1 Department of Physiology, Faculty of Medicine and Dentistry, The University of
Western Ontario, London, Ontario, N6A 5C1, Canada
2 Department of Biochemistry, Faculty of Medicine and Dentistry, The University
of Western Ontario, London, Ontario, N6A 5C1, Canada
3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
Author for correspondence (e-mail:
tunderhi{at}uwo.ca)
Accepted 31 March 2003
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Summary |
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Key words: Myogenesis, p38 MAPK, Limb mesenchyme, Chondrogenesis
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Introduction |
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Several extracellular factors modulate expression of the MRFs and/or MEF2
factors. Signal transducers such as the mitogen-activated protein kinases
(MAPKs), including the extracellular signal-regulated kinases (ERK1 and 2),
the Jun-N-terminal kinases (JNK1, 2, and 3) and the p38 isoforms (,
ß,
and
) (Chang and
Karin, 2001
; Kyriakis and
Avruch, 2001
; Obata et al.,
2000
; Pearson et al.,
2001
), have all been widely studied with respect to their
importance during myogenesis. Evidence from a number of studies strongly
supports a requirement for p38 MAPK during myogenic progression. Transcripts
for the upstream activator of p38, MKK6, are most abundant in skeletal muscle,
and the p38 transcripts, particularly those encoding the
isoform, are
highly expressed in this tissue. Moreover, overexpression of p38 isoforms or
upstream activators causes an upregulation of myogenic markers, enhances
muscle reporter activity and accelerates myotube formation
(Wu et al., 2000
). In a
similar manner, forced p38 induction can restore MyoD function and enhance
MEF2 activity in rhabdomyosarcoma cells deficient for p38 MAPK activation,
resulting in terminal differentiation
(Puri et al., 2000
). In
addition to the effects of p38 activation, independent groups have
demonstrated an inhibition of muscle differentiation in C2C12 and L6 cells
(Cuenda and Cohen, 1999
;
Wu et al., 2000
), human
primary myocytes (Wu et al.,
2000
) and rhabdomyosarcoma cells
(Puri et al., 2000
) in
response to specific p38 inhibitors. This apparent requirement for p38 in
myogenesis is consistent with the demonstrated p38
- and ß-specific
induction of the transcription factor MEF2C
(Wu et al., 2000
;
Yang et al., 1999
).
Clearly, evidence supports an essential role for p38 signaling during
myogenesis. To date, however, biochemical dissection of the myogenic pathway
has largely been done using homogeneous cell populations derived from adult
muscle, including the mouse C2C12 and rat L6 cells, both derivatives of
satellite cells from adult muscle fibers
(Cabane et al., 2003;
Conejo and Lorenzo, 2001
;
Cuenda and Cohen, 1999
;
Gallea et al., 2001
;
Li et al., 2000
;
Puri et al., 2000
;
Zetser et al., 1999
).
Recently, however, p38 inhibitors were shown to reduce the expression of MyoD
target genes in mouse embryonic fibroblasts. Importantly, this same study
suggests distinct subprograms of myogenesis, which may differentially involve
p38 (Bergstrom et al., 2002
).
To date, most of the studies that have implicated p38 in myogenesis focus on
differentiation of precursors and acquisition of the myoblast phenotype. In
the present study, we provide evidence to suggest that in primary limb
mesenchymal cultures, p38 inhibition dramatically advances later stages of the
myogenic program. This is revealed by the rapid alignment, aggregation and
fusion of myocytes to form functional, twitching skeletal muscle. Given the
extent of muscle seen in the limb mesenchymal cultures treated with p38
inhibitors, we believe that the role of p38 signaling in myogenesis is not as
clear as originally thought.
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Materials and Methods |
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Generation of G8-ßgeo and C2C12-ßgeo cells
G8 embryonic myoblasts (American type-culture collection, ATCC) were
maintained in Dulbecco's Modified Eagle's Media supplemented with 10% fetal
bovine serum (FBS) and 10% horse serum
(Christian et al., 1977).
Cultures were subcultured prior to reaching
80% confluence to minimize
the loss of myoblasts. For generation of G8-ßgeo cells and
C2C12-ßgeo cells, G8 and C2C12 cells were each infected with
MSV-tk-ßgeo. MSV-tk-ßgeo retroviral particles were generated by
co-transfection of MSV-tk-ßgeo with pSV
2 into COS cells. 48 hours
after transfection, the supernatant from the cultures was collected and
filtered. To infect G8 or C2C12 cells, the supernatant was added directly to
each cell culture for 3 hours in the presence of 10 µg/ml polybrene. Within
24 hours of infection, two volumes of media were added to each cell culture.
One day post-infection, the media was exchanged for fresh media containing 600
µg/ml active G418. Cells were subcultured three times during the next 10
days of selection in G418. At the end of the culture period >95% of the
cells within each culture (G8 and C2C12 cells) stained positive for
ß-galactosidase.
Cell mixing experiments were performed by adding G8-ßgeo or C2C12-ßgeo cells to resuspended primary cells such that 5% of the entire cell suspension consisted of the tagged G8 or C2C12 cells. These mixtures were used to seed 24-well culture plates in 10 µl volumes.
Immunofluorescence, in situ ß-galactosidase and alcian blue
staining of cultures
The supernatant from a mouse myeloma cell line containing an anti-MyHC
monoclonal antibody was used to detect the myogenic cells within the primary
limb bud cultures (Bader et al.,
1982). Detection of MyHc-positive cells was carried out as
previously described (Ridgeway et al.,
2000
). To follow localization of LacZ-expressing cells in primary
cultures, cells were briefly fixed and stained with Magenta Gal (BioShop Inc.)
as previously described (Weston et al.,
2000
). Alcian blue staining was performed on fixed cultures also
as described previously (Weston et al.,
2000
).
Transient transfections and reporter assays
For transfection purposes, cells were resuspended at
2.5x107 cells/ml and mixed with a DNA/FuGene6 mixture in a
2:1 ratio. FuGene6-DNA mixtures were prepared according to the manufacturer's
instructions (Roche Biomolecular, Laval, Quebec, Canada). Briefly, 1 µg of
reporter, 1 µg of expression vector and 0.05 µg of pRLSV40 (Promega)
were mixed for a total of 2 µg DNA in 100 µl of media and FuGene6.
50 ml of the DNA mixture was transferred into a sterile 1.5 ml eppendorf tube,
followed by 100 µl of cells. Cells were gently triturated, and 10 µl was
used to seed a single well of a 24-well culture dish. After 1.5 hours in a
humidified CO2 incubator, 1 ml of media was added to each well. 24
hours after transfection, the media was replaced and the appropriate
supplements were added. G8 cells were transfected as described above except
that monolayer cultures were transfected into 12-well plates, whereas C2C12
cells were transfected with jetPEI using conditions outlined by the
manufacturer (Polyplus Transfection Inc., Illkirch, France). For experiments
involving characterization of SB202190 activity, supplements were added
immediately following transfection.
Analysis of reporter gene activity was carried out using the Dual Luciferase Assay System according to the manufacturer's instructions (Promega, Madison, WS). Briefly, approximately 48 hours post transfection, cells were washed once with PBS and lysed in 100 µl of passive lysis buffer for 20 minutes. Firefly and renilla luciferase activities were determined by using 40 µl of the cell lysate in a 96-well format Molecular Devices luminometer.
Western blot analysis
For western blot analysis, 10 individual limb mesenchymal cultures were
established in each well of a 6-well culture dish and were treated with
SB202190 (10 µM) daily, starting 24 hours after culture initiation. Lysates
were collected immediately prior to SB202190 addition, and after 4 and 8 days
of treatment, by adding 150 µl lysis buffer (Cell Signaling Technology,
Beverly, MA) to each well. G8 and C2C12 cells were grown to confluence in
6-well culture dishes, lysed with 150 µl lysis buffer/well, and samples
from 2 wells were pooled. Cleared lysates containing approximately 15 µg of
protein were separated by SDS-PAGE gels and transferred to nitrocellulose.
Antibodies for p38 (pan) and p38 (Cell Signaling Technology) were each
used at a 1:1000 dilution, followed by a 1:3000 dilution of a secondary
anti-rabbit IgG-HRP antibody (Santa Cruz, Santa Cruz, CA). The ß-actin
antibody (Sigma) was diluted 1:10,000 followed by incubation with an
anti-mouse IgG-HRP antibody (Santa Cruz) at a 1:3000 dilution. HRP was
detected using chemiluminescence according to the manufacturer's instructions
(Amersham Biosciences, Piscataway, NJ).
Expression plasmids and reporter constructs
To generate pGL3(4X48), a fragment containing the reiterated (4X48) Sox9
binding sequence upstream of the mouse Col2a1 minimal promoter (-89
to +6) was liberated from the 4X48-p89 plasmid
(Lefebvre et al., 1996) and
subcloned into pGL3-basic as described previously
(Weston et al., 2002
). The
cardiac actin reporter (pGL3-c-actin-Luc) was generated by subcloning a
fragment of the cardiac actin promoter from -440 to +6 into pGL3-basic. The
myogenin promoter-luciferase construct was made by subcloning a 1.14 kb
fragment of the myogenin promoter containing the region from pGZ1092,
(Yee and Rigby, 1993
) from
plasmid pGBB into pGL3-basic. The pCMV-GAL4-MEF2A and pCMV-GAL4-MEF2C
constructs were as described previously
(Yang et al., 1999
) and were
co-transfected into cells with the pG5-Luc reporter containing five copies of
a GAL4 DNA-binding element upstream of a TATA box and the luciferase gene
(Stratagene, La Jolla, CA). MSV-tk-ßgeo is a replication-defective
retrovirus derived from the Mouse Sarcoma Virus containing a
ß-galactosidase-neomycin fusion gene. The p38 MAPK-responsive
transactivation system (Stratagene) consists of an expression vector encoding
a GAL4-CHOP10 fusion protein. This vector was co-transfected with pG5 into
primary cells, G8 or C2C12 cells in the presence or absence of a
constitutively active version of MKK6, termed MKK6E, both with and without
expression vectors for p38
and p38ß.
Northern blot analysis
Northern blots were carried out using total RNA from limb mesenchymal
cultures as previously described (Weston
et al., 2000). Briefly, total RNA was extracted from cells 1, 2, 3
or 4 days after cultures were initiated. Cells were treated with media alone
or with SB202190-containing media. Blots were probed with radiolabelled DNA
fragments derived from cDNAs for Col2a1, Mef2c or myogenin.
Subsequently, blots were re-probed with a probe to the18S rRNA to normalize
for loading.
Statistical analysis
All luciferase assays were performed a minimum of three times using
separate preparations of cells each time. Each transfection or treatment was
carried out in four separate wells for all experiments. All luciferase
reporter data were analyzed by a one-way analysis of variance (ANOVA),
followed by a Bonferroni post-test for multiple comparisons. Statistical
analysis was carried out using GraphPad Prism, Version 2.0 (GraphPad Software
Inc., San Diego, CA). One of at least two representative experiments is shown
for all luciferase results.
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Results |
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|
Although most of our studies focused on the effects of 10 µM SB202190,
concentrations as low as 1 µM elicited the same responses, albeit to a
lesser extent. The effects of SB203580 on muscle formation are
indistinguishable from those of SB202190, whereas the inactive analog of these
inhibitors (SB202474) has no noticeable effect, even at concentrations as high
as 20 µM (data not shown). At the concentrations used throughout this study
(1-10 µM), SB202190 and SB203580 are believed to selectively inhibit the
and ß isoforms of p38, leaving the other two isoforms (
and
) fully active (Davies et al.,
2000
). Thus, activation of p38
and ß, either alone or
in combination, appears to have an inhibitory effect on myogenesis.
Myogenic effects of p38 inhibition require factors present in limb
mesenchymal cultures
The effects of SB202190 on myogenesis in primary cultures are reproduced in
G8 myoblasts and in C2C12 cells that have been introduced into the primary
limb bud cultures (Fig. 2).
C2C12 cells are derived from adult muscle
(Yaffe and Saxel, 1977), and
G8 cells, while embryonic in origin, are from a clone isolated from a myogenic
cell line that arose spontaneously in a culture of hind limb muscle cells from
a fetal mouse (Christian et al.,
1977
). There is a major distinction between the primary cultures
used here and G8 cells, as we dissect limb buds long before the emergence of
functional muscle (E11.5), whereas G8 cells are from developed muscle of an
older embryo. When cultured on their own, G8-ßgeo cells show no
noticeable response to SB202190 (Fig.
2A,B). However, when they are introduced into the primary limb
mesenchymal cultures, initially comprising 5% of the total cells in the
cultures, these tagged cells respond to SB202190 in a manner very similar to
myocytes of the developing limb, becoming elongated and highly organized in
parallel arrays of myocytes. In treated cultures, these tagged cells resemble
the MyHc-positive cells from primary cultures that were treated with SB202190
(compare Fig. 2D with
SB202190-treated cultures in Fig.
1).
|
C2C12-ßgeo cells behave similarly to G8-ßgeo cells in primary cultures. Normally, C2C12 cells require serum withdrawal to progress through the myogenic program, and this progression is blocked by p38 inhibition. In the primary cultures, however, the tagged C2C12 cells were induced to form myotubes upon SB202190 treatment, even in the presence of serum (Fig. 2G,H). When cultured on their own in the presence of serum, no obvious change is observed in response to SB202190 (Fig. 2E,F). Thus, combined with the effects on G8-ßgeo cells, these results highlight the influence of the microenvironment on the progression of myogenic cells.
To extend our analysis of the cartilage and muscle phenotypes observed
following treatment with SB202190, we followed the endogenous activity of
cartilage- and muscle-specific genes by northern blot analysis, and monitored
the activity of transiently transfected cartilage- and muscle-specific
reporters (Fig. 3). As
expected, the normal increase in type II collagen (Col2a1) expression
over time in primary cultures is completely blocked in SB202190-treated
cultures (Fig. 3A), whereas
expression of myogenin and Mef2c is increased in cultures
treated with the inhibitor (Fig.
3A). Repression of cartilage-specific genes and activation of
muscle-specific genes was observed using the pGL3(4X48) reporter, which is
activated during chondrocyte differentiation
(Lefebvre et al., 1996), and
the muscle-specific reporters pGL3-E4-Luc (an E box reporter),
pGL3-myogenin-Luc (a myogenin-promoter-based reporter) and pGL3-c-actin-Luc (a
cardiac-actin-promoter-based reporter). SB202190 inhibited activity of
pGL3(4X48) in a dose-dependent manner (Fig.
3B), but enhanced the activity of all of the muscle reporters
(Fig. 3C). We also examined the
activities of MEF2A and MEF2C by co-transfecting constructs containing the
DNA-binding domain of GAL4 fused to each MEF2 (GAL4-MEF2A and GAL4-MEF2C) with
the pG5-Luc reporter containing Gal4 response elements. SB202190 induced
luciferase activity in cells co-transfected with GAL4-MEF2A or GAL4-MEF2C
(Fig. 3D), implicating these
two MEFs in the myogenic response to p38 inhibition and further demonstrating
the myogenic response to SB202190.
|
To confirm the presence of p38 in all three cell types, western blots were
carried out using p38- (pan) and p38-specific antibodies. p38 is
clearly present in limb mesenchymal cultures at comparable levels over 8 days,
and levels are not noticeably affected by SB202190
(Fig. 4A). Similarly, G8 and
C2C12 cells express p38 at levels detectable by western blot analysis
(Fig. 4A). To assess the
ability of SB202190 to block p38 activity, pFA-CHOP, an expression vector
containing the GAL4 DNA-binding domain fused to the transactivation domain of
CHOP10, was co-transfected with a Gal4-reporter gene, pG5-Luc.
CHOP10, a transcription factor, is a known target of p38
, whose
phosphorylation by p38 is blocked by the SB203580 inhibitor in other cell
types (Wang and Ron, 1996
).
The activity of the Gal4 reporter is also attenuated in the limb mesenchymal
cultures by SB202190 (10 µM). Specifically, the increase in luciferase
activity caused by co-transfection with MKK6E (a constitutively active version
of MKK6) is completely blocked by SB202190. As expected, the ability of MKK6E
to increase reporter gene activity is further enhanced by co-transfection of
expression vectors encoding p38
and p38ß, and this activity is
also substantially attenuated by the addition of 10 µM SB202190
(Fig. 4B). These results
provide convincing evidence that this inhibitor can effectively attenuate p38
activity in the limb mesenchymal cultures. Similar results are observed in G8
and C2C12 cells, where addition of 10 µM SB2020190 was able to reduce
MKK6E-induced reporter gene activity to control levels
(Fig. 4C).
|
Inhibition of p38 enhances myogenesis of somitic mesoderm-derived
cells
During limb development, myogenic cells originate from the somites, whereas
cartilage progenitors arise from the progress zone, a region at the distal tip
of the limb bud. The increase in muscle formation caused by SB202190 could be
due to enhanced myogenesis of somite-derived cells or the redirection of cells
originally fated to become chondrocytes. Both possibilities could account for
an increase in muscle that appears to be at the expense of cartilage. To
identify which cells contribute to the increased muscle, cells of the proximal
portion of the limb bud were cultured separately from those of the distal
portion (Fig. 5). The distal
region of the limb bud at this stage (Fig.
5A) contains fewer somite-derived myogenic cells compared with the
proximal portion (Fig. 5G),
although there are more prechondrogenic cells in the distal cultures (compare
Fig. 5E with K). SB202190
attenuates chondrogenesis in both cultures
(Fig. 5F,L). If the effects of
p38 inhibition were the result of prechondrogenic cells being redirected to
the myogenic lineage, the dramatic myogenic effect of SB202190 would be
observed within the distal cultures. In contrast, however, the magnitude of
the myogenic response is directly proportional to the number of somitic cells
present in the culture at the time of initiation. Specifically, more muscle
was seen in the proximal cultures after 6 days of SB202190 treatment
(Fig. 5I) compared with the
distal cultures (Fig. 5C). This
suggests that p38 inhibition promotes myogenesis of somite-derived cells as
opposed to redirecting the chondrogenic cells derived from the distal tip of
the limb bud. Further support for this comes from the almost complete lack of
MyHC-positive cells both in the presence and absence of SB202190 in primary
cultures derived from E10 embryos (data not shown). At E10, very few
somite-derived cells have migrated into the developing limb
(Martin, 1990).
|
Inhibition of p38 induces the rapid re-organization of myocytes.
To further examine the effects of SB202190 on myogenesis, cells were fixed
and analyzed for MyHC expression at earlier time points following treatment
(Fig. 6). The increased muscle
formation appears to be due to the rapid advancement of pre-existing myocytes.
Even 2 hours after treatment with SB202190, MyHC-positive cells aggregate
together and polarize, forming distinct foci of myocytes within 6 hours. The
appearance of these discrete aggregations of bipolar cells is striking by 24
hours. These dramatic changes do not appear to be accompanied by changes in
the number of MyHC-positive cells. Muscle cells expressing MyHC were counted
12 hours after treatment, at which time the effects of SB202190 became visibly
apparent, but not to the extent to which aggregation and fusion of myocytes
makes the counting of individual cells impossible. There was no significant
difference in the number of MyHC-positive cells between SB202190-treated and
untreated cells (data not shown). Thus, the enhanced muscle formation is
probably not due to increased proliferation of myogenic precursors or of
terminally differentiated myocyte, but rather the advanced progression of
pre-existing myocytes. p38 inhibition in these cultures may therefore advance
the post-differentiation stages of muscle formation by facilitating the
polarization, fusion and aggregation of myocytes.
|
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Discussion |
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Importance of the extracellular environment in myogenesis
The ability of p38 inhibition to activate myogenic markers in limb
mesenchymal cultures is surprising given that in a variety of cell types the
exact opposite was found. Moreover, here we demonstrate activation of MEF2A
and C by p38 inhibition, despite the well-documented phosphorylation and
subsequent activation of these factors by p38 in other systems
(Yang et al., 1999;
Zetser et al., 1999
;
Zhao et al., 1999
). Thus, the
role of p38 signaling in activating the myogenic program within the primary
cultures used here seems exactly opposite to that described for other
populations of homogeneous myogenic cells. Given the ability of SB202190 to
enhance muscle formation of G8 and C2C12 cells only after they are co-cultured
with the primary limb mesenchymal cultures, it appears to be important to
study myogenesis in the context of other factors that are non-myogenic in
origin. A major difference between the primary limb mesenchymal cultures and
the C2C12 and G8 clonal populations is the heterogeneity of the primary
cultures, resulting in the production of a number of factors by non-myogenic
cells that are absent from clonal populations but probably important for
myogenesis. The contribution of signals from non-myogenic cells is well
documented and includes such factors as WNTs, sonic hedgehog (SHH), fibroblast
growth factors (FGFs) and BMPs (reviewed in
Blagden and Hughes, 1999
).
In general, limb development relies on the concerted action of multiple
factors secreted from local signaling centers to direct the commitment and
differentiation of precursor cells. This is especially true for myogenic cells
since, despite their somitic origin, limb muscle progenitors enter the
skeletal myogenic program only after they reach the limb bud and are under the
influence of local extrinsic factors that control the specification,
differentiation and patterning of these cells. The importance of the local
cellular environment in mediating muscle development and patterning in the
limb was recently further highlighted in a study that utilized retroviral
vectors to analyze the fate of somitic-derived myogenic precursors in the
chick (Kardon et al., 2002).
To study myogenesis in the context of embryonic development, heterogeneous
limb mesenchymal cultures may provide a more relevant in vitro model system,
in that they contain factors that are normally present during in vivo
development of muscle.
A potential dual role for p38 in myogenesis
Despite the differences between our culture system and those used by
others, the opposing effects seen in response to p38 inhibition may be
explained by a dual role for p38 signaling at distinct stages of myogenesis,
much like the factor MyoD was recently shown to regulate discrete subprograms
of gene expression during muscle formation
(Bergstrom et al., 2002). It is
interesting to note that the same study reveals only a subset of
MyoD-regulated genes required p38 kinase activity. Thus, the possibility that
p38 signaling differentially modulates subsets of the myogenic program is
worth pursuing. Our analysis thus far has focused on the effects of SB202190
on differentiated muscle cells (e.g. cells already expressing MyHC) and thus
do not preclude a possible requirement for p38 signaling in steps leading up
to myocyte formation.
It is possible that p38 signaling is required early on to induce myocyte differentiation, but also acts to prevent the premature progression of those cells. In this context, p38 signaling would be active during the differentiation stage, but would subsequently be suppressed for those differentiated cells to elongate, polarize, aggregate, and fuse. Very little is currently known about these later stages of muscle development. Given the dramatic and rapid phenotypic changes observed after addition of SB202190, it seems likely that p38 is important in these post-differentiation events. Further study of the phenotypic changes caused by SB202190 would improve our current understanding of the mechanisms of polarization, aggregation and fusion of myocytes. In this respect, treatment of the limb mesenchymal cultures with p38 inhibitors provides an excellent model system for characterizing these aspects of myogenesis.
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Acknowledgments |
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Footnotes |
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References |
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Bader, D., Masaki, T. and Fischman, D. A. (1982). Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro. J. Cell Biol. 95,763 -770.[Abstract]
Bergstrom, D. A., Penn, B. H., Strand, A., Perry, R. L., Rudnicki, M. A. and Tapscott, S. J. (2002). Promoter-specific regulation of MyoD binding and signal transduction cooperate to pattern gene expression. Mol. Cell 9,587 -600.[Medline]
Blagden, C. S. and Hughes, S. M. (1999). Extrinsic influences on limb muscle organisation. Cell Tissue Res. 296,141 -150.[CrossRef][Medline]
Braun, T., Buschhausen-Denker, G., Bober, E., Tannich, E. and Arnold, H. H. (1989). A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. EMBO J. 8,701 -709.[Abstract]
Braun, T., Bober, E., Winter, B., Rosenthal, N. and Arnold, H. H. (1990). Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12. EMBO J. 9,821 -831.[Abstract]
Cabane, C., Englaro, W., Yeow, K., Ragno, M. and Derijard,
B. (2003). Regulation of C2C12 myogenic terminal
differentiation by MKK3/p38alpha pathway. Am. J. Physiol. Cell
Physiol. 284,C658
-C666.
Cash, D. E., Bock, C. B., Schughart, K., Linney, E. and
Underhill, T. M. (1997). Retinoic acid receptor alpha
function in vertebrate limb skeletogenesis: a modulator of chondrogenesis.
J. Cell Biol. 136,445
-457.
Chang, L. and Karin, M. (2001). Mammalian MAP kinase signalling cascades. Nature 410, 37-40.[CrossRef][Medline]
Christian, C. N., Nelson, P. G., Peacock, J. and Nirenberg, M. (1977). Synapse formation between two clonal cell lines. Science 196,995 -998.[Medline]
Conejo, R. and Lorenzo, M. (2001). Insulin signaling leading to proliferation, survival, and membrane ruffling in C2C12 myoblasts. J. Cell Physiol. 187,96 -108.[CrossRef][Medline]
Cuenda, A. and Cohen, P. (1999).
Stress-activated protein kinase-2/p38 and a rapamycin-sensitive pathway are
required for C2C12 myogenesis. J. Biol. Chem.
274,4341
-4346.
Davies, S. P., Reddy, H., Caivano, M. and Cohen, P. (2000). Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351,95 -105.[CrossRef][Medline]
Davis, R. L., Weintraub, H. and Lassar, A. B. (1987). Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell 51,987 -1000.[Medline]
Edmondson, D. G. and Olson, E. N. (1989). A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev. 3,628 -640.[Abstract]
Gallea, S., Lallemand, F., Atfi, A., Rawadi, G., Ramez, V., Spinella-Jaegle, S., Kawai, S., Faucheu, C., Huet, L., Baron, R. et al. (2001). Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells. Bone 28,491 -498.[CrossRef][Medline]
Gossett, L. A., Kelvin, D. J., Sternberg, E. A. and Olson, E. N. (1989). A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol. Cell Biol. 9,5022 -5033.[Medline]
Kardon, G., Campbell, J. K. and Tabin, C. (2002). Local extrinsic signals determine muscle and endothelial cell fate and patterning in the vertebrate limb. Dev. Cell 3,533 -545.[Medline]
Kaushal, S., Schneider, J. W., Nadal-Ginard, B. and Mahdavi, V. (1994). Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD. Science 266,1236 -1240.[Medline]
Kyriakis, J. M. and Avruch, J. (2001).
Mammalian mitogen-activated protein kinase signal transduction pathways
activated by stress and inflammation. Physiol. Rev.
81,807
-869.
Lassar, A. B., Buskin, J. N., Lockshon, D., Davis, R. L., Apone, S., Hauschka, S. D. and Weintraub, H. (1989). MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell 58,823 -831.[Medline]
Lefebvre, V., Zhou, G., Mukhopadhyay, K., Smith, C. N., Zhang, Z., Eberspaecher, H., Zhou, X., Sinha, S., Maity, S. N. and de Crombrugghe, B. (1996). An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes. Mol. Cell Biol. 16,4512 -4523.[Abstract]
Li, Y., Jiang, B., Ensign, W. Y., Vogt, P. K. and Han, J. (2000). Myogenic differentiation requires signalling through both phosphatidylinositol 3-kinase and p38 MAP kinase. Cell Signal. 12,751 -757.[CrossRef][Medline]
Martin, P. (1990). Tissue patterning in the developing mouse limb. Int. J. Dev. Biol. 34,323 -336.[Medline]
Miner, J. H. and Wold, B. (1990). Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc. Natl. Acad. Sci. USA 87,1089 -1093.[Abstract]
Molkentin, J. D., Black, B. L., Martin, J. F. and Olson, E. N. (1995). Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell 83,1125 -1136.[Medline]
Obata, T., Brown, G. E. and Yaffe, M. B. (2000). MAP kinase pathways activated by stress: the p38 MAPK pathway. Crit. Care Med. 28,N67 -N77.[Medline]
Pearson, G., Robinson, F., Beers Gibson, T., Xu, B. E.,
Karandikar, M., Berman, K. and Cobb, M. H. (2001).
Mitogen-activated protein (MAP) kinase pathways: regulation and physiological
functions. Endocr. Rev.
22,153
-183.
Perry, R. L. and Rudnicki, M. A. (2000). Molecular mechanisms regulating myogenic determination and differentiation. Front Biosci. 5,D750 -D767.[Medline]
Puri, P. L., Wu, Z., Zhang, P., Wood, L. D., Bhakta, K. S., Han,
J., Feramisco, J. R., Karin, M. and Wang, J. Y. (2000).
Induction of terminal differentiation by constitutive activation of p38 MAP
kinase in human rhabdomyosarcoma cells. Genes Dev.
14,574
-584.
Rhodes, S. J. and Konieczny, S. F. (1989). Identification of MRF4: a new member of the muscle regulatory factor gene family. Genes Dev. 3,2050 -2061.[Abstract]
Ridgeway, A. G., Wilton, S. and Skerjanc, I. S.
(2000). Myocyte enhancer factor 2C and myogenin up-regulate each
other's expression and induce the development of skeletal muscle in P19 cells.
J. Biol. Chem. 275,41
-46.
Wang, X. Z. and Ron, D. (1996). Stress-induced phosphorylation and activation of the transcription factor CHOP (GADD153) by p38 MAP Kinase. Science 272,1347 -1349.[Abstract]
Weston, A. D., Rosen, V., Chandraratna, R. A. and Underhill, T.
M. (2000). Regulation of skeletal progenitor differentiation
by the BMP and retinoid signaling pathways. J. Cell
Biol. 148,679
-690.
Weston, A. D., Chandraratna, R. A., Torchia, J. and Underhill,
T. M. (2002). Requirement for RAR-mediated gene repression in
skeletal progenitor differentiation. J. Cell Biol.
158, 39-51.
Wright, W. E., Sassoon, D. A. and Lin, V. K. (1989). Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell 56,607 -617.[Medline]
Wu, Z., Woodring, P. J., Bhakta, K. S., Tamura, K., Wen, F.,
Feramisco, J. R., Karin, M., Wang, J. Y. and Puri, P. L.
(2000). p38 and extracellular signal-regulated kinases regulate
the myogenic program at multiple steps. Mol. Cell.
Biol. 20,3951
-3964.
Yaffe, D. and Saxel, O. (1977). Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270,725 -727.[Medline]
Yang, S. H., Galanis, A. and Sharrocks, A. D.
(1999). Targeting of p38 mitogen-activated protein kinases to
MEF2 transcription factors. Mol. Cell. Biol.
19,4028
-4038.
Yee, S. P. and Rigby, P. W. (1993). The regulation of myogenin gene expression during the embryonic development of the mouse. Genes Dev. 7,1277 -1289.[Abstract]
Zetser, A., Gredinger, E. and Bengal, E.
(1999). p38 mitogen-activated protein kinase pathway promotes
skeletal muscle differentiation. Participation of the Mef2c transcription
factor. J. Biol. Chem.
274,5193
-5200.
Zhao, M., New, L., Kravchenko, V. V., Kato, Y., Gram, H., di
Padova, F., Olson, E. N., Ulevitch, R. J. and Han, J. (1999).
Regulation of the MEF2 family of transcription factors by p38. Mol.
Cell. Biol. 19,21
-30.