Insulin-like Growth Factor (IGF) I Down-Regulates Type 1 IGF Receptor (IGF 1R) and Reduces the IGF I Response in A549 Non-Small-Cell Lung Cancer and Saos-2/B-10 Osteoblastic Osteosarcoma Cells

The insulin-like growth factor type 1 receptor (IGF 1R) mediates the acute metabolic effects of IGF I as well as IGF I-stimulated cell proliferation and protection from apoptosis. IGF binding proteins (IGFBPs) can modulate these responses. We, therefore, investigated whether intrinsic IGFBPs interfere with IGF I-induced regulation of IGF 1R expression and with the biological response to IGF I in two human tumor cell lines, the non-small-cell lung cancer cell line A549 and the osteoblastic osteosarcoma cell line Saos-2/B-10. We compared the growth rates, IGFBP production, IGF I binding characteristics, IGF 1R protein and mRNA levels, and the acute IGF I response (stimulation of glycogen synthesis) after pretreatment of the cells in serum-free medium with or without added IGF I or medium supplemented with 5% fetal calf serum (FCS). In contrast to A549 cells, which produce IGF I and significant amounts of IGFBPs, survival and proliferation of Saos-2/B-10 cells, which do not produce IGF I or significant amounts of IGFBPs, depended on the addition of exogenous IGF I. IGF I increased the concentration of IGFBP-2 and -3 and decreased the concentration of IGFBP-4 in the medium of A549 cells. As compared to FCS, IGF I pretreatment in both cell lines decreased the number of specific IGF I binding sites, down-regulated total and membrane IGF 1R protein, and largely reduced or abolished the acute IGF I response without affecting IGF 1R mRNA levels. The data suggest that the IGF 1R protein of the two cell lines is translationally and/or posttranslationally down-regulated by its ligand in the presence and in the absence of locally produced IGFBPs and that the cell lines have retained this negative feedback to counteract IGF I stimulation.     

Structural analogs of human insulin-like growth factor (IGF) I with altered affinity for type 2 IGF receptors

We have used site-directed mutagenesis of a synthetic gene for insulin-like growth factor (IGF) I to prepare three analogs in which specific residues in the A region are replaced with the corresponding residues in the A chain of insulin. The analogs are [Ile41, Glu45, Gln46, Thr49, Ser50, Ile51, Ser53, Tyr55, Gln56]IGF I (A chain mutant), in which residue 41 is changed from threonine to isoleucine and residues 42 to 56 of the A region are replaced, [Thr49, Ser50, Ile51]IGF I, and [Tyr55, Gln56]IGF I. These analogs are all equipotent to IGF I at the type 1 IGF receptor in human placental membranes, and in stimulating the incorporation of [3H]thymidine into DNA in the rat vascular smooth muscle cell line A10. However, the A chain mutant and [Thr49, Ser50, Ile51]IGF I have greater than 20-fold lower relative affinity for the type 2 IGF receptor of rat liver membranes, respectively. In contrast, [Tyr55, Gln56]IGF I has 7-fold higher affinity than IGF I for the type 2 IGF receptor. Residues 49, 50, and 51 in IGF I are Phe-Arg-Ser and are strictly conserved in IGF II. Residues 55 and 56 of IGF I and the corresponding residues in IGF II are Arg-Arg and Ala-Leu, respectively. Thus, the presence of the charged residues at these positions in IGF I appears to be responsible, in part, for the lower affinity of IGF I for the type 2 IGF receptor. In addition to the alterations in affinity for the type 2 IGF receptor, the A chain mutant has a 7-fold increase in affinity for insulin receptors, and [Thr49, Ser50, Ile51]IGF I has a 4-fold lower affinity for acid-stable human serum binding protein. These data strongly suggest that specific determinants in the A region of IGF I are important for maintaining binding to the type 2 IGF receptor, and that these determinants are different from those required for maintaining high affinity for the type 1 IGF receptor.     

Type I IGF receptors of Leydig cells are upregulated by human chorionic gonadotropin

The effects of human chorionic gonadotropin (hCG) on type I insulin-like growth factor (IGF) receptors of purified Leydig cells were investigated. Sprague-Dawley rats (50 day-old) were treated with a single injection of hCG 10 units intraperitoneally, type I IGF receptors were then determined daily for 4 days. HCG caused a rapid increase in type I IGF receptors within 24 h, which returned to basal by 72 h. There was no significant change in binding affinity. Our present study indicates that type I IGF receptors of Leydig cells are up regulated by hCG, and this may be one mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.     

IGF I induces differentiation in a transformed human keratinocyte line

A comparison of normal epithelial cells with their transformed counterparts could lead to the definition of parameters related to growth and differentiation which are altered by viral transformation and which may be relevant to malignant changes in vivo. Using the SV40-transformed human keratinocyte line, SVK14, which exhibits characteristics of simple, nonkeratinizing epithelia, we have shown that IGF I stimulation of these cells results in extensive multilayering, increased cell size, accumulation of involucrin, modulation of keratin 18 and expression of keratins 14 and 10, whilst T-antigen expression is maintained in the multilayered cells. Since T-antigen expression is correlated directly with impairment of stratification and differentiation, it is interesting that treatment of SVK14 with a single growth factor. IGF I, results in molecular events characteristic of differentiating normal keratinocytes.     

Insulin and insulin-like growth factor I (IGF I) in early mouse embryogenesis

Growth factors have an important role in the regulation of cell growth, division and differentiation. They are also involved in the regulation of embryonic growth and differentiation. Insulin and insulin-like growth factor I (IGF I) play an important part in these events in the later stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that insulin and IGF I are also secreted by embryonic tissues during the prepancreatic stage of mouse development. We found measurable amounts of insulin and IGF I in 8- to 12-day-old mouse embryos. We also showed that embryonic cells derived from 8-, 9- and 10-day-old mouse embryos secrete insulin, IGF I and/or related molecules. Furthermore, the same growth factors, when added to the culture of 9-day-old mouse embryonic cells, stimulate their proliferation. These results lead to the conclusion that insulin can stimulate the growth of embryonic cells during the period when pancreas is not yet formed, which is indirect evidence for a paracrine (or autocrine) type of action.     

Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

Background

Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling.

Methodology/Principal Findings

A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists.

Conclusions/Significance

The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.     

A novel fetal insulin-like growth factor (IGF) I receptor. Mechanism for increased IGF I- and insulin-stimulated tyrosine kinase activity in fetal muscle

Insulin and insulin-like growth factor (IGF) I receptors from fetal and adult rat skeletal muscle were compared in order to gain insight into the evolving functions of the hormones during development. Basal, insulin-stimulated, and IGF I-stimulated receptor phosphorylation and tyrosine kinase activity are severalfold higher in partially purified receptor preparations from fetal muscle in comparison with equal numbers of receptors from adult muscle. There are distinct insulin and IGF I receptors with Mr 95,000 beta subunits in adult muscle, as evidenced by hormone dose-response curves, immunoprecipitation with specific antibodies, binding to insulin and IGF I affinity columns, and analysis of tryptic phosphopeptides. In addition to these two receptor species, fetal muscle contains a receptor with a Mr 105,000 beta subunit. The fetal receptor is structurally more closely related to the IGF-I receptor than the insulin receptor on the basis of its precipitation with specific antibodies, binding to an IGF I affinity column, and tryptic phosphopeptide map. The fetal receptor does not appear to bind insulin but, unlike the IGF-I receptor, its phosphorylation is stimulated by low physiological concentrations of both insulin and IGF I. This could be explained by the cross-phosphorylation of fetal receptors by activated insulin receptors. Expression of the fetal receptor is highest in the fetus and decreases markedly during the first 2 weeks of postnatal life. The fetal receptor appears to account for the high tyrosine kinase activity of fetal muscle and may be an important mediator of responses to both insulin and IGF I early in development.     

Insulin-like growth factor I (IGF I) receptor autophosphorylation and kinase activity. Effect of a human polyclonal antibody (pIgG)

IGF I receptor is a tyrosine kinase capable of phosphorylating the receptor itself and other substrates. A high degree of homology does exist in tyrosine kinase domain among receptors for several polypeptide growth factor receptors and this enzymic activity has been indicated as a possible mediator of biological action. Nevertheless growth factor receptors possess peculiar specificities both in their functions and tissue distribution. A human polyclonal IgG (pIgG), previously characterized as anti insulin receptor antibody, able to inhibit insulin receptor kinase activity, was used to further investigate subunit homologies and differences in antigenicity and functional regulation between IGF I and insulin receptors, IGF I receptor tyrosine kinase was stimulated by a IGF I analog (aIGF I), produced by DNA recombinant technology, pIgG was able to inhibit IGF I receptor kinase activity, thus revealing antigenic homologies between the kinase domains of insulin and IGF I receptors. However the more pronounced inhibition of IGF I receptor-compared with insulin receptor kinase activity by pIgG suggests the existence of different regulatory mechanisms.     

Phosphorylation of insulin-like growth factor I receptor by insulin receptor tyrosine kinase in intact cultured skeletal muscle cells

The interaction between insulin and insulin-like growth factor I (IGF I) receptors was examined by determining the ability of each receptor type to phosphorylate tyrosine residues on the other receptor in intact L6 skeletal muscle cells. This was made possible through a sequential immunoprecipitation method with two different antibodies that effectively separated the phosphorylated insulin and IGF I receptors. After incubation of intact L6 cells with various concentrations of insulin or IGF I in the presence of [32P]orthophosphate, insulin receptors were precipitated with one of two human polyclonal anti-insulin receptor antibodies (B2 or B9). Phosphorylated IGF I receptors remained in solution and were subsequently precipitated by anti-phosphotyrosine antibodies. The identities of the insulin and IGF I receptor beta-subunits in the two immunoprecipitates were confirmed by binding affinity, by phosphopeptide mapping after trypsin digestion, and by the distinct patterns of expression of the two receptors during differentiation. Stimulated phosphorylation of the beta-subunit of the insulin receptor correlated with occupancy of the beta-subunit of the insulin receptor by either insulin or IGF I as determined by affinity cross-linking. Similarly, stimulation of phosphorylation of the beta-subunit of the IGF I receptor by IGF I correlated with IGF I receptor occupancy. In contrast, insulin stimulated phosphorylation of the beta-subunit of the IGF I receptor at hormone concentrations that were associated with significant occupancy of the insulin receptor but negligible IGF I receptor occupancy. These findings indicate that the IGF I receptor can be a substrate for the hormone-activated insulin receptor tyrosine kinase activity in intact L6 skeletal muscle cells.     

Regulation of hepatic expression of IGF I and fetal IGF binding protein mRNA in streptozotocin-diabetic rats

Hepatic mRNA levels of insulin-like growth factor I (IGF I) and of the fetal, nonglycosylated 32 kDa IGF-binding protein (BP) were analysed in diabetic, diabetic insulin- and IGF I-treated rats as well as in age-matched, healthy control animals. IGF ImRNA levels are reduced in diabetic rats and increased by insulin treatment. In contrast, the infusion of IGF I does not significantly upregulate IGF I mRNA levels. Fetal IGF BP mRNA expression is very low in healthy control animals, but high levels are found in diabetic rats. Insulin therapy lowers fetal IGF BP mRNA levels, whereas IGF I has no effect. We propose that insulin is a major regulator of the 32 kDa IGF BP levels in adult rats.     

Increase of IGF I binding to erythrocyte receptors during the TRH-test: correlation between IGF I binding and thyroid hormone values

The effects of TRH on insulin-like growth factor I receptors were investigated on erythrocytes from 7 GH-deficient children having plasma GH levels less than 10 ng/ml during two provocation tests. Intravenous injection of synthetic TRH (0.2 mg/m2) was followed by a marked increase of IGF I binding on erythrocytes, from 3.9% +/- 0.3% to 5.9% +/- 0.3% (P less than 0.005) after 1 hour and 7.3% +/- 0.4% (P less than 0.005) after 2 hours. The IGF I binding variations were due to an increase in both the receptor affinity and the number of sites. The levels of plasma GH, IGF I, T3, T4, free T4, TSH and prolactin having been determined during the TRH test at 0, 1 hour, and 2 hours after the injection, the increase in the IGF I binding to erythrocytes at the same time correlated with the rise of thyroid hormones: triiodothyronine T3 (P less than 0.001) and thyroxine T4 (P less than 0.005) and not with the level of the other hormones. These findings suggest that thyroid hormones play a role in the regulation of insulin-like growth factor I receptors.     

Madin-Darby canine kidney cells display type I and type II insulin-like growth factor (IGF) receptors

Using affinity cross-linking techniques, we report the presence of type I IGF and type II IGF receptors in Madin-Darby canine kidney cells, a line of cells lacking insulin receptors. The IGF receptors were further characterized by competition binding studies and found to be similar to IGF receptors in other tissue types. In Madin-Darby canine kidney cells, the type I IGF receptor binds IGF-I greater than IGF-II greater than insulin and the type II IGF receptor binds IGF-II and IGF-I with approximately the same affinity, but does not bind insulin.     

Insulin-like growth factors (IGF I and IGF II) mimic the effect of insulin on plasma protein synthesis and glycogen deposition in cultured hepatocytes

Monolayer cultures of chick embryo hepatocytes were used to compare the effects of insulin-like growth factors, IGF I and IGF II, with insulin on two hepatic functions: plasma protein production and glycogen deposition. Just as with exposure of the cells to insulin, addition of either IGF I or IGF II to the otherwise hormone-free medium elicited a dramatic change in the production of secretory proteins as well as the development of glycogen deposits equivalent to in vivo (fed) levels. No major differences between insulin, IGF I or IGF II were observed in terms of the degree of stimulation or potency of the hormones.     

The binding sites of insulin-like growth factor I (IGF I) to type I IGF receptor and to a monoclonal antibody. Mapping by chemical modification of tyrosine residues

The surface topography of IGF I(insulin-like growth factor I) was investigated by chemical modification of amino acid residues in free IGF I and bound to type I IGF receptor or to monoclonal antibody MAB43. Tyrosine residues were modified either by chloramine-T or lactoperoxidase catalyzed iodination. In the free IGF I molecule, all 3 tyrosine residues, A19 (Tyr-60), B25 (Tyr-24), and C2 (Tyr-31), were iodinated. Monoclonal antibody MAB43 protected IGF I against modification at tyrosine residue A19, and in the type I IGF receptor-IGF I complex, all 3 tyrosine residues were shielded against iodine incorporation. These results allow the prediction of the binding domains in the IGF I molecule. The minimal receptor binding site in IGF I would include amino acid residues B25 to C2 and, possibly, the C-terminal part of the A-domain with tyrosine residue A19.     

Insulin-like growth factor I (IGF I) induces cortisol production in bovine adrenocortical cells in primary culture

The effects of a physiological dose of IGF I (40 ng/ml approximately 5 x 10(-9) M) on steroidogenesis were studied in bovine adrenal fasciculata cells cultured in serum-free McCoy's medium. They were compared with those of a single dose of ACTH (0.25 ng/ml approximately 10(-10) M) at approximately the concentration inducing half-maximal stimulation. With IGF I, steroidogenesis commenced after 48 h culture and progressively increased throughout the 96-h test period. Expressed as stimulated level/control level ratios, glucocorticoid (cortisol + corticosterone) responses to IGF I after 4 days' culture (2.41 +/- 0.20 (SEM) n = 9) were similar to those obtained with ACTH (2.59 +/- 0.18, n = 9). A combination of the two peptides had a synergistic effect (5.95 +/- 0.79, n = 5). The cortisol/corticosterone ratio increased in the presence of IGF I from 1 +/- 0.19 to 1.76 +/- 0.45 (n = 7, P less than 0.02), although less so than in the presence of ACTH (5.50 +/- 0.98). Moreover, cortisol production was accompanied by androstenedione production (2.36 ng/10(6) cells, n = 3) similar to that induced by ACTH (2.10 ng/10(6) cells, n = 3). These findings together suggest stimulation of 17 alpha-hydroxylase activity. Cell multiplication was unaffected by IGF I. [3H]Thymidine incorporation into DNA reached only 193% +/- 17 (SEM) (n = 4) of control levels, whereas with ACTH it dropped to 60% +/- 5. Our findings show that IGF I alone has no mitogenic effect on adrenocortical cells in vitro, but that it is capable of inducing differentiated steroidogenesis.     

17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor

BACKGROUND: Human MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17beta-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains. RESULTS: The MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains. CONCLUSIONS: The results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer.     

Type I and II insulin-like growth factor receptors on human phytohemagglutinin-activated T lymphocytes

Human T cells activated with mitogens, antigens, or antibodies to the T-cell receptor complex acquire a cascade of new receptors, including the receptors for interleukin-2, transferrin, and insulin. We investigated whether receptors for insulin-like growth factors (IGF) also were expressed on activated T cells. Based on competitive binding studies, immunoprecipitation of labeled cell surface receptors and blocking of radiolabeled peptide binding by a specific monoclonal antibody (alpha IR-3) to the type I IGF receptor, as well as affinity crosslinking of radiolabeled peptides to their receptors, we concluded that both type I and type II IGF receptors are expressed on activated T cells. A specific binding site for IGF-II also was observed on the type I IGF receptor which was not inhibited by alpha IR-3. Receptors for IGF were more numerous on activated T cells than on resting T cells, and their peak expression appeared by the peak of DNA synthesis. Thus, human activated T cells were shown to express both type I and II IGF receptors which could potentially play a role in the regulation of T-cell proliferation, differentiation, and function.     

A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity.

We obtained 20 mouse monoclonal antibodies specific for human type I insulin-like growth factor (IGF) receptors, using transfected cells expressing high levels of receptors (IGF-1R/3T3 cells) as immunogen. The antibodies immunoprecipitated receptor.125I-IGF-I complexes and biosynthetically labeled receptors from IGF-1R/3T3 cells but did not react with human insulin receptors or rat type I IGF receptors. Several antibodies stimulated DNA synthesis in IGF-1R/3T3 cells, but the maximum stimulation was only 25% of that produced by IGF-I. The antibodies fell into seven groups recognizing distinct epitopes and with different effects on receptor function. All the antibodies reacted with the extracellular portion of the receptor, and epitopes were localized to specific domains by investigating their reaction with a series of chimeric IGF/insulin receptor constructs. Binding of IGF-I was inhibited up to 90% by antibody 24-60 reacting in the region 184-283, and by antibody 24-57 reacting in the region 440-586. IGF-I binding was stimulated up to 2.5-fold by antibodies 4-52 and 16-13 reacting in the region 62-184, and by antibody 26-3 reacting downstream of 283. The latter two groups of antibodies also dramatically stimulated insulin binding to intact IGF-1R/3T3 cells (by up to 50-fold), and potentiated insulin stimulation of DNA synthesis. Scatchard analysis indicated that in the presence of these antibodies, the affinity of the type I IGF receptor for insulin was comparable with that of the insulin receptor. These data indicate that regions both within and outside the cysteine-rich domain of the receptor alpha-subunit are important in determining the affinity and specificity of ligand binding. These antibodies promise to be valuable tools in resolving issues of IGF-I receptor heterogeneity and in studying the structure and function of classical type I receptors and insulin/IGF receptor hybrids.     

Preferential enhancement of myoblast differentiation by insulin-like growth factors (IGF I and IGF II) in primary cultures of chicken embryonic cells

Cells prepared from the body walls of chicken embryos were plated in the absence of serum. Insulin-like growth factors (IGFs) barely stimulated cell replication, but preferentially enhanced the differentiation of muscle cells. Myoblast fusion was favoured in the presence of IGF (or insulin). Concomitantly, acetylcholinesterase activity increased. IGF I and IGF II were equipotent and active in low physiological concentrations, in contrast to insulin, which was known for a long time to exert such effects at pharmacological concentrations.