Protein kinase C regulates endocytosis and recycling of E-cadherin

Tam Luan Le1,2, Shannon R. Joseph1, Alpha S. Yap1,3, and Jennifer L. Stow1,2

1 Institute for Molecular Bioscience, 2 Department of Biochemistry, and 3 Department of Physiology and Pharmacology, University of Queensland, Brisbane 4072, Queensland, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

E-cadherin is a major component of adherens junctions in epithelial cells. We showed previously that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. In the present study, we investigated the potential role of protein kinase C (PKC) in regulating the trafficking of surface E-cadherin in Madin-Darby canine kidney cells. Using surface biotinylation and immunofluorescence, we found that treatment of cells with phorbol esters increased the rate of endocytosis of E-cadherin, resulting in accumulation of E-cadherin in apically localized early or recycling endosomes. The recycling of E-cadherin back to the surface was also decreased in the presence of phorbol esters. Phorbol ester-induced endocytosis of E-cadherin was blocked by specific inhibitors, implicating novel PKC isozymes, such as PKC-epsilon in this pathway. PKC activation led to changes in the actin cytoskeleton facilitating E-cadherin endocytosis. Depolymerization of actin increased endocytosis of E-cadherin, whereas the PKC-induced uptake of E-cadherin was blocked by the actin stabilizer jasplakinolide. Our findings show that PKC regulates vital steps of E-cadherin trafficking, its endocytosis, and its recycling.

trafficking; cell-cell adhesion; actin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADHERENS JUNCTIONS on the lateral surfaces of epithelial cells mediate cell-cell contact and have a central role in establishing and maintaining cell polarity. Members of the cadherin superfamily function as primary mediators of Ca2+-dependent cell-cell adhesion, linking adjacent cells through homophilic binding of cadherin ectodomains (16). E-cadherin subserves this key adhesion role in epithelial cells. Cadherin-mediated adhesion must be dynamic to accommodate epithelial growth and remodeling during development and for wound healing and turnover of epithelia in mature tissues (17, 44, 46). Cell-cell adhesion in epithelia is disrupted during tumorigenesis, where the loss of cadherin function augurs transformation to a metastatic phenotype (20).

The adhesive strength of cell-cell contacts depends on factors such as dimerization and lateral clustering of cadherin molecules (39, 47). Cadherins also form high-affinity complexes with catenins and other molecules, which participate in the overall function and stability of the adherens junctions (34). E-cadherin is bound to beta -catenin and thence to alpha -catenin, which links the complex to the actin cytoskeleton (25, 34, 38). Cadherin-catenin complexes form detergent-resistant plaques when bound stably to actin cables at the points of cell-cell contact (2, 33). Tracking of fluorescence-tagged E-cadherin in forming epithelial monolayers revealed the dynamic nature of the cadherin complexes associated with circumferential actin during contact formation and compaction (1). Rho family GTPases---Rho, Rac, and Cdc42---produce different configurations of actin in cells (18) and have been implicated in remodeling actin for regulation of cell-cell contacts. Expression of constitutively active Rac1 accumulates cadherins, catenins, and actin at junctions, resulting in E-cadherin-mediated adhesion in both mammalian cells and Drosophila (9, 12, 37). The actin cytoskeleton therefore has roles in stabilizing cell-cell contacts and also perhaps in more dynamic movement of cadherins in and out of adherens junctions.

The trafficking of E-cadherin---its delivery to or removal from the cell surface---is one aspect of cadherin biology that is not yet thoroughly understood. There is a growing body of evidence to show that cell surface E-cadherin can be endocytosed and trafficked back into the cell. A variety of studies have shown that cadherins are internalized after the disruption of junctions, for instance, by Ca2+ depletion (4, 6, 22, 23). In a previous study we (26) also established, importantly, that E-cadherin undergoes endocytosis and recycling in Madin-Darby canine kidney (MDCK) cell monolayers under physiological conditions. The adhesive strength at cell-cell contacts depends, in part, on the amount of E-cadherin at the cell surface (47). On the basis of the finding that recycling of internalized E-cadherin is required to form cell-cell contacts (26), we proposed that such a recycling pathway provides one mechanism for dynamically regulating cadherin-based adhesion, by balancing the relative amounts of surface-exposed and sequestered intracellular E-cadherin. Such a mechanism has, in fact, been recorded during gastrulation in sea urchin embryos, where E-cadherin is seen to move in and out of the cell to accommodate epithelial-mesenchymal conversions (29). Parallel pathways also exist for the endocytosis and recycling of integrins and thereby for the regulation of integrin-based contact with substrata (32). Recycling might thus be a common means of regulating adhesion at the cell surface. How the endocytosis and recycling of E-cadherin are regulated is therefore of prime importance to the control of cell-cell adhesion.

The protein kinase C (PKC) family of serine-threonine protein kinases includes multiple isoforms, or isozymes, which are classified into three main groups, conventional, novel, or atypical, depending on their requirements for Ca2+ and diacylglycerol (30). Phorbol 12-myristate 13-acetate (PMA) is a well-known tumor-promoting phorbol ester and a commonly used activator of conventional and novel forms of PKC. The PKC family is ubiquitous, and individual isozymes have been broadly implicated in many cell functions, including cytoskeletal rearrangements, membrane traffic, ion transport, and cell adhesion (24, 31). PKC has demonstrated roles in regulating endocytosis (21, 41, 43). PKC has been implicated in signaling events that control the internalization of several cell surface receptors, usually via clathrin-mediated pathways, and in other pathways such as phagocytosis (27) and the uptake of fluid phase markers. The use of phorbol esters to activate PKC produces disparate effects in different cell types and even reveals independent mechanisms for regulating endocytosis at the apical or basolateral membrane domains of polarized cells. PMA activation of PKC in T84 cells increased basolateral, but not apical, uptake of fluid phase markers (43), whereas, in MDCK cells, phorbol esters increased both fluid phase uptake and clathrin-mediated uptake from the apical but not basolateral membrane (21, 41). PKC activation also stimulated transcytosis and delivery of receptors to the apical membrane in MDCK cells (10).

In the present study, regulation of the endocytosis and recycling of E-cadherin in confluent MDCK cells under physiological conditions was investigated. Novel findings are presented to show that the bidirectional movement of E-cadherin between the cell surface and endosomes is regulated and that PKC and actin are implicated in this regulation. This is the first demonstration that two linked trafficking steps of E-cadherin are regulated by PKC in a fashion poised to regulate cadherin function at the cell surface.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. MDCK cells (strain II) were grown and passaged as previously described in DMEM (Life Technologies, Grand Island, NY) with 10% FCS and 2 mM glutamine in 5% CO2-95% air (26). Cells used in experiments were plated at confluent density on semipermeable polycarbonate filters (Transwell; Corning Costar, Cambridge, MA) or plated onto glass coverslips and maintained for 1-3 days before being used. For most experiments, cells were incubated in medium containing 10 µM cycloheximide to block protein synthesis to clear newly synthesized E-cadherin, leaving only endocytosed E-cadherin as the intracellular pool. Cells were incubated in medium containing 0.1 µM PMA to induce PKC activation (11). The PKC inhibitor bisindolylmaleimide (Gö-6850; Calbiochem, CA) or the indolocarbazole PKC inhibitor (Gö-6976; Calbiochem, CA) was added at 5 µM (final concentration) to culture medium for various times at 37°C. In some experiments, cells were incubated in medium containing 1 µM jasplakinolide (Jas; Molecular Probes, Eugene, OR) or in medium containing 10 µM cytochalasin D (CyD; Sigma, St. Louis, MO) before processing.

Antibodies. A mouse E-cadherin antibody (3B8) raised against MDCK E-cadherin was used for immunofluorescence experiments. For immunoblotting, we used a mouse monoclonal antibody against human E-cadherin (Transduction Laboratories, Lexington, KY). Other primary antibodies used included mouse monoclonals against alpha -catenin and beta -catenin (Transduction Laboratories) and a mouse monoclonal antibody directed against beta -actin (a gift of Dr. R. Weinberger, New Children's Hospital, Westmead, Australia). Secondary antibody conjugates used were sheep anti-mouse IgG-Cy3 (Jackson Immunoresearch Labs, West Grove, PA), goat anti-mouse IgG-Alexa 488 (Molecular Probes), and horseradish peroxidase-labeled sheep anti-mouse IgG (Amrad Laboratories).

Immunofluorescence staining. Cells were fixed in 4% paraformaldehyde in PBS for 90 min and then permeabilized for 5 min in PBS containing 0.1% Triton X-100. Cells were incubated with primary antibodies followed by incubation in secondary antibodies with PBS containing BSA as a blocking buffer. Cells were mounted in 50% glycerol-1% n-propyl gallate in PBS and viewed by epifluorescence on an Olympus Provis X-70 microscope. Images were collected with an CCD300ET-RCX camera using NIH image software. Cells grown on Transwell filters were examined with a Bio-Rad MRC-600 confocal laser scanning microscope, and X-Y and X-Z sections were generated with Bio-Rad MRC-600 cmos software.

Fluid-phase endocytosis. Rhodamine-dextran (lysine-fixable, anionic rhodamine-conjugated dextran, molecular weight 10,000; Molecular Probes) was added to the medium in upper or lower chambers of Transwell units and used to measure fluid phase uptake from the apical or basal side of monolayers. After various times at 37°C, cells were placed on ice and washed three times with ice-cold serum-free medium, followed by fixation and mounting on coverslips for confocal microscopy. In some experiments, cells were also fixed and double labeled with E-cadherin antibodies.

Cell surface biotinylation. MDCK cells grown on filters were incubated on ice for 1 h with 1.5 mg/ml sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin; Pierce, Rockford, IL) applied to the basal side of the filter, followed by washing with sulfo-NHS-SS-biotin blocking reagent (50 mM NH4Cl in PBS containing 1 mM MgCl2 and 0.1 mM CaCl2) to quench free sulfo-NHS-SS-biotin, followed finally by several further washes in PBS. Cells were then scraped off filters and lysed in 500 µl of RIPA buffer (25 mM Tris · HCl pH 7.4 with 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 1% deoxycholate) with protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany). Cell extracts were centrifuged, and the supernatants were incubated with streptavidin beads (Sigma) to collect biotinylated proteins, which were then analyzed by SDS-PAGE and immunoblotting to identify E-cadherin. Staining of transfer membranes with 0.1% Coomassie brilliant blue ensured even protein transfer and protein loading. Proteins detected by immunoblotting were visualized with chemiluminescence (Pierce). Different luminescence exposures were collected, and exposures in the linear range were used.

Biotinylation assay for endocytosis and recycling. Surface biotinylation was adapted to measure the endocytosis of E-cadherin as described previously (26). Confluent MDCK cells grown on Transwell filters were pretreated with cycloheximide and biotinylated at 0°C, followed by washing and quenching of free biotin. The cells were then incubated in culture medium at 37°C for various times to allow uptake. Monolayers were then glutathione stripped as originally described by Graeve et al. (15) by incubation in two 20-min washes of glutathione solution (50 mM glutathione, 75 mM NaCl, 75 mM NaOH, and 1% BSA) at 0°C, which removed all cell surface biotin groups. The effectiveness of glutathione stripping at removing all biotinyl groups from surface proteins was demonstrated in cells surface-biotinylated at 0°C for 1 h followed immediately by glutathione stripping. No E-cadherin was recovered with streptavidin beads from these samples in the recovered biotinylated fraction (e.g., see Fig. 1A, lane 2). The remaining biotinylated proteins, sequestered inside cells after endocytosis, were protected from glutathione stripping and were collected on streptavidin beads and analyzed by immunoblotting. In some experiments cells were incubated at 18°C to accumulate internalized E-cadherin (26).


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Fig. 1.   Dose-dependent internalization of surface-biotinylated E-cadherin in the presence of phorbol 12-myristate 13-acetate (PMA). A: Madin-Darby canine kidney (MDCK) cells were surface-biotinylated at 0°C and then incubated at 37°C in the presence of different concentrations of PMA (1 nM-10 µM) for 30 min (lanes 4-9). Surface-biotinylated proteins were recovered on streptavidin beads and analyzed by SDS-PAGE and immunoblotting to detect E-cadherin. Biotinylated cell surface proteins were recovered from the cell extracts (lane 1). Glutathione stripping (g.s.) immediately after biotinylation at 0°C completely removed biotin from surface proteins (lane 2). E-cadherin sequestered in an internal pool was recovered after glutathione stripping (lanes 3-9). Cells incubated in the presence of 1 nM-0.1 µM PMA displayed a dose-dependent increase in the amount of internalized E-cadherin. Higher concentrations of PMA (1-10 µM) resulted in inhibition of E-cadherin uptake (lanes 8 and 9). B: amounts of biotinylated E-cadherin sequestered in the presence of different concentrations of PMA in 3 separate experiments were quantitated by densitometry and plotted. This emphasizes that peak accumulation of E-cadherin was obtained with PMA at 0.1 µM.

To measure recycling of endocytosed proteins accumulated at 18°C, cells were glutathione stripped at 0°C and then returned to 37°C for various times in normal medium. Cells were then washed quickly in PBS and incubated with 0.01% trypsin (Sigma) in Ca2+-free PBS for 20 min followed by addition of 100-fold excess soybean trypsin inhibitor to inhibit further protease digestion. Trypsinized, biotinylated cell surface proteins were recovered on streptavidin beads, and the cells were then lysed in RIPA buffer and the remaining cell-associated biotinylated proteins were recovered on streptavidin beads. The extracellular fragment of E-cadherin released by trypsin and cell-associated E-cadherin were analyzed by SDS-PAGE and immunoblotting.

Immunoprecipitation. Confluent monolayers of MDCK cells were solubilized in cold RIPA buffer containing protease inhibitors on ice. RIPA-soluble cell extracts were obtained by centrifugation and incubated with E-cadherin antibody for 90 min and then with washed protein A Sepharose beads (Pierce) for a further 90 min. Precipitates were recovered by centrifugation and then washed several times with RIPA buffer and 20 mM Tris · HCl pH 7.4 before solubilization in concentrated SDS-PAGE sample buffer. Bound E-cadherin and actin were then analyzed by SDS-PAGE and immunoblotting.

Cell fractionation. Confluent monolayers of cells were scraped into ice-cold homogenization buffer (1 mM EDTA, 10 mM Tris pH 7.4, containing protease inhibitors). Cells were lysed by repeated passage through a 27.5-G needle. Nuclei and unbroken cells were removed by low-speed centrifugation. The resulting postnuclear supernatant was ultracentrifuged at 100,000 g for 90 min. The pellet constituting the total microsomal membrane fraction was resuspended in homogenization buffer. The supernatant was recentrifuged at 100,000 g for 45 min to finally remove membranes, and this supernatant was collected as the cytosol fraction. Protein concentrations of the membranes and cytosol were determined with the bicinchoninic acid (BCA) protein assay kit (Pierce). Membrane and cytosol fractions were analyzed by SDS-PAGE and immunoblotting.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Accumulation of intracellular E-cadherin in PMA-treated MDCK cells. We previously established (26) a biotinylation assay for measuring the internalization of surface-biotinylated E-cadherin in confluent MDCK cell monolayers. At steady state, relatively small but uniform amounts of E-cadherin are endocytosed (Fig. 1A, lane 1) and are measured as a pool of biotinylated E-cadherin that remains sequestered after stripping cell surface biotin (Fig. 1A, lane 3). In all experiments, cells are pretreated with cycloheximide to ensure that all E-cadherin in the biosynthetic pathway is cleared, leaving endocytosed E-cadherin as the only intracellular pool. In these experiments, cells were treated with a phorbol ester (PMA) at a range of concentrations (1 nM-1 µM) to activate PKC and then assayed for E-cadherin endocytosis. Cells incubated in the presence of PMA displayed a progressive, dose-dependent increase in the amount of internalized E-cadherin (Fig. 1A, lanes 4-7). PMA at 0.1 µM induced maximal sequestration of internalized E-cadherin (>4-fold increase over control cells; Fig. 1). At higher concentrations of PMA (1-10 µM) the amount of internalized E-cadherin was reduced, consistent with the downregulation of PKC often observed with prolonged exposure or at high concentrations of PMA (10).

The uptake of surface-biotinylated E-cadherin was then followed over a time course in the presence or absence of 0.1 µM PMA (Fig. 2A). In control cells a small, constitutive pool of surface-biotinylated E-cadherin was internalized, and this remained constant over time, as a result of recycling (Ref. 26; Fig. 2A, lanes 3-6). In contrast, in PMA-treated cells, the size of the intracellular pool of E-cadherin increased over time (Fig. 2A, lanes 7-10), so that by 60 min, there was a greater than fivefold accumulation of intracellular E-cadherin (Fig. 2A, lanes 7-10). The increased accumulation of E-cadherin by PMA was also seen in cells incubated at 18°C, which itself serves to accumulate endosomal E-cadherin (26). As seen in Fig. 2B, PMA-treated cells displayed a more rapid accumulation of internalized E-cadherin during the first 30 min, resulting in a larger internal pool of E-cadherin after 60 min (Fig. 2B, lanes 7 and 8). Thus PMA treatment enhances the uptake of surface E-cadherin; this increased uptake contributes to a greater and progressive accumulation of E-cadherin in internal endosomes.


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Fig. 2.   Internalization of surface-biotinylated E-cadherin. A: time-course of E-cadherin uptake. Cycloheximide-treated cells were surface-biotinylated at 0°C (lane 1) and then incubated at 37°C for periods of 0-60 min (lanes 3-10) in the absence (-) or presence (+) of PMA. A constant amount of surface-biotinylated E-cadherin was sequestered at chase times from 15 to 60 min (lanes 3-6), whereas in PMA-treated cells, there was a progressive increase in the amount of intracellular E-cadherin (lanes 7-10). B: when the same experiments were carried out on MDCK cells incubated at 18°C, in normal cells there was a steady accumulation of internalized, surface-biotinylated E-cadherin. However, in cells incubated in the presence of PMA, internalized E-cadherin accumulated more rapidly over the chase period (15-60 min).

Selective PKC inhibition retards PMA-induced E-cadherin endocytosis. Phorbol esters are known to have their effects by acting on multiple targets and signaling pathways. To establish whether the effects of PMA on E-cadherin endocytosis are working primarily through PKC, we used PKC inhibitors to abrogate the effects of PMA in the biotinylation assay. The uptake of surface-biotinylated E-cadherin in PMA-treated cells was measured over 90 min at 37°C in the presence of specific PKC inhibitors, Gö-6850 (5 µM), which acts to inhibit both conventional and novel PKC isoforms by competing for ATP binding sites (14, 45), and Gö-6976, which is a more selective inhibitor for the conventional PKC isoforms (28). Cells incubated in the presence of PMA alone showed a progressive increase in internalized E-cadherin; addition of Gö-6850 significantly reduced internalization throughout the time course. Cells incubated in the presence of Gö-6850 alone or with PMA plus Gö-6850 did not accumulate E-cadherin intracellularly over 90 min, showing that this inhibitor blocked the effect of PMA (Fig. 3, A and C). The other inhibitor, Gö-6976, had no such effect and did not alter the PMA-induced accumulation of intracellular E-cadherin (Fig. 3, B and C). The selective abrogation of PMA-induced uptake by Gö-6850, but not by Gö-6976, implicates the novel class of PKC isozymes in this regulation. Additional PKC isoforms or other regulators may also affect E-cadherin internalization because Gö-6850 produced an incomplete block of PMA activity in this assay.


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Fig. 3.   Detection of protein kinase C (PKC) isoforms and effect of inhibitors. A: cycloheximide-treated cells were surface-biotinylated at 0°C (control, con) followed by incubation at 37°C in media with PMA or with PMA (0.1 µM) + Gö-6850 (5 µM) for various times (15-90 min), followed by glutathione stripping and recovery of internalized, surface-biotinylated E-cadherin on streptavidin beads. In cells incubated with PMA there was a steady accumulation of surface-biotinylated E-cadherin; Gö-6850 reduced uptake and accumulation of E-cadherin. B: inhibitor Gö-6976 had no significant effect on PMA-induced internalization of surface-biotinylated E-cadherin. C: accumulation of surface-biotinylated E-cadherin in the presence of PMA, Gö-6850, PMA + Gö-6850, and PMA + Gö-6976 was quantitated by densitometry and graphed. PMA alone causes an accumulation (>4-fold) of intracellular E-cadherin, and this was blocked by the addition of Gö-6850 but not by Gö-6976. D: detection and translocation of PKC. Distribution of different PKC isoforms in response to PMA was examined by immunoblotting in cytosol (C) and membrane (M) fractions of MDCK cell homogenates from control or PMA-treated cells (15 min). PKC-alpha and -gamma were found predominantly in cytosol, whereas PKC-µ and -epsilon were in both membrane and cytosol fractions. In response to PMA, all 4 isoforms translocated to the membrane fractions.

To establish whether candidate PKC isoforms are present in this system, MDCK cell fractions were screened with isoform-specific antibodies. Several isoforms of PKC (alpha , gamma , epsilon , and µ) were detected with specific antibodies, noting that the conventional isoforms alpha  and gamma  are restricted to cytosol fractions and the novel isoforms epsilon  and µ were found on both membrane and cytosol fractions in control cells (Fig. 3D). In cells treated with PMA before fractionation, all four isoforms translocated to membrane fractions within a time frame (15 min) concurrent with the stimulation of E-cadherin endocytosis (Fig. 3D). Thus novel class isoforms of PKC associate with membranes and are potentially available for regulating trafficking in these cells.

PMA inhibits recycling of endocytosed E-cadherin. To test the influence of PMA on the recycling of surface E-cadherin, the exit of internalized, surface-biotinylated E-cadherin back to the cell surface was measured (Fig. 4A). Cells were surface-biotinylated at 0°C and then incubated at 18°C for 60 min to accumulate internalized, labeled E-cadherin. After glutathione stripping, cells were returned to 37°C to resume trafficking in the presence or absence of PMA for various periods (0-30 min). Cells were then sequentially treated with glutathione at each chase time to remove biotinyl groups from endocytosed proteins as they returned to the cell surface. The remaining unrecycled, biotinylated proteins at each time point were recovered and immunoblotted for E-cadherin. The amount of internalized E-cadherin accumulated in control cells at 18°C progressively decreased as cells were rewarmed to 37°C to allow recycling (Fig. 4A, lanes 3-6), so that by 30 min the majority (90%) of internal E-cadherin had returned to the cell surface and was stripped (Fig. 4A, lane 6). In contrast, PMA-treated cells displayed a much slower decrease in the internal pool of E-cadherin throughout the release period, with ~40% of the accumulated pool still remaining after 30-min treatment with PMA (Fig. 4A, lane 10). These results show that PMA retards E-cadherin release from endosomes.


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Fig. 4.   Recycling of E-cadherin. A: cycloheximide-treated cells were surface-biotinylated on ice and then incubated at 18°C for 2 h to allow for endocytosis and accumulation of E-cadherin (lanes 3 and 7). After glutathione stripping, cells were then returned to 37°C in the absence (-) or presence (+) of PMA for chase periods of 10, 15, and 30 min (lanes 3-10). At each chase time, cells were glutathione stripped and remaining biotinylated proteins were recovered. In the absence of PMA, the internal E-cadherin pool diminished over time (lanes 4-6) as E-cadherin was returned to the cell surface. PMA caused retention of E-cadherin in the internal pool (lanes 8-10). B: in similar experiments, after accumulation of surface-biotinylated E-cadherin and subsequent glutathione stripping to remove all surface-biotinylated proteins, cells were then incubated at 37°C in the presence (+) or absence (-) of PMA during chase times (0-30 min). At each time point cells were gently trypsinized to strip cell surface proteins. Biotinylated proteins from both the cell-associated (B, top) and trypsin-released fractions in the medium (B, bottom) were recovered on streptavidin beads and analyzed by SDS-PAGE. Intact E-cadherin (120 kDa) or its trypsin-cleaved 82-kDa ectodomain was detected by immunoblotting with a specific antibody raised against the NH2-terminus of E-cadherin. Top, internalized E-cadherin accumulated at 18°C (lane 3) gradually disappeared from the internal pool over 30 min (lanes 4-6). In the presence of PMA internal E-cadherin was retained (lanes 7 and 8). Bottom: the 82-kDa ectodomain of biotinylated E-cadherin was released from recycled E-cadherin during 15- to 30-min chases; in PMA-treated cells, less ectodomain was recovered and it only appeared after 30 min at 37°C (lane 8). C: immunofluorescence of E-cadherin in control cells (a) and cells treated with PMA for 30 min (b) showing staining of vesicular endosomes and the cell surface; cells then incubated for an additional 30 min in PMA (c) accumulated E-cadherin in endosomes, whereas cells incubated for an additional 30 min in PMA + Gö-6850 (d) show depletion of endosomal staining.

A second type of experiment also confirmed this finding. E-cadherin recycled to the cell surface was cleaved with trypsin to release an 82-kDa extracellular soluble fragment into the medium. Internalized, biotinylated E-cadherin was gradually depleted during chase times with the concomitant appearance of the 82-kDa ectodomain in the medium (Fig. 4B, lanes 3-6), and we found that, in the presence of PMA, both the depletion of intact E-cadherin and the appearance of the ectodomain were significantly decreased (Fig. 4B, lanes 7 and 8).

Finally, the accumulation of E-cadherin endosomes and its exit from this store were observed in cells by immunofluorescence. PMA treatment for 30 min greatly increased the staining of E-cadherin in vesicular endosomes (Fig. 4C) compared with control cells, which had only faint vesicular staining. When cells were incubated for a further 30 min in the presence of PMA, they maintained this intracellular store of E-cadherin, which continued to accumulate (Fig. 4C; also shown biochemically in Fig. 3). However, when this additional incubation contained both PMA and Gö-6850, the endosomal staining and initial accumulation of E-cadherin disappeared (Fig. 4C). By blocking the effect of PMA, Gö-6850 allowed the endosomal E-cadherin to exit the endosomes and be recycled, implicating a novel class PKC in regulation of recycling of E-cadherin. Together these experiments show that PMA separately regulates the recycling arm of the surface E-cadherin trafficking pathway, with the effect of the Gö-6850 inhibitor implicating PKC in this regulation. Reduced traffic back out to the cell surface is also a contributing factor, along with increased uptake, in the accumulation of E-cadherin in endosomes of PMA-treated cells.

PMA increases uptake of E-cadherin into apical recycling endosomes. The effect of PMA on the distribution of E-cadherin in fixed, filter-grown MDCK cells was studied by confocal microscopy. At steady state, E-cadherin in confluent MDCK cells is localized mostly at the lateral cell surface with minimal staining in small intracellular vesicles (Fig. 5, see also Fig. 4C; Ref. 26). PMA treatment (30 min) produced significantly more vesicular staining of E-cadherin than in untreated cells, with no discernable change in the level of cell surface staining (Fig. 5, a and b). Intracellular E-cadherin in PMA-treated cells was localized in more numerous and larger structures, which were found in X-Z image views to be located near the apical cell surface (Fig. 5d). Increased intracellular staining is thus consistent with increased endocytosis of surface E-cadherin in the presence of PMA.


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Fig. 5.   E-cadherin and rhodamine-dextran distribution in confluent MDCK cells. Immunofluorescence staining of E-cadherin in cycloheximide-treated, confluent cells in the presence or absence of PMA. a, b, e, f, i, and j: Confocal image cross sections. c, d, g, and h: X-Z sections of cell monolayers grown on filters. a: In control cells, E-cadherin staining is mostly localized to the lateral cell surfaces with some small intracellular vesicles. b: After incubation with PMA, intense intracellular staining of E-cadherin was found concentrated in vesicles underlying the apical membrane. c and d: In the X-Z sections the apical position of the endosomes can be seen along with staining of the lateral and basal membranes. e and f: Rhodamine-dextran taken up from the apical pole is localized in apical endosomes. PMA had no effect on this uptake. i and j: Double labeling revealed that E-cadherin- and dextran-positive vesicles did not overlap. k and l: FITC-phalloidin staining of F-actin in the plane of the lateral adhesion junctions shows that cell morphology and the cortical actin staining are preserved in monolayers incubated in the presence of PMA.

To determine whether PMA induces a general increase in membrane turnover or more selective upregulation of E-cadherin endocytosis, we examined the uptake of rhodamine-dextran as a fluorescent marker of fluid phase endocytosis. The amount and distribution of intracellular rhodamine-dextran endocytosed from either side of cells treated with PMA were similar to those in control cells at 30 min. Figure 5 shows internalized dextran localized in apically oriented compartments in a position similar to that of internalized E-cadherin in PMA-treated cells. However, double labeling revealed that E-cadherin and dextran were sequestered in separate nonoverlapping endosomes in the apical pole of MDCK cells (Fig. 5, i and j). In fully confluent monolayers phalloidin staining showed that PMA, under these conditions, did not induce significant changes in cell shape or in the actin cytoskeleton (Fig. 5, k and l). Hence, PMA treatment increased the sequestration of E-cadherin in specific compartments that were largely separate from the regulation of, and pathway for, fluid phase uptake.

Depolymerization of F-actin is required for E-cadherin endocytosis. Actin-binding proteins are commonly implicated as downstream effectors of PKC and are also known to be involved in endocytosis. Experiments were carried out to examine the role of actin polymerization in PKC-regulated E-cadherin trafficking. The uptake of surface-biotinylated E-cadherin was measured in the presence of the drugs CyD and Jas, which act to prevent actin polymerization and to stabilize F-actin, respectively (42). Treatment with CyD alone significantly enhanced the internalization of E-cadherin above controls to ~80% of the levels obtained with PMA (Fig. 6). Preincubation of cells with CyD and PMA together did not produce an additive effect. Stabilization of F-actin with Jas alone had no effect on E-cadherin uptake; however, pretreatment with Jas fully abrogated the subsequent PMA response, resulting in only control levels of E-cadherin uptake (Fig. 6). Immunofluorescence staining for E-cadherin also confirmed that preincubation of cells with Jas blocked the accumulation of E-cadherin in apical endosomes, which were otherwise present in cells treated with PMA alone (Fig. 7). Because Jas competes with the binding sites for phalloidin (42), the F-actin changes accompanying these treatments could not be demonstrated in stained cells. Together these results show that PMA is acting largely or wholly through actin to regulate the uptake of E-cadherin. Moreover, depolymerization of F-actin is required for endocytosis induced by PMA.


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Fig. 6.   E-cadherin internalization and actin polymerization. A: cycloheximide-treated cells were surface-biotinylated at 0°C and then incubated in normal medium (Con) or medium containing jasplakinolide (Jas), PMA, or cytochalasin D (CyD) to allow internalization, followed by glutathione stripping. Some cells were preincubated with either CyD or Jas before surface biotinylation and internalization in the additional presence of PMA. B: relative amounts of internalized E-cadherin shown on the graph were measured by SDS-PAGE, immunoblotting, and densitometry. Both PMA and CyD increased internalization of E-cadherin; only Jas blocked the PMA-induced uptake. Data are means ± SE from 3 separate experiments.



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Fig. 7.   F-actin blocks PMA-induced E-cadherin uptake. Confluent MDCK monolayers were treated with Jas for 30 min and then with Jas + PMA before fixation, staining for E-cadherin, and confocal analysis in apical and basolateral (B/L) planes. a and b: Cells incubated with PMA alone showed E-cadherin staining in a typical circumferential pattern at the lateral cell surfaces and in intracellular vesicles near the apical cell surface. c and d: Cells pretreated with Jas showed E-cadherin staining at the lateral membrane, but only occasional vesicles were labeled e and f: PMA increased apical vesicular staining, whereas in g and h, cells preincubated with PMA and Jas showed no such vesicular accumulation.

E-cadherin at the adherens junctions is bound to the actin cytoskeleton. The effect of PMA on the association of E-cadherin with actin was investigated by immunoprecipitation. E-cadherin was immunoprecipitated from extracts of cells incubated with or without PMA, and immunoprecipitates were analyzed to detect E-cadherin and any coprecipitated beta -actin. beta -Actin was coimmunoprecipitated with E-cadherin in control cells, and immunoprecipitates from PMA-treated cells showed relatively reduced amounts of beta -actin (Fig. 8A). Densitometric analysis of these gels shows that there is a 50% reduction in the actin coprecipitated with E-cadherin in PMA-treated cells.


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Fig. 8.   E-cadherin association with actin. A: immunoprecipitation of E-cadherin. Cells were incubated with or without (control) PMA for 45 min at 37°C followed by cell lysis in RIPA buffer and immunoprecipitation with the E-cadherin antibody (lanes 1-4). Immunoprecipitates (p) were analyzed by SDS-PAGE and immunoblotting to detect both E-cadherin and associated beta -actin. The amount of coimmunoprecipitated beta -actin was decreased in PMA-treated cells (lanes 2 and 4). B: confluent cells incubated in the presence or absence of PMA for 45 min were extracted with a buffer containing 1% Triton X-100. The resulting soluble extract (s) and insoluble pellet (i) were separated on SDS-PAGE and immunoblotted for E-cadherin. E-cadherin shifted from the Triton X-100-insoluble (fraction bound to cytoskeleton) to the soluble pool in the presence of PMA consistent with its partial release from F-actin.

Finally, to examine the association of E-cadherin with F-actin the relative detergent solubility of E-cadherin was examined in control and PMA-treated cells. E-cadherin firmly bound to the cytoskeleton is normally insoluble in 1% Triton X-100 at room temperature (40), leaving a relatively large proportion of E-cadherin to be recovered in the insoluble pellet after extraction of cells (Fig. 8B). Extraction of PMA-treated cells under these conditions resulted in a shift of E-cadherin from the insoluble pellet (cytoskeleton associated) to the soluble supernatant (Fig. 8B). This suggests that PMA induces a shift of E-cadherin to a less tightly bound state. Both the results of the immunoprecipitation and the increase in the solubility of E-cadherin are consistent with PMA, causing some depolymerization or destabilization of F-actin, resulting in disengagement of E-cadherin complexes from the cytoskeleton.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that both the endocytosis of cell surface E-cadherin and its recycling back to the cell surface are regulated events. Treatment of cells with the phorbol ester PMA increased the uptake of surface-biotinylated E-cadherin and decreased its recycling back to the cell surface, suggesting that PMA affects at least two transport steps. The net effect was a PMA-induced intracellular accumulation of surface E-cadherin, which was demonstrated by two independent methods: 1) the sequestration of increased amounts of surface-biotinylated E-cadherin and 2) increased staining of E-cadherin in endosomal compartments in PMA-treated cells. In several experiments, results obtained with the biotinylation assay were consistent with the finding that PMA increased either the rate or volume of E-cadherin endocytosis, which contributed to an accumulating intracellular pool instead of maintaining the relatively small steady-state level of intracellular E-cadherin. PMA produced a substantial increase (>4-fold) in the amount of E-cadherin sequestered inside cells, supporting the concept that PKC might play a significant role in regulating E-cadherin function and cell adhesion.

The use of specific PKC inhibitors verified that PMA is acting primarily through PKC to have its effect on E-cadherin trafficking. There are multiple phorbol ester-sensitive isozymes of PKC, all of which have pleiotropic distributions and functions in different cell types (30). The inhibitor Gö-6850 was found to largely block PMA-induced endocytosis in the biotinylation assay and the exit of E-cadherin from endosomes. Gö-6850 inhibits novel class, Ca2+-independent PKC isozymes, and immunoblotting of MDCK fractions revealed the presence and membrane translocation of PKC-epsilon as a possible candidate for the regulation of both endo- and exocytosis of E-cadherin. Although our studies do not provide specific evidence to implicate PKC-epsilon as a regulator in E-cadherin recycling, it has been implicated in other trafficking pathways such as the endocytosis of fluid phase markers in polarized T84 cells (43) and in transcytosis in MDCK cells (10).

Two independent steps of E-cadherin trafficking---its internalization into endosomes and its exit from them---were altered by PMA. Decreased exocytosis or recycling also contributed to the intracellular accumulation of E-cadherin in PMA-treated cells. The recycling of internalized E-cadherin was measured biochemically, first by depletion of the internal biotinylated pool and second by trypsin cleavage and recovery of a recycled, biotinylated ectodomain, and in both cases we found that PMA caused a significant (~50%) reduction in the rate of E-cadherin recycling. There was no evidence to suggest that E-cadherin was being degraded in the presence of PMA; over the periods tested, there was no net loss of biotinylated E-cadherin in biochemical assays or of stained E-cadherin in cells. The depletion of E-cadherin from endosomes was sensitive to Gö-6850, implicating PKC in the exocytic step of E-cadherin recycling. In other cases, phorbol esters are capable of altering the fate or routing of endocytosed proteins, such as epidermal growth factor receptor, which is diverted from a degradative to a recycling pathway by PKC activation (7).

E-cadherin was localized in large, prominent vesicles in PMA-treated cells, and by confocal imaging these were found to be located mostly near the apical pole of the cell. The position of stained vesicles did not change in PMA-treated cells, suggesting that PMA increased the loading of the vesicles but did not reroute endocytosed E-cadherin to different compartments. Some of the vesicles in control cells and/or cells at 18°C (26) and those in PMA-treated cells (data not shown) also contain Rab 5 and are thus early endosomes. In MDCK cells, the appearance and apical position of the E-cadherin-labeled vesicles are consistent with the similarly positioned, apical recycling compartment that was first noted in studies tracking the transcytosis of polymeric immunoglobulin receptor (5, 8). This receptor accumulates in the recycling vesicles at 17°C (8), consistent with the 18°C accumulation of E-cadherin. The apical recycling compartment also receives transferrin receptor recycling from the basolateral membrane (5). As with previous findings (5, 8), E-cadherin in this compartment did not colocalize with fluid phase markers taken up from the apical membrane. We also found that PMA did not alter fluid phase uptake from the basolateral membrane in MDCK cells, as assessed by levels of FITC-dextran uptake. This is in agreement with previous results in MDCK cells (21) but differs from phorbol ester-induced fluid phase uptake in intestinal epithelial cells, which was found to occur at the apical, not basolateral, membrane (43). Thus E-cadherin is accumulated in an apical recycling compartment by PKC activation in MDCK cells, and the regulated accumulation of E-cadherin is selective, occurring without any increase in general membrane internalization.

That endocytosis and recycling are poised to regulate cell surface adhesion events is now evident in several systems. Recent work showing that there is regulated trafficking of beta 1-integrin on and off the cell surface in mammary epithelial cells, and that this controls cell spreading and motility (32), has close parallels with the recycling of cadherins. Moreover, beta 1-integrin recycling is regulated by the expression and activation of PKC-alpha (32). This provides an even stronger basis for comparison with the regulation of E-cadherin recycling in epithelial cells, which we show here is also mediated by PKCs, although of a different class. Regulation of both pathways in and out of endosomes allows cognate control of the level of surface-exposed adhesive proteins by rapid movement off the cell surface or out of a recycling compartment. The permeability of endothelial layers in culture is regulated by PKC by being coupled to the internalization of cadherin-based junctions (4). PKC and other regulators might be expected to have their most dynamic and important roles in epithelia during morphogenesis (44), where the levels of surface-cadherin recycling are more active, as they are in preconfluent cells (26). PKC did not unduly affect adhesion or polarity in MDCK cell monolayers in our experiments because of the short treatment times and the relative stability of cadherins in the adherens junctions of fully confluent cells.

PKC has many possible downstream effectors and targets for phosphorylation. Both cadherins and catenins themselves have consensus phosphorylation sites, and serine/threonine phosphorylation of cadherins has a role in regulating its adhesive function (35). However, in many cases the effects of PKC on endocytosis of receptors have been shown to be independent of the phosphorylation of the receptors themselves (10, 13). The actin cytoskeleton is a common target for activated PKC, which is able to modulate actin assembly, either directly or via the phosphorylation of actin-assembly proteins such as myristoylated alanine-rich C kinase substrate (MARCKS) (19). The cadherin-catenin complex at the adherens junction is tightly coupled, via alpha -catenin, to F-actin in the cortical web. The PKC regulation of actin dynamics could be involved in E-cadherin trafficking in two ways, first by modulating the association of the cadherin-catenin complex with F-actin and second by controlling actin in one of its several purported roles as part of the endocytosis machinery (reviewed in Ref. 36).

Our data suggest that depolymerization of F-actin and dissociation of E-cadherin from F-actin could both occur as part of the PMA response. Cytochalasin enhanced the uptake of surface E-cadherin, whereas stabilization of F-actin with Jas was able to significantly abrogate the PMA-induced uptake of E-cadherin; these two effects together suggest that the dynamic disassembly of actin occurs and is required for E-cadherin trafficking. Jas had no effect on endocytosis by itself, consistent with previous studies in MDCK cells from our laboratory (42). Finally, the diminished amount of actin coprecipitated with E-cadherin together with the bulk shift of E-cadherin to a detergent-soluble status are indications that some dissociation of E-cadherin from cortical actin also occurs in response to PMA. Other studies have also provided evidence linking cadherin trafficking to actin dynamics. In response to low calcium, keratinocytes internalize E-cadherin into large perinuclear structures, and this uptake was increased by expression of constitutively active Rac1, which concomitantly decreased cell-cell contact (3). Rac1 and Rho A mutants have also been shown to modulate the coendocytosis of the growth factor receptor c-met and E-cadherin (22).

Our data establish that the movement of surface E-cadherin in a physiological recycling pathway is indeed regulated. It is clear, for the first time, that the movements of E-cadherin into and out of an apical recycling endosomal compartment in MDCK cells are both regulated events and, as such, offer potential for regulating cadherin-based functions. Activation of novel class PKC(s) is implicated in this trafficking of E-cadherin. Regulation of F-actin stability and E-cadherin trafficking via PKC potentially allows for simultaneous regulation of cell-cell contact, cell motility, and growth in response to growth factor signaling.


    ACKNOWLEDGEMENTS

The authors thank Juliana Venturato for assistance with cell culture and members of the Stow laboratory for helpful discussions. We thank various colleagues for providing antibodies, and we thank Trevor Biden for helpful comments and suggestions.


    FOOTNOTES

This work was supported by grant and fellowship funding from the National Health and Medical Research Council (NHMRC) to J. L. Stow and by funding to A. S. Yap from the NHMRC and a Wellcome Trust Senior Research Fellowship. This work was also supported by funding from the Australian Research Council as part of the Special Research Center for Functional and Applied Genomics.

Address for reprint requests and other correspondence: J. L. Stow, Inst. for Molecular Biosciences, Univ. of Queensland, Brisbane, 4072 QLD, Australia (E-mail: j.stow{at}imb.uq.edu.au).

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. Section 1734 solely to indicate this fact.

March 20, 2002;10.1152/ajpcell.00566.2001

Received 27 November 2001; accepted in final form 14 March 2002.


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