Nuclear accumulation of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells.

In an effort to determine the localization of fibroblast growth factor (FGF) receptors (FGFR) that could mediate the intracellular action of FGF-2, we discovered the presence of high-affinity. FGF-2 binding sites in the nuclei of bovine adrenal medullary cells (BAMC). Western blot analysis demonstrated the presence of 103-, 118-, and 145-kDa forms of FGFR1 in nuclei isolated from BAMC. 125I-FGF-2 cross-linking to nuclear extracts followed by FGFR1 immunoprecipitation showed that FGFR1 can account for the nuclear FGF-2 binding sites. Nuclear FGFR1 has kinase activity and undergoes autophosphorylation. Immunocytochemistry with the use of confocal and electron microscopes demonstrated the presence of FGFR1 within the nuclear interior. Nuclear subfractionation followed by Western blot or immunoelectron microscopic analysis showed that the nuclear FGFR1 is contained in the nuclear matrix and the nucleoplasm. Agents that induce translocation of endogenous FGF-2 to the nucleus (forskolin, carbachol, or angiotensin II) increased the intranuclear accumulation of FGFR1. This accumulation was accompanied by an overall increase in FGF-2-inducible tyrosine kinase activity. Our findings suggest a novel mode for growth factor action whereby growth factor receptors translocate to the nucleus in parallel with their ligand and act as direct mediators of nuclear responses to cell stimulation.     

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

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

Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

Bone morphogenetic protein-7 stimulates initial dendritic growth in sympathetic neurons through an intracellular fibroblast growth factor signaling pathway.

Bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor (TGF)-beta superfamily of signaling cytokines, induces dendritic growth in rat sympathetic neurons. In this study, we present evidence that the recently discovered integrative nuclear FGFR1 signaling (INFS) pathway is involved in dendrite outgrowth mediated by BMP-7. Immunocytochemical analysis of expressed fibroblast growth factors (FGFs) showed that little FGF-2 was detected in control neurons, but the expression of this molecule in the cytoplasm and nucleus increased within 6 h after BMP-7 treatment. In contrast, FGF-1 was constitutively present in the peripheral cytoplasm and in neurites under control conditions, and its distribution did not change with BMP-7 exposure. The high-affinity receptor FGFR1 was present in low amounts in control neurons and was associated with the cytoplasm, the plasma membrane, and the nucleus. Twenty-four hours of BMP-7 treatment elicited an increase in FGFR1 nuclear localization. Overexpressed constructs of FGFR1 that lack the tyrosine kinase domain, and have been shown to act in a dominant-negative manner on FGFR1 signaling, inhibited BMP-7 mediated initial dendrite outgrowth in transfected neurons by approximately 50%. However, targeted inhibition of extracellular FGF-2 by overexpression of a secreted receptor mutant FGFR1(TM-) lacking the transmembrane domain failed to affect BMP-7 induced dendritic growth, as did treatment with the extracellular FGFR antagonist inositol hexakisphosphate. These results suggest that the INFS, which has already been implicated in a broad range of activities in other cell types, may also be required for BMP-7 to stimulate dendritic development.     

Cellular distribution of lens epithelium-derived growth factor (LEDGF) in the rat eye: loss of LEDGF from nuclei of differentiating cells

Lens epithelium-derived growth factor (LEDGF) enhances the survival and growth of cells. To understand LEDGF's spatial localization and its putative function(s) during proliferation and differentiation, we localized LEDGF during terminal differentiation in whole rat lenses, lens epithelial cell (LEC) explants stimulated with FGF-2, and insulin, iris, human LECs with lentoids. In addition, intracellular localization of LEDGF was performed in other ocular tissues: ciliary body, retina, and cornea. We found the immunopositivity of nuclear LEDGF decreased in LECs of the equatorial region. In contrast, immunopositivity of LEDGF was detected in the cytoplasm of LECs and superficial fiber cells. After treating LEC explants with FGF-2 and insulin, which are known to be differentiating factors for LECs, the nuclei of these cells showed no LEDGF immunopositivity, but explants did express p57(kip2), a differentiation marker protein. Also, immunopositive LEDGF was not detected in the nuclei of differentiated cells, lentoid body, and corneal epithelial cells. This demonstrated that the loss of LEDGF from the nucleus may be associated with the process of terminal differentiation that might be in some way common with the biochemical mechanisms of apoptosis. The spatial and temporal distribution of LEDGF in the present study also provides a vision for further investigation as to how this protein is involved in cell fate determination.     

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.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

Nuclear trafficking of FGFR1: a role for the transmembrane domain

Several members of the fibroblast growth factor (FGF) family lack signal peptide (SP) sequences and are present only in trace amounts outside the cell. However, these proteins contain nuclear localization signals (NLS) and accumulate in the cell nucleus. Our studies have shown that full length FGF receptor 1 (FGFR1) accumulates within the nuclear interior in parallel with FGF-2. We tested the hypothesis that an atypical transmembrane domain (TM) plays a role in FGFR1 trafficking into the nuclear interior. With FGFR1 destined for constitutive fusion with the plasma membrane due to its SP, how the receptor may enter the nucleus is unclear. Sequence analysis identified that FGFR1 has an atypical TM containing short stretches of hydrophobic amino acids (a.a.) interrupted by polar a.a. The beta-sheet is the predicted conformation of the FGFR1 TM, in contrast to the alpha-helical conformation of other single TM tyrosine kinase receptors, including FGFR4. Receptor trafficking in live cells was studied by confocal microscopy via C-terminal FGFR1 fusions to enhanced green fluorescent protein (EGFP) and confirmed by subcellular fractionation and Western immunoblotting. Nuclear entry of FGFR1-EGFP was independent of karyokinessis, and was observed in rapidly proliferating human TE671 cells, in slower proliferating glioma SF763 and post-mitotic bovine adrenal medullary cells (BAMC). In contrast, a chimeric FGFR1/R4-EGFP, where the TM of FGFR1 was replaced with that of FGFR4, was associated with membranes (golgi-ER, plasma, and nuclear), but was absent from the nucleus and cytosol. FGFR1delta-EGFP mutants, with hydrophobic TM a.a. replaced with polar a.a., showed reduced association with membranes and increased cytosolic/nuclear accumulation with an increase in TM hydrophilicity. FGFR1(TM-)-EGFP (TM deleted), was detected in the golgi-ER vesicles, cytosol, and nuclear interior; thus demonstrating that the FGFR1 TM does not function as a NLS. To test whether cytosolic FGFR1 provides a source of nuclear FGFR1, cells were transfected with FGFR1(SP-) (SP was deleted), resulting in cytosolic, non-membrane, protein accumulation in the cytosol and the cell nucleus. Our results indicate that an unstable association with cellular membranes is responsible for the release of FGFR1 into the cytosol and cytosolic FGFR1 constitutes the source of the nuclear receptor.     

PGF2alpha increases FGF-2 and FGFR2 trafficking in Py1a rat osteoblasts via clathrin independent and importin beta dependent pathway

Previous studies showed that prostaglandin F2alpha (PGF2alpha) stimulated fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor 2 (FGFR2) cytosolic and nuclear accumulation, however, the endocytic pathway has not been elucidated. This study demonstrates that although PGF2alpha increased the formation of clathrin-coated structures in Py1a rat osteoblasts, they were not involved in FGF-2 and FGFR2 trafficking. PGF2alpha increased binding of FGF-2 and FGFR2 and co-localization of reactive sites in addition to nuclear translocation at the nuclear pore complex level. FGF-2 and FGFR2 were in close spatial correlation with importin beta, further supporting nuclear import of the FGF-2/FGFR2 complex. Immunogold and immunofluorescence techniques as well as Western blotting demonstrated increased importin beta protein labeling in response to PGF2alpha. Similar to PGF2alpha, phorbol 12-myristate 13-acetate (PMA) also increased importin beta protein. These data strongly suggest that prostaglandins may regulate osteoblast metabolism via FGF-2/FGFR2/importin beta nuclear trafficking.     

Nuclear localization of basic fibroblast growth factor is mediated by heparan sulfate proteoglycans through protein kinase C signaling

Understanding the process of wound healing will provide valuable insight for the development of new strategies to treat diseases associated with improper regeneration, such as blindness induced by corneal scarring. Heparan sulfate proteoglycans (HSPG) are not normally expressed in the corneal stroma, but their presence at sites of injury suggests their involvement in the wound healing response. Primary cultured corneal stromal fibroblasts constitutively express HSPG and represent an injured phenotype. Recently, nuclear localization of HSPG was shown to increase in corneal stromal fibroblasts plated on fibronectin (FN), an extracellular matrix protein whose appearance in the corneal stroma correlates with injury. One possible role for the nuclear localization of HSPG is to function as a shuttle for the nuclear transport of heparin-binding growth factors, such as basic fibroblast growth factor (FGF-2). Once in the nucleus, these growth factors might directly modulate cellular activities. To investigate this hypothesis, cells were treated with (125)I-labelled FGF-2 under various conditions and fractionated. Our results show that nuclear localization of FGF-2 was increased in cells plated on FN compared to those on collagen type I (CO). Interestingly, FGF-2-stimulated proliferation was increased in cells plated on FN compared to CO and this effect was absent in the presence of heparinase III. Furthermore, pre-treatment with heparinase III decreased nuclear FGF-2, and CHO cells defective in the ability to properly synthesize heparan sulfate chains showed reduced nuclear FGF-2 indicating that the heparan sulfate chains of HSPG are critical for this process. HSPG signaling, particularly through the cytoplasmic tails of syndecans, was investigated as a potential mechanism for the nuclear localization of FGF-2. Treatment with phorbol 12-myristate-13-acetate (PMA), under conditions that caused downregulation of protein kinase Calpha (PKCalpha), decreased nuclear FGF-2. Using pharmacological inhibitors of specific PKC isozymes, we elucidated a potential mode of regulation whereby PKCalpha mediates the nuclear localization of FGF-2 and PKCdelta inhibits it. Our studies suggest a novel mechanism in which FGF-2 translocates to the nucleus in response to injury.     

A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.     

Nuclear and nucleolar localization of 18-kDa fibroblast growth factor-2 is controlled by C-terminal signals

Members of high (22-, 22.5-, 24-, and 34-kDa) and low (18-kDa) molecular mass forms of fibroblast growth factor-2 (FGF-2) regulate cell proliferation, differentiation, and migration. FGF-2s have been previously shown to accumulate in the nucleus and nucleolus. Although high molecular weight forms of FGF-2 contain at least one nuclear localization signal (NLS) in their N-terminal extension, the 18-kDa FGF-2 does not contain a standard NLS. To determine signals controlling the nuclear and subnuclear localization of the 18-kDa FGF-2, its full-length cDNA was fused to that of green fluorescent protein (GFP). The fusion protein was primarily localized to the nucleus of COS-7 and HeLa cells and accumulated in the nucleolus. The subcellular distribution was confirmed using wild type FGF-2 and FGF-2 tagged with a FLAG epitope. A 17-amino acid sequence containing two groups of basic amino acid residues separated by eight amino acid residues directed GFP and a GFP dimer into the nucleus. We systematically mutated the basic amino acid residues in this nonclassical NLS and determined the effect on nuclear and nucleolar accumulation of 18-kDa FGF-2. Lys(119) and Arg(129) are the key amino acid residues in both nuclear and nucleolar localization, whereas Lys(128) regulates only nucleolar localization of 18-kDa FGF-2. Together, these results demonstrate that the 18-kDa FGF-2 harbors a C-terminal nonclassical bipartite NLS, a portion of which also regulates its nucleolar localization.     

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.     

Inhibitory role of the somatostatin receptor SST2 on the intracrine-regulated cell proliferation induced by the 210-amino acid fibroblast growth factor-2 isoform: implication of JAK2

The fibroblast growth factor (FGF)-2 isoform of 210 amino acids (HMW FGF-2) contains a nuclear localization sequence (NLS) and is targeted to the nucleus. This FGF-2 isoform allows cells to grow in low serum concentrations through still unknown mechanisms called intracrine regulations. Different peptide hormones and cytokines have been found to be nuclearized through NLS and to induce cell proliferation. The existence of molecules acting as negative regulators of the intracrine-induced cell growth has not been explored. Pancreatic cells AR4-2J were stably transfected to express selectively the HMW FGF-2. We demonstrated that activation of the somatostatin receptor subtype SST2 by the somatostatin analogue RC-160 in serum-deprived medium inhibits the mitogenic effect of the HMW FGF-2, without affecting growth of control cells. The signaling pathway implicates Galphai/JAK2/SHP-1. The Galphai inhibitor pertussis toxin and the JAK2 inhibitor AG490 abrogate the inhibitory effect of RC-160 on HMW FGF-2-induced cell growth. Co-immunoprecipitation studies demonstrate the constitutive association of JAK2 and SHP-1, and RC-160 induces a rapid activation of both proteins followed by the dissociation of the complex. AG490 prevents the RC-160 induced SHP-1 activation indicating the implication of JAK2 in this process. JAK2 and SHP-1 are immunoprecipitated with SST2 in basal conditions indicating the existence of a functional signaling complex at the receptor level. In summary, these data provide the following evidence: 1) the intracrine-induced proliferation can be reversed by extracellular acting polypeptides; 2) SST2 inhibitory signaling may involve the JAK2/SHP-1 pathway.     

Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in normal human seminiferous epithelium

 Fibroblast growth factor 2 (FGF-2), which occurs in various isoforms both species and tissue specifically, regulates cell proliferation and differentiation via a dual receptor system consisting of heparan sulphate proteoglycans and receptor tyrosine kinases (FGFRs). This study demonstrates for the first time the distribution pattern of FGF-2 and the receptors FGFR 1–4 in the normal seminiferous epithelium of adult men. In western blot analyses, the polyclonal antibody, anti-FGF-2, shows two immunoreactive bands at 18 and 24 kDa. On paraffin sections, positive immunoreaction occurs within the cytoplasm of spermatogonia. The distribution pattern of the polyclonal anti-FGFR 1–4 antibodies is as follows: anti-FGFR-1 (one 68-kDa band) stains nuclei and cytoplasm of spermatogonia; anti-FGFR-3 (five bands at 68, 78, 105, 125 and 145 kDa) stains the nuclei of all germ cells except those of elongated spermatids; and anti-FGFR-4 (one 48-kDa band) stains the cytoplasm of primary pachytene spermatocytes. We were unable to demonstrate FGFR-2 immunoreactivity either in western blot analysis or on paraffin sections. This distribution pattern suggests that FGF-2 in spermatogonia is involved in the autocrine and paracrine regulation of the proliferation and differentiation of spermatogonia and spermatocytes via the receptors FGFR-1, FGFR-3 and FGFR-4. Accepted: 23 December 1997     

Translocation of fibroblast growth factor-10 and its receptor into nuclei of human urothelial cells

Fibroblast growth factor-10 (FGF-10), a mitogen for the epithelial cells lining the lower urinary tract, has been identified inside urothelial cells, despite its acknowledged role as an extracellular signaling ligand. Recombinant (r)FGF-10 was determined by fluorescence microscopy optical sectioning to localize strongly to nuclei inside cultured urothelial cells. To clarify the possible role of a nuclear localization signal (NLS) in this translocation, a variant of rFGF-10 was constructed which lacked this sequence. rFGF-10(no NLS) was found in cytoplasm to a far greater degree than rFGF-10, identifying this motif as a possible NLS. Furthermore, this variant displayed poor or non-existent bioactivity compared to the wild-type protein in triggering mitogenesis in quiescent urothelial cells. The presence of rFGF-10(no NLS) in the nucleus suggested that additional interactions were also responsible for the nuclear accumulation of rFGF-10. The FGF-10 receptor was observed in cell nuclei regardless of the presence or concentration of exogenous rFGF-10 ligand. Co-localization studies between rFGF-10 and the FGF-10 receptor revealed a strong intracellular relationship between the two. This co-localization was seen in nuclei for both rFGF-10 and for rFGF-10(no NLS), although the correlation was weaker for rFGF-10(no NLS). These data show that an NLS-like motif of rFGF-10 is a partial determinant of its intracellular distribution and is necessary for its mitogenic activity. These advancements in the understanding of the activity of FGF-10 present an opportunity to engineer the growth factor as a therapeutic agent for the healing of damaged urothelial tissue.