Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat

Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter–IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma.

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson–Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

soc-2 encodes a leucine-rich repeat protein implicated in fibroblast growth factor receptor signaling

Activation of fibroblast growth factor (FGF) receptors elicits diverse cellular responses including growth, mitogenesis, migration, and differentiation. The intracellular signaling pathways that mediate these important processes are not well understood. In Caenorhabditis elegans, suppressors of clr-1 identify genes, termed soc genes, that potentially mediate or activate signaling through the EGL-15 FGF receptor. We demonstrate that three soc genes, soc-1, soc-2, and sem-5, suppress the activity of an activated form of the EGL-15 FGF receptor, consistent with the soc genes functioning downstream of EGL-15. We show that soc-2 encodes a protein composed almost entirely of leucine-rich repeats, a domain implicated in protein–protein interactions. We identified a putative human homolog, SHOC-2, which is 54% identical to SOC-2. We find that shoc-2 maps to 10q25, shoc-2 mRNA is expressed in all tissues assayed, and SHOC-2 protein is cytoplasmically localized. Within the leucine-rich repeats of both SOC-2 and SHOC-2 are two YXNX motifs that are potential tyrosine-phosphorylated docking sites for the SEM-5/GRB2 Src homology 2 domain. However, phosphorylation of these residues is not required for SOC-2 function in vivo, and SHOC-2 is not observed to be tyrosine phosphorylated in response to FGF stimulation. We conclude that this genetic system has allowed for the identification of a conserved gene implicated in mediating FGF receptor signaling in C. elegans.

Overexpression of transforming growth factor β1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor β1 (TGF-β1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-β1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-β1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-β1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-β1 in vascular disease.

Nerve growth factor inhibits sympathetic neurons’ response to an injury cytokine

Axonal damage to adult peripheral neurons causes changes in neuronal gene expression. For example, axotomized sympathetic, sensory, and motor neurons begin to express galanin mRNA and protein, and recent evidence suggests that galanin plays a role in peripheral nerve regeneration. Previous studies in sympathetic and sensory neurons have established that galanin expression is triggered by two consequences of nerve transection: the induction of leukemia inhibitory factor (LIF) and the reduction in the availability of the target-derived factor, nerve growth factor. It is shown in the present study that no stimulation of galanin expression occurs following direct application of LIF to intact neurons in the superior cervical sympathetic ganglion. Injection of animals with an antiserum to nerve growth factor concomitant with the application of LIF, on the other hand, does stimulate galanin expression. The data suggest that the response of neurons to an injury factor, LIF, is affected by whether the neurons still receive trophic signals from their targets.

Patterns of brain angiogenesis after vascular endothelial growth factor administration in vitro and in vivo

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell mitogen that has been shown to induce vasculogenesis and angiogenesis in many organ systems and tumors. Considering the importance of VEGF to embryonic vascularization and survival, the effects of administered VEGF on developing or adult cerebrovasculature are unknown: can VEGF alter brain angiogenesis or mature cerebrovascular patterns? To examine these questions we exposed fetal, newborn, and adult rat cortical slice explants to graduated doses of recombinant VEGF. The effects of another known angiogenic factor, basic fibroblast growth factor (bFGF), were evaluated in a comparable manner. In addition, we infused VEGF via minipump into the adult cortex. Significant angiogenic effects were found in all VEGF experiments in a dose-responsive manner that were abolished by the addition of VEGF neutralizing antibody. Fetal and newborn explants had a highly complex network of branched vessels that immunoexpressed the flt-1 VEGF receptor, and flk-1 VEGF receptor expression was determined by reverse transcription–PCR. Adult explants had enlarged, dilated vessels that appeared to be an expansion of the existing network. All bFGF-treated explants had substantially fewer vascular profiles. VEGF infusions produced both a remarkable localized neovascularization and, unexpectedly, the expression of flt-1 on reactive astrocytes but not on endothelial cells. The preponderance of neovascularization in vitro and in vivo, however, lacked the blood–brain barrier (BBB) phenotype marker, GLUT-1, suggesting that in brain the angiogenic role of VEGF may differ from a potential BBB functional role, i.e., transport and permeability. VEGF may serve an important capacity in neovascularization or BBB alterations after brain injury.

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.

Rsk-2 activity is necessary for epidermal growth factor-induced phosphorylation of CREB protein and transcription of c-fos gene

Activation by growth factors of the Ras-dependent signaling cascade results in the induction of p90 ribosomal S6 kinases (p90rsk). These are translocated into the nucleus upon phosphorylation by mitogen-activated protein kinases, with which p90rsk are physically associated in the cytoplasm. In humans there are three isoforms of the p90rsk family, Rsk-1, Rsk-2, and Rsk-3, which are products of distinct genes. Although these isoforms are structurally very similar, little is known about their functional specificity. Recently, mutations in the Rsk-2 gene have been associated with the Coffin–Lowry syndrome (CLS). We have studied a fibroblast cell line established from a CLS patient that bears a nonfunctional Rsk-2. Here we document that in CLS fibroblasts there is a drastic attenuation in the induced Ser-133 phosphorylation of transcription factor CREB (cAMP response element-binding protein) in response to epidermal growth factor stimulation. The effect is specific, since response to serum, cAMP, and UV light is unaltered. Furthermore, epidermal growth factor-induced expression of c-fos is severely impaired in CLS fibroblasts despite normal phosphorylation of serum response factor and Elk-1. Finally, coexpression of Rsk-2 in transfected cells results in the activation of the c-fos promoter via the cAMP-responsive element. Thus, we establish a link in the transduction of a specific growth factor signal to changes in gene expression via the phosphorylation of CREB by Rsk-2.

Inhibition of myogenesis by transforming growth factor β is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm

Transforming growth factor β (TGF-β) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-β. Within 30 min of treatment, TGF-β induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-β. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-β, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-β. We propose a mechanism in which the inhibition of myogenesis by TGF-β is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.

Gene therapy in allergic encephalomyelitis using myelin basic protein-specific T cells engineered to express latent transforming growth factor-β1

A myelin basic protein (MBP)-specific BALB/c T helper 1 (Th1) clone was transduced with cDNA for murine latent transforming growth factor-β1 (TGF-β1) by coculture with fibroblasts producing a genetically engineered retrovirus. When SJL x BALB/c F1 mice, immunized 12–15 days earlier with proteolipid protein in complete Freund’s adjuvant, were injected with 3 × 106 cells from MBP-activated untransduced cloned Th1 cells, the severity of experimental allergic encephalomyelitis (EAE) was slightly increased. In contrast, MBP-activated (but not resting) latent TGF-β1-transduced T cells significantly delayed and ameliorated EAE development. This protective effect was negated by simultaneously injected anti-TGF-β1. The transduced cells secreted 2–4 ng/ml of latent TGF-β1 into their culture medium, whereas control cells secreted barely detectable amounts. mRNA profiles for tumor necrosis factor, lymphotoxin, and interferon-γ were similar before and after transduction; interleukin-4 and -10 were absent. TGF-β1-transduced and antigen-activated BALB/c Th1 clones, specific for hemocyanin or ovalbumin, did not ameliorate EAE. Spinal cords from mice, taken 12 days after receiving TGF-β1-transduced, antigen-activated cells, contained detectable amounts of TGF-β1 cDNA. We conclude that latent TGF-β1-transduced, self-reactive T cell clones may be useful in the therapy of autoimmune diseases.

SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor β signaling

Transforming growth factor β (TGF-β) regulates a variety of physiologic processes, including growth inhibition, differentiation, and induction of apoptosis. Some TGF-β-initiated signals are conveyed through Smad3; TGF-β binding to its receptors induces phosphorylation of Smad3, which then migrates to the nucleus where it functions as a transcription factor. We describe here the association of Smad3 with the nuclear protooncogene protein SnoN. Overexpression of SnoN represses transcriptional activation by Smad3. Activation of TGF-β signaling leads to rapid degradation of SnoN and, to a lesser extent, of the related Ski protein, and this degradation is likely mediated by cellular proteasomes. These results demonstrate the existence of a cascade of the TGF-β signaling pathway, which, upon TGF-β stimulation, leads to the destruction of protooncoproteins that antagonize the activation of the TGF-β signaling.

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.