- Email: [email protected]
Signaling via Fibroblast Growth Factor Receptor-1 Is Dependent on Extracellular Matrix in Capillary Endothelial Cell Differentiation
Experimental Cell Research 248, 203–213 (1999) Article ID excr.1999.4400, available online at http://www.idealibrary.com on
Signaling via Fibroblast Growth Factor Receptor-1 Is Dependent on Extracellular Matrix in Capillary Endothelial Cell Differentiation Shigeru Kanda,* ,1 Bianca Tomasini-Johansson,* ,2 Peter Klint,* Johan Dixelius,* Kristofer Rubin,* and Lena Claesson-Welsh* ,3 *Department of Medical Biochemistry and Microbiology, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden
Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2K b-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-b 1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell–matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell–matrix interactions, possibly involving the a 3b 1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells. © 1999 Academic Press
INTRODUCTION
Angiogenesis plays a key role in physiological processes such as development, ovulation, and wound healing but also in the progression of diseases, including arthritis, retinopathy, and tumor growth and metastasis (for a review, see Ref. [1]). Angiogenesis can be 1 Present address: Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852, Japan. 2 Present address: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, Box 575, 751 23 Uppsala, Sweden. 3 To whom correspondence and reprint requests should be addressed. Fax: 146-18-471 4975. E-mail: [email protected]
divided into a series of sequential events, including focal digestion of the basement membrane, migration, proliferation, and tube formation (for reviews, see [2– 4]). Establishment of a functional vessel also requires production and remodeling of basement membrane, polarization of the endothelial cells, and recruitment of pericytes. Growth factors regulate angiogenesis by activation of cell surface receptor tyrosine kinases, which through binding and activation of Src homology-2 (SH2) domain-containing proteins convey a signal to the interior of the cells, resulting in cellular responses such as proliferation and differentiation (for a review, see [5]). To study signal transduction by receptor tyrosine kinases in endothelial cells, we established a capillary endothelial cell line [6] from brains of the H-2K b-tsA58 transgenic mouse (“immortomouse”) [7]. The endothelial cells were able to produce plasminogen activator activity, migrate, proliferate, and differentiate, i.e., form tubes in collagen gels, in response to fibroblast growth factor-2 (FGF-2) stimulation. In agreement with the general scheme for activation of receptor tyrosine kinases, FGFR-1 is known to dimerize in conjunction with ligand binding, through a process facilitated by heparan sulfate proteoglycans, leading to activation of the tyrosine kinase and autophosphorylation (for a review, see [8]). Autophosphorylated tyrosine residues may be critical for regulation of tyrosine kinase activity or may serve as binding sites for SH2 proteins. This far, seven autophosphorylation sites have been identified in FGFR-1 [9]. Two phosphorylation sites, Y653 and Y654, are located in the second tyrosine kinase domain; these sites may play a role in regulation of the kinase activity. Phosphorylated Y766 is known to serve as a binding site for phospholipase C-g (PLC-g) [10]. The functions of the other sites in FGFR-1 signal transduction have not yet been elucidated. Recent reports show that extracellular matrix (ECM)– integrin interactions play significant roles in the angiogenic process in vitro and in vivo (for a review, see [11]). Laminin, one of the main components of the basement membrane, has a particular role in differen-
203
0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
204
KANDA ET AL.
tiation [12]. Fibronectin stimulates the survival of endothelial cells [13], and the a vb 3-integrin has been reported to play an important role for invasion, migration, and survival of endothelial cells [14]. Integrins constitute a family of a and b heterodimeric transmembrane glycoproteins which mediate intracellular and cell to ECM adhesion [15]. Different combinations of a and b chains give rise to integrins that have specificity for different ECM proteins. In vitro, upon ECM binding to their extracellular domain, integrins associate in focal adhesions. At these sites integrins provide a linkage between the ECM and the cytoskeleton, to which integrin cytoplasmic tails connect. It has been shown that binding of integrins to their ECM ligands, with subsequent focal adhesion formation, leads to signal transduction, in part converging with those initiated by growth factors [16, 17]. Signaling molecules, including growth factor receptors and SH2 domain proteins, are recruited into the focal adhesion [18, 19]. Focal adhesion kinase (FAK), Grb2, members of the small GTPase protein family Rho, insulin receptor substrate-1, and the Src family kinases are known to be involved in integrin-coupled signal transduction [20]. Consequently, there are strong indications for crosstalk between signal transduction pathways initiated by growth factor receptors and integrins [17]. Thus, the platelet-derived growth factor (PDGF) b-receptor is tyrosine phosphorylated following attachment of fibronectin to its integrin receptor [21]. In this report, we characterize the FGF-2-induced tube formation of H-2K b-tsA58 brain endothelial cells and show that the b 1-integrin is involved in differentiation of the endothelial cells. Cells grown on fibronectin spread and proliferated in response to FGF-2 treatment, but failed to differentiate. On the other hand, cells cultured between two layers of collagen gel did not spread and failed to proliferate in response to FGF-2 stimulation. Instead, the rounded cells aggregated and subsequently formed tubes. Our results indicate that receptor tyrosine kinase signal transduction is modulated by signals initiated by integrin–ECM interactions. MATERIALS AND METHODS Cell culture. A capillary endothelial cell line isolated from H-2K btsA58 transgenic mouse brain (denoted immortomouse brain endothelial cells; IBEC) was cultured on fibronectin-coated dishes (coated at 37°C for 18 h using 20 mg/ml human plasma fibronectin in Ham’s F12 medium) at 33°C in Ham’s F12 medium (Life Technologies, Inc.) supplemented with 20% heat-inactivated fetal bovine serum (Life Technologies), 75 mg/ml endothelial cell growth supplement (Sigma), 20 U/ml recombinant mouse interferon-g (IFN-g; Genzyme), 10 ng/ml human recombinant epidermal growth factor, a kind gift from Amgen, Inc., Thousand Oaks, CA), and 5 mg/ml bovine pancreas insulin (Sigma) (denoted growth medium). IBE cells expressing a chimeric molecule composed of the extracellular domain of the platelet-derived growth factor receptor-a (PDGFR-a) fused to the intracellular domain of FGFR-1 has been described before [6]. Prior to
assays, cells were kept in Ham’s F12 containing 0.25% bovine serum albumin (BSA; fatty acid-free; Sigma) (assay medium), lacking IFN-g and the growth stimulatory supplements, except when otherwise indicated. Tube formation assay in collagen gel. The tube formation assay in three-dimensional collagen gels was performed as described previously [6]. Briefly, cells were trypsinized, centrifuged, and resuspended in F12 medium containing 0.25% BSA. The mixture of type I collagen (Celltrix Pharmaceuticals), 10 3 F12 medium, and concentrated buffer (260 mM NaHCO 3, 200 mM Hepes, and 50 mM NaOH) (8:1:1) was put into the wells of 12-well plates and allowed to gel at 37°C. In indicated experiments, 100 mg/ml (final concentration) of human plasma fibronectin (Sigma) or laminin from Engelbreth– Holm–Swarm mouse sarcoma (i.e., laminin-1; Sigma) was mixed in the collagen gels. The cells were inoculated onto the collagen gels with indicated supplements at a density of 1.8 3 10 5 cells/cm 2 and cultured for 3 h at 33°C. When the effect of cell density on tube formation was examined, densities of 0.9 and 0.45 3 10 5 cells/cm 2 were also used. After attachment of the cells, the medium was removed and a second collagen layer was added. After gelation at 37°C, F12 medium containing 0.25% BSA with or without indicated supplements was added and the cultures were incubated at 33°C. The cells between collagen gel layers were observed using a phasecontrast microscope. Detection of S-phase cells. Cells were inoculated onto fibronectincoated or collagen-gel-containing wells at a density of 1.8 3 10 5 cells/cm 2 in F12 medium containing 0.25% BSA with or without ligand and cultured at 33°C for 4 h. Bromodeoxyuridine (BrdU) at 10 mg/ml was added and the cells were cultured for an additional 4 h. Cells were fixed in 70% ethanol at 4°C for 20 min. After the cells were washed, 0.02 M NaOH was added and incubated for 1 min, followed by five washes with PBS. Anti-BrdU monoclonal antibody (Becton– Dickinson) was added to the cells and incubated for 60 min at room temperature. The cells were washed three times and then incubated with peroxidase-conjugated anti-mouse IgG for 60 min. Nuclei labeled with BrdU were visualized by incubation with diaminobenzidine solution, and counterstaining of whole nuclei was performed with a light hematoxylin staining. Labeling index was expressed as a percentage of labeled nuclei/total nuclei. TUNEL. Cells were inoculated for 5 h at 33°C on type I collagen gels in Ham’s F12 medium containing 20 U/ml aprotinin and 0.25% BSA with or without growth factors. The cells were harvested, fixed, and prepared according to the instructions of the “In Situ Cell Death Detection Kit, Fluorescein” (Boehringer Mannheim), which is a terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) kit containing fluorescein-conjugated dUTP. Briefly, cells were fixed in 4% paraformaldehyde, washed twice in PBS, permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate, washed twice in PBS, and incubated 1 h at 37°C in the presence of the kit reagents. The cells were washed once in PBS and analyzed using a flow cytometer (FACScan), using a 488-nm laser for excitation. Data for light scattering and green fluorescence were collected. Propidium staining. Cells undergoing tube formation in threedimensional collagen gels were fixed in methanol at 220°C for 10 min, 20 h after seeding on collagen. After three washes with PBS, the upper collagen layer was removed. Subsequently, RNase A (Sigma; 1 mg/ml) and propidium iodide (Molecular Probes; 20 mg/ml) were added to the collagen gel and incubated 2 h at 4°C. Cells were analyzed using a fluorescence microscope and photographed. Metabolic labeling and immunoprecipitation of integrins. IBE cells (2 3 10 6 cells/well in 2 ml) were plated in methionine- and cysteine-free MCDB medium supplemented with 20 U/ml aprotinin, on collagen gels or on fibronectin-coated 3-cm dishes, in the presence of 50 mCi/ml [ 35S]methionine/cysteine mix (Amersham Pharmacia Biotech), with or without 10 ng/ml FGF-2. Cells were labeled for 8 –10 h and extracted for 10 min on ice with 1 ml 1% Triton X-100, 20 mM Tris–HCl, pH 7.4, 0.65 mM MgSO 4, 1.2 mM CaCl 2 in the presence of 5 mg/ml leupeptin, 5 mg/ml pepstatin, and 4 mM PefaBlock.
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
205
FIG. 1. (A) Kinetics of tube formation of murine brain capillary endothelial (IBE) cells in three-dimensional type I collagen gels. Cells were inoculated onto a layer of collagen gel in the absence (a, c, e, g) or presence (b, d, f, h) of 5 ng/ml FGF-2. After 3 h, medium was removed and a second layer of collagen was added. Medium without (a, c, e, g) or with (b, d, f, h) FGF-2 was added after gelation of the second layer of collagen. Pictures were taken at 0 (a, b), 2 (c, d), 8 (e, f), and 18 h (g, h) after addition of the top layer of collagen. Bar, 50 mm. (B) Cell-density-dependent formation of tubes by IBE cells in collagen gels. Tube formation was assayed in the absence (a, c, e) or presence (b, d, f) of 5 ng/ml FGF-2. Cells were inoculated onto the first layer of collagen gels at densities of 0.2 3 10 5 (a, b), 0.9 3 10 5 (c, d), and 1.8 3 10 5 cells/cm 2 (e, f). Bar, 50 mm. (C) Morphology of IBE cells grown on fibronectin. Cells were inoculated and cultured on fibronectin-coated dishes, in the absence (a) or presence (b) of 5 ng/ml FGF-2. Bar, 25 mm. (D) Kinetics of tube formation of IBE cells ectopically expressing the chimeric receptor PDGFR-a/FGFR-1, in three-dimensional collagen gels. Cells were plated in the absence (a, c, e, g) or presence (b, d, f, h) of 5 ng/ml PDGF-AA and incubated in the continued absence or presence of PDGF-AA as described for A, for 0 (a, b), 2 (c, d), 8 (e, f), and 18 h (g, h) after addition of the top layer of collagen. Bar, 50 mm.
206
KANDA ET AL.
FIG. 2. (A) Fluorescence-activated cell sorting of TUNEL-stained IBE cells. Cells on collagen gels in the presence (1) or absence (2) of FGF-2 (top) were harvested 7 h after seeding on collagen, fixed, labeled with fluorescein-conjugated dUTP (TUNEL), and analyzed by flow cytometry. Alternatively, collagen-gel-seeded IBE cells extopically expressing the chimeric receptor (PDGFR-a/FGFR-1) were treated in the absence or presence of 5 ng/ml PDGF-AA, harvested at 7 h, and analyzed for TUNEL-positive cells. The forward scatter represents cell size and the fluorescence intensity represents the degree of labeling. The histograms are divided in two sections, representing apoptotic (Ap) and nonapoptotic (NA) cells, based on the degree of DNA stand breaks. (B) Propidium iodide staining of IBE cells cultured in three-dimensional collagen gels. IBE cells cultured in a three-dimensional collagen gel for 20 h were fixed, and the upper collagen layer was removed. Cells were stained with propidium iodide to visualize cells undergoing morphological changes typical for apoptosis. (Top) Cells cultured in the presence of FGF-2; (bottom) cells cultured in the absence of FGF-2. Large arrow indicates a cell with fragmented nucleus; small arrow indicates a cell with condensed nucleus.
The lysates were spun and precleared by addition of 30 ml normal rabbit serum and 30 ml protein A– or protein G–Sepharose for 1 h at 10°C. The precleared cell lysates were aliquoted and incubated overnight at 10°C with various antibodies. Protein A– or protein G–Sepharose beads (30 ml of a 1:1 slurry in PBS) were added for 1 h at 10°C. The Sepharose pellets were washed four times with extraction buffer containing 0.5 M NaCl and once with 0.1% Triton buffer, 0.5 M NaCl before addition of SDS–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer. Samples were heated to 95°C for 10 min and run on 6 or 7.5% SDS–PAGE and analyzed by fluorography. Anti-integrin antibodies were as follows: Rabbit polyclonal b 1 IgG was raised and isolated as previously described [22, 23] with the modification that purified rat hepatocytes were used as starting material in the purification of the b 1 subunit. Rabbit anti-human a 3 antiserum, kindly provided by Dr. Mats Johansson (University of Uppsala, Sweden), was raised against a synthetic peptide (KRARTRALYEAKRQKAEMSQPSETERLTDDY) corresponding to a major portion of the human a3 cytoplasmic domain. This antiserum immu
noprecipitates a 3-integrin from mouse 3T3/Ha cells (Dr. Mats Johansson, personal communication). Rabbit anti-mouse a 2 antiserum was raised against a peptide (CNDEMDETTELNS) corresponding to the C-terminal part of the mouse a 2 cytoplasmic domain. This antiserum immunoprecipitates a 2-integrin from mouse and rat fibroblasts (unpublished observation) as well as surface-labeled a 2b 1 from mouse fibroblasts (data not shown). Rabbit anti-human a 5 antiserum (cytoplasmic domain) was from Chemicon. Supernatants from the antia 6-integrin antibody producing hybridoma GoH6 was kindly provided by Dr. Arnoud Sonnenberg (University of Amsterdam, The Netherlands) [24]. Cell attachment assay. The attachment assay was performed as previously described [25]. Briefly, non-tissue-culture-treated 96-well plates (Dynatech Laboratories) were coated with 30 mg/ml human fibronectin [26], EHS laminin [27], bovine skin collagen type 1 (CELLON), or BSA (Boehringer Mannheim) in PBS, during an overnight incubation at 10°C. Following one wash with PBS, 2% BSA was
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
207
ping of the membrane by soaking in 62.5 mM Tris–HCl, pH 6.8, containing 2% SDS and 0.7% 2-mercaptoethanol at 50°C for 15 min was performed.
RESULTS
Morphological Changes of Capillary Endothelial Cells in Response to FGF-2 Are Dependent on the Extracellular Matrix and Dimensions
FIG. 2—Continued
added to the wells and incubated for 1 h at room temperature. Cells were detached and resuspended to 30,000 cells/ml in serum-free Ham’s F12 medium containing 0.2% BSA, and 100 ml per well was added to the PBS-washed plate. Following 45 min at 37°C, the unattached cells were washed three times with Ham’s F12, and attached cells were fixed in 4% paraformaldehyde in PBS, stained with 0.5% toluidine blue in 4% paraformaldehyde, and solubilized in 2% SDS. The absorbance at 562 nm was measured in a vertical pathway spectrophotometer. Treatment of cells with anti-b1-integrin antibody. Cells were harvested by trypsinization, neutralized by soybean trypsin inhibitor, and suspended in Ham’s F12 containing 0.25% BSA with nonimmune rabbit IgG or anti-b 1 polyclonal antibody. Cells were inoculated at a density of 2.1 3 10 4 cells/cm 2 on collagen-gel-containing 12-well dishes. The collagen-gel cultures were covered with a second layer of collagen and after gelation of the second layer, medium containing nonimmune rabbit IgG or anti-b 1-integrin polyclonal antibody was added, and the culture was continued overnight and subsequently photographed. Immunoblot analysis of FAK. IBE cells were harvested by trypsin, neutralized by soybean trypsin inhibitor, and inoculated on fibronectin- or collagen-coated dishes (6-cm diameter) in Ham’s F12 containing 20 U/ml aprotinin and 0.25% BSA, then cultured for indicated periods in the presence or absence of FGF-2. Cells were washed once with Tris-buffered saline, pH 7.5, containing 100 mM orthovanadate and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM Na fluoride, 2 mM EDTA, 30 mM sodium pyrophosphate, 100 mM orthovanadate, 2 mM PMSF, and 100 U/ml aprotinin) on ice. After centrifugation to remove insoluble material, the cell lysate was passed through a 27-gauge needle twice and immunoprecipitated with monoclonal antibody against FAK (2A7; kindly provided by Dr. T. Parsons, Department of Microbiology, Health Science Center, University of Virginia, Charlottesville 22908) on ice overnight, followed by adsorption to protein A–Sepharose beads. Beads were washed three times with RIPA buffer, and samples heated in SDS sample buffer were separated on 7.5% polyacrylamide gels. After electrophoretic transfer onto a nitrocellulose membrane (Hybond-C Extra; Amersham), the membrane was blocked with 3% BSA in PBS containing 0.05% Tween 20 overnight. Then, the blot was probed with monoclonal antibody against phosphotyrosine, PY-20 (Transduction Laboratory) or the BC3 polyclonal antibody against FAK, kindly provided by Dr. T. Parsons. Reactive sites were detected through enhanced chemiluminescence. Between two probings, strip-
We isolated an endothelial cell line from brain tissue of a transgenic mouse strain denoted immortomouse [6]. All tissues in this mouse express a temperaturesensitive version of simian virus 40 T under the control of the histocompatibility-2 (H-2) promoter, which allows temperature- and interferon-g-regulated expression of the oncogene [7]. The endothelial cells, which we denote IBEC, respond to FGF-2 with induction of lumen-containing vessel-like structures, when cultured in three-dimensional collagen gels, as demonstrated through confocal and electron microscopy [6, 28]. This differentiation process is accompanied by formation of tight junctions and apical pinocytosis [28]. In order to define the signal transduction pathways involved in differentiation, the kinetics of tube formation were analyzed (Fig. 1). Cells were seeded in the middle of a three-dimensional type I collagen gel and cultured at 33°C in the presence and absence of FGF-2 in serum-free medium. Figure 1A, a and b, shows that independent of ligand stimulation, cells failed to spread in the collagen gel. FGF-2-stimulated cells started to aggregate within 2 h (Fig. 1A, d). In cultures stimulated for 8 h (f), cell aggregates started to sprout and fuse to form tubes. After 18 h (h), the majority of the cells in the culture were engaged in aggregates or tubes. Cell aggregation and tube formation were celldensity dependent, since cells seeded at low density formed neither aggregates nor tubes (Fig. 1B).
FIG. 3. Labeling index of endothelial cells cultured on fibronectin or collagen. Parental IBE cells or IBE cells ectopically expressing the chimeric receptor (PDGFR-a/FGFR-1) were plated on fibronectin (FN)-coated or type I collagen (CO) gel-containing wells in the presence (1) or absence (2) of FGF-2 or PDGF-AA, as indicated. After 4 h, BrdU was added and the cultures were continued for an additional 4 h. The cultures were washed and fixed and nuclei that had incorporated BrdU were visualized by immunoperoxidase staining. Labeling index was expressed as percentage of labeled nuclei/total nuclei. Data are means 6SD for triplicate wells.
208
KANDA ET AL.
FIG. 4. Immunoprecipitation analysis of integrin expression in IBE cells. IBE cells cultured on collagen gels (C) or on fibronectin-coated dishes (F) were metabolically labeled with 35S for 8 h in the presence or absence of 5 ng/ml FGF-2. Cell lysates were immunoprecipitated overnight with nonimmune serum (NRS), with polyclonal rabbit antiserum to rat b1, or with antisera raised against peptides comprising the cytoplasmic tails of integrins a3 and a5. Samples were analyzed by SDS–PAGE (6% acrylamide gel) and fluorography.
We compared cultures in collagen gels with cells cultured on fibronectin; under the latter condition, cells were able to spread and form a monolayer (Fig. 1C). Cells growing on fibronectin never formed aggregates or tubes when stimulated with FGF-2. The IBE cells express mainly FGF receptor-1, but also small levels of FGF receptor-2 and -4 [6]. We created an engineered version of IBEC expressing a chimeric receptor construct composed of the PDGF a-receptor extracellular domain fused to the FGF receptor-1 intracellular domain. The chimeric receptorexpressing IBE cells formed aggregates and tubes in response to PDGF-AA, with the same kinetics and morphology as the FGF-2 response seen in untransfected cells (Fig. 1D). Since the cells lack an endogenous PDGF a-receptor [6], these results indicate that the FGF receptor-1 intracellular domain transduces signals for differentiation of endothelial cells.
fragmentation. In the absence of FGF-2, the fraction of apoptotic cells increased to over 90% (Fig. 2A, top). A similar pattern was observed when collagen cultures of IBE cells expressing the chimeric PDGFR-a/FGFR-1 receptor, treated with PDGF-AA, were analyzed (Fig. 2A, bottom). These data indicate that cellular survival in the presence of growth factor was dependent on the FGFR-1 intracellular domain. We further treated IBEC cultured in three-dimensional collagen gels with propidium iodide, which intercalates DNA, in order to visualize morphological changes typical for apoptotic cells (Fig. 2B). After 20 h culture, cells deprived of FGF-2 contained condensed, sometimes fragmented nuclei (arrows in the lower image). These results indicate that the cells in the collagen gel were undergoing apoptosis and that FGF receptor-1 is able to transduce signals for inhibition of apoptosis of capillary endothelial cells.
Cells on Collagen Undergo Apoptosis in the Absence of FGF-2
Signals for Growth of Endothelial Cells Are Modulated by Extracellular Matrix
Cells grown in collagen gels in the absence of FGF-2 started to die within 8 h of culture (Fig. 1A). We performed flow cytometric analysis of DNA fragmentation in IBE cells cultured on collagen gels for 5 h, detected by staining of fragmented DNA using TUNEL in the presence of fluorescein-conjugated dUTP. In the presence of FGF-2, 0.1% of the cells showed signs of DNA
To analyze the effect of extracellular matrix on FGF2-stimulated biological responses of the endothelial cells, we measured labeling index of the cells grown on fibronectin or collagen. Figure 3 shows that the labeling index was increased by FGF-2 stimulation when cells were cultured on fibronectin-coated wells, but not when cells were cultured on collagen gel-containing
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
209
wells. A similar pattern was observed when cells expressing the chimeric PDGFR-a/FGFR-1, stimulated with PDGF-AA, were analyzed (Fig. 3). These data suggest that fibronectin mediates connection of FGF receptor-1 signal transduction to pathways for growth rather than differentiation. In cells on collagen on the other hand, FGFR-1 activity can be correlated with differentiation but not with proliferation. Analysis of the Expression of Integrins in the Brain Capillary Endothelial Cells In order to investigate the capacities of the IBE cells to interact with different extracellular matrixes, we examined the pattern of integrin expression in IBE cells grown under different conditions. Analysis by [ 35S]methionine labeling of the cells and immunoprecipitation with polyclonal anti-rat b 1 -integrin IgG showed major labeled polypeptides with M r ’s of 122,000 –133,000 and 140,000 (Fig. 4). The protein bands were broad and the components most likely represent a-integrin subunits and their precursors. It should be noted that no polypeptides in the M r 180,000 –200,000 region could be detected, indicating low or no synthesis of a 1b 1 and a 2b 1. To further investigate which a subunits were expressed on the IBE cells, immunoprecipitations with specific anti-a subunit antibodies were performed. Anti-a 3 and -a 5 subunit antibodies precipitated polypeptides with M r ’s 135,000 and 145,000, demonstrating active synthesis of a 3b 1 and a 5b 1 in IBE cells. A polyclonal anti-mouse a 2 antiserum did not immunoprecipitate any labeled polypeptides in the M r region
FIG. 6. Effect of the anti-b 1 integrin antibody on tube formation of IBE cells in three-dimensional collagen gels. Cells were cultured in three-dimensional collagen gels in the presence of nonimmune IgG (a and b) or anti-b 1-integrin antibody (c and d) in the absence (a, c) or presence (b, d) of 5 ng/ml FGF-2 for 18 h.
of 150,000 (data not shown). This is in agreement with the fact that no peptides in the M r region of 150,000 could be detected after immunoprecipitation with antib 1-integrin IgG (Fig. 4). The anti-a 6-integrin antibody GoH6 similarly failed to immunoprecipitate labeled polypeptides having M r ’s expected for a 6 (data not shown). These results suggest that the IBE cells have low or no synthesis of the a 2b 1- and a 6b 1-integrins. As judged from several experiments, no consistent difference in integrin synthesis was detected after stimulation of the cells with FGF-2, irrespective of whether cells were plated on fibronectin or collagen. Effect of Anti-b 1-Integrin Antibody on Differentiation of Endothelial Cells
FIG. 5. Attachment of IBE cells to different extracellular matrixes. IBE cells were seeded on BSA-treated plastic coated with fibronectin, laminin-1 (EHS laminin), collagen I, or BSA. Unattached cells were washed off after 45 min incubation at 37°C and attached cells were fixed, stained with toluidine blue, and solubilized in SDS and the absorbance at 562 nm was measured spectrophotometrically.
Integrin receptors for collagen (a 1b 1 and a 2b 1) were not detected on the IBE cells, which is in agreement with the fact that the cells failed to attach to collagen (Fig. 5). On the other hand, fibronectin (a 5b 1) and laminin (a 3b 1) receptors were present and cells attached well to these matrixes (Fig. 5). To test the effect of inhibition of these receptors, we employed a neutralizing anti-b 1-integrin antibody. Figure 6 shows that the anti-b 1-integrin antibody efficiently inhibited tube formation of endothelial cells in three-dimensional collagen gels. Kinetic analysis showed that aggregation of cells still occurred but that the subsequent tube formation of the capillary endothelial cells was attenuated (data not shown). The antibody had, however, no effect on cell survival. Cell death in unstimulated cells and stimulation of cell survival by FGF-2 were still evident (Figs. 6c and 6d). This result indicates that tube formation but not survival of cells grown in collagen was dependent on signal transducion pathways involving b 1-integrins.
210
KANDA ET AL.
Laminin Promotes Tube Formation of Endothelial Cells The results in Figs. 4 and 5 showed that laminin receptors were expressed on the IBE cells. Inclusion of 100 mg/ml EHS laminin (i.e., laminin 1) in the threedimensional collagen gel led to spontaneous tube formation (Figs. 7c and 7d), independent of FGF-2. Although laminin induced tube formation, it failed to protect the cells from apoptosis (Fig. 7c), which is in agreement with the finding that the neutralizing b 1integrin antibody inhibited tube formation, but not cell survival, in the presence of FGF-2 (Fig. 6). The stimulation of tube formation by laminin in the absence of FGF-2 was not the result of the contaminating FGF, since the laminin we used had no stimulatory effect on the kinase activity of FGF receptor-1 (data not shown). In contrast, when cells were grown in fibronectin (100 mg/ml)-containing collagen gels in the presence of FGF-2, there was marked cell aggregation but no tube formation (Figs. 7e and 7f). These results indicate that the a 5b 1-integrin ligated to fibronectin is inhibitory for differentiation of endothelial cells, whereas the a 3b 1integrin ligated to laminin is stimulatory. Tyrosine Phosphorylation of Focal Adhesion Kinase Integrin-mediated signaling is known to involve tyrosine phosphorylation and activation of FAK. We examined whether culture of IBEC on collagen involved FAK tyrosine phosphorylation. Since the cells fail to attach to collagen (Fig. 5), the low level of FAK tyrosine phosphorylation in unstimulated cells after 1 h of culture was expected (Fig. 8, lane 1). Stimulation of FGF-2 during 1 h increased the level of tyrosine-phosphorylated FAK only marginally (Fig. 8, lane 2). Cultures incubated for 7 h in the absence of FGF-2 showed increased phosphorylation of FAK, which was further increased with FGF-2 treatment to fivefold over that of the untreated, 1-h culture. These data indicate that the IBE cells produce a matrix component, possibly laminin, in the basal condition and with increased efficiency in the presence of FGF-2 and agree with the notion that differentiation of endothelial cells involves integrin signaling. Whether FAK itself is critical in the differentiation process remains to be demonstrated. DISCUSSION
In this report, we show that tube formation of murine brain endothelial cells occurs in response to FGF-2 when the cells are cultured in three-dimensional collagen gels. This process is accompanied by cell survival, and removal of FGF-2 leads to apoptosis of the cells. The signal transduction pathways mediating tube formation and survival appear to be distinct, since tube formation involved b 1 -integrin–ECM interactions, whereas cell survival did not. Inclusion of laminin in
FIG. 7. Effects of laminin and fibronectin on tube formation of IBE cells. Cells were cultured in three-dimensional collagen gels, supplemented or not with laminin or fibronectin, in the absence (a, c, e) or presence (b, d, f) of 5 ng/ml FGF-2 for 18 h. (a and b) Collagen gels alone, (c and d) 100 mg/ml laminin-1-containing collagen gels, and (e and f) 100 mg/ml fibronectin-containing collagen gels.
the collagen gel stimulated spontaneous tube formation, but did not protect against apoptosis. Failure to attach has previously been shown to lead to programmed cell death, apoptosis (see [16, 29] for reviews), and to be coupled to cellular programs of different kinds. Cell survival, such as that induced by FGF-2 treatment of cells in collagen, has been reported for many cell types treated with serum or cytokines and may directly or indirectly involve integrin–ECM interactions. Thus, interactions between fibronectin and integrins (i.e., a 5b 1) are accompanied by cell survival through increased expression levels of Bcl-2, which is known to protect against apoptosis [30, 31]. We did not detect any changes in expression levels of Bcl-2 family members in IBE cells during tube formation (data not shown); potential posttranslational modifications were not examined. Neither culture on collagen nor FGF-2 treatment appeared to affect expression of integrins. Integrins have a slow turnover rate and fluctuations in expression levels may not be detectable under the conditions used. Since the control cells cultured in the absence of FGF-2 underwent apoptosis, it was, however, not possible to compare time points later than 8 –10 h after inoculation of the cells. An analysis of FGF-2-induced modulation of integrins on microvascular endothelial cells [32] showed maximal difference in integrin expression 72 h after exposure to the growth factor. On the other hand, available data indicate that adhesion of integrins to their ligands is not constitutive, but is
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
211
FIG. 8. Immunoblot analysis of tyrosine phosphorylation of FAK. IBE cells were plated on collagen-gel-containing dishes in the presence or absence of 5 ng/ml FGF-2 for indicated periods. Cell lysates were immunoprecipitated overnight at 4°C, using the anti-FAK 2A7 monoclonal antibody. Precipitated proteins were subjected to SDS–PAGE, followed by transfer onto nitrocellulose. The membrane was probed with anti-phosphotyrosine antibody (PY20) (left). After stripping, the same membrane was blotted with anti-FAK BC3 polyclonal antibody (right). Proteins were visualized using enhanced chemiluminescence. The relative levels of tyrosine-phosphorylated FAK in different lanes on the left were estimated by relating the optical densities of the tyrosine-phosphorylated bands to the FAK protein bands.
regulated by intracellular signal transduction pathways (see [33] for a review). Our data suggest that the IBE cells do not express the collagen- and lamininbinding integrins a 1b 1 and a 2b 1. This is based on the fact that IBE cells failed to attach to collagen and that synthesis of these integrins was not detected by immunoprecipitation. The IBE cells attached efficiently to fibronectin and laminin, however, and synthesis of the integrins a 3b 1 and a 5b 1, but not of a 6b 1, could be demonstrated. Together, these results suggest that the IBE cells use a 3b 1 as a laminin receptor. It is possible that different growth conditions allowed specific production of extracellular matrix, and preliminary data indicate that the IBEC produce laminin (data not shown). Deposition of laminin by the cells would allow interaction with the a 3b 1-integrin receptor expressed on the IBE cells. Previous reports show the importance of the b 1integrin and laminin in differentiation of different cell types, among them endothelial cells (see [12] for a review). Human umbilical vein endothelial cells have been shown to rapidly form vessel-like, lumen-containing structures when grown in laminin-enriched Matrigel, a solid gel of basement membrane proteins and growth factors [34, 35]. Grant et al. [35] showed that laminin had multiple effects in endothelial cell differentiation; an RGD-containing laminin-derived peptide induced cell attachment, whereas a YIGSR peptide induced cell– cell interactions and tube formation in human umbilical vein endothelial cells. The protein tyrosine kinase FAK is activated in response to clustering of certain integrins, such as a 3b 1 ([36]; see [16] for a review). We observed tyrosine phosphorylation of FAK, which is presumed to represent its activation,
several hours after inoculation of the IBE cells on collagen, potentially due to clustering of a 3b 1-integrin expressed on these cells. A role for the a 3b 1-integrin in endothelial cell differentiation was further supported by the data showing inhibition of tube formation by a neutralizing anti-b 1 antibody. Which are the molecular mechanisms behind the differentiation-promoting effects of laminin? Studies of the neuronal cell line PC12, which undergoes differentiation involving neurite outgrowth in culture, has implicated the Ras pathway in differentiation [37]. Signal transduction by at least certain integrins leads to activation of the Ras pathway, via binding of the Grb2/ mSos1 complex to FAK [16, 38]. FGFR-1 is also capable of activating the Ras pathway [39], which points to the possibility that an integration of integrin– growth factor receptor signal is critical to drive differentiation. In this context, it is interesting that FGFR-1 and its downstream signaling molecules, including PLC-g and Ras, are recruited into focal adhesion complexes in endothelial cells, together with FAK, after clustering of integrins [18]. This accumulation of growth factor receptor and signaling molecules implies a possibility for direct interactions and interplay between, e.g., FAK and the FGFR-1, with potentially unique consequences for downstream signaling. Future studies on forced expression of kinase-inactive FAK in the IBE cells should allow conclusions as to the role of FAK in endothelial cell differentiation. This study was supported by grants from The Swedish Cancer Foundation (Project 3820-B96-01XAB to L.C.W. and 2729 B97 08XBC to K.R.) and the King Gustaf V Fund.
212
KANDA ET AL.
REFERENCES 1.
Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nat. Med. 1, 27–31.
2.
Ausprunk, D. H., and Folkman, J. (1977). Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res. 14, 53– 65.
3.
D’Amore, P., and Thompson, R. W. (1987). Mechanisms of angiogenesis. Annu. Rev. Physiol. 49, 453– 464.
4.
Jain, R. K., Schlenger, K., Hockel, M., and Yuan, F. (1997). Quantitative angiogenesis assays: Progress and problems. Nat. Med. 3, 1203–1208.
5.
Pawson, T. (1995). Protein modules and signalling networks. Nature 373, 573–580.
6.
Kanda, S., Landgren, E., Ljungstro¨m, M., and ClaessonWelsh, L. (1996). Fibroblast growth factor receptor 1-induced differentiation of endothelial cell line established from tsA58 large T transgenic mice. Cell Growth Differ. 7, 383– 395.
7.
Noble, M., Groves, A. K., Ataliotis, P., and Jat, P. S. (1992). From chance to choice in the generation of neural cell lines. Brain Pathol. 2, 39 – 46.
8.
Schlessinger, J., Lax, I., and Lemmon, M. (1995). Regulation of growth factor activation by proteoglycans: What is the role of the low affinity receptors? Cell 83, 357–360.
9.
Mohammadi, M., Dikic, I., Sorokin, A., Burgess, W. H., Jaye, M., and Schlessinger, J. (1996). Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol. Cell. Biol. 16, 977– 989.
10.
Mohammadi, M., Honegger, A. M., Rotin, D., Fischer, R., Berliot, F., Li, W., Dionne, C. A., Jaye, M., Rubinstein, M., and Schlessinger, J. (1991). A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-g1. Mol. Cell. Biol. 11, 5068 –5078.
11.
Stro¨mblad, S., and Cheresh, D. A. (1996). Cell adhesion and angiogenesis. Trends Cell Biol. 6, 462– 468.
12.
Lin, C. Q., and Bissell, M. J. (1993). Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J. 7, 737– 743.
13.
Varner, J. A., Emerson, D. A., and Juliano, R. L. (1995). Integrin a 5 b 1 expression negatively regulates cell growth: Reversal by attachment to fibronectin. Mol. Biol. Cell 6, 725–740.
14.
Brooks, P. C., Montgomery, A. M. P., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., and Cheresh, D. A. (1994). Integrin a vb 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157–1164.
15.
Hynes, R. O. (1992). Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25.
16.
Clark, E. A., and Brugge, J. S. (1995). Integrins and signal transduction pathways: The road taken. Science 268, 233– 239.
17.
Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995). Integrins: Emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11, 549 –599.
18.
Plopper, G. E., McNamee, H. P., Dike, L. E., Bojanowski, K., and Ingber, D. E. (1995). Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol. Biol. Cell 6, 1349 –1365.
19.
Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995). Integrin function: Molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131, 791– 805.
20.
Parsons, J. T. (1996). Integrin-mediated signalling: Regulation by protein tyrosine kinases and small GTP-binding proteins. Curr. Opin. Cell Biol. 8, 146 –152.
21.
Sundberg, C., and Rubin, K. (1996). Stimulation of b 1 integrin on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF b-receptors. J. Cell Biol. 132, 741– 752.
22.
Gullberg, D., Terracio, L., Borg, T. K., and Rubin, K. (1989). Identification of integrin-like matrix receptors with affinity for interstitial collagens. J. Biol. Chem. 264, 12686 – 12694. ¨ brink, B. (1986). 23. Rubin, K., Gullberg, D., Borg, T. K., and O Hepatocyte adhesion to collagen. Isolation of membrane glycoproteins involved in adhesion to collagen. Exp. Cell Res. 164, 127–138. 24.
Kennel, S. J., Foote, L. J., Falcioni, R., Sonnenberg, A., Stringer, C. D., Crouse, C., and Hemler, M. E. (1989). Analysis of the tumor-associated antigen TSP-180. Identity with a6b4 in the integrin superfamily. J. Biol. Chem. 264, 15515– 15521.
25.
Grzesiak, J. J., Davis, G. E., Kirchhofer, D., and Pierschbacher, M. D. (1992). Regulation of alpha 2 beta 1-mediated fibroblast migration on type I collagen by shifts in the concentrations of extracellular Mg 21 and Ca 21 . J. Cell Biol. 117, 1109 –1117.
26.
Miekka, S. I., Ingham, K. C., and Menache, D. (1982). Rapid methods for isolation of human plasma fibronectin. Thromb. Res. 27, 1–14.
27.
Kleinman, H. K., McGarvey, M. L., Liotta, L. A., Robey, P. G., Tryggvason, K., and Martin, G. R. (1982). Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21, 6188 – 6193.
28.
Rahmanian, M., Pertoft, H., Kanda, S., Christofferson, R., Claesson-Welsh, L., and Heldin, P. (1997). Hyaluronan oligosaccharides induce tube formation of a brain endothelial cell line in vitro. Exp. Cell Res. 237, 223–230.
29.
Ruoslahti, E., and Reed, J. C. (1994). Anchorage dependence, integrins, and apoptosis. Cell 77, 477– 478.
30.
Zhang, Z., Vuori, K., Reed, J. C., and Ruoslathi, E. (1995). The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc. Natl. Acad. Sci. USA 92, 6161– 6165.
31.
Adams, J. M., and Cory, S. (1998). The Bcl-2 protein family: Arbiters of cell survival. Science 28, 1322–1326.
32.
Klein, S., Giancotti, F. G., Presta, M., Albelda, S. M., Buck, C. A., and Rifkin, D. B. (1993). Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells. Mol. Biol. Cell 4, 973–982.
33.
Diamond, M. S., and Springer, T. A. (1994). The dynamic regulation of integrin adhesiveness. Curr. Biol. 4, 506 –517.
34.
Kubota, Y., Kleinman, H. K., Martin, G. R., and Lawley, T. J. (1988). Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107, 1589 –1598.
35.
Grant, D. S., Tashiro, K., Segui-Real, B., Yamada, Y., Martin, G. R., and Kleinman, H. K. (1989). Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58, 933– 943.
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION 36.
Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R., and Parsons, J. T. (1994). Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2dependent binding of pp60src. Mol. Cell. Biol. 14, 1680 – 1688. 37. Marshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179 –185. Received October 20, 1998 Revised version received December 30, 1998
38.
213
Schlaepfer, D. D., Broome, M. A., and Hunter, T. (1997). Fibronectin-stimulated signaling from a focal adhesion kinase-cSrc complex: Involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol. Cell. Biol. 17, 1702–1713. 39. Klint, P., Kanda, S., and Claesson-Welsh, L. (1995). Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells. J. Biol. Chem. 270, 23337–23344.
Signaling via Fibroblast Growth Factor Receptor-1 Is Dependent on Extracellular Matrix in Capillary Endothelial Cell Differentiation Shigeru Kanda,* ,1 Bianca Tomasini-Johansson,* ,2 Peter Klint,* Johan Dixelius,* Kristofer Rubin,* and Lena Claesson-Welsh* ,3 *Department of Medical Biochemistry and Microbiology, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden
Differentiation of endothelial cells, i.e., formation of a vessel lumen, is a prerequisite for angiogenesis. The underlying molecular mechanisms are ill defined. We have studied a brain capillary endothelial cell line (IBEC) established from H-2K b-tsA58 transgenic mice. These cells form hollow tubes in three-dimensional type I collagen gels in response to fibroblast growth factor-2 (FGF-2). Culture of IBEC on collagen gels in the presence of FGF-2 protected cells from apoptosis and allowed tube formation (i.e., differentiation) but not growth of the cells. FGF-induced differentiation, but not cell survival, was inhibited by treatment of the cells with an anti-b 1-integrin IgG. Changes in integrin expression in the collagen-gel cultures could not be detected. Rather, cell–matrix interactions critical for endothelial cell differentiation were created during the culture, as indicated by the gradual increase in tyrosine phosphorylation of focal adhesion kinase in the collagen-gel cultures. Inclusion of laminin in the collagen gels led to FGF-2-independent formation of tube structures, but cells were not protected from apoptosis. These data indicate that FGF receptor-1 signal transduction in this cell model results in cell survival. Through mechanisms dependent on cell–matrix interactions, possibly involving the a 3b 1-integrin and laminin produced by the collagen-cultured IBE cells, FGF stimulation also leads to differentiation of the cells. © 1999 Academic Press
INTRODUCTION
Angiogenesis plays a key role in physiological processes such as development, ovulation, and wound healing but also in the progression of diseases, including arthritis, retinopathy, and tumor growth and metastasis (for a review, see Ref. [1]). Angiogenesis can be 1 Present address: Department of Urology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852, Japan. 2 Present address: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Biomedical Center, Box 575, 751 23 Uppsala, Sweden. 3 To whom correspondence and reprint requests should be addressed. Fax: 146-18-471 4975. E-mail: [email protected]
divided into a series of sequential events, including focal digestion of the basement membrane, migration, proliferation, and tube formation (for reviews, see [2– 4]). Establishment of a functional vessel also requires production and remodeling of basement membrane, polarization of the endothelial cells, and recruitment of pericytes. Growth factors regulate angiogenesis by activation of cell surface receptor tyrosine kinases, which through binding and activation of Src homology-2 (SH2) domain-containing proteins convey a signal to the interior of the cells, resulting in cellular responses such as proliferation and differentiation (for a review, see [5]). To study signal transduction by receptor tyrosine kinases in endothelial cells, we established a capillary endothelial cell line [6] from brains of the H-2K b-tsA58 transgenic mouse (“immortomouse”) [7]. The endothelial cells were able to produce plasminogen activator activity, migrate, proliferate, and differentiate, i.e., form tubes in collagen gels, in response to fibroblast growth factor-2 (FGF-2) stimulation. In agreement with the general scheme for activation of receptor tyrosine kinases, FGFR-1 is known to dimerize in conjunction with ligand binding, through a process facilitated by heparan sulfate proteoglycans, leading to activation of the tyrosine kinase and autophosphorylation (for a review, see [8]). Autophosphorylated tyrosine residues may be critical for regulation of tyrosine kinase activity or may serve as binding sites for SH2 proteins. This far, seven autophosphorylation sites have been identified in FGFR-1 [9]. Two phosphorylation sites, Y653 and Y654, are located in the second tyrosine kinase domain; these sites may play a role in regulation of the kinase activity. Phosphorylated Y766 is known to serve as a binding site for phospholipase C-g (PLC-g) [10]. The functions of the other sites in FGFR-1 signal transduction have not yet been elucidated. Recent reports show that extracellular matrix (ECM)– integrin interactions play significant roles in the angiogenic process in vitro and in vivo (for a review, see [11]). Laminin, one of the main components of the basement membrane, has a particular role in differen-
203
0014-4827/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
204
KANDA ET AL.
tiation [12]. Fibronectin stimulates the survival of endothelial cells [13], and the a vb 3-integrin has been reported to play an important role for invasion, migration, and survival of endothelial cells [14]. Integrins constitute a family of a and b heterodimeric transmembrane glycoproteins which mediate intracellular and cell to ECM adhesion [15]. Different combinations of a and b chains give rise to integrins that have specificity for different ECM proteins. In vitro, upon ECM binding to their extracellular domain, integrins associate in focal adhesions. At these sites integrins provide a linkage between the ECM and the cytoskeleton, to which integrin cytoplasmic tails connect. It has been shown that binding of integrins to their ECM ligands, with subsequent focal adhesion formation, leads to signal transduction, in part converging with those initiated by growth factors [16, 17]. Signaling molecules, including growth factor receptors and SH2 domain proteins, are recruited into the focal adhesion [18, 19]. Focal adhesion kinase (FAK), Grb2, members of the small GTPase protein family Rho, insulin receptor substrate-1, and the Src family kinases are known to be involved in integrin-coupled signal transduction [20]. Consequently, there are strong indications for crosstalk between signal transduction pathways initiated by growth factor receptors and integrins [17]. Thus, the platelet-derived growth factor (PDGF) b-receptor is tyrosine phosphorylated following attachment of fibronectin to its integrin receptor [21]. In this report, we characterize the FGF-2-induced tube formation of H-2K b-tsA58 brain endothelial cells and show that the b 1-integrin is involved in differentiation of the endothelial cells. Cells grown on fibronectin spread and proliferated in response to FGF-2 treatment, but failed to differentiate. On the other hand, cells cultured between two layers of collagen gel did not spread and failed to proliferate in response to FGF-2 stimulation. Instead, the rounded cells aggregated and subsequently formed tubes. Our results indicate that receptor tyrosine kinase signal transduction is modulated by signals initiated by integrin–ECM interactions. MATERIALS AND METHODS Cell culture. A capillary endothelial cell line isolated from H-2K btsA58 transgenic mouse brain (denoted immortomouse brain endothelial cells; IBEC) was cultured on fibronectin-coated dishes (coated at 37°C for 18 h using 20 mg/ml human plasma fibronectin in Ham’s F12 medium) at 33°C in Ham’s F12 medium (Life Technologies, Inc.) supplemented with 20% heat-inactivated fetal bovine serum (Life Technologies), 75 mg/ml endothelial cell growth supplement (Sigma), 20 U/ml recombinant mouse interferon-g (IFN-g; Genzyme), 10 ng/ml human recombinant epidermal growth factor, a kind gift from Amgen, Inc., Thousand Oaks, CA), and 5 mg/ml bovine pancreas insulin (Sigma) (denoted growth medium). IBE cells expressing a chimeric molecule composed of the extracellular domain of the platelet-derived growth factor receptor-a (PDGFR-a) fused to the intracellular domain of FGFR-1 has been described before [6]. Prior to
assays, cells were kept in Ham’s F12 containing 0.25% bovine serum albumin (BSA; fatty acid-free; Sigma) (assay medium), lacking IFN-g and the growth stimulatory supplements, except when otherwise indicated. Tube formation assay in collagen gel. The tube formation assay in three-dimensional collagen gels was performed as described previously [6]. Briefly, cells were trypsinized, centrifuged, and resuspended in F12 medium containing 0.25% BSA. The mixture of type I collagen (Celltrix Pharmaceuticals), 10 3 F12 medium, and concentrated buffer (260 mM NaHCO 3, 200 mM Hepes, and 50 mM NaOH) (8:1:1) was put into the wells of 12-well plates and allowed to gel at 37°C. In indicated experiments, 100 mg/ml (final concentration) of human plasma fibronectin (Sigma) or laminin from Engelbreth– Holm–Swarm mouse sarcoma (i.e., laminin-1; Sigma) was mixed in the collagen gels. The cells were inoculated onto the collagen gels with indicated supplements at a density of 1.8 3 10 5 cells/cm 2 and cultured for 3 h at 33°C. When the effect of cell density on tube formation was examined, densities of 0.9 and 0.45 3 10 5 cells/cm 2 were also used. After attachment of the cells, the medium was removed and a second collagen layer was added. After gelation at 37°C, F12 medium containing 0.25% BSA with or without indicated supplements was added and the cultures were incubated at 33°C. The cells between collagen gel layers were observed using a phasecontrast microscope. Detection of S-phase cells. Cells were inoculated onto fibronectincoated or collagen-gel-containing wells at a density of 1.8 3 10 5 cells/cm 2 in F12 medium containing 0.25% BSA with or without ligand and cultured at 33°C for 4 h. Bromodeoxyuridine (BrdU) at 10 mg/ml was added and the cells were cultured for an additional 4 h. Cells were fixed in 70% ethanol at 4°C for 20 min. After the cells were washed, 0.02 M NaOH was added and incubated for 1 min, followed by five washes with PBS. Anti-BrdU monoclonal antibody (Becton– Dickinson) was added to the cells and incubated for 60 min at room temperature. The cells were washed three times and then incubated with peroxidase-conjugated anti-mouse IgG for 60 min. Nuclei labeled with BrdU were visualized by incubation with diaminobenzidine solution, and counterstaining of whole nuclei was performed with a light hematoxylin staining. Labeling index was expressed as a percentage of labeled nuclei/total nuclei. TUNEL. Cells were inoculated for 5 h at 33°C on type I collagen gels in Ham’s F12 medium containing 20 U/ml aprotinin and 0.25% BSA with or without growth factors. The cells were harvested, fixed, and prepared according to the instructions of the “In Situ Cell Death Detection Kit, Fluorescein” (Boehringer Mannheim), which is a terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) kit containing fluorescein-conjugated dUTP. Briefly, cells were fixed in 4% paraformaldehyde, washed twice in PBS, permeabilized in 0.1% Triton X-100 and 0.1% sodium citrate, washed twice in PBS, and incubated 1 h at 37°C in the presence of the kit reagents. The cells were washed once in PBS and analyzed using a flow cytometer (FACScan), using a 488-nm laser for excitation. Data for light scattering and green fluorescence were collected. Propidium staining. Cells undergoing tube formation in threedimensional collagen gels were fixed in methanol at 220°C for 10 min, 20 h after seeding on collagen. After three washes with PBS, the upper collagen layer was removed. Subsequently, RNase A (Sigma; 1 mg/ml) and propidium iodide (Molecular Probes; 20 mg/ml) were added to the collagen gel and incubated 2 h at 4°C. Cells were analyzed using a fluorescence microscope and photographed. Metabolic labeling and immunoprecipitation of integrins. IBE cells (2 3 10 6 cells/well in 2 ml) were plated in methionine- and cysteine-free MCDB medium supplemented with 20 U/ml aprotinin, on collagen gels or on fibronectin-coated 3-cm dishes, in the presence of 50 mCi/ml [ 35S]methionine/cysteine mix (Amersham Pharmacia Biotech), with or without 10 ng/ml FGF-2. Cells were labeled for 8 –10 h and extracted for 10 min on ice with 1 ml 1% Triton X-100, 20 mM Tris–HCl, pH 7.4, 0.65 mM MgSO 4, 1.2 mM CaCl 2 in the presence of 5 mg/ml leupeptin, 5 mg/ml pepstatin, and 4 mM PefaBlock.
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
205
FIG. 1. (A) Kinetics of tube formation of murine brain capillary endothelial (IBE) cells in three-dimensional type I collagen gels. Cells were inoculated onto a layer of collagen gel in the absence (a, c, e, g) or presence (b, d, f, h) of 5 ng/ml FGF-2. After 3 h, medium was removed and a second layer of collagen was added. Medium without (a, c, e, g) or with (b, d, f, h) FGF-2 was added after gelation of the second layer of collagen. Pictures were taken at 0 (a, b), 2 (c, d), 8 (e, f), and 18 h (g, h) after addition of the top layer of collagen. Bar, 50 mm. (B) Cell-density-dependent formation of tubes by IBE cells in collagen gels. Tube formation was assayed in the absence (a, c, e) or presence (b, d, f) of 5 ng/ml FGF-2. Cells were inoculated onto the first layer of collagen gels at densities of 0.2 3 10 5 (a, b), 0.9 3 10 5 (c, d), and 1.8 3 10 5 cells/cm 2 (e, f). Bar, 50 mm. (C) Morphology of IBE cells grown on fibronectin. Cells were inoculated and cultured on fibronectin-coated dishes, in the absence (a) or presence (b) of 5 ng/ml FGF-2. Bar, 25 mm. (D) Kinetics of tube formation of IBE cells ectopically expressing the chimeric receptor PDGFR-a/FGFR-1, in three-dimensional collagen gels. Cells were plated in the absence (a, c, e, g) or presence (b, d, f, h) of 5 ng/ml PDGF-AA and incubated in the continued absence or presence of PDGF-AA as described for A, for 0 (a, b), 2 (c, d), 8 (e, f), and 18 h (g, h) after addition of the top layer of collagen. Bar, 50 mm.
206
KANDA ET AL.
FIG. 2. (A) Fluorescence-activated cell sorting of TUNEL-stained IBE cells. Cells on collagen gels in the presence (1) or absence (2) of FGF-2 (top) were harvested 7 h after seeding on collagen, fixed, labeled with fluorescein-conjugated dUTP (TUNEL), and analyzed by flow cytometry. Alternatively, collagen-gel-seeded IBE cells extopically expressing the chimeric receptor (PDGFR-a/FGFR-1) were treated in the absence or presence of 5 ng/ml PDGF-AA, harvested at 7 h, and analyzed for TUNEL-positive cells. The forward scatter represents cell size and the fluorescence intensity represents the degree of labeling. The histograms are divided in two sections, representing apoptotic (Ap) and nonapoptotic (NA) cells, based on the degree of DNA stand breaks. (B) Propidium iodide staining of IBE cells cultured in three-dimensional collagen gels. IBE cells cultured in a three-dimensional collagen gel for 20 h were fixed, and the upper collagen layer was removed. Cells were stained with propidium iodide to visualize cells undergoing morphological changes typical for apoptosis. (Top) Cells cultured in the presence of FGF-2; (bottom) cells cultured in the absence of FGF-2. Large arrow indicates a cell with fragmented nucleus; small arrow indicates a cell with condensed nucleus.
The lysates were spun and precleared by addition of 30 ml normal rabbit serum and 30 ml protein A– or protein G–Sepharose for 1 h at 10°C. The precleared cell lysates were aliquoted and incubated overnight at 10°C with various antibodies. Protein A– or protein G–Sepharose beads (30 ml of a 1:1 slurry in PBS) were added for 1 h at 10°C. The Sepharose pellets were washed four times with extraction buffer containing 0.5 M NaCl and once with 0.1% Triton buffer, 0.5 M NaCl before addition of SDS–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer. Samples were heated to 95°C for 10 min and run on 6 or 7.5% SDS–PAGE and analyzed by fluorography. Anti-integrin antibodies were as follows: Rabbit polyclonal b 1 IgG was raised and isolated as previously described [22, 23] with the modification that purified rat hepatocytes were used as starting material in the purification of the b 1 subunit. Rabbit anti-human a 3 antiserum, kindly provided by Dr. Mats Johansson (University of Uppsala, Sweden), was raised against a synthetic peptide (KRARTRALYEAKRQKAEMSQPSETERLTDDY) corresponding to a major portion of the human a3 cytoplasmic domain. This antiserum immu
noprecipitates a 3-integrin from mouse 3T3/Ha cells (Dr. Mats Johansson, personal communication). Rabbit anti-mouse a 2 antiserum was raised against a peptide (CNDEMDETTELNS) corresponding to the C-terminal part of the mouse a 2 cytoplasmic domain. This antiserum immunoprecipitates a 2-integrin from mouse and rat fibroblasts (unpublished observation) as well as surface-labeled a 2b 1 from mouse fibroblasts (data not shown). Rabbit anti-human a 5 antiserum (cytoplasmic domain) was from Chemicon. Supernatants from the antia 6-integrin antibody producing hybridoma GoH6 was kindly provided by Dr. Arnoud Sonnenberg (University of Amsterdam, The Netherlands) [24]. Cell attachment assay. The attachment assay was performed as previously described [25]. Briefly, non-tissue-culture-treated 96-well plates (Dynatech Laboratories) were coated with 30 mg/ml human fibronectin [26], EHS laminin [27], bovine skin collagen type 1 (CELLON), or BSA (Boehringer Mannheim) in PBS, during an overnight incubation at 10°C. Following one wash with PBS, 2% BSA was
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
207
ping of the membrane by soaking in 62.5 mM Tris–HCl, pH 6.8, containing 2% SDS and 0.7% 2-mercaptoethanol at 50°C for 15 min was performed.
RESULTS
Morphological Changes of Capillary Endothelial Cells in Response to FGF-2 Are Dependent on the Extracellular Matrix and Dimensions
FIG. 2—Continued
added to the wells and incubated for 1 h at room temperature. Cells were detached and resuspended to 30,000 cells/ml in serum-free Ham’s F12 medium containing 0.2% BSA, and 100 ml per well was added to the PBS-washed plate. Following 45 min at 37°C, the unattached cells were washed three times with Ham’s F12, and attached cells were fixed in 4% paraformaldehyde in PBS, stained with 0.5% toluidine blue in 4% paraformaldehyde, and solubilized in 2% SDS. The absorbance at 562 nm was measured in a vertical pathway spectrophotometer. Treatment of cells with anti-b1-integrin antibody. Cells were harvested by trypsinization, neutralized by soybean trypsin inhibitor, and suspended in Ham’s F12 containing 0.25% BSA with nonimmune rabbit IgG or anti-b 1 polyclonal antibody. Cells were inoculated at a density of 2.1 3 10 4 cells/cm 2 on collagen-gel-containing 12-well dishes. The collagen-gel cultures were covered with a second layer of collagen and after gelation of the second layer, medium containing nonimmune rabbit IgG or anti-b 1-integrin polyclonal antibody was added, and the culture was continued overnight and subsequently photographed. Immunoblot analysis of FAK. IBE cells were harvested by trypsin, neutralized by soybean trypsin inhibitor, and inoculated on fibronectin- or collagen-coated dishes (6-cm diameter) in Ham’s F12 containing 20 U/ml aprotinin and 0.25% BSA, then cultured for indicated periods in the presence or absence of FGF-2. Cells were washed once with Tris-buffered saline, pH 7.5, containing 100 mM orthovanadate and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 50 mM Na fluoride, 2 mM EDTA, 30 mM sodium pyrophosphate, 100 mM orthovanadate, 2 mM PMSF, and 100 U/ml aprotinin) on ice. After centrifugation to remove insoluble material, the cell lysate was passed through a 27-gauge needle twice and immunoprecipitated with monoclonal antibody against FAK (2A7; kindly provided by Dr. T. Parsons, Department of Microbiology, Health Science Center, University of Virginia, Charlottesville 22908) on ice overnight, followed by adsorption to protein A–Sepharose beads. Beads were washed three times with RIPA buffer, and samples heated in SDS sample buffer were separated on 7.5% polyacrylamide gels. After electrophoretic transfer onto a nitrocellulose membrane (Hybond-C Extra; Amersham), the membrane was blocked with 3% BSA in PBS containing 0.05% Tween 20 overnight. Then, the blot was probed with monoclonal antibody against phosphotyrosine, PY-20 (Transduction Laboratory) or the BC3 polyclonal antibody against FAK, kindly provided by Dr. T. Parsons. Reactive sites were detected through enhanced chemiluminescence. Between two probings, strip-
We isolated an endothelial cell line from brain tissue of a transgenic mouse strain denoted immortomouse [6]. All tissues in this mouse express a temperaturesensitive version of simian virus 40 T under the control of the histocompatibility-2 (H-2) promoter, which allows temperature- and interferon-g-regulated expression of the oncogene [7]. The endothelial cells, which we denote IBEC, respond to FGF-2 with induction of lumen-containing vessel-like structures, when cultured in three-dimensional collagen gels, as demonstrated through confocal and electron microscopy [6, 28]. This differentiation process is accompanied by formation of tight junctions and apical pinocytosis [28]. In order to define the signal transduction pathways involved in differentiation, the kinetics of tube formation were analyzed (Fig. 1). Cells were seeded in the middle of a three-dimensional type I collagen gel and cultured at 33°C in the presence and absence of FGF-2 in serum-free medium. Figure 1A, a and b, shows that independent of ligand stimulation, cells failed to spread in the collagen gel. FGF-2-stimulated cells started to aggregate within 2 h (Fig. 1A, d). In cultures stimulated for 8 h (f), cell aggregates started to sprout and fuse to form tubes. After 18 h (h), the majority of the cells in the culture were engaged in aggregates or tubes. Cell aggregation and tube formation were celldensity dependent, since cells seeded at low density formed neither aggregates nor tubes (Fig. 1B).
FIG. 3. Labeling index of endothelial cells cultured on fibronectin or collagen. Parental IBE cells or IBE cells ectopically expressing the chimeric receptor (PDGFR-a/FGFR-1) were plated on fibronectin (FN)-coated or type I collagen (CO) gel-containing wells in the presence (1) or absence (2) of FGF-2 or PDGF-AA, as indicated. After 4 h, BrdU was added and the cultures were continued for an additional 4 h. The cultures were washed and fixed and nuclei that had incorporated BrdU were visualized by immunoperoxidase staining. Labeling index was expressed as percentage of labeled nuclei/total nuclei. Data are means 6SD for triplicate wells.
208
KANDA ET AL.
FIG. 4. Immunoprecipitation analysis of integrin expression in IBE cells. IBE cells cultured on collagen gels (C) or on fibronectin-coated dishes (F) were metabolically labeled with 35S for 8 h in the presence or absence of 5 ng/ml FGF-2. Cell lysates were immunoprecipitated overnight with nonimmune serum (NRS), with polyclonal rabbit antiserum to rat b1, or with antisera raised against peptides comprising the cytoplasmic tails of integrins a3 and a5. Samples were analyzed by SDS–PAGE (6% acrylamide gel) and fluorography.
We compared cultures in collagen gels with cells cultured on fibronectin; under the latter condition, cells were able to spread and form a monolayer (Fig. 1C). Cells growing on fibronectin never formed aggregates or tubes when stimulated with FGF-2. The IBE cells express mainly FGF receptor-1, but also small levels of FGF receptor-2 and -4 [6]. We created an engineered version of IBEC expressing a chimeric receptor construct composed of the PDGF a-receptor extracellular domain fused to the FGF receptor-1 intracellular domain. The chimeric receptorexpressing IBE cells formed aggregates and tubes in response to PDGF-AA, with the same kinetics and morphology as the FGF-2 response seen in untransfected cells (Fig. 1D). Since the cells lack an endogenous PDGF a-receptor [6], these results indicate that the FGF receptor-1 intracellular domain transduces signals for differentiation of endothelial cells.
fragmentation. In the absence of FGF-2, the fraction of apoptotic cells increased to over 90% (Fig. 2A, top). A similar pattern was observed when collagen cultures of IBE cells expressing the chimeric PDGFR-a/FGFR-1 receptor, treated with PDGF-AA, were analyzed (Fig. 2A, bottom). These data indicate that cellular survival in the presence of growth factor was dependent on the FGFR-1 intracellular domain. We further treated IBEC cultured in three-dimensional collagen gels with propidium iodide, which intercalates DNA, in order to visualize morphological changes typical for apoptotic cells (Fig. 2B). After 20 h culture, cells deprived of FGF-2 contained condensed, sometimes fragmented nuclei (arrows in the lower image). These results indicate that the cells in the collagen gel were undergoing apoptosis and that FGF receptor-1 is able to transduce signals for inhibition of apoptosis of capillary endothelial cells.
Cells on Collagen Undergo Apoptosis in the Absence of FGF-2
Signals for Growth of Endothelial Cells Are Modulated by Extracellular Matrix
Cells grown in collagen gels in the absence of FGF-2 started to die within 8 h of culture (Fig. 1A). We performed flow cytometric analysis of DNA fragmentation in IBE cells cultured on collagen gels for 5 h, detected by staining of fragmented DNA using TUNEL in the presence of fluorescein-conjugated dUTP. In the presence of FGF-2, 0.1% of the cells showed signs of DNA
To analyze the effect of extracellular matrix on FGF2-stimulated biological responses of the endothelial cells, we measured labeling index of the cells grown on fibronectin or collagen. Figure 3 shows that the labeling index was increased by FGF-2 stimulation when cells were cultured on fibronectin-coated wells, but not when cells were cultured on collagen gel-containing
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
209
wells. A similar pattern was observed when cells expressing the chimeric PDGFR-a/FGFR-1, stimulated with PDGF-AA, were analyzed (Fig. 3). These data suggest that fibronectin mediates connection of FGF receptor-1 signal transduction to pathways for growth rather than differentiation. In cells on collagen on the other hand, FGFR-1 activity can be correlated with differentiation but not with proliferation. Analysis of the Expression of Integrins in the Brain Capillary Endothelial Cells In order to investigate the capacities of the IBE cells to interact with different extracellular matrixes, we examined the pattern of integrin expression in IBE cells grown under different conditions. Analysis by [ 35S]methionine labeling of the cells and immunoprecipitation with polyclonal anti-rat b 1 -integrin IgG showed major labeled polypeptides with M r ’s of 122,000 –133,000 and 140,000 (Fig. 4). The protein bands were broad and the components most likely represent a-integrin subunits and their precursors. It should be noted that no polypeptides in the M r 180,000 –200,000 region could be detected, indicating low or no synthesis of a 1b 1 and a 2b 1. To further investigate which a subunits were expressed on the IBE cells, immunoprecipitations with specific anti-a subunit antibodies were performed. Anti-a 3 and -a 5 subunit antibodies precipitated polypeptides with M r ’s 135,000 and 145,000, demonstrating active synthesis of a 3b 1 and a 5b 1 in IBE cells. A polyclonal anti-mouse a 2 antiserum did not immunoprecipitate any labeled polypeptides in the M r region
FIG. 6. Effect of the anti-b 1 integrin antibody on tube formation of IBE cells in three-dimensional collagen gels. Cells were cultured in three-dimensional collagen gels in the presence of nonimmune IgG (a and b) or anti-b 1-integrin antibody (c and d) in the absence (a, c) or presence (b, d) of 5 ng/ml FGF-2 for 18 h.
of 150,000 (data not shown). This is in agreement with the fact that no peptides in the M r region of 150,000 could be detected after immunoprecipitation with antib 1-integrin IgG (Fig. 4). The anti-a 6-integrin antibody GoH6 similarly failed to immunoprecipitate labeled polypeptides having M r ’s expected for a 6 (data not shown). These results suggest that the IBE cells have low or no synthesis of the a 2b 1- and a 6b 1-integrins. As judged from several experiments, no consistent difference in integrin synthesis was detected after stimulation of the cells with FGF-2, irrespective of whether cells were plated on fibronectin or collagen. Effect of Anti-b 1-Integrin Antibody on Differentiation of Endothelial Cells
FIG. 5. Attachment of IBE cells to different extracellular matrixes. IBE cells were seeded on BSA-treated plastic coated with fibronectin, laminin-1 (EHS laminin), collagen I, or BSA. Unattached cells were washed off after 45 min incubation at 37°C and attached cells were fixed, stained with toluidine blue, and solubilized in SDS and the absorbance at 562 nm was measured spectrophotometrically.
Integrin receptors for collagen (a 1b 1 and a 2b 1) were not detected on the IBE cells, which is in agreement with the fact that the cells failed to attach to collagen (Fig. 5). On the other hand, fibronectin (a 5b 1) and laminin (a 3b 1) receptors were present and cells attached well to these matrixes (Fig. 5). To test the effect of inhibition of these receptors, we employed a neutralizing anti-b 1-integrin antibody. Figure 6 shows that the anti-b 1-integrin antibody efficiently inhibited tube formation of endothelial cells in three-dimensional collagen gels. Kinetic analysis showed that aggregation of cells still occurred but that the subsequent tube formation of the capillary endothelial cells was attenuated (data not shown). The antibody had, however, no effect on cell survival. Cell death in unstimulated cells and stimulation of cell survival by FGF-2 were still evident (Figs. 6c and 6d). This result indicates that tube formation but not survival of cells grown in collagen was dependent on signal transducion pathways involving b 1-integrins.
210
KANDA ET AL.
Laminin Promotes Tube Formation of Endothelial Cells The results in Figs. 4 and 5 showed that laminin receptors were expressed on the IBE cells. Inclusion of 100 mg/ml EHS laminin (i.e., laminin 1) in the threedimensional collagen gel led to spontaneous tube formation (Figs. 7c and 7d), independent of FGF-2. Although laminin induced tube formation, it failed to protect the cells from apoptosis (Fig. 7c), which is in agreement with the finding that the neutralizing b 1integrin antibody inhibited tube formation, but not cell survival, in the presence of FGF-2 (Fig. 6). The stimulation of tube formation by laminin in the absence of FGF-2 was not the result of the contaminating FGF, since the laminin we used had no stimulatory effect on the kinase activity of FGF receptor-1 (data not shown). In contrast, when cells were grown in fibronectin (100 mg/ml)-containing collagen gels in the presence of FGF-2, there was marked cell aggregation but no tube formation (Figs. 7e and 7f). These results indicate that the a 5b 1-integrin ligated to fibronectin is inhibitory for differentiation of endothelial cells, whereas the a 3b 1integrin ligated to laminin is stimulatory. Tyrosine Phosphorylation of Focal Adhesion Kinase Integrin-mediated signaling is known to involve tyrosine phosphorylation and activation of FAK. We examined whether culture of IBEC on collagen involved FAK tyrosine phosphorylation. Since the cells fail to attach to collagen (Fig. 5), the low level of FAK tyrosine phosphorylation in unstimulated cells after 1 h of culture was expected (Fig. 8, lane 1). Stimulation of FGF-2 during 1 h increased the level of tyrosine-phosphorylated FAK only marginally (Fig. 8, lane 2). Cultures incubated for 7 h in the absence of FGF-2 showed increased phosphorylation of FAK, which was further increased with FGF-2 treatment to fivefold over that of the untreated, 1-h culture. These data indicate that the IBE cells produce a matrix component, possibly laminin, in the basal condition and with increased efficiency in the presence of FGF-2 and agree with the notion that differentiation of endothelial cells involves integrin signaling. Whether FAK itself is critical in the differentiation process remains to be demonstrated. DISCUSSION
In this report, we show that tube formation of murine brain endothelial cells occurs in response to FGF-2 when the cells are cultured in three-dimensional collagen gels. This process is accompanied by cell survival, and removal of FGF-2 leads to apoptosis of the cells. The signal transduction pathways mediating tube formation and survival appear to be distinct, since tube formation involved b 1 -integrin–ECM interactions, whereas cell survival did not. Inclusion of laminin in
FIG. 7. Effects of laminin and fibronectin on tube formation of IBE cells. Cells were cultured in three-dimensional collagen gels, supplemented or not with laminin or fibronectin, in the absence (a, c, e) or presence (b, d, f) of 5 ng/ml FGF-2 for 18 h. (a and b) Collagen gels alone, (c and d) 100 mg/ml laminin-1-containing collagen gels, and (e and f) 100 mg/ml fibronectin-containing collagen gels.
the collagen gel stimulated spontaneous tube formation, but did not protect against apoptosis. Failure to attach has previously been shown to lead to programmed cell death, apoptosis (see [16, 29] for reviews), and to be coupled to cellular programs of different kinds. Cell survival, such as that induced by FGF-2 treatment of cells in collagen, has been reported for many cell types treated with serum or cytokines and may directly or indirectly involve integrin–ECM interactions. Thus, interactions between fibronectin and integrins (i.e., a 5b 1) are accompanied by cell survival through increased expression levels of Bcl-2, which is known to protect against apoptosis [30, 31]. We did not detect any changes in expression levels of Bcl-2 family members in IBE cells during tube formation (data not shown); potential posttranslational modifications were not examined. Neither culture on collagen nor FGF-2 treatment appeared to affect expression of integrins. Integrins have a slow turnover rate and fluctuations in expression levels may not be detectable under the conditions used. Since the control cells cultured in the absence of FGF-2 underwent apoptosis, it was, however, not possible to compare time points later than 8 –10 h after inoculation of the cells. An analysis of FGF-2-induced modulation of integrins on microvascular endothelial cells [32] showed maximal difference in integrin expression 72 h after exposure to the growth factor. On the other hand, available data indicate that adhesion of integrins to their ligands is not constitutive, but is
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION
211
FIG. 8. Immunoblot analysis of tyrosine phosphorylation of FAK. IBE cells were plated on collagen-gel-containing dishes in the presence or absence of 5 ng/ml FGF-2 for indicated periods. Cell lysates were immunoprecipitated overnight at 4°C, using the anti-FAK 2A7 monoclonal antibody. Precipitated proteins were subjected to SDS–PAGE, followed by transfer onto nitrocellulose. The membrane was probed with anti-phosphotyrosine antibody (PY20) (left). After stripping, the same membrane was blotted with anti-FAK BC3 polyclonal antibody (right). Proteins were visualized using enhanced chemiluminescence. The relative levels of tyrosine-phosphorylated FAK in different lanes on the left were estimated by relating the optical densities of the tyrosine-phosphorylated bands to the FAK protein bands.
regulated by intracellular signal transduction pathways (see [33] for a review). Our data suggest that the IBE cells do not express the collagen- and lamininbinding integrins a 1b 1 and a 2b 1. This is based on the fact that IBE cells failed to attach to collagen and that synthesis of these integrins was not detected by immunoprecipitation. The IBE cells attached efficiently to fibronectin and laminin, however, and synthesis of the integrins a 3b 1 and a 5b 1, but not of a 6b 1, could be demonstrated. Together, these results suggest that the IBE cells use a 3b 1 as a laminin receptor. It is possible that different growth conditions allowed specific production of extracellular matrix, and preliminary data indicate that the IBEC produce laminin (data not shown). Deposition of laminin by the cells would allow interaction with the a 3b 1-integrin receptor expressed on the IBE cells. Previous reports show the importance of the b 1integrin and laminin in differentiation of different cell types, among them endothelial cells (see [12] for a review). Human umbilical vein endothelial cells have been shown to rapidly form vessel-like, lumen-containing structures when grown in laminin-enriched Matrigel, a solid gel of basement membrane proteins and growth factors [34, 35]. Grant et al. [35] showed that laminin had multiple effects in endothelial cell differentiation; an RGD-containing laminin-derived peptide induced cell attachment, whereas a YIGSR peptide induced cell– cell interactions and tube formation in human umbilical vein endothelial cells. The protein tyrosine kinase FAK is activated in response to clustering of certain integrins, such as a 3b 1 ([36]; see [16] for a review). We observed tyrosine phosphorylation of FAK, which is presumed to represent its activation,
several hours after inoculation of the IBE cells on collagen, potentially due to clustering of a 3b 1-integrin expressed on these cells. A role for the a 3b 1-integrin in endothelial cell differentiation was further supported by the data showing inhibition of tube formation by a neutralizing anti-b 1 antibody. Which are the molecular mechanisms behind the differentiation-promoting effects of laminin? Studies of the neuronal cell line PC12, which undergoes differentiation involving neurite outgrowth in culture, has implicated the Ras pathway in differentiation [37]. Signal transduction by at least certain integrins leads to activation of the Ras pathway, via binding of the Grb2/ mSos1 complex to FAK [16, 38]. FGFR-1 is also capable of activating the Ras pathway [39], which points to the possibility that an integration of integrin– growth factor receptor signal is critical to drive differentiation. In this context, it is interesting that FGFR-1 and its downstream signaling molecules, including PLC-g and Ras, are recruited into focal adhesion complexes in endothelial cells, together with FAK, after clustering of integrins [18]. This accumulation of growth factor receptor and signaling molecules implies a possibility for direct interactions and interplay between, e.g., FAK and the FGFR-1, with potentially unique consequences for downstream signaling. Future studies on forced expression of kinase-inactive FAK in the IBE cells should allow conclusions as to the role of FAK in endothelial cell differentiation. This study was supported by grants from The Swedish Cancer Foundation (Project 3820-B96-01XAB to L.C.W. and 2729 B97 08XBC to K.R.) and the King Gustaf V Fund.
212
KANDA ET AL.
REFERENCES 1.
Folkman, J. (1995). Angiogenesis in cancer, vascular, rheumatoid and other diseases. Nat. Med. 1, 27–31.
2.
Ausprunk, D. H., and Folkman, J. (1977). Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res. 14, 53– 65.
3.
D’Amore, P., and Thompson, R. W. (1987). Mechanisms of angiogenesis. Annu. Rev. Physiol. 49, 453– 464.
4.
Jain, R. K., Schlenger, K., Hockel, M., and Yuan, F. (1997). Quantitative angiogenesis assays: Progress and problems. Nat. Med. 3, 1203–1208.
5.
Pawson, T. (1995). Protein modules and signalling networks. Nature 373, 573–580.
6.
Kanda, S., Landgren, E., Ljungstro¨m, M., and ClaessonWelsh, L. (1996). Fibroblast growth factor receptor 1-induced differentiation of endothelial cell line established from tsA58 large T transgenic mice. Cell Growth Differ. 7, 383– 395.
7.
Noble, M., Groves, A. K., Ataliotis, P., and Jat, P. S. (1992). From chance to choice in the generation of neural cell lines. Brain Pathol. 2, 39 – 46.
8.
Schlessinger, J., Lax, I., and Lemmon, M. (1995). Regulation of growth factor activation by proteoglycans: What is the role of the low affinity receptors? Cell 83, 357–360.
9.
Mohammadi, M., Dikic, I., Sorokin, A., Burgess, W. H., Jaye, M., and Schlessinger, J. (1996). Identification of six novel autophosphorylation sites on fibroblast growth factor receptor 1 and elucidation of their importance in receptor activation and signal transduction. Mol. Cell. Biol. 16, 977– 989.
10.
Mohammadi, M., Honegger, A. M., Rotin, D., Fischer, R., Berliot, F., Li, W., Dionne, C. A., Jaye, M., Rubinstein, M., and Schlessinger, J. (1991). A tyrosine-phosphorylated carboxy-terminal peptide of the fibroblast growth factor receptor (Flg) is a binding site for the SH2 domain of phospholipase C-g1. Mol. Cell. Biol. 11, 5068 –5078.
11.
Stro¨mblad, S., and Cheresh, D. A. (1996). Cell adhesion and angiogenesis. Trends Cell Biol. 6, 462– 468.
12.
Lin, C. Q., and Bissell, M. J. (1993). Multi-faceted regulation of cell differentiation by extracellular matrix. FASEB J. 7, 737– 743.
13.
Varner, J. A., Emerson, D. A., and Juliano, R. L. (1995). Integrin a 5 b 1 expression negatively regulates cell growth: Reversal by attachment to fibronectin. Mol. Biol. Cell 6, 725–740.
14.
Brooks, P. C., Montgomery, A. M. P., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., and Cheresh, D. A. (1994). Integrin a vb 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157–1164.
15.
Hynes, R. O. (1992). Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25.
16.
Clark, E. A., and Brugge, J. S. (1995). Integrins and signal transduction pathways: The road taken. Science 268, 233– 239.
17.
Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995). Integrins: Emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11, 549 –599.
18.
Plopper, G. E., McNamee, H. P., Dike, L. E., Bojanowski, K., and Ingber, D. E. (1995). Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol. Biol. Cell 6, 1349 –1365.
19.
Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995). Integrin function: Molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131, 791– 805.
20.
Parsons, J. T. (1996). Integrin-mediated signalling: Regulation by protein tyrosine kinases and small GTP-binding proteins. Curr. Opin. Cell Biol. 8, 146 –152.
21.
Sundberg, C., and Rubin, K. (1996). Stimulation of b 1 integrin on fibroblasts induces PDGF independent tyrosine phosphorylation of PDGF b-receptors. J. Cell Biol. 132, 741– 752.
22.
Gullberg, D., Terracio, L., Borg, T. K., and Rubin, K. (1989). Identification of integrin-like matrix receptors with affinity for interstitial collagens. J. Biol. Chem. 264, 12686 – 12694. ¨ brink, B. (1986). 23. Rubin, K., Gullberg, D., Borg, T. K., and O Hepatocyte adhesion to collagen. Isolation of membrane glycoproteins involved in adhesion to collagen. Exp. Cell Res. 164, 127–138. 24.
Kennel, S. J., Foote, L. J., Falcioni, R., Sonnenberg, A., Stringer, C. D., Crouse, C., and Hemler, M. E. (1989). Analysis of the tumor-associated antigen TSP-180. Identity with a6b4 in the integrin superfamily. J. Biol. Chem. 264, 15515– 15521.
25.
Grzesiak, J. J., Davis, G. E., Kirchhofer, D., and Pierschbacher, M. D. (1992). Regulation of alpha 2 beta 1-mediated fibroblast migration on type I collagen by shifts in the concentrations of extracellular Mg 21 and Ca 21 . J. Cell Biol. 117, 1109 –1117.
26.
Miekka, S. I., Ingham, K. C., and Menache, D. (1982). Rapid methods for isolation of human plasma fibronectin. Thromb. Res. 27, 1–14.
27.
Kleinman, H. K., McGarvey, M. L., Liotta, L. A., Robey, P. G., Tryggvason, K., and Martin, G. R. (1982). Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21, 6188 – 6193.
28.
Rahmanian, M., Pertoft, H., Kanda, S., Christofferson, R., Claesson-Welsh, L., and Heldin, P. (1997). Hyaluronan oligosaccharides induce tube formation of a brain endothelial cell line in vitro. Exp. Cell Res. 237, 223–230.
29.
Ruoslahti, E., and Reed, J. C. (1994). Anchorage dependence, integrins, and apoptosis. Cell 77, 477– 478.
30.
Zhang, Z., Vuori, K., Reed, J. C., and Ruoslathi, E. (1995). The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc. Natl. Acad. Sci. USA 92, 6161– 6165.
31.
Adams, J. M., and Cory, S. (1998). The Bcl-2 protein family: Arbiters of cell survival. Science 28, 1322–1326.
32.
Klein, S., Giancotti, F. G., Presta, M., Albelda, S. M., Buck, C. A., and Rifkin, D. B. (1993). Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells. Mol. Biol. Cell 4, 973–982.
33.
Diamond, M. S., and Springer, T. A. (1994). The dynamic regulation of integrin adhesiveness. Curr. Biol. 4, 506 –517.
34.
Kubota, Y., Kleinman, H. K., Martin, G. R., and Lawley, T. J. (1988). Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107, 1589 –1598.
35.
Grant, D. S., Tashiro, K., Segui-Real, B., Yamada, Y., Martin, G. R., and Kleinman, H. K. (1989). Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58, 933– 943.
FGFR-1 AND EXTRACELLULAR MATRIX IN DIFFERENTIATION 36.
Schaller, M. D., Hildebrand, J. D., Shannon, J. D., Fox, J. W., Vines, R. R., and Parsons, J. T. (1994). Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2dependent binding of pp60src. Mol. Cell. Biol. 14, 1680 – 1688. 37. Marshall, C. J. (1995). Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179 –185. Received October 20, 1998 Revised version received December 30, 1998
38.
213
Schlaepfer, D. D., Broome, M. A., and Hunter, T. (1997). Fibronectin-stimulated signaling from a focal adhesion kinase-cSrc complex: Involvement of the Grb2, p130cas, and Nck adaptor proteins. Mol. Cell. Biol. 17, 1702–1713. 39. Klint, P., Kanda, S., and Claesson-Welsh, L. (1995). Shc and a novel 89-kDa component couple to the Grb2-Sos complex in fibroblast growth factor-2-stimulated cells. J. Biol. Chem. 270, 23337–23344.