Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee 38163
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ABSTRACT |
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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--difluoromethylornithine; integrins; paxillin; focal adhesions; cell spreading; attachment; migration
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INTRODUCTION |
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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--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
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 - and
-subunits. Currently, 15
- and 8
-subunits have been described, and the combination of a particular
-subunit with a particular
-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.
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MATERIALS AND METHODS |
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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. [-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,
2-integrin,
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 [-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, 2-integrin,
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 2-integrin and
polyclonal anti-mouse
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.
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RESULTS |
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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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Immunohistochemical localization of integrins.
Figure 2A shows localization
of 2- and
1-integrins in the cells
attached to the Matrigel. Strong colocalization (yellow) of
2- and
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
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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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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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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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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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 2- and
1-integrins
revealed that although levels of
2- (Fig. 2A)
and
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
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 2- and
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.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52784 and by the Thomas A. Gerwin Endowment.
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FOOTNOTES |
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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.
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