Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins.

The insulin-like growth factor-binding proteins (IGFBPs) comprise a family of six related peptides that interact with high affinity with IGFs. IGFBPs compete with IGF receptors for IGF binding, and as a consequence of this competition they can affect cell growth. In addition, IGF-independent regulatory mechanisms of IGFBPs have been described. Despite their common property to interact with IGFs every IGFBP is expressed in a tightly regulated time- and tissue-specific manner suggesting that each protein may have its own distinct functions. Several transgenic mouse models overexpressing IGFBP-1, -2, -3, or -4 were developed in the past few years. Brain abnormalities were a common feature of IGFBP-1 transgenic models. Individual strains showed alterations in glucose homeostasis, reproductive performance, and a reduction of somatic growth as the most prominent phenotypes. The latter was also the main effect observed in IGFBP-2 transgenic mice. The overexpression of IGFBP-3 under the control of an ubiquitous promoter resulted in selective organomegaly, whereas mammary gland-targeted expression of this protein caused an altered involution after pregnancy in this organ. Tissue-specific overexpression of IGFBP-4 resulted in hypoplasia and reduced weight of smooth muscle-rich tissues such as bladder, aorta, and stomach. This review summarizes the current knowledge about the actions of IGFBPs in vivo based on the presently established transgenic mice.     

Interaction of Insulin-Like Growth Factor-Binding Protein 2 with α2-Macroglobulin in the Circulation

Insulin-like growth factors (IGFs) play active role in mitogenic and metabolic processes. In the peripheral circulation, they are mostly bound to specific IGF-binding proteins (IGFBPs). Proteolysis of IGFBPs releases free, active IGFs. IGFBP-2 is the second most abundant of the six binding proteins and its concentration increases in catabolic states. The possible interaction between IGFBP-2 and other proteins in the circulation was investigated in this study. Our results showed that IGFBP-2 associates with α2-macroglobulin (α2M), a protease inhibitor. Formation of IGFBP-2/α2M complexes most likely contributes to the regulation of IGFBP-2 proteolysis and, thus, the activity of IGFs.     

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.     

Perlustrin, a Haliotis laevigata (abalone) nacre protein, is homologous to the insulin-like growth factor binding protein N-terminal module of vertebrates.

The 84-amino-acid-long sequence of perlustrin showed homology of the abalone nacre protein to the N-terminal domain of mammalian insulin-like growth factor binding proteins (IGFBPs). Despite the evolutionary distance between mollusks and mammals, the sequence identity was 40% including 12 conserved cysteines. However, the residues which were suggested recently to bind IGF-II in a complex with IGFBP-5 were conserved only partially. Nevertheless, perlustrin bound human IGFs with K(D) approximately 10(-7) M. This was the same affinity range as measured before for the interaction of isolated IGFBP-5 N-terminal domains with IGFs. Moreover, perlustrin bound bovine insulin with only approximately two- to sevenfold lower affinity than IGFs. Sequence similarity and growth factor binding identified perlustrin unequivocally as a member of the IGFBP family, the first found in an invertebrate biomineral. Nacre is known to contain proteinaceous factors which promote bone formation in vitro and in vivo. Bone contains IGFBPs which influence bone metabolism in many ways by modulating either IGF effects or IGF independently. Thus, perlustrin may provide a first clue at the molecular level to what these two phylogenetically rather distant biomineralization systems have in common.     

The interaction of Insulin-like Growth Factors (IGFs) with Insulin-like Growth Factor Binding Proteins (IGFBPs): a review

Summary The interactions between insulin-like growth factor binding proteins (IGFBPs) and insulin-like growth factors (IGFs) are important in regulating the availability of IGFs to the type 1 IGF receptor (IGF1R) but there is still a lack of information regarding the mechanism of IGF binding by IGFBPs. While many studies have defined general regions of the IGFBPs that interact with IGFs, only recently have specific IGF binding residues been described. This review will outline the structural evidence describing residues of both the IGFBPs and IGFs involved in the IGFBP·IGF interaction.     

Differential regulation of insulin-like growth factor binding protein (IGFBP)-2 mRNA in liver and bone cells by insulin and retinoic acid in vitro.

Isolated cells produce insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). Two distinct cell types were studied with regard to IGFBP-2 expression: (i) rat hepatocytes, which produce IGF I at a high rate and thus regulate its plasma concentration; and (ii) rat osteoblasts, which are targets of IGF I action. IGFBP-2 expression is low in hepatocytes prepared from normal adult rats and high in calvaria cells from newborn rats. Retinoic acid stimulates IGFBP-2 production by liver cells. Insulin suppresses both basal and retinoic acid-induced IGFBP-2 mRNA expression in hepatocytes and has no such effect on osteoblasts. Retinoic acid and insulin regulate IGFBP-2 expression in a tissue-specific manner.     

Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3.

Insulin-like growth factors (IGFs) are important growth regulators of both normal and malignant prostate cells. Their action is regulated by six insulin-like growth factor binding proteins (IGFBPs). The proteolytic cleavage of IGFBPs by various proteases decreases dramatically their affinity for their ligands and therefore enhances the bioavailability of IGFs. To elucidate the putative biological role of prostatic kallikreins hK2 and hK3 (prostate-specific antigen) in tumour progression, we analyzed the degradation of IGFBP-2, -3, -4 and -5 by these two tissue kallikreins. We found that hK3, already characterized as an IGFBP-3 degrading protease, cleaved IGFBP-4 but not IGFBP-2 and -5, whereas hK2 cleaved all of the IGFBPs much more effectively, and at concentrations far lower than those reported for other IGFBP-degrading proteases. The proteolytic patterns after cleavage of IGFBPs by hK2 and hK3 were similar and were not modified in the presence of IGF-I. Heparin, but not other glycosaminoglycans, enhanced dramatically the ability of hK3 but not hK2 to degrade IGFBP-3 and IGFBP-4. More importantly, the IGFBP fragments generated by hK2 and hK3 had no IGF-binding capacity, as assessed by Western ligand blotting. Our results suggest that the prostatic kallikreins hK2 and hK3 may influence specifically the tumoral growth of prostate cells through the degradation of IGFBPs, to increase IGF bioavailability.     

Interaction of insulin-like growth factor II (IGF-II) with multiple plasma proteins: high affinity binding of plasminogen to IGF-II and IGF-binding protein-3

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.     

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions.

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.     

Expression of insulin-like growth factor binding proteins in human breast cancer correlates with estrogen receptor status

The insulin-like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF-dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP-4 mRNA was detected in all BCCLs studied. IGFBP-5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP-6 mRNA was detected in only two ER negative BCCLs. We also found that E₂ treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47-D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP-1 message. IGFBP-3 expression was higher in ER negative tumors, while that of IGFBP-4 and -5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.     

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.     

The N-terminal subdomain of insulin-like growth factor (IGF) binding protein 6. Structure and interaction with IGFs

Insulin-like growth factor binding proteins (IGFBPs) modulate the activity and distribution of insulin-like growth factors (IGFs). IGFBP-6 differs from other IGFBPs in being a relatively specific inhibitor of IGF-II actions. Another distinctive feature of IGFBP-6 is its unique N-terminal disulfide linkages; the N-domains of IGFBPs 1-5 contain six disulfides and share a conserved GCGCC motif, but IGFBP-6 lacks the two adjacent cysteines in this motif, so its first three N-terminal disulfide linkages differ from those of the other IGFBPs. The contributions of the N- and C-domains of IGFBP-6 to its IGF binding properties and their structure-function relationships have been characterized in part, but the structure and function of the distinctive N-terminal subdomain of IGFBP-6 are unknown. Here we report the solution structure of a polypeptide corresponding to residues 1-45 of the N-terminal subdomain of IGFBP-6 (NN-BP-6). The extended structure of the N-terminal subdomain of IGFBP-6 is very different from that of the short two-stranded beta-sheet of the N-terminal subdomain of IGFBP-4 and, by implication, the other IGFBPs. NN-BP-6 contains a potential cation-binding motif; lanthanide ion binding was observed, but no significant interaction was found with physiologically relevant metal ions like calcium or magnesium. However, this subdomain of IGFBP-6 has a higher affinity for IGF-II than IGF-I, suggesting that it may contribute to the marked IGF-II binding preference of IGFBP-6. The extended structure and flexibility of this subdomain of IGFBP-6 could play a role in enhancing the rate of ligand association and thereby be significant in IGF recognition.     

Insulin-like Growth Factor Binding Protein6 Associated with Neuronal Apoptosis Following Intracerebral Hemorrhage in Rats

The insulin-like growth factor (IGF) system is linked to CNS pathological states. The functions of IGFs are modulated by a family of binding proteins termed insulin-like growth factor binding proteins (IGFBPs). Here, we demonstrate that IGFBP-6 may be associated with neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant upregulation of IGFBP-6 in neurons adjacent to the hematoma following ICH with the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing IGFBP-6 level was found to be accompanied by the upregulation of Bax, Bcl-2, and active caspase-3. Besides, IGFBP-6 co-localized well with active caspase-3 in neurons, indicating its potential role in neuronal apoptosis. Knocking down IGFBP-6 by RNA-interference in PC12 cells reduced active caspase-3 expression. Thus, IGFBP-6 may play a role in promoting the brain secondary damage following ICH.     

Interaction between fibrinogen and insulin-like growth factor-binding protein-1 in human plasma under physiological conditions

Fibrinogen is a plasma glycoprotein and one of the principle participants in blood coagulation. It interacts with many proteins during formation of a blood clot, including insulin-like growth factors (IGFs) and their binding proteins (IGFBP). Fibrinogen complexes were found as minor fractions in fibrinogen preparations independently of the coagulation process, and their presence influences the kinetics of polymerization. The idea of this work was to investigate whether fibrinogen in human plasma interacts with IGFBPs independently of the tissue injury or coagulation process. The results have shown that fibrinogen forms complexes with IGFBP-1 under physiological conditions. Several experimental approaches have confirmed that complexes are co-isolated with fibrinogen from plasma, they are relatively stable, and they appear as a general feature of human plasma. Several other experiments excluded the possibility that alpha-2 macroglobulin/IGFBP-1 complexes or IGFBP-1 oligomers contributed to IGFBP-1 immunoreactivity. The role of fibrinogen/IGFBP-1 complexes is still unknown. Further investigation in individuals expressing both impaired glucose control and coagulopathy could contribute to identification and understanding of their possible physiological role.