The role of the IGF axis in hepatocarcinogenesis.

Primary hepatocellular carcinoma (HCC) is one of the most common forms of malignant cancer with the fourth highest mortality rate worldwide. Major risk factors for the development of HCC include chronic infections with the hepatitis B or C virus, alcohol consumption, exposure to dietary aflatoxin B1, hereditary liver disease or liver cirrhosis of any etiology. Recent studies have discovered changes in the insulin-like growth factor (IGF) axis that affect the molecular pathogenesis of HCC, including the autocrine production of IGFs, IGF binding proteins (IGFBPs), IGFBP proteases, and IGF receptor expression. Characteristic alterations detected in HCC and hepatoma cell lines comprise the overexpression of IGF-II and the IGF-I receptor emerging as critical events in malignant transformation and growth of tumors. Simultaneous reduction of IGFBP expression and the increase in proteolytic cleavage of IGFBPs result in an excess of bioactive IGFs. Finally, defective functions of the IGF-II/mannose 6-phosphate receptor involved in degradation of IGF II, the activation of the growth inhibitor TGF-beta1, and the lysosomal targeting of cathepsin proteases capable to degrade extracellular matrix proteins may contribute to the development of HCC.     

IGF binding proteins and their functions

The insulin-like growth factor (IGF) binding proteins (IGFBPs) have several functions, including transporting the IGFs in the circulation, mediating IGF transport out of the vascular compartment, localizing the IGFs to specific cell types, and modulating both IGF binding to receptors and growth-promoting actions. The functions of IGFBPs appear to be altered by posttranslational modifications. IGFBP-3, -4, -5, and -6 have been shown to be glycosylated. Likewise all the IGFBPs have a complex disulfide bond structure that is required for maintenance of normal IGF binding. IGFBP-2, -3, -4, and -5 are proteolytically cleaved, and specific proteases have been characterized for IGFBP-3, -4, and -5. Interestingly, attachment of IGF-I or II to IGFBP-4 results in enhancement of proteolysis, whereas attachment of either growth factor to IGFBP-5 results in inhibition of proteolytic cleavage. Cleavage of IGFBP-3 results in the appearance of a 31 kDa fragment that is 50-fold reduced in its affinity for the IGF-I or IGF-II. In spite of the reduction in its affinity, this fragment is capable of potentiating the effect of IGF-I on cell growth responses; therefore, proteolysis may be a specific mechanism that alters IGFBP modulation of IGF actions. Other processes that result in a reduction in IGF binding protein affinity are associated with potentiation of cellular responses to IGF-I and -II. Specifically, the binding of IGFBP-3 to cell surfaces is associated with its ability to enhance IGF action and with a ten- to 12-fold reduction in its affinity for IGF-I and IGF-II. Likewise, binding of IGFBP-5 to extracellular matrix (ECM) results in an eightfold reduction in its affinity and a 60% increase in cell growth in response to IGF-I. Another post-translational modification that modifies IGFBP activity is phosphorylation. IGFBP-1, -2, -3, and -5 have been shown to be phosphorylated. Phosphorylation of IGFBP-1 results in a sixfold enhancement in its affinity for IGF-I and -II. Following this enhancement of IGFBP-1 affinity, this binding protein loses its capacity to potentiate IGF-I growth-promoting activity. Future studies using site-directed mutagenesis to modify these proteins should enable us to determine the effect of these posttranslational modifications on the ability of IGFBPs to modulate IGF biologic activity. © 1993 Wiley-Liss, Inc.     

Insulin-like growth factor binding protein (IGFBP)-3-bound IGF-I and IGFBP-3-bound IGF-II in growth hormone deficiency

In blood, circulating IGFs are bound to six high-affinity IGFBPs, which modulate IGF delivery to target cells. Serum IGFs and IGFBP-3, the main carrier of IGFs, are upregulated by GH. The functional role of serum IGFBP-3-bound IGFs is not well understood, but they constitute the main reservoir of IGFs in the circulation. We have used an equation derived from the law of mass action to estimate serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II, as well as serum free IGF-I and free IGF-II, in 129 control children and adolescents (48 girls and 81 boys) and in 13 patients with GHD. Levels of serum total IGF-I, total IGF-II, IGFBP-1, IGFBP-2 and IGFBP-3 were determined experimentally, while those of IGFBP-4, IGFBP-5 and IGFPB-6, as well as the 12 affinity constants of association of the two IGFs with the six IGFBPs, were taken from published values. A correction for in vivo proteolysis of serum IGFBP-3 was also considered. In controls, serum total IGF-I, total IGF-II, IGFBP-3, IGFBP-3-bound IGF-I, IGFBP-3-bound IGF-II and free IGF-I increased linearly with age, from less than 1 to 15 years, in the two sexes. The concentrations of serum free IGF-I and free IGF-II were approximately two orders of magnitude below published values, as well as below the affinity constant of association of IGF-I with the type-1 IGF receptor. Therefore, it is unlikely that these levels can interact with the receptor. In the 13 patients with GHD, mean (+/- SD) SDS of serum IGFBP-3-bound IGF-I was -2.89 +/- 0.97. It was significantly lower than serum total IGF-I, free IGF-I or IGFBP-3 SDSs (-2.35 +/- 0.83, -1.12 +/- 0.78 and -2.55 +/- 1.07, respectively, p = 0.0001). The mean SDS of serum total IGF-II, IGFBP-3-bound IGF-II and free IGF-II were -1.25 +/- 0.68, -2.03 +/- 0.87 and 0.59 +/- 1.10, respectively, in GHD. In control subjects, 89.8 +/- 4.47% of serum total IGF-I and 77.3 +/- 9.4% of serum total IGF-II were bound to serum IGFBP-3. In patients with GHD, the mean serum IGFBP-3-bound IGF-I and IGFBP-3-bound IGF-II were 8.63 +/- 8. 53 and 19.1 +/- 14.7% below the respective means of control subjects (p < 0.02). In conclusion, in GHD there was a relative change in the distribution of serum IGFs among IGFBPs, due to the combined effects of the decrease in both total IGF-I and IGFBP-3. As a result, serum IGFBP-3-bound IGF-I and IGFBP-3 bound IGF-II, the main reservoirs of serum IGFs, were severely affected. This suggests that the decrease in serum IGFPB-3-bound IGF-I and IGFBP-3-bound IGF-II might have a negative effect for growth promotion and other biological effects of IGF-I and IGF-II. Finally, the estimation of serum IGFBP-3-bound IGF-I, or the percentage of total IGF-I and IGF-II bound to IGFBP-3, might be useful markers in the diagnosis of GHD.     

Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of six homologous proteins with high binding affinity for IGF-I and IGF-II. Information from NMR and mutagenesis studies is advancing knowledge of the key residues involved in these interactions. IGF binding may be modulated by IGFBP modifications, such as phosphorylation and proteolysis, and by cell or matrix association of the IGFBPs. All six IGFBPs have been shown to inhibit IGF action, but stimulatory effects have also been established for IGFBP-1, -3, and -5. These generally involve a decrease in IGFBP affinity and may require cell association of the IGFBP, but precise mechanisms are unknown. The same three IGFBPs have well established effects that are independent of type I IGF receptor signaling. IGFBP-1 exerts these effects by signaling through alpha(5)beta(1)-integrin, whereas IGFBP-3 and -5 may have specific cell-surface receptors with serine kinase activity. The regulation of cell sensitivity to inhibitory IGFBP signaling may play a role in the growth control of malignant cells.     

Biosensor measurement of the interaction kinetics between insulin-like growth factors and their binding proteins.

The binding kinetics of human insulin-like growth factor binding protein (IGFBP) 1-6 for recombinant human insulin-like growth factor (IGF) I and II were measured and compared in the present study using surface plasmon resonance biosensor technique. Different concentrations of IGFBPs (5-100 nM) were allowed to interact with the immobilized IGF-I or IGF-II on sensor chip surface. Both des(1-3)IGF-I and insulin are known to bind weakly to the IGFBPs and therefore are used as negative controls for the binding experiments. The resultant sensorgrams were analyzed by using simple 1:1 binding model to derive both the association rate (k(a)) and dissociation rate (k(d)) constants for IGFBP-IGF interactions. The k(a) values of IGFBPs are in the range of 1x10(4) to 9x10(5) M(-1) s(-1) for IGF-I and 7x10(3) to 1.7x10(6) M(-1) s(-1) for IGF-II, respectively. The orders of k(a) for both IGF-I and IGF-II are IGFBP-3>IGFBP-5>IGFBP-6>IGFBP-4>IGFBP-2>++ +IGFBP-1. The k(d) values of IGFBPs are in the range of 1.5x10(-5) to 2x10(-4) s(-1) for IGF-I and 3.6x10(-5) to 3.7x10(-4) s(-1) for IGF-II, respectively. The order of k(d) for IGF-I is IGFBP-6>IGFBP-5>IGFBP-4>IGFBP-3>IGFBP-2>++ +IGFBP-1 and that for IGF-II is IGFBP-5>IGFBP-6>IGFBP-2>IGFBP-4>IGFBP-3>++ +IGFBP-1, respectively. The equilibrium affinity constants (K(A)) were calculated based on the ratio of k(a)/k(d) and were more precise than the published literature values based on competitive radioligand binding assays. The systematic study enables a direct comparison on the IGF-binding properties among the various IGFBPs, and the kinetic data provide additional information to delineate the physiological role of different IGFBPs in vivo.     

C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II

IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.     

Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis

During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.     

The role of the insulin-like growth factors in the central nervous system

Increasing evidence strongly supports a role for insulin-like growth factor-I (IGF-I) in central nervous system (CNS) development. IGF-I, IGF-II, the type IIGF receptor (the cell surface tyrosine kinase receptor that mediates IGF signals), and some IGF binding proteins (IGFBPs; secreted proteins that modulate IGF actions) are expressed in many regions of the CNS beginningin utero. The expression pattern of IGF system proteins during brain growth suggests highly regulated and developmentally timed IGF actions on specific neural cell populations. IGF-I expression is predominantly in neurons and, in many brain regions, peaks in a fashion temporally coincident with periods in development when neuron progenitor proliferation and/or neuritic outgrowth occurs. In contrast, IGF-II expression is confined mainly to cells of mesenchymal and neural crest origin. While expression of type I IGF receptors appears ubiquitous, that of IGFBPs is characterized by regional and developmental specificity, and often occurs coordinately with peaks of IGF expression. In vitro IGF-I has been shown to stimulate the proliferation of neuron progenitors and/or the survival of neurons and oligodendrocytes, and in some cultured neurons, to stimulate function. Transgenic (Tg) mice that overexpress IGF-I in the brain exhibit postnatal brain overgrowth without anatomic abnormality (20–85% increases in weight, depending on the magnitude of expression). In contrast, Tg mice that exhibit ectopic brain expression of IGFBP-1, an inhibitor of IGF action when present in molar excess, manifest postnatal brain growth retardation, and mice with ablated IGF-I gene expression, accomplished by homologous recombination, have brains that are 60% of normal size as adults. Taken together, these in vivo studies indicate that IGF-I can influence the development of most, if not all, brain regions, and suggest that the cerebral cortex and cerebellum are especially sensitive to IGF-I actions. IGF-I’s growth-promoting in vivo actions result from its capacity to increase neuron number, at least in certain populations, and from its potent stimulation of myelination. These IGF-I actions, taken together with its neuroprotective effects following CNS and peripheral nerve injury, suggest that it may be of therapeutic benefit in a wide variety of disorders affecting the nervous system.     

The Insulin-like Growth Factor System and Its Biological Actions in Fish

The insulin-like growth factor (IGF) system plays an importantrole in controlling animal development and growth. There arethree components to the IGF system: ligands (IGF-I and IGF-II),receptors (type I and type II IGF receptors) and IGF-bindingproteins (IGFBPs). These members of the IGF system are expresseddifferentially and their expression patterns implicate themin critical developmental events. Even though IGFs have beenproven essential for normal fetal growth through experimentsusing IGF "knock-out" mice, the precise role of each memberof the IGF system is not fully understood. Recent studies suggestthat the IGF system has been highly conserved in teleost fish.This paper provides an overview of what is currently known aboutthe IGF system in teleost fish. Several aspects of the IGF systemare discussed, including the structural and functional aspectsof fish IGFs, the fish IGF receptors and IGFBPs. The biologicalactions of fish IGFs are described in reference to their rolesin growth, development, reproduction and osmoregulation. Finally,some of the unique advantages of using teleost fish as experimentalmodels for defining the developmental role of the IGF systemand the underlying molecular mechanisms are discussed     

Insulin-like growth factor binding protein-6: the "forgotten" binding protein?

Insulin-like growth factor binding protein-6 (IGFBP-6) differs from IGFBPs 1-5 in that it binds IGF-II with marked preferential affinity over IGF-I. Human and rat IGFBP-6 lack 2 and 4 N-terminal cysteines and therefore the Gly-Cys-Gly-Cys-Cys motif present in IGFBPs 1-5. IGFBP-6 is O-glycolsylated, and five serine/threonine glycosylation sites in the non-conserved mid-region of human IGFBP-6 have been identified. O-Glycosylation inhibits proteolysis of IGFBP-6 by chymotrypsin and trypsin, but has no effect on high affinity IGF binding. IGFBP-6 is a relatively specific inhibitor of IGF-II actions; it has not been shown to potentiate IGF actions. IGFBP-6 is only cell-associated to a very limited extent, if at all. IGFBP-6 is often expressed in non-proliferative, quiescent states in vitro and differentiating agents increase its expression. IGFBP-6 expression is associated with inhibition of growth of tumour cells in vitro and in vivo. Although many questions remain regarding the biological role of IGFBP-6, its major function appears to be the regulation of IGF-II actions. This could be especially significant since IGF-II has been implicated as an autocrine tumour growth factor.     

Characteristics of binding of insulin-like growth factor (IGF)-I and IGF-II analogues to the type 1 IGF receptor determined by BIAcore analysis.

Insulin-like growth factor (IGF) binding to the type 1 IGF receptor (IGF1R) elicits mitogenic effects, promotion of differentiation and protection from apoptosis. This study has systematically measured IGF1R binding affinities of IGF-I, IGF-II and 14 IGF analogues to a recombinant high-affinity form of the IGF1R using BIAcore technology. The analogues assessed could be divided into two groups: (a) those designed to investigate binding of IGF-binding protein, which exhibited IGF1R-binding affinities similar to those of IGF-I or IGF-II; (b) those generated to probe IGF1R interactions with greatly reduced IGF1R-binding affinities. The relative binding affinities of IGF-I analogues and IGF-I for the IGF1R determined by BIAcore analysis agreed closely with existing data from receptor-binding assays using cells or tissue membranes, demonstrating that BIAcore technology is a powerful tool for measuring affinities of IGFs for IGF1R. In parallel studies, IGF1R-binding affinities were related to ability to protect against serum withdrawal-induced apoptosis in three different assays including Hoechst 33258 staining, cell survival, and DNA fragmentation assays using the rat pheochromocytoma cell line, PC12. In this model system, IGF-I and IGF-II at low nanomolar concentrations are able to prevent apoptosis completely. We conclude that ability to protect against apoptosis is directly related to ability to bind the IGF1R.     

Tissue-specific expression of messenger ribonucleic acids for insulin-like growth factors and insulin-like growth factor-binding proteins during perinatal development of the rat uterus

Insulin-like growth factor (IGF)-I and IGF-II play a number of important roles in growth and differentiation, and IGF-binding proteins (IGFBPs) modulate IGF biological activity. IGF-I has been shown previously to be essential for normal uterine development. Therefore, we used in situ hybridization assays to characterize the unique tissue- and developmental stage-specific pattern of expression for each IGF and IGFBP gene in the rat uterus during perinatal development (gestational day [GD]-20 to postnatal day [PND]-24). IGF-I and IGFBP-1 mRNAs were expressed in all uterine tissues throughout this period. IGFBP-3 mRNA was not detectable at GD-20 but became detectable beginning at PND-5, and the signal intensity appeared to increase during stromal and muscle development. IGFBP-4 mRNA was abundant throughout perinatal development in the myometrium and in the stroma, particularly near the luminal epithelium. IGFBP-5 mRNA was abundantly expressed in myometrium throughout perinatal development. IGFBP-6 mRNA was detected throughout perinatal development in both the stroma and myometrium in a diffuse expression pattern. IGF-II and IGFBP-2 mRNAs were not detected in perinatal uteri. Our results suggest that coordinated temporal and spatial expression of IGF-I and its binding proteins (IGFBP-1,-3,-4,-5, and -6) could play important roles in perinatal rodent uterine development.     

Production of insulin-like growth factor II (IGF-II) and different forms of IGF-binding proteins by HT-29 human colon carcinoma cell line.

The serum-free medium conditioned by the human colon cancer cell line HT-29 contains insulin-like growth factors (IGF) that are entirely complexed to binding proteins (IGF-BP). Gel filtration in acid conditions of the cell-conditioned medium permits separation of IGF-BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF-BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF-I and IGF-II radioreceptor assays, IGF-I radioimmunoassay (RIA), and competitive protein-binding assay specific for IGF-II, it is shown that the IGF-type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF-II. Its concentration is approximately 6 ng/10(6) HT-29 cells with 60% present as a high-molecular-weight form of IGF-II. This large 15 K IGF molecule is devoided of any IGF-binding activity and might represent incomplete processing of pro-IGF-II peptide. By contrast, the level of IGF-I detected by RIA is barely measurable and considered negligible (0.57 pg/10(6) HT-29 cells). Although these IGF-II-like peptides exhibit a growth-promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF-I or IGF-II, 3H-thymidine incorporation into DNA of HT-29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT-29-D4 clonal cells, derived from the parental HT-29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.     

A novel binding site for the human insulin-like growth factor-II (IGF-II)/mannose 6-phosphate receptor on IGF-II

The mammalian insulin-like growth factor (IGF)-II/cation-independent mannose 6-phosphate receptor (IGF2R) binds IGF-II with high affinity. By targeting IGF-II to lysosomal degradation, it plays a role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Loss of IGF2R function is associated with tumor progression; therefore, the IGF2R is often referred to as a tumor suppressor. The interaction between IGF2R and IGF-II involves domains 11 and 13 of the 15 extracellular domains of the receptor. Recently, a hydrophobic binding region was identified on domain 11 of the IGF2R. In contrast, relatively little is known about the residues of IGF-II that are involved in IGF2R binding and the determinants of IGF2R specificity for IGF-II over the structurally related IGF-I. Using a series of novel IGF-II analogues and surface plasmon resonance assays, this study revealed a novel binding surface on IGF-II critical for IGF2R binding. The hydrophobic residues Phe(19) and Leu(53) are critical for IGF2R binding, as are residues Thr(16) and Asp(52). Furthermore, Thr(16) was identified as playing a major role in determining why IGF-II, but not IGF-I, binds with high affinity to the IGF2R.     

Gene therapy vector-mediated expression of insulin-like growth factors protects cardiomyocytes from apoptosis and enhances neovascularization

IGF-I and IGF-II are single-chain polypeptide growth factors that regulate pleiotropic cellular responses. We have characterized the effect of recombinant IGF proteins, as well as third-generation adenoviral vectors encoding either IGF-I or IGF-II genes, on cardiomyocyte apoptosis and on angiogenesis. We found that endothelial cells cultured in the presence of the extracellular protein laminin exhibit a robust response to IGF-I and -II proteins via enhanced cell migration and angiogenic outgrowth. Furthermore, IGF vectors greatly enhanced neovascularization in an in vivo Matrigel model. Transduction of cardiomyocytes with the IGF adenoviral vectors resulted in a dose- and time-dependent increase in the expression of IGF-I or IGF-II protein. This correlated with abrogation of apoptosis induced by ischemia-reoxygenation, ceramide, or heat shock with optimal inhibition of approximately 80%. We conclude that gene transfer of IGF-I and IGF-II is a plausible strategy for the local delivery of IGFs to treat ischemic heart disease and heart failure by stimulating angiogenesis and protecting cardiomyocytes from cell death.     

Role of insulin-like growth factors and their binding proteins in growth control and carcinogenesis

Interest in the role of the insulin-like growth factor (IGF) axis in growth control and carcinogenesis has recently been increased by the finding of elevated serum insulin-like growth factor I (IGF-I) levels in association with three of the most prevalent cancers in the United States: prostate cancer, colorectal cancer, and lung cancer. IGFs serve as endocrine, autocrine, and paracrine stimulators of mitogenesis, survival, and cellular transformation. These actions are mediated through the type 1 IGF-receptor (IGF-1R), a tyrosine kinase that resembles the insulin receptor. The availability of free IGF for interaction with the IGF-1R is modulated by the insulin-like growth factor-binding proteins (IGFBPs). IGFBPs, especially IGFBP-3, also have IGF-independent effects on cell growth. IGF-independent growth inhibition by IGFBP-3 is believed to occur through IGFBP-3-specific cell surface association proteins or receptors and involves nuclear translocation. IGFBP-3-mediated apoptosis is controlled by numerous cell cycle regulators in both normal and disease processes. IGFBP activity is also regulated by IGFBP proteases, which affect the relative affinities of IGFBPs, IGFs and IGF-1R. Perturbations in each level of the IGF axis have been implicated in cancer formation and progression in various cell types.