IGFBP-5 induces epithelial and fibroblast responses consistent with the fibrotic response.

Fibrosis involves activation of fibroblasts, increased production of collagen and fibronectin and transdifferentiation into contractile myofibroblasts. The process resembles aspects of wound-healing but remains unresolved and can be life-threatening when manifest in the kidneys, lungs and liver, in particular. The causes are largely unknown, but recent suggestions that repetitive micro-injury results in the eventual failure of epithelial cell repair due to replicative senescence are gaining favour. This is consistent with the onset of fibrotic diseases in middle age. Because epithelial injury often involves blood loss, inflammatory responses associated with the fibrotic response have been considered as therapeutic targets. However, this has proved largely unsuccessful and focus is now switching to earlier events in the process. These include EMT (epithelial-mesenchymal transition) and fibroblast activation in the absence of inflammation. TGFbeta1 (transforming growth factor-beta1) induces both EMT and fibroblast activation and is considered to be a major pro-fibrotic factor. Recently, IGFBP-5 [IGF (insulin-like growth factor)-binding protein-5] has also been shown to induce similar effects on TGFbeta1, and is strongly implicated in the process of senescence. It also stimulates migration of peripheral blood mononuclear cells, implicating it in the inflammatory response. In this paper, we examine the evidence for a role of IGFBP-5 in fibrosis and highlight its structural relationship with other matrix proteins and growth factors also implicated in tissue remodelling.

IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin.

We have used Surface Plasmon Resonance (SPR) - based biosensor technology to investigate the interaction of the six high affinity insulin-like growth factor binding proteins (IGFBP 1-6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN ((9-10)FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein. Using site-directed mutagenesis of (9-10)FNIII, we show both the "synergy" and RGD sites within these FN domains are required for maximum binding of both IGFBPs. We discuss the possible biological consequences of our results.

Epithelial injury induces an innate repair mechanism linked to cellular senescence and fibrosis involving IGF-binding protein-5.

Fibrosis is associated with epithelial repair. It involves the activation of fibroblasts, increased production of extracellular matrix proteins and transdifferentiation to contractile, myofibroblasts that aid in wound contraction. This provisional matrix plugs the injured epithelium and provides a scaffold for epithelial cell migration, involving an epithelial-mesenchymal transition (EMT). When epithelial injury involves blood loss, this leads to platelet activation, the production of several growth factors and an acute inflammatory response. Under normal circumstances, the epithelial barrier is repaired and the inflammatory response resolves. However, in fibrotic disease, the fibroblast response continues, resulting in unresolved wound healing. The fibrotic diseases range from scleroderma, where the problem may be restricted to the skin and where it is not life-threatening, through to systemic forms that can manifest as, for example, idiopathic pulmonary fibrosis, in which death is inevitable within 3-5 years. Anti-inflammatory treatments have failed to ameliorate the disease condition and focus has instead turned to transforming growth factor-beta1 (TGFB1), since it induces many of the processes involved, including fibroblast activation and EMT. Most recently, however, a new player in this process has been described, IGF-binding protein-5 (IGFBP5). IGFBP5 has also been shown to induce similar effects to TGFB1, but, in addition, it is strongly implicated in the process of senescence which is now believed to be a significant factor in these diseases. We examine the evidence for this role of IGFBP5 and identify some of the therapeutic targets which might be used to ameliorate these diseases of unknown cause.

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study.

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.

Insulin-like growth factor-binding protein-5 (IGFBP-5): a critical member of the IGF axis.

The six members of the insulin-like growth factor-binding protein family (IGFBP-1-6) are important components of the IGF (insulin-like growth factor) axis. In this capacity, they serve to regulate the activity of both IGF-I and -II polypeptide growth factors. The IGFBPs are able to enhance or inhibit the activity of IGFs in a cell- and tissue-specific manner. One of these proteins, IGFBP-5, also has an important role in controlling cell survival, differentiation and apoptosis. In this review, we report on the structural and functional features of the protein which are important for these effects. We also examine the regulation of IGFBP-5 expression and comment on its potential role in tumour biology, with special reference to work with breast cancer cells.

Insulin-like growth factor-binding protein-5 activates plasminogen by interaction with tissue plasminogen activator, independently of its ability to bind to plasminogen activator inhibitor-1, insulin-like growth factor-I, or heparin.

Transgenic mice expressing IGFBP-5 in the mammary gland exhibit increased cell death and plasmin generation. Because IGFBP-5 has been reported to bind to plasminogen activator inhibitor-1 (PAI-1), we determined the effects of this interaction in HC11 cells. PAI-1 prevented plasmin generation from plasminogen and inhibited cleavage of focal adhesions, expression of caspase 3, and cell death. IGFBP-5 could in turn prevent the effects of PAI-1. IGFBP-5 mutants with reduced affinity for IGF-I (N-term) or deficient in heparin binding (HEP- and C-term E and F) were also effective. This was surprising because IGFBP-5 reportedly interacts with PAI-1 via its heparin-binding domain. Biosensor analysis confirmed that, although wild-type IGFBP-5 and N-term both bound to PAI-1, the C-term E had greatly decreased interaction with PAI-1. This suggests that IGFBP-5 does not antagonize the actions of PAI-1 by a direct molecular interaction. In a cell-free system, using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to activate plasminogen, PAI-1 inhibited plasmin generation induced by both activators, whereas IGFBP-5 prevented the effects of PAI-1 on tPA but not uPA. Furthermore, we noted that IGFBP-5 activated plasminogen to a greater extent than could be explained solely by inhibition of PAI-1, suggesting that IGFBP-5 could directly activate tPA. Indeed, IGFBP-5 and the C-term E and F were all able to enhance the activity of tPA but not uPA. These data demonstrate that IGFBP-5 can enhance the activity of tPA and that this can result in cell death induced by cleavage of focal adhesions. Thus IGFBP-5 can induce cell death by both sequestering IGF-I and enhancing plasmin generation.

Insulin-like growth factor binding protein (IGFBP)-5 is upregulated during both differentiation and apoptosis in primary cultures of mouse mammary epithelial cells.

We have previously demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) is upregulated following treatment of the mouse mammary epithelial cell line HC11 with lactogenic hormones (dexamethasone, insulin, and prolactin-DIP). In addition, we have also shown that IGFBP-5 is upregulated in mammary epithelial cells in vivo during involution of the rodent mammary gland. We have, therefore, postulated that there may be a dual regulation of IGFBP-5 expression during the temporally separated processes of differentiation and apoptosis of mammary epithelial cells. To test this hypothesis further, we have used a phenotypically differentiated model, which comprises primary cultures of mouse mammary epithelial cells grown on a layer of EHS (Engelbreth-Holm-Swarm) extracellular matrix. We show that lactogenic hormone treatment (hydrocortisone, insulin, and prolactin-HIP) of these cultures induces the upregulation of IGFBP-5 thus replicating the results obtained with the HC11 cell line. In addition, following the induction of apoptosis in primary cultures of mammary epithelial cells by treatment with TGFbeta-3, IGFBP-5 expression is also upregulated. In parallel with this upregulation of IGFBP-5, there is also an increase in the levels of cleaved caspase-3, a well-characterized marker of cellular apoptosis. These findings confirm previous in vivo work demonstrating an increase in IGFBP-5 expression during involution of the mouse mammary gland. When HC11 cells are cultured under serum-free conditions (a well-characterized apoptotic insult in cell culture), there is also an increase in cleaved caspase-3 levels. Unexpectedly, in the presence of TGFbeta-3, caspase-3 levels are attenuated. In the presence of DIP, caspase-3 levels are also decreased in HC11 cells. As described previously, TGFbeta-3 inhibits beta-casein synthesis in HC11 cells. In the HC11 cell line (in contrast to primary cultures of mammary epithelial cells), there is no evidence for TGFbeta-3 induction of IGFBP-5 under either serum-free or DIP-supplemented conditions. We believe our data with primary cultures of mammary epithelial cells support the hypothesis of dual regulation of IGFBP-5 expression during both differentiation and apoptosis in the mammary gland and emphasizes the importance of using appropriate cell culture models to investigate such phenomena in this tissue. We discuss the possible implications of our observations in relation to the physiological processes of pregnancy, lactation, and involution in the mammary gland and the associated changes in mammary epithelial cell function.

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action.

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201-218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (K(D)) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity.

Molecular recognition characteristics in the insulin-like growth factor (IGF)-insulin-like growth factor binding protein -3/5 (IGFBP-3/5) heparin axis.

Insulin-like growth factor binding proteins (IGFBPs) -3 and -5 are known to interact with various components of the extracellular matrix (ECM; e.g. heparin and heparan sulphate) and this interaction is believed to affect the affinity of both IGFBP species for their cognate ligands--IGF-I and -II. There is little detail on the nature of the molecular complex formed between ECM components, IGFBPs and IGFs although the glycosaminoglycan (GAG) heparin has been reported to reduce the affinity of IGFBP-5 for IGF-I. In order to investigate this phenomenon further, we have undertaken an extensive surface plasmon resonance based biosensor study to report the affinity of IGFBP-3 and -5 for binding heparin (22 and 7 nM respectively). We have also shown that pre-complexation of IGFBP with IGF-I and -II inhibits the subsequent association of IGFBP with heparin and conversely that heparin complexation of IGFBP-3 and -5 inhibits IGFBP binding to biosensor surfaces containing immobilised IGF-I. In addition we have used both IGF-I and heparin coated biosensor surfaces in an attempt to build ternary IGF-IGFBP-heparin complexes in order to gain some insight into the nature of inhibition by heparin of IGFI-IGFBP complex formation. Our data lead us to conclude that the inhibition by heparin is partly competitive in nature, and that ternary complexes of IGF-IGFBP-heparin are either unable to form, or only form unstable transient complexes. The potential biological significance of our data is highlighted by the demonstration that IGF-I and IGF-II can displace endogenous IGFBP-5 from monolayer cultures of the mouse mammary epithelial cell line HC11.

The role of IGFBP-5 in mammary gland development and involution.

Insulin-like growth factor-I (IGF-I) plays an important role as a survival factor during mammary gland development and remodelling during involution of the mature/lactating mammary gland, and elevated concentrations have been associated with increased risk of breast cancer. The actions of IGF-I are modulated by a family of binding proteins (IGFBPs) and we have shown that IGFBP-5 is associated with cell death in the mammary gland and more recently provided the first evidence that it is causally related to apoptosis of the mammary gland. A transgenic mouse expressing IGFBP-5 on a mammary-specific promoter led to impaired mammary development involving inhibition of IGF-signalling and involving members of the Bcl-2 family. Subsequent studies in vitro and in vivo using exogenous IGFBP-5 treatment have added support to this concept. Although the effects of IGFBP-5 did appear to involve inhibition of IGF action, a role for IGF-independent effects cannot be ruled out. Such IGF-independent effects involve potential interactions with components of the extracellular matrix involved in tissue remodelling including plasminogen activator inhibitor-1 (PAI-1). In addition, intracellular events involving nuclear localisation of IGFBP-5 have been shown to have the ability to inhibit cell proliferation. Thus, IGFBP-5 seems important for regulating both apoptosis and cell proliferation in the mammary gland during development and post-lactation involution.

Major components of the insulin-like growth factor axis are expressed early in chicken embryogenesis, with IGF binding protein ( IGFBP) -5 expression subject to regulation by Sonic Hedgehog.

Using whole-mount in situ hybridisation techniques, we have examined the expression of major components of the insulin-like growth factor (IGF) axis in early development of the chicken embryo, including both IGF-I and -II, the type 1 IGF receptor ( IGFR), and two of the IGF binding proteins, ( IGFBP) -2 and -5. We report that these genes fall into two distinct groups with respect to expression pattern, with IGFBP-2 displaying broad overlap of mRNA expression with IGFR and IGF-I during early development, whereas the expression profile of IGFBP-5 most closely resembled that of IGF-II. Comparison between different stages revealed IGFBP-2 mRNA was detected as early as stage 3, whereas IGFBP-5 was first seen at stage 4. In addition, we detected expression domains of IGFBP-5, and to a lesser extent IGFBP-2, which did not overlap with either IGFR or IGF expression patterns. This could indicate IGF independent actions of the IGFBPs during early embryonic development. A striking observation concerning the expression profiles of both IGF-II and IGFBP-5 at early stages of chick embryogenesis is that both these genes are expressed asymmetrically in a pattern similar to that of Sonic Hedgehog (Shh). Furthermore, using cyclopamine, we have demonstrated that IGFBP-5 expression in the early embryo is regulated by Shh. Taken together, these results describe an important role for the IGF system in the very early stages of the developing chicken embryo, and imply that IGFBP-2 and -5 are fundamental developmental factors, with the latter involved in Shh signalling pathways.

Specific amino acid substitutions determine the differential contribution of the N- and C-terminal domains of insulin-like growth factor (IGF)-binding protein-5 in binding IGF-I.

We have previously reported that two highly conserved amino acids in the C-terminal domain of rat insulin-like growth factor-binding protein (IGFBP)-5, Gly(203) and Gln(209), are involved in binding to insulin-like growth factor (IGF)-1. Here we report that mutagenesis of both amino acids simultaneously (C-Term mutant) results in a cumulative effect and an even greater reduction in IGF-I binding: 30-fold measured by solution phase IGF binding assay and 10-fold by biosensor analysis. We compared these reductions in ligand binding to the effects of specific mutations of five amino acids in the N-terminal domain (N-Term mutant), which had previously been shown by others to cause a very large reduction in IGF-I binding (). Our results confirm this as the major IGF-binding site. To prove that the mutations in either N- or C-Term were specific for IGF-I binding, we carried out CD spectroscopy and showed that these alterations did not lead to gross conformational changes in protein structure for either mutant. Combining these mutations in both domains (N+C-Term mutant) has a cumulative effect and leads to a 126-fold reduction in IGF-I binding as measured by biosensor. Furthermore, the equivalent mutations in the C terminus of rat IGFBP-2 (C-Term 2) also results in a significant reduction in IGF-I binding, suggesting that the highly conserved Gly and Gln residues have a conserved IGF-I binding function in all six IGFBPs. Finally, although these residues lie within a major heparin-binding site in IGFBP-5 and -3, we also show that the mutations in C-Term have no effect on heparin binding.

Growth hormone, acting in part through the insulin-like growth factor axis, rescues developmental, but not metabolic, activity in the mammary gland of mice expressing a single allele of the prolactin receptor.

The heterozygous prolactin (PRL) receptor (PRLR(+/-)) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. PRL and GH, acting through the IGF system, have interactive effects to enhance epithelial cell survival. Thus, we propose that a reduction in the expression of the PRLR may lead to increased IGFBP-5 expression (proapoptotic) and that GH may rescue mammary development by increasing IGF-I, an important mitogen and survival factor for the mammary epithelium. Mammary IGF-binding protein-5 (IGFBP-5) concentrations and plasmin activity in PRLR(+/-) mice were increased on d 2 postpartum, indicative of increased cell death and extracellular matrix remodeling. After GH treatment, a restoration of mammary alveolar development and a reduction in the activities of IGFBP-5 and plasmin were observed. Despite the severely impaired mammary development in PRLR(+/-) mice, both mRNA and protein expression for caseins and acetyl-coenzyme A (acetyl-CoA) carboxylase and acetyl-CoA caboxylase-alpha mRNA increased at parturition, although not to the extent in wild-type animals. Surprisingly, GH treatment actually led to a further decrease in milk protein and acetyl-CoA carboxylase-alphaexpression when expressed per cell. This was confirmed by the smaller alveolar size, the relative paucity of milk in the mammary glands of GH-treated animals, and the inability of their pups to gain weight. In a subsequent study IGFBP-5 was administered to wild-type mice and produced a 45% decrease in mammary DNA content, a 30% decrease in parenchymal tissue, and impaired lactation. These results suggest that GH can improve mammary development in PRLR(+/-) mice, but that it fails to enhance metabolic activity. This may be due to the maintenance by GH/IGF-I of a proliferative, rather than a differentiative, phenotype.

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice.

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.

Sensitivity of hybrid ovine/rat GH receptors to oGH and rat GH in transfected FDC-P1 mouse myeloid cells in vitro.

We have described previously the properties of two mutant ovine growth hormone receptor extracellular domain (oGHR-ECD) proteins which were created by substituting sequences from the rat GHR at two different locations within the framework of the oGHR-ECD. The first mutation occurred at a region close to the N-terminus of oGHR-ECD between residues Thr 28 and Leu34 and created the protein T28E/N29S/N33K/L34P-oGHR-ECD, where the ovine specific residues T, N, N and L are replaced by their equivalent residues E, S, K and P from the rat protein. This site lies N-terminal to the first element of beta-strand structure in the GHR-ECD and we designated this protein as Site-A mutant. The second mutation was made between residues Serl21 andAsp 124 of oGHR-ECD to produce the protein S121T/E123D/D124E-oGHR-ECD where ovine specific residues are again replaced with the equivalent residues from the rat GHR-ECD. This region lies in a loop structure which joins the two beta-barrel domains of the GHR-ECD. This protein is designated as Site-B mutant. A subsequent analysis confirmed the N-terminal region between residues 28-34 of oGHR-ECD as an important epitope defined by antiserum raised to oGHR-ECD. Also of interest was the finding that mutation at both Sites A and B in oGHR-ECD compromised the affinity of the protein for bovine GH (50-fold for Site-A and 4-fold for Site-B). A comparison of the affinity of wild type oGHR for the highly homologous bovine GH with its affinity for rat GH indicated a 10-fold higher affinity for the ruminant GH than for rat GH. Mutation at Site-A of oGHR-ECD reduced the affinity for rat GH a further 3-fold. However, mutation at Site-B of oGHR-ECD increased the affinity for rat GH 2-3 fold. This indicated that the substitution of rat GHR residues for ovine GHR residues in this part of the protein had a beneficial effect in relation to affinity for rat GH and that this region of the GHR-ECD may contain important specificity determinants. In order to test whether these observed differences in affinity for bovine and rat GH affinities have any biological relevance, we have produced the same ovine --> rat mutations in the context of the full length ovine GHR. Transfection of the cDNAs encoding the wt or mutant GHRs into the mouse myeloid pre-B cell line FDC-P1 to create stably transfected clonal lines, has allowed an examination of the relative activities of ovine and rat GH, using a robust and high throughput bio-assay based on the reduction of a cell permeable tetrazolium salt. In the current manuscript, we report that the decrease in binding affinity previously reported for Site-A and Site-B mutant oGHR extracellular domain proteins is not reflected in compromised biological activities when the same mutations are expressed in the context of the full length oGHR protein. We discuss these findings in the context of the relationships between affinity and activity at the GHR.