Molecular Biology of the CellVol. 4, 953-961, September 1993
The Extracellular Matrix as a Cell Survival FactorJere E. Meredith, Jr., Babak Fazeli, and Martin A. Schwartz*The Scripps Research Institute, Committee on Vascular Biology, La Jolla, California 92037
Submitted June 23, 1993; Accepted July 27, 1993
Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathwayinduced in a variety of cell types. In many cases, PCD is induced by the withdrawal ofspecific hormones or growth factors that function as survival factors. In this study, wehave investigated the potential role of the extracellular matrix (ECM) as a cell survivalfactor. Our results indicate that in the absence of any ECM interactions, human endothelialcells rapidly undergo PCD, as determined by cell morphology, nuclei fragmentation, DNAdegradation, protein cross-linking, and the expression of the PCD-specific gene TRPM-2.PCD was blocked by plating cells on an immobilized integrin f1 antibody but not byantibodies to either the class I histocompatability antigen (HLA) or vascular cell adhesionmolecule-1 (VCAM-1), suggesting that integrin-mediated signals were required for main-taining cell viability. Treatment of the cells in suspension with the tyrosine phosphataseinhibitor sodium orthovanadate also blocked PCD. When other cell types were examined,some, but not all, underwent rapid cell death when deprived of adhesion to the ECM.These results suggest that in addition to regulating cell growth and differentiation, theECM also functions as a survival factor for many cell types.
INTRODUCTIONProgrammed cell death (PCD), or apoptosis, is the pro-cess whereby cells are induced to activate their owndeath or cell suicide. PCD occurs in a wide variety ofcell types and is required during the development ofmany tissues (reviewed in Ellis et al., 1991). The term"apoptosis" has been used historically to refer to theunique morphology of cells undergoing PCD. Apoptoticcells appear shrunken, with extensive membrane bleb-bing and nuclear fragmentation (reviewed in Wyllie etal., 1980). Apoptotic cells ultimately fragment intomembrane-bound vesicles or apoptotic bodies that con-tain cellular remnants of proteins and fragmentedchromatin. These apoptotic bodies are eventuallyphagocytosed by neighboring cells and scavengingmacrophages (Wyllie et al., 1980). In contrast, cells un-dergoing pathological death or necrosis swell and thenrupture, releasing their cellular contents, thereby elic-iting inflammatory reactions (Wyllie et al., 1980).Concomitant with changes in morphology, cells un-
dergoing PCD also actively degrade their DNA and formextensive protein cross-links. Studies have shown thatDNA degradation requires the activation of an endog-enous deoxyribonuclease (Wyllie, 1980; Compton,
* Corresponding author.
1992). In some, but not all, cases of PCD (e.g., in glu-cocorticoid-treated thymocytes), this deoxyribonucleaseis specific for intemucleosomal DNA such that the de-graded DNA will form a 200-bp ladder pattern whenseparated by gel electrophoresis (Wyllie, 1980). In thy-mocytes, DNA degradation appears to be a key step incommitting a cell to the PCD pathway (McConkey etal., 1989). PCD-induced protein cross-linking appearsto be dependent on the activation of an endogenoustransglutaminase(s) required for the formation of apop-totic envelopes. (Fesus et al., 1987).
In many models of PCD, cells are induced to die asa result of changes in environmental stimuli. For ex-ample, hormone depletion will induce PCD in hormone-dependent tissues such as the prostate gland (Kyprianouand Isaacs, 1988), mammary gland (Strange et al., 1992),uterine epithelium (Rotello et al., 1989), and breast can-cer cells (Bardon et al., 1987). In insects, changes insteroid hormone levels control the massive cell deathaccompanying metamorphosis (Ellis et al., 1991).Growth factors also regulate programmed cell death.Treatment of endothelial cells with serum-free mediumwill induce PCD (Araki et al., 1990). Certain neuronsdeprived of growth factors, such as nerve growth factor,will also initiate PCD (Ellis et al., 1991). And some ma-ture T cells are dependent on interleukin-2 to preventPCD (Duke and Cohen, 1986). In general, these ex-
1993 by The American Society for Cell Biology 953
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amples of PCD suggest that the absence of a "survivalfactor," such as a particular hormone or growth factor,will induce a cell to initiate its own death.
Like hormones and growth factors, the extracellularmatrix (ECM) plays an important role in the regulationof cell growth, differentiation, and behavior (reviewedin Daniels and Solursh, 1991; Shimizu and Shaw, 1991).ECM-cell interactions are mediated to a large extent bythe integrins, a family of more than 20 different af3heterodimers (reviewed in Hynes, 1992). Recent workhas demonstrated that, in addition to their role as adhe-sion receptors, integrins also function as signaling re-ceptors. Integrins have been found to regulate manyintracellular signaling pathways such as tyrosine phos-phorylation, cytoplasmic alkalization, intracellular Ca21fluctuations, and inositol lipid metabolism (reviewed inJuliano and Haskill, 1993; Schwartz, 1993). The recentidentification of a nonreceptor focal adhesion tyrosinekinase (FAK) involved in adhesion-dependent phos-phorylation suggests that the FAK kinase may be in-volved in integrin signaling (Guan and Shalloway, 1992;Schaller et al., 1992).Based on the importance of growth factors and hor-
mones in maintaining cell viability and the increasingevidence that integrins function in an analogous mannerto control both signaling pathways and cellular function,we have investigated the role of the ECM as a cell sur-vival factor. In this study we report that the ECM isrequired for preventing PCD and that integrin-mediatedevents are involved.
MATERIALS AND METHODSChemicalsAntibody TS2/16 (IgGl) to the integrin fl subunit was a gift fromDr. Martin Hemler (Harvard Medical School, Boston, MA), antibody489 to VCAM-1 (IgGl) was a gift from Dr. John Harlan, and antibodyW6/32 to HLA (IgG2a) was a gift from Dr. David Cheresh (The ScrippsResearch Institute, La Jolla, CA). Fibronectin was prepared from humanplasma by affinity chromatography on gelatin-Sepharose (Miekka etal., 1982). Herbimycin A (GIBCO Laboratories, Grand Island, NY)was used at a final concentration of 1 Mug/ml. All other chemicals werepurchased from Sigma Chemical (St. Louis, MO) unless stated oth-erwise.
CellsHuman umbilical vein endothelial cells (HUVECs) were obtained fromDr. David Loskuoff (The Scripps Research Institute, La Jolla, CA).Cells were subcultured by using phosphate-buffered saline (PBS)/2mM EDTA and were plated on tissue culture plastic in growth mediumconsisting of M199 medium (GIBCO Laboratories) supplemented with20% fetal bovine serum (FBS; GIBCO Laboratories), 23 Mug/ml en-dothelial cell growth supplement (ECGS; Upstate Biotechnology, LakePlacid, NY), 4 mM glutamine (GIBCO Laboratories), and 68 ng/mlheparin (Sigma Chemical). Cells were used between passages 2 and10. Serum-free medium (SFM) consisted of M199 medium supple-mented with 0.1% endotoxin- and protease-free bovine serum albumin(BSA) (Calbiochem, San Diego, CA) and growth media supplementG, containing insulin, selenium, and transferrin (GIBCO Laboratories).Defined medium consisted of SFM with 10 ng/ml basic fibroblastgrowth factor (bFGF) (Upstate Biotechnology), 90 ng/ml heparin,
and 10,ug/ml high-density lipoprotein (Biomedical Technologies,Stoughton, MA). Incubation of cells in defined medium gave essentiallythe same results as incubation in serum-containing growth medium.For interleukin la (IL-1a)-treatment, cells were incubated with 50ng/ml IL-la (R & D Systems, Minneapolis, MN) in growth mediumfor 24 h before the experiment.Human peritoneal mesothelial cells (LP-9) were a generous gift of
Dr. James Rheinwald (Biosurface Technology, Cambridge, MA). Cellswere maintained on tissue culture plastic in M199 medium supple-mented with 20% FBS, 30 jig/ml ECGS, and 0.4,ug/ml hydrocortisone(Sigma Chemical) and were detached with PBS/2 mM EDTA. Humanureteral epithelial cells were a gift of Dr. Ada Elgavish (University ofAlabama at Birmingham School of Medicine, Birmingham, AL). Cellswere detached with PBS/2 mM EDTA. The human gut epithelial cellline Caco-2 was a gift of Dr. Martin Kagnoff (University of Californiaat San Diego, San Diego, CA). Cells were maintained on tissue cultureplastic in Dulbecco's modified Eagles medium (GIBCO Laboratories)with 10% FBS and were detached by trypsinization. Human cruciateligament fibroblasts were a gift from Dr. Virgil Woods (University ofCalifornia at San Diego, San Diego, CA) and were grown in Dulbecco'smodified Eagle's medium with 10% FBS.
Suspension and Coated DishesSuspension culture dishes were made by coating tissue culture plasticwith heat-denatured 10 mg/ml BSA in PBS for 5 min, followed by 5ml (per 10-cm dish) of melted 2% agarose (BioRad Laboratories, Rich-mond, CA) in M199. The agarose completely prevented cells fromattaching to the dish. Cells in serum-free medium were sometimesincubated in dishes blocked with denatured BSA without agarose,which also blocked cell attachment. No differences were observedbetween the two types of suspension dish.
Fibronectin (FN)-coated dishes were made by incubating bacterio-logical plastic dishes with FN (50 Mg/ml in PBS) for 1-2 h. Disheswere then rinsed and blocked with denatured BSA. For antibodies,the dishes were first coated with 50 Mg/ml goat anti-mouse IgG (SigmaChemical) in PBS for 1-2 h, rinsed, blocked with denatured BSA, andthen incubated with the specific antibody. Anti-f, (TS2/16) and anti-HLA (W6/32) were used at 1:150 dilutions of ascites in PBS. Anti-VCAM-1 (489) was used at 20 Mg/ml in PBS.
Nuclear Fragmentation and DNA DegradationNuclear fragmentation was detected by acridine orange staining. Cellswere stained with 10 MAg/ml acridine orange (Sigma Chemical) in PBSfor 1-2 min. Cells were then viewed using the lOX and 40X dryobjectives of a diaphot microscope (Nikon, Garden City, NY) with axenon arc light source. A total of 200 cells were scored, and the fractionwith fragmented nuclei was calculated.DNA degradation was detected by gel electrophoresis essentially
as described (Wyllie, 1980). Cells were pelleted at 400 X g and washedtwice with ice cold TBS (137 mM NaCl, 2.7 mM KCl, and 25 mMtris(hydroxymethyl) aminomethane [Tris], pH 7.4). Pellets were re-suspended in 50 Ml TE (1 mM EDTA and 10 mM Tris, pH 8.0) andlysed with 0.5 ml of extraction buffer (10 mM Tris, pH 8, 0.1 M EDTA,and 0.5% SDS) with 0.5 mg/mi proteinase K. Samples were incubatedovernight at 50C. Samples were then extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) and then precipitated with 0.2M NaCl and 2 volumes of ethanol. DNA was recovered by centrif-ugation, and pellets were washed with 70% ethanol, air dried, andresuspended in 25 Mul TE with 20 Mug/ml RNAse A. Samples were thenincubated at 37C for 1 h. DNA was again extracted, precipitated,and washed as above, and then resuspended in 20 Mul TE. Finally,DNA samples were separated on 1.2% agarose gels with 0.25 Mg/mlethidium bromide, visualized by UV fluorescence and photographed.DNA degradation was quantitated by extraction with nonionic de-
tergent essentially as described (Wyllie, 1980). Approximately 1-5X 106 cells were harvested by centrifugation at 400 X g and washedtwice with ice cold TBS. Cells were then lysed in 0.5 ml extraction
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ECM as a Cell Survival Factor
buffer (10 mM Tris, pH 8, 20 mM EDTA, 0.5% Triton X-100) for 30min on ice. Lysates were spun at 14000 X g for 15 min to separatethe intact chromatin (pellet) from the degraded DNA (supematant).After centrifugation, the supernatants were removed and saved, andthe pellets were resuspended in 0.5 ml extraction buffer. The amountof DNA in the pellet and supematant fractions was determined bythe diphenylamine reaction, as described (Burton, 1956). The extentof DNA degradation was then expressed as the percentage of totalDNA found in the supernatant.
Protein Cross-linkingCells were rinsed once in cold PBS, extracted with 10 mM Tris, pH7, 2% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride (PMSF), 10 Mg/ml aprotinin and leupeptin,and then heated to 90C for 5 min. Protein concentration was deter-mined by the method of Lowry using BSA as a standard. Tris (100mM, pH 6.8), 10% glycerol, and 20 mM dithiothreitol (DTT) wereadded, and the samples were again heated to 90C for 5 min. Samplesnormalized for protein concentration were run on a 7% SDS poly-acrylamide gel and stained with Coomassie brilliant blue.
Northern Blot AnalysisTotal RNA was prepared from HUVECs using RNAzol B (BiotecxLaboratories, Houston, TX). Total RNA, 15 Mg per sample, was de-natured with formaldehyde and blotted onto nylon membranes(Amersham, Arlington Heights, IL) according to Sambrook et al. (1989).Duplicate samples were stained with ethidium bromide to determineRNA integrity. Filters were hybridized using [a-32P]dCTP randomprimed probes for the testosterone-repressed prostate message 2(TRPM-2) (Bandyk et al., 1990) and glyceraldehyde-3-phosphate de-hydrogenase (GAPDH) (Fort et al., 1985). TRPM-2 was a generousgift of Dr. Ralph Buttyan (Columbia University, New York) andGAPDH was a gift of Dr. Ron Bowditch (The Scripps Research In-stitute, La Jolla, CA).
Anti-Phosphotyrosine ImmunoblotsCells were rinsed once in cold PBS and extracted with 10 mM Tris,pH 7, 2% SDS, 1 mM PMSF, 1 mM sodium orthovanadate, 10 mMsodium fluoride, 10 Mug/ml aprotinin and leupeptin. Samples wereimmediately heated to 90C for 5 min. Protein concentrations weredetermined by the method of Lowry using BSA as a standard. Tris(100 mM, pH 6.8) 10% glycerol, and 20 mM DTT were added, andthe samples again were heated to 90C for 5 min. Samples normalizedfor protein concentration were run on a 6% SDS polyacrylamide geland electrophoretically transferred to nitrocellulose paper using theBioRad mini transblot system. Blots were blocked with 10% goat serumin pH 7.0 Tris-buffered saline with 0.5% Tween 20, incubated for 4-6 h with 1 MAg/ml PY20 anti-phosphotyrosine antibody (Zymed, SouthSan Francisco, CA), rinsed, and incubated with a 1:4000 dilution ofhorseradish peroxidase (HRP)-conjugated goat anti-nrouse IgG (Cal-biochem). Blots were again rinsed and visualized using chemilumi-nescence (ECL kit, Amersham) according to the manufacturer's in-structions. Note that the HRP-conjugated secondary antibody alsononspecifically recognized the prestained high molecular weightstandards (GIBCO Laboratories). We found, however, that bindingof the secondary antibody to phosphotyrosine-containing proteinswas highly specific as it was completely dependent on PY20 and wasblocked by addition of soluble phosphotyrosine.
RESULTSEndothelial Cells in Suspension UndergoProgrammed Cell DeathWe have investigated...