Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3.

The presumed open reading frame for mouse FGF3, starting at the most 5' AUG codon, predicts a hydrophobic N-terminus characteristic of a signal peptide for secretion. However, in reticulocyte lysates and transfected COS-1 cells, the full-length Fgf-3 cDNA is translated almost exclusively from an upstream CUG codon. The resultant products are distributed in both the nucleus and the secretory pathway, implying that the single CUG-initiated form of FGF3 has dual fates. By analysing a series of deletion and replacement mutants and by linking parts of FGF3 to a heterologous protein, we show that secretion is mediated by cleavage adjacent to the previously defined signal peptide, whereas nuclear localization is determined primarily by a classical but relatively weak bipartite motif. In the context of FGF3, nuclear localization also requires the N-terminal sequences which lie upstream of the signal peptide. Thus, the subcellular fate of FGF3 is determined by the competing effects of signals for secretion and nuclear localization within the same protein, rather than by alternative initiation or processing.     

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.     

Control of the intracellular signaling induced by fibroblast growth factors (FGF) over the proliferation and survival of retinal pigment epithelium cells: example of the signaling regulation of growth factors endogenous to the retina ]

Retinal pigmented epithelium (RPE) cells are of central importance in the maintenance of neural retinal function. RPE cell apoptosis is responsible for the development of a variety of retinal degeneration. The role of FGF2 was investigated on RPE cell proliferation and apoptosis in vitro. In the absence of serum, RPE cells died by apoptosis, while the addition of FGF2 greatly reduces apoptosis over a 7-day culture period. This is due to an autocrine loop involving secretion of endogenous FGF1 in the mechanism that govern FGF2-induced resistance to apoptosis. FGF2 induces long-term activation of FGFR1 and ERK1/2, and production of the anti-apoptotic protein BcL-x. Because FGF1 has no classical signal sequence to direct its secretion, we investigated the effects of FGF1 secretion on RPE proliferation and apoptosis in the absence of exogenous FGF2. Forced secretion of endogenous FGF1 by adding a signal peptide to the FGF1 molecule induces FGF1 secretion and cell proliferation in the presence of serum, while in FGF1 stops to be secreted and cell die in the absence of serum. Conversely, in cells cultured in the presence of serum, FGF1 without signal peptide is not secreted, but is secreted and rescue RPE cell from apoptosis when cells are cultured without serum. Thus, the proliferation and survival activities of endogenous FGF1 depend on the secretion of FGF1 which is determined by the cell environment.     

Fibroblast growth factor 3, a protein with a dual subcellular fate, is interacting with human ribosomal protein S2

The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.     

Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2

Members of the FGF family of growth factors localize to the nuclei in a variety of different cell types. To determine whether FGF receptors are also present within nuclei and if this localization is regulated by FGFs, nuclei were prepared from quiescent and FGF-2-treated Swiss 3T3 fibroblasts and examined for the presence of FGF receptors by immunoblotting with an antibody produced against the extracellular domain of FGF receptor-1 (FGFR-1). Little or no FGFR-1 is detected in nuclei prepared from quiescent cells. When cells are treated with FGF- 2, however, there is a time- and dose-dependent increase in the association of FGFR-1 immunoreactivity with the nucleus. In contrast, treatment with either EGF or 10% serum does not increase the association of FGFR-1 with the nucleus. When cell surface proteins are labeled with biotin, a biotinylated FGFR-1 is detected in the nuclear fraction prepared from FGF-2-treated, but not untreated, cells indicating that the nuclear-associated FGFR-1 immunoreactivity derives from the cell surface. The presence of FGFR-1 in the nuclei of FGF-2- treated cells was confirmed by immunostaining with a panel of different FGFR-1 antibodies, including one directed against the COOH-terminal domain of the protein. Fractionation of nuclei from FGF-2-treated cells indicates that nuclear FGFR-1 is localized to the nuclear matrix, suggesting that the receptor may play a role in regulating gene activity.     

Interaction between ERK and GSK3beta mediates basic fibroblast growth factor-induced apoptosis in SK-N-MC neuroblastoma cells

The Ewing's sarcoma family of tumors (ESFT) includes Ewing's sarcoma (ES), Askin's tumor of the chest wall, and peripheral primitive neuroectodermal tumor. Basic fibroblast growth factor (FGF2) suppresses the growth of ESFT cells and causes their apoptosis. The underlying mechanism is unclear. Using a human peripheral primitive neuroectodermal tumor cell line, SK-N-MC, we demonstrated FGF2 stimulated phosphorylation of ERK1 and ERK2 (pERK1/2) and GSK3beta (pGSK3beta(Tyr-216)), all of which were primarily retained in the cytoplasm. FGF2 promoted the association between ERK and pGSK3beta(Tyr-216). Inhibitors for GSK3beta (TDZD and LiCl) and ERK (PD98059) protected cells from FGF2-induced apoptosis. On the other hand, inhibitors of GSK3beta, but not PD98059 decreased ERK/pGSK3beta(Tyr-216) association and caused a nuclear translocation of pERK1/2. Similarly, expression of a kinase-deficient (K85R) GSK3beta or GSK3beta-small interfering RNA inhibited FGF2-regulated ERK/pGSK3beta(Tyr-216) association and translocated pERK to the nucleus. Both K85R GSK3beta and small interfering RNA offered protection against FGF2-induced cell death. In contrast, overexpression of wild-type GSK3beta sensitized cells to FGF2 cytotoxicity. Hydrogen peroxide and ethanol enhanced FGF2-stimulated pGSK3beta(Tyr-216), ERK/pGSK3beta(Tyr-216) association, and cytoplasmic retention of pERK1/2. As a result, they potentiated FGF2-induced cell death. Taken together, our results suggested that FGF2-induced accumulation of pERK1/2 in the cytoplasm is toxic for SK-N-MC cells. The formation of an ERK.GSK3beta complex retained pERK1/2 in the cytoplasm. In contrast, disruption of the ERK.GSK3beta complex resulted in nuclear translocation of pERK1/2 and offered protection.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

Pivotal Role of Translokin/CEP57 in the Unconventional Secretion Versus Nuclear Translocation of FGF2

Intracellular trafficking of fibroblast growth factor 2 (FGF2) exhibits two unusual features: (i) it is secreted despite the lack of signal peptide and (ii) it can translocate to the nucleus after interaction with high- and low-affinity receptors on the cell surface, although it does not possess any classical nuclear localization signal. This nuclear translocation constitutes an important part of the response to the growth factor. Previously, we identified Translokin/CEP57, an FGF2 binding partner, as an intracellular mediator of FGF2 trafficking, which is essential for the nuclear translocation of the growth factor. Here, we report the identification of four Translokin partners: sorting nexin 6, Ran-binding protein M and the kinesins KIF3A and KIF3B. These proteins, through their interaction with Translokin, are involved in two exclusive complexes allowing the bidirectional trafficking of FGF2. Thus, Translokin plays a pivotal role in this original mechanism. In addition, we show that FGF2 secretion is regulated by a negative loop, retro-controlled by FGF receptor and involving FGF2 itself.     

Activation of multiple signaling pathways is critical for fibroblast growth factor 2- and vascular endothelial growth factor-stimulated ovine fetoplacental endothelial cell proliferation

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by N(G)-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.     

Heparin affin regulatory peptide/pleiotrophin mediates fibroblast growth factor 2 stimulatory effects on human prostate cancer cells

Fibroblast growth factor 2 (FGF2) is a pleiotropic growth factor that has been implicated in prostate cancer formation and progression. In the present study we found that exogenous FGF2 significantly increased human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) or pleiotrophin seems to be an important mediator of FGF2 stimulatory effects, since the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, FGF2, through FGF receptors (FGFRs), significantly induced HARP expression and secretion by LNCaP cells and increased luciferase activity of a reporter gene vector carrying the full-length promoter of HARP gene. Using a combination of Western blot analyses, as well as genetic and pharmacological inhibitors, we found that activation of FGFR by FGF2 in LNCaP cells leads to NAD(P)H oxidase-dependent hydrogen peroxide production, phosphorylation of ERK1/2 and p38, activation of AP-1, increased expression and secretion of HARP, and, finally, increased cell proliferation and migration. These results establish the role and the mode of activity of FGF2 in LNCaP cells and support an interventional role of HARP in FGF2 effects, providing new insights on the interplay among growth factor pathways within prostate cancer cells.     

Delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein

Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.     

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.     

Localization of rRNA genes in the nuclear space of Calliphora erythrocephala Mg. nurse cells during polytenization

Multicolor 3D fluorescence in situ hybridization was used to study arrangement of rRNA genes in Calliphora erythrocephala nurse cell nuclei with different levels of polyteny. It has been shown that the rRNA genes are exclusively localized to chromosome 6, suggesting that chromosome 6 is the only C. erythrocephala chromosome responsible for nucleolar formation. We have also described changes in localization of ribosomal genes within the chromosome territory during polytenization, namely, that rDNA signals are detected in the peripheral region of chromosome territory starting from the stage of polytene chromosomes. In addition, it has emerged that large nucleolus associated with chromosome 6 starts to develop in the central nuclear region in the C. erythrocephala nurse cell nuclei at the stage of a primary reticular structure. The central position and nucleolar structure are retained at the stages when chromosome 6 occupies the central position, that is, at the stages of polytene and bloblike chromosomes. When the nucleus restores a reticular structure but at a higher polyteny level, the displacement of chromosome 6 to the nuclear periphery is accompanied by disruption of the large nucleolus into micronucleoli. The micronucleoli are distributed in the nuclear space retaining their association with the nucleolar-organizing regions of chromosome 6. Thus, our data suggest that the large-scale alterations in the organization of chromosome 6 and the nucleolus during polytenization are the correlated processes directly dependent on the rRNA gene activity. The earlier described dynamics of nucleolar-organizing chromosome territory and nucleolus in the nuclear space is likely to be associated with the change in the total expression activity of the nucleus, which complies with the hypothesis on the correlation between spatial nuclear organization and expression regulation of genetic material.     

Retention of fibroblast growth factor 3 in the Golgi complex may regulate its export from cells.

The fibroblast growth factors (FGFs) fall into two distinct groups with respect to their mode of release from cells. Whereas FGF1 and FGF2 lack conventional signal peptides, the remaining members have typical features of secreted proteins. However, the behavior of mouse FGF3 is anomalous, since, despite entering the secretory pathway and undergoing primary glycosylation, its release from transfected COS-1 cells is very inefficient compared with that of FGF4 and FGF5. To investigate the unusual properties of FGF3, we analyzed the processing, secretion, and intracellular localization of a series of site-directed mutants as well as chimeras produced by fusing parts of FGF3, FGF4, and FGF5. Wild-type FGF3 was shown to accumulate in an immature form in the Golgi complex, from where it is slowly released into the extracellular matrix. Removing or relocating the Asn-linked glycosylation site further impaired its release, and exchanging the signal peptide or carboxy terminus had little effect. In contrast, a chimeric protein with an amino terminus from FGF5 was efficiently secreted and biologically active in cell transformation assays. The data suggest that a structural feature of FGF3 involving the amino-terminal region and glycosylation site has a significant bearing on its passage through the Golgi complex and may regulate the secretion of the ligand.     

Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis

The liver and intestine play crucial roles in maintaining bile acid homeostasis. Here, we demonstrate that fibroblast growth factor 15 (FGF15) signals from intestine to liver to repress the gene encoding cholesterol 7alpha-hydroxylase (CYP7A1), which catalyzes the first and rate-limiting step in the classical bile acid synthetic pathway. FGF15 expression is stimulated in the small intestine by the nuclear bile acid receptor FXR and represses Cyp7a1 in liver through a mechanism that involves FGF receptor 4 (FGFR4) and the orphan nuclear receptor SHP. Mice lacking FGF15 have increased hepatic CYP7A1 mRNA and protein levels and corresponding increases in CYP7A1 enzyme activity and fecal bile acid excretion. These studies define FGF15 and FGFR4 as components of a gut-liver signaling pathway that synergizes with SHP to regulate bile acid synthesis.     

Spatial repositioning of centromeric domains during regrowth of axons in nuclei of murine dorsal root ganglion neurons in vitro

Specific chromatin domains within interphase nuclei are organized in cell type specific distributions and are rearranged in association with changes in cell function. Axotomy leads to changes in gene expression. Dorsal root ganglion (DRG) neurons in vitro are a model for axotomy because they are detached from their axons in preparation for the culturing procedure. In a test of the hypothesis that neurons regrowing in vitro undergo rearrangement of specific chromatin domains, changes in the distribution of centromere-associated kinetochores proteins within DRG neurons were assessed as a function of time in vitro. Comparison of the kinetochore distributions in neurons in situ to those 24 h after placement into culture showed that the mean proportion (±S.E.M.) of kinetochore signals in the karyoplasm decreased from 41.0 ± 1.8% to 28.6 ± 3.3%, while the proportion at the nucleolus increased from 35.2 ± 2.0% to 48.4 ± 2.9%. This indicated redistribution of centromeric domains to the nucleolus. Between 1 day and 16 days in vitro, signals were redistributed to the nuclear periphery, indicated by an increase in the proportion of signals in this nuclear compartment from 23.0 ± 4.3% to 37.6 ± 3.4% and a decrease in the proportion of signals from 48.4 ± 2.9% to 23.0 ± 2.3% at the nucleolus. The results indicate that neurite regrowth following axotomy is associated with changes in nuclear topology. The reorganization that occurs within 24 h is speculated to be associated with a recapitulation of a cytoskeletal development program, while later changes in centromeric distributions may be related to cues elicited by in vitro conditions. © 1996 John Wiley & Sons, Inc.