John H. Shand

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, Gly203 and Gln209, 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 (1). 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.

The esterification of cholesterol in the yolk sac membrane of the chick embryo.

The uptake of lipid from the yolk by the yolk sac membrane of the chick embryo is accompanied by the rapid esterification of a large proportion of the yolk cholesterol. This could arise from enhanced acyl-CoA:cholesterol acyltransferase (ACAT) activity and/or inhibition of cholesteryl ester hydrolase (CEH) activity. The activity of ACAT was therefore measured in microsomes obtained from yolk sac membranes at various stages of development. A high level of activity (up to 929 pmol of cholesteryl oleate formed per min per mg protein) was found during the second half of this period. Supplementation with exogenous cholesterol stimulated ACAT activity in microsomes obtained from the tissue at the earlier, but not at the later, stages of development suggesting that the enzyme became saturated with microsomal cholesterol as development proceeded. Correlating with this, the concentration of cholesterol in the microsomes increased 4-fold between 9 and 20 d of development. The activity of CEH was very low in the microsomes and could not be detected in the cytosolic fraction. The activity of a protein, which has been shown to function as an inhibitor of CEH, was found to be present at all stages of development. The high activity of ACAT, together with the low activity of CEH and an active CEH inhibitor protein is a combination well suited to promote an essentially unidirectional conversion of cholesterol to cholesteryl ester. This process may be a major determinant of the rate of lipid transfer from the yolk to the embryo.

IGFBP5 induces cell adhesion, increases cell survival and inhibits cell migration in MCF-7 human breast cancer cells

Maintenance of tissue boundaries is crucial for control of metastasis. We describe a new signalling pathway in which epithelial cell disruption can be minimised and thereby restricts epithelial–mesenchymal transgressions. This involves the release of insulin-like growth factor (IGF)-binding protein 5 (IGFBP5) from apoptotic cells, which increases the adhesion of epithelial cells on mesenchymal but not epithelial extracellular matrix (ECM), and involves the direct interaction of IGFBP5 and α2β1 integrins. IGFBP5 also induced cell adhesion to vitronectin in the absence of αVβ3 integrin, the vitronectin receptor, again through an α2β1-integrin-dependent action, suggesting that IGFBP5 can induce spreading on matrices, even in the absence of the integrins normally used in this process. Using IGFBP5 mutants we demonstrate that the effect is IGF-independent but requires the heparin-binding domain in the C-terminus of IGFBP5. A truncated mutant containing only the C-terminal of IGFBP5 also induced adhesion. Adhesion induced by IGFBP5 was dependent on Cdc42 and resulted in activation of integrin-linked kinase (ILK) and Akt. Consistent with these changes, IGFBP5 facilitated prolonged cell survival in nutrient-poor conditions and decreased phosphorylation of the stress-activated kinase p38 MAPK (MAPK14). Whereas IGFBP5 enhanced adhesion, it inhibited cell migration, although this was not evident using the truncated C-terminal mutant, suggesting that effects of IGFBP5 on adhesion and migration involve different mechanisms. We anticipate that these responses to IGFBP5 would reduce the metastatic potential of cells.

The esterification of cholesterol in the liver of the chick embryo.

The development of the chick embryo was characterised by the accumulation of large droplets of lipid in the cytoplasm of the embryonic liver, as revealed by electron microscopy. Analysis of the lipid composition of the livers indicated that this accumulation resulted from a dramatic increase in the cholesteryl ester content of the tissue during the the latter part of the embryonic period. This lipid is apparently derived from yolk cholesterol and may be taken up by the liver in the form of lipoprotein remnants. Significant levels of acyl-CoA: cholesterol acyltransferase (ACAT) activity were expressed in the liver throughout the second half of the developmental period, and this activity was maximal at the time when lipid transfer from the yolk was most intensive. The activity of microsomal cholesterol ester hydrolase (CEH) was very low throughout development, and no CEH activity was detected in the cytosolic fraction. In addition, substantial amounts of a cytosolic protein which inhibits CEH activity were present. Thus the relative activities of these enzymic systems are consistent with the net accumulation of cholesteryl ester which occurs in the liver during development.

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.

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.

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.

Synthesis of C20 and C22 polyunsaturated fatty acids by the placenta of the sheep.

Incubation in vitro of sheep placental homogenates with [1-14C] oleic, linoleic and linolenic acids resulted in extensive amounts of radioactivity becoming associated with C20 and C22 polyunsaturated fatty acid products. The fatty acids of the fetal plasma displayed substantial levels of both C20 and C22 polyunsaturated fatty acids, including the n-9 eicosatrienoic fatty acid associated with essential fatty acid deficiency, but extremely low levels of linoleic and linolenic acids. A role for the placenta in the provision to the fetus of a range of C20 and C22 polyunsaturated fatty acids derived from C18 fatty acid precursors is implicated.

The Effects of Simvastatin and Cholestyramine, Alone and in Combination, on Hepatic Cholesterol Metabolism in the Male Rat

The influence of dietary simvastatin, cholestyramine, and the combination of simvastatin plus cholestyramine on hepatic cholesterol metabolism has been investigated in male rats. Recovery from the effects of the drugs was also investigated by refeeding normal chow for 24 h. Both drugs, alone and in combination, increased 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activityin vitro, but activity returned toward control values, after drug withdrawal. Acyl-CoA:cholesterol acyltransferase (ACAT) was significantly reduced (P<0.001) by simvastatin (−75%), cholestyramine (−71%), and by the drug combination (−81%), due both to a decrease in microsomal cholesterol and to nonsubstrate-dependent modulation of enzyme activity. Refeeding control diet increased ACAT activity but not to control levels. The enhanced activity arose partly from higher microsomal cholesterol and partly from increases in total enzyme activity. Cytosolic neutral cholesteryl ester hydrolase (CEH) activity was substantially elevated by simvastatin (3-fold) and by the drug combination (6-fold), whereas the effect of cholestyramine was smaller (1.5-fold). Normal chow for 24 h only partially returned cytosolic CEH activity to control values. Microsomal CEH activity was increased by simvastatin, alone and in combination with cholestyramine (1.4 to 1.7-fold), and was also enhanced, in the cholestyramine-treated animals, following drug withdrawal. Removal of simvastatin did not allow recovery of this enzyme activity, while withdrawal of the drug combination led to values 29% below controls. The results indicate that in the rat, simvastatin and cholestyramine alter both ACAT and CEH activity, as well as inhibiting HMG-CoA reductase activity.

Incorporation of Linoleic and Arachidonic Acids into Ovine Placental Phospholipids in vitro

The incorporation of [1-14C]-linoleic acid and [1-14C]-arachidonic acid into the lipids of ovine placental homogenate was studied. Both fatty acids were incorporated mainly into the phospholipid fraction with much lower levels being found in the diacylglycerols and triacylglycerols, particularly when the substrate was [1-14C]-arachidonic acid. Within the phospholipid fraction, both substrates were incorporated largely into phosphatidic acid although significant levels of incorporation were found in the phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol fractions. The incorporation of [1-14C]-arachidonic acid into phosphatidylcholine was lower and into phosphatidylethanolamine and phosphatidylinositol was higher than those levels found when the substrate used was [1-14C]-linoleic acid. While some of the trienoic and tetraenoic, but not pentaenoic, desaturation products of [1-14C]-linoleic acid were detected in phosphatidylcholine and phosphatidylethanolamine, the highest levels of incorporation of these metabolites were into phosphatidic acid. Similarly, the pentaenoic products of [1-14C]-arachidonic acid were found mainly in phosphatidic acid although significantly higher levels were observed in phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol. The results suggest that in ovine placental tissue polyunsaturated fatty acids, whether supplied pre-formed or made available by desaturation and elongation processes, could potentially be incorporated into phospholipids by the de novo route via phosphatidic acid.