David G. Fernig

Fibroblast growth factors and their receptors: An information network controlling tissue growth, morphogenesis and repair

The stimulation of cellular metabolism by the nine fibroblast growth factors (FGFs) is mediated by a dual-receptor system. This comprises a family of four receptor tyrosine kinases (FGFR) and heparan sulphate proteoglycans (HSPG). The stimulation of cell division by FGFs has an obligate requirement for both partners of the dual-receptor system. The binding of the nine FGFs to the FGFRs is marked by a pattern of overlapping specificity despite alternative splicing events generating a large number of FGFR proteins. Thus many of the FGFR isoforms bind several FGFs. It is likely that each FGF requires a different pattern of sulphation within the heparan sulphate chains for binding. Therefore, the HSPG receptors may provide additional specificity, allowing a cell to fine tune its response to the FGFs present in the extracellular milieu. The HSPG receptors also control the availability of FGFs and hence regulate the transport of FGFs within a tissue. FGF-stimulated cell division would appear to have a mandatory requirement for the FGFs to be translocated to the nucleus via the cytosol after interacting with the dual-receptor system. The consequences of the potential direct action of FGFs in stimulating cell division are examined in the light of current models of signal transduction.

Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity.

Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458–20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression.

Hepatocyte growth factor/scatter factor binds with high affinity to dermatan sulfate

We have demonstrated by affinity chromatography that hepatocyte growth factor/scatter factor (HGF/SF) binds strongly to dermatan sulfate (DS), with a similar ionic strength dependence to that previously seen with heparan sulfate (HS). Analysis of binding kinetics on a biosensor yields an equilibrium dissociation constant,K D , of 19.7 nm. This corresponds to a 10–100-fold weaker interaction than that with HS, primarily due to a faster dissociation rate of the complex. The smallest DS oligosaccharide with significant affinity for HGF/SF by affinity chromatography appears to be an octasaccharide. A sequence comprising unsulfated iduronate residues in combination with 4-O-sulfated N-acetylgalactosamine is sufficient for high affinity binding. The presence of 2-O-sulfation on the iduronate residues does not appear to be inhibitory. These observations concur with our previous suggestions, from analyses of HS binding (Lyon, M., Deakin, J. A., Mizuno, K., Nakamura, T., and Gallagher, J.T. (1994) J. Biol. Chem. 269, 11216–11223), that N-sulfation of hexosamines and 2-O-sulfation of iduronates are not absolute requirements for glycosaminoglycan binding to HGF/SF. This is the first described example of a high affinity interaction between a growth factor and DS, and is likely to have significant implications for the biological activity of this paracrine-acting factor.

Human Lactoferrin Interacts with Soluble CD14 and Inhibits Expression of Endothelial Adhesion Molecules, E-Selectin and ICAM-1, Induced by the CD14-Lipopolysaccharide Complex

ABSTRACT Lipopolysaccharides (LPS), either in the free form or complexed to CD14, a LPS receptor, are elicitors of the immune system. Lactoferrin (Lf), a LPS-chelating glycoprotein, protects animals against septic shock. Since optimal protection requires administration of Lf prior to lethal doses of LPS, we hypothesized that interactions between Lf and soluble CD14 (sCD14) exist. In a first step, human sCD14 and human Lf (hLf) were used to determine the kinetic binding parameters of hLf to free sCD14 in an optical biosensor. The results demonstrated that hLf bound specifically and with a high affinity (Kd= 16 ± 7 nM) to sCD14. Affinity chromatography studies showed that hLf interacted not only with free sCD14 but also, though with different binding properties, with sCD14 complexed to LPS or lipid A–2-keto-3-deoxyoctonic acid–heptose. In a second step, we have investigated whether the capacity of hLf to interact with sCD14 could modulate the expression of endothelial-leukocyte adhesion molecule 1 (E-selectin) or intercellular adhesion molecule 1 (ICAM-1) induced by the sCD14-LPS complex on human umbilical vein endothelial cells (HUVEC). Our experiments show that hLf significantly inhibited both E-selectin and ICAM-1 expressions at the surface of HUVEC. In conclusion, these observations suggest that the anti-inflammatory effects of hLf are due not only to the ability of the molecule to chelate LPS but also to its ability to interact with sCD14 and with the sCD14 complexed to LPS, thus modifying the activation of endothelial cells.

Interactions of Multiple Heparin Binding Growth Factors with Neuropilin-1 and Potentiation of the Activity of Fibroblast Growth Factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000–1,600,000 m–1 s–1). These results suggest that Npn-1 possesses a “heparin” mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.

Programmed cell death in bovine mammary tissue during lactation and involution

Cessation of milk removal causes mammary tissue involution, which in rodents is characterized by extensive tissue degeneration and loss of the majority of luminal epithelial cells by apoptosis. In contrast, bovine mammary tissue shows little histological evidence of tissue remodelling between lactations. In this study, we combined histology with molecular biology to examine the cellular and molecular changes in bovine mammary tissue on cessation of milking. Oligonucleosomal laddering of genomic DNA extracted from lactating tissue indicated that a proportion of cells were dying by apoptosis. This was confirmed by terminal deoxynucleotide transferase‐mediated deoxyuridine nick end‐labelling of apoptotic cells in lactating tissue sections (TUNEL). One week after cessation of milking, alpha‐lactalbumin and alpha S1‐casein messenger RNA (mRNA) abundance had decreased by 99 and 85%, respectively, whereas lactoferrin mRNA had increased 20‐fold. Drying off was also accompanied by an increase in oligonucleosomal laddering of genomic DNA, and by an increase in the proportion of TUNEL‐positive cells, which were localized preferentially in regions where alveolar structure had deteriorated. Therefore, termination of lactation was associated with partial loss of the mammary cell population and dedifferentiation of the remainder.

A systems biology approach for the investigation of the heparin/heparan sulfate interactome

A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.

Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import

The Galβ1–3GalNAcα (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 μg/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40–100 μg/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.

Fibroblast growth factor-2 binds to small heparin-derived oligosaccharides and stimulates a sustained phosphorylation of p42/44 mitogen-activated protein kinase and proliferation of rat mammary fibroblasts

We examine the relationship between the chain length of heparin-derived oligosaccharides, fibroblast growth factor (FGF)-2 binding kinetics and the ability of the oligosaccharides to allow FGF-2-induced proliferation of chlorate-treated rat mammary fibroblasts. First, using an optical biosensor, we show that FGF-2 did not bind disaccharides, but definitively bound to tetrasaccharides. As the chain length increased from tetrasaccharide to octasaccharide, there was a substantial increase in k(ass) (564000 M(-1) x s(-1) to 2000000 M(-1) x s(-1), respectively) and affinity (K(d) 77 nM to 11 nM, respectively) for FGF-2. From decasaccharides and longer, the k(ass) and affinity for FGF-2 was reduced substantially (tetradecasaccharide k(ass) 470000 M(-1) x s(-1), K(d) 30 nM). In chlorate-treated, and hence sulphated, glycosaminoglycan-deficient cells, FGF-2 alone or in the presence of disaccharides did not stimulate DNA synthesis and it only elicited an early transient dual phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). In the same cells FGF-2 in the presence of tetrasaccharides and longer oligosaccharides was able to restore DNA synthesis and enable the sustained dual phosphorylation of p42/44(MAPK). However, the oligosaccharides from tetrasaccharides to octasaccharides were less potent in proliferation assays than deca- and longer oligosaccharides. Therefore, there was no correlation between the binding parameters and the potency of the oligosaccharides in DNA synthesis assays. These results demonstrate that tetrasaccharides are able to bind FGF-2 and enable FGF-2 to stimulate cell proliferation, which sets important boundary conditions for models of the FGF-2-heparan sulphate-FGF receptor complex.

Cathepsin L Digestion of Nanobioconjugates upon Endocytosis

Understanding the dynamic fate and interactions of bioconjugated nanoparticles within living cells and organisms is a prerequisite for their use as in situ sensors or actuators. While recent research has provided indications on the effect of size, shape, and surface properties of nanoparticles on their internalization by living cells, the biochemical fate of the nanoparticles after internalization has been essentially unknown. Here we show that, upon internalization in a wide range of mammalian cells, biological molecules attached to the nanoparticles are degraded within the endosomal compartments through peptide cleavage by the protease cathepsin L. Importantly, using bioinformatics tools, we show that cathepsin L is able to cleave more than a third of the human proteome, indicating that this degradation process is likely to happen to most nanoparticles conjugated with peptides and proteins and cannot be ignored in the design of nanomaterials for intracellular applications. Preservation of the bioconjugates can be achieved by a combination of cathepsin inhibition and endosome disruption.