Insulin-like growth factors in sheep thyroid cells: action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 micrograms of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions, [125I]IGF-I bound to a moiety of approximate Mr = 135,000 and [125I]IGF-II to a moiety of approximate Mr = 260,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr = 34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation

The insulin-like growth factors (IGFs) I and II exert pleiotropic effects on diverse cell types through interaction with specific high affinity cell surface receptors and with locally produced binding proteins. In skeletal muscle and in myoblast cell lines, the functions of IGF-I and -II are complex. Both growth factors appear capable of stimulating cellular proliferation and differentiation, as well as exerting insulin-like effects on intermediary metabolism. We have demonstrated recently that the expression of IGF-II and its receptor is induced during the terminal differentiation of the myoblast cell line, C2, and have suggested that IGF-II may be an autocrine growth factor in these cells (Tollefsen, S.E., Sadow, J.L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1543-1547). We now have examined this cell line for expression of other components involved in IGF signaling. The synthesis of IGF-I is low during myoblast proliferation; IGF-I mRNA can be detected only through use of a sensitive solution hybridization assay. Typical IGF-I receptors can be measured in myoblasts, whereas IGF binding proteins cannot be detected in proliferating cells or in conditioned culture medium. During myogenic differentiation, IGF-I mRNA levels increase transiently by 6-10-fold within 48-72 h. The expression of IGF-I mRNA is accompanied by a 2.5-fold accumulation of IGF-I in the culture medium. IGF-I receptors also increase transiently, doubling by 48 h after the onset of differentiation. By contrast, secretion of a Mr 29,000 IGF binding protein is induced 30-fold to 100 ng/ml within 16 h and continues to increase throughout differentiation. These studies demonstrate that several components critical to IGF action are produced in a fusing skeletal muscle cell line in a differentiation-dependent manner and suggest that both IGF-I and IGF-II may be autocrine factors for muscle.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Production of insulin-like growth factor binding-proteins by bovine adrenomedullary cells: differential regulation by IGF-I and dexamethasone

In the present study we examined the production of insulin-like growth factor binding proteins (IGFBPs), in chromaffin cells, a model system for sympathetic neurons. Four IGFBPs of approximately 27, approximately 31, approximately 36 and a doublet of approximately 45-50 kDa, detected in Western ligand blots of conditioned medium, were identified in Western immunoblots as IGFBP-4, IGFBP-5, IGFBP-2 and IGFBP-3, respectively. In ligand blots IGFBP-3 and IGFBP-4 appeared as the most prominent species. IGF-I (1 nM) enhanced release of IGFBP-3 while dexamethasone (1 nM) diminished release of IGFBP-4. No significant proteolytic degradation of the IGFBPs was demonstrated. Cycloheximide completely attenuated release of the IGFBPs, indicating dependency on new synthesis of the proteins. These findings are consistent with autocrine modulation of the IGF system in bovine adrenomedullary chromaffin cells by IGFBPs. Furthermore, the specific stimulatory and inhibitory effects of IGF-I and dexamethasone, respectively, on release of the predominant species of IGFBP-3 and IGFBP-4, suggested that IGFBP production may be selectively modulated in a positive and negative manner.     

Regulated autocrine growth of CHO cells

The goal of this work was to engineer a CHO cell line capable ofautocrine growth in a fully defined protein-free medium. Thiswas accomplished by stable integration of the genes encodinginsulin-like growth factor I (IGF-I) and transferrin into thegenome of a CHO-K1 cell line. Thelac operator/repressorsystem was used to regulate the expression of the IGF-I gene with thelac operator sequence being placed upstream ofthe coding sequence for IGF-I. The expression of thelacrepressor protein was driven by a modified metallothioneinpromoter allowing repressor expression to be regulated by theculture medium. The cell line calledSuper CHO^r (r for regulated) was able to grow in protein-free medium in an autocrine fashion with a doubling time of 20–24 hr,either attached to microcarriers or as aggregate suspensioncultures. Upon addition of metal to the culture medium, therepressor protein was produced and bound to the operatorsequences shutting down the expression of IGF-I and arrestingthe growth of the cells. Expression of the human growth hormone(hGH) gene and production of hGH was induced by the presence ofmetal ions. It was possible to release the cells from growtharrest in the presence of metal by the addition of isopropyl-D-thiogalactopyranoside (IPTG), which prevented bindingof the repressor to its operator sequences. The ability to growCHO cells in fully defined protein-free medium and to be able toregulate their growth rate offers a number of advantages for theuse of these cells as hosts for the production of recombinantDNA derived proteins.     

Discrete alterations of the insulin-like growth factor I molecule which alter its affinity for insulin-like growth factor-binding proteins result in changes in bioactivity.

Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity. They are present in extracellular fluids and modulate the interactions of their ligands with the type 1 IGF cell surface receptor. These studies utilized IGF-I analogs that have reduced binding affinity for either the type 1 IGF receptor or binding proteins to study the ligand specificity of IGF-BP-1 and the role of IGF-BP-1 in modulating the biological activity of IGF-I. The data indicate that the regions of IGF-I which are responsible for binding to IGF-BP-1 and to human serum-binding proteins are distinct but overlapping and are clearly distinct from the type I receptor binding sites. In the absence of exogenously added IGF-BP-1, the analogs with reduced affinity for IGF-BP-1 are more potent than IGF-I in stimulating DNA synthesis by porcine aortic smooth muscle cells. In contrast, when cells are concomitantly exposed to IGF-BP-1, two of the analogs with reduced affinity for binding protein give only 40-65% of the maximal IGF-I response. [Leu24, 1-62]IGF-I, which has a 100-fold reduced affinity for the type 1 IGF receptor, gave a value that was 62% of the maximal IGF-BP-1 potentiated response. A second biological response, that of stimulating binding protein secretion by IGF-I, was also examined. [Leu24, 1-62]IGF-I is more potent than IGF-I whereas the activity of the analogs with lower affinity for IGF-BP-1 is significantly reduced. Thus, the ability to activate DNA synthesis and binding protein secretion maximally in the presence of IGF-BP-1 is dependent on the affinity of IGFs for both type 1 receptors and binding proteins.     

The role of the IGF-I receptor in the growth and transformation of mammalian cells

Abstract. Recent developments in the molecular biology of the insulin-like growth factor I (IGF-I) receptor have clarified its role in cellular growth and transformation. Although cells homozygous for a targeted disruption of the IGF-I receptor genes can grow in serum-supplemented medium, the IGF-I receptor is required for optimal growth, and is required equally in all phases of the cell cycle. The receptor plays an even more stringent role in cellular transformation and tumorigenicity, which seem to be dependent on its normal expression in several cell types. The expression of both the IGF-I receptor and its ligands is regulated by other growth factors (especially PDGF and EGF), by oncogenes (like SV40 T antigen and c-myb) and by tumour suppressor genes (like WT1 and RB). The picture emerging from these studies is that several transforming agents may exert their growth promoting effects through the direct or indirect activation of the IGF autocrine loop.     

IGF-I causes an ultrasensitive reduction in GH mRNA levels via an extracellular mechanism involving IGF binding proteins

IGF-I-dependent decreases in endogenous GH mRNA expression were studied in individual rat MtT/S somatotroph cells using in situ hybridization. It was first shown that increasing IGF-I concentrations (0-90 nM) decreased GH mRNA levels in a ultrasensitive manner when averaged over the entire population, such that the decrease occurred over a narrow range of IGF-I concentration with an EC50 of 7.1 nM. The degree of ultrasensitivity of the population average was expressed by calculating the Hill coefficient (nA), which had a value of -2.0. GH mRNA levels in individual dispersed cells from these cultures were then measured. These results were first summed for all cells to show that the average response of the population remained ultrasensitive (nA = -2.6, EC50 = 8.1 nM). Then, parameters for individual cells of the population were calculated using mathematical modeling of the distribution of individual cell GH mRNA levels after treatment with 0-90 nM IGF-I. Solution of the data from the individual cells yielded a Hill coefficient (nI = -0.65) and a heterogeneity coefficient (mI = -1.2) indicative of individual cell responsiveness to IGF-I that was not ultrasensitive and very heterogeneous. These results suggested that ultrasensitivity in the population may likely be caused by an extracellular mechanism regulating IGF-I concentrations, such as IGF binding proteins. Increasing concentrations of long (Arg)3IGF-1, an analog that binds the IGF type-1 receptor but not IGF binding proteins, showed a linear inhibition of GH mRNA levels. Treatment with IGF binding protein ligand inhibitor, an IGF-I analog that binds to IGF binding proteins but not the IGF type-1 receptor, decreased GH mRNA levels in the absence of exogenous IGF-I. Thus, IGF binding proteins provide the extracellular sequestration of IGF-I necessary for the precise and ultrasensitive regulation of GH mRNA levels in the entire cell population, although expression within individual cells is regulated in a graded fashion.     

Changes in IGF-I receptor and IGF-I mRNA during differentiation of 3T3-L1 preadipocytes

Insulin-like growth factor-1 (IGF-I) is an essential factor for the differentiation of preadipocytes into adipocytes. We investigated the expression of IGF-I receptor and IGF-I RNA messenger during 3T3-L1 preadipocyte differentiation. Levels of IGF-I receptor decreased in the mature adipocytes compared to cells before the initiation of differentiation. In addition, cultures not induced to differentiate showed a decrease on the receptor levels after 4 days in the presence of insulin compared to cultures without treatment. The levels of the IGF-I RNA messenger were shown to be higher in mature adipocytes compared to preadipocytes. We propose an autocrine and/or paracrine action of IGF-I in this adipocyte differentiation model, where IGF-I produced by the differentiating preadipocytes acts over their adjacent cells and, in this way, diminishes the expression of IGF-I receptor.     

The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells

Insulin-like growth factors I and II (IGF-I and II) and insulin are chemotactic agents for the human melanoma cell line A2058. As shown in this report, the motility receptor mediating this response is the heterodimeric type I IGF receptor. These three factors are able to compete with 125I-labeled IGF-I for binding to the cell surface with IC50 values equal to approximately 2 (IGF-I), approximately 150 (IGF-II), and approximately 300 nM (insulin). Cross-linking of 125I-IGF-I to the cell surface with disuccinimidyl suberate followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveals a 130-kDa protein (reduced) consistent with the alpha component of a type I receptor and a 38-kDa protein which does not bind insulin, and thus could be another IGF-I cell surface binding protein. The anti-IGF-I receptor monoclonal antibody (alpha IR-3) also competes with labeled IGF-I in binding experiments. In contrast, a control monoclonal antibody, matched to alpha IR-3 with respect to IgG subclass, has no significant effect on IGF-I binding. While alpha IR-3 inhibits the motility induced by IGF-I, IGF-II, and insulin, pertussis toxin (0.01-1.0 micrograms/ml) has no significant effect on the motility induced by the insulin-like growth factors or insulin on this cell line. Therefore, the type I IGF receptor appears to mediate a highly potent pertussis toxin-insensitive motility response to IGF-I, IGF-II, and insulin. In contrast, motility induced by the autocrine motility factor, a cytokine produced by the A2058 cells, is not affected by alpha IR-3 but is extremely sensitive to pertussis toxin. When mixtures of autocrine motility factor and IGF-I are employed to induce chemotaxis, the resulting motility is greater than that induced by either agent alone. These data indicate that motility in this melanoma cell line can be initiated through multiple receptors that stimulate the cells by separate transduction pathways. This capability to respond to multiple stimuli could enhance the metastatic potential.     

Structural analogs of human insulin-like growth factor I with reduced affinity for serum binding proteins and the type 2 insulin-like growth factor receptor

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.     

PEGylation of lysine residues reduces the pro-migratory activity of IGF-I

Background

The insulin-like growth factor (IGF) system is composed of ligands and receptors which regulate cell proliferation, survival, differentiation and migration. Some of these functions involve regulation by the extracellular milieu, including binding proteins and other extracellular matrix proteins. However, the functions and exact nature of these interactions remain incomplete.

Methods

IGF-I variants PEGylated at lysines K27, K65 and K68, were assessed for binding to IGFBPs using BIAcore, and for phosphorylation of the IGF-IR. Furthermore, functional consequences of PEGylation were investigated using cell viability and migration assays. In addition, downstream signaling pathways were analyzed using phospho-AKT and phospho-ERK1/2 assays.

Results

IGF-I PEGylated at lysines 27 (PEG-K27), 65 (PEG-K65) or 68 (PEG-K68) was employed. Receptor phosphorylation was similarly reduced 2-fold with PEG-K65 and PEG-K68 in 3T3 fibroblasts and MCF-7 breast cancer cells, whereas PEG-K27 showed a more than 10- and 3-fold lower activation for 3T3 and MCF-7 cells, respectively. In addition, all PEG-IGF-I variants had a 10-fold reduced association rate to IGF binding proteins (IGFBPs). Functionally, all PEG variants lost their ability to induce cell migration in the presence of IGFBP-3/vitronectin (VN) complexes, whereas cell viability was fully preserved. Analysis of downstream signaling revealed that AKT was preferentially affected upon treatment with PEG-IGF-I variants whereas MAPK signaling was unaffected by PEGylation.

Conclusion

PEGylation of IGF-I has an impact on cell migration but not on cell viability.

General significance

PEG-IGF-I may differentially modulate IGF-I mediated functions that are dependent on receptor interaction as well as key extracellular proteins such as VN and IGFBPs.     

Secretion of polypeptides related to epidermal growth factor and insulinlike growth factor I by a human teratocarcinoma cell line

Summary To identify polypeptide growth factors for human teratocarcinoma cells, we studied the malignant ovarian teratoma-derived cell line, PA-1, that grew autonomously in serum-free medium. Medium conditioned by undifferentiated PA-1 cells strongly stimulated proliferation of the mouse mammary tumor cell line, GR 2H6, which is responsive to epidermal growth factor (EGF) and insulinlike growth factor-I (IGF-I). After ammonium sulfate precipitation, PA-1 conditioned medium was analyzed by anion exchange chromatography and bioassay of elution fractions on GR 2H6 cells that were grown in medium deficient in either EGF or insulin. The results demonstrated that PA-1 CM contained factors that can substitute for EGF and IGF-I in stimulating growth of GR 2H6 cells. Western blots of peak mitogenic fractions revealed low molecular weight polypeptides that were immunoreactive with either anti-EGF or anti-IGF-I antibodies. Indirect immunofluorescence staining of PA-1 cells with monoclonal antibodies localized receptors for each growth factor, and binding of human EGF and IGF-I to these cells was quantified by radioreceptor assays. Secretion of factors closely related to EGF and IGF-I by PA-1 cells under serum-free conditions may provide a novel model system to study molecular mechanisms of autocrine growth stimulation in teratocarcinomas.     

Interaction of secreted insulin-like growth factor-I (IGF-I) with cell surface receptors is the dominant mechanism of IGF-I's autocrine actions.

In a prior report we presented evidence that insulin-like growth factor-I (IGF-I) can act in an autocrine fashion by demonstrating that FRTL-5 cells transfected with hIGF-IA fusion genes express and secrete biologically active IGF-I that renders the stimulation of DNA synthesis in FRTL-5 cells independent of their requirement for exogenous IGFs or insulin. To determine if IGF-I's autocrine actions require secretion or can be mediated by interactions with intracellular receptors, we have created a new line of FRTL-5 cells that express a mutant IGF-IA precursor containing the endoplasmic reticulum retention amino acid sequence, Lys-Asp-Glu-Leu (KDEL), at its carboxyl terminus. The mutant IGF-IA/KDEL precursor expressed by stably transfected FRTL-5 cells was shown to be retained intracellularly and to have biological activity comparable with mature IGF-I, as judged by the activity of partially purified IGF-IA/KDEL in wild type FRTL-5 cells. Expression of IGF-IA/KDEL in FRTL-5 cells, however, neither augmented TSH-stimulated DNA synthesis nor stimulated IGF-binding protein-5 expression, as does IGF-IA expression in transfected FRTL-5 cells and the addition of exogenous IGF-I to wild type FRTL-5 cells. IGF-IA/KDEL expression, however, desensitized FRTL-5 cells to the actions of exogenous IGF-I despite having only minimal effects on cell surface type I receptor number, suggesting that intracellular IGF-I is capable of significant biological actions. The failure of IGF-IA/KDEL to replicate the actions of secreted IGF-I, taken together with the findings that a monoclonal antibody against IGF-I blocked IGF-I's actions in IGF-I-secreting transfected FRTL-5 cells, provides evidence that IGF-I secretion and interaction with cell surface type I IGF receptors is the dominant mechanism of IGF-I's autocrine actions.