JOURNAL OF CELLULAR PHYSIOLOGY 152:410-421(1992)

Competition Between Cell-Substratum Interactions and Cell-Cell Interactions PIERRE S. TUNG, KRYSTYNA BURDZY, KATHERINE WONG, AND IRVING B. FRITZ* Banting and Best Department of Medical Research, Univenity of Toronto, C.H. Best Institute, Toronto, Ontario M 5 C 1 L6, Canada Clusterin, a glycoprotein which elicits the aggregation of a wide variety of cells (Fritz, I.B., and Burdy, K.: J. Cell Physiol., 140:18-28, 19891, has been utilized to investigate some of the factors modulating the competition between cell-substratum interactions and cell-cell interactions. We compared the responses to clusterin by anchorage-independent cells (erythrocytes) with those by anchoragedependent TM4 cells (a cell line derived from neonatal mouse testis cells). Cells were maintained in culture in the presence of various substrata chosen to enhance cell-substratum interactions (laminin-coated wells), or to diminish cell-substratum interactions (agarose-coated wells). Results obtained showed that the aggregation uf erythrocytes elicited by clusterin was independent of the nature of the substratum. In contrast, clusterin addition resulted in aggregation of anchorage-dependent TM4 cells only when TM4 cell-substratum interactions were weak. Thus, clusterin did not aggregate TM4 cells plated upon a laminin substratum, but readily aggregated TM4 cells plated upon an agarose-coated substratum, independent of the sequence of addition of cells and clusterin to the culture dish. W e utilized YIGSR, a peptide which competes with laminin for laminin receptors, to determine the possible rule of laminin receptors on TM4 cells in the competition between cell-substratum interactions and cell-cell interactions. Thr presence of YlGSR did not alter responses of erythrocytes to clusterin under all conditions examined. In contrast, the responses of TM4 cells to clusterin were greatly changed. YIGSR addition resulted in the inhibition of aggregation of TM4 cells otherwise elicited by clusterin. YIGSR also prevented attachment of TM4 cells to a laminin-coated surface, but this was reversed by the presence of clusterin. We discuss the possible roles of clusterin and laminin in altering the balance in the competition between cell to cell interactions and cell to substratum 0 1992 Wiiey-Liss, Inr. interactions.

During embryogenesis and development, cell interactions are of fundamental importance, and are thought to play a significant role in the maintenance of tissue histotype, long after commitment has occurred during differentiation (Bernfield and Banerjee, 1978; Hay, 1981; Alberts et al., 1983; Edelman and Thiery, 1985; Fritz and Tung, 1986; McClay and Ettensohn, 1987).In intact organisms, it is often difficult to assess the extent to which interactions of cells with the substratum may influence cell behavior. In cells in culture, however, there is no doubt concerning the impressive effects of substratum on cell properties, ranging from influences on adhesiveness and morphology t o controls of gene expression and functions (for reviews, see Ekblom et al., 1986; McClay and Ettensohn, 1987; Kleinman et al., 1988). Interactions of cells with each other, and with the substratum, generate extracellular signals which influence the cytoskeleton, which in turn is an important determinant in generating the polarized epithelial cell phenotype (Rodriquez-Boulanand Nelson, 1989;Schoenberger and Matlin, 1991). In the polarized cell, receptors to laminin and other extracellular matrix (ECM) components are localized on the basal plasma mem0 1992 WILEY-LISS. INC

brane surface, adjacent to the basal lamella; cell adhesion molecules and junctional complex proteins are localized on the lateral plasma membrane surface; and various ion channel or transporter proteins are preferentially localized on the apical plasma membrane surface (Simons and Fuller, 1985; Rodriquez-Boulan and Nelson, 1989). Mechanisms responsible for generating and maintaining this remarkable phenotype are only partially understood. Signals received during cell-substratum interactions, via integrin receptors (for reviews, see Ruoslahti and Pierschbacher, 1987; Ruoslahti, 1991; Quaranta and Jones, 19911, are different from signals received during cell to cell interactions (Edelman, 1985; McClay and Ettensohn, 1987). There can be some overlap, however, since specific integrin

Received October 25,1991: accepted February 25,1992.

*To whom reprint requestsicorrspondence should be addressed a t Department of Molecular and Cellular Physiology, AFRC Institute of Animal Physiology and Genetics Research, Babraham Hall, Cambridge CB2 4AT, United Kingdom.

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receptors bind to intercellular adhesion molecules, identified in cells in the immune system (Springer, 1990). Both types of cell interactions are able to induce the reorganization of membrane proteins, but to varying extents (see Fig. 2 in Rodriquez-Boulan and Nelson, 1989). These phenomena are clearly of fundamental importance, since many-indeed most-biological processes are dependent upon the generation and maintenance of polarity. For examples, the roles of cell polarity in embryogenesis and in vectorial transport systems have recently been reviewed (Rodriquez-Boulan and Nelson, 1989). In investigations reported in this communication, we have examined some of the factors which modulate the balance in the competition between cell-substratum interactions and cell-cell interactions. We have approached the problem by comparing the behavior of anchorage-dependent cells with that of anchorage-independent cells in culture under conditions which have been manipulated to alter the degree of cell adhesion or cohesion, respectively. In experiments designed to alter the degree of cellcell cohesiveness, we have utilized clusterin, a glycoprotein which elicits the aggregation of several different types of cells (Fritz e t al., 1983; Blaschuk et al., 1983; Fritz and Burdzy, 1989; Fritz e t al., 1991). Clusterin is a widely distributed protein which has recently been shown to influence many systems, including not only cell aggregation but also the inhibition of complement-mediated lysis, the transfer of lipids between lipoprotein particles, and the involution of prostatic tissue following castration (for reviews, see Sylvester et al., 1991; Jenne and Tschopp, 1992). Mechanisms of action of clusterin are not understood, but this protein has been implicated in a wide variety of membranemediated events. For experiments described in this communication, we employed clusterin simply as a useful reagent for enhancing the aggregation of cells. In experiments designed to alter the degree of cell adhesion to the substratum, we have compared the effects of plating cells onto a surface coated with laminin, a component in ECM to which many cells form a strong attachment, with the effects of plating cells onto a surface coated with agarose, a gel to which most cells have a very weak attachment. In other experiments designed to evaluate the competition between cell-substratum interactions and cellcell interactions, we have determined the influences of the pentapeptide YIGSR in systems described. This pentapeptide, located in the cysteine-rich domain of the B1 chain of laminin, competes with laminin for the laminin receptor (Kleinman and Weeks, 1989; Graf et al., 1987). Results obtained with these approaches demonstrate that the aggregation of anchorage-independent cells (erythrocytes) elicited by clusterin is independent of the nature of the substratum, and that it is not influenced by the presence of YIGSR. In contrast, the aggregation of anchorage-dependent epithelialtype cells (TM4 cells) elicited by clusterin is greatly dependent upon the nature of the substratum and the presence of YIGSR. These observations are interpreted to indicate that increased cell-cell interactions elicited by clusterin become dominant only when cell-substratum interactions are weak. In addition, we present data

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supporting the view that laminin is implicated not only in cell-substratum interactions, but also in cell-cell interactions.

MATERIALS AND METHODS Materials Ram rete testis fluid (RTF) was a generous gift from Dr. Michel Courot, Nouzilly, France. Clusterin was isolated and purified from RTF by immunoaffinity chromatography, using monoclonal antibodies and procedures previously described (Blaschuk et al., 1983). YIGSR pentapeptide, YIGSE pentapeptide (used as a n “irrelevant peptide”), laminin, and rabbit antiserum against laminin were generously provided by Dr. H. Kleinman of the National Institutes of Health (Bethesda, MD). Agarose was supplied by Bethesda Research Laboratories (Gaithersburg, MD). All incubation media and trypsin were purchased from GIBCO (Grand Island, NY). Soybean trypsin inhibitor, human fibronectin, and bovine serum albumin, fraction V (BSA),were obtained from Sigma Chemicals (St. Louis, MO). Modified Eagle’s minimal essential medium (MEM) was prepared as previously described (Tung et al., 1975). U-bottom wells of polyvinyl chloride microtitration plates (96-well) were purchased from Dynatech (Alexandria, VA). Linbro flat-bottom cell culture plates (24-well) were supplied by Flow Laboratories (McLean, VA). Bacterial culture dishes were purchased from Becton Dickinson (Lincoln Park, NJ). Cell preparation TM4 cells, originally isolated from testes of neonatal mice (Mather, 1980),were grown a t 37°C in 1:l (vol/vol) mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium (F12-DMEM), containing 5%horse serum and 2.5% new born calf serum. Cells were trypsinized and subcultured every 72 h, as described previously (Fritz et al., 1983). Sheep red blood cells (SRBC) were freshly isolated from whole sheep blood and washed twice with phosphate buffered saline (PBS). In some experiments, these cells were used directly after having been diluted to a density of 1 x 106/ml in Dulbecco’s PBS. In other experiments to be designated, SRBC were fixed in 0.5% glutaraldehyde for 30 min and then washed twice with PBS. Fixed SRBC were stored at 4°C and used within 2 weeks. Coating of cell culture dishes Agarose in water suspension was solubilized in hot distilled water a t a concentration of 0.67% (wtivol), and 1 ml of hot agarose solution was transferred into wells in Linbro 24-well plates. The solution was immediately withdrawn, leaving a film covering the bottom of the well (Tung and Fritz, 1991).The coated wells were then stored a t 4°C for later use within 4 weeks. Laminin (100 kg/ml) was solubilized in 20 mM sodium carbonate buffered at pH 9.6. Linbro 24-well plates were coated with 1ml/well of laminin solution at 4°C overnight. After being coated wells were washed three times in PBS, sterilized under ultraviolet light (UV), and saturated in MEM for 3 h (Tung and Fritz, 1986a,b).

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Cell incubation procedures Procedures for cell incubation under rotation were essentially the same as those previously reported (Fritz et al., 1983). Briefly, TM4 cells were detached by 0.1% trypsin in Ca2+-Mg2+-freeHanks' balanced salt solution (HBSS?for 2 min. Cells were then washed, first in F12-DMEM with serum containing 0.1% trypsin inhibitor, and subsequently in F12-DMEM without serum but containing 0.1% soybean trypsin inhibitor, and 1% BSA. Dispersed TM4 cells or SRBC were incubated in U-bottom microtitration plates. Each well contained a final volume of 100 pl PBS, and cell suspensions to yield a final density of 1 x lo4 cells per well. In most experiments, additions of clusterin, RTF, and peptides were made prior to the addition of cells. In some experiments to be designated, clusterin was added a t varying periods after cells were added. Plates were rotated a t a n angle of 30" and at a speed of 12 rpm in chambers maintained at 37"C, having a humidified atmosphere containing 95% air and 5% C02. In other experiments designated, cells were incubated without rotation in flat-bottom culture plates. In these cases, TM4 subcultures were digested with 0.25% trypsin in Ca" -Mg2+-free HBSS a t 37°C for 20 min; washed as above with medium containing 0.1% soybean trypsin inhibitor and then with HBSS alone; and cells were finally resuspended in MEM a t densities desired unless otherwise stated. Cells were plated a t a density of 1 x 105iwell (200 mm2) in 0.5 ml MEM in wells of cell culture plates described, and were incubated without rotation under conditions identical to those described in the preceding paragraph. In all experiments, triplicate wells were employed for each treatment described. Scoring and semi-quantitative assessment of cell aggregation Qualitative scoring of the degree of aggregation of TM4 cells and SRBC was performed as previously described for cells incubated in U-wells in microtitration plates under rotation (Fritz et al., 1983). The numerical scores denoting the degree of aggregation were the same as those previously defined (Fritz and Burdzy, 1989). Independent evaluations by each of three investigators resulted in nearly identical scores by these criteria. For cells incubated without rotation in flat-bottom cell culture wells, semi-quantitative assessments for cell aggregation were performed a s follows. Undisturbed culture plates were observed at 100 x magnification with a Nikon inverted microscope. In each well, five microscopic fields, each equal to 1mm2 in surface area, were randomly chosen. The numbers of cells attached to the bottom, and numbers of unaggregated single cells within the field, were counted separately. The sum of cells in each category in five fields was multiplied by 40 to represent the total number of cells in the well (200 mm'). The number of aggregated cells was estimated by determining the difference between the total number of cells plated per well, and the sum of attached plus single, suspended cells counted.

Immunofluorescent technique TM4 cells in suspension culture were aggregated by treatment with clusterin (50 pgiml). Twenty-four hours after plating, the non-adhering aggregates formed were frozen, and cryostat sections (5 pm thickness) were cut. The sections were air-dried for 15 min and treated with acetone at -10°C for 7 min. Preparations were then washed with multiple changes of PBS at 4°C. The specimens were incubated with rabbit antiserum against laminin in serial dilutions in PBS, and were again washed with PBS containing 0.05% Tween 20. Aliquots of affinity-purified F(ab'), fragments of goat immunoglobulin, conjugated with fluorescein isothiocyanate (Cappel Laboratories, West Chester, PA) diluted 1:20 with PBS, were added to the washed specimens and incubated for 30 min. This was followed by three additional washes with PBS containing 0.05% Tween 20. The preparation was then mounted and examined with fluorescent microscopy by the same procedures a s those previously utilized (Tung and Fritz, 1978).

RESULTS Influences of clusterin on the aggregation of anchorage-dependent vs. anchorage-independent cells Addition of clusterin at time zero, prior to seeding of anchorage-dependent TM4 cells onto uncoated microplate wells, resulted in a nearly complete aggregation of all cells (Fig. l), in confirmation of previous findings (Fritz and Burdzy, 1989). However, subsequent addition of clusterin, 5-10 min after plating the TM4 cells, did not elicit cell aggregation (Figs. 1,2). In contrast, the degree of aggregation of anchorage-independent SRBC elicited by clusterin was identical when clusterin was added before or after SRBC suspensions were introduced into the medium (Figs. 1, 2). Similar results were observed in freshly prepared, intact nonfixed SRBC and in glutaraldehyde-fixed SRBC (data not shown). YIGSR inhibits the aggregation of anchorage-dependent TM4 cells otherwise elicited by clusterin Addition of clusterin (50 pg!ml) at time zero resulted in a marked aggregation of TM4 cells, evident at 4 h and a t 24 h. However, addition of clusterin (50 pgiml) in the presence of YIGSR (100 pgiml) elicited little if any cell aggregation (Fig. 3A). These inhibitory effects of YIGSR were detected as early as 4 h after incubation, but YIGSR alone had no effects on cell aggregation at all concentrations examined (data not shown). In clusterin-treated TM4 cells, the degree of inhibition of cell aggregation by YIGSR was proportional to the concentrations of YIGSR in the incubation medium (Fig. 3A). In contrast, addition of the irrelevant peptide YIGSE (up to 100 pgiml) had no effect on the degree of cell aggregation elicited by clusterin (Fig. 3A?. Parallel experiments, using anchorage-independent SRBC, were performed under otherwise identical conditions. Addition of YIGSR pentapeptide, a t concentrations up to 300 pgiml, did not appreciably inhibit the

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Fig. 1. Photomicrographs showing effects of clusterin on the aggregation of TM4 cells (A-C) and sheep red blood cells (SRBC) (D-F). A , D Control cells seeded in phosphate buffered saline (PBS) with no other additions; B,E: clusterin (50 pg/ml) present in minimal essential

medium (MEM) at time cells were seeded; C,F: clusterin added 10 min after cells were seeded. All cells were incubated a t 37°C with rotation for 24 h. Experimental observations presented are representative of results obtained with three different cell preparations. X 120.

aggregation of SRBC elicited by clusterin (Fig. 3B). Similar results, indicating little if any inhibitory effect, were observed when comparable levels of YIGSE were added to the incubation medium (Fig. 3B). In parallel experiments, fresh, nun-fixed SRBC were used under otherwise identical conditions. Results with these prep-

arations were identical to those reported with fixed SRBC (data not shown). Combined observations indicate that the aggregation of anchorage-dependent TM4 cells elicited by clusterin is inhibited by YIGSR to a far greater extent than is the aggregation of anchorageindependent SRBC.

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TIME OF ADDITION OF CLUSTERIN (mln) Fig. 2. Effects of varying the times of addition of clusterin on the degree of aggregation of TM4 cells and fixed sheep red blood cells (SRBC).Clusterin (50 Kgiml) was added at times indicated after seeding, and scoring of aggregation was performed for 24 h as previously described (Fritz and Burdzy, 1989). Cells were seeded in uncoated U-bottom microtitration wells, incubated under rotation, and the preparations were then observed microscopically at x 50 magnification. Results, shown from a single experiment on triplicate samples from preparations of TM4 cells or fixed SRBC, are representative of observations obtained with three different cell preparations. Identical results to those shown with fixed SRBC were observed with fresh, non-fixed SRBC preparations.

Effects of agarose-coated substratum on the aggregation of anchorage-dependent TM4 cells elicited by clusterin To minimize cell-substratum interactions, we plated TM4 cells in MEM in agarose-coated cell culture plates. When plates were incubated, without rotation for 24 h, 32% of untreated TM4 cells spontaneously aggregated and remained unattached to the substratum (Fig. 4A). Addition of clusterin (50 pgiml) before or after plating enhanced this aggregation of cells, resulting in a clustering together of 96% of TM4 cells plated (Figs. 4B,5). In contrast, addition of BSA (50 pgiml) resulted in only 32%cell aggregation, a level similar to that observed in cells plated in MEM containing YIGSE or YIGSR alone (Fig. 5 ) .Addition of YIGSR to the culture medium moderately reduced the degree of' cell aggregation otherwise elicited by clusterin, with only 52%of cells in the well becoming aggregated (Figs. 4C, 5 ) . This effect was reversible, as shown by the complete aggregation by clusterin of TM4 cells previously incubated in the presence of YIGSR, and then washed (data not shown). Addition of YIGSE did not alter the responses of TM4 cells to clusterin (compare Fig. 4B with Fig. 4D; and Fig. 5). Similar results were observed in TM4 cells plated in bacterial culture dishes, to which TM4 cells attach poorly (data not shown). Effects of YIGSR on attachment and aggregation of TM4 cells plated on uncoated culture dishes Untreated TM4 cells plated onto uncoated culture dishes attached to the substratum, and formed a monolayer within 24 h (Fig. 6A). When cells were treated with clusterin, 60% formed aggregates (Figs. 5, 6B). Many of these aggregates partially attached to the sub-

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LOG CONCENTRATION OF PEPTIDE (mlcrogramslml) Fig. 3. A Aggregation of living TM4 cells, and B: glutaraldehydefixed sheep red blood cells (SRBC) by clusterin (50 Kg/ml) in the presence of YIGSR, or YIGSE, the irrelevant pentapeptide a t designated concentrations. Cclls were seeded in uncoated U-bottom microtitration wells and incubated under rotation, and the preparations were observed 24 h after plating. Results shown are from a single experiment on triplicate samples from one preparation of TM4 cells or fixed SRBC, respectively. The experimental observations presented are representative of those obtained with three different preparations of each cell type. Identical results to those shown for fixed SRBC were obtained with preparations of fresh non-fixed SRBC.

stratum, showing some degree of spreading (Fig. 6B). This appeared to be an intermediate type of aggregation. Addition of BSA instead of clusterin resulted in an aggregation of only 8% of cells, while the majority of cells attached and formed a monolayer in a manner

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Fig. 4. Phase contrast photomicrographs showing effects of clusterin on the aggregation of TM4 cells plated upon agarose-coated substratum, and cultured in minimal essential medium (MEMI in the presence or absence of YIGSR pentapeptide. Each well (bottom area 200 mm2/well) contained 1 x los cells in 0.5 ml MEM, and was incubated without rotation for 24 h. A Cells incubated in MEM alone; B: cells treated with clusterin (50 pgiml); C: cells treated with both clusterin and YIGSR pentapeptide (100 pgimli; D cells treated with both clus-

terin and the irrelevant peptide YIGSE (100 pgiml). The experimental observations presented are representative of those obtained with three different cell preparations. The photomicrograph B shows TM4 cells in which clusterin was added prior to TM4 cells. Identical results to those shown in “B” were obtained in preparations in which clusterin was added 5-60 min after cells were added (photomicrographs not shown). x250.

comparable to that of control cells (Fig. 6A). Similarly, YIGSE addition did not influence cell aggregation (Fig. 5). Addition of both clusterin (50 pgiml) and YIGSR (100 pgiml) to the culture medium resulted in a reduced degree of cell attachment, and also in a diminution of cell aggregation. In the presence of clusterin plus YIGSR only 28% of the cells became aggregated (Fig. 5). In contrast, cells treated with clusterin and the “irrelevant peptide” YIGSE had an intermediate type of aggregation indistinguishable from cells treated with clusterin alone (Fig. 6B, D). Under conditions described, YIGSR markedly inhibited cell attachment and YIGSR also inhibited the aggregation of cells otherwise elicited by clusterin. In contrast, neither YIGSE nor albumin had any inhibitory effects on clusterin actions. Effects of YIGSR were readily reversible, since washed cell preparations, previously incubated in the presence of YIGSR, aggregated when incubated in the

presence of clusterin on uncoated culture dishes (data not shown).

Effects of YIGSR on aggregation of TM4 cells plated onto a laminin-coatedsubstratum When TM4 cells were plated onto laminin-coated wells, and incubated for 24 h without rotation, only 2% of cells aggregated, while the majority of cells readily attached and formed homogenous monolayers (Figs. 5, 7A). The same phenomenon occurred in cells incubated in the presence of clusterin (Fig. 7B) or YIGSE (Fig. 5). In TM4 cells plated onto laminin-coated wells, addition of clusterin to the incubation medium prior to plating elicited minimal aggregation (6%). Instead, the majority of TM4 cells attached, spread, and formed homogenous monolayers (Figs. 5,7B), in a manner comparable to that of control cells (compare Fig. 7B with Fig. 7A). Thus, no effects of clusterin on the aggregation of TM4

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trast, when clusterin and YIGSR pentapeptide were simultaneously in the medium with TM4 cells, the degree of aggregation was reduced to 40% of the value observed in TM4 cells incubated in the presence of clusterin alone (Fig. 5,8D). In parallel experiments, TM4 cells were plated onto fibronectin-coated surfaces, and placed in culture in the presence or absence of clusterin or YIGSR. As previously reported, cells in culture in MEM alone on a fibronectin substratum readily attached to fibronectin, spread, and formed a confluent monolayer within 24 h (Tung and Fritz, 1986a). Addition of either YIGSR (100 pg/ml) or clusterin (50 p,g/ml) resulted in a slight diminution of cell attachment and monolayer formation. Addition of both YIGSR and clusterin together also resulted in a diminution of cell attachment and monolayer formation to the same extent a s that observed when each was added singly (data not shown). These observations indicate that cells plated on fibronectincoated surfaces resnonded differentlv to clusterin and YIGSR than did TM4 cells plated on& a laminin-coated substratum (Table 1). ~

Fig. 5. Influence of various types of substrata upon the aggregation of TM4 cells seeded upon coated or uncoated culture plates in minimal essential medium (MEM) in the presence or absence of clusterin, YIGSR, and YIGSE. TM4 cells were plated upon each substratum indicated, as described in Materials and Methods. Results shown are means, plus or minus the standard deviation, from a single experiment from one preparation of TM4 cells, with triplicate vessels for each bar. Experimental observations presented are representative of those obtained with three different cell preparations. A, YIGSR (100 pgiinl) alone; B, YIGSE (100pgiml); C, clusterin alone (50 pgiml); D, clusterin (50 pgiml) plus YIGSR (100 pg/ml).

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cells could be detected in cells plated upon a laminincoated substratum. Addition of YIGSR, however, markedly reduced cell attachment (Fig. 7C). As anticipated from previously published information, this resulted in the formation of sparse monolayers, while the majority of cells remained unattached and unaggregated (Fig. 7 0 . In contrast, addition of both clusterin and YIGSR to the culture medium unexpectedly resulted in the formation of monolayers (Fig. 7D), and the relatively few cells remaining in suspension did not aggregate (Figs. 5, 7D). Thus, inhibition by YIGSR of monolayer formation by cells incubated on a laminin-coated substratum was reversed by simultaneous treatment with clusterin, and the aggregation otherwise elicited by clusterin was not detectable under these conditions. Neither YIGSE nor albumin had any discernible effects on the behavior of TM4 cells plated onto a laminin-coated surface, in the presence or absence of clusterin (data not shown).

Location of laminin on surfaces of aggregated TM4 cells YIGSR addition resulted in a n inhibition of clusterin-induced aggregation of TM4 cells plated onto uncoated or agarose-coated culture dishes (Figs. 3-6, Table 1).Since YIGSR is known to act by competing with laminin for binding to laminin receptors, i t was of interest to determine whether laminin was present a t sites of cell contact in clusterin-aggregated cells. Immunofluorescent microphotographs of deposited rabbit antiserum against laminin indicated the presence of laminin around most but not all of the borders of TM4 cells aggregated by the addition of clusterin (Fig. 9). Since the presence of laminin a t many sites of contact suggested that laminin might be involved in the aggregation of TM4 cells elicited by clusterin, we examined the possible influences of antiserum against laminin on these processes. Addition of antisera against laminin, at levels up to lo%, did not prevent the aggregation elicited by clusterin of TM4 cells plated onto uncoated or agarose-coated tissue culture vessels (data not shown). We conclude that the inhibition of aggregation observed in YIGSR-treated cells in culture under similar conditions (Figs. 3: 5) must therefore involve sites of interactions of YIGSR with components on lateral surfaces of plasma membranes which are not reactive with specific polyclonal antibodies against laminin.

Preincubation with YIGSR pentapeptide did not inhibit the actions of clusterin on cell aggregation Dispersed TM4 cells were preincubated in PBS with gentle agitation for 1 h at 37"C,in the presence of varying concentrations of YIGSR or YIGSE. These cell preparations were then washed three times with PBS, and subsequently plated in MEM onto agarose-coated wells in the presence or absence of clusterin (50 p,g/ml). Preincubation, even with high concentrations of YIGSE or YIGSR (up to 100 pgiml), exerted no inhibitory or cytotoxic effects on the degree of aggregation by washed TM4 cells incubated with clusterin (Fig. 8A-C). In con-

DISCUSSION The extent of cell aggregation elicited by clusterin has been shown to be influenced greatly by the type of cells, and by the nature of interactions between cells and substratum. In the case of anchorage-independent erythrocytes, clusterin addition always resulted in aggregation, regardless of the type of substratum employed. In anchorage-dependent TM4 cells, however, clusterin elicited aggregation only in cells not readily adhering to the substratum. Thus, clusterin did not aggregate TM4 cells when clusterin was added after the cells had begun to attach to the surface of uncoated culture dishes (Figs. l A , 2A), or at any time if the

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Fig. 6. Phase contrast photomicrographs showing effects of clusterin on the aggregation of TM4 cells plated upon uncoated cell culture plates, and maintained in culture in the presence or absence of YIGSR pentapeptide. All cells were seeded at a density of 1 x 10ii0.5 mliwell in minimal essential medium (MEM), and were incubated without rotation fnr 24 h. A: Untreated cells plated in MEM; B: cells treated

with clusterin (50 pgiml) alone; C: cells treated with both clusterin (50 pgiml) and YIGSR pentapeptide (100 pg/mI); D: cells treated with both clusterin (50 pg/ml) and YIGSE (100 pgirnlJ. For details, see Materials and Methods. The experimental observations presented are representative of those obtained with three different cell preparations. X 250.

substratum consisted of a laminin-coated surface (Fig. 7A,B). In contrast, clusterin elicited the aggregation of SRBC before or after erythrocytes were added to laminin-coated or uncoated surfaces (Figs. 1, 2B). Presumably, SRBC do not bind to a substratum because these cells do not contain receptors to ECM components. By varying the nature of the substratum, we have found conditions under which the responses to clusterin by TM4 cells appeared similar to responses by erythrocytes. When TM4 cells were plated onto a n agarosecoated substratum, they attached poorly, and readily aggregated in the presence of clusterin added before or after cells were seeded (Fig. 5 ) . On the other hand, strong cell-substratum interactions between TM4 cells and laminin totally prevented the aggregating actions of clusterin on TM4 cells (Figs. 5, 7). From combined observations, we infer that the net effect of clusterin on cell aggregation depends upon the equilibrium between cell-cell and cell-substratum interactions, having greatest effects on cell aggregation when adhesive interactions between cells and substratum are weakest.

TM4 cells, isolated from testes of neonatal mice, are thought to be of Sertoli cell origin (Mather, 1980). We have previously shown t h a t sparsely plated primary cultures of Sertoli cells spread moderately when explanted onto uncoated polystyrene or glass surfaces. As Sertoli cells in culture generate colonies or monolayers, they form characteristic “epithelial-type” contacts, becoming arranged in a cobblestone-like association pattern (Tung et al., 1988). In contrast, when cells are plated upon air-dried Matrigel, a preparation of reconstituted basement membrane (Kleinmann e t al., 19881, the Sertoli cells display a markedly different behavior during the transitional remodeling phase. They migrate rapidly, send forth elaborate extensions between distant Sertoli cells, and form complex contacts with the substratum as well as with each other. Thus, Sertoli cells sparsely plated upon air-dried Matrigel, associate to generate a fibroblast-like pattern rather than a cobblestone-like pattern of cell borders formed by cells plated upon uncoated polystyrene surfaces (Tung et al., 1988).These observations support the concept t h a t the

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Fig. 7. Phase contrast photomicrographs showing effects of clusterin on the aggregation of TM4 cells plated upon a laminin-coated substratum and maintained in culture in the presence or the absence of YIGSR. All cells were seeded at a density of 1 x 105/0.5mliwells, and were incubated without rotation for 24 h. A: Untreated cells seeded in

minimal essential medium (MEMI alone; B: cells treated with clusterin (50 pgiml) alone; C: cells treated with YIGSR pentapeptide (100 pgiml) alone; D cells treated with both clusterin and YIGSR pentapeptide. The experimental observations presented are representative of those obtained with three different cell preparations. ~ 2 5 0 .

balance between cell-cell interactions and cell-substratum interactions is of considerable importance in determining the behavior and properties of cells in culture. The peptide YIGSR has been shown to compete for laminin receptors on the cell surface, thereby inhibiting laminin-mediated cell attachment to the substratum (Graf et al., 1987; Kleinman and Weeks, 1989). When we utilized YIGSR in investigations on the binding of TM4 cells to various substrata, we therefore anticipated that YIGSR would enhance the aggregation of TM4 cells, especially in the presence of clusterin. To our surprise, YIGSR decreased the aggregation of TM4 cells elicited by clusterin. This occurred in preparations plated upon uncoated (Figs. 3A, 6 ) or agarose-coated surfaces (Figs. 4, 5, Table 1).The inhibition of clusterin-elicited aggregation of TM4 cells by YIGSR appeared to be relatively specific, since the “irrelevant peptide” YIGSE did not influence clusterin actions on cell aggregation (Figs. 3A, 6), and YIGSR did not alter the effects of clusterin on the clustering of anchorageindependent erythrocytes. Combined data therefore suggest that YIGSR acts to decrease interactions be-

tween anchorage-dependent cells which otherwise aggregate in the presence of clusterin. Could these YIGSR effects involve inhibition of cell interactions involving intercellular adhesion molecules, such as those reported in the immune system (Springer, 1990)? If so, YIGSR could act on lateral surfaces of plasma membranes via mechanisms not directly related to the competition between YlGSR and laminin for laminin receptor sites on the basal surfaces of cells. It is conceivable that homologous sites might be involved in cell-cell interactions along lateral surfaces. Alternatively, intercellular adhesion molecules (ICAM) might be involved in interactions with specific integrins possibly present on TM4 cells (Ruoslahti, 1991). The absence of such sites in SRBC may be correlated with the lack of effects of YIGSR on the aggregation of erythrocytes incubated in the presence or absence of clusterin (Fig. 3B). Preincubation of TM4 cells with YIGSR, followed by washing of the cells, failed to indicate any evidence of sufficient binding of YIGSR to influence the aggregation of TM4 cells in response to subsequent addition of

419

CELL-CELL VS. CELL-SUBSTRATUMINTERACTIONS

Fig. 8. Phase contrast photomicrographs showing effects of YIGSR preincubation on the effects of clusterin on the aggregation of TM4 cells. All cells were seeded in agarose-coated wells at a density of 1 X 105/0.5ml/well in minimal essential medium (MEM), and incubated without rotation for 24 h. A: Cells preincubated with YIGSR pentapeptide (100Fgiml), washed, and then seeded in the presence of clusterin (50 Fgiml); B: cells treated with clusterin without preincuba-

tion; C: cells preincubated with YIGSE pentapeptide (100 pg/ml), washed, and then seeded in the presence of clusterin; D: cells treated simultaneously with both clusterin and YIGSR pentapeptide, without preincubation. The experimental observations presented are representative of those obtained with three different cell preparations. ~250.

TABLE 1. Summary of oualitative responses to clusterin of anchoraee-deoendent TM4 cells dated on different substrata' Appearance of TM4 cells in culture on substrata indicated

Additions to medium (MEMj Albumin (50 pg/ml)

Uncoated culture dishes A iQ)

M

8

+++

60

+ +++

nr

YIGSE (100 pgiml) Clusterin (50 kg/mll (+ YISGE) YIGSR (100 pg/mlj Clusterin + YIGSR

4.1 28

++

Agarosecoated surface A (5%)

Laminincoated surfaces

Fibronectincoated surfaces

M

A (%I

M

A (%)

M

32

0