Departments of Surgery, Yale University School of Medicine and Connecticut Veterans Affairs Health Care System, New Haven, Connecticut 06511
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ABSTRACT |
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The signals involved in restitution during mucosal healing are poorly understood. We compared focal adhesion kinase (FAK) and paxillin protein and phosphorylation, extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 activation, as well as FAK and paxillin organization in static and migrating human intestinal Caco-2 cells on matrix proteins and anionically derivatized polystyrene dishes (tissue culture plastic). We also studied effects of FAK, ERK, and p38 blockade in a monolayer-wounding model. Compared with static cells, cells migrating across matrix proteins matrix-dependently decreased membrane/cytoskeletal FAK and paxillin and cytosolic FAK. Tyrosine phosphorylated FAK and paxillin changed proportionately to FAK and paxillin protein. Conversely, cells migrating on plastic increased FAK and paxillin protein and phosphorylation. Migration matrix-dependently activated p38 and inactivated ERK1 and ERK2. Total p38, ERK1, and ERK2 did not change. Caco-2 motility was inhibited by transfection of FRNK (the COOH-terminal region of FAK) and PD-98059, a mitogen-activated protein kinase-ERK kinase inhibitor, but not by SB-203580, a p38 inhibitor, suggesting that FAK and ERK modulate Caco-2 migration. In contrast to adhesion-induced phosphorylation, matrix may regulate motile intestinal epithelial cells by altering amounts and distribution of focal adhesion plaque proteins available for phosphorylation as well as by p38 activation and ERK inactivation. Motility across plastic differs from migration across matrix.
focal adhesion kinase; mitogen-activated protein kinase; epithelium; intestine; migration; restitution; signal transduction
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INTRODUCTION |
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MUCOSAL INJURY AND REPAIR are common to gastrointestinal pathological conditions as diverse as peptic or infectious ulcer disease, inflammatory bowel disease, hypoperfusion injuries, surgical anastomosis, and stercoral ulcers. Repair of such epithelial injury involves epithelial cell migration and proliferation. Cell-extracellular matrix (ECM) interaction is crucial for multiple biological functions in the gut, including intestinal epithelial cell morphogenesis (42), migration, proliferation, and differentiation (4, 5). In particular, restitutive cell migration, which involves specialized cell-matrix interactions that generate physical forces to move the mucosal cells across the defect, is critical to the resurfacing of the wound. The matrix composition of gut mucosal wounds may vary in different pathological conditions. The wound may simply denude the epithelium and leave intact the basement membrane (composed chiefly of collagen IV and laminin) or strip the basement membrane, exposing interstitial matrix proteins such as collagen I, collagen III, and tissue fibronectin. Bleeding also deposits serum fibronectin and fibrinogen clot in the wound (2). Finally, scar formation after healing may result in collagen deposition (27). Despite the obvious importance of interactions between epithelial cells and matrix proteins during intestinal wound healing, the signal transduction events involved in this process remain poorly understood.
The effects of matrix on intestinal epithelial cell biology during cell migration may be mediated at least in part by intracellular signals related to integrins, the best-characterized cell receptors for matrix proteins (2). Although the mechanisms of integrin-mediated signal transduction are still not completely understood, tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK) and FAK-associated proteins such as paxillin and p130, may be critical for focal adhesion assembly and cell adhesion (8, 44) as well as cell spreading (38). Tyrosine phosphorylation of FAK and paxillin is thought to assist in ECM-stimulated cytoskeletal remodeling and stabilization of adhesion (4). The primary role of FAK was previously thought to facilitate focal adhesion complex formation on the basis of observations that activated FAK localizes at focal contacts. However, recent studies suggest that FAK may be more likely to regulate the turnover of focal adhesion complex proteins and cell motility. Indeed, activity of FAK correlates with endothelial cell motility (39), and inhibition of FAK results in decreased endothelial cell motility (15). FAK activity also correlates with keratinocyte migration in repairing burn wounds, suggesting that FAK-mediated motility may also be important in wound-healing processes (14). Our preliminary observations have indicated cytoskeletal FAK autophosphorylation and intracellular tyrosine kinase activation in migrating human intestinal epithelial (Caco-2) cells (28, 29).
Paxillin may be a substrate for FAK or FAK-associated kinases since
paxillin localizes to focal adhesion complexes and is tyrosine
phosphorylated concomitantly with FAK (50). Indeed, it had
been proposed that p125 FAK is localized to focal adhesions by direct
association with paxillin, but tyrosine phosphorylation of FAK and
paxillin are not necessary for this association (48). In
preliminary investigations, we have recently observed that 1-integrin receptor binding may stimulate Caco-2 cell
proliferation and differentiation via intracellular tyrosine
phosphorylation and paxillin translocation (41). Although
FAK and associated adapter proteins such as paxillin may indeed be
involved in cell motility, the manner in which this occurs remains
unclear. For instance, a recent study indicated that FAK and paxillin
phosphorylation mediates insulin-like growth factor (IGF)-I-stimulated
cell and growth cone motility in SH-SY5Y cells (24), but
another investigator reported insulin-stimulated
dephosphorylation of FAK and paxillin in Chinese hamster ovary
cells during cell motility (21).
FAK activation and autophosphorylation have also been proposed to play a role in activation of several downstream signaling pathways in cell adhesion (43). In particular, integrin binding to matrix proteins has been reported to stimulate mitogen-activated protein kinase (MAPK) activity (54), and recent observations have suggested that the extracellular signal-regulated kinase (ERK) and p38 MAPK pathways may be involved in intestinal epithelial wound-induced (12, 16) and gastric ulcer healing signal transduction (32).
Since intestinal epithelial cell motility appears to modulate and perhaps be modulated by these signal transduction pathways and since variations in the ECM across which restitutive motility occurs are critical to the pathobiology of mucosal wound healing, we therefore sought to compare FAK- and paxillin-signaling events and activation of the MAPK family members ERK1, ERK2, and p38 MAPK in static and migrating human Caco-2 intestinal epithelial cells cultured on collagen I, laminin, fibronectin, and tissue culture plastic. We chose to study these matrix proteins because collagen I represents the dominant collagen of the interstitial matrix beneath the basement membrane, laminin is the dominant noncollagenous constituent of the basement membrane itself, and serum fibronectin is a primary constituent of blood clots, which often form on bleeding mucosal lesions. Tissue culture plastic is polystyrene that has been treated by vacuum gas plasma to incorporate a variety of anionic functional groups, thus rendering it hydrophilic and providing a nonspecific surface to which cells may attach. This contrasts with bacteriological plastic polystyrene, which is more hydrophobic and generally shows markedly reduced cell attachment unless otherwise precoated, as with matrix proteins in this study. Indeed, Caco-2 cells do not adhere to bacteriological plastic at all unless otherwise precoated (unpublished observations). Uncoated tissue culture plastic, which is commonly used for mammalian cell culture, was used as an irrelevant control substrate here to contrast with the effects of specific matrix proteins. We generated homogenous populations of migrating and nonmigrating cells for biochemical analysis using differential density seeding, a technique previously described by Batt and Roberts in fibroblasts (6) and subsequently applied by us to Caco-2 cells (28, 29).
In additional studies, we further examined the potential function of FAK, MAPK, and p38 during cell motility. We performed migration assays after transfection with FRNK (the COOH-terminal region of FAK that competes with FAK for localization to focal contacts but does not have kinase activity), to inhibit FAK activity, and after treatment with the MAPK-ERK kinase (MEK) inhibitor PD-98059 and the p38 inhibitor SB-203580.
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MATERIALS AND METHODS |
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Cells and cell culture. Human Caco-2 intestinal epithelial cells were maintained using standard cell culture techniques in DMEM with 10% FBS, 10 µg/ml transferrin, 25 mmol/l glucose, 4 mmol/l glutamine, 1 mmol/l pyruvate, 15 mmol/l HEPES, 100 U/ml penicillin G, and 100 mg/ml streptomycin. Homogenous static and migrating cell populations for biochemical study were generated as previously described (28, 29). Briefly, Caco-2 cells were seeded at 13,000/cm2 and 750/cm2 into 35 × 10-mm- and 100 × 20-mm-style bacteriological culture dishes (Easy Grip petri dish), respectively, after these dishes had been precoated with purified matrix proteins (Sigma, St. Louis, MO) as previously described (4). To allow for the substantially lower adhesion observed on tissue culture plastic, we simultaneously seeded cells at 26,000/cm2 and 6,000/cm2 into 35 × 10-mm- and 100 × 20-mm-style tissue culture plastic without matrix precoating, respectively, on the basis of preliminary studies demonstrating equivalent levels of subconfluence in the matrix-precoated dishes and the uncoated tissue culture plastic at 4 days after seeding at these cell numbers. At the time point at which these two cell populations were then studied (4 days after seeding), cells cultured in 35 × 10-mm dishes are nearly confluent, but cells cultured in the 100 × 200-mm dishes are chiefly motile. For instance, in one typical series of studies, 89 ± 4.7% of the cells cultured on 100 × 20-mm dishes exhibited free edges and lamellipodial extension, and only 7.2 ± 0.7% of the cells cultured in 35 × 10-mm dishes exhibited these properties (P < 0.001).
Cell proliferation assays. For assays of cell proliferation in the low- and high-density cell populations, cells were seeded in parallel into 16 dishes each onto collagen I as described above. Cell number was assessed 24, 48, 72, and 96 h after initial seeding by trypsinizing to a single cell suspension and counting with an automated cell counter (Coulter Counter ZM; Coulter).
Brush border enzyme assays. Alkaline phosphatase and dipeptidyl dipeptidase specific activities were assayed as described previously (3, 17). Briefly, after bicinchoninic acid (BCA) protein assay (52), protein-matched aliquots of cell lysates were assayed for alkaline phosphatase and dipeptidyl dipeptidase by digestion of specific synthetic substrates. Alkaline phosphatase was assayed by p-nitrophenyl phosphate disodium digestion, which was measured spectrophotometrically at 410 nm. Dipeptidyl peptidase was assessed by Ala-p-nitroanilide digestion and quantitated at 380 nm.
Cell collection and fractionation.
For study of FAK and paxillin, both static and migrating cells were
washed twice with ice-cold PBS and scraped into 400 µl of lysis
buffer containing (in mmol/l) 25 Tris · HCl (pH 7.6), 5 EGTA,
0.7 CaCl2, 1 phenylmethylsulfonyl fluoride (PMSF), 0.1 sodium phospho-tosyl-1-lysine chloromethyl ketone (TLCK), 1 sodium vanadate, and 1 dithiothreitol, with 10 µmol/l leupeptin. The whole cell lysates were fractionated as described by Bissonnette et al.
(7) for Caco-2 cell subcellular fractionation and
subsequently also used by our group for this purpose (17,
28, 29). Briefly, after sonication with a
sonic Dismembrator 60 (Fisher Scientific, Fair Lawn, NJ) at
setting 10 for 10 s, the lysates were centrifuged at
13,000 rpm for 10 min at 4°C. The supernatant corresponded to the
cytosolic fraction. The pellet was resuspended in lysis buffer
supplemented with 0.3% Triton X-100 and then vortexed at setting
10 for 30 s and centrifuged at 13,000 rpm for 10 min at 4°C
to generate the membrane/cytoskeletal fraction. Substantial enrichment
of the cytosolic and membrane/cytoskeletal fractions was confirmed by
partitioning of the cytosolic protein heat shock protein 70 (HSP70)
into the cytosolically enriched fraction (33, 36) and of surface biotinylated 1-integrin
subunit into the membrane/cytoskeletally enriched fraction. For
instance, densitometric analysis of four separate fractionations from
four separate cell lysates indicated that HSP70 recovery from the
cytosolic fraction exceeds that from the membrane/cytoskeletal fraction
by >9.5-fold (n = 4, P < 0.0001).
Similar analysis demonstrated that biotinylated
1-integrin subunit protein was recovered from the
membrane/cytoskeletal fraction to a degree more than ninefold in excess
of that recovered from the cytosolic fraction (n = 4, P < 0.01, data not shown).
Immunoprecipitation of FAK and paxillin. Protein samples (400 µg for FAK and 200 µg for paxillin) of cell lysates were preincubated for 1 h at 4°C with second antibody (rabbit anti-mouse IgG; Sigma) and protein A Sepharose CL4B (1:1 slurry in PBS; Pharmacia, Piscataway, NJ) to remove protein in the cell lysates that nonspecifically bind to the secondary antibody and protein A Sepharose. Samples were then centrifuged at 5,000 rpm for 2 min at 4°C. Supernatants were saved and incubated with monoclonal antibody to FAK and paxillin (Transduction Laboratories, Lexington, KY) overnight. Rabbit anti-mouse IgG and protein A Sepharose CL4B were then added to each sample to collect FAK and paxillin protein. After incubating for another 2 h at 4°C, samples were centrifuged at 5,000 rpm for 2 min at 4°C and rinsed 3 times with 0.5 ml of immunoprecipitation buffer before resolution on 3.5% stacking/7.5% separating SDS-polyacrylamide gels. Gels were transferred overnight at 4°C, 0.1 A onto Hybond enhanced chemiluminescence (ECL) nitrocellulose (Amersham, Arlington Heights, IL) before immunodetection. To validate our immunoprecipitation techniques, we also performed a second immunoprecipitation from the remaining lysate to determine whether FAK or phosphorylated FAK remained in the cell lysates after the first immunoprecipitation. However, we did not find any detectable FAK in these reimmunoprecipitates (data not shown).
Immunodetection of phosphotyrosine and FAK/paxillin protein.
For immunodetection of phosphotyrosine, blots were blocked for 1 h
at room temperature with 5% nonfat dry milk in wash buffer (TBS-T)
containing 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20. After
five additional rinses with TBS-T, blots were incubated with a
horseradish peroxidase-conjugated phosphotyrosine antibody (RC20,
Transduction Laboratories) in TBS-T at 1:2,500 dilution for 1 h at
room temperature. After rinsing with TBS-T, blots were detected via the
ECL method and exposed to Hyperfilm ECL (Amersham). For FAK and
paxillin protein itself, blots immunodetected for phosphotyrosine were
stripped in 100 mM Tris·HCl, 0.003% SDS, and 2 mM
-mercaptoethanol at 60°C for 30 min with occasional agitation.
Blots were washed for 2 × 10 min at room temperature using large
volumes of TBS-T. Blots were reblocked with 5% nonfat dry milk at room
temperature for 1 h. After five washes with TBS-T, blots were
incubated with monoclonal anti-FAK or anti-paxillin (Transduction
Laboratories) at 1: 1,000 dilution in TBS-T for 1 h at room
temperature. After five additional rinses with TBS-T, blots were
incubated with horseradish peroxidase-conjugated secondary antibody at
1:5,000 dilution in TBS-T for 30 min at room temperature. Blots were
visualized by the ECL system.
Western blots for detecting MAPK and p38. Twenty micrograms of each cell lysate were separated by discontinuous 10% SDS-PAGE, transferred to nitrocellulose membranes, and probed for phosphorylated ERK1, ERK2, and p38 with a monoclonal phospho-p44/42 MAPK antibody and polyclonal phospho-p38 MAPK antibody (New England Biolabs, Beverly, MA) according to the manufacturer's protocol. For total MAPK, the same blots immunodetected for activated MAPK were stripped and reprobed with total MAPK antibody (New England Biolabs).
Densitometry. All blot results were quantitated densitometrically using a Microtek ILXE ScanMaker and an IBM-based densitometric software package (SigmaScan/Image, Jandel Scientific). Multiple exposures were taken of each blot. After densitometric analysis of blots exposed within the linear range of the film and the scanner for phosphorylated and total protein in each experiment, we normalized all data from each gel to static cells grown on tissue culture plastic to control for exposure differences. The ratio of phosphorylated FAK to total FAK was then calculated as the ratio of the normalized densitometric quantitation of the phosphorylated FAK band to the normalized densitometric quantitation of the total protein FAK band. Similar calculations were performed for paxillin.
Immunofluorescent staining. Caco-2 cells were similarly seeded at 13,000/cm2 and 750/cm2 into 4-well cell culture slides (Becton Dickinson, Bedford, MA) coated with matrix proteins to produce confluent and nonconfluent cells characterized by lamellipodial extensions. After washing three times with PBS at room temperature, the cells were fixed with 0.01 M sodium periodate, 0.75 M lysine, 0.0375 M sodium phosphate buffer, pH 7.4, and 2% paraformaldehyde on ice for 30 min. Cells were washed once with PBS and then permeabilized with 0.2% Triton X-100 on ice for 20 min before six additional PBS washes. After incubating with 3% BSA and 0.05% NaN3 in PBS for 2 h to quench nonspecific reactive sites, cells were incubated with primary antibody diluted in 1% BSA in PBS for 2 h. Cells were then washed with 1% BSA and then incubated with Texas red-conjugated secondary sheep anti-mouse IgG (Amersham) for 2 h. After washing three times with 1% BSA and once with PBS, cells were covered with Crystal/Mount media (Biomeda, Foster, CA). FAK and paxillin staining patterns were visualized by fluorescent microscopy and photographed (Zeiss).
Caco-2 migration assays. For PD-98053 and SB-203580 studies, Caco-2 cells were seeded into 30 × 35-mm tissue culture dishes precoated with collagen I. After cells reached confluence, a linear wound was created in the cell monolayer by manual scratching with a pipette tip as described by Sieg et al. (45). After washing once with PBS, cells were incubated in normal Caco-2 medium containing DMSO as a control or medium containing 5 µM and 10 µM PD-98059 and SB-203580, respectively. Hydroxyurea (30 mM) was added to all cell culture media during these motility assays to prevent Caco-2 proliferation (51). Cells that had moved across the wound edge into the defect were counted at 24 and 48 h after treatments in at least 40 high-power fields (40× magnification). In further studies, FRNK epitope-tagged with a triple COOH-terminal hemagglutinin (HA) and HA-tagged wild-type FAK were transfected into Caco-2 cells when cells reached 30-50% confluence using the Lipofectamine Plus system according to the manufacturer's protocol using 1 µg/dish of DNA (GIBCO Life Technologies, Gaithersburg, MD). These constructs were a generous gift from Dr. David D. Schlaepfer. Two days later all monolayers were confluent, and the monolayers were wounded as described above. Immunofluorescent staining with primary monoclonal anti-HA (Upstate) and Texas red-conjugated secondary sheep anti-mouse IgG (Amersham) was performed 48 h after wounding. HA-staining and nonstaining cells per field (40× magnification) were counted in both the wound area and the confluent area behind the wound.
Statistical analysis. Unless specified, data from at least five similar blots were subjected to densitometric analysis as described and normalized to respective control values. Results are expressed as means ± SE. Data were expressed as a percentage of that seen in static cells grown on tissue culture plastic in each figure. Differences between migrating cells and static cells were analyzed using paired t-tests, and data comparing cells on various matrix proteins were analyzed using one-way ANOVA, with P < 0.05 being set a priori as the threshold for statistical significance.
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RESULTS |
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Caco-2 proliferation and differentiation in this model.
Because Caco-2 cell proliferation and differentiation might also
modulate intracellular signaling, we first assessed cell proliferation
and the expression of two commonly used markers of Caco-2
differentiation in the low-density and high-density cell populations.
Studies of cell proliferation demonstrated equivalent increases in cell
number in the two cell populations over the 4 days of study
(n = 4, P = not significant at each
time point; Fig. 1A).
Similarly, assays of the specific activity of alkaline phosphatase and
dipeptidyl dipeptidase at 2 and 4 days after seeding onto collagen I
demonstrated no significant differences in the specific activity of
either marker between the two cell populations, although each
population exhibited increased expression of each marker at day 4 compared with day 2 (n = 4, P < 0.05 for comparisons between days 2 and
4, P = not significant for comparisons
between the two cell populations; Fig. 1B)
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Basal FAK and tyrosine-phosphorylated FAK in static Caco-2 cells.
Our initial analysis of the data demonstrated differences in basal FAK
protein in static Caco-2 cells cultured on the four substrates studied.
Static cells cultured on collagen I exhibited the highest basal FAK in
both the membrane/cytoskeletal and cytosolic fractions. Compared with
tissue culture plastic, basal membrane/cytoskeletal FAK was increased
by 31.6 ± 14.1% (n = 5, P < 0.05) in cells grown on collagen I. No significant differences were
found among tissue culture plastic, laminin, and fibronectin (Fig.
2A). Similarly, cytosolic
basal FAK was increased by 43.9 ± 11.2% (n = 5, P < 0.05) in cells cultured on collagen I and
29.7 ± 6.9% (n = 5, P < 0.05)
in cells cultured on laminin compared with cells cultured on tissue
culture plastic (Fig. 3A). The
apparent tendency of static cells cultured on each matrix to exhibit
more phosphorylated FAK than cells cultured on tissue culture plastic
did not achieve statistical significance for either
membrane/cytoskeletal or cytosolic pools of phosphorylated FAK (data
not shown).
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Membrane/cytoskeletal pools of FAK protein and tyrosine-phosphorylated FAK in migrating cells. In addition to matrix-dependent alterations in FAK protein in static Caco-2 cells, we also observed further matrix-dependent modulation of FAK protein and phosphorylation during Caco-2 motility. Compared with static cells on the same substrates, both membrane/cytoskeletal FAK and phosphorylation were decreased in cells migrating across each matrix protein, with the greatest effects on collagen I and fibronectin and a lesser effect on laminin. However, cells migrating across tissue culture plastic exhibited the reverse effect (Fig. 2).
Membrane/cytoskeletal FAK protein was substantially decreased in migrating cells compared with static cells on each matrix studied. For instance, on collagen I, membrane/cytoskeletal FAK protein was 70.2 ± 12.1% of similar FAK protein levels in static cells on plastic vs. 131.4 ± 14.2% for static cells (n = 5, P < 0.001 migrating cells vs. static cells). Membrane/cytoskeletal FAK was similarly decreased in motile cells on laminin (71.3 ± 17.4% of static plastic control levels vs. 95.7 ± 17.8% of static cells, P < 0.05 migrating cells vs. static cells) and fibronectin (36.9 ± 7.0% of static plastic control levels vs. 95.6 ± 8.7% of static cells, P < 0.05 migrating cells vs. static cells) compared with static cells. The relatively more substantial decrease in FAK during motility across collagen I or fibronectin was statistically different from the lesser decrease during motility across laminin (P < 0.05). In contrast to the observed decrease in membrane/cytoskeletal FAK protein in cells migrating on matrix proteins, membrane/cytoskeletal FAK protein actually increased to 150.5 ± 11.5% of similar levels in static cells on plastic (n = 5, P < 0.05) (Fig. 2A). Analysis of intracellular pools of phosphorylated FAK yielded results similar to those observed in studies of FAK protein. For example, on collagen I, phosphorylated membrane/cytoskeletal FAK was 64.5 ± 5.7% of similar FAK protein levels in static cells on plastic vs. 118.6 ± 16.4% for static cells (n = 5, P < 0.001, migrating cells vs. static cells). Membrane/cytoskeletal FAK phosphorylation was similarly decreased in motile cells on laminin (48.5 ± 9.4% of static plastic control levels vs. 108.5 ± 10.3% of static cells, P < 0.05 migrating cells vs. static cells) and fibronectin (53.0 ± 12.7% of static plastic control levels vs. 122.1 ± 18.7% of static cells, P < 0.05 migrating cells vs. static cells) compared with static cells.Cytosolic pools of FAK protein and tyrosine-phosphorylated FAK in migrating cells. Studies of cytosolic pools of FAK protein and tyrosine-phosphorylated protein demonstrated effects of cell motility similar to those observed in the membrane/cytoskeletal fraction (Fig. 3). For instance, on collagen I, cytosolic FAK protein was 86.4 ± 15.6% of similar FAK protein levels in static cells on plastic vs. 143.9 ± 15.1% for static cells (n = 5, P < 0.001 migrating cells vs. static cells). Cytosolic FAK was similarly decreased in motile cells on laminin (87.4 ± 15.2.4% of static plastic control levels vs. 129.3 ± 13.7% of static cells, P < 0.05 migrating cells vs. static cells) and fibronectin (63.2 ± 15.1% of static plastic control levels vs. 103.8 ± 11.2% of static cells, P < 0.05 migrating cells vs. static cells) compared with static cells. However, cells migrating across tissue culture plastic again exhibited increased cytosolic FAK (26.4 ± 8.7%) compared with static cells on tissue culture plastic. The apparent difference between cytosolic FAK on collagen I and cytosolic FAK on fibronectin did not achieve statistical significance. However, the difference between the effects of motility on cytosolic FAK protein on each matrix was significantly different from the effects of motility across tissue culture plastic on cytosolic FAK protein (n = 5, P < 0.01). Cytosolic tyrosine-phosphorylated FAK also decreased significantly in migrating cells compared with static cells on the same matrix protein. On collagen I, cytosolic FAK phosphorylation was 79.1 ± 11.9% of similar FAK phosphorylation level of static cells on plastic vs. 127.3 ± 15.0% for static cells (n = 5, P < 0.001, migrating cells vs. static cells). Cytosolic FAK phosphorylation was similarly decreased in motile cells on laminin (58.6 ± 13.1% of static plastic control levels vs. 128.1 ± 19.3% of static cells, P < 0.05 migrating cells vs. static cells) and fibronectin (61.4 ± 4.4% of static plastic control levels vs. 129.1 ± 13.3% of static cells, P < 0.05 migrating cells vs. static cells) compared with static cells. Phosphorylated FAK in cells migrating across tissue culture plastic were substantially increased in both the membrane/cytoskeletal and cytosolic fractions (54.7 ± 8.6% and 45.8 ± 12.4%, respectively, n = 5, P < 0.05) compared with static cells (Figs. 2B and 3B).
Ratio of tyrosine-phosphorylated FAK to total FAK protein. Since activated FAK phosphorylates itself, the ratio of tyrosine-phosphorylated FAK to total FAK protein may correlate with the proportion of FAK that is activated (28, 31). The ratio of membrane/cytoskeletal phosphorylated FAK to total FAK increased 56.6 ± 15.8% (n = 5, P < 0.05) in cells migrating on collagen I and 39.2 ± 13.6% (n = 5, P < 0.05) in cells migrating on fibronectin compared with static cells on the same matrix protein. However, cells migrating on laminin and tissue culture plastic did not exhibit significant alterations in the ratio of phosphorylated FAK to total FAK. The ratio of tyrosine-phosphorylated FAK to FAK protein in the cytosolic fraction did not differ significantly between migrating and static cells for any substrate studied (data not shown). The ratio of phosphorylated paxillin to total paxillin did not differ between migrating cells and static cells (data not shown).
Paxillin and tyrosine-phosphorylated paxillin.
Parallel studies of total paxillin protein and tyrosine-phosphorylated
paxillin demonstrated that both paxillin protein and tyrosine-phosphorylated paxillin decreased in both fractions in migrating cells grown on matrix proteins compared with static cells,
whereas cells cultured on tissue culture plastic exhibited the opposite
results (Fig. 4). Basal paxillin protein
and phosphorylation did not appear substantially different among matrix
proteins or tissue culture plastic (Fig. 4). Compared with static
cells, membrane/cytoskeletal paxillin protein was significantly
decreased in cells migrating on the same matrix protein studied. For
instance, on collagen I, membrane/cytoskeletal paxillin protein was
53.8 ± 13.8% of similar paxillin protein level of static cells
on plastic vs. 108 ± 13.3% for static cells (n = 5, P < 0.001 migrating cells vs. static cells).
Similarly, membrane/cytoskeletal paxillin protein was also decreased in
motile cells on laminin (57.9 ± 8.1% of static plastic control
levels vs. 117.8 ± 10.3% of static cells, P < 0.05 migrating cells vs. static cells) and fibronectin (73.9 ± 7.8% of static plastic control levels vs. 104.8 ± 5.3% of
static cells, P < 0.05 migrating cells vs. static
cells) compared with static cells (Fig. 4A). Like paxillin
itself, membrane/cytoskeletal paxillin phosphorylation also decreased
significantly in cells migrating on collagen I, on laminin, and on
fibronectin compared with static cells on the same matrix protein (Fig.
4B). Thus motility-associated alterations in
membrane/cytoskeletal pools of total paxillin and tyrosine-phosphorylated paxillin varied in a matrix-dependent manner
similar to FAK. The greatest decrease was seen in cells migrating on
laminin and the least on fibronectin (P < 0.05). In
contrast, as for FAK, paxillin and tyrosine-phosphorylated paxillin in
cells migrating on tissue culture plastic appeared to increase by
20.4 ± 6.2% and 24.6 ± 5.1% compared with static cells
(n = 5, P < 0.05). Cytosolic pools of
paxillin protein and tyrosine-phosphorylated paxillin did not change
substantially with cell motility (data not shown).
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Subcellular localization of FAK and paxillin during Caco-2
motility.
To investigate the organization of FAK and paxillin during Caco-2 cell
motility, the distribution of these two proteins in migrating Caco-2
cells was examined by immunofluorescent staining. FAK organization in
motile and confluent cells on collagen I and tissue culture plastic is
shown in Fig. 5. These images all
represent the same magnification, but confluent cells appear much
smaller because of decreased cell spreading and lack of lamellipodial extension (2). In confluent cells cultured on collagen I
(Fig. 5B), FAK protein was expressed in a perinuclear
endoplasmic reticulum (ER)-Golgi pattern as well in a dense punctate
pattern along areas of cell-cell contact (Fig. 5B). In
motile cells on collagen I, the cell borders contacting other cells
stained positively for FAK protein, but this staining appeared to
decrease gradually toward the free edge and was virtually absent from
migrating lamellipodia. (Fig. 5A). The
organization of FAK in cells cultured on fibronectin and laminin was
similar to that on collagen I (data not shown). Compared with cells
migrating across matrix proteins, motile cells on tissue culture
plastic (Fig. 5C) appeared to exhibit stronger and more
distinct FAK organization. Punctate FAK staining was more obvious at
cell borders involved in cell-cell contact in motile cells on plastic
than in motile cells on collagen I. As on collagen I, FAK staining
within migrating lamellipodia was substantially decreased compared with
other areas of the cells, but faint FAK staining could be seen even in
the migrating lamellipodia in cells on plastic (Fig.
5C). Confluent cells on tissue culture plastic
appeared to exhibit less distinct or organized FAK staining than
confluent cells on collagen I or motile cells on plastic, although some
punctate staining at areas of cell-cell contact could still be
discerned (Fig. 5D).
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p38 MAPK activation.
To elucidate the activation of p38 MAPK during Caco-2 cell motility on
matrix proteins and tissue culture plastic, both total p38 protein and
phosphorylated (active) p38 were investigated in both migrating and
static cells. In contrast to our observations with FAK and paxillin,
Western blotting for total p38 protein did not demonstrate substantial
differences between migrating and static cells or among matrix
substrates (n = 6, P > 0.05; Fig.
7). In contrast, blotting with antibody
specific for phosphorylated p38 revealed that basal amounts of
phosphorylated p38 in static cells cultured on plastic were
40-50% higher than that observed in static cells cultured on
matrix proteins (n = 6, P < 0.05 plastic vs. matrix proteins; Fig. 7).
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ERK MAPK activation.
Parallel studies of ERK1 and ERK2 also demonstrated no differences
between static and migrating Caco-2 ERK1 or ERK2 protein or among the
substrates studied (n = 6, P > 0.05, data not shown). However, phosphorylated ERK1 and ERK2 appeared to
decrease in migrating cells compared with nonmigrating cells on each
matrix studied (Fig. 8). For instance, in
migrating cells on collagen I, phosphorylated ERK1 was 55.2 ± 10.6% of similar ERK1 phosphorylation levels in static cells on
plastic, whereas in static cells on collagen I, ERK1 phosphorylation
was 113.3 ± 25.8% of ERK1 phosphorylation observed in static
cells on plastic (n = 5, P < 0.001 migrating cells vs. static cells). Phosphorylated ERK1 was similarly
decreased in motile cells on laminin (65.2 ± 16.5% of static
plastic control levels) compared with static cells on laminin
(91.3 ± 11.5% of static cells on plastic, n = 6, P < 0.05 migrating cells vs. static cells). Similar
results were obtained on fibronectin (61.9 ± 12.5% of static
plastic control levels for motile cells on fibronectin vs. 119.1 ± 15.2% of static cells, P < 0.05 migrating cells
vs. static cells) compared with static cells. Similar to ERK1,
phosphorylated ERK2 was 59.1 ± 9.7% of similar ERK2
phosphorylation level of static cells on plastic vs. 129.1 ± 16.6% for static cells (n = 5, P < 0.001 migrating cells vs. static cells). Also, phosphorylated ERK2 was
similarly decreased in motile cells on laminin (54.6 ± 7.6% of
static plastic control levels vs. 100.9 ± 8.1% of static cells,
P < 0.05 migrating cells vs. static cells) and
fibronectin (61.2 ± 7.8% of static plastic control levels for
motile cells on fibronectin vs. 113.1 ± 10.4% of static plastic
cells for static cells on fibronectin, P < 0.05 migrating cells vs. static cells; Fig. 8).
|
FRNK inhibits FAK-mediated Caco-2 migration.
Transient transfection of FRNK into Caco-2 cells appeared to potently
inhibit Caco-2 migration in a monolayer cell wounding model. We
performed transient transfections of HA-tagged FRNK and HA-tagged
wild-type FAK into Caco-2 monolayers 48 h before wounding, stained
for HA 48 h later, and used cells not staining for HA (thus
presumably not expressing either HA-FRNK or HA-FAK) as internal
controls. The ratio of HA-staining to unstained cells in the areas of
the monolayers distant from the wounds was ~18-25% in different
experiments and did not vary significantly between FAK transfectants
and FRNK transfectants (Fig. 9). The
ratio of HA-staining cells to unstained cells among cells migrating
into the monolayer wound in cells transfected with HA-tagged wild-type FAK was also not different from staining ratios behind the wounds (Fig.
9). However, the proportion of migrating cells in the FRNK-transfected dishes that stained for HA-FRNK was less than half that observed in the
same cell monolayers distant from the wounds (9.1 ± 1.3% vs.
20.0 ± 2.9% n = 4, P < 0.002;
Fig. 9). Similar results were obtained in parallel studies performed
24 h after wounding (data not shown).
|
Effect of MEK and p38 inhibitors on Caco-2 migration.
PD-98059 inhibited Caco-2 migration in a dose-dependent manner.
Compared with a DMSO vehicle control group, PD-98059 decreased the
number of cells migrating into the wound defect at 48 h after wounding by 32.1 ± 7.6% and 74.8 ± 11.3% at 5 µM and 10 µM, respectively. SB-203580 (5-10 µM) did not appear to
influence Caco-2 migration (Fig. 10).
Similar results were obtained in parallel studies performed 24 h
after wounding (data not shown).
|
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DISCUSSION |
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Gut mucosal wound healing requires an exquisite balance of proliferation, differentiation, and migration likely to have an equally complex regulation. Human Caco-2 intestinal epithelial cells represent an excellent in vitro model of these processes (4, 5, 19) and have a similar integrin repertoire to normal intestinal mucosa (Refs. 2 and 22 and unpublished observations). We believe that the observations we have made reflect differences between migrating and nonmigrating cells rather than epiphenomena of differences in cell proliferation because proliferation rates are equivalent in the two cell populations that we studied (Caco-2 cells at near confluence do not display contact inhibition). Furthermore, blocking cell proliferation with 30 mM hydroxyurea did not alter the differences between static and motile cells in FAK, ERK1, ERK2, or p38 signaling in these cell preparations (C. F. Yu and M. D. Basson, unpublished data).
Caco-2 differentiation might also alter intracellular signaling. Since Caco-2 cells differentiate over time after seeding, we also designed our model to study cells at two different levels of subconfluence that had been seeded simultaneously. Although confluence initiates a substantial increase in Caco-2 differentiation, the nonmotile cell population had essentially just reached 92% confluence at the time of cell harvest. Thus minimal differences in differentiation between the two cell populations would be expected to occur. Levy et al. (25) have previously demonstrated that FAK phosphorylation decreases in differentiated 2-wk postconfluent Caco-2 cells, which differs from our observations of increased FAK phosphorylation in static Caco-2 cells. Furthermore, although it is always possible that other differentiation markers might be induced at different rates, we observed equivalent expression of alkaline phosphatase and dipeptidyl dipeptidase, common markers of Caco-2 cell differentiation, in these cell preparations at 2 and 4 days after seeding. In addition, preliminary studies of the effects of inducing Caco-2 differentiation with 10 mM sodium butyrate suggest that butyrate decreases FAK expression at the protein level but increases p38 phosphorylation in confluent cells on collagen I, each the opposite effect of what would be expected if our present results reflected the effects of increased cell differentiation in our static cell population (C. F. Yu and M. D. Basson, unpublished data).
FAK activation and the tyrosine phosphorylation of associated proteins
such as paxillin are among the earliest events detected in response to
cell adhesion and integrin binding (10). Activation of the
MAPK family (including ERK1, ERK2, and p38) also occurs in response to
integrin binding to matrix proteins, although it is not clear that FAK
activation is required for MAPK activation in all cell types
(26, 43). Our own preliminary observations suggest that adhesion induces similar signals in Caco-2 cells (40). The effects of motility on cell signaling are less
well characterized than the effects of adhesion, but our present data suggest that human Caco-2 intestinal epithelial motility is associated with substantial modulation of these pathways in a matrix-specific manner. We observed decreases in both total protein pools and phosphorylated protein of the integrin-associated tyrosine kinase FAK
and the adapter protein paxillin, as well as decreased ERK1 and ERK2
activation and increased p38 activation. The alterations in FAK and
paxillin phosphorylation reflected both substantial changes in total
FAK and paxillin protein available to be phosphorylated and (for some
matrix substrates) the proportion of FAK and paxillin actually
phosphorylated. In general, our observations of motility-associated decreases in phosphorylated FAK and paxillin appeared to mirror similar
changes in FAK and paxillin protein, suggesting that regulation of FAK
and paxillin protein synthesis or degradation may play an important
role in regulating intestinal epithelial signal transduction during
cell motility. However, motility also significantly increased the
proportion of FAK that was phosphorylated (calculated as the ratio of
phosphorylated FAK to total FAK) from within the total FAK
intracellular pool, suggesting that FAK may be regulated during cell
motility across matrix proteins at both the protein level and the level
of FAK phosphorylation and activation. In contrast, neither matrix nor
motility altered ERK1, ERK2, and p38 total protein, but motility
altered activation of each signal transduction element. Polk et al.
(35) have previously reported that only confluent
nonmotile IEC-6 cells (compared with motile nonconfluent IEC-6 cells)
display activation of another intracellular signaling pathway
[epidermal growth factor (EGF)-responsive phospholipase C-1
activation].
These effects all occurred in a matrix-dependent manner. In particular, motility on collagen I appeared most potent in influencing FAK and paxillin signals, whereas MAPK signaling seems more sensitive to fibronectin than to collagen I or laminin. The basement membrane protein laminin tended to be intermediate between collagen I and fibronectin. In parallel experiments, motility-associated alterations in FAK, ERK1, ERK2, and p38 were similar on tissue fibronectin to those that we describe here on serum fibronectin (data not shown). Although the motile state was characterized by relative activation of available FAK pools on both collagen I and fibronectin, no FAK activation (assessed by the ratio of phosphorylated to total FAK) occurred on laminin, a constituent of the basement membrane on which intestinal epithelial cells rest in the absence of mucosal injury.
Several mechanisms could contribute to the matrix specificity we observed. These include differing amounts or matrix specificities of the available matrix receptors (13), matrix-specific reorganization of the available receptors during motility (4), differing intracellular signal transduction activity of the receptors that are available, and matrix-dependent cross talk with signals from growth factor receptors (53). In addition to integrin receptors, such nonintegrin receptors as the 67-kDa laminin receptor (30), nonintegrin laminin receptors of ~30-50 kDa (46), and matrix metalloproteinases (11) could also modulate intestinal epithelial cell adhesion to and motility across matrix proteins and could also contribute to signal transduction during motility. Interestingly, among the matrix substrates studied, FAK membrane/cytoskeletal and cytosolic protein and paxillin membrane/cytoskeletal protein changes with motility tended to be most pronounced on collagen I, which is the most rapid substrate for Caco-2 migration across these different substrates (4) and Caco-2 proliferation during static culture on these substrates (5). Signal transduction events associated with motility across tissue culture plastic differed radically from signals associated with motility across matrix proteins.
Concomitant with the observed decrease in FAK protein and phosphorylation in motile cells on matrix proteins, FAK staining disappeared in the lamellipodial edge of cells migrating on matrix proteins, consistent with the hypothesis that focal contact turnover may play a role in cell migration. In contrast, although immunoprecipitation studies suggested decreased paxillin in migrating cells on matrix proteins and immunofluorescent staining appeared to suggest decreased paxillin staining in the ER-Golgi distribution in these cells, paxillin staining actually appeared most intense at the lamellipodial edges of cells migrating on matrix proteins. The function of this paxillin in the absence of colocalizing FAK awaits further study. A recent study has shown that paxillin binding to FAK is not required for cell migration (45). However, these data suggest the possibility that redistribution of paxillin during intestinal epithelial cell motility may complement changes in total paxillin protein in determining the role of paxillin signaling during intestinal epithelial cell motility. Previous studies of migrating SH-SY5Y neural cells (24) demonstrated a similar pattern of lamellipodial paxillin staining oriented in the direction of lamellipodial extension but also documented FAK staining in the lamellipodia. However, these investigators studied IGF-I-stimulated lamellipodial extension and worked only on a poly-L-lysine/laminin dried matrix, so differences in cell type, matrix substrate, and growth factor stimulation may account for the differences in FAK organization results between that study and the present work.
The present data extend previous preliminary observations (28, 29) but differ from a previous report by Levy et al. (25) that overconfluent Caco-2 cells 2 wk after seeding exhibit increased FAK protein and decreased FAK phosphorylation. However, Caco-2 cells differentiate spontaneously over time (25), so these previous observations (as Levy suggested) are likely to reflect Caco-2 differentiation rather than the effects of motility on the Caco-2 cells. Tyrosine-phosphorylated FAK and paxillin are also greater in confluent (presumably nonmotile) BALB/3T3 fibroblasts on tissue culture plastic (6). This disparity could reflect differences between fibroblasts and epithelial cells or differences in the model studied. For instance, the fibroblasts were studied 2.5 h after seeding, when signaling related to initial cell adhesion and spreading might be expected to dominate intracellular signal transduction. We evaluated migrating and confluent cells 4 days after seeding.
We also found that the MAPK family also appears to be influenced by motility in a matrix-dependent manner. The MAPK family, including the ERKs, c-Jun amino terminal kinase, and p38, modulate diverse aspects of cell biology, including proliferation, differentiation, and adaptation to external stress (9). Because of the pleiotropic function of these kinases, their activation must be strictly regulated. Unlike FAK and paxillin, the MAPK were primarily modulated by activation rather than total protein during Caco-2 motility. Compared with static cells of the same age after passage, p38 activation was increased but ERK1 and ERK2 activation were decreased in motile cells.
The p38 activation during Caco-2 motility is consistent with previous comparisons of confluent and nonconfluent IEC-6 cells (12) as well as the responsiveness of p38 activation to ultraviolet irradiation in confluent and nonconfluent 3T3 and HaCa T cells (24). For ERK1 and ERK2, however, the present results differ from previous comparisons of MAPK activation in undifferentiated subconfluent Caco-2 cells and postconfluent differentiated Caco-2 (25) and IEC-6 (12, 16) cells. In our study, ERK1 and ERK2 were less active in motile cells than static confluent cells, whereas these previous comparisons of nonconfluent and postconfluent intestinal epithelial cell lines demonstrated increased activity of ERK1 and ERK2 in nonconfluent cells compared with confluent or overconfluent and differentiated cells. The differences between the previous Caco-2 results (25) and our own may reflect the differences between allowing Caco-2 cells to become confluent and differentiate over time, as Levy et al. (25) did and comparing static and motile Caco-2 cells of the same age after passage. The previous IEC-6 cell studies (12, 16) focused on transient ERK1 and ERK2 activation within 5 min that returned to baseline by 15 min, so that comparison between these transient effects on ERK activation and the chronic 4-day model studied here is difficult.
Pai et al. (32) reported increased ERK1 and ERK2
activities in vivo in epithelium at the margin of experimental gastric ulcers during healing. Although these results also appear to contrast directly with our in vitro observations, the differences may well be
explained by the manifest differences between our isolated cell culture
model, which addresses only the direct effects of cell spreading and
motility, and the complexities of a healing wound in vivo. For
instance, growth factors such as EGF and transforming growth factor-
are increased during mucosal injury and repair in vivo (4)
and also activate ERK1 and ERK2 in vitro (12). Since such
growth factors activate FAK (24) and MAPK
(1), cross talk at multiple levels is highly likely
between matrix adhesion receptors, growth factor receptors, and
mechanicochemical signals. In addition, the matrix of a healing wound
may differ from that in static uninjured mucosa, and such variations in
the matrix environment may themselves regulate intestinal epithelial biology. Not only do different matrix proteins directly influence cell
motility, differentiation, and proliferation (3), but these matrix proteins influence growth factor receptor expression in
intestinal epithelial cells, adding another layer of cross talk. For
example, laminin may inhibit EGF- and IGF-I-stimulated proliferation
through downregulation of the expression of the receptors for these
mitogens, whereas collagen I may stimulate enterocyte proliferation by
promoting EGF and IGF-I receptor expression (30). The pH
(34) and O2 tension (47) are also
altered in vivo in the healing wound. Thus the present study of the
effects of cell motility on purified matrix substrates in serum-free
conditions may be reconciled with the studies of Pai et al.
(32) by distinguishing between the direct effects of
cell-matrix interactions during cell motility and the complex indirect
effects observed during mucosal healing in vivo.
What function could the signals we have described here serve in this setting? Inhibition of ERK1 and ERK2 activation by the MEK inhibitor PD-98059 inhibited Caco-2 motility, suggesting the possibility that even though ERK1 and ERK2 activity is decreased in migrating cells, the decreased ERK1 and ERK2 activity that is still present in motile Caco-2 cells may actually contribute to their motility. The apparent discrepancy could stem from the fact that total cellular ERK activity as measured here could be the summation of different intracellular ERK pools. Immunofluorescent stains suggest that Caco-2 motility may be associated with reorganization of activated ERK within the cell (C. F. Yu and M. D. Basson, unpublished observations). Alternatively, ERK activity could have different functions in migrating cells than in static nonmotile cells. In contrast, inhibition of the p38 signals using SB-203580 did not inhibit Caco-2 motility, suggesting that the motility-associated alterations in p38 activation may be involved in some other aspect of the motile cell phenotype.
Inhibition of FAK activity by transfection with FRNK, the COOH-terminal region of FAK that competes with FAK for localization to focal contacts but does not have kinase activity (45), also appeared to inhibit Caco-2 cell motility across collagen I, suggesting that FAK activity may in some way also regulate Caco-2 motility. Although cells migrating on matrix proteins exhibited substantially decreased FAK protein levels compared with static cells, it is noteworthy that the ratio of phosphorylated FAK to total FAK actually increased in motile cells on collagen I and fibronectin, suggesting that the (smaller) amount of total FAK protein in these motile cells was actually proportionally more activated. FRNK transfection could be expected to inhibit localization of wild-type FAK to the focal adhesion complex and its subsequent activation by cell-matrix engagement. Thus these results raise the possibility that FRNK might inhibit Caco-2 motility at the level of proportional FAK phosphorylation independently of total FAK protein levels.
Early observations suggested that FAK is associated with MAPK activation during adhesion through integrin-mediated signaling, binding Src family proteins, and Ras signal pathway activation (43). However, integrin-mediated MAPK activity in fibroblasts is independent of Ras and FAK (26). Whether the effect of FAK on Caco-2 motility occurs via FAK-regulated MAPK activation remains an open question. However, disparities between motility-associated changes in phosphorylated FAK and p38 on tissue culture plastic suggest that some factor other than FAK also influences p38 activity in these cells. Furthermore, comparison of the patterns of matrix specificity for ERK1 and ERK2 in this study with those for FAK also suggests an incomplete correlation. It therefore seems likely that MAPK/p38 regulation during Caco-2 motility is multifactorial, reflecting not only the downstream effects of FAK-related signal transduction but also other as yet less well characterized pathways such as Raf-1 (37). A more precise demonstration of this point might be achieved by cotransfection of FRNK and epitope-tagged ERK into static and migrating Caco-2 cells to actually quantitate whether FRNK expression alters the activity of the expressed epitope-tagged ERK. However, our preliminary studies in this regard suggest that differences in transfection efficiency between static and migrating Caco-2 cells render such studies difficult to interpret (data not shown).
Although this study focused on the possibility that the matrix might influence motile Caco-2 cells by modulating integrin-mediated FAK and MAPK signals, other extracellular stimuli and intracellular signal transduction events may also be involved, including nonintegrin matrix receptors (18), Src kinases (20), growth factors (1), and mechanicochemical signals associated with matrix-dependent changes in cell spreading that may initiate signals via cytoskeletal reorganization without requiring integrin binding (3, 17, 49). However, to the extent to which FAK, paxillin, MAPK, and p38 are significant mediators of Caco-2 cell biology, these results suggest that human intestinal epithelial Caco-2 cell motility may be associated with matrix-dependent alterations in intracellular signal transduction events that may influence the migratory phenotype in intestinal epithelial restitution. In motile Caco-2 cells on matrix proteins, FAK, paxillin protein, and phosphorylation were decreased and ERK1 and ERK2 were inactivated but p38 was activated. These signals were most pronounced on interstitial wound matrix proteins that support the most rapid Caco-2 motility (4). Our results further suggest that ERK1 or ERK2 and FAK may modulate Caco-2 migration, and the function of the p38 signal awaits further study. The interplay between matrix proteins and other biological features of the healing mucosal wound awaits further investigation, as does the role of other signal transduction pathways. Furthermore, to the extent to which purified ECM proteins in vitro resemble the complex ECM gel found in vivo, these observations suggest that tissue culture plastic may not be an ideal substrate for the study of the biology of signal transduction during cell motility.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R29-DK-47051 (M. D. Basson).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. D. Basson, Dept. of Surgery, Yale Univ. School of Medicine, 333 Cedar St., P. O. Box 208062, New Haven, CT 06520-8062 (E-mail: marc.basson{at}yale.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. §1734 solely to indicate this fact.
Received Received 4 August 1999; accepted in final form 28 January 2000.; accepted in final form .
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