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.

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.

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.

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.

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Transforming growth factor β-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells

Drosophila Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the Drosophila transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-β signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-β receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-β treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-β-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-β in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-β signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-β receptor complex is an essential step in signal transduction by TGF-β for both inhibition of cell proliferation and activation of the PAI-1 promoter.

Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.

Insulin and insulin-like growth factor-I acutely inhibit surface translocation of growth hormone receptors in osteoblasts: A novel mechanism of growth hormone receptor regulation

We previously have demonstrated that insulin and insulin-like growth factor-I (IGF-I) down-regulate growth hormone (GH) binding in osteoblasts by reducing the number of surface GH receptors (GHRs). The present study was undertaken to investigate the mechanism of GHR down-regulation. Treatment with 5 nM insulin or IGF-I for 18 hr significantly decreased surface GH binding to 26.4 ± 2.9% and 23.0 ± 2.7% of control (mean ± SE; P < 0.05), respectively. No corresponding reductions in the mRNA level and total cellular content of GHR were found, nor was the rate of receptor internalization affected. The effects on GHR translocation were assessed by measuring the reappearance of GH binding of whole cells after trypsinization to remove the surface receptors. GH binding of control cultures significantly increased (P < 0.05) over 2 hr after trypsinization, whereas no recovery of binding activity was detected in insulin and IGF-I-treated cultures, indicating that GHR translocation was impaired. Studies on the time course of GHR down-regulation revealed that surface GH binding was reduced significantly by 3-hr treatment (P ≤ 0.0005), whereas GHR translocation was completely abolished by 75–90 min with insulin and IGF-I. The inhibition of receptor translocation by insulin, but not IGF-I, was attenuated by wortmannin. In conclusion, insulin and IGF-I down-regulated GH binding in osteoblasts by acutely impairing GHR translocation, with their effects exerted through distinct postreceptor signaling pathways.

Fluorescence in situ hybridization analysis of keratinocyte growth factor gene amplification and dispersion in evolution of great apes and humans

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15–21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12–13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.