c),
with an excess of fibers containing more than 20 nuclei
(p < 0.05) (Fig. 2d, e). Thus, our in vitro model confirmed
a hypertrophic effect of the NGF/p75NTR axis due to both
increased fusion rate and increased number of fibers [
but we also demonstrated an effect on the expression of
MyHC isoforms, claiming for a putative role of NGF in
the contractile properties of skeletal muscle fibers in vivo
It has been recently reported that the chronic
up-regulation of NFκB relates to impairment of the myogenic
process in skeletal muscle or to muscle atrophy [
]. Nonetheless, we and others demonstrated that the
transient NFκB activation plays a role during L6C5 and
C2C12 “in vitro” differentiation [
], or during growth
factors-promoted survival and/or differentiation of
myogenic cells [
]. Actually, L6C5 myogenic cultures
supplemented with NGF showed a significant increase in
NFκB activity compared to the control at 2 days in DM
(p < 0.05), while no effects were detected in proliferating
myoblasts (data not shown), sub-confluent myoblasts or
at a later differentiation stage (Fig. 3a). This result
demonstrated that, as in other cell types, also in myogenic
cell line neurotrophins transiently regulate the activity
of NFκB via p75NTR receptor [
]. We then verified
in L6C5 cellular model the possible correlation between
NFκB activity, modulation of sHSPs and resistance to
apoptosis. Hsp27 and αB-Cry are key components of
the myofibril structure, with a prominent role in skeletal
muscle physio-pathology [
] and in exercise-related
adaptation to damaging contraction [
]. We found
that NGF induced a significant reduction of αB-Cry
expression at 5 days in DM and an increase of Hsp27
levels after 2 days in DM (p < 0.05) (Fig. 3b–d). This result
mirror our previous data on rat muscle regeneration
in vivo [
] suggesting an anticipatory effect of NGF in
the progression throughout the differentiation process,
consistent with the inhibitory and promoting effects
exerted during myogenic differentiation by αB-Cry and
Hsp27, respectively [
14, 15, 38
]. However, in contrast
with previous results by our group [
8, 11, 17
] and by
] on the relevance of the neurotrophin/p75NTR
axis in the promotion of myofibres survival, in the
present study we did not find any protective effects of NGF
towards spontaneous or H2O2-induced apoptosis, neither
in proliferating nor in differentiating L6C5 cells. Indeed,
as already demonstrated in a fibroblast derived cell line
constitutively expressing rat p75 , in our “in vitro”
model NGF did not modulate neither the total number
of TUNEL-positive apoptotic cells, nor the expression
and/or cleavage of Bcl-2 and Caspase-3 in differentiating
cells (Additional file 2: Fig. S2) and in myoblasts (data not
As summarized in Fig. 3e, our results demonstrate that
p75NTR is sufficient to mediate the NGF modulation of
L6C5 cells differentiation inducing structural, metabolic
and functional changes as well as NGF direct, or
indirect effect on αB-Cry and Hsp27, essential for the
myogenic program and myofibrillar stabilization. Moreover,
we show that the NGF-mediated hypertrophic at the late
stage of differentiation correlates to an increased
expression of MyHC isoforms.
(See figure on previous page.)
Fig. 3 Effects of NGF supplementation on NFκB, αB-crystallin and Hsp27 during L6C5 in vitro differentiation. a NFκB activity measurement in
myoblasts and differentiating L6C5 cells (2, 5 and 9 days) grown with or without NGF supplementation. Nuclear protein extract from Jurkat cells
stimulated by TPA and calcium ionophore was used as positive control (2 days: − NGF vs + NGF, 2.2 vs 3.0, p < 0.05). b, c Quantitative analysis of
αB-Cry and Hsp27 expression during differentiation process (2, 5 and 9 days) of myogenic cells supplemented with or without NGF (αB-Cry: NGF
1.3 ± 0.08 vs Ctrl 2.0 ± 0.11, p < 0.05; Hsp27: NGF 1.0 ± 0.03 vs Ctrl 0.53 ± 0.04, p < 0.05). The histograms represent the mean ± SD of experiments
repeated at least three times. *p < 0.05. d Representative western blot of αB-crystallin and Hsp27 expression. The β-actin was used as housekeeping
for both markers. e Proposed mechanism for NGF-p75NTR signaling pathways in L6C5 myogenic cells: during myoblast proliferation and
serum-deprivation condition, the supplementation with NGF can sustain the activity of key enzymes in carbohydrate metabolism, such as citrate synthase and
glyceraldehyde-phosphate-dehydrogenase, through the activation of the JNK pathway. During the early stage of myoblast fusion, NGF transiently
up-regulates NFκB activity and, directly or indirectly trough a NFκB-mediated mechanism, anticipates the myogenic progression by modulating
αBcry and Hsp27 expression and promoting, at a late differentiation stage, myonuclear fusion and the accumulation and stabilization of the MyHC
myofibrillar component. CS citrate synthase, GAPDH Glyceraldehyde-phosphate-dehydrogenase, NGF nerve growth factor, αBcry αB-crystallin, JNK
c-Jun N-terminal kinases, NFκB nuclear factor kappa-light-chain-enhancer of activated B cells, p75NTR neurotrophin receptor p75
Although the results on the NGF-mediated effects on the
enzymatic activities, NFκB activation and sHSPs
expression are solid, the study did not reveal the causal
relationship among these factors.
Additional file 1: Figure S1. TrkA and p75NTR expression in L6C5
myoblasts and myotubes. a, b Relative mRNA levels of TrkA and p75NTR in L6C5
cells at different times since the seeding (proliferating = 24, 48 h; 2, 5, 9,
and 12 days of differentiation). Proliferating (Pr) and differentiated (Dif )
PC12 cells were used as positive control for the expression of TrkA and
p75NTR receptors. c Western blot analysis of TrkA and p75NTR in
proliferating and differentiated L6C5 cells. RNA extraction and quantitative RT-PCR
was performed as already described [
]. Primers for PCR amplification
were as follows: housekeeping gene
glyceraldehyde-3-phosphatedehydrogenase (GAPDH): 5′-ACCACAGTCCATGCCATCAC-3′ and
5′-TCCACCACCCTGTTGCTGTA-3′; Neurotrophic tyrosine kinase receptor type 1 (TrkA):
5′-CCTGATGCCTTCCATTTCAC -3′ and 5′-TGACATTGACCAGAGTTAGCC-3′;
Nerve growth factor receptor (p75NTR): 5′-CAAGGAGACATGTTCCACAG-3′
and 5′GGATCTCTTCGCATTCAGCA-3′. L6C5-P proliferating myoblasts in
GM, L6C5-D differentiating cultures in DM.
Additional file 2: Figure S2. Effect of NGF supplementation on
spontaneous or H2O2-induced apoptosis during L6C5 in vitro
differentiation. a TUNEL assay (Roche applied sciences) and b Bcl-2 and Caspase-3
protein expression in L6C5 cells growing in DM NGF-supplemented under
standard and oxidative stress condition (100 μM H2O2). For the analysis of
H2O2-induced apoptosis, cells under differentiation (48 h before, or 2, 5 or
9 days from DM addiction) in presence or in absence of NGF (20 ng/ml)
were treated with H2O2 100 µM for the last 1-h of culture. The histogram
represents the mean ± SD of experiments repeated at least three times.
*p < 0.05 compared with control (Ctrl). §p < 0.05 compared with control
NGF-supplemented (Ctrl + NGF).
ALT: alanine transglutaminase; αB-Cry: αB-crystallin; CS: citrate synthase;
DMEM: Dulbecco modified eagle medium; DM: differentiation medium;
GAPDH: glyceraldehyde-phosphate-dehydrogenase; GH: growth hormone;
GM: growing medium; HAD: 3-OH acylCoA dehydrogenase; JNK: c-Jun
N-terminal kinase; LDH: lactate dehydrogenase; MTS:
3-(4,5-dimethylthiazol1)-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), inner salt;
MyHC: myosin heavy chain; NFκB: nuclear factor κB; NGF: nerve growth factor;
p75NTR: p75 neurotrophin receptor; sHSPs: small heat shock proteins; TrkA:
tyrosin kinase receptor A; TPA: 12-otetradecanoylphorbol-13-acetate.
ADP, ID, GD, CF, NM and DC have conceived the work, designed methodology,
interpreted data and written the manuscript; RC, LDL and SS participated in
data interpretation and manuscript writing. All authors read and approved the
We thank Prof. Jose Viňa for the helpful discussion of the results and Dr. Paola
Teti from the University of Rome Foro Italico for the English spelling revision.
The authors declare that they have no competing interests.
Availability of data and materials
Some of the data has been included as additional supplementary material.
We will however readily share our datasets and spreadsheets per individual
Consent for publication
Ethics approval and consent to participate
The work has been supported by grant from University of Rome Foro Italico,
Research Grant 2015.
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published maps and institutional affiliations.
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