Insulin-like Growth Factor Receptor Type Research Articles

Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Delta). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Delta allele has no phenotype; however, paternal inheritance results in biallelic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Delta allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.

Alcohol‐Induced Modulation of the Insulin‐Like Growth Factor System in Early Chick Embryo Cranial Tissue

Background: Fetal alcohol exposure has been shown to reduce fetal/embryonic growth. The insulin‐like growth factor (IGF) system plays a major role in normal growth and development of the embryo. The purpose of this study was to gain a better understanding of the effects of alcohol (ethanol, EtOH) exposure on the insulin‐like growth factors, their binding proteins, and receptors during embryonic development.Methods: After the administration of either alcohol or chick Ringer's solution to individual eggs at the start of incubation, type‐1 IGF receptors, IGF‐binding proteins (IGFBPs) as well as IGF‐1 and IGF‐2 levels were measured in chick embryo craniums on days 5, 6, 7, and 8 of incubation.Results: Levels of the IGF‐1 receptor protein were not significantly different between treatment groups on any day studied. In EtOH‐treated embryos, the 30 kDa IGFBP levels were significantly higher than vehicle levels on days 5 and 6. On day 6, IGF‐1 levels were significantly lower in the alcohol‐treated embryos compared with levels in vehicle‐treated embryos of the same age. By day 8 of incubation, IGF‐1 levels were significantly higher and the 30 kDa IGFBP levels were significantly lower in the alcohol‐treated group compared with vehicles. These results indicate an initial EtOH‐associated reduction in the amount of IGF‐1 available to bind to its receptor (bioavailability), followed by increased IGF‐1 bioavailability by day 8.Conclusions: The elevated IGFBP levels and reduced IGF‐1 levels on days 5 and 6 of incubation are congruent with an overall reduction in the bioavailability of IGF‐1 during this period and correlate with the decreased embryo weight observed in the alcohol‐treated embryos. An increased bioavailability of IGF‐1 observed by day 8 may represent a rebound effect and is associated with increases in ornithine decarboxylase activity, a marker of increased growth.

Alcohol-induced modulation of the insulin-like growth factor system in early chick embryo cranial tissue.

Fetal alcohol exposure has been shown to reduce fetal/embryonic growth. The insulin-like growth factor (IGF) system plays a major role in normal growth and development of the embryo. The purpose of this study was to gain a better understanding of the effects of alcohol (ethanol, EtOH) exposure on the insulin-like growth factors, their binding proteins, and receptors during embryonic development. After the administration of either alcohol or chick Ringer's solution to individual eggs at the start of incubation, type-1 IGF receptors, IGF-binding proteins (IGFBPs) as well as IGF-1 and IGF-2 levels were measured in chick embryo craniums on days 5, 6, 7, and 8 of incubation. Levels of the IGF-1 receptor protein were not significantly different between treatment groups on any day studied. In EtOH-treated embryos, the 30 kDa IGFBP levels were significantly higher than vehicle levels on days 5 and 6. On day 6, IGF-1 levels were significantly lower in the alcohol-treated embryos compared with levels in vehicle-treated embryos of the same age. By day 8 of incubation, IGF-1 levels were significantly higher and the 30 kDa IGFBP levels were significantly lower in the alcohol-treated group compared with vehicles. These results indicate an initial EtOH-associated reduction in the amount of IGF-1 available to bind to its receptor (bioavailability), followed by increased IGF-1 bioavailability by day 8. The elevated IGFBP levels and reduced IGF-1 levels on days 5 and 6 of incubation are congruent with an overall reduction in the bioavailability of IGF-1 during this period and correlate with the decreased embryo weight observed in the alcohol-treated embryos. An increased bioavailability of IGF-1 observed by day 8 may represent a rebound effect and is associated with increases in ornithine decarboxylase activity, a marker of increased growth.

Inverse Relationship between Microsatellite Instability and K- ras and p53 Gene Alterations in Colon Cancer

Some studies have shown an inverse relationship between microsatellite instability in colon cancer and mutations in p53 and K-ras, whereas others have not. We therefore evaluated these features in a population-based sample of 496 individuals with colon cancer. Microsatellite instability was determined by a panel of 10 tetranucleotide repeats, the Bethesda consensus panel of mono- and dinucleotide repeats, and coding mononucleotide repeats in transforming growth factor-beta receptor type II, hMSH3, BAX, hMSH6, and insulin-like growth factor receptor type II. Mutations in codons 12 and 13 in K-ras were evaluated by sequencing. p53 overexpression (as detected by immunohistochemistry) was used as an indicator of p53 mutation; this was evaluated in 275 of the tumors. K-ras mutations were present in 33.2% of tumors, p53 overexpression in 51.5%, and microsatellite instability (as determined by the Bethesda consensus panel) in 12.5%. K-ras mutations were significantly less common in unstable tumors than stable tumors (11.8% versus 36.9%, P: < 0.001). p53 overexpression was significantly less common in unstable tumors than stable tumors (20.0% versus 55.7%, P: < 0.001). These inverse relationships between microsatellite instability and ras gene mutations and p53 overexpression were shown to be independent of tumor site in logistic regression analyses. All other measures of instability also showed statistically significant inverse relationships independent of tumor site with alterations in ras and p53, and instability results determined by the panel of 10 tetranucleotide repeats were highly significantly related to those determined by the Bethesda consensus panel. Coding mononucleotide repeat mutations were significantly more common in unstable tumors than stable tumors (85.7% versus 1.0%, P: < 0.001). We conclude that there is an inverse relationship between microsatellite instability and mutations in p53 and K-ras, and that the molecular profile of colon cancers with microsatellite instability is characterized by relatively infrequent mutations in K-ras and p53 and relatively frequent mutations in coding mononucleotide repeats.

Insulin-like growth factor binding protein 5 and type-1 insulin-like growth factor receptor are differentially regulated during apoptosis in cerebellar granule cells.

Neuronal apoptosis is considered to play a significant role in several neuropathological conditions. However, the molecular mechanisms underlying neuronal apoptosis are poorly understood. Insulin-like growth factor (IGF) signalling is considered to be an important regulator of neuronal differentiation, survival and apoptosis. We have examined the expression of two members of the IGF system, insulin-like growth factor binding protein 5 (IGFBP-5) and the type-1 IGF receptor (IGF1R), during apoptosis of rat cerebellar granule cells (CGCs) in vitro. We describe a prominent downregulation of IGFBP-5 mRNA and protein expression. We also show that IGF-I increases IGFBP-5 expression in CGCs and that the downregulation of IGFBP-5 mRNA can be suppressed by inhibiting mRNA synthesis with actinomycin D. The expression of IGF1R mRNA showed a transient upregulation during potassium chloride (KCl) deprivation induced apoptosis, in contrast to the IGF1R protein level, which was downregulated during KCl deprivation. Our results provide insight into the expression of IGF-related genes during neuronal apoptosis, and indicate that they mediate a protective response to the withdrawal of trophic stimulation. It seems that the expression of IGFBP-5 and IGF1R is regulated to maximize the availability of IGF and the activity of IGF-triggered survival signalling.

Crystal Structure of the First Three Domains of the Type-1 Insulin-like Growth Factor Receptor

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-IR (L1-Cys-rich-L2) determined to 2.6 A resolution. The L domains each consist of a single-stranded right-handed beta-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1-462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.

Antagonists of Growth Hormone-Releasing Hormone Inhibit the Growth of U-87MG Human Glioblastoma in Nude Mice

Antagonists of growth hormone-releasing hormone(GH-RH) inhibit the growth of various cancers by mechanisms that involve the suppression of the insulin-like growth factor (IGF) -I and/or IGF-II. In view of the importance of the IGF system in glioma tumorigenesis, the effects of GH-RH antagonists MZ-5-156 and JV-1-36 were investigated in nude mice bearing subcutaneous and orthotopic xenografts of U-87MG human glioblastomas. After 4 weeks of therapy with MZ-5-156 or JV-1-36 at the dose of 20 µmg/day per animal, the final volume of subcutaneous U-87MG tumors was significantly (P < .01) decreased by 84% and 76%, respectively, as compared with controls. Treatment with GHRH antagonists also reduced tumor weight and the levels of mRNA for IGF receptor type I (IGFR-I). A reduction in the mRNA levels for IGF-II was found in tumors of mice treated with MZ-5-156. Treatment with MZ-5-156 or JV-1-36 also extended the survival of nude mice implanted orthotopically with U-87MG glioblastomas by 81% (P < .005) and 18%, respectively, as compared with the controls. Exposure in vitro to GH-RH antagonists MZ-5-156 or JV-1-36 at 1 MM concentration for 24 hours decreased the tumorigenicity of U-87MG cells in nude mice by 10% to 30% and extended the latency period for the development of subcutaneous palpable tumors by 31% to 56%, as compared with the controls. Exposure of U-87MG cells to GH-RH antagonists in vitro also resulted in a time-dependent increase in the mRNA levels of IGFR-II or a decrease in the mRNA levels of IGFR-I. mRNA for GH-RH was detected in U87MG cells and xenografts implying that GH-RH may play a role in the pathogenesis of this tumor. Our results suggest that GH-RH antagonists MZ-5-156 and JV-136 inhibit the growth of U-87MG human glioblastoma by mechanisms that involve the suppression of IGF system. Antagonistic analogs of GH-RH merit further development for the treatment of malignant glioblastoma.

Crystal structure of the first three domains of the type-1 insulin-like growth factor receptor

The type-1 insulin-like growth-factor receptor (IGF-1R) and insulin receptor (IR) are closely related members of the tyrosine-kinase receptor superfamily. IR is essential for glucose homeostasis, whereas IGF-1R is involved in both normal growth and development and malignant transformation. Homologues of these receptors are found in animals as simple as cnidarians. The epidermal growth-factor receptor (EGFR) family is closely related to the IR family and has significant sequence identity to the extracellular portion we describe here. We now present the structure of the first three domains of IGF-IR (L1-Cys-rich-L2) determined to 2.6 A resolution. The L domains each consist of a single-stranded right-handed beta-helix. The Cys-rich region is composed of eight disulphide-bonded modules, seven of which form a rod-shaped domain with modules associated in an unusual manner. The three domains surround a central space of sufficient size to accommodate a ligand molecule. Although the fragment (residues 1-462) does not bind ligand, many of the determinants responsible for hormone binding and ligand specificity map to this central site. This structure therefore shows how the IR subfamily might interact with their ligands.

Insulin-like and fibroblast growth factors and their receptors are differentially expressed in the oviducts of the common marmoset monkey (Callithrix jacchus) during the ovulatory cycle.

It is suggested that growth factors support the process of maturation and differentiation in the mammalian oviduct. Fibroblast growth factors (FGF) and insulin-like growth factors (IGF) are possible influences on these processes. The present study describes for the first time the expression of FGF-1 and -2 and their receptors as well as IGF-1 and -2 and the corresponding IGF receptor type 1 in the oviduct of the New World monkey Callithrix jacchus. Because of the limited RNA yields from oviducts, reverse transcription-polymerase chain reaction (RT-PCR) was performed to estimate expression levels. Expression patterns were found to be similar for all examined growth factors and receptors: the highest mRNA contents were obtained at the late proliferative and early to midsecretory phases, compared with lower levels during the early proliferative phase. Elevated amounts of these RNAs were correlated with high serum estradiol but not with progesterone concentrations. Each PCR product showed a high degree of homology (> 92%) to the known human sequences. Immunohistochemical analysis indicated greater specific staining for FGF-1 and -2 and IGF-1 before ovulation on the luminal epithelial surface of marmoset oviducts in comparison to the other cycle phases. Differences in staining intensity were not observed between the ampulla and isthmus. In summary, the marmoset oviduct expresses all components of functional FGF and IGF systems, thus suggesting auto-/paracrine effects of these growth factors within the primate oviduct, possibly under the control of estrogenic hormones.

Up-regulation of insulin-like growth factor binding protein-5 is independent of muscle cell differentiation, sensitive to rapamycin, but insensitive to wortmannin and LY294002.

Skeletal myoblast differentiation is stimulated by insulin-like growth factors (IGFs). The autocrine action of IGFs is mediated through the type-1 IGF receptor (IGFR-1) and modulated by IGF binding proteins (IGFBPs) secreted by the cells. The mouse C2 myoblast cell line stably transfected with a vector producing IGF-II antisense RNA was used to show that specific IGFBP expression changes with the state of the cells: high levels of IGFBP-2 messenger RNA (mRNA) were found only in proliferating myoblasts, whereas IGFBP-3 mRNA was induced in quiescent cells. Secretion of IGFBP5 was strongly stimulated during differentiation. Insulin and IGF dose-response experiments showed that up-regulation of IGFBP-5 resulted from IGFR-1 activation. Drugs interfering with IGFR-1 signaling and inhibiting myoblast differentiation had different effects on IGFBP-5 up-regulation. Two phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmaninn and LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], failed to alter IGFBP-5 up-regulation, which persisted in the absence of differentiation. Rapamycin which indirectly prevents activation of the p70 ribosomal protein-S6 kinase (p70S6k), suppressed IGFBP-5 induction. Because the PI3-kinase inhibitors block p70S6k, neither kinase would be required for IGFR-1-dependent IGFBP-5 induction. In C2 anti-IGF-II myoblasts, IGFBP-5 induction is therefore rapamycin-sensitive and independent of differentiation.

Retrovirally mediated overexpression of insulin-like growth factor binding protein 4: evidence that insulin-like growth factor is required for skeletal muscle differentiation.

Recent studies have indicated that the insulin-like growth factors (IGFs) stimulate skeletal myoblast proliferation and differentiation. However, the question of whether IGFs are required for myoblast differentiation has not been resolved. To address this issue directly, we used a retroviral vector (LBP4SN) to develop a subline of mouse C2 myoblasts (C2-BP4) that constitutively overexpress IGF binding protein-4 (IGFBP-4). A control C2 myoblast subline (C2-LNL6) was also developed by using the LNL6 control retroviral vector. C2-BP4 myoblasts expressed sixfold higher levels of IGFBP-4 protein than C2-LNL6 myoblasts. 125I-IGF-I cross linking indicated that IGFBP-4 overexpression reduced IGF access to the type-1 IGF receptor tenfold. At low plating densities, myoblast proliferation was inhibited, and myoblast differentiation was abolished in C2-BP4 cultures compared with C2-LNL6 cultures. At high plating densities in which nuclear numbers were equal in the two sets of cultures, C2-BP4 myoblast differentiation was inhibited completely. Differentiation was restored in C2-BP4 cells by treatment with high levels of exogenous IGF-I or with des(1-3)IGF-I, an analog of IGF-I with reduced affinity for IGFBPs. These findings confirm the hypothesis that positive differentiation signals from the IGFs are necessary for C2 myoblast differentiation, and they suggest that the present model of myogenic differentiation, which involves only negative external control of differentiation by mitogens, may be incomplete.

Type-1 insulin-like growth factor receptor reexpression in the malignant phenotype of SV40-T-immortalized human prostate epithelial cells enhances apoptosis.

The authors have previously shown that the type 1 insulin-like growth factor receptor (IGF-1R) is decreased in the transformation from benign to malignant human prostate epithelial cells in vivo. Further, in a well-described human SV40-T immortalized human epithelial cell system beginning with the immortalized, but rarely tumorigenic P69SV40-T cell line, to the highly tumorigenic and metastatic M12 subline, there is a similar decrease in IGF-1R number from 2.0 x 10(4) receptors per cell to 1.1 x 10(3) receptors per cell. When the IGF-1R was reexpressed in the M12 subline using a retroviral expression vector, M12-LISN, to a receptor number similar to that of the P69SV40-T parental cell line, the authors demonstrated a marked decrease in colony formation in soft agar in the M12-LISN cells vs the M12 control cells (p < or = 0.01), and a decrease in vivo tumor growth and metastases when injected either subcutaneously or an intraprostatic location (p < or = 0.01). This decrease in tumor volume was not because of a decrease in proliferative capacity, but was associated with an increase in apoptosis in baseline cultures and in response to the apoptotic-inducing agents 6-hydroxyurea, retinoic acid, and transforming growth factor beta 1.

La PI3 kinase, une activité critique pour la différenciation des cellules musculaires IGFs dépendante

Evidence has accumulated that suggests that insulin-like growth factors (IGFs) exert a positive influence on myoblast differentiation. We have undertaken to study the signalling events required for differentiation resulting from type-1 IGF receptor stimulation in C2 myoblasts, where autocrine production of IGF-II was abolished by means of antisense RNA. Exposure of the cells to IGFs leads to a rapid and sustained activation of phosphatidyl-inositol 3-kinase followed by the expression of Myod, myogenin and differentiation. The fungal metabolite, wortmannin, inhibits both PI 3-kinase and muscle differentiation with an IC 50 in the nanomolar range. IGFs are also known to cause a rapid activation of MAP kinase. However, the synthetic inhibitor of MEK, PD098059, which prevents MAP kinase activation, does not affect myoblast differentiation. These results provide evidence that PI3-kinase, but not MAP kinase, is required for insulin-like growth factor receptor-dependent differentiation of muscle cells.

Evolution of Insulin-like Growth Factor (IGF) Function: Production and Characterization of Recombinant Hagfish IGF

While there is considerable structural evidence that insulin-like growth factors (IGFs) share a long evolutionary history, little is known about the conservation of IGF function. In order to address this, we have made recombinant hagfish IGF, hence allowing characterization of an IGF from a representative of the primitive vertebrate class, Agnatha. The production of recombinant hagfish IGF has been complicated by a number of factors including the requirement of a longer leader peptide for fusion protein expression, reduced solubility of the protein, as well as problems in the refolding procedure. However, we were able to produce a small quantity of hagfish IGF with an N-terminal glycine addition which is biologically active. Furthermore, N-terminal amino acid sequencing and mass spectrometry confirm that we have produced hagfish IGF.In vitroassessment of recombinant hagfish IGF in cultured cells indicates that hagfish IGF indeed shares functional properties with mammalian IGFs. Thus, hagfish IGF stimulates protein synthesis in rat myoblasts, but 20- and 5-fold more peptide, respectively, is required to achieve the same half-maximal responses as with human IGF-I (hIGF-I) or IGF-II (hIGF-II). Hagfish IGF also competes for binding to the type-1 IGF receptor present both on rat myoblasts and on salmon embryo fibroblasts, though with somewhat lower affinity than either hIGF-I or hIGF-II. However, studies investigating binding to the IGF-II-specific type-2 receptor suggest that hagfish IGF may in fact be more closely related to IGF-I than to IGF-II. These results indicate that motifs important for functions associated with mammalian IGFs appear to have evolved prior to the Agnathans diverging from the main line of vertebrate evolution 550 million years ago. Accordingly, we now have functional as well as structural evidence that the IGFs have a long evolutionary history.

Overexpression of the type-1 insulin-like growth factor receptor increases ligand-dependent proliferation and differentiation in bovine skeletal myogenic cultures

Previous studies demonstrated that overexpression of the type-1 insulin-like growth factor (IGF) receptor (IGF-1R) in skeletal myogenic cell lines increased proliferation and differentiation responses to IGF. However, it was unclear if such manipulations in primary, untransformed skeletal myogenic cells would result in modulation of these responses, which may be more stringently regulated in primary cells than in myogenic cell lines. In this study, low passage untransformed fetal bovine myogenic cultures were infected with a replication-deficient retroviral expression vector (LISN) coding for the human IGF-1R or with a control retroviral vector (LNL6). Bovine myogenic cultures infected with the LISN vector (Bov-LISN) displayed ten times more IGF-1Rs than controls (Bov-LNL6). Bov-LISN myogenic cultures exhibited elevated rates of IGF-I-stimulated proliferation and increased rates of terminal differentiation which were reduced to control levels by the anti-human IGF-1R antibody alpha IR3. These findings indicate overexpression of the IGF-1R can enhance IGF sensitivity and thereby modify the proliferation and differentiation behavior of untransformed low passage myoblasts. Such manipulations may be useful to increase muscle mass in clinical or agricultural applications.

Effect of in utero ethanol exposure on the postnatal ontogeny of insulin-like growth factor-1, and type-1 and type-2 insulin-like growth factor receptors in the rat brain

There is convincing evidence that alcohol consumption during pregnancy causes major CNS abnormalities; however, the molecular and cellular basis of these dysfunctions is currently not understood. This study examined the effects of prenatal ethanol exposure on the expression of insulin-like growth factor-1 messenger RNA and type-1 and type-2 receptor protein and messenger RNA expression in the developing rat brain. Mothers were maintained on an ethanol containing liquid diet from day 2 of pregnancy through parturition and the offspring were killed at birth, 10, 20 and 40 days of age. Insulin-like growth factor-1 messenger RNA, and insulin-like growth factor receptors demonstrated developmentally dependent expression in specific brain regions throughout the postnatal period of CNS maturation. Insulin-like growth factor-1 gene expression in the brain, as analysed by dot-blot hybridization, was greatest at birth, and decreased 61% in ad libitum and pair-fed animals by 20 days of age. In contrast, ethanol-treated animals exhibited only a 25% decrease in insulin-like growth factor-1 messenger RNA levels during the same period. This delay in insulin-like growth factor-1 messenger RNA maturation may be related to a developmental delay in CNS development in the prenatally ethanol exposed offspring. Prenatal ethanol exposure did not alter the observed localization of insulin-like growth factor-1 messenger RNA. While alterations were observed in long-term insulin-like growth factor-1 messenger RNA regulation, quantitative receptor autoradiography and in situ hybridization demonstrated no alterations in either type-1 or type-2 insulin-like growth factor receptor populations in ethanol-treated animals. Changes in hepatic and plasma insulin-like growth factor-1 and insulin-like growth factor-binding protein regulation have also been observed in these animals, suggesting changes in protein translation and the autocrine/paracrine actions of this peptide. The present study demonstrated that insulin-like growth factor-1 messenger RNA and insulin-like growth factor receptors are regionally expressed during early postnatal development and that ethanol administration influenced the long-term regulation of insulin-like growth factor messenger RNA levels in the brain without affecting either its localization or insulin-like growth factor receptor populations. These results, in combination with previous results from our laboratory on the effects of prenatal ethanol exposure on plasma insulin-like growth factor-1 and insulin-like growth factor-binding proteins, provide additional evidence that even modest levels of prenatal ethanol exposure enhance the possibility of alterations in growth promoting peptides in the fetus and neonate, leading to an increased risk of fetal alcohol effects in the offspring.

Type‐1 insulin‐like growth factor receptor overexpression produces dual effects on myoblast proliferation and differentiation

Using a retroviral vector, we developed a line of C2 mouse skeletal myoblasts, C2-LISN, which expressed high levels of the human type-1 insulin-like growth factor (IGF) receptor. When switched to low serum medium, C2-LISN myoblasts underwent terminal differentiation extremely rapidly compared to control C2 myoblasts. In high serum conditions which were not permissive for differentiation, C2-LISN myoblasts expressed ten-fold higher levels of the myogenic transcription factor myogenin than did control C2 myoblasts. When cultured in low serum medium with both transforming growth factor-beta (TGF-beta) and high concentrations of IGF-I, C2-LISN myoblasts failed to differentiate and grew to very high saturation densities, forming multilayers. Upon removal of TGF-beta, multilayered C2-LISN myoblasts differentiated within 2 days. These results demonstrate that overexpression of the type-1 IGF receptor can amplify signals which stimulate myogenic differentiation. Overexpressed type-1 IGF receptors can also mediate strong mitogenic signals if differentiation is inhibited by TGF-beta. The C2-LISN myoblast cell line may be a useful model to investigate the intracellular pathways which stimulate myogenic differentiation. Additionally, overexpression of the type-1 IGF receptor could provide a strategy to expand populations of differentiation-competent myoblasts for experimental or clinical applications.

Overexpression of the Human Type-1 Insulin-like Growth Factor Receptor in Rat L6 Myoblasts Induces Ligand-Dependent Cell Proliferation and Inhibition of Differentiation

The human type-1 insulin-like growth factor receptor was overexpressed in rat L6 skeletal myoblasts using a retroviral expression vector. At low concentrations of insulin-like growth factor-I (1-10 ng/ml), dose-dependent stimulation of myoblast differentiation was similar in control L6 myoblasts and L6 myoblasts which overexpressed the type-1 insulin-like growth factor receptor. At high concentrations of insulin-like growth factor-I (50-100 ng/ml), L6 myoblasts which overexpressed the receptor did not undergo terminal differentiation and proliferated to form multilayers, while differentiation of control myoblasts was further stimulated. These findings indicate that overexpression of the type-1 IGF receptor can stimulate proliferation and inhibit differentiation in skeletal myoblasts in a ligand-dependent manner.

Ligand‐dependent inhibition of myoblast differentiation by overexpression of the type‐1 insulin‐like growth factor receptor

The insulin-like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2-LISN) which expressed 7.5 times higher levels of type-1 IGF receptors than control (P2-LNL6) myoblasts, which were infected with a virus lacking the type-1 IGF receptor sequence. Overexpression of the type-1 IGF receptor caused the IGF dose-response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF-I concentrations, complete inhibition of P2-LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type-1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2-LISN myoblasts constitutively expressed high levels of type-1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF-I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF-BP) release into the culture medium. These results indicate that the type-1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation-inducing and differentiation-inhibitory, which predominate at different ligand concentrations.

Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts.

Protein synthesis in rat L6 myoblasts is stimulated and protein breakdown inhibited in a co-ordinate manner by insulin-like growth factors (IGF) or insulin. For both processes, bovine IGF-1 was somewhat more potent than human IGF-1, which was effective at a tenth the concentration of insulin, rat IGF-2 or human IGF-2. A similar order of potency is noted when DNA synthesis or protein accumulation is monitored over a 24 h period, but between 20- and 50-fold higher concentrations of each growth factor are required than those needed to produce effects in the 4 h protein-synthesis or -breakdown measurements. Binding experiments with labelled human or bovine IGF-1 as ligand demonstrated competition at concentrations of IGF-2, especially human IGF-2, lower than that of either IGF-1 preparation. This pattern was much more pronounced when the radioligand was either human IGF-2 or rat IGF-2. Insulin competed 10-15% for the binding of labelled IGF-1, but not at all with labelled IGF-2. Ligand-receptor cross-linking experiments showed that labelled bovine IGF-1 bound approximately equally to the type 1 IGF receptor (Mr 130000 after reduction) and to the type 2 IGF receptor (Mr 270000 after reduction), and that unlabelled IGF-1 competed equally with radioligand binding to both receptors. On the other hand, rat IGF-2 competed more effectively for binding to the type-2 receptor, and insulin competed only for binding to the type-1 receptor. Further cross-linking experiments with rat IGF-2 as radioligand demonstrated binding only to the type-2 receptor and to proteins with Mr values after reduction of 230000 and 200000. This binding was prevented by high rat IGF-2 concentrations, less effectively by bovine IGF-1 and not at all by insulin. The apparently conflicting biological potencies and receptor binding of the different growth factors can be explained if all the biological actions are mediated via the type-1 IGF receptor, rather than through the abundant type-2 receptor.