Selectivity in glycosaminoglycan binding dictates the distribution and diffusion of fibroblast growth factors in the pericellular matrix

The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in pericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the pericellular matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular matrix has long-range structure.     

Glypican-1, phosphacan/receptor protein-tyrosine phosphatase-ζ/β and its ligand,tenascin-C,are expressed by neural stem cells and neural cells derived from embryonic stem cells

The heparan sulfate proteoglycan glypican-1, the chondroitin sulfate proteoglycan phosphacan/RPTP (receptor protein-tyrosine phosphatase)-ζ/β and the extracellular matrix protein tenascin-C were all found to be expressed by neural stem cells and by neural cells derived from them. Expression of proteoglycans and tenascin-C increased after retinoic acid induction of SSEA1-positive ES (embryonic stem) cells to nestin-positive neural stem cells, and after neural differentiation, proteoglycans and tenascin-C are expressed by both neurons and astrocytes, where they surround cell bodies and processes and in certain cases show distinctive expression patterns. With the exception of tenascin-C (whose expression may decrease somewhat), expression levels do not change noticeably during the following 2 weeks in culture. The significant expression, by neural stem cells and neurons and astrocytes derived from them, of two major heparan sulfate and chondroitin sulfate proteoglycans of nervous tissue and of tenascin-C, a high-affinity ligand of phosphacan/RPTP-ζ/β, indicates that an understanding of their specific functional roles in stem cell neurobiology will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration.     

Lack of acidic fibroblast growth factor activation by heparan sulfate species from diabetic rat skin

The glucosaminoglycans isolated from the skin of control and streptozotocin-diabetic rats were fractionated on ion-exchange chromatography into a heparan sulfate (HS)-like and a heparin-like species. In addition, a low sulfated fraction was isolated from the diabetics. The HS and heparin-like fractions isolated from the diabetics (in contrast to the low sulfated fractions) retained high affinity for the acidic (FGF-1) and basic (FGF-2) fibroblast growth factors. In culture, the fractions purified from the control rats and the heparin-like material isolated from the diabetics mediated the biological activity of both FGFs in a dose-dependent manner. By contrast, the diabetic HS-like fractions promoted the biological activity of FGF-2 but not of FGF-1. The results support the idea that the structural motives in HS required for FGF-1 and FGF-2 mediated receptor signalling are different. They may be relevant to the impaired wound healing observed in the disease. This revised version was published online in November 2006 with corrections to the Cover Date.     

Perlecan and tumor angiogenesis.

Perlecan is a major heparan sulfate proteoglycan (HSPG) of basement membranes (BMs) and connective tissues. The core protein of perlecan is divided into five domains based on sequence homology to other known proteins. Commonly, the N-terminal domain I of mammalian perlecan is substituted with three HS chains that can bind a number of matrix molecules, cytokines, and growth factors. Perlecan is essential for metazoan life, as shown by genetic manipulations of nematodes, insects, and mice. There are also known human mutations that can be lethal. In vertebrates, new functions of perlecan emerged with the acquisition of a closed vascular system and skeletal connective tissues. Many of perlecan's functions may be related to the binding and presentation of growth factors to high-affinity tyrosine kinase (TK) receptors. Data are accumulating, as discussed here, that similar growth factor-mediated processes may have unwanted promoting effects on tumor cell proliferation and tumor angiogenesis. Understanding of these attributes at the molecular level may offer opportunities for therapeutic intervention.     

Cytokines and growth factors cross-link heparan sulfate

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors.     

Intracellular trafficking in neurones and glia of fibroblast growth factor-2, fibroblast growth factor receptor 1 and heparan sulphate proteoglycans in the injured adult rat cerebral cortex

The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.     

Immunoreactive fibroblast growth factor (FGF) in a transplantable chondrosarcoma: inhibition of tumor growth by antibodies to FGF

Using a radioimmunoassay for bovine pituitary fibroblast growth factor (FGF), we have established the presence of the immunoreactive mitogen in extracts of a transplantable mouse chondrosarcoma. Both neutral and acidic extracts of the tumor contain an immunoreactive FGF (ir-FGF) that cross-reacts in a parallel and dose-dependent fashion in the radioimmunoassay. The ir-FGF is retained on heparin-Sepharose affinity columns and can be detected in the same molecular weight forms as rat pituitary FGF. Mice (C57/Bl) inoculated with the tumor (10 mg, im) show a decreased rate of tumor growth when passively immunized with the antiserum to FGF. The results establish the presence of FGF in this tumor and implicate its role in the etiology of its development.     

Heparin binding preference and structures in the fibroblast growth factor family parallel their evolutionary diversification

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans.     

Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: Implications for potentiation of growth factor action by heparin

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1887): J Cell Physiol 131:123–130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factros for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates form the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoplycan. Subsequent studies of dissociation kinetic demonstrated that bFGF dissociates from the FGFR at least 10-fold slwer than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interactio fo FGF with FGFR, possibly by the formatin of a ternary complex. © Wiley-Liss, Inc.     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.     

Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory system

Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure‐specific monoclonal antibodies (HepSS‐1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS‐1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin‐A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events.     

FGF对周围神经损伤后脊髓前角运动神经元的保护作用

目的：研究周围神经损伤后,局应用碱性成纤维细胞生长因子（basic fibroblast growth factor ,bFGF）对脊髓前角运动神经元的保护作用。方法：切为SD大鼠右侧坐骨神经,近端套接单盲硅胶管后,管内立好注入20μlbFGF（浓度为4000u/ml）或等量生理盐水,术后2周,观察L3－L5脊央前角外侧群大、中小型神经元乙酰胆碱酯酶（acetyl cholinesterase,AChE）,酸性磷酸酶（acid phosphatas,Acp)和一氧化氮合酶（nitric oxide synthase,NOS)的阳性细胞数、反应强度和平均灰度。结果：坐骨神经损伤后,脊髓腰节段运动神经元因受损伤的影响发明明显改变。表现为AChE降低,Acp,NOS活性升高,局部应用bFGF后,大型运动神经元(α运动神经元）阳性细胞数,反应强度和平均灰度接近正常侧,与对照组比较具有显性意义（P＜0．01）。结论：bFGF可以有效地防止周围神经损伤引起的脊髓前角α运动神经元退变,促进其恢复。     

Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan,a heparan sulfate proteoglycan

In this study, the internalization mechanism of basic fibroblast growth factor (bFGF) at the blood-brain barrier (BBB) was investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin-resistant binding of [125I]bFGF to TM-BBB4 cells was found to be time-, temperature-, osmolarity- and concentration-dependent. Kinetic analysis of the cell-surface binding of [125I]bFGF to TM-BBB4 cells revealed saturable binding with a half-saturation constant of 76 +/- 24 nm and a maximal binding capacity of 183 +/- 17 pmol/mg protein. The heparin-resistant binding of [125I]bFGF to TM-BBB4 was significantly inhibited by a cationic polypeptide poly-L-lysine (300 micro m), and compounds which contain a sulfate moiety, e.g. heparin and chondroitin sulfate-B (each 10 micro g/mL). Moreover, the heparin-resistant binding of [125I]bFGF in TM-BBB4 cells was significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG-mediated endocytosis. RT-PCR analysis revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine-kinase receptors for bFGF) mRNA are expressed in TM-BBB4 cells. Moreover, immunohistochemical analysis demonstrated that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes.     

Fibroblast growth factors in the management of spinal cord injury

Spinal cord injury (SCI) possesses a significant health and economic burden worldwide. Traumatic SCI is a devastating condition that evolves through two successive stages. Throughout each of these stages, disturbances in ionic homeostasis, local oedema, ischaemia, focal haemorrhage, free radicals stress and inflammatory response were observed. Although there are no fully restorative cures available for SCI patients, various molecular, cellular and rehabilitative therapies, such as limiting local inflammation, preventing secondary cell death and enhancing the plasticity of local circuits in the spinal cord, were described. Current preclinical studies have showed that fibroblast growth factors (FGFs) alone or combination therapies utilizing cell transplantation and biomaterial scaffolds are proven effective for treating SCI in animal models. More importantly, some studies further demonstrated a paucity of clinical transfer usage to promote functional recovery of numerous patients with SCI. In this review, we focus on the therapeutic capacity and pitfalls of the FGF family and its clinical application for treating SCI, including the signalling component of the FGF pathway and the role in the central nervous system, the pathophysiology of SCI and the targets for FGF treatment. We also discuss the challenges and potential for the clinical translation of FGF‐based approaches into treatments for SCI.     

Fibroblast growth factors and their receptors in the central nervous system

Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that participates in many developmental and repair processes of virtually all mammalian tissues. Among the 23 FGF members, ten have been identified in the brain. Four FGF receptors (FGFRs), receptor tyrosine kinases, are known so far. Ligand binding of these receptors greatly depends on the presence of heparan sulfate proteoglycans, which act as low affinity FGFRs. Ligand binding specificity of FGFRs depends on the third extracellular Ig-like domain, which is subject to alternative splicing. Activation of FGFRs triggers several intracellular signaling cascades. These include phosphorylation of src and PLC leading finally to activation of PKC, as well as activation of Crk and Shc. SNT/FRS2 serves as an alternative link of FGFRs to the activation of PKC and, in addition, activates the Ras signaling cascade. In the CNS, FGFs are widely expressed; FGF-2 is predominantly synthesized by astrocytes, whereas other FGF family members, e.g., FGF-5, FGF-8, and FGF-9, are primarily synthesized by neurons. During CNS development FGFs play important roles in neurogenesis, axon growth, and differentiation. In addition, FGFs are major determinants of neuronal survival both during development and during adulthood. Adult neurogenesis depends greatly on FGF-2. Finally, FGF-1 and FGF-2 seem to be involved in the regulation of synaptic plasticity and processes attributed to learning and memory.     

Production of sulfated proteoglycans by human breast cancer cell lines: Binding to fibroblast growth factor-2

The cellular distribution and nature of proteoglycans synthesised by human breast cancer cells in culture were studied. Proteoglycans were labelled with [³⁵S] sulfate, purified, and characterised after ion-exchange chromatography followed by gel-filtration chromatography and treatment with glycosaminoglycan degrading enzymes. Proteoglycans were isolated from the culture medium and from cell layers of the hormono-dependent well-differentiated MCF-7 cell line, the hormono-independent poorly-differentiated MDA-MB-231 and the HBL-100 cell line which is derived from non malignant breast epithelium. HBL-100 and MDA-MB-231 cells produced larger amounts of proteoglycans which had a lower degree of sulfation than MCF-7 cells. Gel-filtration chromatography on Sepharose CL-6B indicated that HBL-100 and MDA-MB-231 cells accumulated cell surface heparan sulfate proteoglycans (HSPG), with a high apparent molecular weight (K_av 0.1). In contrast, the MCF-7 cell monolayers synthesised small sulfated macromolecules (K_av 0.4) which possessed mostly chondroitin sulfate chains. Moreover, considerable differences in the nature of the sulfated proteoglycans released into the culture medium of these breast epithelial cell lines were observed. MCF-7 cells released into the culture medium HSPG as the main proteoglycan component while MDA-MB-231 and HBL-100 cells released mainly chondroitin sulfate proteoglycans. In these three cell lines, medium-released sulfated macromolecules have a higher hydrodynamic size than cell-associated ones. Proteoglycans purified by ion-exchange chromatography were tested for their ability to bind ¹²⁵I FGF-2. We demonstrated that HBL-100 and MDA-MB-231 cells bind more FGF-2 to their heparan sulfate proteoglycans than MCF-7 cells. Taken together, these results suggest that differences in proteoglycan synthesis of human breast epithelial cells could be responsible for differences in their proliferative and/or invasive properties. J. Cell. Biochem. 64:605–617. © 1997 Wiley-Liss, Inc.     

Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth

Midkine, a heparin-binding growth factor, was found to be expressed in neural precursor cells, which consist of neural stem cells and the progenitor cells. When embryonic brain cells were allowed to form neurospheres enriched in neural precursor cells, numbers were significantly smaller from the midkine-deficient brain than from the wild-type brain. Dissociated neurosphere cells yielded nestin-positive neural precursor cells and differentiated neuronal cells upon culture on a substratum. Neural precursor cells from the midkine-deficient brain spread poorly and grew less effectively on a substratum coated with poly-l-lysine than the cells on midkine-coated substratum. Neural precursor cells from the wild-type brain spread and grew well on both the substrata. Differentiation to neurons and glia cells was not affected by the absence of midkine. Heparitinase digestion of dissociated neurosphere cells resulted in poor growth of neural precursor cells, while chondroitinase digestion had no effect. These results indicate that midkine is involved in the growth of neural precursor cells and suggest that the interaction with heparan sulfate proteoglycans is important in midkine action to these cells.