Matthew M. Rechler

Insulin-like growth factor binding proteins

Publisher Summary This chapter discusses the insulin-like growth factor (IGF) binding proteins—namely, IGF-I and IGF-II. The IGFs are purified from human plasma and cell culture medium by virtue of their ability to stimulate the growth of cartilage or cultured fibroblasts, or their insulin-like activity. The IGFs are synthesized in many fetal and postnatal tissues and are capable of acting locally. IGF-I and IGF-II are single-chain 7.5-kDa polypeptides that are chemically related to insulin. They bind with high affinity to IGF-I receptors that are thought to mediate most of their biological actions. The IGF-I receptor is a homolog of the insulin receptor, having a heterotetrameric structure and a tyrosine kinase domain in the cytoplasmic portion of the β-subunit that phosphorylates the receptor and other substrates, and presumably is involved in transmembrane signaling. Hybrid receptors consisting of one αβ IGF-I receptor heterodimer and one αβ insulin receptor heterodimer are described. IGF-II also binds with high affinity to the IGF-II/Mannose 6-phosphate receptor. IGF-II added with the IGF-II/Mannose 6-phosphate receptor stimulated the activation of the guanosine triphosphate (GTP) binding protein G i-2 . The IGFs participate in the physiological growth of the developing child, fetus, and embryo.

Direct Demonstration of Separate Receptors for Growth and Metabolic Activities of Insulin and Multiplication-stimulating Activity (an Insulinlike Growth Factor) Using Antibodies to the Insulin Receptor

Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [(3)H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each others specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 mug/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K(5) and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 mug/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike effects, and Fab fragments of antireceptor antibody did not stimulate thymidine incorporation. These data demonstrate directly that the insulin receptor mediates the metabolic effects of insulin and MSA, whereas the growth-promoting action of both peptides is mediated by the MSA receptor or other growth factors.

Autonomous growth of a human neuroblastoma cell line is mediated by insulin-like growth factor II.

Insulin-like growth factor II (IGF-II) mRNA was increased in two of eight neuroblastomas and in eight of eight pheochromocytomas, tumors of the adrenal medulla that occur in childhood and adulthood, respectively. RNA encoding the type I IGF receptor, the receptor thought to mediate the mitogenic effects of IGF-I and IGF-II, also was uniformly expressed in these cells. To assess the role of IGF-II in the growth of these tumor cells, we have used the SK-N-AS cultured neuroblastoma cell line, which can be continuously propagated in mitogen-free medium, as a model system. Our results strongly suggest that IGF-II, synthesized by SK-N-AS cells and acting through type I IGF receptors, contributes to the autonomous growth of this tumor cell line. (a) SK-N-AS cells synthesized large amounts of IGF-II RNA and secreted greater than 50 ng/ml of IGF-II (as determined by specific radioimmuno- and radioreceptor assays). Little, if any, IGF-I RNA or immunoreactive IGF-I were detected. (b) SK-N-AS cells possess type I IGF receptors. (c) Exogenous IGF-I and IGF-II stimulated DNA synthesis in SK-N-AS cells, and this stimulation was abolished by a blocking antibody to the type I IGF receptor. (d) This anti-receptor antibody also abolished the multiplication of SK-N-AS cells in the absence of added mitogens. We conclude that IGF-II is an autocrine growth factor for SK-N-AS cells and suggest that this mechanism may contribute to the growth of some adrenal medullary tumors.

Insulin-like growth factor (IGF)-binding protein-3 mutants that do not bind IGF-I or IGF-II stimulate apoptosis in human prostate cancer cells.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can stimulate apoptosis and inhibit cell proliferation directly and independently of binding IGFs or indirectly by forming complexes with IGF-I and IGF-II that prevent them from activating the IGF-I receptor to stimulate cell survival and proliferation. To date, IGF-independent actions only have been demonstrated in a limited number of cells that do not synthesize or respond to IGFs. To assess the general importance of IGF-independent mechanisms, we have generated human IGFBP-3 mutants that cannot bind IGF-I or IGF-II by substituting alanine for six residues in the proposed IGF binding site, Ile56/Tyr57/Arg75/Leu77/Leu80/Leu81, and expressing the 6m-hIGFBP-3 mutant construct in Chinese hamster ovary cells. Binding of both IGF-I and IGF-II to 6m-hIGFBP-3 was reduced >80-fold. The nonbinding 6m-hIGFBP-3 mutant still was able to inhibit DNA synthesis in a mink lung epithelial cell line in which inhibition by wild-type hIGFBP-3 previously had been shown to be exclusively IGF-independent. 6m-hIGFBP-3 only can act by IGF-independent mechanisms since it is unable to form complexes with the IGFs that inhibit their action. We next compared the ability of wild-type and 6m-hIGFBP-3 to stimulate apoptosis in serum-deprived PC-3 human prostate cancer cells. PC-3 cells are known to synthesize and respond to IGF-II, so that IGFBP-3 could potentially act by either IGF-dependent or IGF-independent mechanisms. In fact, 6m-hIGFBP-3 stimulated PC-3 cell death and stimulated apoptosis-induced DNA fragmentation to the same extent and with the same concentration dependence as wild-type hIGFBP-3. These results indicate that IGF-independent mechanisms are major contributors to IGFBP-3-induced apoptosis in PC-3 cells and may play a wider role in the antiproliferative and antitumorigenic actions of IGFBP-3.

Insulin-like growth factors in health and disease

Abstract ▪ The insulin-like growth factor (IGF) family of peptides, binding proteins, and receptors are ubiquitous and important for normal human growth and development. Modern techniques including...

Modulation of insulinlike growth factor I binding to human fibroblast monolayer cultures by insulinlike growth factor carrier proteins released to the incubation media.

The relative contributions of type I and type II insulinlike growth factor (IGF) receptors and IGF carrier proteins to the binding of IGF-I tracer to cultured human fibroblasts were determined in competitive binding experiments that used unlabeled insulin and synthetic insulin-IGF-I hybrid molecules containing the A chain of insulin and the B domain of IGF-I. Whereas insulin binds only to type I IGF receptors, the B-IGF-I hybrids bind to type I receptors and IGF carrier proteins but not to type II receptors. In suspended human fibroblasts, IGF-I tracer binds predominantly to type I IGF receptors (inhibition by IGF-I much greater than insulin greater than B-IGF-I hybrid molecules). By contrast, in fibroblast monolayers, IGF-I binding was minimally inhibited by insulin or hybrid molecules, suggesting predominant binding to the type II IGF receptor. The type I receptor appears to be masked on fibroblast monolayers, and to require suspension or detergent solubilization of the cells to be demonstrated. In the course of the monolayers binding experiments, we noted that low concentrations of unlabeled IGF-I (5-10 ng/ml) or B-IGF-I hybrids (100 ng/ml) paradoxically increased IGF-I tracer binding up to twofold. We postulated that during the binding incubation (5 h, 15 degrees C), IGF-I tracer partitioned between binding sites on the cell surface and IGF carrier proteins released to the incubation media. Preferential occupancy of binding sites in the media by unlabeled ligand increased the tracer available to bind to the cells. In support of this hypothesis, carrier proteins were demonstrated in the media at the end of the binding incubation with fibroblast monolayers, and the concentration of unsaturated binding sites in the media correlated inversely with tracer binding to the cells. Thus carrier proteins released to the media during the binding incubation modulate the binding of IGF-I tracer to cell receptors, suggesting that the carrier proteins may play an important role in regulating cellular responsiveness to the IGFs.

The Coactivator p300 Directly Acetylates the Forkhead Transcription Factor Foxo1 and Stimulates Foxo1-Induced Transcription

The FOXO (Forkhead box class O) subgroup of forkhead transcription factors controls the expression of many genes involved in fundamental cellular processes. Until recently, studies conducted on posttranslational modifications of Forkhead proteins were restricted to their phosphorylation. In this report, we show that the coactivator p300 directly acetylates lysines in the carboxyl-terminal region of Foxo1 in vivo and in vitro, and potently stimulates Foxo1-induced transcription of IGF-binding protein-1 in transient transfection experiments. The intrinsic acetyltransferase activity of p300 is required for both activities. Our results suggest that acetylation of Foxo1 by p300 is responsible, at least in part, for its increased transactivation potency, although acetylation of histones cannot be excluded. Insulin, the major negative regulator of Foxo1-stimulated transcription, potently enhances p300 acetylation of Foxo1. Three consensus protein kinase B/Akt phosphorylation sites whose phosphorylation is stimulated by insulin are required for insulin-induced acetylation of Foxo1. In contrast to its importance in regulating the transcriptional activity of Foxo1 in the absence of insulin, acetylation plays only a minor role compared with phosphorylation in insulin inhibition of Foxo1 transcriptional activity.

Regulatory Actions of Insulin-like Growth Factor-binding Proteins

The six insulin-like growth factor-binding proteins (IGFBPs) are important regulators of insulin-like growth factor (IGF) action. Circulating high molecular weight complexes that contain IGF and IGFBP-3 restrict IGF bioavailability, and excess IGFBPs inhibit IGF action by forming biologically inactive complexes. IGFs can be released from these complexes by proteolysis. Potentiation of IGF activity might occur under specific circumstances, and involves the slow dissociation of IGFs from IGFBP complexes localized in the pericellular space, whose affinity has been reduced by dephosphorylation or association with the cell surface or extracellular matrix. Several IGFBPs or IGFBP fragments also have activities that do not involve IGFs or IGF receptors. The mechanisms by which IGFBPs regulate IGF action and exert their independent actions will be examined.

3 Somatomedin/insulin-like growth factor tissue receptors

Summary There are two types of Sm/IGF receptors based on results of competitive binding experiments and investigations of receptor structure. The type I receptor preferentially interacts with IGF I rather than IGF II and interacts weakly with insulin. This receptor has a binding subunit of M r = 130 000 which is disulphide bonded to form larger structures of M r greater than 300 000. The type II receptor prefers IGF II to IGF I and does not interact with insulin. Its binding subunit is not linked by disulphide bonds to other membrane components ( M r = 260 000 with reduction, 220 000 without reduction). Subunit organization of the type I receptor appears to be similar to that of the insulin receptor but it is unlikely that the insulin and Sm/IGF binding sites are on a common a subunit. The type I receptor is down-regulated by IGFs and insulin. A rapid increase in ligand binding to the type II receptor by insulin has been described in intact rat adipocytes. The original idea that an IGF receptor mediates the growth-promoting action of both IGFs and insulin while acute metabolic effects of insulin and IGFs are mediated by the insulin receptor is an oversimplification. There now are clear examples of insulin stimulating growth by acting through the insulin receptor and, conversely, instances of IGF stimulating glucose transport by acting through an IGF receptor. Radioreceptor assays which measure IGF I in preference to IGF II (human placental membrane) and which measure IGF II in preference to IGF I (rat liver and rat placental membranes) have been utilized for clinical measurements of Sm/IGF levels, but are less specific than the respective radioimmunoassays. With the demonstration of Sm/IGF receptors on circulating human mononuclear cells and human skin fibroblasts, it is expected that these, systems will be useful for investigations of patients with possible end-organ resistance to Sm/IGF.

Editorial: Growth Inhibition by Insulin-Like Growth Factor (IGF) Binding Protein-3—What’s IGF Got To Do with It?

Life has suddenly become more interesting for the insulinlike growth factor binding proteins (IGFBPs). For years the IGFBPs were relegated to preventing IGF-I or IGF-II from binding to IGF-I receptors and activating the signaling pathways that stimulated cell proliferation or survival (1, 2). They did this job well because they bound IGFs with higher affinity than did the receptors, forming inactive complexes that could not bind to the IGF-I receptor. First came the suggestion that they could potentiate IGF action, albeit only under precisely defined in vitro conditions (2). Now, recent studies with IGFBP-3, the most abundant IGFBP in the circulation, have opened new vistas for the proteins. Recent papers from several laboratories have reported that IGFBP-3 can: 1) inhibit growth without binding IGF-I and blocking its access to the IGF-I receptor; 2) travel to the cell nucleus instead of remaining outside the cell like a pariah; 3) induce apoptosis; and 4) mediate the potent growth inhibitory actions of transforming growth factor(TGF-) b and possibly the induction of apoptosis by the tumor suppressor, p53. And if this were not enough, new cousins (IGFBPs 7–10) have been discovered. These are heady times, indeed. There were earlier premonitions that IGFBP-3 might have a secret life as an inhibitor of cell proliferation independent of IGFs and IGF-I receptors by a mechanism that did not involve sequestering IGF-I. Liu et al. (3) reported that rat IGFBP-3 inhibited the stimulation of chick embryo fibroblast (CEF) DNA synthesis by a serum fraction from which IGFs had been removed by acid gel-filtration. But the skeptics countered that other growth factors in serum might have stimulated the synthesis of endogenous IGFs, so that sequestration remained a possibility. Support came from Oh et al. (4), who showed that exogenous IGFBP-3 inhibited constitutively activated DNA synthesis in a human breast cancer cell line, and from Cohen et al. (5), who demonstrated that stable transfection of Balb c/3T3 mouse fibroblasts with human IGFBP-3 complementary DNA (cDNA) decreased the rate of cell proliferation. The key to the latter experiment was that addition of insulin, which has mitogenic activity in these cells but does not bind to and therefore can not be inhibited by IGFBP-3, could not overcome the decrease in cell proliferation. Still another blow to the skeptics came when Lalou et al. (6) showed that a 16-kDa fragment of IGFBP-3, generated by limited proteolysis of human recombinant IGFBP-3 with plasmin, inhibited IGF-Iand insulin-stimulated DNA synthesis in CEFs. Although both peptides act through the IGF-I receptor, the critical point is that the 16-kDa IGFBP-3 fragment has negligible binding affinity for IGF-I and presumably does not bind insulin, so that sequestration of the growth peptides was unlikely to explain the inhibition of cell proliferation. The best was yet to come. Studies using a fibroblast cell line developed from mice with a targeted disruption of the IGF-I receptor (R cells) (7) established that the growth inhibitory effects of IGFBP-3 did not involve IGF binding to the IGF-I receptor. First, Valentinis et al. (8) reported that stable transfection of human IGFBP-3 cDNA slowed the proliferation of R cells, as it had in Balb c/3T3 fibroblasts. And in the present issue, Zadeh and Binoux (9) demonstrate that the 16-kDa IGFBP-3 fragment inhibited the stimulation of DNA synthesis in R cells by basic fibroblast growth factor (bFGF). Even if bFGF induced IGF-I synthesis in these experiments, the IGF-I could not have stimulated DNA synthesis through the IGF-I receptor. (Only a diehard skeptic would suggest that IGF-I still might act through another receptor pathway, such as the insulin receptor). If IGFBP-3 is not acting by simply preventing IGF-I from binding to the IGF-I receptor, how does it inhibit growth? The final answer is not in, but some important clues are available. First, it has been appreciated for several years that IGFBP-3 associates with different cells and that this binding was decreased by incubation with heparin (10, 11). Although these experiments were initially interpreted as indicating that heparin competitively inhibited the binding of IGFBP-3 to a cell-associated heparan sulfate proteoglycan, this does not appear to be the case because complete enzymatic removal of heparan sulfate and other glycosaminoglycans from the cell surface did not affect IGFBP-3 binding (12). An alternative explanation, that the binding of heparin to IGFBP-3 induced a conformational change in the protein that decreased its ability to bind to putative IGFBP-3 receptors on the cell surface, was proposed. A heparin binding domain (XBBBXXBX, where B is a basic amino acid and X is a nonbasic amino acid) is present in a highly basic region (residues 214–232) in the COOH-terminal portion of IGFBP-3. Identification of the putative IGFBP-3 receptors remains elusive. Although several cell-associated proteins that bind IGFBP-3 have been described (13–15), the specificity of the binding and whether these IGFBP-3-binding proteins have a functional role in growth inhibition by IGFBP-3 must be established before they can be considered signaling IGFBP-3 receptors. Nonetheless, the ability of IGFBP-3 to bind to cells is strongly correlated with its ability to cause growth inhibition, suggesting that IGFBP-3 must interact with specific cell receptors before growth inhibition can occur. The strongest evidence for this hypothesis comes from the concomiReceived May 8, 1997. Address all correspondence and requests for reprints to: Dr. Matthew M. Rechler, National Institutes of Health, Building 10, Room 8D-14, 10 Center Drive, MSC 1758, Bethesda, Maryland 20892-1758. E-mail: mrechler@helix.nih.gov. 0013-7227/97/