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Geriatric Research, Education, and Clinical Center, Veterans Administration Puget Sound Health Care System, Tacoma, Washington 98493; and the Division of Gerontology and Geriatric Medicine, Departments of Medicine (K.L.H., L.S.Q.) and Pathology (H.M.P.W., K.S.), University of Washington, Seattle, Washington 98195
Address all correspondence and requests for reprints to: LeBris S. Quinn, Ph.D., 151 Veterans Administration Puget Sound, American Lake, Tacoma, Washington 98493. E-mail: quinnl{at}u.washington.edu
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Introduction |
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Primary cultures from developing skeletal muscle as well as cultured myogenic cell lines such as rat L6 and mouse C2 undergo proliferation and differentiation similar to those observed in vivo, allowing analysis of the molecular mechanisms controlling myogenesis (7, 12, 13). Such studies have generally indicated that mitogenic growth factors such as fibroblast growth factor-2 repress differentiation by inhibiting both the expression and DNA-binding activity of muscle-specific basic helix-loop-helix transcription factors such as MyoD1 and myogenin (7, 12, 13). Conversely, these muscle regulatory factors repress proliferation by effects on cell cycle regulatory molecules, such as induction of the cyclin-dependent kinase inhibitor p21 (12, 14, 15). Thus, although myoblast proliferation and differentiation are both necessary for myogenesis, these are intrinsically opposing pathways in a single myoblast, as most factors that stimulate proliferation inhibit differentiation and vice versa (12, 13). However, IGF-I and IGF-II are unique among growth factors in that they stimulate both proliferation and differentiation in myoblasts (13). A number of studies have shown that both responses are mediated by the same receptor, the type 1 IGF receptor (IGF-1R) (13, 16, 17, 18). Although somewhat divergent postreceptor signaling pathways for myoblast differentiation and proliferation are beginning to be dissected (13, 19, 20, 21, 22, 23, 24), the question of what determines whether myoblasts respond to the IGFs by proliferating or differentiating (i.e. which of these signal transduction pathways will predominate) still remains.
One hypothesis that has been advanced is that myoblast responses to the IGFs are determined by the timing of the IGF stimulus (13, 25, 26). In both mouse C2 and rat L6 myogenic cultures, IGF exposure early in culture (within 24 h of a switch to low serum medium) evokes proliferation responses, whereas IGF exposure at later time points evokes a differentiation response (13, 25, 26). However, the molecular basis of this differential response is unclear.
In culture, the pattern of IGF-binding protein (IGFBP) expression changes during the course of myogenic differentiation from high IGFBP-4 levels during the proliferative phase to high IGFBP-5 levels during differentiation, which suggested that these molecules could play a role in specifying the nature of the response to the IGFs (13, 27, 28). However, functional studies of IGFBP-4 and -5 in myogenesis have indicated that these molecules are generally inhibitory to both IGF-mediated proliferation and differentiation (28, 29, 30, 31, 32) and therefore do not support the concept that they are involved in determining the nature of the response to IGF by myoblasts.
In a previous publication (33), we characterized the expression of IGFBP-related protein 1 (IGFBP-rP1) during myogenesis in vitro. IGFBP-rP1 is a member of a newly described family of secreted cellular regulators (IGFBP-rP-1 to -4) that contain significant N-terminal homology to the IGFBPs, but lack the C-terminal region that is homologous among IGFBP-1 to -6 (34, 35). IGFBP-rP1 binds insulin and the IGFs at approximately 5- to 25-fold lower affinity than that of the IGFBPs for the IGFs (36, 37). However, it is unclear whether this binding is involved in the actions of IGFBP-rP1 on cellular processes (34, 36). During differentiation of C2 myoblasts, IGFBP-rP1 messenger RNA (mRNA) is highly expressed during the first 24 h after a switch to low serum medium, but expression declines rapidly thereafter, slightly before the onset of differentiation (33). Moreover, transforming growth factor-ß (TGFß), an inhibitor of myoblast differentiation, stimulates and prolongs the expression of IGFBP-rP1 (33). This pattern of expression suggested that IGFBP-rP1 may be involved in stimulating myoblast proliferation responses to the IGFs or in repressing myoblast differentiation responses to the IGFs. In the present study we overexpressed IGFBP-rP1 in C2 myoblasts using a retroviral expression vector to determine the role of this molecule in myoblast proliferation and differentiation responses to the IGFs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vector construction and preparation of cell populations
The full-length human mac25/IGFBP-rP1 cDNA (
874 bp), was
ligated into the BamHI site of the Moloney murine leukemia
virus-based retroviral plasmid pLXSN (40). The resulting pLrP1SN
plasmid expressed IGFBP-rP1 from the viral long terminal repeat, and
G418 resistance was conferred by the neomycin phosphotransferase gene
controlled by an internal simian virus 40 promoter (Fig. 1
). Both pLrP1SN and pLXSN (control) plasmids
were transiently transfected into the ecotropic packaging cell line,
PE501 (40); media from the transfected PE501 cells were used to infect
amphotropic PA317 packaging cells (41), which were cultured in 10%
FCS/DMEM plus the selection agent G418 (Geneticin, Life Technologies, Inc., Grand Island, NY) at 1.0 mg/ml and stored
frozen in 10%FCS/DMEM containing 10% dimethylsulfoxide in liquid
N2. Culture media from each set of PA317 cells,
containing replication-deficient retroviruses carrying the LrP1SN and
LXSN vectors, were used to infect mouse C2 myoblasts, to produce C2-rP1
and C2-LX myoblast populations. Viruses were added to C2 myoblasts in
10%FCS/DMEM at a 1:1 ratio; 24 h later, C2 myoblasts were
administered the selection agent, G418, at 1 mg/ml. C2 myoblasts were
maintained and passaged in these conditions until all myoblasts in
sister cultures that did not receive virus were eliminated. The
efficiency of infection by both vectors was approximately 75% of the
initial C2 myoblast population. C2-LX and C2-rP1 myoblasts were
maintained as nonclonal cell populations and stored frozen in aliquots
in liquid N2 as described above for experimental
analyses.
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-32P]deoxy-CTP
(NEN-DuPont) using a random priming kit (Prime-a-Gene, Promega Corp., Madison, WI). Membranes were hybridized with the
radiolabeled probes for 16 h at 42 C, then washed for 30 min in
2 x SSC at room temperature, for 30 min in 2 x SSC and 1%
SDS at room temperature, and stringently washed at 60 C in 0.2% SSC
and 1% SDS for 30 min. Blots were exposed to Kodak XAR2
film (Eastman Kodak Co., Rochester, NY) for 5 days with
one intensifier screen at -70 C. Bands in the resulting
autoradiographs were quantified using an image analyzer equipped with
MCID version 4.2 software (Imaging Research, Inc., St.
Catherines, Canada). For Western blot analyses of secreted IGFBP-rP1, C2-LX and C2-rP1 myoblasts were plated at 4 x 105 cells/35-mm culture dish (Falcon, Franklin Lakes, NJ) in 10% FCS/DMEM. The next day, cells were rinsed once with DMEM, the medium was replaced with serum-free DMEM, and medium was collected after 24 h. Medium was mixed with 4 x nonreducing SDS sample buffer (for a final concentration of 0.5 M Tris, 1% SDS, and 10% glycerol, pH 6.8) and stored at -20 C. Before electrophoresis, samples were heated to 95 C for 2 min. Twenty-five microliters of each sample were loaded onto 12.5% SDS-PAGE minigels (apparatus from Bio-Rad Laboratories, Inc., Richmond, CA). A positive control for IGFBP-rP1, medium from P69 human prostate epithelial cells (43) as well as mol wt standards (Bio-Rad Laboratories, Inc.) were also loaded onto the gels. After electrophoresis, proteins were transferred to nitrocellulose using a Bio-Rad Laboratories, Inc., blotting apparatus, and stained for IGFBP-rP1 using a rabbit antihuman IGFBP-rP1 antibody (38). Briefly, blots were incubated at room temperature in Tris-buffered saline (TBS; 50 mM Tris and 0.9% saline, pH 7.4) containing 0.05% Tween-20 (TBS-Tw) and 10% H2O2 for 30 min to reduce endogenous peroxidase activity, then blocked overnight at 4 C with TBS-Tw plus 5% nonfat dry milk (Carnation, Solon, OH) and 1% BSA (RIA grade; Sigma). Blots were incubated with rabbit anti-IGFBP-rP1 at 1:2500 in TBS-Tw plus 5% nonfat dry milk for 1 h at room temperature, rinsed three times for 15 min each time with TBS-Tw, then incubated with peroxidase-labeled goat antirabbit IgG (affinity purified; Life Technologies, Inc.) at 1:3000 (1 h, room temperature), and rinsed again as described above. Blots were processed using an enhanced chemiluminescence (ECL) kit from Amersham Pharmacia Biotech (Arlington Heights, IL). Band intensities were quantified by densitometry as described above.
Assays for myoblast differentiation
Western blot and immunocytochemical analyses of MHC expression
were used to quantify terminal differentiation (17). For Western blots,
C2-LX and C2-rP1 myoblasts were plated at confluent densities (5 x
105 cells/35-mm culture dish or
105 cells/well in 24-well plates) in 10%
FCS/DMEM, and 16 h after plating were changed to 0.5% FCS/DMEM
with or without exogenous growth factors, with or without the
mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor PD098059
(New England Biolabs, Inc., Beverly, MA) or with or
without the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor
LY294002 (Calbiochem, San Diego, CA) the following day.
The growth factors used were human recombinant IGF-I and IGF-II (R&D
Systems, Minneapolis, MN), human recombinant insulin
(Sigma), and des (1, 2, 3)IGF-I (DSL, Webster, TX), applied
at the concentrations specified in the figures. Sister cultures were
treated identically and used to quantify DNA content per well in
24-well plates. However, DNA content did not vary appreciably even with
growth factor administration, as cells were plated at confluence. For
Western blot analyses of MHC accretion, at 72 h after the change
to low serum medium (with or without factors or inhibitors) with no
further medium changes, cultures were rinsed three times with DMEM,
then harvested using SDS sample buffer containing 1%
ß-mercaptoethanol. Sister cultures in 24-well plates were rinsed as
described above, fixed with cold 70% ethanol/formalin/acetic acid
(20:2:1), rinsed again, and reacted with 1 µg/ml Hoechst 33258 dye
(bisbenzimide, Sigma) in 1 x SSC for 3 min. The DNA
content per well was quantified using a Wallac, Inc.,
Victor2 fluorescent microplate reader (Gaithersburg, MD). For analysis
of MHC accumulation, samples were heated to 95 C for 2 min and volumes
were normalized to the DNA content of sister cultures; samples were
then run on 7.5% SDS-PAGE gels, blotted onto nitrocellulose, and
processed using ECL as described above. The primary antibody,
monoclonal MF-20 anti-MHC, was used at a 1:20 dilution, and the
secondary antibody, affinity-purified peroxidase-labeled goat antimouse
IgG (Life Technologies, Inc.), was used at a 1:5000
dilution.
In one series of experiments, a Transwell cell culture plate insert system with 0.4-µm pore size (Costar, Cambridge, MA) was used to determine whether IGFBP-rP1 secreted by C2-rP1 myoblasts would affect C2-LX myoblast differentiation. C2-LX or C2-rP1 myoblasts (secreting cells) were plated into the lower chambers of the apparatus at a density of 1.9 x 105 cells/3.8 cm2 in 2 ml 10% FCS/DMEM. C2-LX myoblasts (test cells) were plated at 5 x 104/1.13 cm2 onto the upper chamber of each apparatus. Cultures were changed to 0.5% FCS/DMEM and cultured for 72 h, and test cells in the upper chambers were harvested for Western blot analysis of MHC accumulation as described above.
Immunocytochemical analysis of differentiation was also performed. C2-LX and C2-rP1 myoblasts were plated at 4 x 105 cells/35-mm dish in 10% FCS/DMEM, changed to 0.5% FCS/DMEM with or without 30 ng/ml IGF-I 16 h after plating, and cultured for 72 h. Cultures were rinsed three times, fixed with cold ethanol/formalin/acetic acid, rinsed again with TBS, and blocked with TBS containing 1% normal goat serum (NGS; Life Technologies, Inc.) overnight at 4 C to reduce nonspecific staining. The cultures were stained with MF-20 anti-MHC antibody (1:5 in TBS-NGS, 1 h at room temperature), rinsed three times with TBS-TW, and reacted with affinity-purified goat antimouse IgG labeled with fluorescein isothiocyanate (1:5000 in TBS-NGS, 1 h at room temperature). During the last 10 min of staining with secondary antibody, EtBr for nuclear staining was added at a final concentration of 0.01%. Cultures were rinsed three times, mounted under glass coverslips with glycerol/TBS (9:1), and viewed with epifluorescence optics using a Nikon Optiphot 2 microscope (Tokyo, Japan). MHC staining (green, cytoplasmic) of differentiated mono- and multinucleated muscle cells was easily distinguished from EtBr staining (orange in the fluorescein channel) of all nuclei.
Assays for myoblast proliferation
C2-LX and C2-rP1 myoblast growth rates in 10% FCS/DMEM were
assessed using the Hoechst 33258 DNA fluorescence assay described
above. Cells were plated at 5 x 103
cells/well in 24-well plates, and DNA content per well was assessed
with daily medium changes over 3 days in parallel plates fixed at 24-h
intervals.
To assay proliferative responses to specific growth factors, C2-LX and C2-rP1 myoblasts were plated at subconfluent densities (2.5 x 104 cells/well) in 24-well plates in 10% FCS/DMEM. Sixteen hours after plating, cells were changed to 0.5% FCS/DMEM, with or without IGF-I, IGF-II, insulin, or des(1, 2, 3)IGF-I. Proliferation in response to each of these factors was assessed after 48-h exposure to the factors by two methods. In both techniques, treatments were performed in triplicate in each experiment; the experiments were performed two or three times. DNA content per well was assessed using the Hoechst 33258 fluorescence assay described above and quantified using a fluorescent microplate reader. Additionally, proliferation was assayed using a [3H]thymidine (TdR) incorporation assay. Cells were cultured as described above, and during the last 4 h of culture, [3H]TdR (6.7 Ci/mmol; New England Nuclear, Wilmington, DE) was added to a final concentration of 0.5 µCi/ml. Incorporation of label into DNA was determined using trichloroacetic acid precipitation as described previously (44).
Assay of MAPK phosphorylation
C2-LX and C2rP1 cells were cultured for 72 h in low serum
medium with 0, 5, and 10 µM PD098059, a MAPK kinase (MEK)
inhibitor. Cell lysates were harvested in SDS sample buffer as
described above for differentiation assays. Lysates were resolved on
7.5% SDS-PAGE gels, blotted onto nitrocellulose, and probed using an
anti-phospho-MAPK polyclonal antibody (New England Biolabs, Inc.). Blots were exposed to primary antibody (1:1000)
overnight; secondary antibody (peroxidase-labeled goat antirabbit IgG)
was used at a 1:5000 dilution for 1 h, and blots were visualized
using ECL as described above. Phospho-MAPK control protein (New England Biolabs, Inc.) was run as a standard in phospho-MAPK
blots and comigrated with extracellular signal-regulated kinase-2
(ERK-2) in the experimental lanes at Mr
42,000.
Statistical analysis
The significance of differences between treatments was
determined by Student’s t tests (two-tailed, unpaired).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) revealed
that both control C2-LX myoblasts (infected with the empty vector
coding only for neomycin phosphotransferase) and C2-rP1 myoblasts
(infected with a retroviral expression vector for human IGFBP-rP1)
expressed the endogenous 1.1-kb IGFBP-rP1 transcript. C2-rP1 myoblasts
expressed an additional 4.2-kb IGFBP-rP1 transcript (indicated by the
arrow in Fig. 2) coded by the retroviral expression vector.
The 4.2-kb transcript was expressed at 3 times greater amounts than the
endogenous transcript. Western blot analysis of secreted IGFBP-rP1
present in culture medium collected after 24 h was also used to
compare C2-LX and C2-rP1 myoblasts (Fig. 3).
Immunoreactive IGFBP-rP1 from both populations migrated identically, at
the expected Mr of 31,000. Densitometry revealed
that the signal from C2-rP1 myoblasts was 27 times higher than that
from C2-LX myoblasts, although this should be taken as an
approximation, because the relative immunoreactivity of the antibody
for mouse IGFBP-rP1 (the endogenous protein) vs. the
overexpressed human IGFBP-rP1 is unknown. However, the magnitude of the
estimated overexpression is comparable to that in previous studies
using the LXSN system in C2 myoblasts, in which transgenes expressed by
this vector were measured at 30-fold higher levels than those in
control myoblasts (17).
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). Estimated
doubling times for the two populations were 14.5 ± 1.8 h for
C2-LX myoblasts and 14.8 ± 1.5 h for C2-rP1 myoblasts, which
were not significantly different (by t test) and which are
comparable to that for the nontransduced parental population.
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). As the cells were plated at
confluence, the DNA content did not vary by cell population or by
treatment (not shown). Des(1, 2, 3)IGF-I is an IGF-I analog with markedly
reduced affinity for the IGFBPs, but with close to normal affinity for
the IGF-1R (45). Insulin, which likewise is not bound by IGFBP-1- to 6,
was used at microgram quantities, at which it can bind to the IGF-1R
and elicit proliferation and differentiation responses in myoblasts
(13). These factors were used along with IGF-I and IGF-II to determine
whether IGFBP-rP1 action was similar to that of the IGFBPs. Figure 5
shows that, with the exception of one data point (30 ng/ml IGF-I),
differentiation of C2-rP1 myoblasts at all concentrations of each
factor was significantly reduced (P < 0.05) compared
with that of C2-LX myoblasts. C2-rP1 differentiation levels were also
significantly lower than those of C2-LX myoblasts without the addition
of exogenous IGFs; however, because C2 myoblasts express autocrine
IGF-I and IGF-II (46, 47), a background level of these factors is
present in cultures not exposed to exogenous IGFs. MHC levels in C2-rP1
cultures were approximately half those of C2-LX cultures in all
conditions except des(1, 2, 3)IGF-I exposure, in which C2-rP1 MHC levels
were approximately 75% of the control values (C2-LX). Both C2-LX and
C2-rP1 myoblasts expressed increasing levels of MHC in response to the
addition of each of the factors.
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, markedly lower numbers of differentiated
cells (or nuclei within MHC+ cytoplasm, in the
case of fused cells) appeared in C2-rP1 cultures compared with C2-LX
cultures, with or without 30 ng/ml IGF-I. However, total numbers of
nuclei were similar, and little myoblast or myotube cell death was
observed in either set of cultures.
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). Exposure of the test cells, C2-LX, to
medium from C2-rP1 myoblasts resulted in a 4-fold decrease in MHC
accumulation compared with coculture with C2-LX myoblasts, significant
at P < 0.05 (t test). These findings
indicate that secreted IGFBP-rP1 can act in a paracrine fashion to
inhibit myoblast differentiation.
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). In contrast to the
results comparing differentiation responses, the proliferation
responses of C2-LX and C2-rP1 myoblasts to each of these mitogens over
a 48-h period were not significantly different (by t tests)
with the exception of one data point (100 ng/ml IGF-II measured by
Hoechst fluorescence assay of DNA).
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). In
C2-rP1 cultures, the values for MHC accumulation at 5 and 10
µM PD098059 were not significantly different from the
control (no PD098059) value. In contrast, addition of 5 and 10
µM PD098059 to C2-LX myoblasts caused a progressive
decrease in MHC, which at 10 µM was significantly
different (P < 0.05) from the control value, and
similar to the values for the C2-rP1 myoblasts. PD098059 inhibited ERK
phosphorylation approximately 10-fold in both C2-LX and C2-rP1
myoblasts, as shown by Western blots of cell extracts probed by
anti-phospho-MAPK antibodies (Fig. 9, B and C). In agreement with other
studies using C2 myoblasts (22, 24), in the differentiation-permissive
conditions assayed here, the predominant phosphorylated MAPK species
expressed by both C2-LX and C2-rP1 myoblasts was ERK-2, migrating at
Mr 42,000 (Fig. 9B). Phosphorylated ERK-1,
migrating at Mr 44,000, was a minor species.
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), a figure
similar to the difference in MHC expression without PD098059 between
the two populations in this experiment, in which MHC expression by
C2-rP1 myoblasts was 72% of that of C2-LX myoblasts (Fig. 9A).
However, upon addition of 5 and 10 µM PD098059,
phospho-ERK-2 expression was progressively decreased in both
populations of myoblasts, whereas MHC accretion by C2-rP1 myoblasts did
not decrease in response to PD098059. Also, although PD098959 inhibited
phospho-ERK-2 expression by C2-LX myoblasts approximately 10-fold, MHC
accretion in these cells decreased only 25%. These findings are in
agreement with studies by Sarbassov and Peterson (24), indicating that
inhibition of MAPK phosphorylation results in only partial inhibition
of C2 myoblast differentiation.
Studies by other groups (24) have indicated that the MAPK-dependent
steps in C2 myoblast differentiation are downstream of a PI-3
kinase-dependent step. In agreement with observations by several groups
using a range of myogenic cell types (19, 21, 24), addition of the PI
3-kinase inhibitor LY294002 completely inhibited differentiation of
both C2-LX and C2-rP1 myoblasts (Fig. 9D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retrovirally mediated overexpression of IGFBP-rP1 was chosen in this
study over plasmid transfection, because the extremely high efficiency
of retroviral infection (75% of the initial cell population)
allowed analysis of a representative, nonclonal population and thus
obviated concerns about selection of aberrant cell populations or
subclones. It is unlikely that the retroviral transduction procedure
itself produced spurious results in our study. Control C2-LX cells,
infected with the parental vector devoid of the IGFBP-rP1-coding
sequence, were derived from the same initial population of C2 myoblasts
and selected for antibiotic resistance along with the C2-rP1 cells.
Both lines of myoblasts proliferated at the same rates in high serum
medium, and both lines of myoblasts exhibited similar proliferation
patterns in response to a range of IGF-like mitogens.
It is unclear whether the inhibition of differentiation mediated by IGFBP-rP1 involves binding of IGF and/or insulin. C2-rP1 differentiation levels were significantly lower than those of C2-LX myoblasts both with and without the addition of exogenous IGFs; however, as C2 myoblasts produce endogenous IGF-I and IGF-II (46, 47), it cannot be inferred whether the differentiation-inhibiting effects of IGFBP-rP1 are IGF dependent or IGF independent. Additionally, differentiation of C2-rP1 myoblasts was inhibited compared with that of controls in the presence of both des(1, 2, 3)IGF-I and insulin, IGF-1R-binding ligands for which the IGFBPs have little or no affinity (35, 37, 45). However, as IGFBP-rP1 binds insulin as well as the IGFs (albeit at low affinity) (36, 37, 49), and the affinity of IGFBP-rP1 for des(1, 2, 3)IGF-I has not been determined, it cannot be deduced from these observations whether the mechanism of IGFBP-rP1 action involves binding of the ligands to IGFBP-rP1. Clearly, however, IGFBP-rP1 is not functioning similarly to an IGF- dependent IGFBP, which would be expected to modulate responses to IGF-I and IGF-II, but not to insulin and des(1, 2, 3)IGF-I.
An IGF-independent mechanism of IGFBP-rP1 action is a likely possibility given the rather low affinity of this molecule for insulin and the IGFs (36, 37, 49). Some of the IGFBPs, as well as carboxyl-truncated IGFBP fragments, have been shown to exhibit IGF-independent effects on cells (51, 53). The sequence of IGFBP-rP1 also resembles that of carboxyl-truncated follistatin (52); both intact and truncated follistatin bind the TGFß superfamily member activin (53, 54). Follistatin and a follistatin-like gene have been implicated in early myogenic determination and development (55). TGFß superfamily proteins, in turn, regulate follistatin expression (52, 55). Mutant mice deficient in follistatin exhibit underdeveloped skeletal muscles (56). Conversely, treatment of chick primary myogenic cultures with follistatin potentiated myogenesis and appeared to act via prolongation of myoblast proliferation (57). These observations are consistent with our previous observations (33) that TGFß, an inhibitor of myogenic differentiation, stimulates and prolongs IGFBP-rP1 expression, and with our present findings, which support a role for IGFBP-rP1 in facilitating myoblast proliferation responses.
Several potential mechanisms of IGFBP-rP1 action can be ruled out by our data. Although IGF-stimulated differentiation was depressed by IGFBP-rP1, it cannot be acting by rendering myoblasts insensitive to IGF (i.e. either up-regulating an inhibitory IGFBP or down-regulating the IGF-1R) or by inhibiting endogenous IGF expression, because the IGF dose-response curves for C2-LX and C2-rP1 myoblast proliferation were indistinguishable. IGFBP-rP1 is also unlikely to have suppressed differentiation by altering IGFBP expression, because the differentiation responses to insulin and des(1, 2, 3)IGF-I were suppressed. Finally, IGFBP-rP1 does not appear to inhibit differentiation by up-regulating a proliferation-related pathway, as C2-rP1 myoblast proliferation rates and proliferation responses to specific mitogens were unaffected compared with control myoblasts. Additionally, the MEK inhibitor PD098059, which might be expected to inhibit mitogenic pathways (19), did not rescue differentiation in C2-rP1 myoblasts.
Other groups have shown that IGF-stimulated C2 myoblast differentiation is mediated by both MAPK-dependent and -independent pathways (22, 24). In the current study, we observed that the MEK inhibitor PD098059 reduced the level of C2-LX differentiation, but did not affect C2-rP1 differentiation, suggesting that IGFBP-rP1 acted to inhibit a MAPK-dependent differentiation pathway. C2-rP1 myogenic cultures nevertheless exhibited a low level of differentiation that was stimulated in a dose-dependent manner by the IGFs and insulin. These findings are in agreement with the con-cept that there are MAPK-dependent and -independent postIGF-1R signaling pathways leading to differentiation, and that IGFBP-rP1 specifically inhibited the MAPK-dependent differentiation pathway. IGF-stimulated C2 myoblast proliferation is also mediated by MAPK-dependent and -independent pathways (23). However, it has been unclear what factors determine which of these signaling pathways predominates after activation of the IGF-1R. Our observations suggest that IGFBP-rP1 inhibits the MAPK-dependent differentiation pathway and does not affect MAPK-dependent proliferation pathways, nor does it affect MAPK-dependent pathways that inhibit myoblast differentiation in the presence of high serum and/or mitogens (23).
Our findings suggest that IGFBP-rP1 is an autocrine/paracrine factor expressed by skeletal myoblasts that specifies the proliferative response to the IGFs. Exogenous application of this factor could be of use in extending the myoblast proliferative phase and hence in increasing the amount of muscle produced by a given number of myoblasts. Such modulation could be useful in improving muscle regeneration during aging (4), after injury (3), or in neuromuscular disorders (58), as well as in increasing the efficiency of exercise protocols or meat animal production (5, 8). More work is needed to further elucidate the mechanism of action and patterns of expression of this factor in skeletal muscle development and regeneration.
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Acknowledgments |
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Footnotes |
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Received May 13, 1999.
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