Insulin-like growth factor I (IGF-I) receptor overexpression abolishes the IGF requirement for differentiation and induces a ligand-dependent transformed phenotype in C2 inducible myoblasts.

Insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cell lines, and these actions are mostly mediated through the type I IGF receptor (type I IGF-R). To further investigate the role of this receptor in phenotypic characteristics of C2 murine myoblasts, we overexpressed the human type I IGF-R in the inducible clone of C2 cells, which requires IGFs in the differentiation medium to undergo terminal differentiation. Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells. We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype. It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37-40, 32, 30-31, 28, and 24 kDa. The 30-31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an approximately 400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the alpha 2 beta 2 form of the IGF-I receptor, and two beta subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two beta subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two beta subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

A developmentally regulated form of insulin-like growth factor receptor beta-subunit in C2 myoblasts exhibiting altered requirements for differentiation.

Ligand-dependent autophosphorylation and immunoprecipitation have been used to distinguish insulin and insulin-like growth factor-I (IGF-I) receptor beta-subunits in the permissive and inducible subclones of the C2 myoblast cell line. Permissive myoblasts differentiate spontaneously, whereas myoblasts of the inducible subclone require exogenous IGFs to undergo terminal differentiation. Permissive myoblasts contain beta-subunits of 95 and 101 kilodalton (kDa) mol wt. The 95-kDa subunits are immunoprecipitated with antipeptide antibodies directed against tyrosine kinase (AbP2), juxtamembrane (AbP4), and carboxy-terminal (AbP5) domains of the insulin receptor and insulin receptor monoclonal antibody 29B4. The tryptic phosphopeptide map of the 95-kDa band suggests that it contains both insulin and IGF-I receptor beta-subunits. The 101-kDa subunit is immunoprecipitated by AbP2, AbP4, and AbP5, because it forms a hybrid complex with the 95-kDa protein, but it does not react directly with AbP4, AbP5, or antibody 29B4. Phosphorylation of the 101-kDa subunit is more responsive to IGF-I than to IGF-II or insulin, indicating that it is a second IGF-I receptor beta-subunit. Inducible myoblasts exhibit a single major beta-subunit of 106 kDa mol wt. Its immunoreactivity and phosphopeptide map are virtually identical to those of the 101-kDa IGF-I receptor beta-subunit from permissive cells. However, unlike the 101-kDa beta-subunit, phosphorylation of the 106-kDa protein appears to be more responsive to IGF-II than to either IGF-I or insulin. It is lost upon differentiation of myoblasts into myotubes concomittant with the appearance of 95- and 101-kDa beta-subunits. These data demonstrate 1) an alpha 2 beta 2 IGF receptor that has high sensitivity for IGF-II in inducible, but not in permissive, myoblasts; 2) the beta-subunit of this receptor exhibits different migration in sodium dodecyl sulfate-polyacrylamide gels from either of those found in permissive cells; and 3) expression of this beta-subunit is developmentally regulated. This suggests that the inducible cell beta-subunit is a component of a stage-specific alpha 2 beta 2 IGF receptor subtype that functions as an IGF-II receptor.

Differentiation of adipocyte precursors in a serum-free medium is influenced by glucocorticoids and endogenously produced insulin-like growth factor-I.

Stromal vascular cells from rabbit perirenal adipose tissue differentiated at a high frequency in a chemically-defined serum-free medium containing insulin, transferrin, tri-iodothyronine and dexamethasone. The omission from the culture medium of dexamethasone resulted in a lack of adipose conversion. Addition of IGF-I increased glycerol-3-phosphate dehydrogenase (GPDH) activity. The conditioned media from adipocyte precursor cells contained measurable quantities of immunoreactive IGF-I as determined by RIA after neutralization of IGF binding proteins interference. Dexamethasone increased IGF-I secretion during the first seven days after plating and decreased IGF-I binding to conditioned media. Three molecular forms of IGF binding proteins (IGFBPs) were identified by Western ligand blots in conditioned media, with M(r) = 40,000, 29,000 and 25,000. The major form (M(r) = 29,000) was decreased by dexamethasone. In contrast, the M(r) = 24,000 form was increased. Specific binding of 125I-labelled IGF-I to rabbit adipocyte precursor cells was more effectively inhibited by unlabelled IGF-I than by unlabelled IGF-II or insulin. The electrophoretic migration of cross linked 125I-IGF-I to microsomal membranes revealed a complex with M(r) = 130,000 under reducing conditions corresponding to the alpha-subunit of the IGF-I receptor. The addition of IGF-I monoclonal antibody to rabbit adipocyte precursor cells cultured in serum-free medium significantly inhibited [3H]-thymidine incorporation and significantly decreased (50%) GPDH specific activities. This inhibitory effect was overcome by the addition of exogenous IGF-I. Thus stromal vascular cells isolated from perirenal adipose tissue secrete IGF-I and IGFBPs, possess IGF-I receptors and respond to exogenous and endogenous IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)