Polyamine deficiency alters EGF receptor distribution and signaling effectiveness in IEC-6 cells

Shirley A. McCormack1, Patrick M. Blanner1, Barbara J. Zimmerman1, Ramesh Ray1, Helen M. Poppleton2, Tarun B. Patel2, and L. R. Johnson1

1 Department of Physiology and Biophysics and 2 Department of Pharmacology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with alpha -difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipatated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.

transferrin receptor; cell proliferation; cell migration; actin filaments; epidermal growth factor receptor

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE POLYAMINES PUTRESCINE, spermidine, and spermine are found in all prokaryotic and eukaryotic cells. Their biosynthetic pathway from ornithine is primarily rate limited by ornithine decarboxylase (ODC) and can be specifically and irreversibly inhibited by alpha -difluoromethylornithine (DFMO), an analog of ornithine. The mechanism of action of DFMO as a suicide inhibitor ensures that it does not interfere with other enzymatic reactions in which ornithine is the substrate (18). The polyamines are cations at physiological pH. They are able to bridge distances not possible for inorganic ions because their positive charges are distributed at fixed lengths along a flexible carbon chain rather than localized at a single point. Although definitive modes of action for the polyamines remain unexplained, their essential involvement in many cellular functions has been repeatedly demonstrated. These functions include the maintenance of cell membranes, Ca2+ homeostasis, ion and metabolite transport (31), cell proliferation (18), cell migration (22, 24, 45), transglutaminase activity (23), signal transduction (3), growth-related gene transcription (7), hormone production (32), and other vital physiological processes. In vivo, polyamines are essential for the healing of gastrointestinal lesions caused by a variety of insults (2, 19, 40) and are found at high levels in cancer for which cell proliferation is often uncontrolled (18).

Polyamines are involved in many actions of epidermal growth factor (EGF). For instance, DFMO can prevent the inhibition of parietal cell acid secretion by EGF (44). Polyamines inhibit EGF receptor (EGFR) tyrosine kinase activity in A-431 cells (9). In a colon cancer cell line, Caco-2, polyamine uptake is stimulated by EGF and inhibited by genistein, a tyrosine phosphorylation inhibitor (25). The overexpression of ODC is oncogenic in transfected NIH/3T3 cells (26). In L6 cells and fetal bovine myoblasts, EGF and transforming growth factor-alpha (TGF-alpha ), also a ligand of EGFR, stimulate polyamine biosynthesis, suggesting that the biosynthesis of polyamines is important in the early events induced by EGF (3).

EGFR is composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The intracellular domain contains the tyrosine autophosphorylation sites, the ATP binding site, and regions involved in Ca2+ regulation, internalization, and substrate binding. EGF binds to the extracellular domain, causing dimerization of the receptor and autophosphorylation of the receptor's tyrosine autophosphorylation sites. Autophosphorylation establishes a conformation of the receptor that can interact with and phosphorylate its cellular substrates (21), which then initiate signal transduction through the phosphatidylinositol and Ras pathways.

Unfilled EGFRs are randomly distributed on the cell surface. After binding ligand, the EGF-EGFR complexes cluster in clathrin-coated pits and are internalized. They enter early endosomes near the cell's periphery, then late endosomes in the perinuclear region, and, ultimately, lysosomes where the EGF-EGFR complex is degraded (6, 12, 43). The internalization of ligand-bound EGFR is accompanied by a rapid downregulation of receptors on the cell surface. Unbound EGFR is also internalized, but at a slower rate, and is not followed by downregulation (42). In A-431 cells, internalized EGFR can be autophosphorylated for up to 20 min after the initial binding of EGF to the receptor, showing that EGF does not dissociate immediately from its receptor after internalization. Continued receptor-receptor interactions and tyrosine kinase activity take place for some minutes after the complex has left the cell membrane (33). An autocrine pathway for the activation of EGFR in A-431 cells has also been demonstrated. This pathway involves the endogenous production and secretion of TGF-alpha , which then binds EGFR at the surface, activating only membrane-bound EGFR (38). EGF has also been reported to translocate EGFR to the nucleus where it may have direct effects (14).

Activated EGFR tyrosine kinase induces actin polymerization (29, 36, 37). In the gastric mucosa of rats, EGFR tyrosine kinase activity is increased after injury and plays an important role in the early restitution that follows gastric mucosal injury (27). Newly assembled actin filaments localize selectively to tyrosine-phosphorylated EGFR in the plasma membrane rather than to internalized tyrosine-phosphorylated EGFR. Actin, however, is phosphorylated on serine, not tyrosine, residues and therefore is not a direct substrate of EGFR. Instead, actin may be the target of a serine-threonine kinase at the plasma membrane activated by EGF (39).

In this report, we have described the effects of polyamine deficiency in IEC-6 cells, an epithelial crypt cell of the small intestine in normal rats. Abnormalities in the actin cytoskeleton, in the distribution of EGFR, in tyrosine phosphorylation of the 170-kDa protein, and in EGFR tyrosine kinase activity occurred in polyamine-deficient cells. These changes were associated with, and may explain, the severely reduced ability of EGF to stimulate cell proliferation and migration in polyamine-deficient intestinal epithelial cells.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Medium and salt solutions were obtained from GIBCO (Grand Island, NY). IEC-6 cells were obtained from the American Type Culture Collection (Rockville, MD). Serum, chemicals, and biochemicals were from Sigma (St. Louis, MO). Matrigel and EGF were purchased from Collaborative Research (Bedford, MA). DFMO was supplied without charge by Marion Merrell Dow (Cincinnati, OH). Primary antibodies against EGFR and transferrin receptor (TRFR) were purchased from Sigma, and secondary antibodies were from Boehringer Mannheim (Indianapolis, IN). EGFR antibody for Western analysis was from Calbiochem (La Jolla, CA). Rhodamine phalloidin was obtained from Molecular Probes (Eugene, OR). The phosphotyrosine antibody was from Zymed Laboratories (San Francisco, CA).

Methods

Culture and treatment conditions. IEC-6 cells were plated at 1.5 × 104 cells/cm2 on Matrigel-coated plates or coverslips in Dulbecco's modified Eagle's medium (DMEM) plus 5% dialyzed fetal bovine serum (dFBS), 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate (DMEM-dFBS). Passages 17 through 20 were used. Cells were incubated at 37°C in 90:10 air-CO2 and fed on day 2. Serum was removed on day 3, and cells were processed at confluency 24 h later. (DMEM itself does not contain putrescine.) Three basic experimental groups were used and are as follows: control (without further additions to the plating medium), DFMO (plating medium plus 5 mM DFMO), and DFMO-putrescine (plating medium plus 5 mM DFMO and 10 µM putrescine). EGF was used at 10 ng/ml (1.6 × 10-9 M), unless stated otherwise, for the indicated periods. Migration experiments were plated in triplicate and repeated three times, and slide experiments were plated in duplicate and repeated at least twice. We have previously established that DFMO reduces intracellular putrescine to undetectable levels in 6 h. Other polyamines are also effectively reduced; spermidine is undetectable by day 2, and spermine is reduced 60% by day 4 (22). These reductions occur without an effect on overall protein synthesis (in disintegrations/min of tritiated leucine incorporated per µg protein: 51.46 ± 4.18 for control and 53.18 ± 4.89 for DFMO; 4 experiments, n = 12 experimental repetitions, P < 0.7910, no significant difference). Also, we have shown previously that 10 µM putrescine restores DFMO-inhibited cell migration to control levels at 4 days (45).

Cell proliferation. Cell proliferation was measured over the short term by analyzing cell doubling times and over the long term by 10-day growth curves. The doubling time experiments were carried out over 48 h during which 5% dFBS with or without 5 mM DFMO or 5 mM DFMO plus 10 ng EGF/ml was present throughout. Cells were plated in triplicate in T-25 flasks and counted at 24 and 48 h. The growth experiments were carried out over 10 days for four groups: control, EGF, DFMO, and DFMO-EGF. The cells were plated in triplicate in T-25 flasks and fed every other day. One set of four groups (12 flasks) was taken up and counted by Coulter counter every other day.

Migration studies. Cells were plated in triplicate in 35-mm dishes and treated as described above. Serum was removed on day 3. On day 4, cell migration was assessed by removing cells from an area on the plate with a razor blade, changing the medium, and incubating the cells at 37°C for 3 h to allow migration. The cells were fixed with 3.7% formaldehyde and washed, and the number of cells migrating across the scratch line was quantitated by computer imaging. The procedure has been described in detail previously (22, 23).

Immunocytochemistry. Cells in the various treatment groups were grown on coverslips, serum was removed on day 3, and the periods of incubation with EGF began 18 h later. EGF was added by removing the serumless medium and replacing it with fresh medium still containing the treatments plus 10 ng EGF/ml. Control medium was also removed and replaced. Incubation was continued for the indicated times. The cells were fixed with 3.7% paraformaldehyde in 10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 2 mM MgCl2, pH 6.8, containing 0.1% Triton X-100 and postfixed with 95% ethanol at -20°C for 3 min. Cells were rehydrated for 30 min in Dulbecco's phosphate-buffered saline with 0.1% bovine serum albumin (DPBS-0.1% BSA) and incubated for 1 h with a monoclonal EGFR antibody that recognized the intracellular domain of the receptor. After three washes with DPBS-0.1% BSA, cells were incubated for 1 h with goat anti-mouse immunoglobulin G (IgG) affinity-purified tetramethylrhodamine isothiocyanate-conjugated second antibody. After a further washing with DPBS-0.1% BSA and DPBS, the slides were mounted. In the experiments that used double-staining for EGFR and TRFR, EGF was added for 10 min. The cells were fixed, postfixed, and stained for 1 h each with monoclonal anti-EGFR and TRFR followed by 1 h each with the appropriate second antibody. In the experiments showing EGFR and F-actin, the monolayers were wounded as in the migration experiments and allowed to migrate for 3 h. EGF was added 5, 10, and 180 min before the end of the migration period. The cells were fixed and stained as in the above experiments except that the anti-mouse IgG second antibody was fluorescein isothiocyanate conjugated. F-actin was stained with rhodamine phalloidin. Images were made using a Nikon Diaphot-TMD inverted microscope with a photometrics NU200 charge-coupled device camera system run on a Macintosh computer using the program IP Lab Spectrum from Signal Analytics (Vienna, VA). Images were digitally deconvolved from three images 0.1 µm apart and normalized to optimum intensity.

Western blot analysis. IEC-6 cells were plated in 60-mm dishes at a density of 6.25 × 104 cells/cm2 in DMEM-dFBS with or without 5 mM DFMO or 10 µM putrescine. Serum was removed on day 3. On day 4, 30 ng EGF/ml was added to the plates for 2, 5, 10, 20, 40, 60, 120, and 240 min. The sample medium was removed, and 400 µl of Laemmli buffer [125 mM tris(hydroxymethyl)aminomethane at pH 6.8, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% beta -mercaptoethanol, 200 µM phenylmethylsulfonyl fluoride, 200 µM Na3VO4, 80 µg/ml leupeptin, and 40 µg/ml aprotenin] were added, and the plates were scraped. The extracts were boiled for 10 min and stored at -80°C until use. Protein concentration was determined by the method of Bradford (5) with BSA as standard. To remove SDS, 0.1 M K2HPO4 (pH 7.2) was added to aliquots of the cell lysate. The samples were centrifuged at 10,000 g for 2 min. The supernatant was analyzed for protein content. Total cell protein (100 µg) was separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes for Western blotting. The membranes were probed with a phosphotyrosine antibody. The immune complexes were visualized by a chemiluminescence detection system (DuPont NEN, Boston, MA) and quantitated by densitometric scanning. The experiments were repeated three times.

Kinase activity of purified EGFR. Kinase activity of EGFR in membranes was determined by measuring the incorporation of [gamma -32P]ATP into angiotensin II at 25°C for 5 min as described by Weber et al. (41). Membranes were preincubated with 4 mM angiotensin II on ice for 10 min in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4, 5 mM MgSO4, 2 mM MnCl2, 10 µg/ml aprotinin, 100 µg/ml leupeptin, 50 µM sodium vanadate, and 100 nM EGF. Phosphorylation was initiated by the addition of 10 µM [gamma -32P]ATP (1.5 µCi; 6,000 Ci/mmol) after membranes were warmed to 25°C for 5 min. Membrane proteins were separated from angiotensin II by precipitation with 2% trichloroacetic acid (TCA) on ice. Supernatants were spotted onto cellulose acetate disks (P81; Whatman) and washed extensively with 5% phosphoric acid to remove unincorporated radioactivity. Radiolabeled angiotensin II was quantified by scintillation counting.

Western blot of total EGFR protein. The three basic treatment groups were assayed without EGF to determine the amount of EGFR present before EGF was added. EGFR protein was immunoprecipitated from the cell extract (500 µg protein) using an anti-EGFR antibody from Calbiochem. Immunoprecipitated EGFR protein was separated on 7.5% SDS-PAGE, and Western blot analysis was carried out as described above using anti-EGFR antibody.

Statistics. Data were tested using analysis of variance and two-tailed, unpaired Student's t-test or Wilcoxon's test where necessary. Results were considered significantly different at or below P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell Growth

Polyamine deficiency significantly slowed doubling time in IEC-6 cells, lengthening it by 14.2 h. When the polyamine-deficient cells were supplemented with putrescine, doubling times were equal to controls. EGF treatment shortened doubling times by 8 h in control cells and in cells treated with DFMO-putrescine but had no effect on doubling time in polyamine-deficient cells (Fig. 1). These results were corroborated by a 10-day growth study that also showed that EGF had no effect on the growth inhibition caused by DFMO (Fig. 2). We have previously shown that putrescine (45) and spermidine (24) are able to maintain growth rates equal to controls if used to supplement DFMO-treated cells.


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Fig. 1.   Cell doubling time in polyamine-deficient cells with and without epidermal growth factor (EGF). Cells were plated in triplicate in 2 sets in T-25 flasks and treated from plating with 5 mM alpha -difluoromethylornithine (DFMO) or with 5 mM DFMO plus 10 µM putrescine (PUT). EGF was added as shown at 10 ng/ml. Flasks were taken up with trypsin-EDTA, and cell number was determined in a Coulter counter at 24 and 48 h postplating. Difference in cell number between the time periods was used to calculate the cell doubling times. Experiment was run twice, and results were combined. Means and SE are shown. Significant differences are shown by the letters: a/b, P < 0.0009; a/c, P < 0.0001; e/f, P < 0.0001.


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Fig. 2.   Cell growth in polyamine-deficient cells with and without EGF. Cells were plated in triplicate in T-25 flasks for each of 4 treatment groups (control, 10 ng EGF/ml, 5 mM DFMO, 5 mM DFMO + 10 ng EGF/ml) and fed every other day for 10 days. At the same time, 1 set of 4 was taken up with trypsin and counted. Experiment was run twice, and results were combined. Means and SE are shown (except where smaller than the symbol). * EGF group was significantly higher than the control group on days 4, 6, and 8 (P < 0.0005).

Cell Migration

DFMO treatment caused an 80% loss in the ability of IEC-6 cells to migrate over a 3-h period, but, when supplemented with putrescine, DFMO-treated cells approached control levels of migration. We have repeatedly shown this effect of putrescine supplementation on cell migration in DFMO-treated cells (22, 45) but included the group in these experiments to provide controls for EGF treatment. EGF stimulated migration in both control and putrescine-supplemented cells by 20-30% and also significantly increased migration in DFMO-treated cells (Fig. 3). This increased level was, however, still only 30% of the control level.


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Fig. 3.   Effect of EGF on cell migration in normal and polyamine- deficient cells. Cells were plated in DMEM-dialyzed fetal bovine serum (dFBS) and treated for 4 days with 5 mM DFMO with or without 10 µM putrescine. Serum was removed on day 3. Cells were removed from an area of each plate on day 4, and fresh medium was added containing 10 ng EGF/ml as shown. After 3 h, monolayers were fixed and the number of migrating cells/mm was determined. Experiment was run twice in triplicate. Means and SE are shown. Significant differences involving EGF are shown by the letters: a/b, P < 0.0398; c/d, P < 0.0003; e/f, P < 0.0092.

EGFR Distribution

The major difference between control cells and polyamine-deficient cells with regard to the location of EGFR before and after binding EGF was the extent to which receptor was found in the cell periphery. EGFR was found in a loosely defined perinuclear area in control cells before exposure to EGF. After EGF exposure, control cells showed wide peripheral staining only briefly, primarily at 5 min (Fig. 4, 1b). Ten minutes after EGF exposure, peripheral staining of EGFR had decreased in control cells (Fig. 4, 1c), and, by 30 min, the distribution of EGFR had returned to its original location (Fig. 4, 1d). DFMO-treated cells, on the other hand, showed widespread punctate staining for EGFR at all times. EGFR was scattered in punctate foci throughout the peripheral cytoplasm with small areas of receptor staining at the nuclear membrane (Fig. 4, 2a). Five minutes after EGF was added to DFMO-treated cells, EGFR staining was present at the nuclear membrane and in the peripheral cytoplasm (Fig. 4, 2b). Ten minutes after EGF exposure, perinuclear and peripheral staining of EGFR remained, with little change in most DFMO-treated cells (Fig. 4, 2c). Thirty minutes after EGF addition, EGFR had substantially returned to its original distribution (Fig. 4, 2d). In general, receptor staining in the DFMO-treated cells supplemented with putrescine closely followed the pattern in control cells (Fig. 4, 3a-d). Because the antibody used bound an intracellular domain of the receptor, cells were permeabilized to allow the antibody access to the intracellular domain; therefore, receptors present at the outer membrane only cannot be separately distinguished.


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Fig. 4.   Differences in EGF receptor (EGFR) distribution between treatment groups before and after incubation with EGF. Cells were plated in DMEM-dFBS and treated for 4 days with 5 mM DFMO with or without 10 µM putrescine. Serum was removed on day 3. On day 4, 10 ng/ml EGF was added for different time periods. Slides were prepared in duplicate for each treatment and time period and stained by immunofluorescence using a monoclonal antibody to EGFR as described in Methods. Images were made as described in Methods. Solid white arrows show perinuclear staining of EGFR. Outlined arrows show peripheral staining of EGFR. 1, Control; 2, DFMO; 3, DFMO-putrescine. a, 0-min EGF; b, 5-min EGF; c, 10-min EGF; and d, 30-min EGF exposure. Bar = 600 µm.

Cells stained for EGFR and TRFR showed that the intracellular location of EGFR in control and putrescine-supplemented cells differed from that in DFMO-treated cells, especially after exposure to EGF (Fig. 5). EGFR was stained with a rhodamine-labeled second antibody and is shown in red, whereas TRFR was stained with a fluorescein-labeled second antibody and is shown in green. Overlapping areas are yellow. In control cells, EGFR and TRFR were concentrated near the nucleus with some overlapping areas (Fig. 5, 1a). Ten minutes after EGF was added, the two receptors were segregated into separate perinuclear areas in control cells. Little staining of either receptor appeared in the cytoplasm (Fig. 5, 2a). DFMO-treated cells showed large areas of EGFR staining in the outer periphery as well as perinuclear staining of EGFR and TRFR with overlap of the two (Fig. 5, 1b). Ten minutes after EGF was added, both receptors were scattered throughout the cell (Fig. 5, 2b). In DFMO-treated, putrescine-supplemented cells, staining of both receptors resembled that in control cells before (Fig. 5, 1c) and after (Fig. 5, 2c) EGF addition. Nonspecific staining by the labeled second antibodies was negligible (Fig. 6).


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Fig. 5.   EGFR distribution compared with transferrin receptor (TRFR). Cells were treated as in Fig. 4. Ten nanograms EGF/ml were added for 10 min. Immunofluorescent staining shows EGFR (red) and TRFR (green). Yellow indicates both receptors. 1, No EGF; 2, with EGF; a, control; b, DFMO; c, DFMO-putrescine. Note the lack of separation of receptors in DFMO-treated cells 10 min after EGF exposure (2b). Bar = 600 µm.


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Fig. 6.   Nonspecific staining by the tetramethylrhodamine isothiocyanate-labeled second antibody (a) and the fluorescein isothiocyanate-labeled second antibody (c) and their corresponding phase-contrast pictures (b and d). Bar = 600 µm.

Association of EGFR With F-Actin

The object of this experiment was to determine whether polyamine deficiency had any effect on the association of EGFR with F-actin after exposure to EGF (Figs. 7-10). EGFR (green) and F-actin (red) are shown combined in columns 1, and their separate images are shown as columns 2 (EGFR) and 3 (F-actin) in black and white. EGF was added at 5 and 10 min from the end of 3-h migration and for the entire 3-h migration period. The apparent effect of polyamine deficiency on the location of the receptor was a delay and reduction in its association with F-actin. The receptor's association with F-actin was evident in most control cells within 5 min of exposure to EGF. In polyamine-deficient cells, the receptor's association with F-actin was restricted to very few cells and did not occur at all until 10 min after exposure to EGF.

In the treatment groups (control, DFMO, and DFMO-putrescine) that migrated for 3 h without EGF, EGFR staining was strongest in cells on the migrating edge (Fig. 7). In control cells, EGFR staining was found in a diffuse perinuclear area (Fig. 7, 2a). In the DFMO group, EGFR staining was present at the nuclear membrane and in the surrounding cytoplasm (Fig. 7, 2b). In the DFMO-putrescine-supplemented group (Fig. 7, 2c), EGFR staining was similar to controls. Control cells contained many actin stress fibers and had strong cortical staining on the migrating edge except where lamellipodia extended beyond the cortex in fine actin filaments (Fig. 7, 3a). DFMO-treated cells had fewer actin stress fibers throughout the cell but prominent cortical staining of actin without lamellipodia on the migrating edge (Fig. 7, 3b). F-actin staining in DFMO-putrescine-treated cells resembled that in control cells (Fig. 7, 3c).


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Fig. 7.   Differences in EGFR distribution relative to F-actin between treatment groups at 0-min EGF exposure. Procedure was the same as for Fig. 4, except slides were stained for EGFR and F-actin. Color images show staining for EGFR and F-actin superimposed. Black-and-white images show staining for the 2 separately. Staining for EGFR is shown as green, and staining for F-actin is shown as red. Yellow indicates an overlap of the 2 stains. Images were made as described in Methods. Solid white arrow shows areas of fine actin filaments extending into lamellipodia. Yellow arrow shows an area of reduced density of actin filaments characteristic of DFMO-treated cells. 1, EGFR and F-actin superimposed; 2, EGFR only; 3, F-actin only. a, Control; b, DFMO; c, DFMO-putrescine. Bar = 600 µm.

When EGF was added 5 min before the end of 3-h migration (Fig. 8), a response was already apparent in control and DFMO-putrescine-supplemented cells. Control cells showed strong stress fibers and fine actin filaments extending beyond the cortex (Fig. 8, 3a), and EGFR could be seen in association with F-actin, especially on the leading edge (Fig. 8, 1a). This was especially clear in the group treated with DFMO-putrescine (Fig. 8, 1c). In the DFMO-treated group, EGFR was not associated with F-actin. EGFR was present at the nuclear membrane and again in the peripheral cytoplasm (Fig. 8, 2b). F-actin, especially in stress fibers, was scarce in this group (Fig. 8, 3b), as before EGF exposure. However, the actin cortex on the leading edge was now discontinuous, and the actin cortex was thinner than before EGF exposure.


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Fig. 8.   Procedure was same as in Fig. 7, except exposure to EGF was for 5 min. Solid white arrow shows areas of fine actin filaments extending into lamellipodia. Outlined arrow shows areas where EGFR is found with F-actin. Yellow arrow shows an area of low density of F-actin characteristic of DFMO-treated cells. All other labels are same as in Fig. 7. Bar = 600 µm.

At 10 min after EGF addition (Fig. 9), the staining of EGFR involved cells farther back from the migrating edge in control cells. EGFR was found near the nucleus and, to a lesser degree than at 5 min, throughout the cell (Fig. 9, 2a). EGFR was associated with fine filament actin on the leading edge at this time (Fig. 9, 1a). Control cells had numerous stress fibers and fine actin filaments, and the migrating edge was uneven with areas in which fine filaments extended outside the actin cortex in lamellipodia (Fig. 9, 3a). In the DFMO-treated cells, EGFR was associated with F-actin in a few cells (Fig. 9, 2b), but actin filaments were few (Fig. 9, 3b), with only the cortical actin staining strongly.


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Fig. 9.   Procedure was same as in Fig. 7, except exposure to EGF was for 10 min. Outlined arrow shows areas where EGFR is found with F-actin. Yellow arrow shows an area of low density of F-actin characteristic of DFMO-treated cells. All other labels are same as in Fig. 7. Bar = 600 µm.

After 3 h of exposure to EGF during migration (Fig. 10), EGFR staining in control cells was localized to areas near the nucleus and in diffuse areas in the peripheral cytoplasm but did not appear to extend into the extreme edges of lamellipodia (Fig. 10, 1a and 2a). Stress fibers were pronounced and extended into long filopodia in which EGFR was absent from the tips (Fig. 10, 3a). In DFMO-treated cells, EGFR was arranged in a ring around the nucleus (Fig. 10, 2b), essentially the same as before EGF exposure (Fig. 7, 2b). This was a frequent pattern in DFMO-treated cells. However, they did show a relatively normal density of F-actin and an increased association of EGFR (Fig. 10, 3b). The DFMO-treated group supplemented with putrescine generally resembled the control group.


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Fig. 10.   Procedure was the same as in Fig. 7, except exposure to EGF was for 180 min. Green arrows show where filopodia begin to be devoid of EGFR. Yellow arrow shows an area of increasing density of F-actin characteristic of DFMO-treated cells treated with EGF over an extended period. All other labels are same as in Fig. 7. Bar = 600 µm.

Protein Tyrosine Phosphorylation and Kinase Activity

In untreated control cells, treatment with EGF stimulated phosphorylation of the 170-kDa receptor protein by approximately sixfold within 2 min (Fig. 11, A and B). A significant increase in protein phosphorylation was sustained for 120 min. The increase returned to unstimulated levels at 240 min. Polyamine depletion dramatically attenuated the ability of EGF to stimulate the phosphorylation of the 170-kDa protein, resulting in an increase in phosphorylation at 2 min that was only half as great in the DFMO-treated cells as in the untreated controls. Furthermore, a significant phosphorylation increase in DFMO-treated cells was sustained for only 40 min. Supplementation of DFMO-treated cells with putrescine restored the ability of EGF to stimulate protein tyrosine phosphorylation to the same degree as in control cells.


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Fig. 11.   A: representative Western blot of EGFR phosphorylation in control, DFMO-treated, and DFMO-putrescine-treated cells after exposure to 30 ng/ml EGF for up to 240 min. After EGF stimulation, proteins were separated on 7.5% polyacrylamide gels by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose. Blots were probed with an anti-phosphotyrosine antibody and visualized by chemiluminescence. Experiment was repeated 3 times with similar results. B: quantitation by densitometric scanning of EGF-stimulated phosphorylation of the 170-kDa protein. Relative degrees of phosphorylation stimulation between the 3 treatment groups after stimulation with 30 ng/ml EGF for up to 240 min are shown. Stimulation of phosphorylation of the 170-kDa protein was significantly less in DFMO-treated groups than in untreated controls until all groups had nearly returned to the unstimulated state. Data are from 3 experiments. * P < 0.05 vs. control levels at corresponding time point. C: quantitation of EGFR kinase activity in purified membranes. Membranes were preincubated with angiotensin II for 10 min on ice with 100 nM EGF. After membranes were warmed to 25°C, phosphosphorylation was initiated by the addition of radiolabeled ATP. Membrane proteins were separated from angiotensin II by precipitation with TCA. Radiolabeled angiotensin II was quantified by scintillation counting. Membranes were prepared from 2 cell experiments and represent 8 samples in control and DFMO-putrescine groups and 7 samples in DFMO groups. * P < 0.05 vs. control and DFMO-putrescine groups. D: Western blot of immunoprecipatated EGFR protein in the 3 treatment groups before the addition of EGF. Equal amounts of protein were applied to the gel for all three groups. C, control; D, DFMO; D/P DFMO-putrescine.

The kinase activity of purified membrane EGFR in DFMO-treated cells was reduced to <50% of that in the controls and was restored by putrescine supplementation (Fig. 11C). The lowered kinase activity was not due to less EGFR protein, as no lowering of the protein was shown by Western blot (Fig. 11D).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell proliferation and cell migration are often mutually exclusive processes, but some growth factors, including EGF, are able to stimulate both. In polyamine-deficient cells, proliferation and migration are greatly reduced and EGF stimulation is inhibited. Extensive cytoskeletal reorganization is necessary for these processes. We have shown that cytoskeletal reorganization following EGF/EGFR binding in polyamine-deficient cells is abnormal, as are the intracellular distribution, tyrosine kinase phosphorylation, and kinase activity of EGFR. These processes, as well as the stimulation of cell proliferation and migration by EGF, can be restored to normal levels by putrescine supplementation, showing the importance of polyamines for these functions.

It is well known that polyamines have the ability to stabilize membranes, bind to phospholipids (1, 16), and decrease the lateral mobility of proteins in the plasma membrane (10, 30). The stabilization is effected from the cytoplasmic side of the membrane only and is not dependent on high-energy phosphates (30). Endosomal, lysosomal, and other membranes of the Golgi apparatus are also involved. The polyamines may modulate the movement of membrane receptor proteins, including protein kinase C and the transmembrane receptors, through this stabilizing ability. An interruption of polyamine biosynthesis could remove their modulating influence, upsetting the integration of cytoskeletal remodeling processes as well as signal transduction from transmembrane receptors.

The pathway taken by EGFR after binding EGF has been described in some detail by others (6, 12, 43). In brief, ligand-bound EGFR is endocytosed from the plasma membrane in clathrin-coated vesicles that bud from the membrane carrying the EGF-EGFR complex into the cytoplasm. Most of the complex then takes a nonrecycling pathway, accumulating in early endosomes within 1-5 min at 37°C. After 10-20 min, the complex appears in late endosomes in a perinuclear area around the centriole (20). After 40-60 min, the complex moves from late endosomes into mature lysosomes where it is degraded, culminating in the downregulation of the receptor (34). The intracellular locations and the time course of EGFR staining following EGF exposure in control cells appeared to follow a similar pathway and schedule. After EGF exposure, receptor staining appeared in the peripheral cytoplasm within 5 min and then returned to the perinuclear area by 30 min, leaving little staining in the periphery. The distribution of EGFR in cells supplemented with DFMO-putrescine was similar to that in the control cells at all time points following EGF exposure. Receptor staining found next to the nucleus before EGF was added may represent unfilled or newly synthesized receptors still in the Golgi apparatus.

In DFMO-treated cells, the distribution of EGFR after EGF binding was markedly different from control and DFMO-putrescine-treated cells. The major difference between the groups was a more extensive peripheral EGFR staining at all times in the DFMO-treated cells before and after EGF exposure. The prolonged time spent by the receptor near the nucleus, which is characteristic of the EGF-EGFR complex and is required for its physiological action (34), did not seem to occur.

A comparison of the intracellular position of EGFR with that of the TRFR 10 min after binding EGF also demonstrated a deviation from normal distribution by EGFR in polyamine-deficient cells. TRFR has been used by many investigators to delineate the endocytic pathway of internalized transmembrane receptors. TRFR is a nutritional, rather than a signaling, receptor. It is constitutively endocytosed, with or without ligand (15, 17). It is not transported into lysosomes and degraded but recycles through the endocytic pathway (34). The recycling pathway taken by TRFR has been distinguished from the nonrecycling pathway taken by EGFR by electron microscopy (15), cell fractionation (11, 13, 42), and fluorescence microscopy (35). In our experiments, exposure to EGF caused separation of the two receptors within 10 min in control and DFMO-putrescine-supplemented cells but not in polyamine-deficient cells, suggesting a failure of normal processing events.

Colocalization of the EGF/EGFR complex with F-actin causes rapid reorganization of the cytoskeleton (28) followed by the activation of signal transduction in A-431 cells (8). Actin filaments in polyamine-deficient cells are not only reduced in the cell interior but are also redistributed to the cortex. This redistribution may reflect the reduction and early fall of phosphorylation of the 170-kDa protein as well as the lack of association of the EGF-EGFR complex with actin in polyamine-deficient cells. It has been suggested that actin filaments may serve as a matrix that provides a high local concentration of substrate enzymes for efficient interaction with EGFR (37).

Few reports explore the action of EGF in relation to the polyamines. Our experiments have shown that polyamine deficiency is associated with the failure of EGF to stimulate processes requiring the cytoskeleton, alterations in the distribution of ligand-stimulated EGFR, and concomitant reduction of 170-kDa protein phosphorylation and kinase activity, all of which can be maintained by putrescine supplementation. Faaland and co-workers (9) have reported that putrescine, spermidine, and spermine inhibited proliferation and tyrosine kinase activity in A-431 cells and that DFMO did not reduce EGFR protein tyrosine kinase activity. However, first, they added the polyamines to cells that were not polyamine deficient, second, dosages and treatment periods differed, and, third, cell lines were basically different, since A-431 cells are a cancer line and IEC-6 cells are not. Regarding the last point, we have shown that polyamine depletion inhibited transglutaminase activity in IEC-6 cells but stimulated it in colon cancer cells (23). Faaland and co-workers (9) concluded that the action of polyamines in A-431 cells probably involves the modulation of EGFR signal transduction pathways. Borowski and co-workers (4), using affinity-purified EGFR kinase from A-431 cells, found that spermidine and other polycationic compounds activated EGFR kinase threefold. They suggested that, because changes in EGFR kinase activity could be substrate specific, an influence on substrate levels or conformation could be an important factor in EGFR kinase activation. In vivo, EGFR tyrosine kinase activity, EGFR tyrosine phosphorylation, and EGFR abundance were all increased 30 min after injury to the gastric mucosa in rats (27). The increase was proportionately more in young than in old rats, and the speed of muscosal repair followed the same pattern. These experiments strongly support the important part played by an activated EGFR tyrosine kinase in the early restitution of gastric mucosal injury. Polyamines were not measured.

With respect to the cytoskeleton, the most obvious effect of polyamine deficiency in IEC-6 cells was increased cortical actin and reduced intracellular actin filaments. In addition, the intracellular distribution of the receptor was altered and its association with actin was delayed. EGFR tyrosine phosphorylation and kinase activity were reduced by 50%, although EGFR total protein was not reduced. These changes inhibited the response of polyamine-deficient cells to EGF stimulation as shown by proliferation or migration. Whether these effects are due to defects in signaling or in cytoskeletal remodeling, it is tempting to suspect that the polyamines may be important for crucial regulating steps in the reorganization of actin, a process that is vital to both proliferation and migration.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the gift of DFMO from Marion Merrell Dow (Cincinnati, OH), the confocal microscopy assistance from Larry Tague, the graphic assistance from Danny Morse and Laura Malinik, and the manuscript preparation by Easter Jenkins.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 (L. R. Johnson) and National Heart, Lung, and Blood Institute Grant HL-48308 (T. B. Patel).

Address for reprint requests: S. A. McCormack, Dept. of Physiology and Biophysics, Univ. of Tennessee, Memphis, 894 Union Ave., Rm. 426 Nash Bldg., Memphis, TN 38163.

Received 29 January 1997; accepted in final form 30 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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