Cells

For most of our studies we used LLC-PK1 CL4 cells stably expressing the AT₁ receptor, as we characterized EMT in detail in this proximal tubular cell line in our previous studies.15 However, the process of EMT was qualitatively and quantitatively similar in wild-type LLC-PK1 cells as well. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (Gibco, Burlington, ON) at 37°C under 5% CO₂ in a humidified incubator. Cells were grown to 30% or 100% confluence, and then treated with vehicle only (4 mmol/L HCl and 0.1% albumin) or 4 ng/ml human recombinant TGF-β1 (Sigma) for the indicated times. In certain experiments, the medium of confluent layers grown on plastic or permeable support (Corning, Acton, MA) was replaced with nominally Ca²⁺-free DMEM (Gibco) without FBS.

Western Blotting

After treatments, cells were scraped into Triton lysis buffer (30 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (pH 7.4), 100 mmol/L NaCl, 1 mmol/L ethylene glycol-bis (2-aminoethylether)-N, N N′ N′-tetraacetic acid (EGTA), 20 mmol/L NaF, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 20 μl/ml Protease Inhibitory Cocktail (Pharmingen BD Biosciences, San Diego, CA), and 1 mmol/L Na₃VO₄). A portion of the samples was saved as total lysates, then the cytosolic (Triton soluble) and cytoskeletal (Triton insoluble) fractions were separated by centrifugation. Samples were dissolved in Laemmli buffer and boiled for 5 minutes. Equal amount of protein were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Immunoblotting was performed as described previously.15 Immunoreactive bands were visualized by enhanced chemiluminescence reaction kit (Amersham).

Preparation of GST-RBD Beads and Measurement of Rho Activity

Rho activity was determined as described previously15,37 using a pull-down affinity assay based on the ability of the Rho-binding domain (RBD) of Rhotekin to capture the GTP-loaded form of Rho. Recombinant glutathione-S-transferase (GST)-RBD fusion protein was prepared from E. coli, and adsorbed onto glutathione-covered beads. Confluent monolayers on 10-cm Petri dishes were serum-starved for 3 hours in normal or calcium-free DMEM, and then exposed to 10 ng/ml TGF-β1 for 10 minutes. Subsequently, cells were washed with ice-cold phosphate-buffered saline (PBS) and scraped into 800 μl lysis buffer supplemented with 0.1% SDS, 0.5% Na-Deoxycholate. The supernatants were incubated with GST-RBD beads for 45 minutes at 4°C. After washing, the beads were boiled in Laemmli buffer. Bead-associated Rho protein was detected by Western blotting.

Wound Assay

Confluent monolayers grown on 25-mm glass coverslips were wounded with a rubber policeman generating an ~3-mm gap. After extensive washing, the cells were placed into DMEM, and treated with 4 ng/ml TGF-β1 or vehicle for 3 days.

Immunofluorescence Microscopy

Cells grown on coverslips were fixed in 4% paraformaldehyde, incubated with PBS containing 100 mmol/L glycine, and washed with PBS. Cells were permeabilized in PBS containing 0.1% Triton X-100, blocked with 5% albumin for 1 hour, and incubated with the primary antibodies for another hour. After extensive washing, fluorescently labeled secondary antibodies were added for 1 hour, the coverslips were washed again and mounted on slides using Fluorescence Mounting Medium (DAKO Diagnostics, Mississauga, ON). Samples were analyzed using a Nikon Eclipse TE200 microscope (×100 objective) (Nikon, Mississauga, ON) and a Hamamatsu cooled CCD camera (C4742–95) (Hamamatsu, Bridgewater, NJ) controlled by the Simple PCI software.

Plasmids

A 765-bp piece of the rat SMA promoter containing several cis-elements including the serum response element binding motifs (CArG A and CArG B boxes), a TGF-β1 control element (TCE), a TATA box, and two E-boxes was ligated into PA₃-Luc luciferase vector (pSMA-Luc). The construct was a kind gift from Dr. R. A. Nemenoff (Department of Medicine, Renal Division, University of Colorado Health Sciences Center, Denver, CO).38 The LEF/TCF reporter plasmid, TopFLASH and its mutant control, FopFLASH were purchased from Upstate Biotechnology (Charlottesville, VA). The thymidine kinase-driven Renilla luciferase vector (pRL-TK, Promega, Madison, WI) was used as an internal control. The construct coding the cytoplasmic tail of N-cadherin ligated to GFP (N-cad-GFP) was a generous gift of Dr. B. Geiger (Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel).39 Dominant-negative TCF4 construct (dN-TCF4) cloned into pcDNA₃ vector was from Drs. O. Tetsu and F. McCormick (Cancer Reserch Institute, University of California, School of Medicine, San Francisco, CA).40 This construct encodes for a Myc-epitope-tagged truncation mutant of TCF-4, in which the region between amino acids 2–53 has been deleted. pEGFP vector was from Clontech Laboratories (Palo Alto, CA) and pcDNA₃ was from Invitrogen (Burlington, ON).

Transient Transfection and Luciferase Assay

Cells plated onto 6-well plates were transfected at 100% or 30% of confluence using FuGENE6 (Roche Molecular Biochemicals, Indianapolis, IN; reagent:DNA ratio 2.5 μl/μg). For promoter activity measurements, cells were co-transfected with 0.5 μg promoter plasmid, 0.05 μg pRL-TK along with 2 μg of the specific inhibitory construct or pcDNA₃ per well. After 16 hours, the cells were washed with Hank’s balanced salt solution (HBSS) and incubated in serum-free normal or low calcium DMEM for 3 hours, followed by treatment with vehicle or 10 ng/ml TGF-β1 for 24 hours. Subsequently, the cells were lysed in 250 μl Passive Lysate Buffer (Promega), exposed to freezing/thawing, and the samples were clarified by centrifugation. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay Kit (Promega) and a Berthold Lumat LB 9507 luminometer according to the manufacturers’ instructions. Results were normalized by dividing the Firefly luciferase activity with the Renilla luciferase activity of the same sample. Each transfection was done in duplicate, and determinations for each group were repeated at least three times.

Retroviral Infection

For generating the pFB-N-cad and pFB-GFP vectors, the coding region of N-cad-GFP and pEGFP plasmids were inserted into the pFB-Neo plasmid (Stratagene, La Jolla, CA) using EcoRI and Xho enzymes. To produce retroviruses, 293T packaging cells were transfected with pFB-N-cad, pFB-GFP, or pFB-Neo vectors, respectively, along with pVpack-GP and pVpack-VSVG. After 24 to 36 hours, supernatants were collected and filtered through a 0.45-μm filter. LLC-PK1 cells plated on 6-well plates were transduced with the virus at 20% confluence. After a 16-hour transduction, cells were further incubated in fresh medium for 36 hours and treated with vehicle or 4 ng/ml TGF-β1. Three days later, cell lysates were prepared and samples were analyzed by Western blot.

Role of Cell-Cell Contacts in TGF-β1-Induced Epithelial-Myofibroblast Transition

We sought to determine whether the ability of TGF-β1 to induce EMT was indeed related to the absence of intercellular contacts. To address this we generated a mechanical wound in otherwise confluent monolayers, and tested the effect of TGF-β1 on SMA expression. Wounding itself did not induce SMA production, while exposure to TGF-β1 resulted in SMA expression (Figure 2, A and B). Strikingly, SMA expression was restricted exclusively to cells located at the wound edge, ie, it occurred in cells, which partially lost their contacts with their neighbors. Similarly, enhanced fibronectin labeling exhibited a strong gradient starting a few cell rows behind the wound and sharply increasing toward the free edge (Figure 2, C and D). The disintegration of the cell-cell contacts in rows near the wound was clearly visualized by β-catenin staining. Cells lining the wound lost their β-catenin labeling at the free edge but kept most of the normal peripheral β-catenin distribution at cell-cell borders. When treated with TGF-β1, β-catenin became redistributed in cells near the wound. This manifested in the widening of the peripheral β-catenin staining, higher cytosolic labeling, and accumulation of β-catenin in the nuclei of cells located at the edge or migrating into the wound (Figure 2, E and F).

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Figure 2

Loss of cell-cell contacts by wounding restores the ability of TGF-β1 to induce EMT. Cells were allowed to grow until 100% confluence and then a wound was generated with a rubber policeman in the monolayer. Subsequently cells were treated with vehicle (Control; A, C, and E) or 4 ng/ml TGF-β1 (B, D, and F) for 3 days, then fixed, permeabilized, and stained for SMA (A and B), fibronectin (C and D), or β-catenin (E and F). The magnification was ×40 for A to D, and ×100 for E and F.

As a further method to test the role of cell-cell contacts in EMT, we used an approach that did not involve mechanical injury. We induced disassembly of mature cell-cell contacts in confluent cultures by Ca²⁺-removal, a maneuver known to disrupt the homotypic adhesion between cadherins.41 Remarkably, Ca²⁺-removal completely restored the ability of TGF-β1 to provoke EMT. During the course of a 3-day experiment in the presence of Ca²⁺, TGF-β1 neither induced SMA expression, nor caused a significant reduction in E-cadherin, as reported above (Figure 3A). Removal of Ca²⁺ without TGF-β1 addition did not cause SMA expression and only marginally affected fibronectin expression, but it induced a substantial reduction in E-cadherin. Importantly, TGF-β1 added in the absence of Ca²⁺ triggered a gradually increasing SMA expression, led to complete elimination of E-cadherin, and strongly stimulated fibronectin production (Figure 3A). In accordance with these findings, in a Ca²⁺-free medium epithelial cells still preserved their polygonal shape. However, exposure of these cells to TGF-β resulted in mesenchymal morphology (Figure 3B). Thus, the absence of stable intercellular junctions, irrespective of whether it is brought about by non-confluence or mechanical or chemical disruption of the cell contacts is a prerequisite for the EMT-inducing effect of TGF-β1.

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Figure 3

Disassembly of intercellular contacts by low calcium restores the transforming effect of TGF-β1 in confluent monolayers. A: Confluent cells were washed and incubated in serum-free normal or Ca²⁺-free DMEM, and 3 hours later treated with vehicle (Control) or 4 ng/ml TGF-β1 for the indicated times. Expression of SMA, E-cadherin, and fibronectin were determined by Western blotting. B: TGF-β1 treatment (24 hours) induced morphological changes in low calcium DMEM (phase-contrast microscopy, magnification, ×40).

We considered the possibility that, in confluent layers, TGF-β1 cannot promote EMT when added from the apical side, because it cannot access the basolateral membrane, where TGF-β1 receptors might reside.29 To test this, we performed control experiments using LLC-PK1 cells grown on permeable support to provide free access to both membrane domains. As shown on Figure 4A, in confluent monolayers neither apically nor basolaterally added TGF-β1 induced SMA expression. However, when cell contacts were disassembled by Ca²⁺-removal, apical or basolateral administration of TGF-β1 provoked a robust and similar SMA response. These data precluded that resistance to TGF-β1 was due to differential accessibility. Next, we asked whether confluent cultures exhibited general unresponsiveness to TGF-β1, or only the transforming effect was suppressed. To address this, we determined if TGF-β1 remained able to stimulate Rho GTPase, an important signal for cytoskeletal remodeling. In agreement with our previous findings,15 TGF-β1 induced a sizable Rho activation in confluent cultures (Figure 4B), which was similar in the presence or absence of Ca²⁺. This suggests that contact disassembly was not required for this response. Consistent with these results, exposure of confluent layers to TGF-β1 stimulated stress fiber formation (Figure 4C). Together these data show that TGF-β1 signaling pathways are functional in confluent LLC-PK1 cells, suggesting that failed transformation is not due to general unresponsiveness, but rather due to the absence of cell contact-regulated permissive factor(s).

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Figure 4

Confluent cells remain responsive to TGF-β1. A: Cells were cultured until confluence on porous tissue culture inserts (pore size, 3 μm) then serum-deprived in normal (+) or low calcium medium (−). After 3 hours, cells were treated with vehicle (−) or 4 ng/ml TGF-β1 from the upper (U) or the lower side (L) for 3 days. Total cell lysates were prepared and SMA expression was examined by Western blots. B: To determine Rho activity were serum-starved for 3 hours in normal (+) or low Ca²⁺ (−) DMEM, and then treated with vehicle (−) or 10 ng/ml TGF-β1 (+) for 10 minutes. After lysis, active Rho was captured with GST-Rhotekin beads and detected by Western blotting. Aliquots of the same lysates were probed for total Rho. C: Confluent cultures were treated with vehicle (Control) or 4 ng/ml TGF-β1. F-actin was visualized by rhodamine phalloidin.

Cell-Cell Contacts Regulate the SMA Promoter

To assess whether cell contacts might impact the regulation of the SMA promoter, we investigated the effect of TGF-β1 on a SMA promoter luciferase construct under conditions where cell junctions were manipulated by the above-described means (Figure 5). In confluent cultures with stable cell contacts (100%, normal Ca²⁺ medium), TGF-β1 induced a modest ~3-fold increase in the activity of the SMA promoter, confirming the basal responsiveness to TGF-β1 in confluent layers. Reduction of Ca²⁺ without TGF-β1 addition resulted in ~4-fold rise in SMA promoter activity. Importantly, TGF-β1 added to confluent layers in which the cell junctions had been disassembled by Ca²⁺-removal elicited a 14-fold increase in SMA promoter activity. Thus, contact disassembly and TGF-β1 acted in a strongly synergistic manner as evidenced by the multiplicative effect. We next tested the impact of subconfluence in the presence of normal Ca²⁺. The basal activity of the SMA promoter was fivefold higher in 30% than in 100% confluent cultures. TGF-β1 addition to 30% confluent cells resulted in a 20-fold increase in SMA promoter activity, compared to confluent, unstimulated cells. These data show that the overall stimulation of the SMA promoter by TGF-β1 strongly depends on the state of cell-cell contacts in both models, irrespective of whether the contacts have been disassembled in already confluent layers or have not been fully formed yet in growing subconfluent cultures.

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Figure 5

Subconfluence or disassembly of cell-cell contacts potentiates the effect of TGF-β1 on SMA promoter activity. LLC-PK1 cells grown to 100% or 30% confluence were co-transfected with pSMA-Luc (0.5 μg/well), and the Renilla luciferase construct (pRL-TK, 0.05 μg/well). After 16 hours, cells were washed and incubated with serum-free normal (+) or low Ca²⁺ (−) DMEM for 4 hours, and then treated with vehicle (−) or 10 ng/ml TGF-β1 (+) for 24 hours. After lysis, Firefly and Renilla luciferase activities were determined by luminometry. Values were normalized to the basal activity measured in the 100% confluent vehicle-treated group in the presence of normal calcium (n = 6).

TGF-β1 Stimulates the Transcriptional Activity of β-Catenin in LLC-PK1 Tubular Cells

Since β-catenin has a dual function as an adherent junction component and a transcriptional co-activator, and it redistributes during EMT in LLC-PK1 cells (15 and Figure 2), it might act as a mediator of contact-dependent transcriptional responses. To address this possibility, we first determined whether TGF-β1 could stimulate β-catenin-dependent transcription. Cells at 30% confluence were transfected with the β-catenin-dependent reporter TopFLASH or its inactive mutant FopFLASH, and then exposed to vehicle or TGF-β1. The basal TopFLASH activity was 10-fold higher than FopFLASH, indicating that TopFLASH expression reflects β-catenin-specific transcription, and that there is readily detectable, β-catenin-dependent transcription even in non-stimulated, subconfluent cells (Figure 6A). Importantly, TGF-β1 induced a twofold increase in TopFLASH activity, while it had no significant effect on the marginal FopFLASH activity. Moreover, LiCl, an inhibitor of β-catenin degradation also stimulated TopFLASH activity. Western blot analysis verified that LiCl indeed increased the steady state level of β-catenin in LLC-PK1 cells (Figure 6A). Together these findings suggest that the continuous degradation of β-catenin limits β-catenin signaling in non-stimulated cells. We then examined the kinetics of the TGF-β1-induced TopFLASH response. Figure 6B shows that the TopFLASH activity gradually increased over the first 24 hours following TGF-β1 treatment, and then reached a plateau.

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Figure 6

TGF-β1 activates β-catenin-dependent TCF/LEF reporter construct TopFLASH. A: Cells grown to 30% confluence on 6-well plates were transfected either with TopFLASH (0.5 μg/well) or its inactive mutant, FopFLASH (0.5 μg/well), along with pRL-TK (0.05 μg/well). Sixteen hours after transfection, the medium was replaced with serum-free DMEM for 4 hours, followed by exposure to vehicle (Control), 10 ng/ml TGF-β1 or 30 mmol/L LiCl. Cells were lysed after 24 hours, and luciferase activities determined. Values were normalized to the relative luciferase activity measured in the TopFLASH-transfected control group, and expressed as means ± SEM (n = 8). The blot shows the effect of LiCl treatment on the steady state level of β-catenin. Equal loading was detected by probing the blot for tubulin. B: Thirty percent confluent cultures transfected with TopFLASH, as in A, were treated with vehicle or 10 ng/ml TGF-β1 and lysed at the indicated time points. C: The effect of subconfluence or the disassembly of cell-cell contacts on TopFLASH activity was examined by transfecting cells at the indicated confluence with DNA mixtures as above. After 16 hours, cells were washed and further incubated in serum-free normal (+) or low calcium medium (−) for 4 hours before treatment with vehicle or 10 ng/ml TGF-β1. Twenty-four hours later, cells were lysed, and luciferase activities were measured. Results are expressed as means ± SEM, normalized to the activity measured in the 30% confluent vehicle-treated group in normal calcium medium (n = 6).

In subsequent experiments, we tested whether the state of cell-cell contacts affected β-catenin-dependent transcription. After reaching confluence, the basal TopFLASH activity was ~60% of the level observed in subconfluent cultures (Figure 6C). Addition of TGF-β1 to confluent layers induced only a slight (non-significant) increase in TopFLASH from this reduced baseline. Disassembly of junctions by removal of Ca²⁺ did not exert a significant effect itself, but it significantly potentiated the TGF-β1-induced increase in TopFLASH activity. Together these results show that TGF-β1 induced β-catenin-dependent transcription, and the amplitude of this effect was dependent on the state of intercellular contacts.

TGF-β1 Rescues β-Catenin But Not E-Cadherin after Destabilization of Cell Contacts

To explore the mechanism whereby TGF-β1 stimulates β-catenin-dependent transcription, we examined the fate of junctional proteins after disassembly of cell contacts. Ca²⁺-removal resulted in a dramatic reduction of E-cadherin and a substantial decrease in β-catenin protein, indicating that dissociation of the contacts promoted the degradation of their components (Figure 7A). In intact monolayers, TGF-β1 did not affect the level of these proteins. However, when TGF-β1 was added to monolayers kept in Ca²⁺-free medium, it exerted grossly different effects on the two junctional proteins: while it did not influence the degradation of E-cadherin, it largely prevented the loss of β-catenin. The massive down-regulation of E-cadherin, the transmembrane binding partner of β-catenin, together with the selective rescue of β-catenin should increase the level of free β-catenin. In agreement with this assumption, in intact cells β-catenin was present both in the cytosolic and the cytoskeletal (Triton-insoluble) fraction, while after low Ca²⁺ plus TGF-β1 treatment the rescued β-catenin was not bound to the cytoskeleton (Figure 7A, right).

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Figure 7

TGF-β1 rescues β-catenin but not E-cadherin from contact disassembly-induced degradation. A: Confluent LLC-PK1 cells were incubated in serum-free normal (+) or low calcium (−) DMEM for 16 hours, and then treated with vehicle (−) or 4 ng/ml TGF-β1 (+) for 24 hours. Subsequently cells were washed and lysed in Triton lysis buffer. Aliquots from total cell lysates and Triton-insoluble extracts were subjected to SDS-PAGE and the components of adherent junction were detected by Western blotting. Equal loading was verified by reprobing the membranes for β-actin. B: Confluent cells were treated with vehicle dimethylsulfoxide (DMSO) or proteasome inhibitor, MG132 (1 μmol/L) followed by incubation in normal (+) or low calcium (−) medium in the presence of DMSO or MG132 for 24 hours. β-catenin and E-cadherin were measured by Western blotting in total cell lysates. The membrane was reprobed for tubulin. C: Confluent cells were incubated in normal or low calcium (−) medium, in the presence or absence of 30 mmol/L LiCl for 30 minutes or 24 hours, as indicated. Subsequently the cells were lysed and β-catenin and tubulin (as loading control) were detected by Western blotting. D: To assess the role of contact disassembly in β-catenin degradation, protein synthesis was blocked in confluent monolayers by 100 μg/ml cycloheximide, and the cells were incubated for 30 minutes or 24 hours in the presence or absence of Ca²⁺. Note that Ca²⁺-removal strongly stimulated the loss of β-catenin also in the presence of cycloheximide, indicating that it accelerated the degradation of β-catenin.

To gain insight into the mechanism responsible for the decrease in β-catenin protein on contact disassembly, we tested if degradation by the proteasomal pathway contributes to this process (Figure 7B). MG132, a potent proteasome inhibitor strongly reduced the Ca²⁺-removal-induced degradation of β-catenin. Furthermore, LiCl, an inhibitor of glycogen synthase kinase-3β, the enzyme which phosphorylates and thereby facilitates proteasomal processing of β-catenin, also strongly reduced the Ca²⁺ removal-induced drop in β-catenin protein (Figure 7C). Together these findings suggest that proteasomal degradation is involved in β-catenin elimination, and TGF-β1 inhibits this process. Interestingly, MG132 did not inhibit the contact disassembly-induced loss of E-cadherin, suggesting that E-cadherin down-regulation may be due to alternative degradation pathways and/or the inhibition of its synthesis. Pharmacological inhibition of β-catenin degradation is expected to reduce the loss of β-catenin even if contact disassembly does not accelerate β-catenin degradation per se, but inhibits β-catenin synthesis. To address whether contact disassembly indeed impacts on β-catenin degradation, we inhibited protein synthesis by cycloheximide, and tested the effect of Ca²⁺ removal under these conditions, when any change should be due to a difference in degradation. Cycloheximide added alone modestly reduced the level of β-catenin after 24 hours. Importantly, Ca²⁺ removal strongly facilitated the loss of β-catenin even in the presence of cycloheximide, indicating that the disruption of the contacts indeed promotes degradation of β-catenin. These results do not exclude the possibility that altered synthesis also contributes to the reduction in β-catenin.

We thank Drs. K. Szaszi and B. Alman and Ms. C. Di Ciano-Oliveira for valuable discussions.