(Received for publication, December 22, 1995; and in revised form, January 25, 1996)
From the
Recent studies indicate that insulin-like growth factor-II (IGF-II) acts as an autocrine differentiation factor for skeletal myoblasts in culture. IGF-II mRNA and protein are induced as early events in muscle differentiation, and the rate and extent of IGF-II secretion correlate with both biochemical and morphological differentiation. Here we show that IGF-II also functions as an essential survival factor during the transition from proliferating to differentiating myoblasts. Stably transfected C2 muscle cell lines were established in which a mouse IGF-II cDNA was expressed in the antisense orientation relative to the constitutively active Moloney sarcoma virus promoter. IGF-II antisense cells proliferated normally in growth medium containing 20% serum but underwent rapid death when placed in low serum differentiation medium. Death was accompanied by characteristic markers of apoptosis with more than 90% of cells showing DNA fragmentation within 12-16 h. Myoblast death was prevented by IGF-I, des [1-3] IGF-I, IGF-II, and insulin with a dose potency consistent with activation of the IGF-I receptor; death also could be blocked by the protein synthesis inhibitor, cycloheximide. Exogenous IGFs additionally stimulated passage through a single cell cycle and subsequently induced terminal differentiation. Cell survival and cell cycle progression also were enhanced by fibroblast growth factor-2 and platelet-derived growth factor-bb, but these peptides did not promote differentiation. Our results define a novel system for studying apoptotic cell death and its prevention by growth factors, underscore the importance of IGF action in minimizing inappropriate cell death, and indicate that shared signal transduction pathways may mediate myoblast survival in vitro.
The traditional view that growth factors inhibit muscle
differentiation (1) has been challenged by recent observations
implicating the insulin-like growth factors (IGF-I and IGF-II) ()in facilitating myoblast differentiation in
vitro(2, 3, 4, 5, 6, 7) ,
in enhancing muscle growth and regeneration in
vivo(8, 9, 10) , and in modulating
muscle mass during fetal development(11, 12) . The two
IGFs comprise a pair of circulating peptides that are related to each
other and to insulin (13) . IGF action is initiated by binding
to the IGF-I receptor (14, 15, 16) , a
heterotetrameric transmembrane protein that is both structurally
similar to the insulin receptor, and uses many of the same
intracellular signaling pathways(16, 17) . IGF action
also is modified by IGF binding proteins (IGFBPs), a family of secreted
proteins that bind both IGF-I and IGF-II with high
affinity(18, 19) . In addition, a number of studies
have indicated that the IGF-II receptor, a single-chain transmembrane
glycoprotein also known as the cation-independent mannose 6-phosphate
receptor and involved in transport of lysosomal enzymes(20) ,
modulates IGF-II action by removing the growth factor from the
extracellular
environment(14, 20, 21, 22) .
In previous studies, we and others found that IGF-II is produced by skeletal myoblasts as an early event in their terminal differentiation (2, 23, 24, 25) and presented evidence implicating IGF-II as an autocrine differentiation factor(2) . Through use of stable C2 cell lines generated to express an IGF-II cDNA in the antisense orientation, we now show that endogenous IGF-II also functions as a critical survival factor during the transition from proliferating to differentiating myoblasts. We have identified IGF-II antisense clones that undergo rapid apoptotic cell death when incubated in low serum differentiation medium. Cell death could be blocked by des [1-3] IGF-I, IGF-I, IGF-II, or insulin with a dose potency appropriate for activation of the IGF-I receptor and also could be prevented by addition of FGF-2 or PDGF-bb to differentiation medium. Our observations thus define a novel autocrine survival role for IGF-II as an early event in muscle differentiation and indicate that shared growth factor signaling pathways may mediate myoblast survival in vitro.
Figure 1:
Schematic
representation of the pEMSVscribe2/IGF-II antisense expression
plasmid. The plasmid was constructed as described under
``Experimental Procedures.'' The box represents
IGF-II sequences, with the coding region indicated by the hatched
box. ATG and TGA codons, the Moloney sarcoma virus promoter (MSV LTR), and simian virus 40 (SV40) polyadenylation
sequences are marked by arrows.
Figure 2:
Detection of IGF-II antisense mRNA in
myoblasts stably transfected with an antisense IGF-II cDNA. A,
the autoradiograph shows results of a ribonuclease protection
experiment performed using total cellular RNA (10 µg/lane) isolated
from confluent C2 myoblasts and C2 cells transfected with an IGF-II
cDNA (IGF-II antisense (AS) lines 3, 12, and 15) or the empty
expression vector (V) and a sense P-labeled
single-stranded RNA probe generated from a mouse IGF-II cDNA
(diagrammed below). Migration of undigested probe and protected
transgene-derived transcripts are indicated by arrows on the left-hand side of the figure. Autoradiographic exposure was
for 16 h at -80 °C with intensifying screens. B,
ethidium bromide stained gel of RNA used in A. C,
schematic representation of the riboprobe.
In C2 cells and other myoblast
lines, IGF-II secretion accompanies
differentiation(2, 23, 24) . To verify the
effectiveness of the antisense transgene in blunting IGF-II expression,
IGF-II levels were measured by radioimmunoassay in conditioned
differentiation medium from both cell lines. The values were
consistently less than or equal to levels found in nonconditioned
medium (1.2 ± 0.2 nM), thus indicating that the
antisense approach was successful in inhibiting growth factor
expression.
Both antisense lines displayed a rapid decline in the
number of adherent, viable myoblasts following transfer into
differentiation medium. Only 50% of cells from antisense line 12
remained attached to the culture dish by 24 h, and only 15% remained by
72 h. A slower fall in cell number was seen with line 3 (Fig. 3). For both lines, cell death was comparable when
myoblasts were incubated in differentiation medium containing
0-2% horse serum. Nuclear staining using the dye
bisbenzamide showed many cells with condensed nuclei, and analysis of
chromosomal DNA extracted from detached cells revealed DNA laddering,
indicating that IGF-II antisense cells were undergoing apoptotic cell
death when incubated in differentiation medium.
In
addition, detached cells were incapable of reattaching to culture
dishes when incubated in growth medium containing 20% serum.
Figure 3: Premature death in myoblasts stably transfected with an IGF-II antisense cDNA. Cell counts were obtained on trypsinized cells or on bisbenzamide stained nuclei of adherent myoblasts from antisense lines 12 (top panel) or 3 (bottom panel) as described under ``Experimental Procedures.'' The results are shown as the means ± S.E. of a minimum of five assays.
Figure 4: Prevention of cell death by added IGF-I or cycloheximide in myoblasts stably transfected with an IGF-II antisense cDNA. A, photomicrographs of IGF-II antisense myoblasts at time of transfer into differentiation medium (t0) or 24 h later (t24), after incubation with added IGF-I (25 nM), cycloheximide (1 µg/ml), or no addition. The results are representative of a minimum of three experiments. B, time course of cell death and its prevention by IGF-I or cycloheximide. Myoblasts from antisense lines 12 (top panel) or 3 (bottom panel) were treated as in A. Cells were trypsinized and counted by hemocytometer. The results are shown as the means ± S.E. of a minimum of five assays. In the absence of error bars, the results are the means of duplicate assays.
Treatment of IGF-II antisense
12 cells with IGF-I also prevented DNA fragmentation. As assessed by
TUNEL assay (Fig. 5A), over 90% of untreated antisense
12 myoblasts incorporated biotinylated dUTP into their nuclei following
12 h in differentiation medium, whereas less than 10% of IGF-I-treated
cells were labeled. A similar decline in the number of labeled nuclei
was seen when antisense cells were treated with cycloheximide. FACS analysis performed on antisense 12 cells treated with IGF-I
or cycloheximide confirmed results obtained with the TUNEL assay.
Untreated antisense cells showed a marked increase in the percentage of
cells showing DNA fragmentation, from
30% at 4 h to
95% at 16
h. By contrast, only 15-25% of cycloheximide treated cells
displayed DNA fragmentation during the same intervals, whereas fewer
than 10% of IGF-I treated cells had a similar pre-G
apoptotic peak (Fig. 5B).
Figure 5:
Prevention of DNA fragmentation by IGF-I
in myoblasts stably transfected with an IGF-II antisense cDNA. A, DNA fragmentation in IGF-II antisense 12 myoblasts was
assessed in the presence or the absence of 25 nM IGF-I by
TUNEL assay as described under ``Experimental Procedures.'' B, DNA fragmentation was analyzed at 4-h intervals over 16 h
in the presence of IGF-I (25 nM), cycloheximide (1 µg/ml),
or no addition by FACS scanning as described under ``Experimental
Procedures.'' The peaks in each of the panels in
the top part of the figure represent (from left to right, respectively) DNA in the pre-G,
G
/G
, and G
/M phases of the cell
cycle. The results are presented as a representative plot (12-h time
point) from each treatment (note the different y axes) or
summarized by showing the percentage of cells at different time points
with fragmented DNA (the pre-G
peak). The population pool
was
10,000 cells for each time point and was taken from a sample
of 1-5
10
cells.
Figure 6: Enhanced DNA synthesis in IGF-I-treated IGF-II antisense myoblasts. Cell cycle progression into S phase was assessed by BrdUrd incorporation into DNA of IGF-I-treated IGF-II antisense myoblasts as described under ``Experimental Procedures.'' A, results of a representative experiment were photographed. B, incorporation of BrdUrd into DNA was assessed at 4-h intervals by counting labeled nuclei. The data are expressed as the percentages of total cells incorporating BrdUrd.
Figure 7: IGF-I, IGF-II, des [1-3] IGF-I, and insulin are capable of preventing cell death in IGF-II antisense myoblasts. Adherent (top panel) or detached (bottom panel) cells from antisense line 12 were counted by hemocytometer following a 24-h treatment in differentiation medium containing no addition, 0.1-4 nM of des [1-3] IGF-I, 1.4-35 nM of IGF-I, 1.3-70 nM of IGF-II, or 16-16,000 nM of insulin. The results are shown of a single assay. All samples were measured in duplicate. This experiment was performed twice with comparable results.
Figure 8: PDGF-bb and FGF-2 prevent cell death in myoblasts stably transfected with an IGF-II antisense cDNA. Myoblasts from antisense line 12 were transferred into differentiation medium containing no addition (Dif Med), IGF-I (25 nM), FGF-2 (10 or 30 ng/ml), PDGF-bb (2 or 10 ng/ml), or EGF (5 or 50 ng/ml). After 24 h, both adherent (top panel) and detached (bottom panel) cells were counted by hemocytometer. The results are shown as the means ± S.E. of a minimum of three assays.
Figure 9: PDGF-bb and FGF-2 stimulate DNA synthesis in IGF-II antisense myoblasts. Cell cycle progression into S phase was assessed at 8-12 h (top panel) and 20-24 h (bottom panel) of growth factor treatment by BrdUrd incorporation into DNA as described under ``Experimental Procedures.'' The data are expressed as the percentages of total cells incorporating the label.
As seen in Fig. 10, treatment of
antisense 12 cells with a single dose of IGF-I at the onset of
incubation in differentiation medium resulted in measurable creatine
kinase activity by 72 h. Creatine kinase values of 2500
milliunits/mg of total protein were obtained, similar to those seen in
nontransfected and nontreated differentiating C2 cells at 72 h.
By contrast, enzymatic activity in FGF-2- or PDGF-bb-treated
antisense myoblasts was at least 20-fold lower and was equivalent to
values seen when C2 cells were incubated in growth medium.
In addition, myotubes were seen only in IGF-I-treated
cells.
Figure 10: PDGF-bb and FGF-2 do not induce creatine kinase enzymatic activity in IGF-II antisense myoblasts. Cytoplasmic protein extracts from IGF-I-, FGF-2-, or PDGF-bb-treated myoblasts were analyzed after a 72-h incubation for creatine kinase activity as described under ``Experimental Procedures.'' The results are expressed relative to total protein concentration. The means ± S.E. of a minimum of three experiments are illustrated.
Previous studies have documented roles for IGF-I and IGF-II in stimulating myoblast proliferation and differentiation in cell culture (reviewed in (3) ) and in enhancing muscle mass in vivo(10, 11, 12) . Cultured myoblasts have been shown to express IGF-II mRNA and protein as an early event in differentiation(2, 23, 24, 25) , and several lines of evidence have implicated IGF-II as an autocrine differentiation factor(2, 38) . In this report, we show that IGF-II also acts as an autocrine survival factor for myoblasts during the transition from proliferating to differentiating cells. By neutralizing IGF-II expression through stable transfection of C2 myoblasts with an expression plasmid containing a mouse IGF-II cDNA in the antisense orientation, we have identified cell lines that undergo rapid apoptotic cell death when cultured in low serum differentiation medium. Myoblast death could be prevented by the addition of IGF-I, des [1-3] IGF-I, IGF-II, or insulin to the medium with a dose potency consistent with activation of the IGF-I receptor. Cell death also could be blocked by FGF-2 and PDGF-bb, indicating that shared growth factor signaling pathways may mediate myoblast survival in this system.
In previous studies using
antisense oligonucleotides to IGF-II mRNA, we found that IGF-II was
needed for terminal differentiation of C2 cells but did not appear to
be required for cell survival(2) . This apparent discrepancy
between past and current observations may reflect differences in
experimental design. Because the oligonucleotides were added to the
incubation medium at the onset of differentiation, it is possible that
some IGF-II mRNA and protein were produced prior to inhibition of gene
expression. In other experiments, Montarras et al.(38) generated C2 cell lines expressing an antisense IGF
cDNA and showed that these cells differentiated poorly, again
confirming the role of endogenous IGF-II in myoblast differentiation.
Similarly, we have identified IGF-II antisense lines that survive in
low serum differentiation medium but do not differentiate, and
preliminary experiments suggest that these cells maintain low level
secretion of IGF-II. Taken together, these results indicate
that IGF-II is required for C2 cell survival in the absence of other
growth factors.
IGF-mediated myoblast survival was accompanied by stimulation of cell proliferation, as indicated by enhanced entry into S phase of the cell cycle and by increased cell number. This proliferative effect appeared to be limited to progression through a single cell cycle, because the fraction of myoblasts in S phase as measured by incorporation of BrdUrd into DNA over 4-h intervals dropped precipitously after 16-20 h and total cell number rose only by a factor of two. Longer incubations with IGF-I led to induction of myoblast differentiation, so that by 72 h myotubes were evident, and creatine kinase activity was increased. This temporal progression from proliferating to terminally differentiated myoblasts has been described previously for rat L6E9 and L6A1 cells treated with IGF-I or IGF-II(5, 39, 40) , although in the latter cell line it has been suggested that IGF-II only stimulates differentiation, whereas IGF-I promotes both replication and differentiation(40) . By contrast, in C2 myoblasts, we found that des [1-3] IGF-I, IGF-I, IGF-II, and insulin all could promote passage through one cell cycle and subsequent differentiation, with a dose potency reflecting affinity for the IGF-I receptor. Because des [1-3] IGF-I binds poorly to IGFBP-5, the single IGFBP produced by C2 myoblasts(41) , our results additionally support a role for this IGFBP in inhibiting IGF action in muscle cells. The differences between our observations and those of Ewton et al.(40) thus may indicate variability in IGF-I receptor number or in types of IGFBPs expressed by C2 and L6A1 cells, respectively.
IGF-I and IGF-II have been shown to function as survival factors for several other cell types(42, 43) . In cultured cerebellar granular neurons, IGF-I prevented cell death induced by low levels of potassium (42) . Other growth factors, including FGF-2 and PDGF, were ineffective (42) , in contrast to our results with C2 myoblasts. IGF-II has been identified as the growth factor required for full tumorigenesis in transgenic mice expressing simian virus 40 T antigen in the islets of Langerhans(44) . In the absence of IGF-II action, these cells show an enhanced rate of death, and tumor formation is reduced(44) . IGF-I and PDGF have been found to blunt apoptosis induced by c-Myc in serum-deprived fibroblasts, an effect that does not require cell cycle progression or ongoing protein synthesis(45) . In other experiments, IGF-I and the IGF-I receptor were shown to be required for survival of cultured hematopoietic cells after trophic factor withdrawal (46) to prevent apoptosis in fibroblasts exposed to the topoisomerase inhibitor, etoposide(45, 47) , and to block the death of a variety of tumor cell lines cultured for short term in vivo(43, 48) . One general conclusion that emerges from these various observations is that IGF action can prevent the premature death of many cell types, a conclusion supported by the marked cellular hypoplasia in tissues of mice lacking a functioning IGF-I receptor(12) .
Recent observations have indicated a
role for phosphatidylinositol 3-kinase in modulating prevention of
apoptosis by growth factors(49) . In the PC12 pheochromocytoma
cell line, the effects of nerve growth factor, EGF, and insulin on cell
survival were abrogated by wortmannin and LY294002(49) ,
inhibitors of the catalytic subunit of this enzyme (50) . In
agreement with these studies, we have found in preliminary experiments
that these agents promote rapid myoblast death even in the presence of
IGF-I but do not block cell survival mediated by cycloheximide. Because in other cell types, phosphatidylinositol 3-kinase has
been implicated in the regulation of mitogenesis(51) , it is
possible that both the anti-apoptotic and proliferative actions of
IGF-I require the same signaling pathway.
In summary, we have identified a new autocrine role for IGF-II in facilitating the transition from proliferating to terminally differentiated myoblasts in vitro by preventing inappropriate cell death. Because it has been shown recently that IGF-II can block the rapid death of primary skeletal myoblasts isolated from mice with muscular dystrophy(52) , elucidation of the signal transduction pathways responsible for these actions may have important clinical implications.