Departments of 1Medicine, 2Cell Biology, and 3Surgery, University of Alabama at Birmingham, and 4Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 35294
Submitted 14 January 2004 ; accepted in final form 2 April 2004
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ABSTRACT |
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cell density; cyclin D1; hepatocyte growth factor; cell cycle; extracellular signal-regulated kinases
Within the cell cycle signal transduction cascade, type D cyclins play a crucial role. Cyclin D1 is thought to be a critical regulator protein of the progression of cells into the proliferative stage of the cell cycle (16, 25, 26). Cyclin D1 accumulation has also been used as a marker for entry of Madin-Darby canine kidney (MDCK) cells into the cell cycle (21). Expression abundance of cyclin D1 is largely dependent on extracellular signals and serves as an early checkpoint for cells entering the cell cycle (7). Equally important in the progression of the cell cycle is the role played by extracellular signal-regulated kinases (ERKs) (30). ERKs, members of the mitogen-activated protein kinase (MAPK) family, exist as two isoforms (ERK1/2 or p44/p42 MAPK) and connect different types of membrane receptors to the nucleus after mitogenic stimulation (6, 24). ERK1 and ERK2 are activated by phosphorylation of threonine and tyrosine residues via ERK kinases (known as MEK1 and MEK2) (13, 20). ERK signaling can be blocked by the MEK inhibitors PD-98059 and U0126 (11). The ERK cascade is commonly activated by growth factors and is also thought to play a role in cyclin D1 expression and cell proliferation (23).
The MDCK cell line has been the most widely used system for studying important and fundamental issues in epithelial cell biology (28). These cells were derived from the kidney tubules of a cocker spaniel in 1958 and display contact inhibition in culture (12). Depending on culture conditions, MDCK cells respond to hepatocyte growth factor (HGF) through the tyrosine kinase receptor c-met receptor by scattering, forming branching tubules, or dedifferentiating (3, 4). In addition to these morphogenic changes, HGF also abrogates contact inhibition of mitosis in high-density MDCK cells (2).
While the capacity of cells to exhibit contact inhibition is widely recognized, the cellular signaling pathways controlling this process in epithelial cells remain largely unknown. In the present study, we systematically examined the role of ERK phosphorylation, cyclin D1 accumulation, and cell proliferation using the MDCK cell model of contact inhibition.
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EXPERIMENTAL PROCEDURES |
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Mitogenesis assay. DNA synthesis was determined by measuring thymidine incorporation with minor modifications of a previously described protocol (2). Briefly, control and HGF-treated MDCK cells at low and high density were pulsed for 1 h at 37°C with 5 µCi/ml [methyl-3H]thymidine (76.0 Ci/mmol) (placed on the basolateral surface of filter-grown cells). After the pulse, cells were rinsed twice in phosphate-buffered saline containing Mg2+ and Ca2+ (PBS+), fixed in methanol-acetic acid-water (50:10:40, vol/vol/vol) for 1 h at 4°C, and rinsed twice more with PBS+. Filters were cut from the support with a scalpel, and methanol-acetic acid-water insoluble radioactivity was measured by liquid scintillation. Cells grown on plastic were solubilized in 1% SDS for 1 h at 37°C, and methanol-acetic acid-water-insoluble radioactivity was measured by liquid scintillation. Experiments were done in triplicate.
Cell counts. Cells were gently rinsed with PBS without Ca2+ and Mg2+ (PBS). Cells were then trypsinized with the use of 0.5 ml of trypsin (0.25%)-EDTA (0.1%) solution in HBSS (Cellgro, Mediatech) for 15 min at 37°C, 5% CO2. Trypsinized cells (0.5 ml) were then added to separate solutions of 4.5 ml of PBS, and cells were counted using a hemocytometer. Cell count experiments were done in triplicate.
Cell lysate preparation. MDCK type II cells grown on filters or plastic wells were used for preparation of cell lysates for Western blot analysis. Nonadherent cells were removed by rinsing the cell monolayer once with ice-cold PBS+. Adherent cells were then exposed to 20 mM Tris·HCl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% deoxycholic acid, and 5 mM EDTA [radioimmunoprecipitation assay (RIPA) buffer] containing inhibitors of proteases (2 mM phenylmethylsulfonyl fluoride, 50 µg/ml pepstatin, 50 µg/ml chymostatin, and 10 µg/ml antipain) for 20 min on ice. Cells were scraped with a rubber policeman from the filter or plastic well to generate total cell lysates. Total cell lysates were sedimented in a 4°C microfuge at 14,000 rpm for 10 min. The protein concentration of each cell lysate was determined by using the bicinchoninic acid determination assay (Pierce Chemical, Rockford, IL). Samples were diluted as necessary with varying amounts of RIPA buffer to obtain equal protein concentrations of 600 µl of each lysate sample. The lysate preparation was completed by adding 200 µl of 4x Laemmli buffer containing 100 mM dithiothreitol and boiling for 5 min.
Electrophoresis and Western blot analysis. Western blot analysis was performed as described previously (4). Briefly, equal amounts of protein in lysates of MDCK cells were run on acrylamide gels at 160 V for 45 min. Proteins were transferred to Immobilon P membranes (Millipore, Bedford, MA). Membranes were blocked for 30 min with PBS containing 5% milk and 0.1% Tween 20 (block solution). For Cyclin D1 analysis, filters were probed with anti-cyclin D1 (COOH terminal) antibody/affinity purified rabbit IgG (1:200; Medical and Biological Laboratories) for 1 h and then washed with PBS containing 0.1% Tween 20 (4 times for 5 min each). Filters were then probed with horseradish peroxidase-labeled goat anti-rabbit at 1:10,000 dilution in block solution for 1 h. For total ERK analysis, filters were probed with anti-ERK1 goat polyclonal IgG (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h and then washed with PBS containing 0.1% Tween 20 (4 times for 5 min each). This antibody recognizes both ERK1 and ERK2 in MDCK cell lysates. Filters were then probed with horseradish peroxidase-labeled donkey anti-goat at 1:5,000 dilution in block solution for 1 h. For active ERK (MAPK) analysis, filters were probed with phospho-p44/42 (ERK1/2) MAPK antibody (1:10,000; Cell Signaling Technologies) for 1 h and then washed with PBS containing 0.1% Tween 20 (4 times for 5 min each). Filters were then probed with horseradish peroxidase-labeled goat anti-rabbit at 1:5,000 dilution in block solution for 1 h. After the secondary antibody had been added, all filters were again washed (4 times for 5 min each) with PBS containing 0.1% Tween 20. Filters were developed by using the enhanced chemiluminescence kit (ECL; Amersham, Piscataway, NJ) and visualized on Kodak X-OMAT film (Eastman Kodak, Rochester, NY). Band densitometry was performed with NIH Image version 1.60 software (NIH Image is in the public domain).
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RESULTS |
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Before exposure to HGF, cyclin D1 levels were higher in low-density cells than in high-density cells (Fig. 3A). After exposure to HGF, cyclin D1 levels increased in high-density cells and returned to the baseline control levels after 48 h of HGF treatment. The levels of cyclin D1 in low-density cells also increased with HGF treatment and remained elevated for up to 48 h of HGF treatment. Measurement of amounts of cyclin D1 by band densitometry in low vs. high density showed higher levels of cyclin D1 in low-density cells than in high-density cells at all time points except 3 h, when the amounts were approximately equal (Fig. 3B). These results demonstrate that cyclin D1 levels were higher in low-density cells than in high-density cells in the absence of HGF treatment and provide an explanation for the lack of proliferation in high-density cells in the absence of HGF.
Before exposure to HGF, phospho-ERK1/2 levels were higher in low-density cells than in high-density cells (despite lower amounts of total ERK1/2 in low-density cells). Levels of phospho-ERK1/2 increased with HGF treatment in both low-density and high-density cells (Fig. 3A). In the high-density cells, phospho-ERK1/2 levels returned to baseline 2448 h after HGF treatment, whereas phospho-ERK1/2 levels remained elevated in the HGF-treated low-density cells. Measurement of the ratio of phospho-ERK to total ERK band densitometry in low- vs. high-density cells showed higher levels of phospho-ERK1/2 in low-density cells than in high-density cells at all time points (Fig. 3B). The data provide evidence supporting the hypothesis that low-density MDCK cells are proliferating, in part, because of increased levels of phospho-ERK1/2.
Effect of PD-98059 and U0126 on HGF-induced cell proliferation of MDCK cells at high and low density. HGF treatment of confluent monolayers of MDCK cells stimulates DNA synthesis and cell division (2). To determine the role of ERK1/2 phosphorylation and cyclin D1 accumulation on HGF-induced loss of contact inhibition in high-density MDCK cells, we examined the effect of 50 µM PD-98059 or U0126 on HGF-induced cell division and DNA synthesis in high-density cells. We also examined the effects of 50 µM PD-98059 and U0126 on cell division and DNA synthesis in low-density cells in the presence and absence of HGF. The number of cells in confluent monolayers of filter-grown MDCK cells treated with fresh medium or fresh medium with HGF in the presence and absence of PD-98059 or U0126 after 48 h was determined by trypsinization and counting. Figure 4A, top, shows that cell numbers increased in the presence of HGF compared with control cells and that PD-98059 did not significantly block the proliferative effect of HGF. In contrast, the results in Fig. 4A, bottom, demonstrate that U0126 blocked the HGF-induced stimulation of cell division in MDCK cell monolayers. We also examined the effects of PD-98059 and U0126 on HGF-induced stimulation of thymidine incorporation into MDCK cell monolayers. In the case of PD-98059, there was minimal inhibition of HGF-induced stimulation of thymidine incorporation (Fig. 4B, top). However, U0126 inhibited the HGF-induced stimulation of thymidine incorporation more effectively than did PD-98059 (Fig. 4B, bottom).
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Maturation of cell-cell contacts inversely correlates with ERK1/2 phosphorylation and cyclin D1 levels.
We hypothesized that the establishment of cell contacts correlates with contact inhibition of mitosis as well as responsiveness to mitogenic stimuli by a mechanism of inhibiting ERK1/2 phosphorylation and subsequent cyclin D1 accumulation. MDCK cells plated at confluence require 72 h of culture to reach a fully polarized phenotype as manifested by tight junction development, adherens junction development, and cilia formation (34). After 72 h in culture, MDCK cell monolayers are contact inhibited in response to fresh medium (2). MDCK cells grown at low density (no contact inhibition) actively divide and are not polarized with regard to adherens and tight junction formation. HGF also causes disruption of cell-cell contacts in MDCK cell monolayers as manifested by an increase in Triton solubility of adherens junction components (suggesting dissociation from the actin cytoskeleton) and increased insulin diffusion across MDCK cell monolayers (5). We performed an analysis of cyclin D1 expression and ERK1/2 phosphorylation in MDCK cells at different stages of cell-cell junction formation with regard to responsiveness to fresh medium alone and fresh medium containing HGF. Figure 6A shows the different stages of formation of tight and adherens junctions in MDCK cells after the cells were plated at confluence on permeable supports. Cell lysates from an aliquot of freshly trypsinized cells were collected and represented unplated cells. MDCK cells were plated at confluence. At 24, 48, and 72 h after plating, the cells were exposed to no medium change (control), fresh medium alone, or fresh medium containing HGF for a period of 6 h. The time 0 cells were cultured for 6 h in medium with or without HGF, and the medium was not changed, because the cells had just been plated in fresh medium. After 6 h, cell lysates were prepared. Western blot analysis showed that levels of cyclin D1 in trypsinized cells were low (Fig. 6B). However, phospho-ERK1/2 levels in freshly trypsinized cells were higher than they were in contact-inhibited monolayers (see 72 h time point for control). Six hours after plating at confluence on filters, the levels of cyclin D1 were dramatically increased and levels of phospho-ERK1/2 remained elevated. The greatest increases in cyclin D1 and phospho-ERK1/2 were recorded in cells plated for 6 h in the presence of HGF. In the cells that were plated for 24, 48, and 72 h and exposed to nothing or to fresh medium for 6 h, the levels of cyclin D1 and phospho-ERK1/2 were found to be similar to those seen in contact-inhibited high-density cells. However, all of the cells plated for 24, 48, and 72 h and then treated with HGF for 6 h exhibited increased levels of cyclin D1 and phospho-ERK1/2. These data show that disruption of cell-cell contacts by trypsinization led to increased phospho-ERK1/2 and cyclin D1 within 6 h after disruption. The levels of these proteins decreased with time as cell-cell contacts reformed. These data also show that HGF reversed the decreases in cyclin D1 and phospho-ERK1/2 as cell-cell contacts formed. Fresh medium alone was not sufficient to reverse the decreases of cyclin D1 and active ERK1/2 as cell contacts formed. This observation argues against the explanation that contact inhibition of mitosis of cells is due to the depletion of growth factors (9, 15, 29). These data provide evidence that the formation of cell-cell contacts decreases phospho-ERK1/2 levels and cyclin D1 accumulation.
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DISCUSSION |
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At high density, contact-inhibited MDCK cells do not proliferate and accordingly exhibit very low levels of phospho-ERK1/2 and cyclin D1. This contact-inhibited state is abrogated by treatment with HGF by a mechanism of ERK1/2 phosphorylation and cyclin D1 accumulation. When plated at low density, MDCK cells have fewer cell contacts and actively proliferate. As they are in various phases of the cell cycle, they demonstrate higher levels of phospho-ERK1/2 and cyclin D1. Interestingly, HGF treatment of low-density cells further increases the levels of phospho-ERK1/2 and cyclin D1 without further stimulating cell proliferation rates. We interpret this finding as evidence that low-density cells have adequate levels of phospho-ERK1/2 and cyclin D1 to promote maximal proliferation rates and that further increases in phospho-ERK1/2 and cyclin D1 cannot increase the proliferation rates. The MEK inhibitor U0126 blocks proliferation of low-density cells, suggesting that the proliferation in low-density cells is dependent on phospho-ERK1/2 and subsequently elevated cyclin D1 levels. Trypsinization of monolayers dissociates both cell-cell contacts (e.g., E-cadherin-mediated adhesion) and cell-matrix contacts (e.g., integrin-mediated adhesion), and in this setting, levels of phospho-ERK1/2 rapidly rise and cyclin D1 levels subsequently increase. Phospho-ERK1/2 and cyclin D1 levels rapidly diminish with the reestablishment of cell contact formation over time. Collectively, these data support our hypothesis that the establishment of cell contacts causes contact inhibition of ERK signaling pathways and cyclin D1 accumulation. In addition, HGF increases phospho-ERK1/2 and cyclin D1 as cell-cell contacts form. Fresh medium alone is not sufficient to reverse the decreases in phospho-ERK1/2 and cyclin D1 as cell contacts form, challenging the traditional concept that contact inhibition of cell proliferation reflects, at least in part, the ability of a cell to deplete the medium locally of growth factors, thereby depriving its neighbors (9, 15, 29).
Vinals and Pouyssegur (32) reported that cell confluence induces cell cycle exit by inhibiting ERK1/2 activity in vascular endothelial cells. In their study, both confluent and low-density endothelial cells expressed low levels of phospho-ERK1/2 and cyclin D1. However, cells in both conditions had been serum starved for 24 h. Both cell culture conditions responded to stimulation with serum, but the low-density cells exhibited a more robust response with regard to ERK phosphorylation and cyclin D1 accumulation. In our study, the MDCK cell model of contact inhibition did not require serum starvation to decrease both phospho-ERK1/2 and cyclin D1 levels and was more representative of the in vivo situation, in which cells are continuously in contact with serum factors.
Cadherin-mediated cell-cell adhesion has received attention regarding a possible role in contact inhibition. Vascular endothelial cell cadherin (VE-cadherin) and -catenin have been implicated in the contact inhibition VEGF-induced proliferation (14). The
-catenin binding site of VE-cadherin appears to be necessary to block VEGF-induced proliferation by cell-cell contact via inactivation of the phosphatase DEP-1. A recent publication (18) provided evidence for epithelial cell cadherin (E-cadherin)-mediated downregulation of ERK signaling in differentiating intestinal epithelial cells. A continued systematic analysis of the role of E-cadherin and cellular phosphatases in contact inhibition of cell proliferation in MDCK cells is underway in our laboratory.
In conclusion, we demonstrate that HGF reverses contact inhibition in high-density MDCK cells through phospho-ERK-dependent cyclin D1 accumulation and propose a mechanism by which the formation of cell-cell contacts in MDCK epithelial cells inhibits cell proliferation, reducing phospho-ERK1/2 levels and subsequent cyclin D1 accumulation. In low-density, nonconfluent MDCK cells, levels of phospho-ERK1/2 and cyclin D1 remain higher and the cells continue in a proliferative state. This modulation of epithelial cell ERK1/2 phosphorylation and proliferation rates by cell-cell contact may play an important role in complex disease-related processes such as epithelial organ regeneration and/or epithelial cell transformation to carcinoma.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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