JOURNAL OF CELLULAR PHYSIOLOGY 146:325-335 (1991)

Regulation of Motility in Bovine Brain Endothelial Cells ELIOT M. ROSEN,* SUSAN JAKEN,WILLIAM CARLEY, PETER M. LUCKETT, EVA SETTER, MADHU BHARGAVA, AND ITZHAK D. GOLDBERG Departments of Therapeutic Radiology (E. M.R., E.S.) and Anesthesiology (W.C.), Yale University School of Medicine, New Haven, Connecticut 06.5 10; W . Alton lones Cell Science Center, Lake Placid, New York 12946 fS./.); Department of Pediatrics, HarborUClA Medical Center, Torrance, California 90502 fP. M.1,); Department of Radiation Oncology, long lsland lewish Medical Center, New Hyde Park, New York 1 1042 fM.B., 1.D.C.J

Scatter factor (SF) is a fibroblast-derived cytokine which stimulates motility of epithelial and vascular endothelial cells. We used a quantitative assay based on migration of cells from microcarrier beads to flat surfaces to study the regulation of motility in bovine brain endothelial cells (BBEC). Peptide growth factors (EGF, ECGF, basic FGF) did not stimulate migration. Tumor promoting phorbol esters (PMA, PDD) markedly stimulated migration, while inactive phorbol esters (4a-PDD, phorbol-I3,20-diacetate) did not affect migration. Both SF- and PMA-stimulated migration were inhibited by 1 ) TGF-beta; 2) protein kinase inhibitors (e.g., staurosporine, K-252a); 3 ) activators of the adenylate cyclase signaling pathway (e.g., dibutyryl cyclic AMP, theophylline);4) cycloheximide; and 5) anti-cytoskeletonagents (e.g., cytochalasin B, colcemid). However, PMA and SF pathways were distinguishable: 1) PMA induced additional migration at saturating SF concentrations; 2) the onset of migration-stimulation was immediate for PMA and delayed for SF; and 3 ) down-modulationof protein kinase C (PKC) ablated PMA but not SF responsiveness. Assessment of PKC by ('H)-phorbol ester (PDBu) binding and by immunoblot showed 1 ) scatter factor does not cause significant redistribution or down-modulation of PDBu binding or alpha-PKC; and 2 ) PDBu mediates redistribution and down-modulation of both binding and alpha-PKC. These findings suggest two pathways for BBEC motility: a PKCdependent pathway and an SF-StimulatediPKC-independent pathway. Scatter factor (SF)is a cytokine which is produced by fibroblasts and causes cohesive sheets of epithelium to spread and separate into isolated cells (Stoker and Perryman, 1985; Stoker et al., 1987). Arterial smooth muscle cells produce an activity similar to fibroblastderived SF (Rosen et al., 1989). SF from ras-transformed mouse 3T3 cells stimulated migration of MadinDarby canine kidney (MDCK) cells in Boyden chamber assays (Stoker, 1989) and in a new assay based on migration of cells off microcarrier beads onto flat culture surfaces (Rosen et al., 1990b). The latter assay was used to show that ras-3T3 SF stimulates migration of bovine artery endothelial cells. Fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, and fibronectin did not affect migration of calf pulmonary artery endothelial cells in this assay. Purification of ras-3T3 SF was reported by Gherardi et al. (1989) and by us (Rosen et al., 1990~). The factor is a heterodimer consisting of 58 and 31 kd disulfide bonded peptide chains. Little is known about the biochemical processes which regulate motility of vascular endothelial cells. Exposure of endothelial cells on beads to SF for 2 hr at 37°C followed by vigorous washing and incubation in fresh medium resulted in significant stimulation of 0 1991 WILEY-LISS. INC.

migration; cycloheximide blocked stimulation of miation (Rosen et al., 1990b). We hypothesize that SF inds to a specific target cell receptor, resulting in activation of signal transduction pathways, protein synthesis, and induction of a motile phenotype. To investigate the regulation of endothelial cell motility, we studied the effects of agents which modulate various intracellular processes on migration of bovine brain endothelial cells in the absence and presence of SF.

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MATERIALS AND METHODS Cells studied Bovine brain endothelial cells (BBEC). BBEC cells were isolated by collagenase digestion of minced bovine brain cortex after removal of the pia and then selected by the D-valine method. While the isolation procedures were similar to procedures used to obtain microvascular endothelium, cultures exhibited morphology more typical of large-vessel endothelium. Cells formed cobblestone monolayers of polygonal Factor VIII-positive cells (+/- sprouting cells) and did not Received July 18, 1990; accepted November 12, 1990. *To whom reprint requestsicorrespondence should be addressed.

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ROSEN ET AL.

overgrow after reaching confluence. BBEC cells were selected for study because these cells 1)responded well to SF, with 5-fold stimulation of migration at relatively low SF concentrations (10-20 unitsiml) (see “Results”) and 2) grew well and exhibited a long in vitro life span in the absence of added growth factors or special culture dish coatings. Cells were studied from passage 15 to 23. Madin-Darby canine kidney e ithelial cells. MDCK cells were obtained from Dr. tephen L. Warren, Department of Pathology, Yale University School of Medicine (New Haven, CT) (Warren and Nelson, 1987). Cell culture techniques. Stock cultures were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.1 mM non-essential amino acids (NEAA),5.0 mgiml D-glucose, 100 u/ml penicillin, 100 Fgiml streptomycin, and 10%(viv) (MDCK)or 20% (viv) (BBEC) fetal calf serum. Medium, NEAA, antibiotics, and serum were obtained from Grand Island Biological Co. (Grand Island, NY .) Cultures were harvested weekly with trypsin, re-inoculated at split ratios of 1:lO (MDCK)or 1:5 (BBEC) (15 ml per 100 mm Petri dish), and incubated at 37°C in a 5% C02-air atmosphere. Sources of growth factors. Mouse epidermal growth factor (EGF) was obtained from SIGMA Chemical Co. (St. Louis, MO). Crude bovine brain endothelial cell growth factor (ECGF) (Thornton et al., 1983) was provided by Dr. Elliot Levine, Wistar Institute (Philadelphia, PA). Recombinant human basic FGF was provided by Dr. Ruth Halaban, Yale University School of Medicine (New Haven, CT). Human transforming growth factor (TGF)-beta was purchased from Collaborative Research.

activity of this preparation was identical with that of purified SF when concentration was expressed as scatter units per ml.) Scatter assay. To quantitate scatter activity, MDCK cells were incubated with serial dilutions (factors of 2) of SF for T = 20 hr and examined for scatter effect (spreading of colonies and separation into single cells) (Stoker and Perryman, 1985; Rosen et al., 1990a). The activity titer at the limiting dilution at which significant scatter was observed was defined as 0.5 scatter units per ml. Titers were reproducible to within 2 112 dilution (factor of 2). Microcarrier bead migration assay. Assays were performed as described previously (Rosen et al., 1990b). Briefly, BBEC cells were seeded into 100 mm Petri dishes containing Cytodex 2 microcarrier beads (Pharmacia No. 17-0484-Ol), at 1 x lo5 cellsiml in 20 ml DMEM containing 20% serum and 5 m /ml beads. The beads were incubated for 2-3 days, a1 owing 1-2 cell doublings, and then washed twice with DMEM-5% serum; cells on the beads were counted (Van Wezel, 1973). The beads were seeded into 2 cm2 wells in 24-well plates at 1 x lo5 cells in 0.5 ml DMEM-5% serum per well. Metabolic agents and/or SF were added to wells in triplicate, and cultures were incubated at 37°C for 18 hr. The beads were removed by rinsing twice with PBS, and cells which had migrated from beads and attached to wells were stained with crystal violet and counted using a l o x objective. Mi ration was expressed as cells per 10 fields (ca. 0.2 cmB1. Each agent was studied on at least two separate occasions. Concentrations of agents were selected based on published data for responsive intact cells. With the exception of cytochalasins B and D at the highest concentraSources of metabolic agents tion studied, metabolic agents did not appear to be Phorbol esters. Phorbol-12-myristate-13-acetate cytotoxic, as judged by 1)morphology of cells remaining (PMA),phorbol-12,13-didecanoate(PDD), 4a-PDD, 4a- on beads; 2) morphology of stained migrated cells; and phorbol, and phorbol-13,20-diacetatewere obtained from 3) ability of cells to exclude Trypan Blue dye. (Note: All assays were performed in 5% serum Preliminary studSIGMA. Protein kinase inhibitors. Staurosporine and K- ies showed that migration was unaffected by serum at 252a were obtained from Calbiochem (La Jolla, CAI. 1-15% but was reduced at 0 and 0.5% (data not shown). H-7 [l-(5-isoquinolinylsulfonyl)-2-methylpiperazinel Assessment of protein kinase C. PKC was aswas purchased from SIGMA. 7,8-Dihydroxychlorpro- sessed by 1)[3Hlphorbolester binding and 2) immunomazine (DHC)was provided by Dr. William Hait, Yale blot. BBEC cells were grown in DMEM-20%in 100 mm dishes to about 50% confluence, rinsed twice with PBS, University School of Medicine (New Haven, CT). Agents that modulate adenylatelguanylate cy- and incubated in DMEM-5%serum. SF (50 units/ml) or clase pathways. Forskolin, dibutyryl cyclic AMP, phorbol-12,13-dibutyrate(PDBu) (200 nM) was added theophylline, and 8-bromo cyclic GMP were obtained for various times before harvesting cells for subsequent from SIGMA. Cholera toxin, 3-isobutyl-l-methyl- assays. Phorbol ester binding. Subcellular fractions were xanthine (IBMX), and H-8 [N-2-(methylamino)ethyl5-isoquinoline sulfonamidel were obtained from Cal- prepared and assayed for 13HlPDBu binding as described previously (Jaken, 1987). Briefly, the cells were biochem. Inhibitors of macromolecule synthesis and cy- washed twice with PBS, washed once with wash buffer toskeleton. Cycloheximide and cytochalasins B and D (50 mM Tris, pH 7.4, 2.5 mM MgC12, 0.25 M sucrose), were purchased from Calbiochem. Hydroxyurea, colce- scraped into lysis buffer (wash buffer plus 1mM EGTA, mid, colchicine, vinblastine, and tubulazole were ob- 1 mM PMSF, 10 pg/ml leupeptin) (0.25 ml per dish), tained from SIGMA. and sonicated. Soluble and particulate fractions were Scatter factor (SF) preparations. SF was par- prepared by centrifuging at 100,OOOg for 60 min. Alitially purified (ca. 1,000-fold) from serum-free condi- quots of soluble (50-70 p. ) and particulate (150-200 tioned medium from ras-transformed NIH/2 mouse 3T3 pg) fractions were assaye in triplicate for [3H]PDBu cells (clone D4) using cation exchange chromatography binding in the presence of 1.6 mM EGTA, 2.0 mM Ca+’, (Rosen et al., 1990~).The specific activity was 500- 100 pgiml phosphatidylserine, and 25 nM [3HlPDBu. 1,000 scatter units (see below) per pg protein, corre- Non-specific binding was estimated in duplicate with sponding to 10-20% purity. (Migration-stimulating 20 pM PDBu.

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7

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ENDOTHELIAL CELL MOTILITY

Immunoblot. Aliquots of soluble (65 pg SF-treated cells; 39 kg PDBu-treated cells) or particulate (200 yg SF-treated cells; 160 yg PDBu-treated cells) fractions were concentrated by TCA precipitation, analyzed by SDS-PAGE, and electrophoretically transferred to nitrocellulose. Immunoblots were blocked with 5% instant nonfat milk in 50 mM Tris-C1 (pH 7.4) containing 0.5 M NaCl (TBS) and washed in TBS. Blots were sequentially stained with alpha-PKC-specific monoclonal antibody (Leach et al., 1988) and alkaline phosphatase-conjugated anti-mouse antibody (Promega), with intervening TBS washes. Alpha-PKC was visualized after incubation with alkaline phosphatase substrate (Promega).

RESULTS Effect of SF on BBEC migration Ras-3T3 SF markedly stimulated migration of BBEC cells (Fig. 1). Responses were detectable at 1 unit/ml and maximal by 20 unitsiml. In ten different sets of assays, migration was stimulated by factors of 2.4 ? 0.5 at 1 unitiml and 5.0 ? 0.9 at 20 unitsiml. Effect of growth factors on BBEC migration None of the factors studied (EGF, ECGF, basic FGF, TGF-beta) stimulated migration of BBEC cells (Table 1).Basal migration was reduced by 29% in the presence of ECGF; basal and SF-stimulated migration were reduced by 30-35% in the presence of 100 ngiml basic FGF. TGF-beta was reported to inhibit basal or FGFstimulated vascular endothelial cell migration in in vitro wounding and Boyden chamber assays (Heimark et al., 1986; Muller et al., 1987; Sat0 and Rifkin, 1989). Similarly, TGF-beta inhibited basal and SF-stimulated BBEC migration in microcarrier bead assays. Inhibition of stimulated migration was observed at 30.1 nglml and reached 65-70% at 21 ng/ml. Metabolic agent studies Studies were erformed at three SF concentrations (0,1,20 unitdm ) corresponding to basal, submaximal, and maximal migration levels, respectively. Results are analyzed relative to: 1) effect of agent on stimuhted migration be., migration with SF minus that without SF); and 2) degree of stimulation ke., ratio of migration +/- metabolic agent). Data are expressed as 7%of 0 agent control where appropriate. Effect of phorbol esters on migration BBEC migration was markedly stimulated in the presence of tumor-promoting phorbol esters (PMA, PDD) which activate protein kinase C (PKC) (Tables 2, 3). PMA stimulated migration at concentrations of 20.05 ng/ml. In different experiments, migration peaked at 5 or 25 ng/ml and decreased at higher concentrations, but remained above control. At 5 ngiml, average degrees of stimulation were 2.8-fold (2.2-3.81, 2.3-fold (1.1-3.6), and 1.6-fold (1.1-2.5) at 0, 1, and 20 unitslml SF, respectively. Thus, PMA induced additional migration at saturatin concentrations of SF. Similar to PMA, PDD stimu ated migration in the presence or absence of SF. However, three phorbol compounds which do not activate PKC (4a-PDD, phorbol-13,20-diacetate, 4a-phorbol) (Ashendel, 1985; Fine

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40

60

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100

80

SCATTER FACTOR (units/mi) Fig. 1. Migration of bovine brain endothelial cells as a function of scatter factor concentration. Microcarrier bead migration assays (T= 18 hr) were performed a s described in Materials and Methods. Points represent mean 1 SEM (N = 3 replicate determinations).

*

TABLE 1. Effect of peptide growth factors on basal and SFstimulated migration of BBEC cells

Migrated cells per 10 fields ~

0 SF

Factor/concentration None Murine EGF, 100 ng/ml Bovine ECGF 7.1 pg/ml 71.3 pg/ml

None Human basic FGF 0.1 ng/ml 1.0 ng/ml 10 ng/ml 100 ng/ml None Human TGF-beta 0.01 ng/ml 0.1 ng/ml 0.5 ng/ml 1.0 ng/ml 5 ng/ml 10 ng/ml

31 f 1 36 6

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96 4 94 k 6

+ +

22 f 2 (.017) 22 1 (.006) 35 i 2

91 6 87 9 156 t 9

31 i 3 31 3= 5 24 35 i f 21 (.009)

115 k 9 (.036) 115 25 121 15 102 k 12 (.025) 199 10

+

50

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45 4 47 f 12 26 f 5 (.02) 23 4 (.01) 22 f 3 i.008) 19 f 2 (.004)

+ +

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19Ok 14 127 20 (.037) 87 f 14 (.005) 75 + _ 6 (