Expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptors in EL4 lymphoma cells overexpressing growth hormone

Almost all of the previous studies with growth hormone (GH) have been done with exogenously supplied GH and, therefore, involve actions of the hormone through its receptor. However, the actions of endogenous or lymphocyte GH are still unclear. In a previous study, we showed that overexpression of GH (GHo) in a lymphoid cell line resulted in protection of the cells to apoptosis mediated by nitric oxide (NO). In the present study, we show that the protection from apoptosis could be transferred to control cells with culture fluids obtained from GHo cells and blocked by antibodies to the insulin-like growth factor-1 (IGF-1) or antibodies to the IGF-1-receptor (IGF-1R). Northern and Western blot analysis detected significantly higher levels of IGF-1 in cells overexpressing GH. An increase in the expression of the IGF-1R in GHo cells was also detected by Western blot analysis, (125)I-IGF-1 binding and analysis of IGF-1R promoter luciferase constructs. Transfection of GHo cells with a dominant negative IGF-1R mutant construct blocked the generation of NO and activation of Akt seen in GHo cells compared to vector alone control EL4 cells. The results suggest that one of the consequences of the overexpression of GH, in cells lacking the GH receptor, is an increase in the expression of IGF-1 and the IGF-1R which mediate the protection of EL4 lymphoma cells from apoptosis.     

Structural differences between insulin and somatomedin-C/insulin-like growth factor-1 receptors revealed by autoantibodies to the insulin receptor

Polyadenylated RNA prepared from first trimester human placenta was translated in a membrane-free cell-free system derived from wheat germ. Analysis of the [³⁵S]methionine-labeled products by SDS-polyacrylamide electrophoresis demonstrated two proteins with apparent M_rs of 14,500 and 16,000 that were specifically immunoprecipitated by antiserum to reduced and carboxylated bovine LH_α, and two different proteins with apparent M_rs of 18,500 and 21,000 that were specifically immunoprecipitated by antiserum to hCG_β. None of these products was sensitive to cleavage by endoglycosidase H, whereas the M_r 21,000 product precipitated by antisera to bovine LH_α and to hCG_α from translations supplemented by canine pancreatic microsomes was processed to a product with M_r 13,000 by endoglycosidase H. We suggest that the two forms of the α and β subunit precursors could arise from the translation of two distinct mRNAs encoding each subunit.     

Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody

Background

Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.

Methodology/Principal Findings

In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.

Conclusion and Significance

The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.     

Novel nuclear localization and potential function of insulin-like growth factor-1 receptor/insulin receptor hybrid in corneal epithelial cells

Background

Type I insulin-like growth factor receptor (IGF-1R) and insulin receptor (INSR) are highly homologous molecules, which can heterodimerize to form an IGF-1R/INSR hybrid (Hybrid-R). The presence and biological significance of the Hybrid-R in human corneal epithelium has not yet been established. In addition, while nuclear localization of IGF-1R was recently reported in cancer cells and human corneal epithelial cells, the function and profile of nuclear IGF-1R is unknown. In this study, we characterized the nuclear localization and function of the Hybrid-R and the role of IGF-1/IGF-1R and Hybrid-R signaling in the human corneal epithelium.

Methodology/Principle Findings

IGF-1-mediated signaling and cell growth were examined in a human telomerized corneal epithelial (hTCEpi) cell line using co-immunoprecipitation, immunoblotting and cell proliferation assays. The presence of Hybrid-R in hTCEpi and primary cultured human corneal epithelial cells was confirmed by immunofluorescence and reciprocal immunoprecipitation of whole cell lysates. We found that IGF-1 stimulated Akt and promoted cell growth through IGF-1R activation, which was independent of the Hybrid-R. The presence of Hybrid-R, but not IGF-1R/IGF-1R, was detected in nuclear extracts. Knockdown of INSR by small interfering RNA resulted in depletion of the INSR/INSR and preferential formation of Hybrid-R. Chromatin-immunoprecipitation sequencing assay with anti-IGF-1R or anti-INSR was subsequently performed to identify potential genomic targets responsible for critical homeostatic regulatory pathways.

Conclusion/Significance

In contrast to previous reports on nuclear localized IGF-1R, this is the first report identifying the nuclear localization of Hybrid-R in an epithelial cell line. The identification of a nuclear Hybrid-R and novel genomic targets suggests that IGF-1R traffics to the nucleus as an IGF-1R/INSR heterotetrameric complex to regulate corneal epithelial homeostatic pathways. The development of novel therapeutic strategies designed to target the IGF-1/IGF-1R pathway must take into account the modulatory roles IGF-1R/INSR play in the epithelial cell nucleus.     

Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2

Insulin and insulin-like growth factor-1 (IGF-1) regulate metabolism and body growth through homologous receptor tyrosine kinases that phosphorylate the insulin receptor substrate (IRS) proteins. IRS-2 is an important IRS protein, as it mediates peripheral insulin action and beta-cell survival. In this study, we show that insulin, IGF-1, or osmotic stress promoted ubiquitin/proteasome-mediated degradation of IRS-2 in 3T3-L1 cells, Fao hepatoma, cells and mouse embryo fibroblasts; however, insulin/IGF-1 did not promote degradation of IRS-1 in 3T3-L1 preadipocytes or mouse embryo fibroblasts. MG132 or lactacystin, specific inhibitors of 26S proteasome, blocked insulin/IGF-1-induced degradation of IRS-2 and enhanced the detection of ubiquitinated IRS-2. Insulin/IGF1-induced ubiquitination and degradation of IRS-2 was blocked by inhibitors of phosphatidylinositol 3-kinase (wortmannin or LY294002) or mTOR (rapamycin). Chronic insulin or IGF-1 treatment of IRS-1-deficient mouse embryo fibroblasts inhibited IRS-2-mediated activation of Akt and ERK1/2, which was reversed by lactacystin pretreatment. By contrast, IRS-1 activation of Akt and ERK1/2 was not inhibited by chronic insulin/IGF-1 stimulation in IRS-2-deficient mouse embryo fibroblasts. Thus, we identified a novel negative feedback mechanism by which the ubiquitin/proteasome-mediated degradation of IRS-2 limits the magnitude and duration of the response to insulin or IGF-1.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Mediation of the actions of insulin and insulin-like growth factor-1 on preimplantation mouse embryos in vitro.

Previous studies showed that both insulin and insulin-like growth factor-1 (IGF-1) stimulate metabolism and growth of preimplantation embryos. Because the effects of insulin occur with very low doses, it was suggested that its effects were mediated by its own receptors. However, the effects of IGF-1 occurred at higher doses, suggestive of cross reaction with the insulin receptor but still in the range for mediation via its own receptor. The aim of this study was to investigate the mediation of the metabolic and growth effects of insulin and IGF-1 using a specific insulin receptor antagonist. The antagonistic B-10 Fab fragment (B-10f) completely blocked stimulation of protein synthesis by both insulin and IGF-1, indicating that the insulin receptor mediates this action of both hormones. Alternately, only insulin's stimulation of inner cell mass mitogenesis and morphological development was inhibited by the B-10 Fab fragment. This showed that growth stimulation by insulin and IGF-1 was mediated via different receptors, insulin through its own receptor and IGF-1 through some other receptor. However, mediation via the IGF-2 receptor is not excluded since IGF-1 stimulates compaction when there is evidence for only the presence of the IGF-2 receptor. In summary, insulin or IGF-1 at physiological concentrations stimulates preimplantation mouse embryos, suggesting an important role for both these growth factors in early development.     

Conversion of growth hormone-secreting cells into prolactin-secreting cells and its promotion by insulin and insulin-like growth factor-1 in vitro

The newly established rat pituitary cell line, MtT/S, has pituitary somatotroph (growth hormone-producing cell)-like characteristics, i.e., the cells produce growth hormone (GH), possess GH-immunopositive secretory granules, and respond to GH-releasing hormone. When MtT/S cells were cultured in regular medium no prolactin (PRL) cells were observed and PRL was not detected, by radioimmunoassay or Western blot analysis, in the medium or the cells. However, GH production and the GH cell population decreased markedly when the cells were incubated with insulin or insulin-like growth factor-1 (IGF-1). After stimulation with insulin or IGF-1 there was a 2-day lag period, then some PRL was detected in the medium; after 5 days a number of PRL cells appeared. Double immunocytochemistry indicated clearly that no cell contained both PRL and GH. These results show that insulin and IGF-1 stimulate conversion of MtT/S cell line GH cells to PRL cells. This suggests that the MtT/S cell line is an excellent model system which shows the GH-cell/PRL-cell lineage.     

Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/insulin-like growth factor-1 signaling

Zinc is an effector of insulin/IGF-1 signaling and has insulinomimetic effects, the molecular basis of which is not understood. The present study establishes the capacity of zinc to inhibit protein tyrosine phosphatases (PTPs) as a cause for these effects and, moreover, demonstrates modulation of the insulin response by changes in intracellular zinc. The inhibition of PTPs by zinc occurs at significantly lower concentrations than previously reported. In vitro, zinc inhibits PTPs 1B and SHP-1 with IC(50) values of 17 and 93 nM, respectively. A fluorescent probe with a similar binding constant [FluoZin-3, K(D)(Zn) = 15 nM] detects corresponding concentrations of zinc within cells. Increase of cellular zinc after incubation with both zinc and the ionophore pyrithione augments protein tyrosine phosphorylation, and in particular the phosphorylation of three activating tyrosine residues of the insulin/IGF-1 receptor. Vice versa, specific chelation of cellular zinc with the membrane-permeable N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine suppresses insulin- and IGF-1-stimulated phosphorylation. In the context of the emerging concept that intracellular zinc is tightly regulated and fluctuates dynamically, these results suggest that a pool of cellular zinc modulates phosphorylation signaling.     

Impact of reduced insulin-like growth factor-1/insulin signaling on aging in mammals: novel findings

Growth hormone deficiency or resistance resulting from spontaneous or experimentally produced mutations in laboratory mice delay aging and increase lifespan. Alterations in insulin-like growth factor-1 (IGF-1) and insulin signaling emerged as likely mechanisms linking growth hormone and aging, and increased longevity was reported in mice with selective deletion of IGF-1 receptor in all tissues or insulin receptor in fat. Recent studies in mice with reduced IGF-1 levels or deletion of pregnancy-associated plasma protein-A, a protease that cleaves one of the IGF-1 binding proteins, strongly support the role of IGF-1 in the control of longevity. Reports of increased lifespan in mice with deletion of insulin receptor substrate (IRS) 1, reduced expression of IRS2, or selective deletion of IRS2 in the brain specifically implicate the IRS-PI3K-Akt-Foxo signaling pathway (which is shared by IGF-1 and insulin) in the control of aging. These important novel findings also strengthen the evidence for evolutionary conservation of mechanisms regulating lifespan in worms, insects and mammals.     

An insulin-like growth factor I/insulin hybrid exhibiting high potency for interaction with the type I insulin-like growth factor and insulin receptors of placental plasma membranes

We have prepared by semisynthetic methods a two-chain insulin/insulin-like growth factor I hybrid that contains a synthetic peptide related to residues 22-41 of insulin-like growth factor I linked via peptide bond to ArgB22 of des-octapeptide-(B23-B30)-insulin and have applied the analog to the analysis of ligand interactions with the type I insulin-like growth factor and insulin receptors of placental plasma membranes. Relative potencies for the inhibition of 125I-labeled insulin-like growth factor I binding to type I insulin-like growth factor receptors were 1.0:0.20:0.003 for insulin-like growth factor I, the hybrid analog, and insulin, respectively. Corresponding relative potencies for the inhibition of 125I-labeled insulin binding to insulin receptors were 0.007:0.28:1 for the three respective peptides. Additional studies identified that the hybrid analog interacts with only one of two populations of insulin-like growth factor I binding sites on placental plasma membranes and permitted the analysis of insulin-like growth factor I interactions with the separate populations of binding sites. We conclude that (a) des-octapeptide-(B23-B30)-insulin can serve well as a scaffold to support structural elements of insulin-like growth factor I and insulin necessary for high affinity binding to their receptors, (b) major aspects of structure relevant to the conferral of receptor binding affinity lie in the COOH-terminal region of the insulin B chain and in the COOH-terminal region of the insulin-like growth factor I B domain and in its C domain, and (c) the evolution of ligand-receptor specificity in these systems has relied as much on restricting interactions (through the selective introduction of negative structural elements) as it has on enhancing interactions (through the introduction of affinity conferring elements of structure).     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

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The sequence determinant causing different folding behaviors of insulin and insulin-like growth factor-1

Although insulin and insulin-like growth factor-1 (IGF-1) belong to the insulin superfamily and share highly homologous sequences, similar tertiary structure, and a common ancestor molecule, amphioxus insulin-like peptide, they have different folding behaviors: IGF-1 folds into two thermodynamically stable tertiary structures (native and swap forms), while insulin folds into one unique stable structure. To further understand which part of the sequence determines their different folding behavior, based on previous reports from the laboratory, two peptide models, [B9A][1-4]porcine insulin precursor (PIP) and [B10E][1-4]PIP, were constructed. The plasmids encoding the peptides were transformed into yeast cells for expression of the peptides; the results showed that the former peptide was expressed as single component, while the latter was expressed as a mixture of two components (isomer 1 and isomer 2). The expression results together with studies of circular dichoism, disulfide rearrangement, and refolding lead us to deduce that isomer 1 corresponds to the swap form and the isomer 2 corresponds to the native form. We further demonstrate that the sequence 1-4 plus B9 of IGF-1 B-domain can make PIP fold into two structures, while sequence 1-5 of insulin B-chain can make IGF-1 fold into one unique structure. In other words, it is the IGF-1 B-domain sequence that 1-4 allows IGF-1 folding into two thermodynamically stable tertiary structures; this sequence plus its residue B9E can change PIP folding behavior from folding into one unique structure to two thermodynamically stable structures, like that of IGF-1.     

Paradoxical biological effects of overexpressed insulin-like growth factor-1 receptors in chinese hamster ovary cells

One major approach to the study of growth factor receptor action has been to overexpress wild-type or mutant receptors in cultured cells and to evaluate biological responses to exogenous ligand. Studies of this type with insulin and insulin-like growth factor-I (IGF-I) receptors often use Chinese hamster ovary (CHO) cells. We have compared the effect of receptor overexpression in CHO cells and in NIH-3T3 fibroblasts in order to assess the suitability of CHO cells for studies of this nature and the contribution of cell type-specific factors to those responses generally assayed. Overexpression of IGF-I receptors in NIH-3T3 cells resulted in increased sensitivity and maximal responsiveness of thymidine incorporation, 2-deoxyglucose uptake, and phosphatidylinositol-3 (PI3) kinase activation to IGF-I stimulation. In CHO cells, on the other hand, overexpression of either IGF-I or insulin receptors increased the sensitivity of thymidine incorporation to ligand, but maximal responsiveness was unchanged or decreased. Overexpression of the insulin receptor increased sensitivity of glucose uptake and the maximal response of PI3 kinase activation to insulin. Overexpression of the IGF-I receptor did not affect sensitivity or maximal responsiveness of glucose uptake or PI3 kinase activation to IGF-I. These data suggest that IGF-I and insulin signal pathways may differ in CHO cells, and that there may even be divergent IGF-I signaling pathways for short vs. long-term effects. Whether this is a result of differences in the number of endogenous receptors, hybrid receptor formation, or defects in post-receptor signaling, the use of CHO cells to assess receptor function must be approached with caution. © Wiley-Liss, Inc.     

Differential activation of insulin receptor substrates 1 and 2 by insulin-like growth factor-activated insulin receptors

The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.     

Receptors for insulin and insulin-like growth factor-1 and insulin receptor substrate-1 mediate pathways that regulate islet function

The insulin/insulin-like growth factor-1 (IGF-1) signalling pathways are present in most mammalian cells and play important roles in the growth and metabolism of tissues. Most proteins in these pathways have also been identified in the beta-cells of the pancreatic islets. Tissue-specific knockout of the insulin receptor (betaIRKO) or IGF-1 receptor (betaIGFRKO) in pancreatic beta-cells leads to altered glucose-sensing and glucose intolerance in adult mice, and betaIRKO mice show an age-dependent decrease in islet size and beta-cell mass. These data indicate that these receptors are important for differentiated function and are unlikely to play a major role in the early growth and/or development of the pancreatic islets. Conventional insulin receptor substrate-1 (IRS-1) knockouts manifest growth retardation and mild insulin resistance. The IRS-1 knockouts also display islet hyperplasia, defects in insulin secretory responses to multiple stimuli both in vivo and in vitro, reduced islet insulin content and an increased number of autophagic vacuoles in the beta-cells. Re-expression of IRS-1 in cultured beta-cells is able to partially restore the insulin content indicating that IRS-1 is involved in the regulation of insulin synthesis. Taken together, these data provide evidence that insulin and IGF-1 receptors and IRS-1, and potentially other proteins in the insulin/IGF-1 signalling pathway, contribute to the regulation of islet hormone secretion and synthesis and therefore in the maintenance of glucose homeostasis.     

Identification of selective basophil chemoattractants in human nasal polyps as insulin-like growth factor-1 and insulin-like growth factor-2

In a search for novel leukocyte chemoattractants at sites of allergic inflammation, we found basophil-selective chemoattractant activity in extracts of human nasal polyps. The extracts were fractionated by reverse phase HPLC, and the resulting fractions were tested for leukocyte-stimulating activity using sensitive shape change assays. The basophil-selective activity detected was not depleted by a poxvirus CC-chemokine-binding protein affinity column. This activity was further purified by HPLC, and proteins in the bioactive fractions were analyzed by tandem electrospray mass spectrometry. Insulin-like growth factor-2 (IGF-2) was identified in these HPLC fractions, and the basophil-stimulating activity was inhibited by an anti-IGF-2-neutralizing Ab. Recombinant IGF-2 induced a substantial shape change response in basophils, but not eosinophils, neutrophils, or monocytes. IGF-2 stimulated chemokinesis of basophils, but not eosinophils or neutrophils, and synergized with eotaxin-1/CCL11 in basophil chemotaxis. IGF-2 also caused up-regulation of basophil CD11b expression and inhibited apoptosis, but did not stimulate degranulation or Ca(2+) flux. Recombinant IGF-1 exhibited similar basophil-selective effects as IGF-2, and both growth factors were detected in nasal polyp extracts by ELISA. This is the first demonstration of chemokinetic factors that increase the motility of basophils, but do not act on other granulocytes or monocytes. IGF-1 and IGF-2 could play a role in the selective recruitment of basophils in vivo.     

胰岛素样生长因子I对心脏的作用

胰岛素样生长因子I(IGF I)是属于胰岛素家族的一种多肽。它可促进心脏生长发育 ,增强心脏功能 ,参与心肌肥厚、心力衰竭和心肌细胞凋亡等病理过程。本文对心脏的IGF I来源及其受体、IGF I的心脏效应及可能的机制进行综述。IGF I对心脏疾病 (心肌肥厚、心力衰竭等 )的防治有潜在的临床应用价值。