Intracellular trafficking of endogenous fibroblast growth factor-2 Simona Taverna, Salvatrice Rigogliuso, Monica Salamone and Maria Letizia Vittorelli Dipartimento di Biologia Cellulare e dello Sviluppo, Universita ` di Palermo, Italy Most proteins destined for secretion into the extracellu- lar matrix are targeted by the presence of N-terminal signal peptides, which direct their translocation into the rough endoplasmic reticulum (rER). They are sub- sequently transferred to the Golgi apparatus and then secreted into the extracellular space. However, a grow- ing number of secreted proteins have been identified that lack N-terminal signal peptides. These proteins do not enter the rER and their secretion is not influenced by drugs such as brefeldin A and monensin, which block secretion by classical mechanisms [1]. Therefore, they are secreted by alternative, unconventional pro- cesses; these proteins include the inflammatory cytokine interleukin-b1, galectins, macrophage migration inhibi- tory factor, acid and basic fibroblast growth factors (FGF-1, FGF-2) and sphingosine kinase 1. The mecha- nisms of their secretion are the subject of numerous studies and different pathways have been described [2]. An unconventional secretion mechanism common to several signalling proteins devoid of the typical signalling sequence appears to be mediated by the shedding of vesicles into the extracellular matrix. These vesicles, also called exovesicles, are observed to bud from the cell membranes and to be released into the Keywords FGF-2; microfilaments; microtubules; secretion of leaderless proteins; shed vesicles Correspondence M. L. Vittorelli, Dipartimento di Biologia Cellulare e dello Sviluppo, Universita ` di Palermo, Viale delle Scienze ed. 16, 90128 Palermo, Italy Fax: +39 0 9165 77430 Tel: +39 0 9165 77407 E-mail: mlvitt@unipa.it (Received 2 August 2007, revised 18 January 2008, accepted 31 January 2008) doi:10.1111/j.1742-4658.2008.06316.x We have previously reported how the release of fibroblast growth factor-2 (FGF-2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF-2 is targeted to the budding vesicles. Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the pres- ence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF-2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in FGF-2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF-2 granules mov- ing to the cell periphery and FM4-64-labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF-2 release, were analyzed for their effects on FGF-2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF-2 movements toward the cell periphery. The FGF-2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF-2 clustered at the cell periphery, but the rate of its release decreased. FGF-2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF-2 translocation to the nucleus and nucleolus. FGF-2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms. Abbreviations CM, conditioned media; FGF-2, fibroblast growth factor-2; FITC, fluorescein isothiocyanate; NLS, nuclear localization sequence; rER, rough endoplasmic reticulum; uPA, urokinase type of plasminogen activator. FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS 1579 extracellular medium. The vesicle diameters are in the range 100–1000 nm; the vesicle composition and func- tion depend on the type of cells from which they have been produced. Vesicles are found to be involved in cell motility and tumour progression mechanisms as well as in bone formation [3]. Involvement of shed ves- icles in cell–cell and cell–matrix interactions is partially mediated by the presence of several membrane-associ- ated proteolytic enzymes [4–6] and signalling molecules [7–9]. However, shed vesicles also channel the secretion of several leaderless proteins that apparently accumu- late in their internal lumen. For example, Cooper and Barondes [10] reported that lectin 14, a signalling pro- tein involved in muscle differentiation, is released as a component of budding vesicles; MacKenzie et al. [11] and Bianco et al. [12] reported that interleukin-b1is secreted as a component of shed vesicles in response to ATP acting on P2X receptors. Pathways targeting specific molecules to the budding vesicles have not been clarified. In a previous study, we investigated the mechanism of FGF-2 secretion and reported that FGF-2 is released from Sk-Hep1 cells and from NIH 3T3 cells transfected with FGF-2 cDNA through vesicle shedding [13]. FGF-2, also known as basic fibroblast growth factor, belongs to the fibroblast growth factor superfamily. FGFs are structurally related heparin-binding growth factors that exhibit almost ubiquitous involvement in vertebrate embryonic and fetal development [14], as well as in many physio-pathological processes occurring in adult organisms [15]. FGF-1 and FGF-2 are secreted from the cell into the extracellular matrix, although they lack the classical secretion sequence. Unlike FGF-1, which appears to be released in response to stress con- ditions as a component of a multiprotein complex [16,17], FGF-2 is secreted constitutively; however, its release by in vitro cultured cells is inhibited by serum deprivation [18]. FGF-2 transmits pro-angiogenic [19,20] and pro-lymphangiogenic [21] signals. It is also involved in inducing smooth muscle and endothelial cell growth and in regulating early development stages of various organs [22], including the brain [23,24]. FGF-2 is also one of the most significant regulators of human embryonic stem cell self-renewal and of cancer tumourigenesis [25]. As a signalling protein, FGF-2 acts both directly at the nuclear level and after secretion. Indeed, after its synthesis, FGF-2 can be secreted [1] or transferred to the nucleus and to the nucleolus where it behaves like a transcriptional factor, inducing cell growth and rRNA synthesis [26]. Five FGF-2 isoforms (18, 22, 22.5, 24 and 34 kDa) have been identified in humans, all of which present some nuclear localization sequences. However, although the 34 kDa isoform presents an arginine-rich repeat domain similar to the nuclear localization sequence (NLS) present in HIV Rev protein, this sequence is absent in isoforms with lower molecular weight; the 34, 24, 22.5 and 22 isoforms present several glycine-arginine repeats identified as an NLS and a nonclassical bipartite NLS in the C-terminus. [27]. The 18 kDa isoform only presents the bipartite NLS. A portion of the bipartite NLS regulates localization of the factor in nucleoli [28]. As a secreted molecule, FGF-2 interacts both with matrix and membrane-bound proteoglycans and with five members of a family of high-affinity tyrosine kinase FGF receptors [29]. Receptor-mediated signalling patterns appear to be utilized not only in paracrine, but also in autocrine mechanisms [30]. According to Bossard et al. [31], receptor-bound FGF-2 is endocytosed and can be transferred to the nucleus along with associated proteoglycans; nuclear Fig. 1. Targeting of endogenous FGF-2 to shed vesicles and nuclei. (A) Colocalization of FGF-2 and b1 integrin in Sk-Hep1 cells cultured in 3D type I collagen gels. Immunostaining of (a) FGF-2, (b) b1 integrin and (c) merging. FGF-2 was detected using Texas red-conjugated sec- ondary antibodies; b1 integrin was detected using FITC-conjugated antibodies. Arrows indicate shed vesicles. Scale bar = 10 lm. (B) Time- course of serum-induced FGF-2 intracellular movements observed by immunolocalization experiments. FGF-2 immunolocalization at 0, 15, 30, 45, 60 min after serum addition (a–e). Top: sections 1 lm from the surface; thin arrows indicate FGF-2 granules. Bottom: sections 3 lm from the surface; thick arrows indicate area of the nuclei in which large variations of FGF-2 concentration are observed. FGF-2 was detected using Texas red-conjugated secondary antibodies. Scale bar = 10 lm. (C) Number of FGF-2 positive granules (diameters in the range 0.01– 1 lm) in immunostained sections 1 lm from the surface of cells fixed 0, 15, 30, 45 and 60 min after serum addition. Granules were counted by IMAGEJ software. Asterisks indicate a significant difference between each time compared to the 30-min value. (D) FGF-2 concentration in nuclei. FGF-2 concentration (absorbance) was evaluated by IMAGEJ software in sections 3 lm from the surface of cells fixed 0, 15, 30, 45 and 60 min after serum addition. Asterisks indicate a significant difference between each time compared to the 30-min value. Values are mean ± SD of 15 measurements from five independent experiments in (B) to (D). (E) Induction of uPA activity in GM7373 cells by SK-Hep1 vesicles. (a) Casein ⁄ plasminogen zymographies for detection of uPA activity, performed as described in the Experimental procedures on extracts of GM7373 cells that had been incubated in 5 mL of media for 16 h with 0, 25, 50 and 100 lg of SK-Hep1 vesicles (lanes 1–4). Asterisks indicate a significant difference between each amount compared to the previous one. (b) Densitometric analysis of lysis bands due to uPA activity. Arbitrary units represent densitometric values of lysis bands, with 100 considered as the basal uPA activity of GM7373 untreated cells. Values are the mean ± SD of 15 measurements from five independent experiments. Intracellular trafficking of FGF-2 S. Taverna et al. 1580 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS accumulation of the endocytosed molecule requires interaction with a protein called ‘translokin’ and depends on the tubulin cytoskeleton. In the present study, we analyzed mechanisms allow- ing translocation of FGF-2 from cytoplasmatic sites to the budding vesicles and ⁄ or to other cell districts. Foetal bovine serum was reported to induce both vesicle shedding [6,32] and FGF-2 secretion [1,18] and, in our previous studies, we observed that the two phe- nomena were clearly associated. Approximately 30 min after the serum was added to starved cells, granules containing FGF-2 appeared in the proximity of the cell membrane and colocalization between FGF-2 and b1 integrin, a molecule known to be clustered in vesicle membranes [6], became evident. Shortly after that, FGF-2 was detected in shed vesicles [13]. a b c d e B C E D A a b c d e a b c a c 25 µg 50 µg 100 µg 0 100 200 300 400 500 UpA activity-arbitrary unit C 25 50 100 ** ** ** b 0 10 20 30 40 50 60 70 Number of FGF-2 granules 0 15 30 45 60 *** ** ** ** 0 20 40 60 80 100 120 140 A in the nuclei 0 15 30 45 60 *** ** * * S. Taverna et al. Intracellular trafficking of FGF-2 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS 1581 We therefore aimed to follow the FGF-2 transloca- tion pathway to the cell membrane by analyzing events following serum addition. To check whether cytoskele- ton components were mediating FGF-2 trafficking, we analyzed the effects of cytoskeleton perturbation on FGF-2 intracellular movements. To improve our anal- ysis of FGF-2 intracellular movements and targeting, we also tested the effects of known FGF-2 secretion inhibitors on both FGF-2 intracellular movements and vesicle shedding. Results Localization of FGF-2 in SK-Hep1 cells and their shed vesicles Figure 1A shows vesicle shedding by SK-Hep1 cells cultured in 3D collagen gels. As previously reported [13], when SK-Hep1 cells are grown in a complete medium, FGF-2 is observed to be localized in cell pro- trusions in association with b1 integrin, a molecule that is also specifically clustered in shed vesicles [6]. In 3D gels, shed vesicles remain trapped in the collagen and can be observed using confocal microscopy. Shed vesicles, testing positive for both FGF-2 and b1 inte- grin antigens, are visible in Fig. 1A (arrows). As previously reported [13], the shedding of FGF-2 positive vesicles occurs in cells showing no signs of apoptosis or necrosis. Cell cultures used for the present experiments were kept in serum-free media for 72 h in most instances and then stimulated by serum addition. However, the percentage of apoptotic ⁄ necrotic cells was always negligible (data not shown). Immunolocalization of FGF-2 in starved cells (i.e. cells kept for 72 h in a serum-free medium) and in cells that, after starvation, were cultured in the presence of 10% fetal bovine serum for varying lengths of time, showed that serum addition resulted in FGF-2 positive granules, which were almost entirely absent in starved cells (Fig. 1B), appearing at the cell periphery. During the first 45 min of cell growth in the complete media, the number of granules localized at the cell periphery progressively increased; however, their number dropped (Fig. 1B,C) after 1 h, indicating that FGF-2 had been released. As previously reported [13], 1 h after adding serum, FGF-2 rich vesicles can be Fig. 2. Involvement of cytoskeleton elements in FGF-2 intracellular trafficking. (A) Effects of nocodazol treatment on tubulin organization and on serum induced FGF-2 movements. (a, b) Immunolocalization of tubulin, respectively, in control cells (a) and in cells fixed 30 min after the addition of 10 l M nocodazol (b). (c, d) FGF-2 immunolocalization in control cells fixed 30 min after serum addition (c) and in cells fixed 30 min after serum and 10 l M nocodazol addition (d). Sections 2 lm from the surface. Tubulin was detected using TRITC-labelled secondary antibodies; FGF-2 was detected using Texas red-conjugated secondary antibodies. Thin arrows indicate FGF-2 granules; thick arrows indicate nucleoli. Scale bar = 10 lm. (B) Effects of cytocalasin B treatment on serum induced FGF-2 movements. (a, b) Immunolocalization of actin respectively in control cells (a) and in cells fixed 30 min after the addition of 1 l M cytochalasin B. (b) Sections 2 lm from the surface. Thin arrows indicate FGF-2 granules; thick arrows indicate nucleoli. Actin was labelled with FITC-phalloidin. FGF-2 was detected using Texas red- conjugated secondary antibodies. Scale bar = 10 lm. (C) FGF-2 concentration in nuclei 30 min after serum addition, in control cells and in cells treated with drugs affecting the cytoskeleton organization. FGF-2 concentration (absorbance) was evaluated by IMAGEJ software, as described in the Experimental procedures, in sections 3 lm from the surface, 30 min after serum (control) or serum and the following drugs had been added to starved cells: 10 l M nocodazol (Nocadazol), 1 lM paclitaxel (Paclitaxel) 1 lM colchicine (Colchicine) and 1 lM cytochala- sin B (Cytochalasin). Asterisks indicate a significant difference between the treated cells and controls. (D) Numbers of FGF-2 positive gran- ules near the cell surface 30 min after serum addition in control cells and in cells treated with drugs affecting the cytoskeleton organization. FGF-2 positive granules (diameters in the range 0.01–1 lm) were counted in immunostained sections 1 lm from the surface by IMAGEJ soft- ware, as described in the Experimental procedures. Control cells (Controls); cells treated with 10 l M nocodazol (Nocadazol); 1 lM paclitaxel (Paclitaxel); 1 l M colchicine (Colchicine); 1 lM Cytochalasin B (Cytochalasin). Drugs and serum were added together. (E) Amount of vesicles recovered from conditioned media of control cells and of cells treated with drugs affecting the cytoskeleton organization. Media were condi- tioned by 3–6 h of growth of 4 · 10 7 control cells grown in complete medium (Control) and by cells treated for the same time with complete medium to which the following drugs had been added: 10 l M nocodazole (Nocadazole); 1 lM paclitaxel (Paclitaxel); 1 lM colchicine (Colchi- cine) or 1 l M cytochalasin B (Cytochalasin). Vesicle amount was evaluated by Bradford microassay method in at least three different experi- ments. (F) Induction of uPA activity in GM7373 cells by vesicles shed by controls and by cells treated with drugs affecting the cytoskeleton organization. Casein ⁄ plasminogen zymographies for detection of uPA activity were performed as described in the Experimental procedures on GM7373 cells that had been incubated for 16 h with vesicles shed by control cells and by cells treated with drugs affecting the cytoskel- eton organization. Vesicles had been obtained from ml of conditioned media. uPA activity of GM7373 cells incubated without vesicles (Med- ium), with SK-Hep1 vesicles shed in media conditioned by 3–6 h of growth of 4 · 10 7 nontreated cells grown in complete medium (Control) and by cells treated for the same length of time with complete medium to which the following drugs had been added: 10 l M nocodazol (Nocadazol); 1 l M paclitaxel (Paclitaxel); 1 lM colchicine (Colchicine) and 1 lM cytochalasin B (Cytochalasin). With respect to cytochalasin B, a comparison between the specific stimulatory activity of control vesicles and of vesicles shed by treated cells is also shown (Cytochalasin sp. ac.) Arbitrary units represent densitometric values of lysis bands with 100 considered as the basal uPA activity of GM7373 cells. Values are the mean ± SD of 15 measurements from five independent experiments. Intracellular trafficking of FGF-2 S. Taverna et al. 1582 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS recovered from cell-conditioned media (CM), whereas FGF-2 is still undetected in vesicle-free CM. Therefore, we suggest that, after 1 h of cell culture in the com- plete medium, FGF-2 positive granules are released as vesicle components. Immunolocalization studies also showed that, when cells were kept in the presence of serum for 30 min or more, the average concentration of FGF-2 also increased in the nucleus; moreover, the factor accumu- lated in specific areas of the cell nucleus that were FGF-2 negative (Fig. 1B,D) in starved cells. Because FGF-2 is known to stimulate rRNA synthesis [26,27], these nuclear areas are considered to correspond to nucleoli. FGF-2 can induce expression of the urokinase type of plasminogen activator (uPA) in endothelial cells [33,34]. We therefore performed assays of vesicle-asso- ciated FGF-2 by testing the ability of vesicles to induce uPA production in GM7373 endothelial cells. We analyzed induction by incubating GM7373 cells with vesicles for 16 h; uPA activity was then measured by casein ⁄ plasminogen zymography of endothelial cell extracts. Induction was dose-dependent (Fig. 1E); moreover, we already knew that the stimulatory effect of SK-Hep1 vesicles on uPA production by GM7373 cells was completely abolished by anti-FGF-2 serum [13]. We therefore considered the specific stimulatory effect of vesicles on uPA production as an indirect method to evaluate their FGF-2 content. Involvement of cytoskeleton in FGF-2 intracellular trafficking To analyze the involvement of cytoskeleton elements in targeting endogenous FGF-2 to the cell nucleus and to the cell periphery, Sk-Hep1 cells were treated with drugs c a b d a b c d ** 0 20 40 60 80 100 120 Proteins in shed vesicles Control Nocodazol Paclitaxel Colchicine Cytochalasin *** 0 20 40 60 80 100 120 140 AB C D E F A in the nuclei Control Nocodazol Paclitaxel Colchicine Cytochalasin 0 20 40 60 80 100 120 140 160 180 UpA activity-arbitrary units Medium Control Nocodazol Paclitaxel Colchicine Cytochalasin Cytochalasin sp. ac *** ** 0 20 40 60 80 100 120 140 Number of FGF-2 granules *** Control Nocodazol Paclitaxel Colchicine Cytochalasin S. Taverna et al. Intracellular trafficking of FGF-2 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS 1583 affecting tubulin or actin organization and FGF-2 im- munolocalization was analyzed by confocal microscopy. The molecules tested were nocodazol and colchicine, which cause microtubule disassembly; paclitaxel, an inhibitor of tubulin depolymerization; and cytochala- sin B, a drug causing actin depolymerization. The serum-induced increase of FGF-2 concentration in the nucleus was less marked in cells treated with the three drugs affecting microtubular organization. These drugs also had clear inhibitory effects on FGF-2 locali- zation in nuclear areas apparently corresponding to nucleoli (Fig. 2A, panel d, thick arrows). On the other hand, FGF-2 export toward the cell membrane was not modified by treatment with drugs affecting micro- tubular organization; in fact, granules at the cell periphery were also clearly visible in treated cells (Fig. 2A, panel d, thin arrows). The effects of cytochalasin B treatment were the reverse. As shown in Fig. 2B, FGF-2 immunolocaliza- tion experiments demonstrated that actin depolymer- ization did not modify nuclear localization of FGF-2 (Fig. 2B, panel d, thick arrows); however, cytochala- sin B treatment blocked FGF-2 movements toward the cell membrane. To obtain a more quantitative picture detailing the effects of drug treatments on cytoskeleton organization and on FGF-2 movements toward the nucleus and the cell periphery, the optical density in cell nuclei and the number of FGF-2 granules at the cell periphery were measured. As shown in Fig. 2C, nocodazol, colchicine and paclitaxel had a significant inhibitory effect on the increased concentration of FGF-2 induced by the serum in the nucleus, whereas cytochalasin B had no effect. By contrast, as shown in Fig. 2D, the number of FGF-2 positive granules observed at the cell periphery 30 min after the serum was added, was not modified by treatment with drugs affecting microtubule organi- zation, whereas it strongly decreased in cytochalasin B treated cells. In brief, and in accordance with the results pre- viously reported by Bossard et al. [31] concerning nuclear and nucleolar localization of exogenous FGF-2, our data suggest that microtubule integrity is needed for nuclear localization of endogenous FGF-2. Actin filament integrity was not shown to be required for targeting the factor to the cell nucleus. By contrast, FGF-2 targeting to the cell periphery was not influenced by treatment with drugs affecting tubulin organization, whereas it was blocked by cytocalasin b treatment. We also tested the effects of drugs that influence the cytoskeleton on cell attitude to shed membrane vesicles and on vesicle capability to induce uPA production by GM7373 cells. None of the three drugs affecting microtubule organization had significant effects on the amount of vesicles shed by treated cells (Fig. 2E), nor on their stimulation of uPA production (Fig. 2F). There is therefore no evidence of microtubule involve- ment in the FGF-2 release mechanism. By contrast, the amount of vesicles shed by cytochalasin B-treated cells (Fig. 2E) was greatly reduced. Moreover, not only the total, but also the specific stimulatory activity of vesicles shed by cytochalasin B treated cells on uPA production decreased (Fig. 2F). Therefore, microfilament organization appears to be required for both FGF-2 targeting to the budding vesi- cles and for vesicle shedding. a b c 2 45 kDa actin 1 18 kDa FGF-2 b a A B C Fig. 3. Immunolocalization of FGF-2 in comparison with actin fila- ments and lipidic structures. (A) Double staining for FGF-2 and actin, 30 min after serum addition. FGF-2 was detected using Texas red-conjugated secondary antibodies. Actin was labelled with (a) phalloidin-labelled actin; (b) monoclonal antibody-labelled FGF-2; and (c) merging. Arrow indicates FGF-2 granules on actin filaments. Scale bar = 5 lm. Sections 1 lm from the surface. (B) Immunopre- cipitation with anti-FGF-2 serum. Immunoprecipitate was immuno- stained with either monoclonal antibodies against actin or monoclonal antibodies against FGF-2. Lane 1, immunoprecipitation from cells cultured in complete medium; lane 2, immunoprecipita- tion from starved cells. (C) Double staining for FGF-2 and lipids. FGF-2 was detected using FITC-conjugated secondary antibodies (green fluorescence); lipids were stained with FM4-64 (red fluores- cence). (a) Cells in serum-free media. (b) Cells 30 min after serum addition. Sections 1 lm from the surface. Scale bar = 10 lm. Intracellular trafficking of FGF-2 S. Taverna et al. 1584 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS Further proof of the interaction between FGF-2 granules and the actin cytoskeleton was obtained by double fluorescence experiments. Actin filaments were labelled with phalloidin and FGF-2 with antibodies. As shown in Fig. 3A, 30 min after serum addition, FGF-2 positive granules were seen to localize on actin filaments (Fig. 3A, arrow). Actin involvement in FGF-2 release was confirmed by immunoprecipitation experiments with anti-FGF-2 serum. However, as shown in Fig. 3B, actin was found to be present only in immunoprecipitates obtained from cells cultured in complete medium, and was absent in immunoprecipitates obtained from starved cells, thus indicating that FGF-2 ⁄ actin association occurs when serum induces FGF-2 intracellular traf- ficking toward the cell periphery. Analysis of the intracellular localization of FGF-2 and of lipidic structures To verify whether FGF-2 intracellular granules are included in lipidic vesicles and whether they are targeted to endolysosomal districts before being secreted, we performed double fluorescence experiments labelling lipids with FM4-64 (a red fluorescent molecule) and FGF-2 with monoclonal antibodies recognized by fluorescein isothiocyanate (FITC)-conjugated second- ary antibodies. After being endocytosed (15 min of cell culture in the presence of the dye), FM4-64 stains intracellular lipidic structures [35]. As shown in Fig. 3C, FGF-2 granules do not colocalize with FM4- 64-labelled intracellular lipidic vesicles in the absence (Fig. 3C, panel a) or in the presence of serum (Fig. 3C, panel b). Therefore, FGF-2 granules appear to move on actin filaments without being enclosed in vesicles. α sub Na-K-ATPasi actin 100 KDa 45 KDa a b c 0 20 40 60 80 100 120 0 20 40 60 80 100 120 A in the nuclei Control Ouabain Control Ouabain Control Ouabain Number of FGF-2 granules ** * 0 20 40 60 80 100 120 UpA activity-arbitrary unit (2) proteins in shed vesicles (1) 1 2 * A B C D E Fig. 4. Effects of ouabain on FGF-2 intracellular trafficking. (A) Im- munolocalization of FGF-2 in controls and in ouabain treated cells. (a) Cells fixed in serum-free media; (b) cells fixed 30 min after serum addition; (c) cells fixed 30 min after addition of serum and 100 l M ouabain. Sections 1 lm from the surface. FGF-2 was detected using Texas red-conjugated secondary antibodies. Scale bar = 10 lm. (B) FGF-2 concentration (absorbance) in nuclei, 30 min after serum addition, in control cells and in cells treated with ouabain. FGF-2 concentration (absorbance) was evaluated by IMAGEJ software in sections 3 lm from the surface, 30 min after serum (Control) or serum and 100 l M ouabain (Ouabain) addition. (C) Numbers of FGF-2 granules at the cell surface 30 min after serum addition, in controls and in ouabain treated cells. FGF-2 posi- tive granules (diameters in the range 0.01–1 lm) were counted in immunostained sections 1 lm from the surface by IMAGEJ software, as described in the Experimental procedures. Controls, control cells to which only serum was added; Ouabain, cells to which serum and 100 l M ouabain were added. Asterisks indicate a significant dif- ference between ouabain treated cells and controls. (D) Amount of vesicle recovered by control and ouabain treated cells and their stimulatory effect on uPA expression by endothelial cells. (1) Amount of vesicles recovered from complete medium conditioned by 3 h of growth of 4 · 10 7 control cells (Control), and by 3 h of growth of 4 · 10 7 cells in complete medium to which 100 lM oua- bain had been added (Ouabain). (2) Induction of uPA activity in GM7373 cells. Casein ⁄ plasminogen zymographies for detection of uPA activity performed as described in the Experimental proce- dures on GM7373 cells that had been incubated for 16 h with vesi- cles obtained from 5 mL of media conditioned by 3 h of growth of 4 · 10 7 control cells grown in complete medium (Control) and by 3 h of growth of 4 · 10 7 cells in complete medium to which 100 l M ouabain had been added (Ouabain). Arbitrary units repre- sent densitometric values of uPA activity, where 100 is considered as the basal uPA activity of GM7373 cells. Asterisks indicate a sig- nificant difference between vesicles shed by Ouabain treated cells and controls. (E) Detection of Na + ⁄ K + -ATPase in shed vesicles. Western blot analysis of Na + ⁄ K + -ATPase in vesicles shed by untreated (Control) and 100 l M ouabain treated cells. Lane 1, vesi- cles shed by untreated cells (30 lg of protein); lane 2, vesicles shed by ouabain treated cells (30 lg of protein). Values are the mean ± SD of 15 measurements from five independent experi- ments. S. Taverna et al. Intracellular trafficking of FGF-2 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS 1585 This result contradicts the hypothesis that shed vesicles, such as exosomes [36], originate from multivesi- cular bodies. Effects of drugs inhibiting FGF-2 release In another group of experiments, we tested two well- known inhibitors of FGF-2 secretion, ouabain and methylamine, for their effects on intracellular FGF-2 trafficking and on vesicle shedding. Effects of ouabain As shown in Fig. 4A, FGF-2 immunolocalization experiments demonstrated that ouabain interferes with FGF-2 intracellular trafficking toward the cell peri- phery whereas it does not interfere with trafficking toward the nucleus (Fig. 4A,B). FGF-2 positive gran- ules, observed at the cell periphery 30 min after serum addition, were virtually absent in cells treated with both ouabain and serum (Fig. 4A,C). Ouabain treatment did not modify the amount of shed vesicles recovered from CM (Fig. 4D, bars marked 1); however, when the stimulatory effect of vesicles on uPA production by GM7373 cells was tested, we observed that vesicles shed by ouabain-trea- ted cells had a decreased specific activity on uPA induction (Fig. 4D, bars marked 2). Therefore, oua- bain interferes with FGF-2 intracellular trafficking toward the cell periphery. As ouabain is known to bind to a subunit of Na + ⁄ K + -ATPase [37,38], we looked for the presence of this protein in vesicles. As shown in Fig. 4E, a molecule of the expected apparent mass (100 kDa) was marked by a monoclonal antibody against the a ATP- ase subunit. The concentration of the recognized anti- gen decreased in vesicles shed by ouabain-treated cells. a A B D C b c 0 20 40 60 80 100 120 140 A at the cell periphery 0 20 40 60 80 100 120 A in the nuclei 0 1 2 3 4 20 40 60 80 100 Arbitrary unit Control Methylamine Control Methylamine Control Methylamine * * * Fig. 5. Effects of methylamine on FGF-2 intracellular trafficking. (A) Immunolocalization of FGF-2 in controls and methylamine treated cells. (a) Cells fixed in serum-free media; (b) cells fixed 30 min after serum addition; (c) cells fixed 30 min after serum and 10 m M methylamine addition. Sections 1 lm from the surface. FGF-2 was detected using Texas red-conjugated secondary antibodies. Scale bar = 10 lm. (B) FGF-2 concentration (absorbance) in nuclei, 30 min after serum or serum and methylamine addition. FGF-2 con- centration (absorbance) was evaluated by IMAGEJ software in sec- tions 1 lm from the surface, 30 min after serum (Control) or serum and 10 m M methylamine (Methylamine) addition. (C) FGF-2 concen- tration (absorbance) at the cell periphery, 30 min after serum and methylamine addition. FGF-2 concentration (absorbance) was evalu- ated by IMAGEJ software in sections 1 lm from the surface, 30 min after serum (Control) or serum and methylamine addition (10 squares of length 2 lm were analyzed in each field). (D) Amount of recovered vesicles and uPA activity induction. (1) Amount of vesi- cles recovered from medium conditioned by 4 · 10 7 cells grown for 3 h in complete medium and by cells treated for 3 h with com- plete medium and 10 m M methylamine. (2) uPA induction by 20 lg of vesicles shed by cells grown in complete medium and by cells treated for 3 h with complete medium and 10 m M methylamine. (3) uPA induction by vesicles obtained from 5 mL of media conditioned by 4 · 10 7 cells grown in complete medium and by cells treated for 3 h with complete medium and 10 m M methylamine. (4) uPA induction by 5 mL of vesicle-free media conditioned by 4 · 10 7 cells grown in complete medium and by cells treated for 3 h with complete medium and 10 m M methylamine. Control, untreated cells; Methylamine, cells treated for 3 h with 10 m M methylamine. Casein ⁄ plasminogen zymographies for detection of uPA activity were performed as described in the Experimental pro- cedures on GM7373 cells that had been incubated for 16 h with vesicles obtained from media conditioned by 3 h of growth of 4 · 10 7 control cells grown in complete medium and by cells trea- ted for 3 h with complete medium and 10 m M methylamine. Arbi- trary units represent densitometric values of uPA activity, where 100 is considered as the basal uPA activity of GM7373 cells. Val- ues are the mean ± SD of 15 measurements each of 10 squares of length 2 lm from three independent experiments. Intracellular trafficking of FGF-2 S. Taverna et al. 1586 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS Effects of methylamine Treatment with methylamine did not affect FGF-2 movements toward the cell nucleus (Fig. 5A,B). As a consequence of methylamine treatment, FGF-2 positive granules were observed to accumulate at the cell periphery and to coalesce with the plasma mem- brane, which became intensely positive for FGF-2 immunoreactive material (Fig. 5A). In methylamine- treated cells, FGF-2 positive granules were not easily counted; therefore, the main immunofluorescence at the cell periphery was evaluated. Namely, in each microscopic field, we selected 10 squares of length 2 lm at the cell periphery and analyzed them for absorbance. Fluorescence at the cell periphery was not significantly modified by methylamine treatment (Fig. 5C). However, the amount of released vesicles diminished (Fig. 5D, bars marked 1). The specific stim- ulatory effect of vesicles on uPA production by GM7373 cells was not modified (Fig. 5D, bars marked 2); however, we observed a decrease in uPA induction caused by adding the total amount of vesi- cles recovered from a fixed volume of CM (Fig. 5D, bars marked 3) to GM7373 cells. A proportional decrease was also observed in uPA induction caused by adding the same volume of vesicle-free CM (Fig. 5D, bars marked 4) to the cells. Therefore, we can conclude that the inhibitory effect of methylamine on FGF-2 secretion is due to this molecule’s ability to inhibit vesicle shedding. Discussion We previously reported that the release of FGF-2, a pro-angiogenic growth factor that lacks the signalling sequence typical of most secreted proteins, is mediated by vesicle-shedding. Neither FGF-2 release, nor vesicle shedding occurred when cells were kept in serum-free media. Thirty minutes after serum addition, FGF-2 granules were seen at the cell periphery and, after 1 h, FGF-2 rich vesicles were recovered from CM; FGF-2 was detected in vesicle-free CM only 2 h later [13]. The present study aimed to analyze intracellular trafficking of endogenous FGF-2, targeting the mole- cule to the budding vesicles and ⁄ or to different cell districts. Our strategy was to follow the changes in FGF-2 intracellular localization shortly after adding serum, both in controls and in cells treated with drugs affecting cytoskeleton organization or FGF-2 release. To make a comparison of the FGF-2 content of vesi- cles shed by control and treated cells, we performed analyses of uPA activity in extracts of GM7373 endo- thelial cells treated with vesicles. Induction of uPA activity in endothelial cells is a typical response elicited by FGF-2 [33,34], and we previously reported that the stimulatory effect of SK-Hep1 vesicles on uPA produc- tion by GM7373 cells was dose-dependent and fully neutralized by monoclonal anti-FGF-2 serum [13]. Assays of cell vitality were performed in control and treated cells and data were collected using cell cultures in which the number of apoptotic or necrotic cells, evaluated by acridine orange and trypan blue staining, was found to be negligible (i.e. less than 3%). When using untreated cells, we observed that some FGF-2 positive granules were detected at the cell periphery as early as 15 min after serum addition; their number then grew for approximately 45 min. However, after 1 h of culturing in complete medium, the number of FGF-2 granules abruptly dropped. The decrease in the number of FGF-2 granules at the cell periphery was coupled with the appearance of FGF-2 rich vesi- cles in CM. Therefore, the results were in agreement with the previously described [13] vesicle-mediated mechanism of FGF-2 release. FGF-2 is a molecule with autocrine, paracrine and intracrine signalling mechanisms. With respect to intra- crine mechanisms, the factor has to be localized in the nucleus. A detectable amount of FGF-2 was also found to be present in the nucleus of starved cells; however, when serum was added, the nuclear concen- tration of the molecule increased considerably. We also noticed that, in starved cells, FGF-2 was undetectable in specific areas of the nucleus apparently correspond- ing to nucleoli. After serum addition, these areas of the cell nucleus became highly positive for FGF-2 immunostaining. To analyze the involvement of cytoskeletal elements in FGF-2 targeting, we tested drugs that alter either tubulin or actin dynamism. Treatment with drugs affecting the organization of microtubules (nocodazol, colchicine and paclitaxel) did not interfere with FGF-2 granule trafficking toward the cell membrane, nor with the amount of vesicles shed into the extracellular med- ium. No differences were observed in the stimulatory effects of vesicles shed by control and treated cells on the uPA activity of endothelial cells. Therefore, the results of these experiments show that FGF-2 export toward the cell periphery is mediated by mechanisms that do not require microtubule organization. By contrast, treatment with drugs that affect micro- tubule dynamics neither allowed for the serum-induced increase of FGF-2 concentration in the cell nucleus, nor the specific localization of FGF-2 in nucleoli. Therefore, nuclear and nucleolar localization of endog- enous FGF-2 appears to follow a pathway requiring maintenance of the microtubule organization. As S. Taverna et al. Intracellular trafficking of FGF-2 FEBS Journal 275 (2008) 1579–1592 ª 2008 The Authors Journal compilation ª 2008 FEBS 1587 reported by Bossard et al. [31], tubulin organization is also needed for nuclear targeting of endocytosed FGF-2. Treatment with cytochalasin B showed that endo- genous FGF-2 export toward the cell periphery requires the integrity of the actin cytoskeleton. These treatments decreased FGF-2 transportation to the cell periphery and to the budding vesicles. Consequently, the specific stimulatory activity of vesicles on GM7373 uPA activity decreased. Interaction between FGF-2 and actin filaments was also demonstrated by coimmu- noprecipitation and coimmunolocalization of the two molecules. Moreover coimmunoprecipitation experi- ments showed that interactions between FGF-2 and actin were induced by serum addition. Cytochalasin B considerably decreased the rate of vesicle shedding. On the other hand, cytochalasin B treatment did not affect FGF-2 movements toward the nucleus and nucleolus. Therefore, the results indicate that FGF-2 movements toward the cell periphery or the nucleus utilize totally different mechanisms. We also determined whether FGF-2 positive gran- ules, observed at the cell periphery, were enclosed in lipidic vesicles. However, colocalization experiments performed using the lipidic colorant FM4-64 and anti- FGF-2 serum showed that FGF-2 granules were not associated with lipidic structures. These results indicate a secretion mechanism that, unlike the production of exosomes [36], does not involve multivescicular bodies. In a different group of experiments, we analyzed FGF-2 intracellular localization, the amount of shed vesicles and the stimulatory effect of vesicles on uPA production, using SK-Hep1 cells treated with drugs known to inhibit FGF-2 secretion. The drugs tested were ouabain and methylamine. Ouabain is a well-known inhibitor of Na + ⁄ K + - ATPase which binds to the a subunit of Na + ⁄ K + - ATPase. However, the molecule was also reported to inhibit FGF-2 secretion [37] and FGF-2 release was reported to be ouabain-insensitive in cells expressing an ouabain resistant ATPase a subunit. A possible explanation of the inhibitory effect of ouabain on FGF-2 secretion may be that the electrochemical gra- dient created by the Na + ⁄ K + -ATPase is a prerequisite for intracellular FGF-2 movements toward the cell periphery or for its secretion. However, FGF-2 and ATPase a subunit were shown to coimmunoprecipi- tate, and it was therefore suggested that, to allow FGF-2 secretion, FGF-2 and ATPase a subunit had to interact [38]. The reasons why this interaction was needed were not clarified. Our results show that oua- bain treatment blocks intracellular movements of FGF-2 positive granules. In treated cells, FGF-2 gran- ules were not observed to accumulate at the cell periphery. The drug did not modify the rate of vesicle shedding; nevertheless, vesicles shed by ouabain-treated cells had a decreased specific stimulatory effect on GM7373 cells, indicating a decreased FGF-2 content. The results obtained by treating cells with ouabain are in agreement with the vesicle-mediated mechanism of FGF-2 secretion that we described; they show that ouabain-mediated inhibition of FGF-2 secretion is associated with a decrease in FGF-2 targeting to budding vesicles. However, the mechanisms by which ouabain affects FGF-2 movements toward the cell periphery require further analysis. A hypothetical explanation of the drug effect is that direct interaction of the ATPase a subunit with FGF-2, inhibited by ouabain, is needed for the two molecules to be transferred to the budding vesicles. This hypothesis is validated by our observation that ouabain treatment caused a decrease in the vesicle concentration of both FGF-2 and of the ATPase a subunit. However, it is dif- ficult to explain how the two molecules come across and are allowed to run into each other. We hypothesized that interaction could occur at the level of multivesicu- lar bodies or in other membrane-bound intracellular districts. However, this hypothesis was contradicted by the results of lipidic staining, which failed to show any colocalization between intracellular FGF-2 granules and lipidic structures. The site of interaction between the ATPase a subunit and FGF-2 therefore remains unknown. To explain how such interaction could occur in the cell cytoplasm, we are led to suspect the existence of a splice variant of the ATPase a subunit lacking the N-terminal signalling sequence that drives the complete protein to the rER. For the Na + ⁄ K + -ATPase a sub- unit, such a variant is not known; however, it was described for an ouabain-sensitive H + ⁄ K + -ATPase a2 subunit. It was suggested that the alternative splicing of this molecule dictates isoform-specific differences in membrane targeting or cytoskeletal association [39]. Different explanations of the effects of ouabain are also possible. For example, FGF-2 trafficking to the budding vesicles might be inhibited by ouabain in response to one of the several signal transduction path- ways modulated by the alkaloid interaction with the Na + ⁄ K + -ATPase a subunits [40]. The results obtained after methylamine treatment clearly support the prominent role of shed vesicles in FGF-2 secretion. Methylamine has been described to inhibit both FGF-2 release [1] and vesicle shedding [41]. The results contained herein indicate that the methylamine inhibitory effect on FGF-2 release was due to its action on vesicle shedding. FGF-2 granules coalesced with the cell membrane but were not Intracellular trafficking of FGF-2 S. 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Importance of vascular phenotype by basic fibroblast growth factor, and influence of the angiogenic factors basic fibroblast growth factor ⁄ fibroblast growth. obtained from 5 mL of media conditioned by 3 h of growth of 4 · 10 7 control cells grown in complete medium (Control) and by 3 h of growth of 4 · 10 7 cells