Extracellular matrix regulates proliferation and phospho lipid turnover in glomerular epithelial cells ANDREY V. CYBULSKY, LEONHARD S. WOLFE,

JOSEPH V. BONVENTRE, AND DAVID J. SALANT

RICHARD

J. QUIGG,

Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1Al; Donner Laboratory of Experimental Neurochemistry, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada; Evans Memorial Department of Clinical Research, The University Hospital, Boston University Medical Center, Boston 02118; and Renal Unit, Medical Services, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114

CYBULSKY, ANDREY V., JOSEPH V. BONVENTRE, RICHARD J. QUIGG, LEONHARD S. WOLFE, AND DAVID J. SALANT. Extracellular matrix regulates proliferation and phospholipid turnover in glomerular epithelial cells. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F326-F337, 1990.-To understand how glomerular epithelial cell (GEC) growth might be regulated in health and disease, we studied the effects of growth factors and extracellular matrix on proliferation and membrane phospholipid turnover in cultured rat GECs. In GECs adherent to type I collagen matrix, epidermal growth factor (EGF), insulin, and serum stimulated DNA synthesis and increased cell number. In addition, GECs proliferated when adherent to type IV collagen, but not to laminin or plastic substrata. Attachment of GECs to the substrata that facilitated proliferation (types I or IV collagen) produced increases in I,%diacylglycerol (DAG), an activator of protein kinase C (PKC). Increased DAG was associated with hydrolysis of inositol phospholipids and an increase in inositol trisphosphate and was not dependent on the presence of growth factors. After PKC downregulation (by preincubation with a high dose of phorbol myristate acetate), DNA synthesis was enhanced in GECs adherent to collagen. Thus contact of GECs with collagen matrices is required for serum, EGF, or insulin to induce proliferation. Collagen matrix also activates phospholipase C. As a result, the DAG-PKC signaling pathway desensitizes GECs to the mitogenic effects of growth factors and might promote cell differentiation. Understanding the interaction between GECs, growth factors, and extracellular matrix may elucidate the mechanisms of proliferation during glomerular injury. collagen;

diacylglycerol;

phospholipase

C; protein

kinase C

GROWTH FACTORS can stimulate the proliferation of cells and maintain cell viability (10, 33). Excessive secretion of growth factors by normal or neoplastic cells may result in a proliferative cellular response or fibrosis (10, 33). The mitogenic action of growth factors is, however, insufficient to account for the generation of cellular architecture in vivo. Thus the action of growth factors must be selectively supported or inhibited by the local cellular environment. For many cells, contact with extracellular matrix proteins may directly or indirectly modulate their proliferation and, in addition, maintain differentiation (21). Several recent studies have provided evidence for in-

POLYPEPTIDE

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$1.50 Copyright

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volvement of inositol phospholipids in cell growth (33). For example, platelet-derived growth factor, the principal mitogen for cells of mesenchymal origin, activates phospholipase C, which leads to hydrolysis of phosphatidylinositol bisphosphate and an increase in inositol trisphosphate (IPa) and intracellular free Ca2+ concentration, as well as a rise in l,%diacylglycerol (DAG), an endogenous activator of protein kinase C (PKC) (29). Also, synergism of polypeptide growth factors with phorbol esters (exogenous PKC activators) has suggested that in certain cells inositol phospholipid hydrolysis may be obligatory for mitogenesis (29, 33). Among the intrinsic components of the renal glomerulus are visceral and parietal glomerular epithelial cells (GECs). Both cell types are of common embryological origin, and in the mature kidney, are in contact with an extracellular matrix (1). Visceral and parietal GECs are normally bathed with an ultrafiltrate of plasma that, except for large proteins, is similar in composition to plasma. Thus glomerular ultrafiltrate probably contains insignificant amounts of epithelial cell mitogens, including epidermal growth factor (EGF) (lo), because the concentration of EGF in human plasma is low (45 pg/ ml; 15,30), and EGF is not synthesized in the glomerulus or proximal nephron in human and mouse kidney (15). The factors responsible for the control and turnover of GECs in normal and diseased states are poorly understood at the present time. In normal kidneys, there appears to be little turnover of GECs (31). Proliferation of parietal and possibly visceral GECs may occur as a result of glomerular injury, e.g., in malignant hypertension (18) and in focal segmental sclerosis and crescentic glomerulonephritides (27). Glomerular crescents have been shown to contain GECs as well as infiltrating cells and extracellular matrix proteins, including basement membrane collagen, interstitial collagen, and fibrin (25, 27). Recently, it has been reported that urines from children with Henoch-Schonlein purpura nephritis (a nephritis often associated with glomerular crescent formation) contain a factor that resembles transforming growth factor-a (16), an epithelial cell mitogen that is structurally and functionally related to EGF (9,lO). Thus the above findings suggest that, in certain forms of the American

Physiological

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glomerular injury, including glomerulonephritis, the presence of epithelial growth factors and extracellular matrix components in the glomerulus might stimulate proliferation of GECs and formation of crescents. In recent years the development of GEC culture techniques has enabled the definition of some factors that are responsible for inducing and controlling GEC proliferation. For example, it has been reported that GECs can proliferate in response to leukotrienes Cq and D, (5) and to EGF (2). Conversely, GEC proliferation can be inhibited with heparin and transforming growth factor,@(2,22). In this study, we examined the effects of growth factors and the extracellular matrix on the proliferation of rat GECs in culture. We determined that contact with collagen matrices is required for GECs to proliferate in response to serum, EGF, or insulin. In addition, collagen matrix induces an increase in DAG and IP3 due to hydrolysis of inositol phospholipids by phospholipase C. The products of phospholipase activation regulate growth-factor-induced proliferation. METHODS

Materials

Tissue culture dishes were obtained from Falcon, Becton Dickinson, Oxnard, CA. Tissue culture media, 0.05% trypsin-0.53 mM EDTA, and fetal calf serum (FCS) were obtained from GIBCO Laboratories, Grand Island, NY, and Burlington, Ontario, Canada. Pepsin-solubilized bovine dermal collagen (Vitrogen) was from Collagen, Palo Alto, CA. NuSerum, EGF, and type IV collagen and laminin (both purified from Englebreth-Holm-Swarm mouse tumor) were purchased from Collaborative Research, Bedford, MA. Insulin and other hormone supplements were obtained from Sigma Chemical, St. Louis, MO. Semipurified fibroblast growth factor (FGF; bovine endothelial mitogen) was from Biomedical Technologies, Stoughton, MA. Bacterial collagenase (type IV) was purchased from Cooper Biomedical, Malvern, PA, or GIBCO. [methyl-3H]thymidine (2 Ci/mmol), [3H]arachidonic acid (100 Ci/mmol), [ myo-3H]inositol (20 Ci/ mmol), [3H]inositol monophosphate (IPI, 1 Ci/mmol), [3H]inositol bisphosphate (IPZ, 1 Ci/mmol), [“H]IP3 (2 Ci/mmol), [ 3H] choline chloride (80 Ci/mmol), and [ 14C]stearic acid (59 mCi/mmol) were purchased from New England Nuclear, Boston, MA, and Mississauga, Ontario, Canada. [3H]ethanolamine hydrochloride (22 Ci/ mmol) was from Amersham Canada, Oakville, Ontario, Canada; dipentadecanoin was purchased from NuCheck Prep, Elysian, MN; other lipid standards were from Sigma. Thin layer chromatography plates (LK5DF) were obtained from Whatman, Clifton, NJ. Dowex (AG l-X8, ZOO-400 mesh, formate form) was from Bio-Rad, Richmond, CA. Phorbol myristate acetate (PMA), l-oleoylZ-acetyl-glycerol (OAG), l-(5-isoquinolinylsulfonyl)-Zmethylpiperazine (H-7), and digitonin were purchased from Sigma. GEC Culture

Primary

cultures of rat GECs were established accordmethods (17, 32). Glomeruli from male

ing to published

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Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were explanted onto culture dishes that were coated with a collagen gel, as detailed below. After 7-9 days, colonies of GECs were marked on the culture dish, and were excised with the collagen. Cells were replated onto collagen-coated multi-well dishes, and wells growing pure colonies of GECs were then expanded. GECs were passaged every 4-5 days. This was carried out by scraping cells with the collagen substratum into a test tube and incubating with 0.2% bacterial collagenase for 20 min at 37OC. Subsequently, cells were incubated with 0.05% trypsin-0.53 mM EDTA for 5-10 min at 37°C and were then resuspended in tissue culture medium (see below) and replated. Studies were done with cells between passages 20 and 60. Characterization of GECs was published previously (32). According to established criteria (17), the cells demonstrated polygonal shape and cobblestone appearance at confluency, cytotoxic susceptibility to low doses of aminonucleoside of puromycin, positive immunofluorescence staining for cytokeratin, and presence of junctional complexes by electron microscopy. Presently, it is not possible to determine specifically whether GECs in culture originate from visceral or parietal epithelium. Extracellular

Matrices

Under standard culture conditions, GECs were grown on a collagen gel matrix (17, 32). The gel was prepared by combining RPMI-1640 (X10) medium, 7.5% NaHC03, pepsin-solubilized bovine dermal collagen (-3 mg/ml in 0.012 N HCl), and 0.1 N NaOH, in proportions of 10:4:8O:lO at 4OC. The mixture was then poured into tissue culture dishes (-0.06 ml/cm2) and allowed to gel at 37OC. In some experiments, the collagen-containing mixture was further diluted with culture medium before gelation. According to the manufacturer, the bovine derma1 collagen preparation consists of 95-98% type I collagen, the remainder being type III. We assessed this collagen preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under reducing conditions. Polyacrylamide gels (7.5%) that were stained with Coomassie Blue demonstrated the presence of three proteins with molecular masses of 115, 125, and >ZOO kDa, corresponding to the a-, ,&, and y-chains of type I collagen. In this manuscript, the collagen preparation will therefore be referred to as “type I collagen gel.” Other extracellular matrix components that were tested for their ability to support GEC proliferation included type IV collagen, laminin, and gelatin. Solutions of these were applied to culture dishes at -0.02 mg/cm2 and were allowed to air-dry at 2ZOC. In some experiments, type I collagen was applied to culture dishes in a similar manner (referred to as “type I collagen film”). Tissue Culture

Media

The standard medium that was used to maintain GEC cultures, Kl, consisted of Dulbecco’s modified Eagle’s medium-Ham’s F-10 (DMEM-F-10; l:l), containing 5.0% NuSerum and hormone supplements including in-

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sulin (5 pg/ml), transferrin (5 ,ug/ml), hydrocortisone (18 rig/ml), prostaglandin E (25 rig/ml), 3,5,3’-triiodothyronine (0.325 rig/ml), and NazSeOs (1.7 ng/m1)(32). According to the manufacturer, NuSerum contains 25% FCS (vol/vol), 5 rig/ml EGF, endothelial cell growth supplement, progesterone, estradiol, testosterone, and the other hormone supplements listed above. GECs were growth-arrested during 72 h of culture in serum-poor medium, which consisted of DMEM-F-10 (1:l) containing 0.5% FCS.

ically prelabeled GECs were plated into matrix-coated or uncoated 35-mm culture dishes to produce near-confluent monolayers (-10” cells/cm2). Just before extraction, culture medium was removed, and 0.5 ml of cold methanol was added to the cells. Cells were scraped from substrata into a glass tube and were then acidified with 0.75 ml of 0.2% formic acid. After addition of 0.75 ml of chloroform-methanol (1:1.2), followed by 0.75 ml of chloroform, the aqueous phase was separated from the organic phase by centrifugation. In cells labeled with [3H] inositol and [3H]ethanolamine, the aqueous phases were collected and evaporated for subsequent measurement of Measurement of GEC Proliferation inositol phosphates and phosphorylethanolamine, reIn the majority of experiments, proliferation was meas- spectively. The organic phase was evaporated in all exured as DNA synthesis by [3H]thymidine incorporation. periments for subsequent measurement of phospholipids Cells were plated into 24-multiwell culture dishes (18- and phospholipid products. In some experiments, lipids mm wells) at densities of 7,500-15,000 cells/well. To were extracted from GECs in suspension in a similar growth-arrest cells, plating density was reduced to 4,OOO- manner. For measurements of polyphosphoinositides, 6,000 cells/well. Preliminary studies determined that, 5- the extraction protocol was modified by inclusion of 6 h after plating, there were no significant differences in EDTA-KCl, as described previously (8). the efficiency of adhesion to various substrata. [3H] Lipid separation. Lipids were separated on heat-actithymidine (0.25 &i/well) was added to culture medium vated silica thin-layer chromatography plates (8). For for 24 h. Cells were then washed four times with phos- separation of polyphosphoinositides, plates were impregphate-buffered saline and lysed in situ with 0.1 N NaOH. nated with 1% potassium oxalate-2 mM EDTA (8). The lysate was added to scintillation fluid and counted Evaporated samples were redissolved in chloroform and in a beta scintillation counter. Preliminary studies verispotted onto individual lanes on the chromatography fied that 3H in the lysate represented [3H]thymidine plates. Appropriate lipid standards were mixed with the incorporation into DNA. After incubation with [3H]thyradioactive samples. To separate DAG, plates were develmidine, some cells were incubated with cold thymidine oped with the organic phase of ethyl acetate-isooctanefor an additional 3 h (to chase unincorporated [3H]acetic acid-water (55:75:8:100) (8). The major phosphothymidine by competition) and were then washed and lipids (phosphatidylcholine, phosphatidylethanolamine, lysed with NaOH, as above. There were no significant and phosphatidylinositol) were separated with chlorodifferences between the radioactivities in NaOH lysates form-methanol-acetic acid-water (50:37.5:3.5:2) (8). Data of cells that were chased with cold thymidine and cells on phosphatidylserine are not included, as it co-migrated that were not. with lysophosphatidylethanolamine, which constitutes a To confirm that DNA synthesis was followed by cell significant amount of GEC phospholipids (unpublished division, in some experiments cell number was deterobservations). Polyphosphoinositides were separated mined by visual counting. GEC on a collagen gel were with chloroform-methanol-4 N NH,OH (45:35:10), as scraped from collagen-coated 35-mm dishes (with the described previously (8). The bands of interest and the collagen) into a test tube and were then incubated with remainder of each lane were scraped separately into collagenase and trypsin-EDTA (as described above) to scintillation vials and, after addition of scintillation fluid, produce a single-cell suspension. GEC on plastic substrawere counted in a beta scintillation counter. Extraction tum were placed into suspension by incubation with trypsin-EDTA. Suspended cells were then counted in a and counting efficiencies have been described previously (8). Results are presented as the radioactivity in DAG as hemacytometer. a percent of total radioactivity or, alternatively, as the radioactivity per number of cells. (The number of cells Measurement of Phospholipase Activation was determined in preliminary experiments by placing Radiolabeling of phospholipids. For determination of cells in suspension and visually counting them in a products of phospholipase activation, fatty acid chains hemacytometer.) Measurement of water-soluble lipid products. 3H-laor polar head groups of GEC phospholipids were labeled beled inositol phosphates were separated by ion-exwith radioactive tracers for 72 h in 35-, 60-, or loo-mm collagen-coated tissue culture dishes. This resulted in the change chromatography by use of Dowex anion-exchange resin (6). Free inositol was eluted from the columns by labeling of major phospholipids to isotopic equilibrium (8). The tracers included [3H]arachidonic acid (1 ,uCi/ use of water (20 ml), glycerophosphoinositol by 5 mM sodium tetraborate-60 mM ammonium formate (15 ml), ml), [14C]stearic acid (1 ,&i/ml), [3H]inositol (5 &i/ml), IP1 by 5 mM sodium tetraborate-150 mM ammonium [3H]ethanolamine (2 &i/ml), and [3H]choline (2 &i/ formate (20 ml), IP2 by 0.1 M formic acid-O.4 M ammoml). nium formate (20 ml), and IP3 by 0.1 M formic acid-l.0 Extraction of lipids and water-soluble lipid products. M ammonium formate (18 ml). Fractions were collected Phospholipids and products of phospholipase activation into scintillation vials, and, after addition of scintillation were measured in GECs attached to various substrata, as described previously (8). In these experiments, isotop- fluid, were counted in a beta scintillation counter. Before Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 17, 2019.

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separation of samples, column calibration was carried out with [3H]inositol, [3H]IP1, [3H]IP2, and [3H]IP3 standards. [3H]phosphorylethanolamine and free [3H] ethanolamine were separated in the presence of unlabeled standards on heat-activated silica thin-layer chromatography plates by use of methanol-0.5% NaClNH,OH (50:50:1) as a solvent. Measurement of DAG fatty acids. GECs were grown to near-confluence (without radioactive tracers) in 60-mm collagen-coated culture dishes. DAG was measured in cells on type I collagen gel and in cells brought into suspension, as described above. Extraction of lipids was carried out as described for radiolabeled cells, except that larger volumes of solvents were used, and a dipentadecanoin internal standard was added to each sample just before extraction. DAG was separated by thin-layer chromatography, as described above. The DAG bands were scraped into glass test tubes, and lipid was extracted twice from the silica gel into chloroform-methanol (2:l). After evaporation, DAG was incubated in 1 N methanolic-HCl for 16 h at 75°C to hydrolyze and methylate fatty acids. Samples were evaporated, redissolved in isooctane, and analyzed by capillary-column gas chromatography (Ultra 1 cross-linked methyl silicone, 50 m, 0.17~pm film thickness; Hewlett-Packard Canada, Mississauga, Ontario, Canada). The column was precalibrated with fatty acid-methyl ester standards, with a temperature program of 185-285°C at 4”C/min. DAG fatty acids were quantitated by comparing peak areas of each fatty acid with that of the internal standard using a Hewlett-Packard Integrator 3390A. Measurement of lipid phosphorus. GECs were grown to near-confluence (without radioactive tracers) in 60or loo-mm collagen-coated culture dishes. Lipid phosphorus was measured in cells on type I collagen gel and in cells brought into suspension. Extraction of lipids was carried out as described for radiolabeled cells, except that larger volumes of solvents were used. Phospholipid separation was carried out by thin-layer chromatography as described above, except that phospholipid standards were not added to samples, but were chromatographed in parallel lanes. The bands of interest (phosphatidylinositol and phosphatidylcholine) were scraped into glass test tubes. Lipids were extracted twice from the silica gel into chloroform-methanol (2:l). After evaporation, lipid phosphorus was measured as described by Bartlett (4). Statistics Data are presented as means t SE. The unpaired Student’s t test was used to determine significant differences between two groups (unless indicated otherwise). If more than two groups were present, analysis of variance was used to establish significant differences among groups. RESULTS

Effect of Growth Factors and Extracellular Matrix on GEC Proliferation The growth characteristics of GECs in culture are presented in Fig. 1. Equal numbers of cells were plated

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100000

--

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0

1

2

3

Day After Plating FIG. 1. Growth characteristics of GECs. GECs were plated onto type I collagen gel or plastic. Culture media contained 0.5% fetal calf serum (FCS; serum-poor medium, SP), 0.5% FCS + epidermal growth factor (EGF, 10 rig/ml), or 5.0% NuSerum and hormone supplements (Kl). Cell number was determined 1, 2, and 3 days after plating. Kl medium and EGF produced significant increases in cell number above serum-poor medium (P < 0.005, P < 0.01, respectively; two-way analysis of variance). Values are means of: SE of 4 experiments at each data point.

onto type I collagen gel or plastic substrata and were then cultured for 53 days in the presence or absence of growth factors. A progressive increase in cell number was observed in GECs cultured under standard conditions, i.e., adherent to collagen in Kl medium (which contains 5.0% NuSerum and hormone supplements; see METHODS). There was no increase in cell number when GECs were adherent to type I collagen gel and cultured in serum-poor medium (0.5% FCS), indicating that proliferation can be arrested in the absence of growth factors. Serum-poor medium supplemented with EGF (10 rig/ml) also produced a progressive increase in cell number, but the increase was smaller than that observed with Kl medium. In contrast, GECs adherent to plastic substratum did not proliferate, despite the presence of growth factors (5.0% NuSerum and hormone supplements) in the culture medium. Examination of cells by light microscopy revealed that the morphology of GECs on plastic was similar to that of cells on collagen. Thus, GECs were able to adhere to plastic and spread, but they were unable to proliferate. These experiments indicate that presence of both collagen matrix and growth factors is required for GECs to proliferate. Collagen matrix is not able to induce proliferation independently, and the presence of growth factors in the absence of collagen matrix is also insufficient to stimulate proliferation. Results similar to those shown in Fig. 1 were observed when GEC proliferation was assessedas DNA synthesis by [3H]thymidine incorporation (Fig. 2). GECs plated onto type I collagen gel were cultured for 72 h in serumpoor medium (0.5% FCS) to arrest DNA synthesis and produce a synchronized population of cells. Addition of Kl medium resulted in a X0-fold stimulation of [3H]thymidine incorporation above that of cells that remained in serum-poor medium (Fig. 2). Addition of EGF

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TABLE 1. Effects of extracellular T- on GEC proliferation

EGF EGF+lnsulin

matrix proteins

[3H] thymidine Incorporation, cpm 24 h-l. well-l

-

l

. 10000

O-24 h

24-48

h

A. Substratum

-

0

0.01

0.1

1.0

10

100

Kl

EGF (rig/ml) FIG. 2. Effect of growth factors on [“Hlthymidine incorporation. GECs were plated onto type I collagen gel and were cultured in serumpoor medium (0.5% FCS) for 72 h. GECs were then maintained in serum-poor medium for another 24 h or were stimulated for 24 h with EGF, insulin (8 pg/ml), EGF + insulin (8 pg/ml), or Kl medium, in the presence of [“Hlthymidine. Values are means t SE of 3-4 experiments performed in triplicate. (The 2 data points at EGF = 0 represent [3H]thymidine incorporation of GECs in serum-poor medium and in serum-poor medium containing insulin.)

to serum-poor medium also stimulated [3H] thymidine incorporation. Approximately twofold stimulation was observed at EGF concentrations of 0.01-0.1 rig/ml, whereas at l-100 rig/ml, stimulation increased to fourfold (Fig. 2). Insulin (8 pg/ml) independently stimulated [3H]thymidine incorporation and enhanced the effect of EGF (Fig. 2). The independent effect of insulin was also observed at concentrations as low as 100 rig/ml (data not shown). As these concentrations are above physiological, insulin is probably acting via an insulin-like growthfactor receptor. To determine the time of onset of DNA synthesis ( [3H] thymidine incorporation), after adherence to substratum, nonsynchronized GECs were placed into suspension and were then plated onto type I collagen gel in Kl medium containing [3H]thymidine. After 3, 6, or 15 h, [3H]thymidine incorporation was not detectable; but after 24 h, [3H]thymidine incorporation was 26,210 t 5,710 counts per minute (cpm)/well (n = 6). Thus, even though adherent cells were nonsynchronized, DNA synthesis did not occur until 115 h after plating. The effects of extracellular matrix proteins on GEC proliferation are presented in Table 1. Cells in Kl medium were plated onto plastic tissue culture dishes that were uncoated or coated with individual extracellular matrix proteins. By analogy to the findings presented in Fig. 1, [3H]thymidine incorporation was low in GECs on plastic substratum from the time of plating until 24 h and did not change significantly at 24-48 h, whereas [3H] thymidine incorporation was elevated in cells adherent to type I collagen gel at O-24 h and increased markedly at 24-48 h (Table 1). Type IV collagen also supported DNA synthesis (Table 1). Because the amount of material available was insufficient to form gels, plates were coated with films of type IV collagen. For comparison, when type I collagen was applied to the culture dishes as a film (in an amount equivalent to the type IV collagen), the magnitude of [3H]thymidine incorporation was comparable to that seen with type IV collagen, indicating that, at similar concentrations, both types of collagens

Type I collagen gel Type I collagen film Type IV collagen film Plastic B. Substratum Type I collagen gel Laminin Plastic

1,792&306* 652t219 652t237 294tl28

9,688+ 1,250” 3,X4+585* 3,478&884* 44ltl64

5,120+609”f 990+280 717tl48

26,129+4,824$ 2,543+928 1,379+380

Values are means t SE of 3 experiments performed in triplicate. GECs were plated into 18-mm tissue culture dishes that were uncoated (plastic) or coated with extracellular matrix proteins, including type I collagen gel (2.4 mg/ml, 0.2 ml/well), type I collagen film, type IV collagen film, and laminin (each -0.05 mg/well). Culture medium was Kl. [“Hlthymidine incorporation was measured during 1st (O-24 h) or 2nd 24 h (24-48 h) after plating. Significant differences were present between groups at O-24 h and 24-48 h [A, P < 0.001; B, P < lo-“; analysis of variance (ANOVA)]. * P c 0.001, t P < 10W5, and $ P c 10m4 vs. plastic. A greater amount of cells was plated at onset of experiments in B than in A, accounting for the higher radioactivity in 23.

1 100

80

60

.

OQ 0

I

1 Collagen

.

I

2

m

1

3

(mg/mI)

3. Effect of collagen concentration on GEC proliferation. Tissue culture dishes were coated with serial dilutions of type I collagen. At higher concentrations collagen formed a gel. GECs were cultured in Kl medium for 48 h. [“Hlthymidine incorporation was measured during the final 24 h. Values are presented as a % of maximum [“Hlthymidine incorporation (at 3 mg/ml collagen) and are means t SE of 3 experiments. FIG.

were able to support GEC proliferation to a similar extent (Table 1). The effect of laminin was examined in a second series of experiments. In contrast to collagen, laminin did not support GEC proliferation, as [3H]thymidine incorporation of cells adherent to laminin-coated plates was not significantly different from that of cells attached to plastic (Table 1). GECs also did not proliferate in tissue culture plates coated with gelatin (data not shown). Although type I collagen gel was the most effective substratum for GEC proliferation, Fig. 3 demonstrates that the amount of proliferation was dependent on the collagen concentration, and was highest when the collagen was present in a sufficiently high concentration to form a gel. It should be noted that, when GECs were

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plated onto plastic and type I collagen was added to Kl medium, there was no increase in [3H]thymidine incorporation (data not shown), indicating that collagen only facilitated proliferation when it constituted the substratum. This result also suggests that facilitation of mitogenesis by collagen matrix was not due to contamination of the collagen preparation with soluble growth factors. To provide further support for this conclusion, we examined whether DNA synthesis could be induced by FGF (the growth factor believed to be associated with extracellular matrices, in particular with the heparan sulfate proteoglycan component; Ref. 34). GECs were plated into plastic culture wells or wells coated with collagen or heparin (heparan sulfate proteoglycans and heparin are thought to bind FGF and increase its availability to FGF receptors; Ref. 34). [ 3H] thymidine incorporation was measured after adding Kl medium with and without semipurified bovine FGF at a concentration that supported proliferation of human umbilical vein endothelial cells (150 pg/ml). In GECs adherent to plastic or heparin substrata, addition of FGF to Kl medium did not induce DNA synthesis, and FGF did not enhance DNA synthesis above Kl medium alone in GECs adherent to collagen (data not shown). Effect of Growth Factors and Extracellular Matrix on Phospholipids To determine the effect of extracellular matrix on DAG, GECs were prelabeled to isotopic equilibrium with [3H] arachidonic acid (in collagen-coated dishes). Radiolabeled GEC were brought into suspension and were then plated onto plastic or onto culture dishes coated with extracellular matrix proteins. The earliest measurements in adherent cells could be carried out 2 h after plating, at which time a majority of cells had adhered and spread on the substrata to produce near-confluent monolayers. At 2 h, [3H]arachidonoyl-DAG in GECs adherent to plastic was similar to the level seen in GECs in suspension just before plating (Table 2). A similar level of DAG was evident in GECs plated onto laminin (Table 2). In contrast, a marked increase in DAG was seen in GECs adherent to type I collagen gel (Table 2). A smaller but significant increase was observed in GECs that were plated onto a type IV collagen film (Table 2). This 2. Effect of substratum on rH/arachidonoyldiacylglycerol

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increase was comparable to that seen in cells attached to type I collagen film (which contained an equal amount of collagen to the type IV film). Thus increases in DAG were associated only with substrata that supported proliferation, and these increases preceded the onset of DNA synthesis by several hours. Similar increases in [3H]arachidonoyl-DAG were seen at 4 h, 6 h (data not shown), and 24 h (Table 3, below) after plating on type I collagen gel. Furthermore, similar differences in DAG between GECs adherent to collagen and plastic were also seen when plating density was reduced by 80% (data not shown). In addition, it should be noted that an increase in DAG was observed only if cells were attached and spread on a collagen substratum (i.e., when GECs in suspension were incubated with type I collagen that was added to medium, there was no rise in DAG; data not shown). We then examined whether the changes in DAG were associated with the action of GEC growth factors. [3H]arachidonate-labeled GECs were plated onto plastic and collagen gel and were maintained in serum-poor medium for 24 h. As before (Table 2), [3H]arachidonoyl-DAG levels were low in the cells on plastic substratum and were elevated in the cells on collagen (Table 3). Stimulation with Kl medium for 10 s or 2 min did not alter DAG levels in GECs attached to plastic or to collagen (Table 3). Al so, in separate experiments no change in DAG was observed at 20 min (data not shown). The effects on DAG of EGF (10 rig/ml) and of EGF + insulin (10 rig/ml and 8 pg/ml, respectively) were examined in a similar manner in GECs adherent to type I collagen gel. As was observed with Kl medium, neither EGF nor EGF + insulin altered [3H] arachidonoyl-DAG levels (Table 4). These studies indicate that an increase in DAG results from association of GECs with collagen substratum, and this increase is independent of the presence of growth factors. To determine the source of increased DAG associated with extracellular matrix, we monitored changes in [3H] inositol-labeled lipids and the appearance of inositol TABLE 3. Effect of substratum and growth factors on diacylglycerol [3H]arachidonoyl-DAG, % of total radioactivity

TABLE

Substratum

Type I collagen gel Type I collagen film Type IV collagen film Laminin Plastic Suspension

[3H]DAG, % of total radioactivity

3.78t0.46* 1.25+0.17”f 1.19&0.44$ 0.59&O. 10

0.43kO.06 0.52t0.22

Values are means f: SE of 3 experiments. [3H]arachidonate-labeled GECs were plated onto various substrata. Lipids were extracted from cells 2 h after plating, and 1,2-diacylglycerol (DAG) was separated by thin-layer chromatography. A significant difference was present between groups (P < 0.0001, ANOVA). * P < 0.001, t P < 0.005, and $ P < 0.05 vs. mastic.

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Type I collagen gel Serum-poor medium Kl medium Plastic Serum-poor medium Kl medium

10 s

2 min

2.84kO.40 2.14t0.31

2.00k0.25 2.02kO.30

0.40t0.03 0.36t0.02

0.4220.06 0.40t0.03

Values are means k SE of 3 experiments performed in duplicate. GECs were prelabeled with [“Hlarachidonic acid, plated onto type I collagen gel or plastic, and maintained in serum-poor medium for 24 h. GECs were then incubated for 10 s or 2 min with serum-poor medium or Kl medium. Lipids were extracted and BAG was separated by thinlayer chromatography. Significant differences in DAG were present between groups at 10 s and 2 min (P < 10m5 at both time points; analysis of variance). In GECs maintained in serum-poor medium, DAG was significantly higher in cells on collagen compared with plastic (P < 0.0001 at 10 s and at 2 min), but there was no significant stimulation of DAG with Kl medium at 10 s or at 2 min.

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4. Effect of EGF and insulin on diacylglycerol [3H]arachidonoyl-DAG, % of total radioactivity

Control EGF EGF + insulin

10 s

2 min

20 min

3.66t0.45 4.68-1-0.35 5.91t0.63

4.51t0.40 4.71t0.63 4.23kO.51

2.89t0.36 2.38kO.10 2.70t0.30

Values are means rt SE of 4 experiments performed in duplicate (control and EGF), or 2 experiments performed in duplicate (EGF + insulin). GECs were prelabeled with [“Hlarachidonic acid, plated onto type I collagen gel, and maintained in serum-poor medium for 24 h. GECs were then stimulated with serum-poor medium (control), or serum-poor medium containing EGF (10 rig/ml) or EGF + insulin (10 rig/ml and 8 ,ug/ml, respectively). Lipids were extracted, and DAG was separated by thin-layer chromatography. No statistically significant differences were present at any time point (ANOVA).

TABLE 5. Effect of substratum on inositol phosphates and inositol lipids Radioactivity, cpm/104 cells Type

Inositol IPl

gel

Plastic

phosphates

m

I& Inositol PIP2 PIP PI

I collagen

324k44 288t94* 165t18*

305t68 71tl1 21t9

9&l?

48-r-6 102214

lipids

45+7$ 1,785+104”f

4,885+165

Values are means t SE of 3 experiments performed in duplicate. PIP2, phosphatidylinositol4,5-bisphosphate; PIP, phosphatidylinositol 4-phosphate; PI, phosphatidylinositol. [“Hlinositol-labeled GECs were plated onto type I collagen gel or plastic. Inositol phosphates and inositol lipids were extracted from cells 2 h after plating and were separated by ion exchange chromatography and thin-layer chromatography, respectively. * P < 0.001, t P < 0.0001, and $ P < 0.002 collagen vs. plastic.

phosphates. Inositol phospholipids were labeled to equilibrium by culturing GECs (on collagen) in Kl medium supplemented with [ 3H] inositol. Radiolabeled GECs were brought into suspension and were then plated onto plastic substratum or onto type I collagen gel in the presence of 10 mM LiCl (to inhibit inositol phosphatases). After 2 h, GECs on plastic demonstrated low levels of [3H]IP3 and [3H]IPz (Table 5). In contrast, in GECs that were plated onto collagen gel, marked increases in [3H]IP3 and [3H] IP2 were evident (Table 5). Although the elevated IP3 may consist of I(1,3,4)P3 in addition to I(1,4,5)P3, it is reasonable to conclude that I(1,4,5)P3 was generated because I(1,3,4)P3 is a metabolite of I(1,4,5)P3 (13). In association with the increases in IP3 and IP2, GECs adherent to collagen demonstrated a large decrease in the radioactivity of inositol phospholipids (Table 5). Radiolabeled GECs in suspension (before plating) demonstrated high amounts of total inositol lipids that were similar to the amounts in GECs plated onto plastic (data not shown). Thus it is reasonable to conclude that DAG originates, at least in part, from hydrolysis of inositol lipids by phospholipase C. Also, activation of phospholipase C in cells adherent to collagen is present as early as 2 h after plating, at which time 13Hlthvmidine incorporation is not yet detectable.

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Further studies were carried out to confirm that increased DAG results from phospholipase C-induced hydrolysis of inositol lipids. In these experiments, GEC phospholipids were dually radiolabeled with [ 3H] arachidonic acid and [ 14C]stearic acid. Dually-prelabeled GECs were brought into suspension and were then plated onto type I collagen gel or plastic substrata. The [3H]arachidonate and [ 14C]stearate content of DAG was then examined and compared with the 3H and 14C content of parent phospholipids. Table 6 demonstrates that the labeling protocol resulted in a high ratio of 3H to 14C radioactivity in phosphatidylinositol and phosphatidylethanolamine (i.e., the 3H radioactivity exceeded the 14C radioactivity in these two phospholipids). In contrast, the 3H radioactivity was below that of 14C in phosphatidylcholine. Similar to the results presented in Tables 2 and 3, the 3H radioactivity in DAG was low in GECs adherent to plastic substratum (Table 6), and the ratio of 3H to 14C in DAG was similar to that in phosphatidylcholine, suggesting that, in GECs adherent to plastic, the DAG pool is in equilibrium with phosphatidylcholine. Although less than in other experiments (Tables 2 and 3), collagen stimulated a greater than twofold increase in [3H]DAG (Table 6). This was associated with a smaller increase in [‘“CIDAG, as well as a decrease in 3H and 14C radioactivity in phosphatidylinositol. It should be noted that the ratio of 3H to 14C in the collagen-stimulated increase in DAG (i.e., 257 vs. 207 cpm; Table 6) is equivalent to the ratio of 3H to 14C in phosphatidylinositol in GECs adherent to plastic (Table 6) and in the portion of phosphatidylinositol that decreased as a result of cell contact with collagen (i.e., 2,011 vs. 1,633 cpm; Table 6). Therefore the DAG that is produced as a result of cell contact with collagen substratum is most likely due primarily to hydrolysis of inositol phospholipids. Phosphatidylethanolamine, although quantitatively more 6. Arachidonate and stearate labeling of phospholipids and diacylglycerol TABLE

Radioactivity, cpm/105 cells [ 3H] arachidonate

[ 14C] stearate

Phosphatidylcholine Collagen Plastic

3,403+515

Collagen Plastic

27,304+1,569 26,399&1,938

7,159+1,167 7,190-c-1,130

3,234+499 Phosphatidylethanolamine

8,900+1,100 9,000+_1,004

Phosphatidylinositol Collagen Plastic

1,203+163* (-2,011) 3,214*659

889+169t 2,522+315

(-1,633)

Diacylglycerol Collagen Plastic

466+31$ 209t21

(+257)

698+36-f' (+207) 491k27

Values are means t SE of 5 experiments performed in duplicate or triplicate. GECs were dually prelabeled with [“Hlarachidonic acid and [‘*C]stearic acid and were then plated onto type I collagen gel or plastic. Lipids were extracted after 4 h for DAG and phospholipid measurements. DAG and phospholipids were separated by thin-layer chromatography. * P c 0.004, t P < 0.0001, and $ P < 0.00001 collagen vs. plastic.

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important than phosphatidylinositol, is a less likely source of DAG (l3), because the distribution of 3H and 14C in this phospholipid is less compatible with that of the collagen-stimulated increase in DAG, whereas phosphatidylcholine is an unlikely source because its labeling pattern is incompatible with the collagen-stimulated increase in DAG. The greater decrease in phosphatidylinositol compared with the increase in DAG (Table 6) may be accounted for by parallel collagen-induced increases in DAG metabolism. To confirm that additional DAG does not arise from other quantitatively more important phospholipids (13), in a another series of experiments we examined whether collagen substratum could induce changes in ethanolamine- or choline-labeled phospholipids. GECs were prelabeled with [3H] ethanolamine or [3H] choline, brought into suspension, and replated on type I collagen gel or onto plastic. In contrast to the changes seen in inositol phospholipids, there were no differences in [3H]phosphatidylethanolamine between GECs adherent to collagen and those on plastic (2,915 t 429 vs. 2,810 t 524 cpm/ lo4 cells, respectively; n = 5 experiments performed in duplicate) or in [3H]phosphatidylcholine (1,990 t 350 vs. 2,240 t 290 cpm/104 cells, respectively; n = 3 experiments performed in duplicate). Furthermore, because phosphatidylethanolamine is rich in [3H]arachidonate, a small amount of hydrolysis could possibly lead to increased DAG without a detectable decrease in the phospholipid. However, we found no differences between collagen and plastic in cellular levels of [3H]phosphorylethanolamine (757 t 268 vs. 935 t 331 cpm/105 cells) or free [3H]ethanolamine (185 t 66 vs. 189 t 67 cpm/105 cells). Thus phospholipid hydrolysis is not generalized, but appears to involve only a subset of cellular phospholipids. Quantitation of Diacylglycerol Fatty Acids and Lipid Phosphorus

Table 7 presents the fatty acid composition of DAG, as determined by gas chromatography. Because there was no significant difference in [3H] arachidonoyl-DAG levels in GECs on plastic substratum and in suspension (Table 2), for technical reasons DAG fatty acids from GECs in suspension (rather than on plastic) were compared with those from GECs on type I collagen gel. (It was not practical to plate enough cells onto plastic culture dishes to obtain an adequate amount of material for TABLE

7. Fatty acids in diacylglycerol

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quantitative measurements.) As in radiolabeled GECs (Tables 2 and 6), the arachidonic acid content of DAG was low in GEC in suspension, and was increased in cells adherent to collagen (Table 7). Other fatty acids in DAG were present in amounts greater than arachidonate, and there were upward trends in the content of stearic, palmitic, and linoleic acids from suspension to collagen (Table 7). The effect of collagen substratum on the masses of arachidonic and stearic acids in DAG (Table 7) is in keeping with the 3H and 14C determinations of DAG levels, respectively (Tables 2, 3, and 6), indicating that changes in radioactivity of DAG reflect changes in lipid mass. To verify that the effects of collagen on [3H]inositollabeled and [3H]choline-labeled phospholipids were indicative of changes in phospholipid mass, we compared the phosphorus content of these lipids in GECs in suspension to GECs attached to a collagen gel matrix. In suspension, lipid phosphorus in phosphatidylinositol was 0.44 t 0.12 pg/6 X lo6 cells (n = 6), and declined to 0.18 t 0.04 pg/6 X lo6 cells on collagen (n = 6, P < 0.03). Lipid phosphorus in phosphatidylcholine was similar in GECs in suspension and on collagen [1.72 t 0.17 (n = 9) vs. 1.70 t 0.20 pg/6 X lo6 cells (n = 9), respectively). Association Between GEC Proliferation and Activation of PKC

An increase in DAG may lead to the activation of PKC (29, 33). Because arachidonoyl-DAG was elevated in GECs adherent to collagen matrix, we examined whether in these cells PKC might be activated to mediate GEC proliferation. First, [3H] thymidine incorporation was measured in the presence or absence of exogenous PKC activators, PMA, and OAG (a synthetic, cell-permeable DAG)(33). Table 8 demonstrates that inclusion of PMA or OAG in Kl medium decreased [3H] thymidine incorporation. Similarly, PMA decreased the proliferative ef8. Effect of phorbol myristate acetate and 1-oleoyl-2-acetylglycerol on GEC proliferation TABLE

[3H]thymidine Incorporation, cpm ‘24 h-l. well-’ O-24 h

24-48

h

-

A) Kl Untreated PMA (30 nM) PMA (300 nM) OAG (100 pg/ml)

4,671+861 1,082+516” 2,158-+62gb 1,617+556”

16,696+558 4,846+392” 6,014+1,024” 9,294+l,409d

B) EGF (10 nglml) GECs, Fatty

pg/2

x lo6

Acid

Palmitic Stearic Oleic Linoleic Arachidonic n

Suspension

Collagen

1.53k0.61 2.23t0.91 4.67t2.53 0.98t0.96 co.2 4

1.94t0.58 2.64kO.70 4.76t2.13 1.81t0.66 1.6420.37 5

Values are means t SE; n, no. of experiments. from GECs in suspension and GECs adherent DAG was separated by thin-layer chromatography. were quantitated by gas chromatography.

Lipids were extracted to type I collagen gel. Fatty acids in DAG

Untreated PMA (30 nM) PMA (300 nM)

1,083+221 585t296 277t174

5,827+772 2,089+606” 885+282f

Values are means t SE of 2-3 experiments performed in tripi= GECs were plated onto type I collagen gel in Kl medium (A) or serumpoor medium containing EGF (10 rig/ml, B), with and without phorbol myristate acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG). [“HIthymidine incorporation was measured during 1st or 2nd 24-h period after plating. A: statistically significant differences were present between groups at O-24 h (P < 0.005), and 24-48 h (P c 10m7; ANOVA). aP < 0.01, b P < 0.02, ’ P c 10F5, and d P < 0.0003 vs. untreated. B: statistically significant differences were present between groups at 24-48 h (P < ANOVA). e P < 0.002 and f P < 0.0001 vs. untreated.

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feet of EGF (Table 8). The anti-proliferative action of PMA was not associated with modulation of the [3H]arachidonoyl-DAG level (Table 9). As exogenous activation of PKC inhibited DNA synthesis, we then examined whether DNA synthesis might be downregulated in GECs adherent to collagen matrix as a result of endogenously enhanced PKC activity secondary to increased levels of DAG. Thus, DNA synthesis was examined in GECs adherent to collagen, following inhibition of PKC. In the first series of experiments, PKC was depleted by an 18-h preincubation with a high concentration of PMA (2 PM; 36). After removing PMA and adding Kl medium, [ 3H] thymidine incorporation of PKC-depleted GECs was significantly increased compared with PKC-replete cells (Fig. 4). To confirm this result, in a second series of experiments growth of GECs was arrested by culture in serum-poor medium for 72 h 9 and DNA synthesis was then stimulated with Kl medium, in the presence or absence of the protein kinase inhibitor H-7 (19). Inhibition of PKC with H-7 also augmented [3H]thymidine incorporation and was more effective than PKC depletion with PMA (Table 10). Thus PKC appears to be activated in GECs adherent to collagen matrix, and PKC desensitizes GECs to mitogenic effects of growth factors. A possible reason for the greater apparent PKC inhibi-

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10. Effect of protein kinase C-inhibitor H- 7 on GEC proliferation TABLE

[ 3H] thymidine Incorporation, cpm .24 h-l . well-’

Kl Kl Kl

(untreated) + H-7 (25 PM) + H-7 (100 PM) 0.5% FCS

20,733+2,208* 35,405&5,271? 36,259+6,788$ 1,350+321

Values are means & SE of 3 experiments performed in triplicate. GECs were plated onto type I collagen gel and were cultured for 72 h in serum-poor medium [0.5% fetal calf serum (FCS)]. Subsequently, cells were cultured for 24 h in serum-poor medium or in Kl medium containing [“Hlthymidine, in the presence or absence of 1-(5-isoquinoylinylsulfonyl) -2-methylpiperazine (H-7). A statistically significant difference was present between groups (P < lo-“; ANOVA). * P c low5 vs. 0.5% FCS. “f P < 0.01, $ P < 0.02 vs. Kl (untreated).

9

9. Effect of phorbol myristate acetate on diacylglycerol

TABLE

[3H]arachidonoyl-DAG, % of total radioactivity

Untreated PMA (30 nM) PMA (300 nM)

20 min

24 h

3.15t0.08 3.21t0.30 5.28t1.55

2.70t0.66 1.89t0.24

2.23t0.36

Values are means t SE of 3 experiments performed in duplicate. GECs (adherent to type I collagen gel) were prelabeled with [3H]arachidonic acid, and were stimulated for 20 min or 24 h with PMA. Lipids were extracted, and DAG was separated by thin-layer chromatography. There were no statistically significant differences between groups at 20 min and at 24 h (ANOVA).

40000 1

m 0-

Control

PKC-depleted

4. [3H]thymidine incorporation in protein kinase C (PKC)depleted GECs. GECs were plated onto type I collagen gel and were cultured for 18 h in Kl medium containing phorbol myristate acetate (PMA, 2 PM) to downregulate PKC (PKC-depleted) or without PMA (control). After washing, GECs were cultured for 24 h in Kl medium containing [“Hlthymidine. Individual values of 5 experiments performed in triplicate and means rt SE of groups are presented. P c 0.05 control vs. PKC-depleted (paired t test). FIG.

tory effect of H-7 may be that depletion of PKC with PMA was incomplete or, alternatively, that during the early part of the 18-h depletion protocol proliferation was suppressed, resulting in a lower number of PKCdepleted GECs compared with untreated control GECs at the end of the downregulation period (i.e., at the start of [3H] thymidine incorporation). Additional experiments were carried out to confirm that prolonged incubation of GECs with a high concentration of PMA resulted in PKC depletion. After the 1% h incubation with PMA (2 PM), GECs were further incubated in Kl medium containing [3H]thymidine, with or without PMA (300 nM; as in Table 8). There was no difference in the 24-h [3H]thymidine incorporation between the two groups of cells; PMA (300 nM): 17,294 t 5,436 cpm/well vs. no PMA: 18,078 t 6,140 cpm/well (n = 3 experiments performed in triplicate). Therefore, the antiproliferative effect of PMA (30 or 300 nM) in PKCreplete GECs (shown in Table 8) was abolished by pretreatment of GECs with a high concentration of PMA. DISCUSSION

In this study we have demonstrated a novel interaction between extracellular matrix components, growth factors, and inositol phospholipids in rat GECs in culture. Our results show that collagen matrices induce mitogenic responsiveness to epithelial growth factors and activate phospholipase C, and that the DAG-PKC signaling pathway modulates the mitogenic effects of growth factors. Increases in cell number and/or DNA synthesis ( [3H] thymidine incorporation) could be induced with serum, EGF, or insulin only in GECs adherent to types I or IV collagen matrices (Figs. 1 and 2), but not in GECs attached to plastic, laminin, or gelatin substrata (even though by light microscopy there did not appear to be impairment of GEC adhesion or spreading on the surfaces that did not support proliferation). GECs cultured on collagen in serum-poor medium (0.5% FCS) did not proliferate; consequently, collagen matrix is required for growth factors to induce mitogenesis, but collagen by itself has no mitogenic activity. Previous studies have shown that other cultured epithelial cells can be maintained in a viable state longer when adherent to collagen substratum compared with plastic (21), but, unlike

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GECs, the requirement for a collagen matrix for proliferation was not essential. In GECs adherent to substrata that facilitated proliferation (types I and IV collagen), [3H]arachidonoyl-DAG levels were elevated compared with cells adherent to laminin or plastic, or in suspension (Table 2). The type I collagen-induced increase in DAG was associated with an increase in [3H] IP3 and [3H] IP2 (Table 5), as well as a decrease in [3H]inositol-labeled phospholipids (Table 5) and in the mass of phosphatidylinositol. These changes are consistent with the activation of a phosphoinositide-directed phospholipase C. The increase in DAG observed in cells adherent to type IV collagen probably also resulted from phospholipase C-induced hydrolysis of inositol lipids, but due to limited availability of type IV collagen, we were unable to measure this directly. DAG was found to be elevated in cells adherent to collagen at 2, 4, 6, and 24 h after plating. These measurements represent steady-state levels in nonsynchronized GECs, and further studies are required to determine if the magnitude of collagen-induced phospholipase C activity varies during the cell cycle. Elevated DAG in cells adherent to collagen was due to a direct effect of the matrix, as it was not dependent on the presence of serum, EGF, or insulin in the culture medium (Tables 3 and 4). These results are consistent with a previous study that failed to demonstrate phospholipase C activation by EGF in renal mesangial cells (26). Also, to our knowledge, our study is the first demonstration that collagen matrices can activate phospholipase C directly, and that matrix-induced phospholipase C activation may be a biochemical signal for control of GEC proliferation (discussed below). It should be noted that, similar to our results, a recent study demonstrated that adherence of BHK cells to fibronectin substratum produced increases in inositol phosphates, compared with BHK cells adherent to plastic (7). However, unlike GECs, BHK cells were unable to spread on plastic, but only on fibronectic, suggesting that production of inositol phosphates may be associated more directly with cell spreading. Because DAG originated as a result of a novel interaction between collagen and phospholipase C, studies were carried out to further characterize the DAG. In GECs adherent to plastic, which were dually labeled with [3H]arachidonic and [14C]stearic acids, the pattern of radioactivity in DAG resembled that of phosphatidylcholine, suggesting that the pool(s) of DAG in these unstimulated cells may be in equilibrium with choline phospholipids. The collagen-induced increase in 1,2-DAG was similar in the distribution of radioactivity to that in phosphatidylinositol, but not in phosphatidylcholine or phosphatidylethanolamine (Table 6). It is believed that phosphatidylinositol is in rapid equilibrium with phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, and, although there may be some quantitative differences in the fatty acid composition of these three inositol lipids (3), the fatty acid composition of phosphatidylinositol bisphosphate is likely to resemble that of phosphatidylinositol. Therefore, these experiments further support the origin of DAG from inositol phospho-

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lipids. Quantitative measurements of DAG fatty acids (Table 7) demonstrated that collagen matrix stimulated an increase in arachidonoyl-DAG, confirming the isotopic measurements. Other fatty acids in DAG were, however, more abundant than arachidonate, and although upward trends were evident in palmitic, stearic, and linoleic acids between GECs in suspension and GECs adherent to collagen, there was no substantial increase in any single fatty acid. These data support the existence of multiple pools of DAG in different subcellular sites, and suggest that only a small arachidonate-rich pool is expanded as a result of phospholipase C activation following adherence of GECs to collagen matrix. Also, this arachidonate-rich DAG pool probably consists of several molecular species of DAG (i.e., several different fatty acyl groups in the ~2-1 position may be paired with arachidonate in the sn-2 position), indicating that in GECs, as in hepatocytes, the parent inositol phospholipids may not be exclusively constituted of arachidonic and stearic acids (3). Activation of the DAG-PKC pathway by extracellular matrices that support GEC proliferation appears to be a signal for the regulation of proliferation. DAGs are endogenous activators of PKC, with DAGs containing an unsaturated fatty acid being most active (29). In the presence of collagen matrix and growth factors, addition of exogenous PKC activators (PMA and OAG) inhibited [3H]thymidine incorporation in GECs (Table 8). More notable was the finding that, after PKC depletion (by 18-h incubation with a high dose of PMA) and after PKC inhibition with H-7, the mitogenic response to growth factors was enhanced (Fig. 4, Table 10). Therefore, in GECs adherent to collagen, PKC or PKC substrates appeared to be endogenously activated in parallel with elevated DAG, and activation of PKC downregulated growth factor-induced proliferation. Thus, collagen matrices both induced responsiveness to mitogenic effects of epithelial growth factors, and, in addition, through the DAG-PKC pathway they activated mechanisms that desensitized GECs to the effects of growth factors. Previously, exogenous activation of PKC has been shown to inhibit proliferation of endothelial and vascular smooth muscle cells (12, 20). Alternatively, in neoplastic cells, steady-state elevations in DAG were associated with increased steady-state PKC activity and increased proliferation (14, 37). The mechanism by which PKC may regulate proliferation in GECs and its subcellular sites of action require further study. PKC might reduce proliferation by decreasing EGF receptor number, affinity, or kinase activity (33), but PKC does not appear to modulate DAG levels (Table 9) (29). Further study is also required to determine if the collagen-induced increases in IP3 and possible associated changes in cytosolic Ca2’ affect GEC proliferation. Studies in cultured cells and in experimental animals have shown that collagen matrices can induce differentiation or maintain differentiated functions of cells (21). We have observed that GECs, when adherent to collagen substratum, can be maintained in long-term culture, and that differentiated morphological features of epithelium are preserved for >65 passages (32 and unpublished

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observations). Cells that are performing differentiated functions have relatively lower rates of proliferation, compared with less-differentiated cells. In GECs, besides reducing the rate of proliferation, activation of PKC by collagen matrix might therefore be a signal for maintenance of differentiation or promotion of differentiated functions, such as synthesis of basement membrane components (1). This area is currently under investigation. The mechanism by which extracellular matrix induces responsiveness to mitogenic effects of growth factors remains to be elucidated. For example, it is possible that, in GECs, collagen matrix induces certain structural alterations that are required for growth factor-induced mitogenesis, but are not detectable by standard light microscopy (23). Alternatively, adherence to matrix may be required to induce expression of early growth-response genes (11) and competence for cell division that would occur after exposure to growth factors. Finally, collagen might increase the expression of growth factor receptors on the GEC plasma membrane, the rate of receptor internalization, or receptor kinase activity. Further investigation is also required to determine how phospholipase C is activated in GECs adherent to collagen substratum. First, there might be a specific interaction of collagen with a membrane receptor that triggers phospholipase C activation. For example, in platelets, types I and IV collagens interact with membrane glycoprotein Ia (28, 39, and this interaction leads to hydrolysis of phosphatidylinositol (28). A protein with related structure and function might be expressed on the membranes of GECs. Second, phospholipase C activation might be due to a modulation of substrate affinity for the enzyme by extracellular matrix or due to matrixinduced redistribution of cell proteins (23)) including phospholipase enzyme and/or substrate among subcellular compartments, such that availability of substrate for the enzyme is increased. Finally, phospholipase C activation might be due to induction of phospholipase C enzyme synthesis by extracellular matrix. One can speculate on how the effects of growth factors and extracellular matrix observed in vitro might control GEC proliferation in vivo. Under normal conditions, GECs are in contact with an extracellular matrix that contains type IV collagen, and in the local environment of the glomerulus there is likely to be a low concentration of epithelial growth factors. The predominant effect of matrix-induced phospholipase C activation in the normal state might be PKC-directed suppression of proliferation and promotion of differentiated functions of GECs. In contrast, in glomerulonephritis, glomeruli may become infiltrated with inflammatory cells, including macrophages or platelets (18), which are sources of epithelial growth factors. In particular, macrophages may secrete transforming growth factor-a (24), and platelets may secrete EGF (30). There may also be accumulation of interstitial and basement membrane collagens in glomeruli (18), possibly altering phospholipase C activity in GECs. As a result, the concentration of factors that facilitate GEC proliferation might increase and lead to altered GEC function and enhanced proliferation. Further studies on the interaction of between GECs and

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matrix components may lead to the definition anisms of glomerular injury in nephritis.

of mech-

We thank Marie-Danielle Cyr and Klara Rostworowski for their expert technical assistance. This work was supported by Research Grants from the Medical Research Council of Canada, the Kidney Foundation of Canada, National Institute of Diabetes and Digestive and Kidney Disease Grants DK-30932, DK-39773, and DK-38965, Research Service Award DK07730 to R. J. Quigg, and a grant-in-aid from the American Heart Association, Massachusetts Affiliate. A. V. Cybulsky holds a Scholarship from the Medical Research Council of Canada and, during part of this work, was a Fellow of the Medical Research Council of Canada. J. V. Bonventre and D. J. Salant are Established Investigators of the American Heart Association. Address for reprint requests: A. V. Cybulsky, Division of Nephrology, Royal Victoria Hospital, 687 Pine Ave. West, Montreal, Quebec H3A lA1, Canada. Received

1 November

1989; accepted

in final

form

7 March

1990.

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