Neuregulin Stimulates Myogenic Differentiation in an
Autocrine Manner*
Daegun
Kim
,
Susun
Chi
,
Kun Ho
Lee
,
Sangmyung
Rhee
,
Yunhee Kim
Kwon§,
Chin Ha
Chung
,
Hyockman
Kwon¶
, and
Man-Sik
Kang
From the
Department of Molecular Biology and Research
Center for Cell Differentiation, Seoul National University,
Seoul 151-742, the § Department of Biology, Kyung Hee
University, Seoul 130-701, and the ¶ Department of Molecular
Biology, Dankook University, 8 Hannam-dong Yongsan-gu,
Seoul 140-714, Korea
 |
ABSTRACT |
During myogenesis, mononucleated myoblasts form
multinucleated myotubes by membrane fusion. Efficiency of this
intercellular process can be maximized by a simultaneous progress, with
a time window, of other neighboring myoblasts in the differentiation program. This phenomenon has been described as the community effect. It
proposes the existence of a molecule that acts as a
differentiation-inducing signal to a group of identical cells. Here ,we
show that neuregulin is a strong candidate for this molecule in
myoblast differentiation. The expression of neuregulin increased
rapidly but transiently at early stage of differentiation of rat L6
cells. Neuregulin showed a potent differentiation-promoting activity in
membrane fusion and expression of myosin heavy chain. The antibodies
raised against neuregulin and its cognate receptor ErbB3, which were capable of neutralizing the signal pathway, inhibited myotube formation
and expression of myosin heavy chain in both L6 cells and primary rat
myoblasts. The progress of differentiation was mostly halted after the
expression of myogenin and cell cycle arrest. These results suggest
that the activation of an autocrine signaling of neuregulin may provide
a basic mechanism for the community effect observed in the
differentiation of the embryonic muscle cells.
 |
INTRODUCTION |
Embryonic induction, a process in which a signal from one group of
cells regulates the development of an adjacent group of cells, provides
a major paradigm for understanding of cellular basis of development.
For certain differentiation processes, however, interactions within a
group of cells in an autocrine manner become a great consideration
(1-3). One of these phenomena, the community effect, has been
described to explain the requirement of identical neighbors for their
completion of differentiation program as observed in muscle development
of amphibian embryos (Refs. 4-9; for review, see Ref. 7). The
formation of skeletal muscle involves an intercellular process, in
which mononucleated myoblasts form multinucleated myotubes by fusion of
cellular membrane. The fusion involves specific cell-cell adhesion
molecules that mediate recognition between myoblasts (10, 11). This
process inevitably proposes the existence of a time window for fusion
to occur, and spatiotemporal coordination becomes an important factor
to accomplish the homogeneity and uniformity of differentiation. The
community effect, presumably mediated by a local signal molecule made
from the differentiating myoblasts, may establish this spatiotemporal
coordination. This model assumes a checkpoint that can be passed by
only when the concentration of this molecule exceeds a certain
threshold level (12, 13).
The NRGs1 (also known as
acetylcholine receptor inducing activity, GGF, heregulin, or neu
differentiation factor) are a group of polypeptide growth factors that
play roles in the developing heart and nervous system. ErbB2, ErbB3,
and ErbB4 are members of a subfamily of receptor tyrosine kinases that
are activated by NRG through direct or indirect interaction (14, 15).
NRG is essential in the developing heart; NRG
/
embryos
fail to form ventricular trabeculae and die in mid-gestation (16). In
the developing nerves, NRG, ErbB2, and ErbB4 are required for the
formation of the sympathetic nervous system, such as neural crest
precursor cells (17). NRGs, secreted from motor neuron terminals, also
regulate the process of neuromuscular synapse formation (18, 19).
Glial growth factor 2 (GGF2), one member of NRG family secreted from
motor neuron terminals, has recently been shown to stimulate myoblast
differentiation in immortalized cell lines (20). The exogenous addition
of GGF2 to the cultured myoblasts stimulates cell fusion and
accumulation of creatine kinase. Based on these observations, Florini
et al. (20) suggested GGF2 as a candidate for myotrophic
agents secreted by nerves. Here, we provide evidence for the existence
of an NRG-ErbB3 autocrine signaling pathway in an early stage of
myoblast differentiation. Disruption of the NRG-ErbB3 signaling by the
antibodies raised against NRG and ErbB3 receptor markedly suppressed
both myotube formation and muscle-specific gene expression in L6 cell
line and rat primary myoblasts. The neutralizing antibodies appeared to
influence mostly at the differentiation stage(s) after the expression
of myogenin and cell cycle arrest. These results are consistent with a
model that NRG plays a role as a local signal molecule that mediates
the community effect observed in the differentiation of the embryonic
muscle cells.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture, Antibody Treatment, and Immunocytochemistry--
The L6 rat myoblast cell line was obtained from the American
Type Culture Collection. Cultures were plated in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum
(proliferation medium (PM)) at 1.5 × 105 cells per
100-mm dish. The cell differentiation was induced by switching the
medium to DMEM containing 5% horse serum (differentiation medium (DM))
at 3 days after plating. In the antibody inhibition experiments, the
anti-ErbB3 antibody (Ab5) or the anti-NRG antibody (Ab2) was included
in DM. To investigate the effects of NRG
1 or NRG
1 on myogenesis,
cells were cultured for 3 days in PM, washed with DMEM, and further
cultured in DMEM containing 0.05% bovine serum albumin in the presence
or absence of NRG. Ab5, Ab2, NRG
1, and NRG
1 were purchased from NeoMarkers.
Primary rat muscle cell cultures were prepared essentially as described
(21). Because the differentiation of primary rat muscle cells does not
require the switching the culture medium to DM, Ab5 or Ab2 was directly
added to the cultures at 4 h after plating for the antibody
inhibition experiments. Fusion indices were measured as described
previously (22). For immunocytochemistry, cells were fixed with 3.7%
paraformaldehyde and incubated for 1 h with MF20, a monoclonal
antibody specific to the skeletal myosin heavy chain (MHC). MHC was
visualized using a horseradish peroxidase-linked system, which employs
diaminebenzoate as a substrate (Vectastain ABC kit, Vector Laboratories
Inc.).
Preparation of Antibodies--
RNA from C2C12 myogenic cell line
was subjected to RT-PCR using oligonucleotide primers corresponding to
the epidermal growth factor (EGF)-like domain of rat NRG
(5'-TCTGGAGAGTATATGTGCAAAGTGATCAGC-3', 3'-GCAGTAGGCCACCACACACATGATGCC-5'). A 480-base pair amplified fragment
was subcloned into pGEM-T (Promega) and sequenced. Its sequence showed
90% identity with that of the rat neu differentiation factor cDNA.
The 480-base pair fragment of NRG cDNA was subcloned into pGEX-4T
(Amersham Pharmacia Biotech). The resulting glutathione S-transferase fusion protein containing the NRG sequence
(amino acids 107-265) was produced in Escherichia coli and
purified using glutathione-Sepharose (Amersham Pharmacia Biotech).
After separation in SDS-polyacrylamide gel electrophoresis, the band
corresponding to a molecular mass of 42 kDa was cut out, minced, and
injected three times into albino rabbit at 4-week intervals. Upon
immunoblot analysis, the resulting antiserum, but not the preimmune
serum, was found to specifically interact with the 42-kDa glutathione S-transferase-NRG fusion protein in the E. coli
lysates. The antiserum also recognized the extracellular domain of NRG
1 and
1 expressed in E. coli as a 30-kDa protein.
Mouse monoclonal antibodies were used to detect ErbB2 (Ab-3; Oncogene),
-tubulin (T4026; Sigma), and myogenin (F5D; Pharmingen). p21 was
detected with a mixed monoclonal antibody (05-345; Upstate Biotechnology). The antibodies against Rb (C-15) and p27 (C-19) were
purchased from Santa Cruz Biotechnology. Creatine kinase (CK) was
detected using a rabbit polyclonal antibody, prepared as described
previously (23).
RNA Analysis by RT-PCR--
RNA was isolated from L6 cells using
the TRIzol (Life Technologies, Inc.) method at the proper time
intervals. First-strand synthesis was done on 1 µg of RNA in a final
volume of 20 µl using random primers and Superscript reverse
transcriptase (Life Technologies, Inc.). One microliter of the sample
was subjected to PCR using the primers corresponding to the EGF-like
domain of rat NRG as above. The level of NRG RNA was measured by
hybridizing with 32P-labeled internal oligonucleotide
probes (5'-ACATCAACATCCACGACTGGGACCAGCCATCT-3'). To ensure that the
assay was in the linear range, the cycle number and amounts of RNA were varied.
Immunoprecipitation Analysis--
Whole cell extracts were
prepared with radioimmune precipitation buffer (50 mM Tris,
pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium
deoxycholate, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM NaF, 1 µg/ml leupeptin) and precleared by
centrifugation. The precleared lysates were incubated with 2 µg of an
anti-ErbB3 antibody (C-17; Santa Cruz Biotechnology) for 2 h at
4 °C, followed by addition of 50 µl of protein-A Sepharose beads
(10% (v/v) suspension). After incubation for 1 h at 4 °C, the
beads were washed three times with radioimmune precipitation buffer.
Bound ErbB3 proteins were subjected to immunoblot analysis using an
anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology). The ErbB3
proteins were then detected by an enhanced chemiluminescence method
(Amersham Pharmacia Biotech).
 |
RESULTS AND DISCUSSION |
Expression of ErbB3 Receptor and Neuregulin in L6
Cells--
Recently, GGF2 has been suggested to act as a myotrophic
agent secreted from nerve cells (20). If the differentiation of L6
myogenic cell is under the influence of NRG, L6 myoblasts should have
receptors for NRG. To determine whether the functional ErbB3, the
cognate receptor of NRG, is indeed expressed during the myogenesis of
L6 cells, immunoprecipitation experiments were performed using an
antibody raised against ErbB3 (Fig.
1a). Consistent with the previous report (18), the terminally differentiated myotubes expressed
ErbB3. ErbB3 was expressed also in the undifferentiated myoblasts,
although to a lower level than in myotubes. Moreover, the ErbB3
receptor in the myoblasts was tyrosine-phosphorylated upon treatment
with NRG
1 or NRG
1, indicating its functional responsiveness.

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Fig. 1.
Expression of ErbB3 receptor and neuregulin
in L6 cells. a, activation of ErbB3 receptors expressed
in myoblast and myotube. ErbB3 in the extracts of L6 cells was
immunoprecipitated with an anti-ErbB3 antibody. Phosphotyrosine levels
of ErbB3 were then determined by immunoblot with an
anti-phosphotyrosine antibody 4G10. b, a transient
expression of neuregulin during myogenesis. The cell extracts were
analyzed by immunoblot with an anti-NRG antibody. Expression of MHC is
shown as a differentiation marker. c, a transient increase
of neuregulin mRNA during myogenesis. The total RNAs of L6 cells
were subjected to RT-PCR as described under "Experimental
Procedures." The RT-PCR products were hybridized with a
32P-labeled internal oligonucleotide probe. Cyclophilin
served as an internal control.
|
|
Interestingly, we observed a low level of tyrosine phosphorylation of
ErbB3 in the control L6 cells that were not treated with NRGs (Fig.
1a). This observation led us to speculate that ErbB3 might
be activated by endogenously expressed NRG. In fact, previous reports
have shown the expression of NRGs in muscle cells. However, most
attention has been focused on the action of these ligands in the
neuromuscular junction, and their role at the early stage of myogenic
differentiation has not been well documented (24, 25). We therefore
examined in detail the expression pattern of NRGs in the course of L6
myogenesis. When the differentiation was induced by replacing the
culture medium with a low mitogen-containing medium, the expression of
NRG with a size of about 65 kDa was dramatically induced 1 day after
the medium change (Fig. 1b). It is striking that the
expression of NRG maintained only for 1 day and rapidly declined
thereafter. As shown in Fig. 1c, the changes in the level of
NRG mRNA closely correlated with the alterations in the NRG protein
level. Thus, the expression of NRG is most likely to be under
transcriptional control, although an increase in NRG mRNA stability
may give rise to a similar result. These results demonstrate that NRG
is transiently induced at the early stage of myogenesis, whereas ErbB3
receptor is constitutively expressed throughout the differentiation
process. Because the anti-NRG antibody used in this immunoblotting
analysis was raised against the EGF-like domain, a region common to
most isoforms of NRG, it remains uncertain which isotype of NRG is
expressed in the L6 myoblasts.
NRG
1 Stimulates the Differentiation of L6 Myoblasts--
In
order to determine whether NRG may play a role as a local signaling
molecule for myogenesis, L6 myoblasts were treated with the recombinant
1-NRG and
1-NRG isoforms, which contain EGF-like domain. Because
NRG have been reported to show a modest mitogenic activity in the
presence of serum components, the cells were treated with NRGs after
the medium change with a serum-free medium (20). As shown in Fig.
2a, NRG
1 stimulated
membrane fusion in a dose-dependent manner. This
stimulatory effect could be seen at its concentrations as low as 0.1 nM (Fig. 2a). At 0.5 nM, the onset
of myoblast fusion in the NRG
1-treated cells was observed about 1 day earlier than that in the control cells (Fig. 2c, lower
panel). NRG
1 at the same concentration also stimulated the
accumulation of muscle-specific proteins, such as MHC, as assessed by
immunostaining (Fig. 2b) and immunoblot analysis using an
anti-MHC antibody (Fig. 2c, upper panel). Similar results
were obtained when the same experiments were performed with NRG
1, although its effect was slightly less potent than that of NRG
1. These results indicate that NRG facilitates both the morphological and
biochemical differentiation of L6 cells at its concentrations comparable to those used previously for determining its effect on
proliferation and differentiation of other cells. For example, some
cellular responses, such as proliferation and differentiation of
mammary epithelial cells, are known to require 0.3 nM of
NRG to be effective (26-28). In addition, recombinant proteins
containing only the EGF-like domain have been reported to stimulate the
NRG receptors at subnanomolar concentrations in vitro (29,
30).

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Fig. 2.
NRG 1 stimulates the
differentiation of L6 myoblasts. L6 cells were cultured for 3 days
in PM and then incubated with or without NRG 1 in DMEM containing
0.05% bovine serum albumin. a and b,
concentration dependence of membrane fusion to NRG 1. Cells were
fixed at 6 days after plating. Fusion index was determined
(a), and the cells were stained with the anti-MHC antibody,
MF20 (b), as described under "Experimental Procedures."
Bars show mean ± S.E. for three independent
experiments. c, time course of the myogenic induction by
NRG 1. Cells were treated with ( ) or without ( ) 0.5 nM NRG- 1. Extent of fusion was determined at the
indicated time points. The results presented are means ± S.E. for
triplicate determinants. Cell extracts were prepared at the indicated
time points and subjected to an immunoblot analysis with the anti-MHC
antibody, MF20.
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|
Inhibition of NRG Receptor Activation Suppresses the
Differentiation of L6 Cells--
If there is a NRG-ErbB3 autocrine
signaling necessary for myogenesis, disruption of this signaling must
suppress the differentiation process. To test this possibility, we took
advantage of the availability of antibodies that neutralize the
NRG-ErbB3 signal. As shown in Fig.
3a, the activation of ErbB3
receptor by exogenous NRG
1 was dramatically suppressed by treatment
of the cells with 10 µg/ml of the anti-ErbB3 monoclonal antibody
(Ab5) or the anti-NRG polyclonal antibody (Ab2). These results indicate
that Ab5 and Ab2 were capable of neutralizing the NRG-ErbB3 signal
pathway effectively. We then examined the effect of Ab5 and Ab2 on
myogenesis. The differentiation of L6 cells was induced by changing the
medium with DM at 3 days after plating, followed by immediate treatment
with either Ab5 or Ab2. Both the formation of myotube and the
expression of MHC were markedly inhibited by the antibodies, whereas
the differentiation was not influenced by the treatment with normal
mouse or rabbit immunoglobulin (Fig. 3, b-d). These results
demonstrate the existence of an autocrine signaling pathway for NRG in
L6 cells, which is necessary to initiate the process of muscle-specific
gene expression and myotube formation.

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Fig. 3.
Inhibition of NRG receptor activation
suppresses the differentiation of L6 cells. a,
inhibition of the NRG 1-mediated tyrosine phosphorylation of ErbB3 by
Ab5 and Ab2 antibodies. L6 myoblasts were incubated with Ab5 or Ab2
antibody (10 µg/ml) for 1 h at 37 °C and then treated with
0.5 nM NRG 1. After incubation for 5 min, tyrosine
phosphorylation of ErbB3 receptor was monitored as described in Fig.
1a. NMIg and NRIg indicate normal
mouse and rabbit Ig, respectively. b-d, inhibition of the
differentiation of L6 myoblast by Ab5 and Ab2 antibodies. L6 cells were
cultured for 3 days in PM and switched to DM. At the time of the medium
change, the indicated antibodies were added at 10 µg/ml. Expression
of MHC (b and d) and membrane fusion
(c) were determined with the cells 5 days after plating.
NMIg, normal mouse Ig; Ab5, anti-ErbB3 monoclonal
antibody; NRIg, normal rabbit Ig; Ab2, anti-NRG
polyclonal antibody.
|
|
Moreover, the extent of inhibition appeared to be precisely correlated
with the potency of neutralizing antibodies. An extensive exposure
visualized a low level of tyrosine phosphorylation of ErbB3, when Ab2,
the ligand-blocking antibody, was incubated with NRG
1, whereas
tyrosine phosphorylation of ErbB3 was not detected with Ab5, the
receptor-blocking antibody (Fig. 3a). These results demonstrate that Ab5 is more potent than Ab2 in neutralizing the NRG-ErbB3 signal pathway. In parallel, Ab5 inhibited the expression of
MHC and membrane fusion of L6 cells more effectively than Ab2 (Fig. 3,
b and c). A low level of MHC was detected upon a
prolonged exposure when the myogenesis of L6 cells was inhibited with
Ab2 but not with Ab5 (Fig. 3b).
Inhibition of NRG Receptor Activation Suppresses the
Differentiation of Rat Primary Myoblasts--
L6 cell is an
established cell line, and it may not truly represent in
vivo situation. In an attempt to investigate whether the autocrine
signaling of NRG also works in the process of muscle differentiation
in vivo, we used primary culture of rat myoblasts. Treatment
of rat primary myoblasts with Ab5 (30 µg/ml) or Ab2 (50 µg/ml)
caused a severe inhibition in expression of MHC and membrane fusion
(Fig. 4). On the other hand, normal mouse
or rabbit immunoglobulin failed to inhibit the differentiation of
myoblasts into myotubes at the same concentrations. Compared with L6
cells, it is noticeable that rat primary myoblasts required higher
concentrations of neutralizing antibodies for inhibition of the
differentiation processes. Moreover, the inhibitory effect on primary
culture was less prominent than that on L6 cell line. It may be
explained by the observation that the primary cultures of rat myoblasts were significantly contaminated by fibroblasts (less than 30% of total
cell number at the time of plating and more than 60% 2 days after
plating), which are known to secrete NRGs (31, 32). Another explanation
could be the heterogeneity in the population of rat primary myoblasts,
a part of which might have already committed to differentiation program
at the time of the preparation of the primary cultures. Nevertheless,
the significant suppression of MHC expression and membrane fusion by
the neutralizing antibodies supports our notion that the activation of
autocrine signaling of NRG is required for muscle differentiation
in vivo.

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Fig. 4.
Inhibition of NRG receptor activation
suppresses the differentiation of rat primary myoblasts. Rat
primary myoblasts were prepared as described under "Experimental
Procedures." Four hours after the cell plating, they were treated
with Ab5 or Ab2 antibody. Expression of MHC (a and c) and
membrane fusion (b) were determined with the cells 3 days
after plating. NMIg, normal mouse Ig (30 µg/ml);
Ab5, anti-ErbB3 monoclonal antibody (30 µg/ml);
NRIg, normal rabbit Ig (50 µg/ml); Ab2,
anti-NRG polyclonal antibody (50 µg/ml).
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Contribution of NRG Signal Pathway to the Myogenic
Program--
The formation of skeletal muscle proceeds through
sequential developmental events following commitment of mesodermal
precursor cells, withdrawal from cell cycle, and terminal
differentiation into multinucleated myotubes. The commitment to the
differentiation pathway is characterized by the expression of myogenin
(33, 34). Induction of p21 and dephosphorylation of Rb are closely related with the exit from cell cycle (35, 36). The induction of
myogenin and the exit from cell cycle are proposed to be prerequisite for terminal differentiation indicated by myotube formation and muscle-specific gene expression, such as MHC and CK. To examine whether
the neutralizing antibodies selectively suppressed specific stage(s) of the myogenic differentiation, immunoblot analysis was
performed using antibodies against myogenin, p21, Rb, MHC, and CK in
the presence and absence of Ab5 or Ab2. Both Ab5 and Ab2 dramatically
suppressed the expression of MHC and CK (Fig. 5). These antibodies also suppressed the
expression of myogenin and p21, but rather moderately. The
phosphorylation of Rb reflects its activity in cell cycle. Rb in the
absence of the neutralizing antibodies migrated as a single band of 100 kDa, indicating that most of Rb is hypophosphorylated when myoblasts
exit from cell cycle (Fig. 5). In contrast, a part of Rb from Ab5- and
Ab2-treated cells showed a retarded mobility, indicating that cell
cycle was not completely arrested. These results demonstrate that the
commitment to the differentiation pathway and the irreversible cell
cycle arrest were partially blocked by the neutralizing antibodies. On
the other hand, the neutralizing antibodies more effectively suppressed
the terminal differentiation characterized by cell fusion and
expression of MHC and CK.

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Fig. 5.
Effect of inhibition of NRG receptor
activation on the expression of various proteins related with
myogenesis. L6 cell extracts were prepared as described in Fig.
3b. Expression of myogenin, p21, Rb, MHC, and CK was
assessed by immunoblot. The cell cycle inhibitor, p27, was also
analyzed as above for an internal control.
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The expression of myogenin is induced by another myogenic factor, myf5,
expressed at the early stage of myogenic differentiation. Myogenin also
promotes its own gene expression by itself. Therefore, the partial
blockage of myogenin expression by the neutralizing antibodies can be
explained by a possibility that the NRG-ErbB3 signaling be involved in
autoregulation of myogenin expression. Concurrently, the NRG-ErbB3
signaling appears to be also required for the ability of myogenin to
induce the terminal differentiation as evidenced by the finding of
complete inhibition of MHC expression and myotube formation by the
neutralizing antibodies. The partial blockage of cell cycle arrest is
also explained by this hypothesis because myogenin is partly
responsible for the completion of cell cycle arrest (34, 37). It is
noticeable that the extents of suppression were closely correlated with
the potencies of neutralizing antibodies. Ab5, the stronger
neutralizing antibody, more effectively suppressed the
hypophosphorylation of Rb and the induction of myogenin and p21.
NRGs play a role as an inducing signal in various vertebrate
development, such as trabeculae formation in heart, growth and differentiation of Schwann cells, morphogenesis of mammary gland, and
formation of neuromuscular synapse (25, 38, 39). These developmental
processes are presumably mediated by local actions of NRGs in paracrine
manner, in which NRG secreted from one group of cells induces the
development of neighboring target cells. Here, we provide evidence for
activation of an NRG autocrine signaling during the differentiation of
embryonic muscle cells. We have shown that the expression of NRG was
transiently but dramatically up-regulated after the cells were switched
to the differentiation state. The receptors for NRG, ErbB2, and ErbB3
were also found to exist and be active in L6 myoblasts. Furthermore, a
substantial inhibition of myogenesis by the antibodies that neutralize
the NRG signal pathway is consistent with the model that locally
synthesized NRGs may regulate the differentiation of embryonic muscle
cells through an autocrine mechanism. NRG has been shown to act in an autocrine manner in other cases. Schwann cells express NRG mRNA, and the production of NRG is required for the proliferation of Schwann
cell in response to transforming growth factor-
, basic fibroblast
growth factor, or hepatocyte growth factor (31). Mature muscle cells
also synthesize NRG and ErbBs, and both ligands and receptors are
localized to synaptic site (24). Although NRG is also secreted from
nerve terminals, NRG-ErbB signaling at the synapse may play a role, at
least in part, as an autocrine mechanism (25).
The neutralizing antibodies against NRG and ErbB3 showed the
differential effect on each event of myogenic progression. They partially suppressed the dephosphorylation of Rb and the expression of
myogenin and p21 but completely inhibited myotube formation and MHC
expression. These results indicate that NRG has a
differentiation-promoting activity, although it is not absolutely
required for the commitment of myoblasts to differentiate. More
importantly, these results also indicate the existence of a checkpoint
between the expression of myogenin and the terminal differentiation. In
Xenopus and mammal, muscle precursor cells of mid-gastrulae
contain transcripts of muscle-specific transcription factors, and the
level of these factors first becomes detectable at this stage (40-43).
But the expression of muscle-specific transcription factors has proven to be insufficient for autonomous differentiation of muscle precursor cells (41). Our study proposes that NRG, produced by the myoblasts at
early stage of differentiation, may be required to proceed through a
checkpoint presumably for the cells located after the expression of
myogenin and the exit from cell cycle. When NRG reaches a threshold
concentration in an autocrine manner, it may precisely coordinate the
asynchronously committed myoblasts for the spatiotemporal progress of
differentiation. It will be interesting to determine the nature of
checkpoint controlled by NRGs.
 |
FOOTNOTES |
*
This work was supported by grants from the Korea Science and
Engineering Foundation through the Research Center for Cell
Differentiation and by the Academic Research Fund (GE96-199) of the
Ministry of Education, Republic of Korea.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
82-2-709-2817; Fax: 82-2-793-0176; E-mail: hmkwon{at}chollian.net.
 |
ABBREVIATIONS |
The abbreviations used are:
NRG, neuregulin;
GGF, glial growth factor;
PM, proliferation medium;
DM, differentiation
medium;
MHC, myosin heavy chain;
EGF, epidermal growth factor;
RT, reverse transcription;
PCR, polymerase chain reaction;
Rb, retinoblastoma protein;
CK, creatine kinase;
DMEM, Dulbecco's modified
Eagle's medium;
Ab, antibody.
 |
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