Mouse proximal tubular cell-cell adhesion inhibits apoptosis by a cadherin-dependent mechanism

Eoin Bergin, Jerrold S. Levine, Jason S. Koh, and Wilfred Lieberthal

Renal Section, Department of Medicine, Evans Department of Clinical Research, Boston University School of Medicine, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Adhesion of epithelial cells to matrix is known to inhibit apoptosis. However, the role of cell-cell adhesion in mediating cell survival remains uncertain. Primary cultures of mouse proximal tubular (MPT) cells were used to examine the role of cell-cell adhesion in promoting survival. When MPT cells were deprived of both cell-matrix and cell-cell adhesion, they died by apoptosis. However, when incubated in agarose-coated culture dishes (to prevent cell-matrix adhesion) and at high cell density (to allow cell-cell interactions), MPT cells adhered to one another and remained viable. Expression of E-cadherin among suspended, aggregating cells increased with time. A His-Ala-Val (HAV)-containing peptide that inhibits homophilic E-cadherin binding prevented cell-cell aggregation and promoted apoptosis of MPT cells in suspension. By contrast, inhibition of potential beta 1-integrin-mediated interactions between cells in suspension did not prevent either aggregation or survival of suspended cells. Aggregation of cells in suspension activated phosphatidylinositol 3-kinase (PI3K), an event that was markedly reduced by the presence of the HAV peptide. LY-294002, an inhibitor of PI3K, also inhibited survival of suspended cells. In summary, we provide novel evidence that MPT cells, when deprived of normal cell-matrix interactions, can adhere to one another in a cadherin-dependent fashion and remain viable. Survival of aggregated cells depends on activation of PI3K.

anoikis; survival; viability; phosphatidylinositol 3-kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

APOPTOSIS, a form of cell death distinct from necrosis, is characterized by cell shrinking, nuclear condensation and fragmentation, and rapid clearance by phagocytosis (25, 31, 32). Apoptosis can be induced by a wide variety of stimuli. In many situations, apoptosis is induced by activation of a receptor-mediated cell death pathway [e.g., Fas or tumor necrosis factor (TNF)] or by cytotoxic stress (32, 34). However, apoptosis can also be precipitated by the loss of survival factors (48). It has become apparent that most, if not all, cells constitutively express the machinery necessary for apoptosis to occur (56), and that the constant presence of signals provided by survival factors is necessary to maintain cell viability (47). This mechanism for inhibiting apoptosis has been termed the default pathway, because it is induced by the absence of a stimulus rather than the presence of an activating event (48).

The first survival factors described were soluble growth factors (9, 48) and cytokines (10, 57). More recently, it has been demonstrated that adherent cells also require normal cell-matrix interactions to remain viable (3, 11, 45, 54). Frisch and Francis (11), the first to describe the phenomenon of apoptosis induced by loss of cell-matrix adhesion, coined the term anoikis (homelessness) to describe this phenomenon (11). However, apart from the cause, anoikis is no different from apoptotic cell death induced by any other apoptotic trigger (22, 32).

Although the importance of cell-matrix interaction in inhibiting the default pathway and promoting cell survival is now well established, the role of cell-cell interactions as a survival factor is less clear. The purpose of this study was to determine whether cell-cell adhesion alone can inhibit apoptosis of mouse proximal tubular (MPT) cells that have lost normal cell-matrix adhesion and, if so, to elucidate the signaling pathways involved. We demonstrate for the first time that cell-cell adhesion between MPT cells inhibits apoptosis by a cadherin-dependent mechanism. We also show that cadherin-mediated cell survival occurs via activation of phosphatidylinositol 3-kinase (PI3K), a lipid kinase that has been shown to mediate the survival activity of soluble growth factors (30, 40) as well as cell-matrix adhesion (36-38).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents

Culture medium, insulin, hydrocortisone, penicillin/streptomycin, LY-294002, poly-L-lysine, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all obtained from Sigma Chemical (St. Louis, MO). Collagenase was purchased from Worthington Biochemical (Freehold, NJ), and soybean trypsin inhibitor was purchased from GIBCO Laboratories, (Grand Island, NY). Agarose was obtained from FMC Bioproducts (Rockland, MD), and Hoechst 33342 was from Calbiochem (La Jolla, CA).

Primary Culture of MPT Cells

Cells were cultured from collagenase-digested fragments of proximal tubules obtained from the cortices of C57Bl/6 mice with a modification of methods previously described (52). Briefly, cortices were minced and incubated with 0.5 mg/ml collagenase, and 0.5 mg/ml soybean trypsin inhibitor in Hanks' solution for 30 min. After removing large undigested fragments by gravity, the suspension was mixed with an equal volume of 10% horse serum in Hanks' solution and then centrifuged at 500 rpm for 7 min at room temperature. The pellets were washed once with DMEM by centrifugation and then suspended in growth medium. The medium used to grow cells was a serum-free mixture of DMEM and Ham's F-12 (1:1) containing 2 mM glutamine, 15 mM HEPES, 5 µg/ml transferrin, 5 µg/ml insulin, 50 mM hydrocortisone, 500 U/ml penicillin, and 50 mg/ml streptomycin. These cells have previously been identified as being predominantly of proximal tubular origin (52).

Experimental Models for Preventing Cell-Matrix and/or Cell-Cell Adhesion

Prevention of both cell-cell and cell-matrix adhesion. To examine the effect of loss of cell-cell as well as normal cell-matrix interactions, confluent MPT cells were harvested by trypsinization and suspended in DMEM at low cell density (~10,000 cells/ml) in polypropylene test tubes that were gently but continuously rotated at 37°C with a tube rotator (Bioquest). The combination of low cell density and continuous rotation prevented sustained cell-cell interactions (determined by examination of cells under phase contrast).

Prevention of cell-matrix adhesion while allowing cell-cell interaction. To examine the consequences of cell-cell contact in the absence of cell-matrix adhesion, MPT cells were incubated in DMEM at high cell density (2 million cells/ml) in culture plates coated with 10% agarose. The high density of cells promoted cell-cell interactions, and the agarose prevented normal adhesion of cells to the culture dish.

Cell Viability

The viability of MPT cells was determined through the use of a modification of the colorimetric MTT assay described by Mossman (41). MTT is a pale yellow tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] that is converted by viable cells to dark blue crystals of MTT formazan (peak absorbance 570-590 nm).

MPT cells were suspended at high density in agarose-coated wells for 24 h under the conditions described in RESULTS (each variable in duplicate). Control cells were suspended in DMEM alone. Then the cells were transferred to Eppendorf tubes, vortexed, and washed once in Krebs-Henseleit buffer (KHB). Cells were then microfuged at 14,000 rpm for 5 min, and the supernatant was removed, leaving a pellet of MPT cells behind. MTT (100 µl of a 1 mg/ml solution, dissolved in KHB containing 10 mM dextrose) was added to each Eppendorf tube and vortexed to suspend the cell pellet. After incubating the cells in MTT at 37°C for 4 h, 100 µl of 10% SDS (in 0.01 N HCl) was added to each sample to dissolve the crystals of formazan, and the samples were incubated at 37°C for another 24 h (41). The final solution (200 µl) was removed from each Eppendorf tube and transferred to ELISA plates into duplicate wells of 100 µl each. MTT solution (100 µl) was used as the blank. The optical densities (OD) of the blank and samples were read with a Dynatech microELISA plate reader with a test wavelength of 570 nm and a reference wavelength of 630 nm (41). The blank was subtracted from each value. Viability was expressed as a percent of control (cells suspended in DMEM alone).

Quantification of Apoptosis

Cells were stained live with H-33342 (1 µg/ml) for 30 min at 37°C. The cells were fixed with 3.7% paraformaldehyde and then resuspended in 10 µl of PBS. The fixed cells were layered onto glass slides coated with 0.1% poly-L-lysine and allowed to air dry. The cells adherent to the glass slide were washed several times with PBS and mounted in polyvinyl alcohol (Gelvatol). Random fields of cells were photographed at ×400 magnification. The proportion of normal and apoptotic nuclei in each field was determined. At least 500 cells were counted in each experiment, and each experiment was repeated at least three times.

Inhibition of Cadherin- and Integrin-Mediated Cell-Cell Adhesion

Cadherin-mediated cell-cell association was inhibited using a 17-mer peptide containing the tripeptide recognition sequence, His-Ala-Val (HAV), which has been shown to be essential for homophilic binding between the extracellular domains of the cadherins (2, 43). The inhibitory HAV-containing peptide (AKYILYS<UNL>HAV</UNL>SSNGEAV) and a control peptide containing the same amino acids in random order (VLYSYHASNIVEKSAGA) were both custom-made by QCB Biochemicals (Hopkington, MA).

A cyclic RGD-containing peptide (cyclo-RGD-fV; kindly provided by Dr. Michael Goligorsky, Dept. of Medicine, State University of New York, Stony Brook, NY) (42) was used to inhibit binding of beta 1-integrins to matrix receptors. This cyclic RGD, at a concentration of 500 µM, inhibited the growth of MPT cells on tissue culture plastic by 93 ± 2% (n = 4). We also used a neutralizing antibody to the beta 1-integrin [kindly provided by Dr. David J. Salant, Dept. of Medicine, Boston University School of Medicine, Boston, MA (44)] to examine a role for integrin-RGD or homophilic integrin binding events in cell-cell aggregation and survival. In control studies, this anti-integrin antibody inhibited growth of MPT cells on culture plastic by 86 ± 4% (n = 4).

Assessment of E-Cadherin Expression

Immunofluorescence microscopy. MPT cell monolayers grown on glass coverslips or suspended at high cell density in agarose-coated wells were fixed in 3.7% paraformaldehyde and permeabilized with 0.2% Triton X-100. Detection of E-cadherin in these cells was performed by indirect immunofluorescence using methods previously described (27). The primary antibody (a rat monoclonal anti-E-cadherin antibody, clone DECMA-1, Sigma) was used at a dilution of 1:100. The secondary anti-rat IgG antibody, labeled with fluorescein (Molecular Probes, Eugene, OR), was used at a dilution of 1:500. The monolayers and cell aggregates were photographed with a Nikon epifluorescence microscope (magnification ×400).

Flow cytometry. MPT cell monolayers were trypsinized and suspended at high density in DMEM in agarose-coated wells for either 1 or 6 h. A separate group of cells was incubated in the same way for 6 h in the presence of the HAV-containing 17-mer peptide (500 µM). At the end of the appropriate time of incubation, all three groups of cells were disaggregated by vigorous vortexing. Cells from each group were divided into two aliquots. One aliquot of each sample of cells was immunostained with anti-cadherin antibody (a rat monoclonal anti-E-cadherin antibody, clone DECMA-1, Sigma) followed by a secondary fluorescein-labeled anti-rat IgG antibody using the same technique described for immunofluorescent microscopy. The second aliquot of cells from each group was incubated with nonimmune serum (instead of the primary antibody) and then stained with the anti-rat IgG secondary antibody. Two additional groups of cells were stained immediately after trypsinization (without any time to aggregate) with either anti-cadherin antibody or nonimmune serum, followed by secondary antibody for both groups. All eight samples were then fixed with 3.7% paraformaldehyde for 10 min, washed once in PBS, and then stored in PBS at 4°C. Flow cytometry was performed on an Epics ESP Flow Cytometer (Coulter Electronics, Hialeah, FL). A constant number of cells (events) was analyzed for each sample (10,000/sample).

Transmission Electron Microscopy

MPT cells were fixed with 2.5% glutaraldehyde in PBS for 1 h at 4°C and washed three times in Sabatini's solution (PBS with 6.8% sucrose). Samples were postfixed with 1% osmium tetroxide (1 h), washed three times in Sabatini's solution, passed through a graded series of alcohols (30, 50, 70, 90, and 100% for 15 min each), and treated with propylene oxide (15 min), a 1:1 Epon-propylene oxide mix (1 h), and three changes in pure Epon (3 h, 3 h, and overnight). Polymerization occurred overnight at 64°C. Ultrathin sections (~50 nm) were cut with an MT2 Sorvall ultramicrotome, stained with lead citrate and uranyl citrate, and examined with a JEOL 100CX transmission electron microscope at 60 kV using a 20-µm objective aperture.

Assay of PI3K Activity

PI3K activity was measured with a minor modification of previously described methods (26). MPT cells were lysed by a buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA (pH 7.6), 0.05 M beta -glycerophosphate, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM sodium orthovanadate, 40 µM phenylmethylsulfonyl fluoride (PMSF), and a complete anti-protease tablet (Boehringer Mannheim, Indianapolis, IN). Cell lysates from different samples were normalized for total cell protein content and then incubated for 24 h with polyclonal anti-PI3K antibodies directed against the 85-kDa regulatory subunit of the p85-p110 isoform (Upstate Biotechnology, Lake Placid, NY). Immune complexes were adsorbed onto protein A Sepharose, washed twice with PBS containing 1% Nonidet P-40 and 1 mM Na3VO4, washed three times with 100 mM Tris, pH 7.4, containing 5 mM LiCl and 1 mM Na3VO4, and finally washed twice with 10 mM Tris, pH 7.4, containing 160 mM NaCl, 5 mM EDTA, and 1 mM Na3VO4. Lysates were incubated with 10 µg phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL) and radiolabeled ATP (20 µCi [gamma -32P]ATP, 20,000 Ci/mmol; DuPont NEN, Boston, MA) in a buffer containing 10 mM HEPES, pH 7.2, 1 mM EGTA, 20 mM MgCl2, and 100 µM cold ATP. The kinase reactions were run for 10 min and then stopped by addition of 2 N HCl. Lipids were extracted and analyzed by TLC.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Loss of Both Cell-Cell and Cell-Matrix Adhesion on MPT cells

To deprive MPT cells of both cell-cell and normal cell-matrix adhesion, cells were suspended in DMEM at low cell density (10,000 cells/ml) and continuously rotated at 37°C (see METHODS). Cells suspended in this manner lost viability in a time-dependent manner. After 3, 6, and 24 h of suspension, cell viability, as measured by the MTT assay, was reduced to 61.8 ± 5 (P < 0.01 vs. control), 49.7 ± 8, and 23.1 ± 8% (P < 0.01 vs. 3 h), respectively (n = 5). Freshly suspended cells and cells deprived of cell-cell and cell-matrix adhesion for 24 h were also stained with H-33342 and examined under fluorescence microscopy. The majority of the nuclei of freshly suspended cells demonstrated normal morphology (low intensity of H-33342 fluorescence with a well-defined chromatin pattern; Fig. 1A). By contrast, the majority of nuclei of cells deprived of cell-cell and cell-matrix adhesion for 24 h demonstrated the characteristic features of apoptosis. The nuclei are intensely fluorescent and have become condensed. Some of these condensed nuclei have undergone fragmentation (Fig. 1B). The absence of any cell-cell contact between cells suspended at low cell density is also evident in Fig. 1B. Thus loss of both cell-cell and cell-matrix contact results in time-dependent loss of viability due to apoptosis.


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Fig. 1.   Cells deprived of cell-cell and cell-matrix adhesion die by apoptosis. Freshly suspended cells (A) and cells deprived of cell-cell and cell-matrix adhesion for 24 h (B) were stained with H-33342 and examined under fluorescence microscopy (magnification ×400). All nuclei of freshly suspended cells (A) demonstrate normal morphology. The intensity of H-33342 fluorescence of these nuclei is relatively low and the chromatin pattern is well defined. By contrast, most of the nuclei of cells deprived of cell-cell and cell-matrix adhesion for 24 h (B) are intensely fluorescent (arrows). The chromatin of these nuclei has also condensed into featureless masses (arrows). Some condensed nuclei have undergone fragmentation (arrowhead). All 3 morphological features, intense fluorescence, condensation, and fragmentation, are characteristic of apoptotic cell death.

Effect of Loss of Normal Cell-Matrix Attachment Alone on MPT Cells

To deprive MPT cells of cell-matrix adhesion while allowing cell-cell interactions to occur, cells were suspended in DMEM at high cell density (~2 million cells/ml) in agarose-coated wells (see METHODS). MPT cells suspended in this manner aggregated spontaneously and, over a period of a few hours, formed tight clusters of cells (Fig. 2A). The majority of cells within these aggregates had normal nuclear morphology on H-33342 staining (Fig. 3A). After suspension in DMEM for 24 h, only 5 ± 2% of cell nuclei demonstrated apoptotic morphology (n = 7) (Fig. 4).


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Fig. 2.   MPT cells deprived of cell-matrix adhesion but not cell-cell contact form cellular aggregates in a calcium- and cadherin-dependent manner. Phase contrast of mouse proximal tubular (MPT) cells suspended at high cell density in agarose-coated wells for 24 h in DMEM (A), DMEM + 2 mM EGTA (B), DMEM + EGTA + 2 mM calcium (C), DMEM + cyclic RGD-containing peptide (D), DMEM + His-Ala-Val (HAV)-containing peptide (E), and DMEM + random 17-mer peptide (F).



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Fig. 3.   Fluorescence microscopy of cells deprived of cell-matrix interactions but allowed cell-cell adhesion. MPT cells were stained with H-33342 after being suspended at high cell density in agarose-coated wells for 24 h. (A) Suspension in DMEM alone: most of the nuclei have normal morphology. (B) DMEM + EGTA: many of the cells demonstrate apoptotic morphology. (C) DMEM + EGTA + calcium: most cells appear normal. (D) DMEM + cyclic RGD peptide: most cells have normal morphology. (E) DMEM + HAV-containing peptide: most of the cells are apoptotic. (F) DMEM + random control peptide: most cells have normal morphology.



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Fig. 4.   Quantification of apoptotic cell death by fluorescence microscopy after H-33342 staining in MPT cells deprived of cell-matrix interactions but not cell-cell adhesion. MPT cells were suspended at high cell density in agarose-coated wells for 24 h. Cells were then stained with H-33342, and the proportions of nuclei with normal and apoptotic morphology were counted (see METHODS). Number of apoptotic cells is expressed as a percent of total number of cells counted. *P < 0.01 vs. DMEM alone; dagger P < 0.02 vs. nonspecific 17-mer peptide.

To examine the role for calcium, aggregated MPT cells were suspended at high density in agarose-coated wells in DMEM containing 2 mM EGTA. MPT cells did not aggregate in the presence of EGTA (Fig. 2B), and many cells were apoptotic (Fig. 3B). The proportion of apoptotic nuclei was markedly increased by suspension in EGTA to 60 ± 12% (P < 0.001, compared with DMEM alone; Fig. 4; n = 5). When an excess of calcium (2 mM) was added to the EGTA-containing DMEM, the effects of the EGTA were reversed; cells aggregated normally (Fig. 2C), and a few cells had apoptotic nuclear morphology (Fig. 3C). By contrast, when cells were suspended at high cell density in DMEM plus EGTA plus additional (2 mM) magnesium, the cells did not aggregate (data not shown).

The viability of MPT cells suspended at high cell density, as measured by the MTT assay (see METHODS; n = 5), was substantially reduced by the presence of EGTA to 26 ± 7% of control (cells suspended in DMEM alone; Fig. 5). The inhibitory effect of EGTA on cell viability was reversed by the addition of excess (2.0 mM) calcium to 109 ± 25% of control (Fig. 5). By contrast, addition of 2 mM magnesium to EGTA-containing DMEM did not reverse the effects of EGTA on viability (27 ± 8% of control; Fig. 5).


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Fig. 5.   Viability of MPT cells deprived of cell-matrix adhesion but allowed cell-cell contact. MPT cells were incubated at high cell density in agarose-coated wells under various conditions for 24 h. Viability of MPT cells was then determined. Data are expressed as percentage of MPT cells suspended in DMEM alone (n = 5). * P < 0.01 compared to DMEM alone; dagger  P < 0.01 compared to EGTA; ¶ P < 0.02 compared to nonspecific 17-mer peptide.

We next examined the role played by cadherin in this process. MPT cells were incubated at high cell density in agarose-coated wells in the presence of either a 17-mer HAV-containing peptide or a control peptide containing the same amino acids in random sequence (see METHODS). The HAV-containing peptide inhibited cell-cell aggregation (Fig. 2E) and promoted apoptosis (Figs. 3E and 4), whereas the control peptide had no effect (Figs. 2F, 3F, and 4). The proportion of apoptotic cells in the presence of the HAV-containing peptide (n = 6) was substantially higher than in DMEM alone or DMEM plus nonspecific peptide (n = 6; Fig. 4; P < 0.01). Viability of cells in suspension was reduced to 24 ± 8% of control by the HAV-containing peptide but was unaffected by the presence of the control random 17-mer peptide (101 ± 18% of control; Fig. 5).

We next examined the role of beta 1-integrin-mediated cell-cell adhesion in cell aggregation and survival. Although suspension of cells at high density on agarose-coated wells prevented normal cell-matrix adhesion, we cannot exclude the possibility that matrix is secreted between suspended cells. We therefore examined the role of binding between beta 1-integrin and its Arg-Gly-Asp (RGD) matrix-receptor sequence on MPT cell-cell aggregation and/or survival. MPT cells were incubated at high cell density in agarose-coated wells in DMEM containing 500 µg/ml of a cyclic RGD-containing peptide (see METHODS). The cyclic RGD-containing peptide did not prevent cell-cell aggregation of cells suspended at high density (Fig. 2D). Also, the majority of cells incubated in suspension in the presence of the cyclic RGD-containing peptide had normal nuclear morphology on H-33342 staining (Figs. 3D and 4). The viability of cells suspended in the presence of the cyclic RGD-containing peptide was comparable to that of cells suspended in DMEM alone (94 ± 8% of control) (Fig. 5). Finally, a neutralizing antibody to the beta 1-integrin (44) (100 µg/ml) had no effect on cell-cell aggregation (data not shown), apoptosis (Fig. 4), or the viability of MPT cells suspended for 24 h (Fig. 5). The failure of this antibody to prevent cell aggregation and survival not only confirms the lack of involvement of integrin-RGD interactions in this process, but also excludes the possibility that homophilic binding of beta 1-integrins on adjacent cells mediates cell-cell adhesion and survival.

Transmission Electron Microscopy

Cells allowed to aggregate at high cell density in agarose-coated wells for 24 h in DMEM appear viable on electron microscopy (Fig. 6A) and demonstrate the presence of highly organized cell-cell contacts (Fig. 6B). By contrast, the majority of cells incubated at high density in DMEM containing the HAV-containing 17-mer peptide demonstrate typical apoptotic morphology on electron microscopy (Fig. 7).


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Fig. 6.   Transmission electron micrograph of MPT cells incubated in DMEM at high density in agarose-treated wells. A: low-power view of MPT cells allowed to aggregate in suspension in DMEM. Cells within aggregate are viable with normal nuclei (arrowheads). B: high-power view of 2 MPT cells within an aggregate of suspended cells. Cells have microvilli, and morphology of nuclei and mitochondria is normal. The cell-cell junction is complex and interdigitated, and a structure resembling a junctional complex is present (arrowheads).



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Fig. 7.   Transmission electron micrograph of MPT cells incubated in DMEM containing HAV peptide. All cells demonstrate characteristic ultrastructural features of apoptosis. The nuclei demonstrate condensation as well as fragmentation of nuclear chromatin. These apoptotic cells have relatively intact plasma membranes and mitochondria. An apoptotic body (nuclear chromatin surrounded by plasma membrane) has been phagocytosed by 1 of the cells (arrowhead).

E-Cadherin Expression in Aggregates of MPT Cells

Immunofluorescence microscopy. Monolayers of confluent MPT cells demonstrate the typical pattern of E-cadherin. After suspension of MPT cells at high cell density for 24 h, the presence of E-cadherin is clearly visible in the same distribution as in monolayers, i.e., at cell-cell borders (Fig. 8). Freshly trypsinized cells showed negligible E-cadherin staining. Moreover, there was no difference in the intensity of E-cadherin immunofluorescence in freshly trypsinized cells vs. cells allowed to aggregate at high cell density in agarose-coated wells for only 1 h (data not shown).


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Fig. 8.   Immunofluorescence microscopy of MPT cells stained with anti-cadherin antibody. A: MPT cell monolayers demonstrate a pattern of E-cadherin staining that is typical for epithelial monolayers, with E-cadherin found predominantly at cell-cell borders. B: in MPT cells suspended at high cell density in DMEM for 24 h, E-cadherin staining is clearly evident between aggregated cells (arrows).

Flow cytometry. MPT cells that were incubated in suspension at high cell density in DMEM for 6 h and immunostained with anti-cadherin antibody were more intensely fluorescent than MPT cells suspended for 1 h (Fig. 9). In control experiments, the fluorescent intensity of cells suspended for 1 or 6 h and stained with nonimmune serum instead of anti-cadherin antibody was no different from that of cells suspended for 1 h and stained with the anti-cadherin antibody (data not shown). The fluorescent intensity of cells incubated for 6 h in DMEM containing the HAV-peptide and stained for cadherin was no different from that of cells incubated for 1 h in DMEM alone. Also, the fluorescent intensity of freshly trypsinized nonsuspended MPT cells, stained with either anti-cadherin antibody or nonimmune serum, was comparable to that of cells stained with anti-cadherin antibody after 1 h of suspension (data not shown). These data confirm the immunofluorescence data shown in Fig. 8, namely, that the process of cell-cell adhesion and aggregation is associated with increased expression of E-cadherin on the surface of MPT cells.


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Fig. 9.   FACS analysis of MPT cells stained with E-cadherin antibody after suspension. MPT cells suspended at high density in DMEM alone for 1 or 6 h and then immunostained with anti-cadherin antibody followed by a fluorescent secondary antibody. Fluorescent intensity of MPT cells is markedly increased after 6 h of suspension and cell-cell aggregation. By contrast, fluorescent intensity of cells incubated in DMEM containing the HAV-containing peptide for 6 h is comparable to that of cells incubated for 1 h in DMEM alone. Thus during suspension and aggregation, E-cadherin becomes expressed on the surface of aggregated cells. The presence of HAV-containing peptide prevents cadherin expression. FITC, fluorescein isothiocyanate.

Effect of Cell-Cell Aggregation on Activity of PI3K

Time course of PI3K activation in MPT cells suspended at high cell density. The activity of PI3K was determined in lysates of MPT cell monolayers as well as in lysates of MPT cells suspended at high density in agarose-coated wells. Monolayers of MPT cells expressed constitutive PI3K activity (Fig. 10A), which is most likely due to out-to-in signaling induced by cell-matrix adhesion (26, 35). This constitutive activity of PI3K was almost completely eliminated by incubating cells in suspension in calcium-free PBS for 2 h (Fig. 10A). Cells suspended in calcium-free PBS for 2 h, which were then resuspended at high density in calcium-containing medium (DMEM) for varying periods of time, demonstrated re-activation of PI3K activity (Fig. 10A). PI3K activity was increased after 5 min of incubation in DMEM, reached a peak at 10 min, and by 1 h of suspension fell to levels comparable to those of confluent monolayers (Fig. 10A). Thus the process of cell-cell adhesion and aggregation activates PI3K.


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Fig. 10.   Effect of suspension and aggregation of MPT cells on phosphatidylinositol 3-kinase (PI3K) activity. A: PI3K activity was measured in lysates of MPT cell monolayers and in MPT cells maintained in suspension at high cell density. Suspended cells were first incubated in calcium- and magnesium-free PBS for 2 h, and then resuspended in DMEM at high cell density for varying periods of time. PI3K present in MPT cell monolayers was reduced after suspension in PBS for 2 h (time 0). PI3K activity was then reactivated by incubation in calcium- and magnesium-containing DMEM at high cell density. PI3K activity is maximal after 10 min of suspension in DMEM and then decreases after 1 h of suspension to a level comparable to that in confluent adherent monolayers. B: PI3K activity was measured in lysates of MPT cell monolayers and suspended MPT cells. After incubating suspended cells in calcium- and magnesium-free PBS for 2 h, the cells were suspended at high density in DMEM that contained 1) no additive (DMEM alone), 2) HAV-containing peptide, 3) nonspecific 17-mer peptide, or 4) cyclic RGD-containing peptide. Reactivation of PI3K after incubation of suspended cells is inhibited by HAV-containing peptide but not by nonspecific 17-mer peptide or cyclic RGD-containing peptide.

Effect of HAV-containing peptide and cyclic RGD peptides on PI3K activity of MPT cells in suspension. As demonstrated in Fig. 9A, PI3K activity was reduced to almost undetectable levels in MPT cells suspended at high density in calcium-free PBS for 2 h, but was then re-activated on resuspension in DMEM for 10 min (Fig. 10B). PI3K activity was markedly inhibited by the presence of the HAV-containing peptide (Fig. 10B). By contrast, neither the random 17-mer peptide nor the cyclic RGD peptide inhibited reactivation of PI3K (Fig. 10B). These data indicate that cadherin-mediated cell-cell aggregation leads to activation of PI3K.

Effect of Inhibition of PI3K on Cell Survival in Cells Suspended at High Density

To determine whether PI3K was necessary for survival of aggregated cells, the effect of the PI3K inhibitor LY-294002 was examined (26, 30). MPT cells were suspended at high density in agarose-coated wells in DMEM alone or DMEM containing LY-294002. After incubation at 37°C for 48 h, the viability of the cells was determined by MTT assay. LY-294002 reduced cell viability in a dose-dependent manner with an IC50 of ~10 µM (Fig. 11).


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Fig. 11.   Effect of inhibition of PI3K activity on survival of suspended MPT cells allowed to aggregate. MPT cells were suspended at high density in DMEM containing LY-294002, a potent PI3K inhibitor. Viability of cells suspended in LY-294002-containing DMEM is expressed as a percent of viability of cells suspended in DMEM alone. LY-294002 inhibited survival effect of cell-cell adhesion in a dose-dependent fashion with an IC50 of about 10 µM.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We demonstrate that MPT cells, when deprived of both cell-matrix and cell-cell adhesion, die by apoptosis (Fig. 1). These data are consistent with the observations of Frisch et al. (11, 13), who were the first to demonstrate that cell-matrix adhesion is an important survival signal. The study of adhesion-related survival signals has focused predominantly on the role of cell-matrix adhesion. In epithelial cells, the beta 1-integrins are primarily responsible for cell-matrix attachment (23, 50). The predominant matrix receptors for beta 1-integrin are peptide sequences containing the tripeptide sequence arginine-glycine-asparagine (RGD) (51). It has become clear that out-to-in signaling, activated by beta 1-integrin-RGD binding plays a critical role in promoting cell survival (12). A recent study has demonstrated that, in injured proximal renal tubular cells undergoing repair and proliferation, inhibition of integrin function with either an RGD-containing peptide or antibody to the beta 1-integrin promotes apoptosis (58).

Available evidence suggests that matrix-dependent survival signaling depends on activation of focal adhesion kinase (FAK), one of a complex of proteins (the focal contact) that mediates attachment of the intracellular domain of the beta 1-integrin to the actin cytoskeleton (4, 13). However, FAK-independent pathways responsible for inhibition of apoptosis initiated by cell-matrix detachment have also been described. Integrin ligation to the adaptor protein Shc may influence cell fate in a FAK-independent manner (55). Likewise, Ras has been demonstrated to mediate survival in response to cell-matrix adhesion via activation of PI3K (49), which in turn activates protein kinase B/Akt (37, 38).

Although the role of cell-matrix attachment in cell survival is well established, much less is known about the part played by cell-cell adhesion in inhibiting apoptosis. While some evidence suggests that cell-cell adhesion can inhibit apoptosis independently of cell-matrix adhesion (21, 24), the extent to which cell-cell adhesion promotes survival remains controversial and may vary depending on the cell type. The purpose of this study was to examine the extent to which cell-cell contact, in the absence of normal cell-matrix adhesion, can mediate survival of renal tubular cells in primary culture and to elucidate the signaling pathways involved.

We developed a model in which MPT cells are allowed to establish contact with one another without being able to establish normal cell-matrix attachment. To this end, MPT cells were incubated in DMEM at high cell density (to promote cell-cell contact) and in agarose-coated culture dishes (to prevent cell-matrix attachment; see METHODS). Under these conditions, we found that MPT cells aggregated spontaneously with one another (Fig. 2A) and remained viable for as long as 72 h. The viability of the cells allowed to aggregate with one another was demonstrated by a number of different techniques, including nuclear morphology (Figs. 3A and 4), MTT assay of cell viability (Fig. 5), and electron microscopy (Fig. 6A). The ultramicroscopic examination of cell aggregates also demonstrated intimate contacts between adjacent cells with structures resembling junctional complexes (Fig. 6B).

We have also demonstrated that the addition of EGTA to DMEM prevented cell aggregation (Fig. 2B), promoted apoptosis (Figs. 3B and 4), and reduced cell viability (Fig. 5). All these effects of EGTA could be prevented by the addition of calcium to the EGTA-containing DMEM (Figs. 2C, 3C, 4, and 5). However, addition of magnesium to EGTA-containing DMEM did not prevent the effects of EGTA on aggregation (not shown) or survival (Fig. 5). These findings suggest that cell-cell aggregation and survival is a calcium-dependent and magnesium-independent process.

The calcium-dependent and magnesium-independent nature of the cell-cell aggregation led us to examine the role of E-cadherin in this process. E-cadherin plays an important role in mediating adhesion of epithelial cells to one another (19). E-cadherin-mediated adhesion occurs by the homophilic binding of the extracellular domains of E-cadherin present on the surface of adjacent cells (20). This process requires the presence of calcium but does not require magnesium (20). A peptide sequence containing 17 amino acids (73 through 89) present at the end of the extracellular domain of E-cadherin has been shown by site-directed mutagenesis to be essential for homophilic cadherin binding (2). Furthermore, a synthetic 17-mer peptide identical to this HAV-containing amino acid sequence has been shown to be an effective inhibitor of cadherin-mediated cell-cell adhesion (2). We examined the effect of this HAV-containing 17-mer peptide and a control peptide containing a random amino acid sequence (see METHODS) on aggregation and survival of MPT cells. We have demonstrated that the HAV-containing peptide inhibited aggregation (Fig. 2E), promoted apoptosis (Figs. 3E, 4, and 7), and reduced viability of MPT cells (Fig. 5) suspended at high density in the absence of cell-matrix attachment. By contrast, the control 17-mer peptide had no effect on cell-cell-mediated aggregation (Fig. 2F), apoptosis (Figs. 3F and 4), or viability (Fig. 5).

We examined the expression of E-cadherin in cell-cell aggregates with immunofluorescence microscopy and flow cytometry. When MPT cells in suspension were allowed to aggregate with one another, expression of E-cadherin between cells was evident in a pattern similar to that seen in confluent monolayers (Fig. 8). Consistent with this finding are flow cytometric data showing that cell-cell aggregation of cells in suspension for 6 h markedly increases the surface expression of E-cadherin (Fig. 9). Furthermore, the surface expression of E-cadherin is prevented if cell-cell aggregation is inhibited by the presence of the HAV-containing peptide (Fig. 9).

Although these data clearly demonstrate that E-cadherin is necessary for adhesion of suspended MPT cells to one another they do not preclude a role for cadherin-independent events in mediating cell survival. We considered the potential role of integrin-mediated signaling in cell survival. Although plating cells on agarose prevents normal cell-matrix interactions, we cannot exclude the possibility that cell matrix is secreted by suspended cells. Thus it is possible that adhesion between beta 1-integrins on suspended cells and RGD receptors present on secreted matrix proteins represents the signaling pathway responsible for the survival of aggregated cells (14, 33). Another potential integrin-mediated mechanism for cell survival associated with cell-cell adhesion is the homophilic binding between beta 1-integrins present on adjacent renal tubular cells. This latter phenomenon has been shown to contribute to cell-cell adhesion in some cell types (5, 16, 29, 53).

It is worth noting that beta 1-integrins and cadherins share parallels other than their roles as adhesion molecules. Both molecules are attached via intermediary molecules to the actin cytoskeleton (1, 39), and both transduce intracellular signals when engaged (1, 6). Furthermore, both adhesion molecules require divalent cations for activity (15, 50). However, while cadherins require only calcium, integrin-mediated adhesion is dependent on the presence of magnesium as well as calcium (8, 18, 28). Because of the dependence of integrins on magnesium, our observation that calcium is sufficient to reverse the inhibitory effects of EGTA on survival in the absence of magnesium argues against a role for integrins in mediating survival of aggregated cells.

However, we did additional experiments to examine whether beta 1-integrins mediate cell-cell aggregation or survival. We excluded any role for beta 1-integrin-RGD interactions by showing that RGD-containing peptides did not prevent any of the following properties of MPT cells in suspension: cell-cell aggregation (Fig. 2D), inhibition of apoptosis (Figs. 3D and 4), or survival (Fig. 5). Furthermore, a neutralizing antibody to beta 1-integrins also had no effect on cell-cell aggregation, survival, or inhibition of apoptosis (Figs. 4 and 5). These data indicate that neither beta 1-integrin-RGD interactions nor homophilic interactions between beta 1-integrin molecules are responsible for signaling events that mediate the survival of aggregated MPT cells.

We have begun to explore the downstream signaling pathways involved in cadherin-mediated survival. We chose to examine the role of PI3K, which has been implicated in the survival pathway induced by growth factors (7, 26, 30, 59) as well as integrin-mediated adhesion (37). We demonstrate that constitutive activity of PI3K is present in confluent monolayers of MPT cells. The constitutive activity of PI3K is markedly reduced by incubation of suspended MPT cells in calcium- and magnesium-free PBS. PI3K activity can then be restored by resuspending the cells in calcium- and magnesium-containing medium (DMEM; Fig. 10A). The role of cadherin in activation of PI3K induced by cell-cell adhesion is supported by the fact that an HAV-containing peptide inhibits re-activation of PI3K (Fig. 10B). We also demonstrate that LY-294002, a specific PI3K inhibitor, inhibits survival of suspended cells in a dose-dependent manner (Fig. 11).

In summary, we demonstrate for the first time that aggregation of suspended renal tubular cells acts as a potent survival factor. Our data indicate that cell-cell aggregation is dependent on cadherin-mediated adhesion and is independent of integrin-mediated adhesion. We also show that cadherin-mediated adhesion and subsequent activation of PI3K are both necessary for survival of aggregated cells. However, we have not definitively demonstrated that E-cadherin-mediated cell-cell adhesion is sufficient for cell survival. It remains possible, although unlikely, that an as yet unrecognized cadherin-independent adhesion event is also necessary for aggregated cells to survive.

Finally, our observations have potential relevance to a number of pathological states. It is feasible, for example, that the viability of renal carcinoma cells could be mediated by cadherin-mediated signaling (24). Furthermore, we suggest that cadherin-mediated adhesion of exfoliated but viable renal tubular cells (17, 31, 46) after acute renal injury could contribute to the formation of cellular casts in acute renal failure.


    ACKNOWLEDGEMENTS

We are grateful to Dr. Yuhui Xu (Dana Farber Cancer Institute, Core Electron Microscopy Facility, Boston, MA) for the electron microscopic studies.


    FOOTNOTES

This work was supported by National Institutes of Health grants DK-375105 (W. Lieberthal), DK-52898 (W. Lieberthal), HL-53031 (W. Lieberthal), AR/AI 42732 (J. S. Levine), and a Clinical Scientist award from the National Kidney Foundation (J. S. Levine).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: W. Lieberthal, Boston Medical Center, Renal Section, Rm. 537, Evans Biomedical Research Center, 650 Albany St., Boston, MA 02118 (E-mail: wliebert{at}bu.edu).

Received 30 March 1999; accepted in final form 30 November 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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