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Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells

Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 51, Issue of December 19, pp. 51911–51919, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells* Received for publication, April 22, 2003, and in revised form, September 26, 2003 Published, JBC Papers in Press, September 30, 2003, DOI 10.1074/jbc.M304192200 Simona Taverna‡, Giulio Ghersi‡, Angela Ginestra‡, Salvatrice Rigogliuso‡, Sonia Pecorella‡, Giovanna Alaimo‡, Francesca Saladino‡, Vincenza Dolo§, Patrizia Dell’Era , Antonio Pavan§, Giuseppe Pizzolanti , Paolo Mignatti**, Marco Presta , and Maria Letizia Vittorelli‡ ‡‡§§ From the ‡Dipartimento Biologia Cellulare e dello Sviluppo, Universita ` di Palermo, Palermo 90128, Italy, §Dipartimento Medicina Sperimentale Universita ` di L’Aquila, L’Aquila 67100, Italy, Dipartimento Scienze Biomediche e Biotecnologie, Universita ` di Brescia, Brescia 25123, Italy, Cattedra di Endocrinologia, Istituto di Clinica Medica, Policlinico di Palermo, Palermo 90128, Italy, **Departments of Surgery and Cell Biology, New York University School of Medicine, New York, New York 10016, and ‡‡Centro di OncoBiologia Sperimentale, Viale delle Scienze, Palermo 90128, Italy cle and endothelial cell growth, is involved in wound healing, Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentia- and plays important roles in the development and differentia- tion, lacks a conventional secretory signal sequence, tion of various organs (2, 3). Elevated levels of FGF-2 have been and its mechanism of release from cells remains unclear. implicated in the pathogenesis of several diseases character- We characterized the role of extracellular vesicle shed- ized by exaggerated neovascularization (4) and in a broad spec- ding in FGF-2 release. Viable cells released membrane trum of cancers (5, 6). vesicles in the presence of serum. However, in serum- FGF-2 exists in five isoforms with molecular masses of 18, free medium vesicle shedding was dramatically down- 22, 22.5, 24, and 34 kDa (7). The 18-kDa polypeptide is local- regulated, and the cells did not release FGF-2 activity ized primarily in the cytosol (8), whereas forms with higher into their conditioned medium. Addition of serum to molecular mass are found predominantly in the nucleus (7, 9). serum-starved cells rapidly induced intracellular FGF-2 All FGF-2 forms entail high affinity interactions with tyrosine clustering under the plasma membrane and into gran- kinase FGF receptors and low-affinity interactions with proteo- ules that colocalized with patches of the cell membrane glycans (HSPGs) containing heparan sulfate polysaccharides with typical features of shed vesicle membranes. Shed (10, 11). vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Although FGF-2 is found associated with the extracellular Addition of vesicles to endothelial cells stimulated che- matrix in vitro and in vivo and exerts its biological activities by motaxis and urokinase plasminogen activator produc- binding to cell membrane receptors, it lacks a conventional tion, which were blocked by anti-FGF-2 antibodies. secretory peptide and is not released through the classical Treatment of intact vesicles with 2.0 M NaCl or hepari- nase, which release FGF-2 from membrane-bound pro- endoplasmic reticulum (ER)-Golgi pathway. It has been pro- teoglycans, did not abolish their stimulatory effect on posed that FGF-2 is released from cells through alternative endothelial cells, indicating that FGF-2 is carried inside pathways including cell death, wounding, or sublethal injury vesicles. The comparison of the stimulatory effects of (12, 13). However, FGF-2 release does not parallel the release shed vesicles and vesicle-free conditioned medium of cytoplasmic markers (14). Inhibitors of protein secretion via showed that vesicles represent a major reservoir of the ER-Golgi complex do not block FGF-2 release. In contrast, FGF-2. Thus, FGF-2 can be released from cells through reagents or treatments that inhibit exo/endocytosis or energy vesicle shedding. production block externalization of FGF-2 in transfected NIH 3T3 or COS-1 cells. A non-classic secretion pathway has there- fore been proposed for FGF-2 release (15–17). Release of 18- Fibroblast growth factor-2 (FGF-2) is the prototype member kDa FGF-2 from transfected COS-1 cells is inhibited by oua- of a family of structurally related heparin binding growth fac- bain, a known inhibitor of Na,K-ATPase (18), and FGF-2 co- tors that have mitogenic activity for several cell types and immunoprecipitates with the  ATPase subunit (19). It has induce mesenchyme formation (1). FGF-2, a potent inducer of therefore been proposed that Na,K-ATPase plays an important blood vessel formation (angiogenesis), stimulates smooth mus- role in FGF-2 secretion (16, 20). Other extracellular proteins devoid of signal sequence in- * This work was supported in part by grants from the Italian Asso- clude FGF-1, interleukin 1, interleukin 1, and lectin 14 ciation for Cancer Research (to M. L. V. and M. P.), by Ministero del (L-14). FGF-1 appears to be released in response to stress lavoro e previdenza sociale N.792 (to A. P.), and by the Italian Ministry conditions as a component of multiprotein aggregates (21, 22), of University and Scientific and Technological Research (to M. L. V., and a member of the S100 family of Ca binding proteins has M. P., and A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be been implicated in its release (23, 24). However heat shock does hereby marked “advertisement” in accordance with 18 U.S.C. Section not affect FGF-2 secretion (15). Secretory pathways via extra- 1734 solely to indicate this fact. cellular vesicle production have been proposed for both inter- §§ To whom correspondence should be addressed. Tel.: 39-091- leukin 1 and L-14. Externalization of L-14, a signaling mole- 6577407; Fax: 39-091-6577430; E-mail: [email protected]. The abbreviations used are: FGF-2, fibroblast growth factor-2; cule highly expressed in muscle cells, is developmentally HPSGs, heparan sulfate-containing proteoglycans; ER, endoplasmic regulated. As myogenic cells differentiate, cytosolic L-14 is reticulum; GFP, green fluorescent protein; FCS, fetal calf serum; PBS, concentrated in the cellular ectoplasm beneath regions of the phosphate-buffered saline; FITC, fluorescein isothiocyanate; ERK, ex- plasma membrane that appear to evaginate and form extracel- tracellular signal-regulated kinase; uPA, urokinase-type plasminogen activator. lular vesicles. It has therefore been suggested that shed mem- This paper is available on line at http://www.jbc.org 51911 This is an Open Access article under the CC BY license. 51912 Shed Vesicles Mediate FGF-2 Release FIG.1. Immunolocalization of FGF-2 in SK-Hep1 cells. Immunolocal- ization of FGF-2 (a and b) and double staining of FGF-2/ -integrin (c and d) and of FGF-2/annexin V (e and f) are shown. Cells were fixed in serum-free me- dia (a, c, and e) and 30 min after serum addition (b, d, and f). Bar  10 m. Squares show selected areas that were magnified on the left bottom (2in a and b; 4in c, d, e, and f). FGF-2 was detected using Texas red-conjugated secondary an- tibodies.  -Integrin was detected using FITC-conjugated secondary antibodies. Biotinylated annexin V added to fixed cells was detected using FITC-conjugated streptavidin. brane vesicles represent a vehicle for L-14 secretion (25). or monocytes (26) are also enriched in phosphatidyl-serine and IL1 is released from activated immune cells after a second- bind annexin V. They were therefore proposed to possess pro- ary stimulus such as extracellular ATP acting on P2X (7) re- coagulant activity. ceptors. Mackenzie et al. (26) reported that human THP-1 In addition to membrane vesicles, exosomes, a population of monocytes shed vesicles from their plasma membranes within exovesicles released by eukaryotic cells, have also been re- 2–5 s after the activation of P2X (7) receptors; 2 min later the ported to vehicle secreted proteins. Exosomes are 40 – 80-nm released vesicles contain IL1. The cytokine is then released extracellular vesicles released by exocytosis of multivesicular in a vesicle-free form at later time points. bodies. These structures are part of the endosomal system and Membrane vesicles originate from the plasma membrane are considered to belong to the category of late endosomes/ through a mechanism morphologically similar to that of virus lysosomes. Exosomes are probably generated by inward bud- budding. The vesicles are relatively large and heterogeneous in ding of the vesicle membrane (39, 40) and are enriched in major size; their diameters ranging from 100 to 1000 nm (27, 28). histocompatibility complex class I and II molecules, in mem- Vesicle shedding is an active process that requires RNA and bers of the tetraspan protein superfamily (41– 43), and in protein synthesis (29) and occurs in viable cells with no signs of HSP70 (43, 44). Two cytosolic proteins found in exosomes, apoptosis or necrosis. Vesicle membranes carry most surface gelactin-3 and annexin II, are also found in the extracellular antigens expressed on the plasma membrane (30); however environment. Because these proteins do not posses a signal they originate from domains of the plasma membrane selec- sequence, it has been suggested that exosomes represent an tively enriched in membrane components including HLA class unconventional secretion pathway for these proteins (45). I molecules, 1 integrins, and membrane-bound matrix metal- Recently, FGF-2 secretion was analyzed in Chinese hamster loproteinase-9 (31). Vesicle shedding has been implicated in ovary cells expressing FGF-2-GFP fusion protein under control cell migration and in tumor progression (32), a function that by the tetracycline resistance transactivator. FGF-2-GFP pro- may be mediated by vesicle membrane-bound proteinases (28, tein was shown to translocate to the outer surface of the plasma 31, 33–36). Vesicles are also shed by several non-tumor cells membrane; the secreted FGF-2-GFP fusion protein then accu- (37, 38) and have been reported to vehicle a variety of regula- mulated in large HSPG-containing protein clusters on the ex- tory factors (25, 26, 29, 30, 36). Vesicles shed by platelets (38) tracellular surface of the plasma membrane (46). In the present Shed Vesicles Mediate FGF-2 Release 51913 paper, we tested the hypothesis that these large protein clus- ters are exovesicles and that vesicle shedding may represent a mechanism for FGF-2 release from the cell. EXPERIMENTAL PROCEDURES Cells and Culture Media—Human SK-Hep1 hepatoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS; Euroclone, Celbio). NIH 3T3 cells transfected with FGF-2 cDNA have been described (15). Transfected and vector- transfected NIH 3T3 cells were grown in Dulbecco’s minimum essential medium (Sigma-Aldrich) supplemented with 0.6 g/liter NaHCO , 500 g/ml geneticin (G418; Invitrogen) and 10% FCS. Bovine GM7373 fetal aortic endothelial cells (47) were grown in Eagle’s minimal essential medium (Sigma) supplemented with 10% FCS, vitamins, and essential and non-essential amino acids. All the cells were negative for myco- plama contamination by the Hoechst 33258 (Sigma) staining assay. Immunofluorescence—SK-Hep1 cells seeded at low density (2.000 cells/well) onto microscope coverslips in 12-well culture plates (Nunc) were grown overnight in complete medium and then in serum-free medium for 1 week with three medium changes. To study the effect of serum on the intracellular distribution of FGF-2, medium supple- mented with 10% FCS was added. After 5, 10, 20, and 30 min of incubation the cells were fixed in 3.7% formaldehyde for 10 min fol- lowed by permeabilization with 0.05% Triton X-100 for 5 min. FGF-2 was detected with mouse monoclonal anti-FGF-2 (0.5 mg/ml, 1:200; Upstate Biotechnology type II) antibody and Texas Red-conjugated anti-mouse antibodies (1:200; Amersham Biosciences); biotinylated an- nexin V was added (2 mg/ml) to fixed cells, washed (five times for 5 min each) with phosphate-buffered saline (PBS), and detected using FITC- streptavidin (Sigma) (1:500); 1-integrins were detected using FITC- conjugated C27 mAb (48). In some experiments isolated vesicles were bound to poly-L-lysine on a coverslip, fixed, permeabilized, and treated with primary and secondary antibodies as described for cells. Immuno- stained vesicles were analyzed by confocal microscopy (Olympus 1X70 with Melles Griot laser system). To detect interactions between vesicle-associated FGF-2 and the cell membrane, purified vesicles (50 g/ml) were added to sparse GM7373 cell cultures pretreated with cycloheximide (10 g/ml) for 3 h. After 3 h of incubation the cells were fixed with 3.7% formaldehyde for 10 min, FIG.2. Time course of vesicle shedding and shed-vesicle sta- followed by permeabilization with 0.01% Triton X-100 for 5 min. FGF-2 bility in SK-Hep1 cells. a, amount of vesicles recovered from medium was detected with mouse anti-FGF-2 monoclonal antibody (0.5 mg/ml, conditioned by 4  10 SK-Hep1 cells. b, amount of vesicles recovered 1:500; Upstate Biotechnology type II) and FITC-conjugated anti-mouse from medium conditioned by 24 h of 4  10 SK-Hep1 cell growth after antibodies (1:500; Amersham Biosciences). different incubation periods at 37 °C. Ultrastructural Analysis—Transmission electron microscopy of ves- icle shedding was carried out using a standard technique. Briefly, cells were fixed with 2% glutaraldehyde in culture flasks, scraped, and Lae ¨ mmli buffer and electrophoresed in SDS-3–12.5% gradient poly- post-fixed with 1% OsO , dehydrated with ethanol, and embedded in acrylamide gels. Epon 812. Samples were sectioned, post-stained with uranyl acetate Western Blotting—After electrophoresis in SDS-polyacrylamide gels, and lead citrate, and examined with an electron microscope (Philips the proteins were blotted onto a nitrocellulose membrane (Hybond; CM10, Eindhoven, Netherlands). Amersham Biosciences) that was saturated with 5% horse serum, 0.1% For scanning electron microscopy cells were grown on coverslips and Tween 20 in PBS for 2 h and then incubated with either monoclonal fixed with 2% glutaraldehyde in PBS for 30 min. Critical point dried anti-FGF-2 (2.5 g/ml; Upstate Biotechnology type II) or anti-HSP70 samples were glued onto stubs, coated with gold in a SKD040 Balzer protein (0.8 g/ml; Sigma H-5147) or anti CD-44 (10 g/ml; Calbiochem Sputterer, and observed using a Philips 505 scanning electron micro- 217604) antibodies followed by horseradish peroxidase-conjugated anti- scope at 10 –30 kV. mouse or anti-rabbit IgG antibodies (1:7500; Sigma) for1hat room Vesicle Purification from Conditioned Medium—Vesicles were puri- temperature. Immunocomplexes were visualized with the ECL Western fied from conditioned medium as described (28). Briefly, medium con- blotting kit (Amersham Biosciences) using Hyperfilms. ditioned by subconfluent healthy cells for 3, 6, or 24 h were centrifuged ERK Phosphorylation—To characterize extracellular signal-regu- at 2000  g and at 4000  g for 15 min. The supernatant was ultra- lated kinase-1/2 (ERK1/2) phosphorylation, GM7373 cells were seeded centrifuged at 105,000  g for 90 min. Pelleted vesicles were resus- in 24-well plates (80,000 cells/cm ) in Dulbecco’s modified Eagle’sme- pended in PBS. dium containing 10% FCS. The cells then were starved for 24 h in 0.5% In some control experiments, vesicles were purified by affinity bind- FCS, followed by treatment with either 50 g/ml SK-Hep1 cell-derived ing of the 4000  g supernatant to biotinylated annexin-V (Pierce) and vesicles or 50 ng/ml human recombinant FGF-2 (49) for the indicated bound to Streptavidin MagneSphere Paramagnetic Particles (SA- time. Cell extracts were analyzed by Western blotting with anti-phos- PMPs; Promega). To remove components that might bind to the mag- pho-ERK1/2 antibody (Santa Cruz Biotechnology, Inc.). Immunocom- netic beads in the absence of annexin-V, conditioned medium was plexes were visualized by chemiluminescence with the Supersignal® pre-adsorbed with SA-PMPs, in the absence of annexin-V. Vesicles bound to SA-PMPs were solubilized by incubation with Ca - and West Pico chemiluminescent substrate (Pierce) according to the manu- Mg -free PBS, 20 mM EDTA for1hat room temperature. The amount facturer’s instructions. To judge uniform loading, the same membrane of isolated vesicles was determined by measuring protein concentration was stripped and re-incubated with anti-ERK antibody (Santa Cruz by the Bradford microassay method (Bio-Rad) using bovine serum al- Biotechnology). 6 2 bumin (Sigma) as a standard. Assay for uPA Activity—1.75  10 GM7373 cells (70,000 cells/cm ) Heparin-Sepharose Adsorption and SDS-PAGE—Purified vesicles were grown for 28 h in 5 ml of medium supplemented with different were sonicated three times with 50 pulses for 10 s each and incubated concentrations of vesicles. Cell cultures were washed twice with PBS at 4 °C overnight in an end-over-end mixer with 40 – 80 l of heparin- and incubated for 18 –24 h in serum-free medium. After harvesting the Sepharose (Amersham Biosciences) equilibrated in PBS. After four culture supernatant, the cells were washed twice with PBS and subse- washes with PBS, the beads were resuspended in 30 l of reducing quently scraped with a rubber policeman. Following centrifugation at 51914 Shed Vesicles Mediate FGF-2 Release FIG.3. Ultrastructural analysis of SK-Hep1 cells. Scanning (a–d) and transmission (e and f) electron micro- scopic analysis of SK-Hep1 cells grown in the absence (a, c, and e) or in the presence of 10% FCS (b, d, and f) is shown. FIG.4. Presence of FGF-2 in SK-Hep1 shed membrane vesicles. A, immunostaining of purified vesicles with biotinylated annexin V and FIG.5. Interaction of vesicle-released FGF-2 with target-cell anti-FGF-2 antibodies. Biotinylated annexin V, added to fixed vesicles, plasma membranes and stimulation of ERK1/2 phosphorylation. was detected using FITC-conjugated streptavidin. FGF-2 was detected A, immunolocalization of released FGF-2. a, untreated cells; b, cells using Texas red-conjugated secondary antibodies. Bar  10 m. a and treated with 50 g/ml Sk-Hep1 vesicles. B, stimulation of ERK1/2 b, negative controls (a, SK-Hep1 vesicles plus FITC streptavidin; b, phosphorylation by shed vesicles. Western blot analysis of GM7373 SK-Hep1 vesicles plus Texas red-conjugated secondary antibodies); c, cells extracts by anti-phospho-ERK1/2 antibody (Ph-ERK ) and by 1/2 immunostaining for annexin V; d, immunostaining for FGF-2. B, West- ERK antibody (ERK ) is shown.  Vesicles, GM7373 cells treated with 2 2 ern blot analysis of vesicles for FGF-2. Lane 1, human recombinant 50 g/ml SK-Hep1 vesicles for the indicated time;  FGF-2, GM7373 FGF-2 (100 ng); lane 2, SK-Hep1 vesicles (recovered from 500 gof cells treated with 50 ng/ml FGF-2 for the indicated time. vesicle proteins submitted to heparin-Sepharose chromatography). g/ml). Five hundred microliters of Dulbecco’s minimum essential me- 800  g for 10 min at 4 °C, protein concentration was measured by the dium containing 10 GM7373 cells was added to the upper chamber, Bradford method. whereas the bottom chamber received 500 l of Dulbecco’s minimum Cell extracts (30 g of proteins) were loaded onto SDS 7.5% poly- essential medium with or without the indicated concentrations of ves- acrylamide gels. Zymography was performed as described (50) with icles, vesicle-free supernatants, and anti-FGF-2 neutralizing antibodies overlay gels containing 3% non-fat dry milk and 40 g/ml bovine plas- (type I; Upstate Biotechnology). After4hof incubation at 37 °C, the minogen (Sigma). Densitometric analysis of the lysis bands was per- inserts (16 mm diameter) were removed and fixed in methanol. The formed using Eastman Kodak Co. Science 1D Image Analyzer software. cells on the upper surface of the filter were removed with a cotton swab, White bands of caseynolysis of images were converted to dark bands to and the cells migrated across the membrane were stained with 1% better display the activity. crystal violet and counted. Cell Migration Assay—Falcon cell culture inserts with 8-m pore Heparinase and NaCl Treatments—Conditioned media were incu- polyethylene terephthalate membranes were coated with gelatin (50 bated either with heparinase III 4 milliunits/ml for2hat37 °C or with Shed Vesicles Mediate FGF-2 Release 51915 FIG.6. Induction of uPA activity in GM7373 cells by SK-Hep1 vesicles and vesicle-free conditioned medium. GM7373 cells were incubated for 18 h with different concentration of vesicles. Endothelial cells were lysed and processed for uPA activity as described. a, uPA activity of GM7373 cells incubated without vesicles (lane 1) or with 25, 50, and 100 g/ml SK-Hep1 vesicles (lanes 2–4). b, effects of differently purified vesicles. Control, uPA activity of untreated GM7373 cells; Vesicles by centrifugation, uPA activity of cells treated with 6 g/ml SK-Hep1 vesicles obtained by ultracentrifugation; Vesicles by SAPMPs, uPA activity of cells treated with 6 g/ml SK-Hep1 vesicles obtained by affinity binding to annexin-V, which had been biotinylated previously and bound to Streptavidin MagneSphere Paramagnetic Particles (SAPMPs; Promega); Residual pellet, uPA activity of cells treated with 6 g/ml pellet material obtained from conditioned media after vesicles absorption with SAPMPs. c, comparison between vesicles and vesicle-free media, conditioned by1hof Sk-Hep1 cell growth. Control, uPA activity of untreated GM7373 cells; CM-vesicles, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures. Vesicles, uPA activity of GM7373 cells growing in the presence of vesicles purified from 5 ml of SK-Hep1 conditioned media (3 g/ml). d, comparison between vesicles and vesicle-free media, conditioned by3hof Sk-Hep1 cell growth. Control, uPA activity of untreated GM7373 cells; control  Ab, uPA activity of GM7373 cells growing in the presence of 2.5 g of inhibitory anti-FGF-2 monoclonal antibodies in 5 ml; CM-vesicles, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures; CM-vesicles  Ab, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures  2.5 g of inhibitory anti-FGF-2 antibodies in 5 ml; Vesicles, uPA activity of GM7373 cells growing in the presence of vesicles purified from 5 ml of SK-Hep1 conditioned media (3 g/ml); Vesicles  Ab, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures  2.5 g of inhibitory anti-FGF-2 antibodies in 5 ml. d.u. represents densitometric values of uPA activity, expressed in arbitrary units, considering 1.0 the basal uPA activity of GM7373 cells. 2 M NaCl (final concentration) for 30 min at 37 °C without or after consistent with the inhibitory effect of serum deprivation on sonication (three times with 80 pulses for 30 s each). After centrifuga- FGF-2 externalization (15). Similarly, serum deprivation in- tion at 100,000  g, pelleted vesicles were resuspended in PBS and hibits vesicle shedding (28, 53) raising the hypothesis that assayed for their ability to stimulate uPA expression in GM7373 cells. FGF-2 externalization and vesicle shedding may represent re- RESULTS lated processes. We therefore characterized the effect of serum on the expression and intracellular localization of FGF-2 in FGF-2 Is Associated with Membrane Vesicles Shed from SK- SK-Hep1 cells. FGF-2 expression was analyzed by Western Hep1 Cells—SK-Hep1 cells produce high levels of FGF-2 (51). blotting of starved cells or cells collected 10 min, 2 h, and 24 h Previous observations (52) have shown that FGF-2 cannot be detected in serum-free medium conditioned by SK-Hep1 cells, after serum addition. FGF-2 levels were not affected by serum 51916 Shed Vesicles Mediate FGF-2 Release FIG.7. FGF-2 is released by FGF-2- transfected NIH-3T3 cells in shed vesicles. A, time course of vesicle shed- ding in by FGF-2-transfected NIH-3T3 cells in the absence or in the presence of 10% FCS. B, immunostaining of purified vesicles with biotinylated annexin V and anti FGF-2 antibodies. a and b, vesicles shed by parental NIH 3T3 cells; c and d, vesicles shed by FGF-2 transfected NIH 3T3 cells; a and c, immunostaining for annexin V; b and d, immunostaining for FGF-2. Biotinylated annexin V, added to fixed vesicles, was detected using FITC- conjugated streptavidin. FGF-2 was de- tected using Texas red-conjugated sec- ondary antibodies. Bar  10 m. C, Western blot analysis of vesicles for FGF-2. Lane 1, NIH 3T3 vesicles (50 g); lane 2, vesicles from FGF-2-transfected NIH 3T3 cells (50 g). (data not shown). Under serum-free conditions, immunofluo- size (300 –900 nm diameter) of the vesicles shed by SK-Hep1 rescence staining with monoclonal anti-FGF-2 antibody fol- cells were similar to those shed by 8701-BC or HT-1080 tumor lowed by confocal microscopy showed that FGF-2 localized in cells (28, 34) and clearly distinct from the significantly smaller the nucleus and in the cell cytoplasm, where it formed small exosomes (40). aggregates (Fig. 1a). Addition of serum caused rapid clustering In another set of experiments, purified vesicles bound to a of FGF-2 in larger patches (Fig. 1b). Under these conditions, poly-L-lysine-coated coverslip were fixed, permeabilized, and FGF-2 immunoreactive material was found to colocalize with immunostained with anti-FGF-2 antibodies. In parallel, vesi- areas of the cell plasma membranes enriched in 1 integrins cles were assessed for their capacity to bind annexin V. As (Fig. 1d) and characterized by an increased capability to bind shown in Fig. 4a, annexin V-binding vesicles stained inten- annexin V (Fig. 1f), in keeping with a localization of the growth sively with anti-FGF-2 antibodies. Accordingly, Western blot- factor in plasma membrane regions involved in vesicle forma- ting of isolated vesicles showed FGF-2 immunoreactive bands tion (26, 38). This colocalization was not observed when cells of 18, 22, and 24 kDa, consistent with the low and high molec- were grown under serum-free conditions (Fig. 1, c and e). ular weight forms of FGF-2 (Fig. 4b). In keeping with previous In agreement with these and previous observations (28), observations with different tumor cell types (28, 31, 34, 35), SK-Hep1 cells did not release vesicles in the absence of serum. SK-Hep1 cell-derived vesicles carried matrix metalloprotein- However, in the presence of FCS SK-Hep1 cells shed a large ase-2 and matrix metalloproteinase-9 and the hyaluronic acid amount of vesicles (Fig. 2). Vesicle accumulation in the condi- receptor, CD-44. In addition, the lack of HSP70 (data not tioned medium occurred rapidly, reaching a plateau 3–6h shown), further distinguished these vesicles from typical exo- after serum addition. Vesicles were produced by viable cell somes (44). cultures in which the number of apoptotic or necrotic cells, FGF-2 Released from SK-Hep1 Vesicles Binds to the GM7373 evaluated by acridine orange and trypan-blue staining, was Cell Membrane and Generates Intracellular Signaling—To as- found to be negligible (less than 3%). To analyze vesicle stabil- sess the biological significance of vesicle-associated FGF-2, we ity conditioned media were incubated at 37 °C for different tested purified vesicles for their ability to deliver FGF-2 to the times before vesicle purification. As shown in Fig. 2b, vesicles endothelial cell surface and activate downstream signaling. appear to have a short half-life, probably because of their After incubation of GM7373 endothelial cells with vesicles pu- content in proteolytic enzymes. rified from SK-Hep1 cell-conditioned medium, the cells, which Scanning and transmission electron microscopic analysis of do not produce FGF-2, stained positively with antibodies to this SK-Hep1 cells confirmed that vesicle shedding is induced by growth factor (Fig. 5A). Downstream signaling triggered by the serum addition (Fig. 3). Indeed, serum-starved cells showed a binding of FGF-2 to its tyrosine-kinase receptors encompasses smooth plasma membrane (a, c, and e), whereas cells grown in the activation of mitogen-activated protein kinase kinase, with the presence of FCS were characterized by a rough membrane consequent phosphorylation of ERKs (54). Accordingly, vesicle- in the process of shedding membrane vesicles (b, d, and f). treated GM7373 cells showed a long lasting increase in ERK1/2 Vesicles appeared to bud from the cell membrane and to en- phosphorylation (Fig. 5B). Vesicle-mediated ERK1/2 phospho- close a small amount of cytoplasmic material (f). As shown in Fig. 3b, vesicle shedding was a generalized phenomenon that was not limited to isolated, damaged cells. The morphology and D. Cassara ` and M. L. Vittorelli, unpublished data. Shed Vesicles Mediate FGF-2 Release 51917 FIG.8. Vesicles shed by FGF-2-transfected NIH 3T3 cells induce uPA activity and have chemotactic effects on GM7373 cells. a, induction of uPA activity in GM7373 cells. GM7373 cells were incubated for 18 h with different concentration of vesicles. Endothelial cells were lysed and processed for uPA activity as described. uPA activity of GM7373 cells incubated without vesicles (lane 1), with 25 or 50 g/ml non-transfected NIH 3T3 vesicles (lanes 2 and 3), with 25, 50, or 100 g/ml transfected NIH 3T3 vesicles (lanes 4, 5, and 6) is shown. b, chemiotactic effects of vesicles shed by FGF-2-producing and non-producing cells. GM7373 endothelial cells were assayed for cell migration in the presence of indicated concentrations of vesicles shed by non-transfected and FGF-2-transfected NIH 3T3. Vesicles were recovered from media of cells grown in serum-free (FCS) or in complete medium (FCS). c, inhibition of vesicle-mediated chemotaxis by anti-FGF-2 antibody. GM7373 cells were incubated with vesicles shed by FGF-2-transfected NIH 3T3 cells (20 g of vesicle protein/ml) with the indicated concentrations of neutralizing anti-FGF-2 antibody and processed as above. b and c, mean and experimental variability are shown. rylation appeared to be delayed compared with activation trig- the vesicle preparation, vesicles were bound to biotinylated an- gered by the recombinant growth factor, probably because of nexin V and recovered by affinity chromatography on streptavi- the slow release of FGF-2 from disrupted vesicles. din-bound magnetic beads. As shown in Fig. 6b, vesicles purified Another typical response elicited by FGF-2 is uPA produc- by this technique had a uPA-inducing activity similar to that of tion (51, 55). The uPA stimulatory activity of vesicle-associated vesicles isolated by ultracentrifugation. FGF-2 was measured by casein/plasminogen zymography of To compare the stimulatory effects of vesicles and of vesicle- endothelial cell extracts. As shown in Fig. 6a, vesicles induced free supernatants, vesicles, recovered from a fixed volume of a dose-dependent increase in cell-associated uPA activity. Both SK-Hep1 conditioned medium, were assessed for their capacity control and vesicle-treated GM7373 cells showed a caseinolytic to induce uPA up-regulation in GM7373 cells. In parallel, the band with an apparent molecular mass of 46 kDa, correspond- same volume of vesicle-free supernatant was tested under the ing to bovine uPA and distinct from human uPA (55 kDa). This same experimental conditions. As shown by Fig. 6c when this observation ruled out the possibility that the increase in uPA comparison was made using media collected after1hof cell activity was mediated by vesicle-associated human uPA re- growth, only vesicles had a clearly detectable stimulatory effect maining bound to the endothelial cell surface. on GM7373 cells, whereas vesicle-free supernatant was inef- To confirm that the uPA-inducing activity was associated with fective. In media collected after3hof cell growth (Fig. 6d) both shed vesicles and was not mediated by co-sedimented material in vesicles and vesicle-free supernatants had some stimulatory 51918 Shed Vesicles Mediate FGF-2 Release FIG.9. Effects of HSPG removal on the uPA stimulatory effect of vesicles on GM7373 cells. a, effects of heparinase III on uPA stimulatory effect of vesicles shed by transfected 3T3 cells. Control, uPA activity of untreated GM7373 cells; Untreated vesicles, uPA activity of cells treated with 20 g/ml of vesicles. Vesicles digested with 4 milliunits/ml of heparinase III, uPA activity of cells treated with 20 g/ml of vesicles digested with heparinase for2hat37 °C. b, effects of 2 M NaCl incubation on uPA stimulatory effect of vesicles shed by Sk-Hep1 cells. Control, uPA activity of untreated GM7373 cells; Untreated vesicles, uPA activity of cells treated with 30 g/ml of vesicles. Vesicles incubated with 2 M NaCl, uPA activity of cells treated with 30 g/ml vesicles incubated for 30 min at 37 °C. Vesicles sonicated and incubated with 2 M NaCl, uPA activity of cells treated with 30 g/ml vesicles purified from sonicated media and incubated as described. activity, nevertheless vesicles still exerted a stimulatory effect FGF-2 Is Localized Inside Shed Vesicles—To assess whether stronger than the corresponding vesicle-free supernatants. To FGF-2 is present inside the vesicles or associated with HSPGs confirm that the uPA-inducing activity of SK-Hep1 cell-derived on their surface, purified vesicles were digested with hepari- vesicles was indeed mediated by vesicle-associated FGF-2, ex- nase III or washed with a high ionic strength solution (2.0 M periments were also performed in the presence of neutralizing NaCl). Both treatments are known to release FGF-2 from anti-FGF-2 antibodies. As shown by Fig. 6d the stimulatory HSPGs (57). The vesicles were then tested for uPA-inducing effects of both vesicles and vesicle-free supernatants were fully activity on GM7373 cells. As shown in Fig. 9, a and b, neither neutralized by anti-FGF-2 monoclonal antibodies (05117; Up- the heparinase III treatment nor the NaCl wash abolished the state Biotechnology). Unrelated monoclonal antibodies belong- ability of vesicles to up-regulate uPA expression in endothelial ing to the same isotype (Sigma monoclonal antibody T9026, cells. When, however, incubation with NaCl was performed against tubulin), tested at the same concentration, did not after sonication, the vesicle stimulatory effect was completely modify the stimulatory effects of vesicles and vesicle-free con- lost (Fig. 9b). These results are consistent with an intravesicu- ditioned media (data not shown). These data indicated that lar localization of the growth factor. vesicle-associated FGF-2 retains the capacity to up-regulate DISCUSSION uPA expression in endothelial cells and that most, if not all of the uPA-inducing activity present in the conditioned medium of FGF-2 is found associated with the extracellular matrix in SK-Hep1 cells is mediated by FGF-2 initially associated with vitro and in vivo and interacts with cell membrane receptors. vesicles. However, it lacks a hydrophobic signal sequence for secretion FGF-2-transfected NIH 3T3 Cells Release Biologically Active through the ER/Golgi system, and the mechanism(s) of its FGF-2 by Shedding Membrane Vesicles—FGF-2-transfected release are not understood. Because reagents or treatments NIH 3T3 cells have been shown to release limited but signifi- that block protein secretion via the ER-Golgi complex do not cant amounts of this growth factor (15). Therefore, we tested affect FGF-2 release (15) (19), three routes have been proposed whether vesicle shedding was also responsible for FGF-2 re- for FGF-2 externalization: cell death (12), sublethal cell injury lease in this cell type. As shown in Fig. 7A, vesicle shedding (13), or exocytosis via ER/Golgi-independent pathway(s) (15, was stimulated by serum in both parental and FGF-2-overex- 19, 46). pressing NIH 3T3 cells. These cells, however, released vesicles In this study we tested the hypothesis that FGF-2 release also in serum-free medium. Immunolocalization experiments from cells occurs by vesicle shedding. Membrane vesicles bud and Western blotting analysis showed the presence of low and from the plasma membrane of viable cells and can be purified high molecular weight FGF-2 isoforms in annexin V-binding from cell-conditioned medium (for a review see Ref. 58). Both vesicles shed by FGF-2-trasfected cells but not by parental cells FGF-2 secretion and vesicle shedding are energy-dependent (Fig. 7, B and C). phenomena (15, 29), and they are not inhibited by reagents In keeping with the data obtained with SK-Hep1 cells, ves- that block protein secretion via the ER-Golgi complex. There- icles purified from the conditioned medium of FGF-2 transfec- fore, the two processes appear to be modulated by the same tants showed the capacity to up-regulate uPA expression in mechanisms. endothelial GM7373 cells (Fig. 8a). In addition, vesicles shed by The data reported show that serum addition to SK-Hep1 FGF-2-producing NIH 3T3 cells induced a chemotactic re- cells that constitutively express high levels of FGF-2 rapidly sponse in endothelial cells, and their effect was abolished by results in vesicle shedding and that the intracellular localiza- neutralizing anti-FGF-2 antibody (Fig. 8, b and c). The speci- tion of FGF-2 is also strongly affected by these culture condi- ficity of these effects was further demonstrated by the lack of tions. Shortly after serum addition to SK-Hep1 cells, FGF-2 activity of vesicles released by vector-transfected NIH 3T3 appears in granules under the cell membrane and colocalizes cells. with patches of the cell membranes that show increased con- Shed Vesicles Mediate FGF-2 Release 51919 10. Johnson, D. E., and Williams, L. T. (1993) Adv. Cancer Res. 60, 1– 41 centration of 1 integrins and annexin V-binding activity. Ves- 11. Rusnati, M., and Presta, M. (1996) Int. J. Clin. Lab. Res. 26, 15–23 icle membranes were shown to be enriched in 1 integrins (31) 12. Schweigerer, L., Neufeld, G., Friedman, J., Abraham, J. A., Fiddes, J. C., and Gospodarowicz, D. (1987) Nature 325, 257–259 and to have annexin V-binding capacity (26, 38). These obser- 13. McNeil, P. L., Muthukrishnan, L., Warder, E., and D’Amore, P. A. (1989) vations indicate that FGF-2 clusters within areas of the cell J. Cell Biol. 109, 811– 822 membrane where vesicle budding occurs, and FGF-2 is subse- 14. Kandel, J., Bossy-Wetzel, E., Radvanyi, F., Klagsbrun, M., Folkman, J., and Hanahan, D. (1991) Cell 66, 1095–1104 quently externalized with shed vesicles. Consistent with this 15. Mignatti, P., Morimoto, T., and Rifkin, D. B. (1992) J. Cell Physiol. 151, 81–93 hypothesis, we found that the 18-, 22-, and 24-kDa FGF-2 16. Florkiewicz, R. Z., Majack, R. A., Buechler, R. D., and Florkiewicz, E. (1995) forms are associated with shed membrane vesicles. J. Cell Physiol. 162, 388 –399 17. Qu, Z., Kayton, R. J., Ahmadi, P., Liebler, J. M., Powers, M. R., Planck, S. R., The same FGF-2 isoforms are also present in vesicles shed by and Rosenbaum, J. T. (1998) J. Histochem. Cytochem. 46, 1119 –1128 NIH 3T3 cells transfected with FGF-2 cDNA. Based on mor- 18. Jorgensen, J. R., and Pedersen, P. A. (2001) Biochemistry 40, 7301–7308 19. Florkiewicz, R. Z., Anchin, J., and Baird, A. (1998) J. Biol. Chem. 273, 544 –551 phology, size, presence of gelatinolytic enzymes, and annexin 20. Dahl, J. P., Binda, A., Canfield, V. A., and Levenson, R. (2000) Biochemistry V-binding activity, vesicles shed by SK-Hep1 and by trans- 39, 14877–14883 fected NIH 3T3 cells are similar to membrane vesicles shed by 21. Jackson, A., Friedman, S., Zhan, X., Engleka, K. A., Forough, R., and Maciag, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10691–10695 several tumor (28, 31, 34, 53) or normal cells (25, 26, 38). Their 22. Jackson, A., Tarantini, F., Gamble, S., Friedman, S., and Maciag, T. (1995) diameters are much larger than those reported for exosomes J. Biol. Chem. 270, 33–36 (40). Moreover, exosomes are enriched in HSP70 (43, 44) 23. Mouta, C. C., LaVallee, T. M., Tarantini, F., Jackson, A., Lathrop, J. T., Hampton, B., Burgess, W. H., and Maciag, T. (1998) J. Biol. Chem. 273, whereas vesicles shed by SK-Hep1 or NIH 3T3 cells do not 22224 –22231 contain this protein. 24. Landriscina, M., Bagala, C., Mandinova, A., Soldi, R., Micucci, I., Bellum, S., Prudovsky, I., and Maciag, T. (2001) J. Biol. Chem. 276, 25549 –25557 Vesicles shed by SK-Hep1 cells or NIH 3T3 cells transfected 25. Cooper, D. N., and Barondes, S. H. (1990) J. Cell Biol. 110, 1681–1691 with FGF-2 cDNA induce increased uPA expression and migra- 26. MacKenzie, A., Wilson, H. L., Kiss-Toth, E., Dower, S. K., North, R. A., and tion (chemotaxis) in vascular endothelial cells, and both these Surprenant, A. (2001) Immunity 15, 825– 835 27. Dainiak, N. (1991) Blood 78, 264 –276 effects are blocked by neutralizing anti-FGF-2 antibody. Our 28. Dolo, V., Ginestra, A., Ghersi, G., Nagase, H., and Vittorelli, M. L. (1994) J. data demonstrate that most of the uPA-inducing activity pres- Submicrosc. Cytol. Pathol. 26, 173–180 ent in the conditioned medium of SK-Hep1 cells is associated 29. Dainiak, N., and Sorba, S. (1991) J. Clin. Invest. 87, 213–220 30. Dolo, V., Pizzurro, P., Ginestra, A., and Vittorelli, M. L. (1995) J. Submicrosc. with shed vesicles. Because of the short half-life of shed vesi- Cytol. Pathol. 27, 535–541 cles, the capability of vesicle-associated FGF-2 to interact and 31. Dolo, V., Ginestra, A., Cassara, D., Violini, S., Lucania, G., Torrisi, M. R., Nagase, H., Canevari, S., Pavan, A., and Vittorelli, M. L. (1998) Cancer Res. activate FGF receptors and the neutralizing capability of anti- 58, 4468 – 4474 FGF-2 antibodies are explained by vesicle disruption. The pos- 32. Poste, G., and Nicolson, G. L. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, sibility that FGF-2 released into the culture medium is bound 399 – 403 33. Zucker, S., Wieman, J. M., Lysik, R. M., Wilkie, D. P., Ramamurthy, N., and by vesicle-associated, low affinity HSPG binding sites is ruled Lane, B. (1987) Biochim. Biophys. Acta 924, 225–237 out by our finding that vesicles treated with heparinase or 2.0 34. Ginestra, A., Monea, S., Seghezzi, G., Dolo, V., Nagase, H., Mignatti, P., and Vittorelli, M. L. (1997) J. Biol. Chem. 272, 17216 –17222 M NaCl completely retained their uPA-inducing activity on 35. Ginestra, A., La Placa, M. D., Saladino, F., Cassara, D., Nagase, H., and endothelial cells, whereas their stimulatory activity was com- Vittorelli, M. L. (1998) Anticancer Res. 18, 3433–3437 pletely lost when they were sonicated before NaCl treatment. 36. D’Angelo, M., Billings, P. C., Pacifici, M., Leboy, P. S., and Kirsch, T. (2001) J. Biol. Chem. 276, 11347–11353 Direct observations of immunolabeled vesicle-associated 37. Trams, E. G., Lauter, C. J., Salem, N., Jr., and Heine, U. (1981) Biochim. FGF-2 on endothelial cell plasma membranes, and signal Biophys. Acta 645, 63–70 transduction kinetic, indicates that FGF-2, present inside the 38. Heijnen, C. J., and Kavelaars, A. (1999) J. Neuroimmunol. 100, 197–202 39. Gruenberg, J., and Maxfield, F. R. (1995) Curr. Opin. Cell Biol. 7, 552–563 vesicles, is delivered to target cells following vesicle break- 40. Denzer, K., Kleijmeer, M. J., Heijnen, H. F., Stoorvogel, W., and Geuze, H. J. down, which occurs spontaneously. Contact of the phospholipid (2000) J. Cell Sci. 113 Pt 19, 3365–3374 41. Raposo, G., Nijman, H. W., Stoorvogel, W., Liejendekker, R., Harding, C. V., vesicle membrane with the cell membrane may also result in Melief, C. J., and Geuze, H. J. (1996) J. Exp. Med. 183, 1161–1172 increased vesicle permeability and release of the encapsulated 42. Zitvogel, L., Regnault, A., Lozier, A., Wolfers, J., Flament, C., Tenza, D., material. This hypothesis is supported by the finding that Ricciardi-Castagnoli, P., Raposo, G., and Amigorena, S. (1998) Nat. Med. 4, 594 – 600 liposome-encapsulated FGF-2 retains the ability to up-regulate 43. Thery, C., Regnault, A., Garin, J., Wolfers, J., Zitvogel, L., Ricciardi- endothelial cell expression of uPA (11). The slow release of Castagnoli, P., Raposo, G., and Amigorena, S. (1999) J. Cell Biol. 147, FGF-2 induced by contact between vesicle and plasma mem- 599 – 610 44. 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Cell Physiol. 147, 311–318 58. Vittorelli, M. L. (2003) Curr. Top. Dev. Biol. 54, 411– 432 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 51, Issue of December 19, pp. 51911–51919, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells* Received for publication, April 22, 2003, and in revised form, September 26, 2003 Published, JBC Papers in Press, September 30, 2003, DOI 10.1074/jbc.M304192200 Simona Taverna‡, Giulio Ghersi‡, Angela Ginestra‡, Salvatrice Rigogliuso‡, Sonia Pecorella‡, Giovanna Alaimo‡, Francesca Saladino‡, Vincenza Dolo§, Patrizia Dell’Era , Antonio Pavan§, Giuseppe Pizzolanti , Paolo Mignatti**, Marco Presta , and Maria Letizia Vittorelli‡ ‡‡§§ From the ‡Dipartimento Biologia Cellulare e dello Sviluppo, Universita ` di Palermo, Palermo 90128, Italy, §Dipartimento Medicina Sperimentale Universita ` di L’Aquila, L’Aquila 67100, Italy, Dipartimento Scienze Biomediche e Biotecnologie, Universita ` di Brescia, Brescia 25123, Italy, Cattedra di Endocrinologia, Istituto di Clinica Medica, Policlinico di Palermo, Palermo 90128, Italy, **Departments of Surgery and Cell Biology, New York University School of Medicine, New York, New York 10016, and ‡‡Centro di OncoBiologia Sperimentale, Viale delle Scienze, Palermo 90128, Italy cle and endothelial cell growth, is involved in wound healing, Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentia- and plays important roles in the development and differentia- tion, lacks a conventional secretory signal sequence, tion of various organs (2, 3). Elevated levels of FGF-2 have been and its mechanism of release from cells remains unclear. implicated in the pathogenesis of several diseases character- We characterized the role of extracellular vesicle shed- ized by exaggerated neovascularization (4) and in a broad spec- ding in FGF-2 release. Viable cells released membrane trum of cancers (5, 6). vesicles in the presence of serum. However, in serum- FGF-2 exists in five isoforms with molecular masses of 18, free medium vesicle shedding was dramatically down- 22, 22.5, 24, and 34 kDa (7). The 18-kDa polypeptide is local- regulated, and the cells did not release FGF-2 activity ized primarily in the cytosol (8), whereas forms with higher into their conditioned medium. Addition of serum to molecular mass are found predominantly in the nucleus (7, 9). serum-starved cells rapidly induced intracellular FGF-2 All FGF-2 forms entail high affinity interactions with tyrosine clustering under the plasma membrane and into gran- kinase FGF receptors and low-affinity interactions with proteo- ules that colocalized with patches of the cell membrane glycans (HSPGs) containing heparan sulfate polysaccharides with typical features of shed vesicle membranes. Shed (10, 11). vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Although FGF-2 is found associated with the extracellular Addition of vesicles to endothelial cells stimulated che- matrix in vitro and in vivo and exerts its biological activities by motaxis and urokinase plasminogen activator produc- binding to cell membrane receptors, it lacks a conventional tion, which were blocked by anti-FGF-2 antibodies. secretory peptide and is not released through the classical Treatment of intact vesicles with 2.0 M NaCl or hepari- nase, which release FGF-2 from membrane-bound pro- endoplasmic reticulum (ER)-Golgi pathway. It has been pro- teoglycans, did not abolish their stimulatory effect on posed that FGF-2 is released from cells through alternative endothelial cells, indicating that FGF-2 is carried inside pathways including cell death, wounding, or sublethal injury vesicles. The comparison of the stimulatory effects of (12, 13). However, FGF-2 release does not parallel the release shed vesicles and vesicle-free conditioned medium of cytoplasmic markers (14). Inhibitors of protein secretion via showed that vesicles represent a major reservoir of the ER-Golgi complex do not block FGF-2 release. In contrast, FGF-2. Thus, FGF-2 can be released from cells through reagents or treatments that inhibit exo/endocytosis or energy vesicle shedding. production block externalization of FGF-2 in transfected NIH 3T3 or COS-1 cells. A non-classic secretion pathway has there- fore been proposed for FGF-2 release (15–17). Release of 18- Fibroblast growth factor-2 (FGF-2) is the prototype member kDa FGF-2 from transfected COS-1 cells is inhibited by oua- of a family of structurally related heparin binding growth fac- bain, a known inhibitor of Na,K-ATPase (18), and FGF-2 co- tors that have mitogenic activity for several cell types and immunoprecipitates with the  ATPase subunit (19). It has induce mesenchyme formation (1). FGF-2, a potent inducer of therefore been proposed that Na,K-ATPase plays an important blood vessel formation (angiogenesis), stimulates smooth mus- role in FGF-2 secretion (16, 20). Other extracellular proteins devoid of signal sequence in- * This work was supported in part by grants from the Italian Asso- clude FGF-1, interleukin 1, interleukin 1, and lectin 14 ciation for Cancer Research (to M. L. V. and M. P.), by Ministero del (L-14). FGF-1 appears to be released in response to stress lavoro e previdenza sociale N.792 (to A. P.), and by the Italian Ministry conditions as a component of multiprotein aggregates (21, 22), of University and Scientific and Technological Research (to M. L. V., and a member of the S100 family of Ca binding proteins has M. P., and A. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be been implicated in its release (23, 24). However heat shock does hereby marked “advertisement” in accordance with 18 U.S.C. Section not affect FGF-2 secretion (15). Secretory pathways via extra- 1734 solely to indicate this fact. cellular vesicle production have been proposed for both inter- §§ To whom correspondence should be addressed. Tel.: 39-091- leukin 1 and L-14. Externalization of L-14, a signaling mole- 6577407; Fax: 39-091-6577430; E-mail: [email protected]. The abbreviations used are: FGF-2, fibroblast growth factor-2; cule highly expressed in muscle cells, is developmentally HPSGs, heparan sulfate-containing proteoglycans; ER, endoplasmic regulated. As myogenic cells differentiate, cytosolic L-14 is reticulum; GFP, green fluorescent protein; FCS, fetal calf serum; PBS, concentrated in the cellular ectoplasm beneath regions of the phosphate-buffered saline; FITC, fluorescein isothiocyanate; ERK, ex- plasma membrane that appear to evaginate and form extracel- tracellular signal-regulated kinase; uPA, urokinase-type plasminogen activator. lular vesicles. It has therefore been suggested that shed mem- This paper is available on line at http://www.jbc.org 51911 This is an Open Access article under the CC BY license. 51912 Shed Vesicles Mediate FGF-2 Release FIG.1. Immunolocalization of FGF-2 in SK-Hep1 cells. Immunolocal- ization of FGF-2 (a and b) and double staining of FGF-2/ -integrin (c and d) and of FGF-2/annexin V (e and f) are shown. Cells were fixed in serum-free me- dia (a, c, and e) and 30 min after serum addition (b, d, and f). Bar  10 m. Squares show selected areas that were magnified on the left bottom (2in a and b; 4in c, d, e, and f). FGF-2 was detected using Texas red-conjugated secondary an- tibodies.  -Integrin was detected using FITC-conjugated secondary antibodies. Biotinylated annexin V added to fixed cells was detected using FITC-conjugated streptavidin. brane vesicles represent a vehicle for L-14 secretion (25). or monocytes (26) are also enriched in phosphatidyl-serine and IL1 is released from activated immune cells after a second- bind annexin V. They were therefore proposed to possess pro- ary stimulus such as extracellular ATP acting on P2X (7) re- coagulant activity. ceptors. Mackenzie et al. (26) reported that human THP-1 In addition to membrane vesicles, exosomes, a population of monocytes shed vesicles from their plasma membranes within exovesicles released by eukaryotic cells, have also been re- 2–5 s after the activation of P2X (7) receptors; 2 min later the ported to vehicle secreted proteins. Exosomes are 40 – 80-nm released vesicles contain IL1. The cytokine is then released extracellular vesicles released by exocytosis of multivesicular in a vesicle-free form at later time points. bodies. These structures are part of the endosomal system and Membrane vesicles originate from the plasma membrane are considered to belong to the category of late endosomes/ through a mechanism morphologically similar to that of virus lysosomes. Exosomes are probably generated by inward bud- budding. The vesicles are relatively large and heterogeneous in ding of the vesicle membrane (39, 40) and are enriched in major size; their diameters ranging from 100 to 1000 nm (27, 28). histocompatibility complex class I and II molecules, in mem- Vesicle shedding is an active process that requires RNA and bers of the tetraspan protein superfamily (41– 43), and in protein synthesis (29) and occurs in viable cells with no signs of HSP70 (43, 44). Two cytosolic proteins found in exosomes, apoptosis or necrosis. Vesicle membranes carry most surface gelactin-3 and annexin II, are also found in the extracellular antigens expressed on the plasma membrane (30); however environment. Because these proteins do not posses a signal they originate from domains of the plasma membrane selec- sequence, it has been suggested that exosomes represent an tively enriched in membrane components including HLA class unconventional secretion pathway for these proteins (45). I molecules, 1 integrins, and membrane-bound matrix metal- Recently, FGF-2 secretion was analyzed in Chinese hamster loproteinase-9 (31). Vesicle shedding has been implicated in ovary cells expressing FGF-2-GFP fusion protein under control cell migration and in tumor progression (32), a function that by the tetracycline resistance transactivator. FGF-2-GFP pro- may be mediated by vesicle membrane-bound proteinases (28, tein was shown to translocate to the outer surface of the plasma 31, 33–36). Vesicles are also shed by several non-tumor cells membrane; the secreted FGF-2-GFP fusion protein then accu- (37, 38) and have been reported to vehicle a variety of regula- mulated in large HSPG-containing protein clusters on the ex- tory factors (25, 26, 29, 30, 36). Vesicles shed by platelets (38) tracellular surface of the plasma membrane (46). In the present Shed Vesicles Mediate FGF-2 Release 51913 paper, we tested the hypothesis that these large protein clus- ters are exovesicles and that vesicle shedding may represent a mechanism for FGF-2 release from the cell. EXPERIMENTAL PROCEDURES Cells and Culture Media—Human SK-Hep1 hepatoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS; Euroclone, Celbio). NIH 3T3 cells transfected with FGF-2 cDNA have been described (15). Transfected and vector- transfected NIH 3T3 cells were grown in Dulbecco’s minimum essential medium (Sigma-Aldrich) supplemented with 0.6 g/liter NaHCO , 500 g/ml geneticin (G418; Invitrogen) and 10% FCS. Bovine GM7373 fetal aortic endothelial cells (47) were grown in Eagle’s minimal essential medium (Sigma) supplemented with 10% FCS, vitamins, and essential and non-essential amino acids. All the cells were negative for myco- plama contamination by the Hoechst 33258 (Sigma) staining assay. Immunofluorescence—SK-Hep1 cells seeded at low density (2.000 cells/well) onto microscope coverslips in 12-well culture plates (Nunc) were grown overnight in complete medium and then in serum-free medium for 1 week with three medium changes. To study the effect of serum on the intracellular distribution of FGF-2, medium supple- mented with 10% FCS was added. After 5, 10, 20, and 30 min of incubation the cells were fixed in 3.7% formaldehyde for 10 min fol- lowed by permeabilization with 0.05% Triton X-100 for 5 min. FGF-2 was detected with mouse monoclonal anti-FGF-2 (0.5 mg/ml, 1:200; Upstate Biotechnology type II) antibody and Texas Red-conjugated anti-mouse antibodies (1:200; Amersham Biosciences); biotinylated an- nexin V was added (2 mg/ml) to fixed cells, washed (five times for 5 min each) with phosphate-buffered saline (PBS), and detected using FITC- streptavidin (Sigma) (1:500); 1-integrins were detected using FITC- conjugated C27 mAb (48). In some experiments isolated vesicles were bound to poly-L-lysine on a coverslip, fixed, permeabilized, and treated with primary and secondary antibodies as described for cells. Immuno- stained vesicles were analyzed by confocal microscopy (Olympus 1X70 with Melles Griot laser system). To detect interactions between vesicle-associated FGF-2 and the cell membrane, purified vesicles (50 g/ml) were added to sparse GM7373 cell cultures pretreated with cycloheximide (10 g/ml) for 3 h. After 3 h of incubation the cells were fixed with 3.7% formaldehyde for 10 min, FIG.2. Time course of vesicle shedding and shed-vesicle sta- followed by permeabilization with 0.01% Triton X-100 for 5 min. FGF-2 bility in SK-Hep1 cells. a, amount of vesicles recovered from medium was detected with mouse anti-FGF-2 monoclonal antibody (0.5 mg/ml, conditioned by 4  10 SK-Hep1 cells. b, amount of vesicles recovered 1:500; Upstate Biotechnology type II) and FITC-conjugated anti-mouse from medium conditioned by 24 h of 4  10 SK-Hep1 cell growth after antibodies (1:500; Amersham Biosciences). different incubation periods at 37 °C. Ultrastructural Analysis—Transmission electron microscopy of ves- icle shedding was carried out using a standard technique. Briefly, cells were fixed with 2% glutaraldehyde in culture flasks, scraped, and Lae ¨ mmli buffer and electrophoresed in SDS-3–12.5% gradient poly- post-fixed with 1% OsO , dehydrated with ethanol, and embedded in acrylamide gels. Epon 812. Samples were sectioned, post-stained with uranyl acetate Western Blotting—After electrophoresis in SDS-polyacrylamide gels, and lead citrate, and examined with an electron microscope (Philips the proteins were blotted onto a nitrocellulose membrane (Hybond; CM10, Eindhoven, Netherlands). Amersham Biosciences) that was saturated with 5% horse serum, 0.1% For scanning electron microscopy cells were grown on coverslips and Tween 20 in PBS for 2 h and then incubated with either monoclonal fixed with 2% glutaraldehyde in PBS for 30 min. Critical point dried anti-FGF-2 (2.5 g/ml; Upstate Biotechnology type II) or anti-HSP70 samples were glued onto stubs, coated with gold in a SKD040 Balzer protein (0.8 g/ml; Sigma H-5147) or anti CD-44 (10 g/ml; Calbiochem Sputterer, and observed using a Philips 505 scanning electron micro- 217604) antibodies followed by horseradish peroxidase-conjugated anti- scope at 10 –30 kV. mouse or anti-rabbit IgG antibodies (1:7500; Sigma) for1hat room Vesicle Purification from Conditioned Medium—Vesicles were puri- temperature. Immunocomplexes were visualized with the ECL Western fied from conditioned medium as described (28). Briefly, medium con- blotting kit (Amersham Biosciences) using Hyperfilms. ditioned by subconfluent healthy cells for 3, 6, or 24 h were centrifuged ERK Phosphorylation—To characterize extracellular signal-regu- at 2000  g and at 4000  g for 15 min. The supernatant was ultra- lated kinase-1/2 (ERK1/2) phosphorylation, GM7373 cells were seeded centrifuged at 105,000  g for 90 min. Pelleted vesicles were resus- in 24-well plates (80,000 cells/cm ) in Dulbecco’s modified Eagle’sme- pended in PBS. dium containing 10% FCS. The cells then were starved for 24 h in 0.5% In some control experiments, vesicles were purified by affinity bind- FCS, followed by treatment with either 50 g/ml SK-Hep1 cell-derived ing of the 4000  g supernatant to biotinylated annexin-V (Pierce) and vesicles or 50 ng/ml human recombinant FGF-2 (49) for the indicated bound to Streptavidin MagneSphere Paramagnetic Particles (SA- time. Cell extracts were analyzed by Western blotting with anti-phos- PMPs; Promega). To remove components that might bind to the mag- pho-ERK1/2 antibody (Santa Cruz Biotechnology, Inc.). Immunocom- netic beads in the absence of annexin-V, conditioned medium was plexes were visualized by chemiluminescence with the Supersignal® pre-adsorbed with SA-PMPs, in the absence of annexin-V. Vesicles bound to SA-PMPs were solubilized by incubation with Ca - and West Pico chemiluminescent substrate (Pierce) according to the manu- Mg -free PBS, 20 mM EDTA for1hat room temperature. The amount facturer’s instructions. To judge uniform loading, the same membrane of isolated vesicles was determined by measuring protein concentration was stripped and re-incubated with anti-ERK antibody (Santa Cruz by the Bradford microassay method (Bio-Rad) using bovine serum al- Biotechnology). 6 2 bumin (Sigma) as a standard. Assay for uPA Activity—1.75  10 GM7373 cells (70,000 cells/cm ) Heparin-Sepharose Adsorption and SDS-PAGE—Purified vesicles were grown for 28 h in 5 ml of medium supplemented with different were sonicated three times with 50 pulses for 10 s each and incubated concentrations of vesicles. Cell cultures were washed twice with PBS at 4 °C overnight in an end-over-end mixer with 40 – 80 l of heparin- and incubated for 18 –24 h in serum-free medium. After harvesting the Sepharose (Amersham Biosciences) equilibrated in PBS. After four culture supernatant, the cells were washed twice with PBS and subse- washes with PBS, the beads were resuspended in 30 l of reducing quently scraped with a rubber policeman. Following centrifugation at 51914 Shed Vesicles Mediate FGF-2 Release FIG.3. Ultrastructural analysis of SK-Hep1 cells. Scanning (a–d) and transmission (e and f) electron micro- scopic analysis of SK-Hep1 cells grown in the absence (a, c, and e) or in the presence of 10% FCS (b, d, and f) is shown. FIG.4. Presence of FGF-2 in SK-Hep1 shed membrane vesicles. A, immunostaining of purified vesicles with biotinylated annexin V and FIG.5. Interaction of vesicle-released FGF-2 with target-cell anti-FGF-2 antibodies. Biotinylated annexin V, added to fixed vesicles, plasma membranes and stimulation of ERK1/2 phosphorylation. was detected using FITC-conjugated streptavidin. FGF-2 was detected A, immunolocalization of released FGF-2. a, untreated cells; b, cells using Texas red-conjugated secondary antibodies. Bar  10 m. a and treated with 50 g/ml Sk-Hep1 vesicles. B, stimulation of ERK1/2 b, negative controls (a, SK-Hep1 vesicles plus FITC streptavidin; b, phosphorylation by shed vesicles. Western blot analysis of GM7373 SK-Hep1 vesicles plus Texas red-conjugated secondary antibodies); c, cells extracts by anti-phospho-ERK1/2 antibody (Ph-ERK ) and by 1/2 immunostaining for annexin V; d, immunostaining for FGF-2. B, West- ERK antibody (ERK ) is shown.  Vesicles, GM7373 cells treated with 2 2 ern blot analysis of vesicles for FGF-2. Lane 1, human recombinant 50 g/ml SK-Hep1 vesicles for the indicated time;  FGF-2, GM7373 FGF-2 (100 ng); lane 2, SK-Hep1 vesicles (recovered from 500 gof cells treated with 50 ng/ml FGF-2 for the indicated time. vesicle proteins submitted to heparin-Sepharose chromatography). g/ml). Five hundred microliters of Dulbecco’s minimum essential me- 800  g for 10 min at 4 °C, protein concentration was measured by the dium containing 10 GM7373 cells was added to the upper chamber, Bradford method. whereas the bottom chamber received 500 l of Dulbecco’s minimum Cell extracts (30 g of proteins) were loaded onto SDS 7.5% poly- essential medium with or without the indicated concentrations of ves- acrylamide gels. Zymography was performed as described (50) with icles, vesicle-free supernatants, and anti-FGF-2 neutralizing antibodies overlay gels containing 3% non-fat dry milk and 40 g/ml bovine plas- (type I; Upstate Biotechnology). After4hof incubation at 37 °C, the minogen (Sigma). Densitometric analysis of the lysis bands was per- inserts (16 mm diameter) were removed and fixed in methanol. The formed using Eastman Kodak Co. Science 1D Image Analyzer software. cells on the upper surface of the filter were removed with a cotton swab, White bands of caseynolysis of images were converted to dark bands to and the cells migrated across the membrane were stained with 1% better display the activity. crystal violet and counted. Cell Migration Assay—Falcon cell culture inserts with 8-m pore Heparinase and NaCl Treatments—Conditioned media were incu- polyethylene terephthalate membranes were coated with gelatin (50 bated either with heparinase III 4 milliunits/ml for2hat37 °C or with Shed Vesicles Mediate FGF-2 Release 51915 FIG.6. Induction of uPA activity in GM7373 cells by SK-Hep1 vesicles and vesicle-free conditioned medium. GM7373 cells were incubated for 18 h with different concentration of vesicles. Endothelial cells were lysed and processed for uPA activity as described. a, uPA activity of GM7373 cells incubated without vesicles (lane 1) or with 25, 50, and 100 g/ml SK-Hep1 vesicles (lanes 2–4). b, effects of differently purified vesicles. Control, uPA activity of untreated GM7373 cells; Vesicles by centrifugation, uPA activity of cells treated with 6 g/ml SK-Hep1 vesicles obtained by ultracentrifugation; Vesicles by SAPMPs, uPA activity of cells treated with 6 g/ml SK-Hep1 vesicles obtained by affinity binding to annexin-V, which had been biotinylated previously and bound to Streptavidin MagneSphere Paramagnetic Particles (SAPMPs; Promega); Residual pellet, uPA activity of cells treated with 6 g/ml pellet material obtained from conditioned media after vesicles absorption with SAPMPs. c, comparison between vesicles and vesicle-free media, conditioned by1hof Sk-Hep1 cell growth. Control, uPA activity of untreated GM7373 cells; CM-vesicles, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures. Vesicles, uPA activity of GM7373 cells growing in the presence of vesicles purified from 5 ml of SK-Hep1 conditioned media (3 g/ml). d, comparison between vesicles and vesicle-free media, conditioned by3hof Sk-Hep1 cell growth. Control, uPA activity of untreated GM7373 cells; control  Ab, uPA activity of GM7373 cells growing in the presence of 2.5 g of inhibitory anti-FGF-2 monoclonal antibodies in 5 ml; CM-vesicles, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures; CM-vesicles  Ab, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures  2.5 g of inhibitory anti-FGF-2 antibodies in 5 ml; Vesicles, uPA activity of GM7373 cells growing in the presence of vesicles purified from 5 ml of SK-Hep1 conditioned media (3 g/ml); Vesicles  Ab, uPA activity of GM7373 cells growing in the presence of vesicle-free conditioned media obtained from 5 ml of SK-Hep1 cultures  2.5 g of inhibitory anti-FGF-2 antibodies in 5 ml. d.u. represents densitometric values of uPA activity, expressed in arbitrary units, considering 1.0 the basal uPA activity of GM7373 cells. 2 M NaCl (final concentration) for 30 min at 37 °C without or after consistent with the inhibitory effect of serum deprivation on sonication (three times with 80 pulses for 30 s each). After centrifuga- FGF-2 externalization (15). Similarly, serum deprivation in- tion at 100,000  g, pelleted vesicles were resuspended in PBS and hibits vesicle shedding (28, 53) raising the hypothesis that assayed for their ability to stimulate uPA expression in GM7373 cells. FGF-2 externalization and vesicle shedding may represent re- RESULTS lated processes. We therefore characterized the effect of serum on the expression and intracellular localization of FGF-2 in FGF-2 Is Associated with Membrane Vesicles Shed from SK- SK-Hep1 cells. FGF-2 expression was analyzed by Western Hep1 Cells—SK-Hep1 cells produce high levels of FGF-2 (51). blotting of starved cells or cells collected 10 min, 2 h, and 24 h Previous observations (52) have shown that FGF-2 cannot be detected in serum-free medium conditioned by SK-Hep1 cells, after serum addition. FGF-2 levels were not affected by serum 51916 Shed Vesicles Mediate FGF-2 Release FIG.7. FGF-2 is released by FGF-2- transfected NIH-3T3 cells in shed vesicles. A, time course of vesicle shed- ding in by FGF-2-transfected NIH-3T3 cells in the absence or in the presence of 10% FCS. B, immunostaining of purified vesicles with biotinylated annexin V and anti FGF-2 antibodies. a and b, vesicles shed by parental NIH 3T3 cells; c and d, vesicles shed by FGF-2 transfected NIH 3T3 cells; a and c, immunostaining for annexin V; b and d, immunostaining for FGF-2. Biotinylated annexin V, added to fixed vesicles, was detected using FITC- conjugated streptavidin. FGF-2 was de- tected using Texas red-conjugated sec- ondary antibodies. Bar  10 m. C, Western blot analysis of vesicles for FGF-2. Lane 1, NIH 3T3 vesicles (50 g); lane 2, vesicles from FGF-2-transfected NIH 3T3 cells (50 g). (data not shown). Under serum-free conditions, immunofluo- size (300 –900 nm diameter) of the vesicles shed by SK-Hep1 rescence staining with monoclonal anti-FGF-2 antibody fol- cells were similar to those shed by 8701-BC or HT-1080 tumor lowed by confocal microscopy showed that FGF-2 localized in cells (28, 34) and clearly distinct from the significantly smaller the nucleus and in the cell cytoplasm, where it formed small exosomes (40). aggregates (Fig. 1a). Addition of serum caused rapid clustering In another set of experiments, purified vesicles bound to a of FGF-2 in larger patches (Fig. 1b). Under these conditions, poly-L-lysine-coated coverslip were fixed, permeabilized, and FGF-2 immunoreactive material was found to colocalize with immunostained with anti-FGF-2 antibodies. In parallel, vesi- areas of the cell plasma membranes enriched in 1 integrins cles were assessed for their capacity to bind annexin V. As (Fig. 1d) and characterized by an increased capability to bind shown in Fig. 4a, annexin V-binding vesicles stained inten- annexin V (Fig. 1f), in keeping with a localization of the growth sively with anti-FGF-2 antibodies. Accordingly, Western blot- factor in plasma membrane regions involved in vesicle forma- ting of isolated vesicles showed FGF-2 immunoreactive bands tion (26, 38). This colocalization was not observed when cells of 18, 22, and 24 kDa, consistent with the low and high molec- were grown under serum-free conditions (Fig. 1, c and e). ular weight forms of FGF-2 (Fig. 4b). In keeping with previous In agreement with these and previous observations (28), observations with different tumor cell types (28, 31, 34, 35), SK-Hep1 cells did not release vesicles in the absence of serum. SK-Hep1 cell-derived vesicles carried matrix metalloprotein- However, in the presence of FCS SK-Hep1 cells shed a large ase-2 and matrix metalloproteinase-9 and the hyaluronic acid amount of vesicles (Fig. 2). Vesicle accumulation in the condi- receptor, CD-44. In addition, the lack of HSP70 (data not tioned medium occurred rapidly, reaching a plateau 3–6h shown), further distinguished these vesicles from typical exo- after serum addition. Vesicles were produced by viable cell somes (44). cultures in which the number of apoptotic or necrotic cells, FGF-2 Released from SK-Hep1 Vesicles Binds to the GM7373 evaluated by acridine orange and trypan-blue staining, was Cell Membrane and Generates Intracellular Signaling—To as- found to be negligible (less than 3%). To analyze vesicle stabil- sess the biological significance of vesicle-associated FGF-2, we ity conditioned media were incubated at 37 °C for different tested purified vesicles for their ability to deliver FGF-2 to the times before vesicle purification. As shown in Fig. 2b, vesicles endothelial cell surface and activate downstream signaling. appear to have a short half-life, probably because of their After incubation of GM7373 endothelial cells with vesicles pu- content in proteolytic enzymes. rified from SK-Hep1 cell-conditioned medium, the cells, which Scanning and transmission electron microscopic analysis of do not produce FGF-2, stained positively with antibodies to this SK-Hep1 cells confirmed that vesicle shedding is induced by growth factor (Fig. 5A). Downstream signaling triggered by the serum addition (Fig. 3). Indeed, serum-starved cells showed a binding of FGF-2 to its tyrosine-kinase receptors encompasses smooth plasma membrane (a, c, and e), whereas cells grown in the activation of mitogen-activated protein kinase kinase, with the presence of FCS were characterized by a rough membrane consequent phosphorylation of ERKs (54). Accordingly, vesicle- in the process of shedding membrane vesicles (b, d, and f). treated GM7373 cells showed a long lasting increase in ERK1/2 Vesicles appeared to bud from the cell membrane and to en- phosphorylation (Fig. 5B). Vesicle-mediated ERK1/2 phospho- close a small amount of cytoplasmic material (f). As shown in Fig. 3b, vesicle shedding was a generalized phenomenon that was not limited to isolated, damaged cells. The morphology and D. Cassara ` and M. L. Vittorelli, unpublished data. Shed Vesicles Mediate FGF-2 Release 51917 FIG.8. Vesicles shed by FGF-2-transfected NIH 3T3 cells induce uPA activity and have chemotactic effects on GM7373 cells. a, induction of uPA activity in GM7373 cells. GM7373 cells were incubated for 18 h with different concentration of vesicles. Endothelial cells were lysed and processed for uPA activity as described. uPA activity of GM7373 cells incubated without vesicles (lane 1), with 25 or 50 g/ml non-transfected NIH 3T3 vesicles (lanes 2 and 3), with 25, 50, or 100 g/ml transfected NIH 3T3 vesicles (lanes 4, 5, and 6) is shown. b, chemiotactic effects of vesicles shed by FGF-2-producing and non-producing cells. GM7373 endothelial cells were assayed for cell migration in the presence of indicated concentrations of vesicles shed by non-transfected and FGF-2-transfected NIH 3T3. Vesicles were recovered from media of cells grown in serum-free (FCS) or in complete medium (FCS). c, inhibition of vesicle-mediated chemotaxis by anti-FGF-2 antibody. GM7373 cells were incubated with vesicles shed by FGF-2-transfected NIH 3T3 cells (20 g of vesicle protein/ml) with the indicated concentrations of neutralizing anti-FGF-2 antibody and processed as above. b and c, mean and experimental variability are shown. rylation appeared to be delayed compared with activation trig- the vesicle preparation, vesicles were bound to biotinylated an- gered by the recombinant growth factor, probably because of nexin V and recovered by affinity chromatography on streptavi- the slow release of FGF-2 from disrupted vesicles. din-bound magnetic beads. As shown in Fig. 6b, vesicles purified Another typical response elicited by FGF-2 is uPA produc- by this technique had a uPA-inducing activity similar to that of tion (51, 55). The uPA stimulatory activity of vesicle-associated vesicles isolated by ultracentrifugation. FGF-2 was measured by casein/plasminogen zymography of To compare the stimulatory effects of vesicles and of vesicle- endothelial cell extracts. As shown in Fig. 6a, vesicles induced free supernatants, vesicles, recovered from a fixed volume of a dose-dependent increase in cell-associated uPA activity. Both SK-Hep1 conditioned medium, were assessed for their capacity control and vesicle-treated GM7373 cells showed a caseinolytic to induce uPA up-regulation in GM7373 cells. In parallel, the band with an apparent molecular mass of 46 kDa, correspond- same volume of vesicle-free supernatant was tested under the ing to bovine uPA and distinct from human uPA (55 kDa). This same experimental conditions. As shown by Fig. 6c when this observation ruled out the possibility that the increase in uPA comparison was made using media collected after1hof cell activity was mediated by vesicle-associated human uPA re- growth, only vesicles had a clearly detectable stimulatory effect maining bound to the endothelial cell surface. on GM7373 cells, whereas vesicle-free supernatant was inef- To confirm that the uPA-inducing activity was associated with fective. In media collected after3hof cell growth (Fig. 6d) both shed vesicles and was not mediated by co-sedimented material in vesicles and vesicle-free supernatants had some stimulatory 51918 Shed Vesicles Mediate FGF-2 Release FIG.9. Effects of HSPG removal on the uPA stimulatory effect of vesicles on GM7373 cells. a, effects of heparinase III on uPA stimulatory effect of vesicles shed by transfected 3T3 cells. Control, uPA activity of untreated GM7373 cells; Untreated vesicles, uPA activity of cells treated with 20 g/ml of vesicles. Vesicles digested with 4 milliunits/ml of heparinase III, uPA activity of cells treated with 20 g/ml of vesicles digested with heparinase for2hat37 °C. b, effects of 2 M NaCl incubation on uPA stimulatory effect of vesicles shed by Sk-Hep1 cells. Control, uPA activity of untreated GM7373 cells; Untreated vesicles, uPA activity of cells treated with 30 g/ml of vesicles. Vesicles incubated with 2 M NaCl, uPA activity of cells treated with 30 g/ml vesicles incubated for 30 min at 37 °C. Vesicles sonicated and incubated with 2 M NaCl, uPA activity of cells treated with 30 g/ml vesicles purified from sonicated media and incubated as described. activity, nevertheless vesicles still exerted a stimulatory effect FGF-2 Is Localized Inside Shed Vesicles—To assess whether stronger than the corresponding vesicle-free supernatants. To FGF-2 is present inside the vesicles or associated with HSPGs confirm that the uPA-inducing activity of SK-Hep1 cell-derived on their surface, purified vesicles were digested with hepari- vesicles was indeed mediated by vesicle-associated FGF-2, ex- nase III or washed with a high ionic strength solution (2.0 M periments were also performed in the presence of neutralizing NaCl). Both treatments are known to release FGF-2 from anti-FGF-2 antibodies. As shown by Fig. 6d the stimulatory HSPGs (57). The vesicles were then tested for uPA-inducing effects of both vesicles and vesicle-free supernatants were fully activity on GM7373 cells. As shown in Fig. 9, a and b, neither neutralized by anti-FGF-2 monoclonal antibodies (05117; Up- the heparinase III treatment nor the NaCl wash abolished the state Biotechnology). Unrelated monoclonal antibodies belong- ability of vesicles to up-regulate uPA expression in endothelial ing to the same isotype (Sigma monoclonal antibody T9026, cells. When, however, incubation with NaCl was performed against tubulin), tested at the same concentration, did not after sonication, the vesicle stimulatory effect was completely modify the stimulatory effects of vesicles and vesicle-free con- lost (Fig. 9b). These results are consistent with an intravesicu- ditioned media (data not shown). These data indicated that lar localization of the growth factor. vesicle-associated FGF-2 retains the capacity to up-regulate DISCUSSION uPA expression in endothelial cells and that most, if not all of the uPA-inducing activity present in the conditioned medium of FGF-2 is found associated with the extracellular matrix in SK-Hep1 cells is mediated by FGF-2 initially associated with vitro and in vivo and interacts with cell membrane receptors. vesicles. However, it lacks a hydrophobic signal sequence for secretion FGF-2-transfected NIH 3T3 Cells Release Biologically Active through the ER/Golgi system, and the mechanism(s) of its FGF-2 by Shedding Membrane Vesicles—FGF-2-transfected release are not understood. Because reagents or treatments NIH 3T3 cells have been shown to release limited but signifi- that block protein secretion via the ER-Golgi complex do not cant amounts of this growth factor (15). Therefore, we tested affect FGF-2 release (15) (19), three routes have been proposed whether vesicle shedding was also responsible for FGF-2 re- for FGF-2 externalization: cell death (12), sublethal cell injury lease in this cell type. As shown in Fig. 7A, vesicle shedding (13), or exocytosis via ER/Golgi-independent pathway(s) (15, was stimulated by serum in both parental and FGF-2-overex- 19, 46). pressing NIH 3T3 cells. These cells, however, released vesicles In this study we tested the hypothesis that FGF-2 release also in serum-free medium. Immunolocalization experiments from cells occurs by vesicle shedding. Membrane vesicles bud and Western blotting analysis showed the presence of low and from the plasma membrane of viable cells and can be purified high molecular weight FGF-2 isoforms in annexin V-binding from cell-conditioned medium (for a review see Ref. 58). Both vesicles shed by FGF-2-trasfected cells but not by parental cells FGF-2 secretion and vesicle shedding are energy-dependent (Fig. 7, B and C). phenomena (15, 29), and they are not inhibited by reagents In keeping with the data obtained with SK-Hep1 cells, ves- that block protein secretion via the ER-Golgi complex. There- icles purified from the conditioned medium of FGF-2 transfec- fore, the two processes appear to be modulated by the same tants showed the capacity to up-regulate uPA expression in mechanisms. endothelial GM7373 cells (Fig. 8a). In addition, vesicles shed by The data reported show that serum addition to SK-Hep1 FGF-2-producing NIH 3T3 cells induced a chemotactic re- cells that constitutively express high levels of FGF-2 rapidly sponse in endothelial cells, and their effect was abolished by results in vesicle shedding and that the intracellular localiza- neutralizing anti-FGF-2 antibody (Fig. 8, b and c). The speci- tion of FGF-2 is also strongly affected by these culture condi- ficity of these effects was further demonstrated by the lack of tions. Shortly after serum addition to SK-Hep1 cells, FGF-2 activity of vesicles released by vector-transfected NIH 3T3 appears in granules under the cell membrane and colocalizes cells. with patches of the cell membranes that show increased con- Shed Vesicles Mediate FGF-2 Release 51919 10. Johnson, D. E., and Williams, L. T. (1993) Adv. Cancer Res. 60, 1– 41 centration of 1 integrins and annexin V-binding activity. Ves- 11. Rusnati, M., and Presta, M. (1996) Int. J. Clin. Lab. Res. 26, 15–23 icle membranes were shown to be enriched in 1 integrins (31) 12. Schweigerer, L., Neufeld, G., Friedman, J., Abraham, J. A., Fiddes, J. C., and Gospodarowicz, D. (1987) Nature 325, 257–259 and to have annexin V-binding capacity (26, 38). These obser- 13. McNeil, P. L., Muthukrishnan, L., Warder, E., and D’Amore, P. A. (1989) vations indicate that FGF-2 clusters within areas of the cell J. Cell Biol. 109, 811– 822 membrane where vesicle budding occurs, and FGF-2 is subse- 14. Kandel, J., Bossy-Wetzel, E., Radvanyi, F., Klagsbrun, M., Folkman, J., and Hanahan, D. (1991) Cell 66, 1095–1104 quently externalized with shed vesicles. Consistent with this 15. Mignatti, P., Morimoto, T., and Rifkin, D. B. (1992) J. Cell Physiol. 151, 81–93 hypothesis, we found that the 18-, 22-, and 24-kDa FGF-2 16. Florkiewicz, R. Z., Majack, R. A., Buechler, R. D., and Florkiewicz, E. (1995) forms are associated with shed membrane vesicles. J. Cell Physiol. 162, 388 –399 17. Qu, Z., Kayton, R. J., Ahmadi, P., Liebler, J. M., Powers, M. R., Planck, S. R., The same FGF-2 isoforms are also present in vesicles shed by and Rosenbaum, J. T. (1998) J. Histochem. Cytochem. 46, 1119 –1128 NIH 3T3 cells transfected with FGF-2 cDNA. Based on mor- 18. Jorgensen, J. R., and Pedersen, P. A. (2001) Biochemistry 40, 7301–7308 19. Florkiewicz, R. Z., Anchin, J., and Baird, A. (1998) J. Biol. Chem. 273, 544 –551 phology, size, presence of gelatinolytic enzymes, and annexin 20. Dahl, J. 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