Leupeptin enhances cell surface localization of fibroblast growth factor receptor 1 in adult sensory neurons by increased recycling

Fibroblast growth factors (FGFs) act as trophic factors during development and regeneration of the nervous system. FGFs mediate their responses by activation of four types of FGF receptors (FGFR1-4). FGFR1 is expressed in adult sensory neurons of dorsal root ganglia (DRG), and overexpression of FGFR1 enhances FGF-2-induced elongative axon growth in vitro. Ligand-induced activation of FGFR1 is followed by endocytosis and rapid lysosomal degradation. We previously reported that the lysosomal inhibitor leupeptin prevents degradation of FGFR1 and promotes FGF-2-induced elongative axon growth of DRG neurons overexpressing FGFR1. Therefore, we analyzed the effects of leupeptin on intracellular sorting of FGFR1 in PC12 pheochromocytoma cells and DRG neurons. Leupeptin increased colocalization of FGFR1 with lysosomes. Furthermore, leupeptin enhanced the cell surface localization of FGFR1 by increased receptor recycling and this effect was abolished by the recycling inhibitor monensin. In addition, a lysine mutant of FGFR1, which is preferentially recycled back to the cell surface, promoted elongative axon growth of DRG neurons similar to leupeptin. In contrast, the lysosomal inhibitor bafilomycin had no effect on surface localization of FGFR1, inhibited axon growth of DRG neurons and abolished the effects of leupeptin on receptor recycling. Together, our results strongly imply that increased recycling of FGFR1 promotes axon elongation, but not axonal branching, of adult DRG neurons in vitro.     

Polysialylation promotes neural cell adhesion molecule-mediated cell migration in a fibroblast growth factor receptor-dependent manner, but independent of adhesion capability

Polysialic acid (PSA), a carbohydrate polymer mainly present in the neural cell adhesion molecule (NCAM), promotes neural plasticity; however, its mode of action in tumor malignancy remains largely unknown. In this study, we investigated the influence of polysialylation on cell migration. PSA consistently promoted cell migration on different extracellular matrices (ECMs) but differentially affected cell adhesion. All of these actions were reversed by endo-N-acetylneuraminidase treatment, and PSA-driven migration was inhibited by the specific fibroblast growth factor receptor (FGFR) inhibitor Su5402. Consistent with this latter observation, PSA-stimulated migration on different ECMs was paralleled by activation of the FGFR and its downstream signaling components, PLC-γ, focal adhesion kinase and extracellular signal-regulated kinase 1/2. In contrast, the pattern of p59(fyn) activation correlated with differential adhesion to different ECMs. Collectively, these results indicate that PSA-conjugated NCAM potentiates signal transduction by the FGFR pathway and thereby enhances cell migration independent of adhesion capability, providing additional insights into the role of PSA in cancer development.     

p38 mitogen-activated protein kinase activation is required for fibroblast growth factor-2-stimulated cell proliferation but not differentiation.

Basic fibroblast growth factor (FGF-2) is a member of a family of polypeptides that have roles in a wide range of biological processes. To determine why different cell types show distinct responses to treatment with FGF-2, the array of FGF receptors present on the surface of a cell which differentiates in response to FGF-2 (PC12 cells) was compared with that present on the surface of a cell that proliferates in response to FGF-2 (Swiss 3T3 fibroblasts). Both cell types express exclusively FGFR1, suggesting that there are cell type-specific FGFR1 signaling pathways. Since mitogen-activated protein kinases function as mediators of cellular responses to a variety of stimuli, the roles of these proteins in FGF-mediated responses were examined. FGF-2 activates extracellular signal-regulated kinases with similar kinetics in both fibroblasts and PC12 cells, and a specific inhibitor of extracellular signal-regulated kinase activation blocks differentiation but has little effect on proliferation. In contrast, while p38 mitogen-activated protein kinase is activated weakly and transiently in PC12 cells treated with FGF-2, a much stronger and sustained activation of this kinase is seen in FGF-2-treated fibroblasts. Furthermore, specific inhibitors of this kinase block proliferation but have no effect on differentiation. This effect on proliferation is specific for FGF-2 since the same concentrations of inhibitors have little or no effect on proliferation induced by serum.     

An NCAM-derived FGF-receptor agonist, the FGL-peptide, induces neurite outgrowth and neuronal survival in primary rat neurons

The Neural Cell Adhesion Molecule (NCAM) plays a crucial role in development of the central nervous system regulating cell migration, differentiation and synaptogenesis. NCAM mediates cell-cell adhesion through homophilic NCAM binding, subsequently resulting in activation of the fibroblast growth factor receptor (FGFR). NCAM-mediated adhesion leads to activation of various intracellular signal transduction pathways, including the Ras-mitogen activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase (PI3K)-Akt pathways. A synthetic peptide derived from the second fibronectin type III module of NCAM, the FGL peptide, binds to and induces phosphorylation of FGFR without prior homophilic NCAM binding. We here present evidence that this peptide is able to mimic NCAM heterophilic binding to the FGFR by inducing neuronal differentiation as reflected by neurite outgrowth through a direct interaction with FGFR in primary cultures of three different neuronal cell types all expressing FGFR subtype 1: dopaminergic, hippocampal and cerebellar granule neurons. Moreover, we show that the FGL peptide promotes neuronal survival upon induction of cell death in the same three cell types. The effects of the FGL peptide are shown to depend on activation of FGFR and the MAPK and PI3K intracellular signalling pathways, all three kinases being necessary for the effects of FGL on neurite outgrowth and neuronal survival.     

Unique intracellular trafficking processes associated with neural cell adhesion molecule and its intracellular signaling

Homophilic binding of the neural cell adhesion molecule (NCAM) results in intracellular signaling, which also involves heterophilic engagement of coreceptors such as the fibroblast growth factor receptor (FGFR) and receptor protein tyrosine phosphatase-α (RPTPα). NCAM's own cellular dynamic itinerary includes endocytosis and recycling to the plasma membrane. Recent works suggest that NCAM could influence the trafficking of other receptor molecules that it associates with, particularly the FGFR. Furthermore, it was demonstrated that NCAM could undergo proteolytic processing upon activation. A processed fragment of NCAM, together with an N-terminal fragment of focal adhesion kinase (FAK), is translocated into the nucleus. Here, the authors discuss these rather unique (though not without precedence and analogues) receptor trafficking activities that are associated with NCAM and NCAM signaling.     

Unique Intracellular Trafficking Processes Associated With Neural Cell Adhesion Molecule and Its Intracellular Signaling

Abstract

Homophilic binding of the neural cell adhesion molecule (NCAM) results in intracellular signaling, which also involves heterophilic engagement of coreceptors such as the fibroblast growth factor receptor (FGFR) and receptor protein tyrosine phosphatase-α (RPTPα). NCAM's own cellular dynamic itinerary includes endocytosis and recycling to the plasma membrane. Recent works suggest that NCAM could influence the trafficking of other receptor molecules that it associates with, particularly the FGFR. Furthermore, it was demonstrated that NCAM could undergo proteolytic processing upon activation. A processed fragment of NCAM, together with an N-terminal fragment of focal adhesion kinase (FAK), is translocated into the nucleus. Here, the authors discuss these rather unique (though not without precedence and analogues) receptor trafficking activities that are associated with NCAM and NCAM signaling.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Differences in signal transduction pathways by which platelet-derived and fibroblast growth factors activate extracellular signal-regulated kinase in differentiating oligodendrocytes

Treatment of cultured rat oligodendroglial progenitors with either platelet-derived growth factor (PDGF) or fibroblast growth factor-2 (FGF-2) activated extracellular signal regulated kinase 2 (ERK2). Activation was transient in response to PDGF, whereas it was greater and more prolonged in response to FGF-2. ERK2 activation by PDGF was preceded by a very rapid, robust and transient tyrosine phosphorylation of the PDGF receptor. Although there was consistently more activation of ERK2 in response to FGF-2 than to PDGF, immunostaining of FGF receptors 1 (FGFR1) and 2 (FGFR2) and their tyrosine phosphorylation in progenitors was very weak, and both receptors were up-regulated during differentiation to oligodendrocytes. Tyrosine phosphorylation of the FGF receptors was maximal from 15 to 60 min of treatment and was sustained for many hours. Binding of radioiodinated FGF-2 to FGFR1 was predominant in progenitors, whereas binding to FGFR2 was predominant in oligodendrocytes. ERK2 activation by PDGF was more sensitive to inhibition of tyrosine kinases, whereas ERK2 activation by FGF-2 was relatively more sensitive to inhibitors of protein kinase C. These differences in signal transduction pathways probably contribute to the different cellular responses of oligodendroglial lineage cells to PDGF and FGF-2, respectively.     

Production and characterization of the extracellular domain of recombinant human fibroblast growth factor receptor 4

Among the members of the fibroblast growth factor receptor family the FGFR4 has demonstrated strong dependence on heparin-like material for its activation by fibroblast growth factors. We have produced and characterized a recombinant human FGFR4 extracellular domain (FGFR4ed), in order to study its biochemical properties in isolated conditions. The FGFR4ed was expressed in an insect cell system and purified from the culture medium by Ni(2+)-affinity and gel filtration chromatography. Pure FGFR4ed was tested for FGF- and heparin-binding by covalent crosslinking experiments and by biosensor analysis. In solution, FGFR4ed formed complexes with acidic FGF (FGF-1) and basic FGF (FGF-2), both in the presence and absence of heparin. Immobilized FGFR4 also bound FGF-8 besides FGF-1 and FGF-2. Furthermore, heparin alone induced receptor oligomerization on the surface of the receptor coupled chip. Thus, the recombinant FGFR4ed revealed properties described for the cellular form of this receptor and can be used for interaction studies.     

Sorting of the FGF receptor 1 in a human glioma cell line

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase promoting tumor growth in a variety of cancers, including glioblastoma. Binding of FGFs triggers the intracellular Ras/Raf/ERK signaling pathway leading to cell proliferation. Down-regulation of FGFR1 and, consequently, inactivation of its signaling pathways represent novel treatment strategies for glioblastoma. In this study, we investigated the internalization and endocytic trafficking of FGFR1 in the human glioma cell line U373. Stimulation with FGF-2 induced cell rounding accompanied by increased BrdU and pERK labeling. The overexpression of FGFR1 (without FGF treatment) resulted in enhanced phosphorylated FGFR1 suggesting receptor autoactivation. Labeled ligand (FGF-2-Cy5.5) was endocytosed in a clathrin- and caveolin-dependent manner. About 25 % of vesicles carrying fluorescently tagged FGFR1 represented early endosomes, 15 % transferrin-positive recycling endosomes and 40 % Lamp1-positive late endosomal/lysosomal vesicles. Stimulation with FGF-2 increased the colocalization rate in each of these vesicle populations. The treatment with the lysosomal inhibitor leupeptin resulted in FGFR1 accumulation in lysosomes, but did not enhance receptor recycling as observed in neurons. Analysis of vesicle distributions revealed an accumulation of recycling endosomes in the perinuclear region. In conclusion, the shuttling of receptor tyrosine kinases can be directly visualized by overexpression of fluorescently tagged receptors which respond to ligand stimulation and follow the recycling and degradation pathways similarly to their endogenous counterparts.     

Differential effects of heparin saccharides on the formation of specific fibroblast growth factor (FGF) and FGF receptor complexes.

Heparan sulfates (HS) play an important role in the control of cell growth and differentiation by virtue of their ability to modulate the activities of heparin-binding growth factors, an issue that is particularly well studied for fibroblast growth factors (FGFs). HS/heparin co-ordinate the interaction of FGFs with their receptors (FGFRs) and are thought to play a critical role in receptor dimerization. Biochemical and crystallographic studies, conducted mainly with FGF-2 or FGF-1 and FGF receptors 1 and 2, suggests that an octasaccharide is the minimal length required for FGF- and FGFR-induced dimerization and subsequent activation. In addition, 6-O-sulfate groups are thought to be essential for binding of HS to FGFR and for receptor dimerization. We show here that oligosaccharides shorter than 8 sugar units support activation of FGFR2 IIIb by FGF-1 and interaction of FGFR4 with FGF-1. In contrast, only relatively long oligosaccharides supported receptor binding and activation in the FGF-1.FGFR1 or FGF-7.FGFR2 IIIb setting. In addition, both 6-O- and 2-O-desulfated heparin activated FGF-1 signaling via FGFR2 IIIb, whereas neither one stimulated FGF-1 signaling via FGFR1 or FGF-7 via FGFR2 IIIb. These findings indicate that the structure of HS required for activating FGFs is dictated by the specific FGF and FGFR combination. These different requirements may reflect the differences in the mode by which a given FGFR interacts with the various FGFs.     

Structural basis for a direct interaction between FGFR1 and NCAM and evidence for a regulatory role of ATP

The neural cell adhesion molecule (NCAM) promotes axonal outgrowth, presumably through an interaction with the fibroblast growth factor receptor (FGFR). NCAM also has a little-understood ATPase activity. We here demonstrate for the first time a direct interaction between NCAM (fibronectin type III [F3] modules 1 and 2) and FGFR1 (Ig modules 2 and 3) by surface plasmon resonance (SPR) analysis. The structure of the NCAM F3 module 2 was determined by NMR and the module was shown by NMR to interact with the FGFR1 Ig module 3 and ATP. The NCAM sites binding to FGFR and ATP were found to overlap and ATP was shown by SPR to inhibit the NCAM-FGFR binding, indicating that ATP probably regulates the NCAM-FGFR interaction. Furthermore, we demonstrate that the NCAM module was able to induce activation (phosphorylation) of FGFR and to stimulate neurite outgrowth. In contrast, ATP inhibited neurite outgrowth induced by the module.     

Fibroblast growth factor receptor-1 signaling in pancreatic islet beta-cells is modulated by the extracellular matrix

Maintenance of pancreatic beta-cell mass depends on extracellular stimuli that promote survival and proliferation. In the islet, these stimuli come from the beta-cell microenvironment and include extracellular matrix deposited by associated vascular endothelial cells. Fibroblast growth factor receptor-1 (FGFR1) has recently been implicated as a signaling pathway that is important for normal beta-cell function. We would like to understand how extracellular matrix and FGFR1 signaling interact to promote beta-cell survival and proliferation. To examine beta-cell-specific receptor responses, we created lentiviral vectors with rat insulin promoter-driven expression of Venus fluorescent protein-tagged full-length (R1betav) and kinase-deficient (KDR1betav) FGFR1. Significant FGF-1-dependent activation of ERK1/2 was observed in betaTC3 cells, dispersed beta-cells, and beta-cells in intact islets. This response was enhanced by R1betav expression and reduced by KDR1betav expression. Plating-dispersed beta-cells on collagen type IV resulted in enhanced expression of endogenous FGFR1 that was associated with sustained activation of ERK1/2. Conversely, plating cells on laminin reduced expression of FGFR1, and this reduction was associated with transient activation of ERK1/2. Addition of neutralizing antibodies to inhibit beta-cell attachment to laminin via alpha(6)-integrin increased high-affinity FGF-1-binding at the plasma membrane and resulted in sustained ERK1/2 activity similar to cells plated on collagen type IV. These data show that the FGF-stimulated beta-cell response is negatively affected by alpha(6)-integrin binding to laminin and suggest regulation associated with vascular endothelial cell remodeling.     

Keratinocyte growth factor/fibroblast growth factor-7-regulated cell migration and invasion through activation of NF-kappaB transcription factors

Keratinocyte growth factor (KGF)/fibroblast growth factor-7 (FGF-7) is a paracrine- and epithelium-specific growth factor produced by cells of mesenchymal origin. It acts exclusively through FGF-7 receptor (FGFR2/IIIb), which is expressed predominantly by epithelial cells, but not by fibroblasts, suggesting that it might function as a paracrine mediator of mesenchymal-epithelial interactions. KGF/FGF-7 plays an essential role in the growth of epithelial cells and is frequently overexpressed in cancers of epithelial origin such as pancreatic cancer, switching paracrine stimulation of KGF/FGF-7 to an autocrine loop. Less is known, however, about the signaling pathways by which KGF/FGF-7 regulates the response of epithelial cells. To delineate the signaling pathways activated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activated nuclear factor kappaB (NF-kappaB), which in turn induced expression of VEGF, MMP-9, and urokinase-type plasminogen activator and increased migration and invasion of KGF/FGF-7-stimulated human pancreatic ductal epithelial cells. Expression of phosphorylation-defective IkappaBalpha (IkappaBalphaS32A,S36A), which blocked NF-kappaB activation, inhibited KGF/FGF-7-induced gene expression and cell migration and invasion. Our results demonstrate for the first time that KGF/FGF-7 induces NF-kappaB activation and that NF-kappaB plays an essential role in regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellular responses. Thus, these findings identify one signaling pathway for KGF/FGF-7-regulated cell migration and invasion and suggest that paracrine sources of KGF/FGF-7 are one of the malignancy-contributing factors from tumor stroma.     

Fibroblast growth factor receptors participate in the control of mitogen-activated protein kinase activity during nerve growth factor-induced neuronal differentiation of PC12 cells.

The current paradigm for the role of nerve growth factor (NGF) or FGF-2 in the differentiation of neuronal cells implies their binding to specific receptors and activation of kinase cascades leading to the expression of differentiation specific genes. We examined herein the hypothesis that FGF receptors (FGFRs) are involved in NGF-induced neuritogenesis of pheochromocytoma-derived PC12 cells. We demonstrate that in PC12 cells, FGFR expression and activity are modulated upon NGF treatment and that a dominant negative FGFR-2 reduces NGF-induced neuritogenesis. Moreover, FGF-2 expression is modulated by NGF, and FGF-2 is detected at the cell surface. Oligonucleotides that specifically inhibit FGF-2 binding to its receptors are able to significantly reduce NGF-induced neurite outgrowth. Finally, the duration of mitogen-activated protein kinase (MAPK) activity upon FGF or NGF stimulation is shortened in FGFR-2 dominant negative cells through inactivation of signaling from the receptor to the Ras/MAPK pathway. In conclusion, these results demonstrate that FGFR activation is involved in neuritogenesis induced by NGF where it contributes to a sustained MAPK activity in response to NGF.     

The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling

Binding of growth factors to specific cell surface receptors is the first step in initiating cell signaling cascades that ultimately result in diverse activities such as proliferation, differentiation, and apoptosis. Dimerization and phosphorylation of tyrosine kinase transmembrane receptors is the typical paradigm for this activation but, for many growth factors, cell surface interactions are not limited to a single receptor type. In particular, heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG) on the cell surface and within the extracellular matrix (ECM), and these molecules have been viewed as accessory co-receptors serving to facilitate tyrosine kinase receptor binding. Recent studies, however, have indicated that HSPG can directly participate in signal transduction in response to FGF-2 binding. Thus, in the present study, we used mathematical modeling to examine whether the kinetics of formation of the various FGF-2 bound complexes on the cell surface correlate with the activation of the downstream mediators of FGF-2 response, Erk1/2. We find that FGF-2 binding to its receptor correlates well with Erk1/2 activation and that HSPG can modulate this response through its ability to stabilize these ligand receptor complexes. Moreover, we also observed that FGF-2 binding to HSPG correlates strongly with Erk1/2 activation under conditions where there is a loss of receptor activity, and we demonstrate that the relative amounts of signaling and non-signaling HSPG on the cell surface, as well as the presence of competing HSPG in the ECM, can impact the signal potential via this pathway. Thus, the selective regulation of specific HSPG might provide a mechanism for fine tuned modulation of heparin-binding growth factor signaling in cells where signal intensity and duration could direct cellular response toward growth, migration or differentiation.     

Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14. Characterization of a new receptor binding partner

Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 binding partner. A kinase-inactive mutant of FGFR1 failed to interact with Grb14, indicating that activation of FGFR1 is necessary for binding. Deletion of the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to phenylalanine abolished binding, and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites. Co-immunoprecipitation and in vitro binding assays demonstrated that binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor activation by fibroblast growth factor-2 (FGF-2). Deletion of the Src homology 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and eliminated dependence on receptor activation. The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb14 bound only FGFR1, suggesting that regions upstream of the SH2 domain confer specificity for FGFR1. Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this phosphorylation. Expression of exogenous Grb14 inhibited FGF-2-induced cell proliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis. These data demonstrate an interaction between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.     

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.     

Role and regulation of the fibroblast growth factor axis in human thyroid follicular cells

Thyroidal levels of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 1 (FGFR1) are elevated in human thyroid hyperplasia. To understand the significance of this, effects of FGFR1 activation on normal human thyrocyte growth and function in vitro and the regulation of FGF-2 and FGFR1 expression have been examined. FGF-2 stimulated cell growth, as measured by cell counting, and inhibited thyroid function as measured by 125I uptake. Sensitivity to FGF-2 disappeared after 7 days, although FGFR1 expression was maintained. Thyroid-stimulating hormone (TSH, 300 mU/l) increased FGFR1 mRNA expression within 4 h and protein expression by 8 h. Exogenous FGF-2 decreased FGFR1 protein. Endogenous FGF-2 levels were low (approximately 1-2 pg/microg protein), and TSH treatment decreased these by 50%. Protein kinase C (PKC) activation increased FGF-2 mRNA and FGF-2 secretion within 2 h. This effect was enhanced (4.4-fold) when cells were cultured in TSH. We conclude that TSH stimulates FGFR1 but not FGF-2 expression. PKC activation stimulates FGF-2 synthesis and secretion, and TSH synergizes with PKC activators. Increases in FGFR1 or FGF-2 or in both may contribute to goitrogenesis.