©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin-like Growth Factor-II Is an Autocrine Survival Factor for Differentiating Myoblasts (*)

(Received for publication, December 22, 1995; and in revised form, January 25, 1996)

Claire E. H. Stewart Peter Rotwein (§)

From the Departments of Biochemistry and Molecular Biophysics and Medicine, Washington University School of Medicine, Saint Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Tissue culture supplies, fetal bovine serum, newborn calf serum, horse serum, Dulbecco's modified Eagle's medium, Earle's balanced salt solution, and G418 were purchased from Life Technologies, Inc. Plasmid pEMSV scribealpha2 was a gift from the late Dr. Harold Weintraub (Fred Hutchinson Cancer Center, Seattle, WA), and pSV2neo was from Dr. Paul Berg (Stanford University, Stanford, CA). Recombinant human IGF-I was a gift from Dr. C. A. Morrison (Ciba Geigy, St. Aubin, Switzerland); recombinant human IGF-II and des [1-3] IGF-I were purchased from GroPep (Adelaide, Australia); recombinant human FGF-2, PDGF-bb, and EGF were purchased from U. S. Biochemical Corp.; insulin, cycloheximide, bisbenzamide, creatine kinase assay reagents, and secondary antibodies were purchased from Sigma. BCA protein quantitation reagents were obtained from Pierce. Restriction enzymes, ligases, and polymerases were purchased from U. S. Biochemical Corp., Promega Biotech (Madison, WI), New England Biolabs (Boston, MA), and Perkin-Elmer (Norwalk, CT). Radionuclides ([alpha-P]CTP) were purchased from Amersham Corp. Plasmid purification kits were obtained from Qiagen (Chatsworth, CA). BrdUrd, terminal deoxynucleotidyl transferase, biotin-16-dUTP, and avidin/fluorescein isothiocyanate were from Boehringer Mannheim. Polyclonal antiserum to BrdUrd was a gift from Dr. Steve Cohn (Washington University Medical School, St. Louis, MO), CY-3-conjugated anti-goat antibody was from Jackson Immunochemicals (West Grove, PA), and the antibody to IGF-II was from Amano Enzymes (Troy, VA). Other chemicals were reagent grade and were purchased from commercial suppliers.

Construction of a Mouse IGF-II Antisense Expression Plasmid

An IGF-II cDNA containing the entire coding region was generated by reverse transcriptase polymerase chain reaction using neonatal mouse liver RNA as a template and IGF-II-specific primers containing EcoRI sites at their 5` ends. After validation by DNA sequencing, the purified cDNA insert was ligated into the unique EcoRI site of pEMSV scribealpha2(26) . Plasmids with inserts in the antisense orientation relative to the Moloney sarcoma virus long terminal repeat (see Fig. 1) were characterized by restriction endonuclease mapping and DNA sequencing.


Figure 1: Schematic representation of the pEMSVscribealpha2/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.



Stable Transfection of C2 Myoblasts

C2 cells (27) were plated at 150,000 cells/100-mm-diameter gelatin-coated tissue culture plate. Cells were washed 24 h later and transfected with 5 µg of DNA at a 10:1 molar ratio (pEMSV/mIGF-II:pSV2 neo) by a modified calcium phosphate precipitation procedure(28) . Two days later, cells were washed and split onto three 150-mm diameter dishes in growth medium containing 400 µg/ml of active G418. Selection proceeded for 2 weeks; medium was changed every 3 days. Twenty colonies were transferred by trypsinization to 12-well cluster dishes and expanded. After screening for production of chimeric mRNA, two colonies were selected for further characterization.

Cell Culture

Transfected cells were routinely plated at 100,000 cells/ml on gelatin-coated plates in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated newborn calf serum, 10% heat-inactivated fetal bovine serum, and 400 µg/ml active G418 until 80% confluency was attained. Differentiation was initiated following washing with Earle's balanced salt solution by changing to medium containing Dulbecco's modified Eagle's medium plus 2% horse serum(23) . Adherent cells were counted in a hemocytometer after trypsinization; alternatively, nuclei were counted after cells were fixed and stained with bisbenzamide. The number of detached cells was determined by counting an aliquot of culture medium in a hemocytometer. Cell viability was established by replating detached cells in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated newborn calf serum, 10% heat-inactivated fetal bovine serum, and 400 µg/ml active G418.

RNA Isolation and Analysis

Total RNA was isolated from cells using a modified guanidinium thiocyanate method (29) and quantitated by spectrophotometry. RNA integrity was assessed by electrophoresis through 1% agarose, 2.2 M formaldehyde gels after staining with ethidium bromide. Solution-hybridization ribonuclease protection assays were performed as described previously (30, 31) . A single-stranded [alpha-P]CTP-labeled IGF-II sense riboprobe was synthesized in vitro(32) using a linearized plasmid template and T7 RNA polymerase.

IGF-II Radioimmunoassay

Conditioned differentiation medium (Dulbecco's modified Eagle's medium plus 2% horse serum) was harvested, clarified by low speed centrifugation, and stored at -20 °C until assayed. IGF-II concentrations were determined by radioimmunoassay following acid ethanol cryoprecipitation(33, 34) . Recombinant human IGF-II was used as standard and tracer in an equilibrium assay established with a monoclonal anti-rat IGF-II antibody at 2.5 ng/tube(35) . The antibody shows 100% cross-reactivity with human IGF-II and <10% reactivity with human IGF-I. Maximum binding of added tracer was between 45 and 50% and recovery was >95%.

Creatine Kinase Assay

Cytoplasmic lysates were collected from differentiating cells by incubation with 50 mM Tris-MES, pH 7.8, 1% Triton X-100 for 10 min at 25 °C. Samples were stored at -80 °C and assayed within 1 week of collection using a commercially available kit (Sigma). Enzymatic activity was normalized to total protein content as determined by the BCA protein assay.

TUNEL Assay for DNA Fragmentation

Cells were grown to 80% confluency and induced to differentiate as described. After 12 h, cells were fixed (100% ethanol), and DNA was labeled by treatment with terminal deoxynucleotidyl transferase and biotinylated dUTP followed by incubation with avidin/fluorescein isothiocyanate. If biotinylated dUTP is enzymatically added to available 3`-OH ends of DNA, it is then detected by the addition of avidin/fluorescein isothiocyanate followed by fluorescence microscopy(36) . The results are expressed as the percentage of total cells with fluorescent nuclei.

Analysis of DNA Fragmentation by FACS

Cells were grown to 80% confluency and transferred to differentiation medium in the presence or the absence of 25 nM IGF-I or 1 µg/ml cycloheximide. DNA fragmentation was assessed at 4-h intervals over a 16-h period. After each incubation, cells were trypsinized, resuspended, and washed in phosphate-buffered saline and fixed at -20 °C in 70% ethanol. Cells were pelleted, washed in phosphate-buffered saline, and resuspended with vortexing in propidium iodide labeling buffer (50 µg/ml propidium iodide, 0.1% sodium citrate, 20 µg/ml ribonuclease A, 0.3% Nonidet P-40, pH 8.3) at 1-5 times 10^6 cells/ml. Stained cells were stored in the dark at 4 °C until assayed using a Becton Dickinson FACStar equipped with ``ModFit LT'' cell cycle analysis software (Verity software house). Because fixation of cells in ethanol is insufficient to preserve fragmented, low molecular weight DNA inside apoptotic cells, this DNA leaks out during washing and staining, giving rise to the appearance of a pre-G(1) peak, which is considered to be a marker of cell death by apoptosis(37) . The extraction of fragmented DNA from apoptotic cells is increased by the addition of phosphate-citric acid buffer(37) . The data are expressed as the percentage of total cells in the pre-G(1) phase of the cell cycle.

Analysis of DNA Synthesis

Cells were grown to 80% confluency and transferred to differentiation medium in the presence or the absence of IGF-I (25 nM), EGF (5 or 50 ng/ml), FGF-2 (10 or 30 ng/ml), PDGF-bb (2 or 10 ng/ml), or cycloheximide (1 µg/ml). DNA synthesis was assessed by pulsing cells with 10 µM BrdUrd for 4-h intervals over a 24-h period. After each incubation cells were fixed (100% ethanol) and permeabilized (0.25% Triton X-100), and BrdUrd sites were unmasked in 1.5 M HCl. Nonspecific binding sites were blocked (1% bovine serum albumin, 0.2% nonfat dry milk, 0.3% Triton X-100), and BrdUrd was detected using a specific primary antibody and a CY-3 conjugated secondary antibody. Cells were co-stained with bisbenzamide and were visualized by fluorescence microscopy. The data are expressed as the percentage of total cells with fluorescent nuclei.


RESULTS

Rapid Cell Death of IGF-II Antisense Myoblasts after Transfer into Differentiation Medium

C2 myoblasts stably transfected with pSV2neo and pEMSValphascribe/IGF-II (Fig. 1) were characterized for expression of the IGF-II transgene by ribonuclease protection assay using a P-labeled single-stranded RNA probe derived from a mouse IGF-II cDNA (Fig. 2), and two lines (antisense 3 and antisense 12) were selected for further analysis. In both lines, cell doubling time in growth medium was similar to that observed in nontransfected C2 cells and in cells stably transfected with the empty expression plasmid (15.5-16 h). (^2)


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 (leq1.2 ± 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.^2 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.^2 In addition, detached cells were incapable of reattaching to culture dishes when incubated in growth medium containing 20% serum.^2


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.



IGF-I and Cycloheximide Prevent Premature Myoblast Death

The addition of IGF-I or cycloheximide to differentiation medium blocked cell death in IGF-II antisense lines 3 and 12. As shown in Fig. 4A, the dramatic decline in the number of adherent cells in antisense line 12 during a 24-h incubation in differentiation medium was prevented by 1 µg/ml cycloheximide or 25 nM IGF-I. Treatment with cycloheximide completely blocked cell death for at least 36 h in both antisense lines, and incubation with IGF-I led to a 25-50% increase in the number of adherent myoblasts (Fig. 4B).


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.^2 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(1) 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(1), G(0)/G(1), and G(2)/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(1) peak). The population pool was 10,000 cells for each time point and was taken from a sample of 1-5 times 10^6 cells.



IGF-I Promotes DNA Synthesis and Cell Replication in IGF-II Antisense Myoblasts

The results shown in Fig. 4and Fig. 5indicated that IGF-I treatment inhibited apoptotic cell death and increased the total the number of viable cells. To assess the mechanisms leading to this rise in cell number, we examined the effects of IGF-I on DNA synthesis. As shown in Fig. 6A, incubation of IGF-II antisense 12 myoblasts with differentiation medium containing IGF-I increased incorporation of the nucleotide analog BrdUrd into chromosomal DNA. More than 60% of IGF-I-treated myoblasts became labeled during the last 4 h of an 8- or 12-h incubation with growth factor. 35% of cells were labeled during the last 4 h of a 16-h treatment, but fewer than 10% were labeled following a 20- or 24-h incubation with IGF-I. By contrast, the fraction of cells entering S phase declined dramatically in antisense myoblasts incubated in differentiation medium alone, dropping from 50% during the first 4 h to 5% by 12-16 h. As expected, cycloheximide treatment blocked progression into S phase, and few nuclei incorporated BrdUrd (Fig. 6B). Similar results were seen when experiments were performed using serum-free differentiation medium.^2


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.



Ligand-induced Activation of the IGF-I Receptor Mediates Survival of IGF-II Antisense Myoblasts

Fig. 7shows a series of dose-response curves examining myoblast survival following a 24-h incubation in differentiation medium containing graded concentrations of IGF-I, IGF-II, the IGF-I analog des [1-3] IGF-I, or insulin. At the highest doses used, all four growth factors inhibited myoblast death and stimulated replication, as indicated both by a decline in the fraction of detached dead cells and a rise in the number of adherent myoblasts. Des [1-3] IGF-I was the most potent agent, with an ED of approximately 0.5 nM, followed by IGF-I (8 nM), IGF-II (20 nM), and insulin (500 nM). These dose-response curves are consistent with mediation of cell survival and proliferation by the IGF-I receptor.


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.



Apoptotic Cell Death Is Prevented in IGF-II Antisense Cells by FGF-2 and PDGF-bb

The addition of FGF-2 or PDGF-bb to the differentiation medium blocked cell death of antisense line 12. As shown in Fig. 8, the dramatic decline in the number of adherent cells during a 24-h incubation in differentiation medium was prevented by 10 or 30 ng/ml FGF-2 or 10 ng/ml PDGF-bb. By contrast, EGF at both doses tested (5 or 50 ng/ml) and PDGF-bb at its lower dose (2 ng/ml) were incapable of preventing myoblast death. Similar rescue profiles were observed when the number of detached, dead cells were counted (Fig. 8). Treatment of antisense 12 cells with FGF-2 or PDGF-bb but not EGF also prevented DNA fragmentation as assessed by TUNEL assay.^2


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.



FGF-2 and PDGF-bb Promote DNA Synthesis and Cell Replication but Not Differentiation in IGF-II Antisense Myoblasts

The results shown in Fig. 8indicated that FGF-2 and PDGF-bb inhibited apoptotic cell death and enhanced the number of surviving cells by 75-100%. To address whether the rise in cell number was attributable to enhanced cell replication, we monitored progression through S phase of the cell cycle by assessing BrdUrd uptake into DNA. As shown in Fig. 9, treatment of antisense 12 cells with BrdUrd for the last 4 h of a 12-h incubation with either growth factor resulted in labeling of 40-60% of the cells. By contrast, EGF had little effect at either dose tested (5 or 50 ng/ml). As demonstrated with IGF-I treatment, the fraction of cells entering S phase dropped dramatically following a 24-h incubation with FGF-2 or PDGF-bb, although 15-20% of the cells still incorporated BrdUrd.


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.^2 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.^2 In addition, myotubes were seen only in IGF-I-treated cells.^2


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.




DISCUSSION

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.^2 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.^2 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.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grant 5 RO1-DK42748 (to P. R.). Oligonucleotides were prepared at the Washington University Protein and Nucleic Acid Laboratory under support of National Institutes of Health Grant DK20579 (Diabetes Research and Training Center). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, Box 8231, Washington University School of Medicine, 660 South Euclid Ave., Saint Louis, MO 63110. Tel.: 314-362-2703; Fax: 314-362-7183.

(^1)
The abbreviations used are: IGF, insulin-like growth factor; FGF-2, fibroblast growth factor-2; PDGF-bb, platelet-derived growth factor-bb; EGF, epidermal growth factor; BrdUrd, bromodeoxyuridine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling; FACS, fluorescent activated cell sorting; MES, 2-(N-morpholino)ethanesulfonic acid; IGFBP, IGF binding protein; des [1-3], desamino 1-3.

(^2)
C. E. H. Stewart and P. Rotwein, unpublished observations.


ACKNOWLEDGEMENTS

We thank the following individuals for gifts of reagents: Dr. Chris Morrison, Dr. Michael F. Fant, the late Dr. Harold Weintraub, and Dr. Paul Berg.


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