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Endocrinology Vol. 141, No. 1 100-110
Copyright © 2000 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor (IGF)-Binding Protein-Related Protein-1: An Autocrine/Paracrine Factor That Inhibits Skeletal Myoblast Differentiation but Permits Proliferation in Response to IGF1

Kathleen L. Haugk, Heather-Marie P. Wilson, Karen Swisshelm and LeBris S. Quinn

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Skeletal myogenic cells respond to the insulin-like growth factors (IGF-I and IGF-II) by differentiating or proliferating, which are mutually exclusive pathways. What determines which of these responses to IGF skeletal myoblast undergo is unclear. IGF-binding protein-related protein 1 (IGFBP-rP1) is a secreted protein with close homology to the IGF-binding proteins (IGFBPs) in the N-terminal region. IGFBP-rP1, previously called mac25 and IGFBP-7, is highly expressed in C2 skeletal myoblasts during the proliferative phase, but is down-regulated during myoblast differentiation. To determine the role of IGFBP-rP1 in myogenesis, IGFBP-rP1 was overexpressed in C2 myoblasts using a retroviral vector. Western blots indicated that the resulting C2-rP1 myoblasts secreted approximately 27-fold higher levels of IGFBP-rP1 than control C2-LX myoblasts that were transduced with a control vector (LXSN). Compared with C2-LX myoblasts, the differentiation responses of C2-rP1 myoblasts to IGF-I, IGF-II, insulin, and des(1–3)IGF-I were significantly reduced (P < 0.05). However, proliferation responses of C2-rP1 and C2-LX myoblasts to these same factors were not significantly different. Exposure of control C2-LX myoblasts to factors secreted by C2-rP1 myoblasts using a transwell coculture system reduced C2-LX myoblast differentiation significantly (P < 0.05). Experiments with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 suggested that IGFBP-rP1 inhibits a MAPK-dependent differentiation pathway. In confirmation of this idea, levels of phosphorylated extracellular signal-regulated kinase-2 (a MAPK) were reduced in C2-rP1 myoblasts compared with those in C2-LX myoblasts. These findings indicate that IGFBP-rP1 may function as an autocrine/paracrine factor that specifies the proliferative response to the IGFs in myogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors (IGF-I and IGF-II) are critical factors in skeletal muscle development (1, 2), regeneration (3, 4), and hypertrophy (5, 6). The nuclei of skeletal muscle fibers are incapable of DNA synthesis; therefore, increases in skeletal muscle nuclear numbers are due to the proliferation and subsequent differentiation of undifferentiated skeletal muscle precursor cells known as myoblasts (7, 8). The importance of proliferating myoblasts to fetal and postnatal muscle growth and to muscle regeneration after traumatic injury has been established for several decades (7, 8, 9). Additionally, recent evidence has indicated that proliferation of adult myoblasts (satellite cells) is required for loading-induced muscle hypertrophy in rodents (5, 10, 11). Therefore, it is important to understand the factors that control skeletal myoblast proliferation and differentiation.

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
PE501 and PA317 packaging cells as well as the pLXSN plasmid were obtained from Dr. A. D. Miller, Fred Hutchinson Cancer Research Center (Seattle, WA). Monoclonal MF-20 antimuscle-specific myosin heavy chain (MHC) antibody was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). Rabbit antihuman IGFBP-rP1 (38) was obtained from Drs. R. Rosenfeld and V. Hwa, Oregon Health Sciences University (Portland, OR). The human IGFBP-rP1 complementary DNA (cDNA) and IGFBP-rP1 probe were obtained as previously described (39). DMEM was purchased from Sigma (St. Louis, MO); FCS was obtained from HyClone Laboratories, Inc. (Logan, UT).

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. 1Go). 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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  		Figure 1. Schematic representation of LXSN (control) and LrP1SN (IGFBP-rP1 expression) vectors. LTR, Long terminal repeat; SV, simian virus 40 promoter, NEO, neomycin phosphotransferase gene; pA, polyadenylation signal. The expected size for the retrovirally directed IGFBP-rP1 mRNA transcript is approximately 4.2 kb.

 
Assays for IGFBP-rP1 expression
For Northern blot analyses, total RNA was extracted from C2-LX and C2-rP1 cultures using a guanidinium thiocyanate-phenol-chloroform extraction protocol (42). Ten micrograms of RNA from C2-LX and C2-rP1 myoblasts were resolved by electrophoresis through a 1.2% agarose/2.2 M formaldehyde gel in 20 mM MOPS (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA. RNA was transferred to a nylon membrane (GeneScreen, NEN-DuPont, Boston, MA) via capillary action in 10 x SSC (standard saline citrate). The RNA was cross-linked to the membrane with a UV Stratalinker 1800 (Stratagene, La Jolla, CA) and hybridized for 16 h at 42 C in 50% formamide, 5 x SSC, 10 x Denhardt’s solution, 1% SDS, 100 µg/ml denatured herring sperm DNA, and 10 mM sodium phosphate buffer (pH 7.0). Northern blots were probed with a 0.88-kb SspI/XbaI human IGFBP-rP1 cDNA (33). Ethidium bromide (EtBr) staining of the 28S ribosomal bands was also compared with correct for loading differences. The hybridization probe was radiolabeled with [{alpha}-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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of IGFBP-rP1 by C2-LX and C2-rP1 myoblasts
Northern blot analysis (Fig. 2Go) 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. 2Go) 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. 3Go). 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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  		Figure 2. Northern blot comparing expression of IGFBP-rP1 mRNA expression by C2-LX myoblasts (left lane) and C2-rP1 myoblasts (right lane). C2-LX myoblasts, transduced with the LXSN empty vector, expressed the endogenous 1.1-kb IGFBP-rP1 transcript. C2-rP1 myoblasts, transduced with the LrP1SN expression vector, expressed the endogenous IGFBP-rP1 transcript and expressed an additional IGFBP-rP1 4.2-kb transcript (arrow) coded by the retroviral vector. A minor band is due to alternative stop codons within the vector. The 4.2-kb transcript is expressed in 3 times greater amounts than the endogenous transcript in C2-rP1 myoblasts.

 


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  		Figure 3. Western blot comparing expression of IGFBP-rP1 by C2-LX and C2-rP1 myoblasts. Samples shown are media collected from the respective cell types after 24-h culture in serum-free medium. The lane labeled P69 is a postive control for IGFBP-rP1 expression, medium from P69 human prostate epithelial cells, which express high levels of IGFBP-rP1. All of the cell types express a single 31-kDa band using rabbit antihuman IGFBP-rP1. C2-rP1 myoblasts express approximately 27-fold higher levels of IGFBP-rP1 than C2-LX myoblasts.

 
Growth rates of C2-LX and C2-rP1 myoblasts in 10% FCS/DMEM were indistinguishable (Fig. 4Go). 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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  		Figure 4. Proliferation rates of C2-LX and C2-rP1 myoblasts in 10% FCS/DMEM with daily medium changes. DNA content per well was assayed by a Hoechst 33258 fluorescence assay. Each point represents the mean ± SEM for three determinations; in most instances, the extent of the error bars is smaller than the data point symbols. Values for C2-LX and C2-rP1 DNA per well were not significantly different at each time point (by t tests). Mean doubling times were calculated as 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).

 
Differentiation responses of C2-LX and C2-rP1 myoblasts
The differentiation responses of C2-LX and C2-rP1 myoblasts to IGF-I, IGF-II, des(1, 2, 3)IGF-I, and insulin were assessed by Western blot analysis of muscle-specific MHC expression, normalized to DNA (Fig. 5Go). 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 5Go 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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  		Figure 5. Differentiation of C2-LX and C2-rP1 myoblasts in response to IGF-I (A), IGF-II (B), des(1 2 3 )IGF-I (C), and insulin (D). C2-rP1 differentiation in response to all factors was significantly reduced (P < 0.05) compared with that of C2-LX myoblasts for all points except 30 ng/ml IGF-I (by t tests). Differentiation was assessed by densitometric quantitation of Western blots for muscle-specific MHC accumulation, normalized to DNA of sister cultures. Each point represents the mean ± SEM of three determinations.

 
Immunocytochemical analysis was used to determine whether the lower levels of MHC expression in C2-rP1 myoblast cultures were due to reduced levels of MHC expression per nucleus or to a reduction in the rate of myoblast differentiation. As shown in Fig. 6Go, 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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  		Figure 6. Fluorescence photomicrographs of C2-LX (A and C) and C2-rP1 (B and D) cultures 3 days after a shift to low serum medium, without (A and B) and with (C and D) 30 ng/ml exogenous IGF-I. Cultures were stained with ethidium bromide to reveal all nuclei, differentiated or undifferentiated. Differentiated cell cytoplasm was stained with antimuscle specific MHC antibody. Differentiated myocytes are mono- or multinucleated. C2-rP1 cultures contain markedly fewer nuclei within MHC+ cytoplasm compared with C2-LX cultures in both conditions.

 
IGFBP-rP1 can act in a paracrine fashion
To determine whether secreted IGFBP-rP1 could act in a paracrine fashion to inhibit differentiation, C2-LX myoblasts were exposed to factors produced by a 4-fold excess of C2-LX or C2-rP1 myoblasts using a Costar Transwell coculture system (Fig. 7Go). 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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  		Figure 7. Effects of medium secreted by C2-LX vs. C2-rP1 myoblasts (secreting cells) on differentiation of C2-LX myoblasts (test cells). Secreting cells were plated in the lower chambers of Costar Transwell coculture apparati in 4-fold excess to the test cells, which were plated in the upper chambers, separated by a membrane with 0.4 µm pore size. MHC accretion by C2-LX test cells was significantly decreased (P < 0.05, by t test) when cultured with C2-rP1 myoblasts compared with that in culture with C2-LX myoblasts.

 
Proliferation responses of C2-LX and C2-rP1 myoblasts
Proliferation of C2-LX and C2-rP1 myoblasts in response to IGF-I, IGF-II, des(1, 2, 3)IGF-I, and insulin was assessed by a Hoechst dye fluorometric DNA assay and also by an assay of [3H]TdR incorporation into DNA, which yielded similar results (Fig. 8Go). 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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  		Figure 8. Proliferation of C2-LX and C2-rP1 myoblasts in response to a range of IGF-like mitogens. Proliferation was assayed by two methods for each factor, Hoechst 33258 fluorescence (A, C, E, and F) and an assay of [3H]TdR incorporation into DNA (B and D), which gave similar results for each factor. Both assays are shown for proliferation responses to IGF-I (A and B) and IGF-II (C and D). The Hoechst assay only is shown for des(1 2 3 )IGF-I (E) and insulin (F). Except for one point (asterisk), no significant differences between C2-LX and C2-rP1 myoblasts were observed in proliferation responses to any of the mitogens tested (by t tests). Each point represents the mean ± SEM of three replicates.

 
Effects of PD098059 on C2-LX and C2-rP1 myoblast differentiation
Both proliferation and differentiation responses to the IGFs in C2 myoblasts may involve MAPK-mediated pathways (22, 24). We reasoned that if IGFBP-rP1 was stimulating a MAPK-dependent proliferation pathway that also repressed differentiation, inhibition of MAPK activation would restore C2-rP1 myoblast differentiation to control levels. Conversely, if IGFBP-rP1 was acting to inhibit a MAPK-dependent differentiation pathway, inhibition of MAPK activation should have little effect on C2-rP1 myoblast differentiation, but would inhibit C2-LX myoblast differentiation. To test these ideas, the MAPK kinase (MEK) inhibitor PD098059 was administered to C2-LX and C2-rP1 myoblasts, and differentiation was assayed by Western blots of MHC accumulation, normalized to DNA (Fig. 9AGo). 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. 9Go, 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. 9BGo). Phosphorylated ERK-1, migrating at Mr 44,000, was a minor species.



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  		Figure 9. Effects of specific signal pathway inhibitors on C2-LX and C2-rP1 myoblast differentiation. A, Effects of the MEK inhibitor PD098059 on differentiation of C2-LX and C2-rP1 myoblasts. Myosin accretion was assessed by Western blots and normalized to DNA signal as described above. MHC/DNA at 10 µM PD098059 was significantly different from that at 0 µM PD098059 (P < 0.05) for C2-LX cells, but not for C2-rP1 cells. B, Western blot showing effects of PD098059 on phospho-MAPK expression in C2-LX and C2-rP1 myoblasts. The predominant phospho-MAPK species expressed was ERK-2, migrating at 42 kDa. This form migrated identically with the phospho-MAPK control protein purchased commercially (not shown). A minor form, ERK-1, migrating at 44 kDa, was also present. C, Quantitation of phospho-ERK-2 band intensities in response to PD098059. At 10 µM, PD098059 inhibited phosph-ERK-2 expression approximately 10-fold in C2-LX and C2-rP1 myoblasts. At baseline (no PD098059), expression of phospho-ERK-2 by C2-rP1 myoblasts was 25% lower than that of C2-LX myoblasts. D, Effects of the PI 3-kinase inhibitor LY294002 on differentiation (assessed by MHC accretion) in C2-LX and C2-rP1 myoblasts. LY294004 at 10 and 25 µM inhibited MHC expression completely in both C2-LX and C2-rP1 myoblasts.

 
Without addition of PD098059, phospho-ERK-2 expression in C2-rP1 myoblasts was 75% of that of C2-LX myoblasts (Fig. 9CGo), 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. 9AGo). 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. 9DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBP-rP1 is a member of a newly characterized family of cellular regulators, IGFBP-rP-1- to 4, which exhibit close homology to the IGFBPs in the N-terminal region, but lack the C-terminal region characteristic of IGFBP-1 to -6 (35). IGFBP-rP1 has been independently cloned in several cellular systems, and therefore has been previously identified as IGFBP-7 (36), mac25 (39, 48), tumor-derived adhesion factor (49), and prostacylin-stimulating factor (50). A limited number of functional studies have been performed, but IGFB-rP1 appears to act as a tumor suppressor in epithelial (39, 48, 51) and osteosarcoma (52) cells and as a proliferation stimulator in fibroblast-like cells (49). In skeletal myogenic cultures, IGFBP-rP1 expression is high in proliferating myoblasts and declines before differentiation, which suggested that it might play a role in stimulating myoblast proliferation and/or repressing differentiation (33). The present study was designed to determine the role of IGFBP-rP1 in skeletal myogenesis. Our data demonstrate that overexpression of IGFBP-rP1 in C2 skeletal myoblasts partially inhibits differentiation, but has no effect on proliferation. Furthermore, experiments using a transwell coculture system indicate that secreted IGFBP-rP1 can act in a paracrine fashion to inhibit muscle differentiation in control myoblasts. Experiments using the MAPK kinase (MEK) inhibitor PD098059 suggested that IGFBP-rP1 may inhibit a MAPK-dependent pathway involved in differentiation. Combined with our previous study showing that IGFBP-rP1 expression correlates negatively with myoblast differentiation (33), these findings indicate that IGFBP-rP1 may function to specify the proliferation response to IGF in skeletal myoblasts.

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.


   Acknowledgments
 
We are grateful to Drs. R. Rosenfeld and V. Hwa (Oregon Health Sciences University) for the gift of anti-IGFBP-rP1 antibody. We also thank Dr. S. Plymate and C. Tomasini-Sprenger (V.A. Puget Sound/University of Washington) for samples of P69 human prostate epithelial cell-conditioned medium for an IGFBP-rP1 Western positive control. Dr. A. D. Miller (Fred Hutchinson Cancer Research Center) provided the LXSN vector and PE501 and PA317 cell lines. Dr. S. E. Damon (V.A. Puget Sound/University of Washington) provided guidance on preparation of the IGFBP-rP1 hybridization probe. J. Woodmansee (V.A. Puget Sound) provided help with the figures, and B. Haugk provided help with programming the microplate reader. C2 myoblasts were gifts from Drs. D. Yaffe (Weizmann Institute of Science) and Z. Yablonka-Reuveni (University of Washington). The MF-20 antibody (contributed by Dr. D. Fischman, Cornell University) was obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Science, Johns Hopkins University School of Medicine, and the Department of Biology, University of Iowa.


   Footnotes
 
1 This work was supported by the USDA (Award 96–35206-3858; to L.S.Q.), a pilot award from Seattle Breast Cancer Foundation Grant 98–3078-90 (to K.S.), a predoctoral fellowship from the U.S. Army Materiel and Command (DAMD 17–96-1–6247; to K.S. and H.M.P.W.), and Contract N01-HD-6–2915 from the NICHHD. Back

Received May 13, 1999.


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 Introduction
 Materials and Methods
 Results
 Discussion
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