Focal adhesion kinase signaling is decreased in polyamine-depleted IEC-6 cells

Ramesh M. Ray, Mary Jane Viar, Shirley A. McCormack, and Leonard R. Johnson

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Polyamines are essential to the migration of epithelial cells in the intestinal mucosa. Cells depleted of polyamines do not attach as rapidly to the extracellular matrix and do not form the actin stress fibers essential for migration. Because both attachment and stress fiber formation depend on integrin signaling and the formation of focal adhesions, we examined these and related processes in polyamine-depleted IEC-6 cells. There was general decreased tyrosine phosphorylation of focal adhesion kinase (FAK), and, specifically, decreased phosphorylation of Tyr-925, the paxillin binding site. In control cells, FAK phosphorylation was rapid after attachment to the extracellular matrix, while attached cells depleted of polyamines had significantly delayed phosphorylation. FAK activity was also significantly inhibited in polyamine-depleted cells as was the phosphorylation of paxillin. Polyamine-depleted cells failed to spread normally after attachment, and immunocytochemistry showed little colocalization of FAK and actin compared with controls. Focal adhesion complex formation was greatly reduced in the absence of polyamines. These data suggest that defective integrin signaling may, at least in part, account for the decreased rates of attachment, actin stress fiber formation, spreading, and migration observed in polyamine-depleted cells.

ornithine decarboxylase; DL-alpha -difluoromethylornithine; integrins; paxillin; focal adhesions; cell spreading; attachment; migration


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE GASTROINTESTINAL MUCOSA is not only responsible for the absorption of nutrients but also acts as a barrier to a broad range of noxious agents present within the lumen. After damage, rapid resealing of the epithelium is essential to prevent compromising the health of the individual. Repair of mucosal erosions has been shown to involve at least two different processes. First, the rapid process of mucosal restitution occurs by the sloughing off of damaged epithelial cells and the migration of viable cells from areas adjacent to, or just beneath, the injured surface to cover the denuded areas (18). Second, lost cells are replaced by the process of cell division, and the mucosa is returned to its normal thickness. Numerous studies by Silen and others (see Refs. 22 and 23 for reviews) have described early mucosal restitution and emphasized its importance in the overall process of healing. Little is known about this early phase except that it involves cell migration and spreading, depends on intact microfilaments (5), and requires polyamines (27).

Studies from our laboratory have shown that inhibiting ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, with DL-alpha -difluoromethylornithine (DFMO), and the subsequent depletion of polyamines, prevents both phases of mucosal healing (27, 28). Oral administration of polyamines to depleted animals restores the normal rate of healing (28). In an in vitro model involving cultured IEC-6 cells, polyamine depletion inhibited migration by disrupting the cytoskeleton (13, 14). Alterations of the cytoskeleton included a striking decrease in actin stress fibers and a pronounced localization of F-actin to the cortex, a similar change in the distribution of tropomyosin (14), and a decrease in nonmuscle myosin II and a change in its distribution (29). These changes may, in part, be due to failure of thymosin beta 4 to sequester G-actin (11). Polyamines are also essential for the normal attachment of cells to the extracellular matrix (19), which explains at least some of the reliance of cell migration on normal polyamine levels. All of the above responses to DFMO are prevented by exogenous polyamines.

The migration of cells depends on their ability to make and break attachments to the extracellular matrix (ECM). The ECM controls cells through signals via a family of cell surface receptors called integrins (6). Integrins are composed of alpha - and beta -subunits. Currently, 15 alpha - and 8 beta -subunits have been described, and the combination of a particular alpha -subunit with a particular beta -subunit accounts for binding and signaling specificity (7). As integrins bind to the ECM, they aggregate and direct the formation of a cytoskeletal and signaling complex that results in the assembly of actin filaments. The reorganization of actin filaments into stress fibers causes a positive feedback, resulting in additional integrin clustering. This results in transmembrane complexes of ECM protein; integrins; cytoskeletal proteins such as talin, paxillin, and vinculin; and actin. These aggregates are termed focal adhesion complexes and can be detected immunocytochemically with antibodies to their constituents.

Various protein tyrosine kinases are constituents of focal adhesions and are activated by integrins (10). Prominent among these is focal adhesion kinase (FAK), which is activated by most integrins. Upon activation, FAK is targeted to the focal adhesion complex, where its additional phosphorylation provides binding sites for other proteins, such as paxillin, to the complex. Paxillin, one of the actin-binding proteins, recruits actin to the focal adhesion complex, resulting in the formation of stress fibers (3). Other proteins involved in actin binding and focal adhesion formation, such as talin and vinculin, are also recruited in a similar manner (7). The formation of focal adhesions results in the organization of stress fibers, which, together with the making and breaking of contacts with the ECM, result in cell migration and the cell spreading that is necessary for growth (8).

Our laboratory has shown that polyamine depletion delays cell attachment to ECM proteins (19). The purpose of the current study is to examine the role of polyamines in the mechanisms involved in cell attachment and those initiated by attachment that lead to cell spreading. The experiments described focus on the formation of focal adhesion complexes, and, specifically, the recruitment of FAK to the membrane, FAK phosphorylation, and the recruitment of paxillin to the focal adhesion complex. In polyamine-depleted cells, each of the processes was decreased, leading to a decrease in the formation of actin stress fibers and to the inhibition of cell spreading. The results indicate that polyamine-depleted cells, even though they are attached to the ECM, lack the integrin signaling necessary to form viable focal adhesions and the stress fibers essential for spreading, migration, and growth.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Medium and other cell culture reagents were obtained from GIBCO BRL (Grand Island, NY). Fetal bovine serum (FBS) and dialyzed fetal bovine serum (dFBS; 1,000 molecular weight cut-off) were from Sigma, St. Louis, MO. [gamma -32P]ATP and an enhanced chemiluminescence Western blot detection system were purchased from NEN (Boston, MA). DFMO was a gift from Merrell Dow Research Institute of Marion Merrell Dow (Cincinnati, OH). FAK, alpha 2-integrin, beta 1-integrin, and PY99 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). FAK [PY925] antibody was purchased from Biosource International. The IEC-6 cell line was obtained from American Type Culture Collection (Rockville, MD) at passage 13 (ATCC CRL 1592). The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (15). The cells are nontumorigenic and retain the undifferentiated character of epithelial stem cells. All other chemicals were of the highest purity commercially available.

Cell culture. The IEC-6 cell stock was maintained in T-150 flasks in a humidified, 37°C incubator in an atmosphere of 90:10 air:CO2. The medium consisted of Dulbecco's modified Eagle's medium (DMEM) with 5% heat-inactivated FBS, 10 µg insulin, and 50 µg gentamicin sulfate/ml. The stock was passaged weekly at 1:10, fed three times per week, and passages 15-20 were used. For the experiments, the cells were taken up with 0.05% trypsin plus 0.53 mM EDTA in Hanks' balanced salts without calcium and magnesium and counted by hemocytometer.

General experimental protocols. The general protocol for the experiments and the methods used were similar to those described previously (12, 19). In brief, IEC-6 cells were plated at 6.25 × 104 cells/cm2 in DMEM supplemented with 5% dFBS, 10 µg insulin, and 50 µg gentamicin sulfate/ml (DMEM/dFBS, control) or in DMEM/dFBS containing 5 mM DFMO or DFMO plus 10 µM putrescine (DFMO/putrescine). Cells were grown at 37°C in a humidified atmosphere of 90% air-10% CO2. They were fed every other day and serum starved during the 24 h before harvesting.

Attachment assay. Cells were plated on day 0 in 150-cm2 flasks at a density of 9.2 × 106 cells/flask. They were grown in DMEM with 5% dFBS, 10 µg insulin, and 50 µg gentamicin sulfate/ml (DMEM/dFBS, control) or in DMEM/dFBS containing 5 mM DFMO. Cells were fed on day 2. On day 5, cells were taken up with 0.05 trypsin plus 0.53 mM EDTA and counted. Cells were resuspended in either control or DFMO-containing medium at a density of 0.8 × 106/ml. These cell suspensions were allowed to recover from the trypsinization for 1 h at 37°C in an atmosphere of 90:10 air:CO2. They were mixed gently every 15 min during this hour of conditioning. At the end of the hour, cells were plated on 100-mm dishes (with or without Matrigel) by adding 10 ml of the cell suspension containing 8 × 106 cells/dish. The dishes were returned to the incubator (37°C, 90:10 air:CO2) for the desired times. After incubation, the dishes were removed from the incubator, and, after gentle mixing, the medium containing unattached cells was removed. The number of unattached cells was determined by counting on a Coulter model Zf (Coulter Electronics, Hialeah, FL). Attachment was expressed as percentage of total cells plated.

Spreading of the attached cells (15-min attachment) was monitored. After being washed with Dulbecco's phosphate-buffered saline (DPBS), attached cells were incubated with fresh DMEM/dFBS (control) or in DMEM/dFBS containing 5 mM DFMO. Plates were marked (defined area) to follow spreading. At various time intervals, cells were photographed using an inverted-phase microscope with an attached charge-coupled device camera. Three pictures were taken from each dish at ×100 magnification.

Cell fractionation. Cells were grown for 4 days as described earlier. Cells were washed twice with DPBS, scraped with a rubber policeman in DPBS, and pelleted by centrifugation. Pellets were resuspended in cell lysis buffer (50 mM Tris · HCl, pH 7.4, 2 mM MgCl2, and 1 mM EDTA) and sonicated. Membrane and cytoplasmic fractions were separated by centrifugation and used for the Western blot analysis of FAK.

Preparation of cell extracts. Attached cells were washed twice with DPBS, and 500 µl of cold cell lysis buffer (10 mM Tris · HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 200 µM Na3VO4, 80 µg/ml leupeptin, and 40 µg/ml aprotinin) was added. The dishes were incubated at 4°C for 30 min on a rotating shaker, and the extract was cleared by centrifugation at 10,000 g for 5 min. The extracts were stored at -80°C as whole cell extract. The protein concentration was determined by the method of Bradford (2), using bovine serum albumin (BSA) as a standard.

Immunoprecipitation. Cell lysates (400 µg of protein) were matched for protein and precleared with 20 µl of protein A/G agarose for 1 h at 4°C. The precleared supernatants were further incubated overnight with 2 µg of antibody recognizing either FAK or paxillin, and the immunocomplexes were preadsorbed to protein A/G agarose. Preadsorbed immunocomplexes were then used to measure FAK autophosphorylation or for Western blot analysis.

In vitro FAK assay. The immunoprecipitated FAK protein was pelleted by centrifugation (10,000 g for 2 min) and then washed three times with kinase assay buffer (KAB; 20 mM Tris · HCl, pH 7.4, 10 mM MgCl2 6H20, 200 µM Na3VO4, 10 mM NaF, and 1 mM dithiothreitol). The pellet was resuspended in 40 µl of KAB containing 20 µM ATP and 10 µCi [gamma -32P]ATP. After 20 min at 30°C, the reaction was terminated by the addition of 40 µl of 2× Laemmli sample buffer. Samples were then boiled for 5 min and subjected to 15% SDS-PAGE. The gels were stained with Coomassie brilliant blue, dried, and exposed for 1-3 h to Kodak X-ray film.

Western blot analysis. Cell extracts were prepared as described above. Total cell protein (50 µg) or immunoprecipitated protein was separated on 15% SDS-PAGE and transferred to nitrocellulose membranes for Western blotting. Equal loading of protein was confirmed by staining the nitrocellulose membrane with Ponceau S. The membranes were then probed with an antibody directed against FAK, alpha 2-integrin, beta 1-integrin, paxillin, FAK[PY925], or PY99. The immunocomplexes were visualized by the enhanced chemiluminescence detection system.

Immunohistochemical localization of integrins, FAK, and actin. Control and DFMO-treated cells were plated in 35-mm dishes (each containing a Matrigel-coated glass coverslip), and 30 and 60 min after the attachment period, plates were washed twice with DPBS to remove unattached cells. Attached cells were fixed with 4.0% formaldehyde, washed with DPBS, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 3% BSA for 20 min. FAK was stained with rabbit polyclonal anti-FAK antibody (UpState Biotechnology, Lake Placid, NY) at 1:60 dilution for 1 h, followed by goat anti-rabbit IgG-FITC (Chemicon International, Tenecula, CA) at 1:60 dilution for 1 h. Integrins were stained with polyclonal anti-goat alpha 2-integrin and polyclonal anti-mouse beta 1-integrin antibodies (Santa Cruz, CA) at 1:100 dilution for 1 h, followed by rabbit anti-goat IgG-Texas red and donkey anti-mouse IgG-FITC (Chemicon) at 1:60 dilution for 1 h. F-actin was stained by Texas red-conjugated phalloidin (Molecular Probes, Eugene, OR) for 45 min. Images were captured by confocal laser scanning microscopy using Bio-Rad MRC-1024 Laser Sharp. The images were obtained sequentially by z-series and processed by Adobe Photoshop.

Statistical analysis. All experiments were repeated three times in triplicate. Blots shown are representative of three experiments. Numerical data are means ± SE of three experiments. Significance was determined by analysis of variance followed by Duncan's multiple range test. The level of confidence chosen was 0.95.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of polyamine depletion on cell attachment. The data in Fig. 1 show the attachment of IEC-6 cells grown for 4 days in either control or DFMO-containing media. Cells were trypsinized, washed, and identical numbers were conditioned for 1 h in an incubator at 37° in either control media or media containing 5 mM DFMO before plating. Time-dependent increases in attachment to Matrigel were observed (Fig. 1). Cells attached rapidly to Matrigel-coated plates; ~75% were attached at 10 min, and 95% were attached within 40 min. Polyamine depletion significantly delayed the attachment of cells. Attachment was inhibited ~40% at 10 min, and the effect of DFMO decreased with time, so that nearly equal percentages of cells were attached by 100-120 min (Fig. 1).


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Fig. 1.   Polyamine depletion delays the attachment of IEC-6 cells. Cells were grown for 4 days with and without DL-alpha -difluoromethylornithine (DFMO; 5 mM). Cell attachment assay to Matrigel-coated plates was performed as described in MATERIALS AND METHODS. Values are means ± SE of 6 observations. *Significantly different from control (P < 0.05).

Immunohistochemical localization of integrins. Figure 2A shows localization of alpha 2- and beta 1-integrins in the cells attached to the Matrigel. Strong colocalization (yellow) of alpha 2- and beta 1-integrins was observed in control cells and cells incubated with DFMO and putrescine. In polyamine-depleted (DFMO) cells, colocalization was decreased. This was prevented in part by the addition of putrescine to the DFMO-containing medium. Western blot analysis of the cell extract showed that the levels of the alpha 2-integrin were not altered by polyamine depletion (Fig. 2B). These data suggest that changes in integrin clustering due to polyamine depletion might alter downstream signaling events, leading to decreased attachment of cells to Matrigel.


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Fig. 2.   Localization of alpha 2- and beta 1-integrins during attachment and spreading of IEC-6 cells. Cells were grown in DMEM/fetal bovine serum (FBS) with or without DFMO (5 mM) and DFMO + putrescine (PUT; 10 µM) for 4 days. Cells were trypsinized and washed with DMEM twice and plated onto Matrigel-coated glass coverslips. After attachment for the indicated time period, cells were fixed, permeabilized, and immunostained for the localization of alpha 2- and beta 1-integrins. Cell extracts prepared from the cells grown as described above were immunoprecipitated using alpha 2-integrin antibodies, separated by SDS-PAGE, and subjected to Western blot analysis. A: localization of alpha 2-integrin (red) and beta 1-integrin (green). B: Western blot of alpha 2-integrin. Results shown are representative of 3 experiments.

Polyamine depletion and FAK. The levels and degree of phosphorylation of FAK in confluent (4-day) cells grown in control, DFMO, and DFMO plus putrescine in whole cell extract and membrane and cytosolic fractions were determined. As shown in Fig. 3A, levels of FAK protein were not affected dramatically upon polyamine depletion, but tyrosine phosphorylation was decreased by 52% in polyamine-depleted cells. Cell fractionation analysis showed, taking loading into consideration, there was little difference in the amount of FAK localized to the membranes of cells treated with DFMO, and cytoplasmic fractions showed nearly the same levels of FAK protein (Fig. 3B). Total amounts of FAK present in the two groups appeared approximately equal. Polyamine depletion had no effect on overall tyrosine phosphorylation of FAK or the phosphorylation of Tyr-925 in the membrane fractions (Fig. 3B). However, the phosphorylation of cytoplasmic FAK was significantly decreased in DFMO-treated cells. The phosphorylation of Tyr-925, which is present in the focal adhesion targeting domain of FAK, was almost completely prevented by polyamine depletion. After accounting for loading, these changes were almost totally prevented by exogenous putrescine.


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Fig. 3.   Effect of polyamine depletion on the levels and phosphorylation states of focal adhesion kinase (FAK) in confluent cells (A) and cytoplasmic and membrane fractions (B). Cells were grown for 4 days in the presence and absence of 5 mM DFMO or DFMO + putrescine (10 µM). Equal amounts of proteins were separated by SDS-PAGE, and Western blot analysis was performed using FAK, PY99, FAK[PY925], and actin antibodies. A representative blot from 3 experiments with densitometry readings is shown. Ip, immunoprecipitate; WB, Western blot.

We next compared levels of FAK and tyrosine-phosphorylated FAK present in total cell extracts from control and polyamine-depleted cells. To control for detachment-induced changes that might be caused by trypsinization, fresh media, and suspension, one portion of cells from each group was conditioned in media for 1 h before plating. The data in Fig. 4 indicate that detachment had no effect on the ability of cells from either group to phosphorylate FAK or to effect the amount of FAK present. Therefore, observed changes are those that occur after plating.


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Fig. 4.   Conditioning cells before attachment does not affect the level and tyrosine phosphorylation of FAK. Control and DFMO-treated cells (4 days) were trypsinized, washed twice with DMEM, resuspended in DMEM/FBS, and preconditioned as described in MATERIALS AND METHODS. Cells were lysed, equal amounts of proteins were separated by SDS-PAGE, and Western blot analysis was performed using PY99 and FAK antibodies. NC, no conditioning; C, conditioning for 1 h. A representative blot from 3 experiments is shown.

Relationship among FAK, paxillin, and cell attachment. Figure 5 depicts phosphorylation of FAK at time 0 (before any attachment) and in cells attached at 10 and 30 min after plating. An obvious finding is that there was no FAK phosphorylation before cells attached to the ECM. Control cells showed significant phosphorylation of FAK after 10 min, while FAK phosphorylation in polyamine-depleted cells was delayed until 30 min.


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Fig. 5.   Tyrosine phosphorylation of FAK during attachment of control and polyamine-depleted cells. Attached cells were lysed at the indicated times, proteins were separated by SDS-PAGE, and Western blot analysis was performed using PY99 and FAK antibodies. A representative blot with densitometry readings from 3 experiments is shown.

Intrinsic FAK tyrosine kinase activity measured in vitro from FAK immune complexes increases rapidly in control cells and is readily apparent in cells attached at 30 min (Fig. 6). Autophosphorylation of FAK in polyamine-depleted cells occurs much more slowly, and by 120 min is not yet equal to that seen at 30 min in control cells. It is important to point out that by 100 min, attachment is complete in both control and polyamine-depleted cells (Fig. 1).


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Fig. 6.   Polyamine depletion decreases the autophosphorylation of FAK protein. FAK protein from control and DFMO-treated cell extracts was prepared 30 and 60 min into the attachment period. FAK was immunoprecipitated and subjected to an in vitro kinase reaction as described in MATERIALS AND METHODS. Phosphorylated FAK was separated by SDS-PAGE, and the gels were dried and exposed to X-ray film. A representative blot from 3 experiments with densitometry readings is shown.

Figure 7 shows that the amount of paxillin, one of the components of focal adhesions that is phosphorylated by FAK, is slightly less in polyamine-depleted cells. However, the proportion of paxillin that is phosphorylated in the DFMO-treated cells is significantly less than that phosphorylated in the control cells. These data suggest that polyamines play a role in the ability of FAK to recruit and phosphorylate other protein components of the focal adhesion complex, and, therefore, that they are involved in regulating signaling within the focal adhesion targeting domain of FAK.


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Fig. 7.   Polyamines are required for tyrosine phosphorylation of paxillin during attachment of IEC-6 cells. Attached cells were lysed at 10 and 30 min, equal amounts of proteins were separated by SDS-PAGE, and Western blot analysis was performed using paxillin and PY99 antibodies. A representative blot from 3 experiments with densitometry is shown.

The spreading of attached cells from one area of a culture plate was followed over time. Cells were first allowed to attach for 15 min. Plates were then washed, and the first photo was taken at time 0 (Fig. 8). Subsequent photos of the same areas were taken 15, 30, and 45 min later. Figure 8 shows that at time 0, more cells were attached in the control culture than were attached in the dish treated with DFMO. However, another important effect of polyamine depletion was evident. Control cells began to spread almost immediately, while spreading of polyamine-depleted cells was significantly delayed. By 45 min, almost all the cells in the control dish had spread and formed numerous confluent areas. There were few areas of confluency in the dish containing polyamine-depleted cells, and most of these cells had not yet spread (Fig. 8).


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Fig. 8.   Polyamine depletion decreases attachment and spreading of IEC-6 cells. Control and DFMO-treated cells were plated on Matrigel-coated plates and allowed to attach for 15 min. Plates were washed after 15 min to remove unattached cells, fresh DMEM/FBS was added to plates and further incubated, and at indicated times, attached cells from a marked area on the plates were photographed. Results shown are representative of 3 experiments.

Localization of FAK and F-actin during attachment and spreading. Control and polyamine-depleted cells were allowed to attach for 30 and 60 min, FAK was localized by anti-FAK antibody, and F-actin was stained with phalloidan-Texas red. Strong colocalization (yellow) of FAK and F-actin was observed around the periphery of control cells at 30 min (Fig. 9, A1). The concentration of both FAK and F-actin to the plasma membrane is also obvious in Fig. 9, B1 and C1, respectively. Cells that had been treated with DFMO and depleted of polyamines showed little colocalization (Fig. 9, A2). These cells showed a strong cytoplasmic staining of FAK, and while FAK was present in the membranes of some cells in reduced amounts (Fig. 9, B2), there was significantly less F-actin present and only faint peripheral F-actin staining in some cells (Fig. 9, C2). Within 60 min, most control cells had spread (Fig. 9, A3). FAK was localized in distinct patches in the plasma membrane (Fig. 9, B3), indicating the formation of focal adhesion complexes as evidenced by the colocalization with F-actin and the formation of stress fibers (Fig. 9, C3). In polyamine-depleted cells, the translocation of FAK from the cytoplasm to the membrane continued through 60 min but did not reach a sufficient level in most cells to form focal adhesions and initiate spreading (Fig. 9, A4 and B4). At 60 min, there was also an increase in the colocalization of FAK and F-actin (Fig. 9, A4), but this was greatly reduced, even compared with control cells at 30 min. The relative absence of focal adhesion complexes in the polyamine-depleted cells at 60 min was further indicated by the almost complete absence of stress fibers at this time (Fig. 9, C4). Supplementation of putrescine with DFMO prevented the changes in the distribution of FAK to the membrane and colocalization of FAK, and actin and cells began to spread well compared with DFMO alone (Fig. 9, A5, B5, and C5).


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Fig. 9.   Localization of FAK and F-actin during attachment and spreading of IEC-6 cells. Cells were grown in DMEM/FBS with or without DFMO (5 mM) or DFMO + putrescine (10 µM) for 4 days. Cells were trypsinized, washed with DMEM twice, and plated onto Matrigel-coated glass coverslips. After attachment for the indicated time period, cells were fixed, permeabilized, and immunostained for the localization of FAK (green) and F-actin (red) (A), FAK (B), and F-actin (C).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Earlier studies from our laboratory have shown that polyamines are essential for normal healing of mucosal stress ulcers (27, 28) and for cell migration essential to the healing of those ulcers (13, 14). We have also shown that polyamine depletion inhibited the attachment of cells to various components of the ECM (19), a process necessary for migration. Additional studies indicated the lack of actin stress fibers and a general disorganization of the cytoskeleton of polyamine-depleted cells (11, 14, 29), elements that are all necessary for cell migration. Together, these results suggest that even though polyamine-depleted cells eventually attach to the ECM, the subsequent signaling necessary for the cytoskeletal organization involved in cell migration does not occur. In the current study, we have examined some of the processes involved in the cytoskeletal remodeling necessary for migration and the influence of polyamines on those processes.

The inhibition of ODC by DFMO causes time-dependent decreases in putrescine, spermidine, and spermine. In IEC-6 cells, putrescine disappears by 3 h, spermidine by 24 h, and spermine remains at levels equal to ~40% of control even after 3 days of incubation with DFMO (13). Thus in our experiments, there is still some intracellular spermine remaining, but because of its strong positive charges, it is probably bound to proteins and other large negatively charged molecules. It may well be that the only active polyamines, in terms of regulating physiological processes, are those that are free. Thus the free polyamine pool may actually decrease shortly after the inhibition of putrescine production (16).

Earlier studies from this laboratory showed that polyamine depletion inhibited the attachment of cells to plastic and various ECMs (Matrigel, laminin, fibronectin, and collagen IV) and that the addition of putrescine prevented this decrease (19). Polyamine depletion delayed the attachment of cells to the ECM, but did not prevent it. Comparison of time courses of attachment of cells to plastic (data not shown) and Matrigel in this study (Fig. 1) showed that a significantly greater percentage of cells was attached to Matrigel compared with plastic. Approximately 95% of the cells (control and DFMO) were attached at 60 min on Matrigel, while on plastic, ~80% of control and only 73% of DFMO-treated cells were attached. These data suggest that ECM increases the attachment of both control and DFMO-treated cells but that signaling events leading to attachment in polyamine-depleted cells occur slowly. Decreased attachment may be due to alterations in the levels of integrins or the clustering of various integrins involved in the formation of the receptor complex necessary to bind the ECM. Immunohistochemical localization of alpha 2- and beta 1-integrins revealed that although levels of alpha 2- (Fig. 2A) and beta 1- (data not shown) integrins were not altered, the colocalization (clustering) of these two partners was decreased in DFMO-treated cells (Fig. 2B). DFMO-treated cells showed less colocalization of integrins compared with control cultures during attachment. Addition of putrescine to DFMO-treated cultures maintained clustering at control levels. Based on these data, we hypothesize that our previous findings of decreased stress fiber formation in polyamine-depleted cells might be due to the lack of polyamine involvement in signaling events, leading to the formation of focal adhesions and the subsequent organization of the actin cytoskeleton.

Our studies focused on FAK because its phosphorylation and recruitment to the focal contacts are key events in integrin signaling, leading to the attachment and spreading of cells. Decreased tyrosine phosphorylation in DFMO-treated confluent IEC-6 cells (Fig. 3A) and in the cytoplasmic fraction of FAK (PY99 and FAK PY925) suggests that polyamine depletion decreased FAK signaling. Addition of putrescine prevented this decrease in the tyrosine phosphorylation of FAK, in general, and Tyr-925, in particular (Fig. 3B). Tyr-925 is present in the focal adhesion targeting domain, and its phosphorylation is essential for binding to paxillin (26). The tyrosine phosphorylation of FAK was delayed in DFMO-treated cells during attachment to the Matrigel compared with control (Fig. 4). Polyamines interact with membrane structures by virtue of their charge (1). In IEC-6 cells, a deficiency of polyamines reduces signaling effectiveness by epidermal growth factor receptor (11). Smith et al. (24) also have reported that divalent cations regulate beta 3-integrins. Therefore, it is reasonable to believe that polyamine depletion might be responsible for the alteration of integrin receptors and subsequent FAK signaling.

Two features of FAK essential to its functioning are its focal adhesion targeting sequence and its autophosphorylation site. The COOH-terminal domain of FAK contains the focal adhesion targeting sequence and binds paxillin (25, 26, 30). The tyrosine residue at position 397 in FAK serves as the major autophosphorylation site that, when phosphorylated, increases the intrinsic kinase activity and provides the docking site for src and other signaling proteins (4). Formation of the FAK-Src complex is believed to direct the phosphorylation of additional focal adhesion-associated substrates such as paxillin and cas130 (21). The NH2 terminus of paxillin contains four tyrosine phosphorylation sites, but the role of tyrosine phosphorylation of paxillin has not been studied extensively. Richardson et al. (17), however, have shown that reduced tyrosine phosphorylation of paxillin is correlated with decreased cell spreading. We have shown (Fig. 3B) that phosphorylation of Tyr-925 in FAK, which is the site for docking to paxillin, is reduced by polyamine depletion. Several FAK mutants, which are defective for binding to paxillin, are also unable to localize to focal adhesion (20). Thomas et al. (25) found that paxillin binding to FAK is essential for maximal phosphorylation of FAK and that FAK also phosphorylates paxillin. FAK is also believed to be targeted to focal adhesions via paxillin binding.

The kinase activity of FAK in DFMO-treated cells was significantly lower than control at 30 min and increased significantly by 120 min (Fig. 6). Immunoprecipitated paxillin from IEC-6 cells 10 and 30 min after attachment showed that the tyrosine phosphorylation of paxillin was greatly reduced at both time points in polyamine-depleted cells (Fig. 7). Thus in the absence of polyamines, reduced paxillin phosphorylation appears to contribute to the decreased recruitment of FAK to adhesion contacts.

Because paxillin and focal adhesions have been shown to be essential for cell spreading (17), we hypothesized that, based on our findings, polyamine-depleted cells should not spread as readily as normal cells. Results in Fig. 8 show that control cells began spreading almost immediately, and almost all had spread by 45 min. In contrast, polyamine-depleted cells spread slowly, and less than half had begun to spread by 45 min. These results, therefore, provide strong support for the significance of our observations that paxillin phosphorylation and FAK signaling effectiveness are compromised by polyamine depletion. These conclusions are reinforced strongly by the immunofluorescence data presented in Fig. 9 that show the decreased formation and altered distribution of focal adhesion complexes in polyamine-depleted cells. Cells grown in the presence of DFMO and putrescine showed increased colocalization of FAK and actin compared with DFMO-treated cells. Stress fibers were also absent from cells depleted of polyamines (Fig. 9), a finding that is a necessary result of the inability to form focal adhesions (7, 9, 13, 30).

In conclusion, we have shown for the first time that integrin signaling is decreased in polyamine-depleted cells. This was evidenced by decreased colocalization of alpha 2- and beta 1-integrins and subsequent decreases in the phosphorylation of FAK, FAK activity, and paxillin phosphorylation. We have also shown that the predicted consequences of these events, namely, that cell attachment, focal adhesion formation, actin stress fiber formation, and cell spreading are deficient in these cells. These findings may explain, in part, the inhibition of cell migration and proliferation that occurs in polyamine-deficient cells.


    ACKNOWLEDGEMENTS

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52784 and by the Thomas A. Gerwin Endowment.


    FOOTNOTES

Address for reprint requests and other correspondence: L. R. Johnson, Dept. of Physiology, Univ. of Tennessee Health Science Center, 894 Union Ave., Memphis, TN 38163 (E-mail: ljohn{at}physio1.utmem.edu).

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.

Received 16 November 2000; accepted in final form 16 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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