Human Caco-2 motility redistributes FAK and paxillin and activates p38 MAPK in a matrix-dependent manner

Cheng Fang Yu, Matthew A. Sanders, and Marc D. Basson

Departments of Surgery, Yale University School of Medicine and Connecticut Veterans Affairs Health Care System, New Haven, Connecticut 06511


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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 beta 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).

For MAPK studies, cells were cultured with serum-free media for 24 h by the time of cell harvest. Cells were then harvested in modified RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 50 mM NaF, 1 mM PMSF, 1 mM Na3VO4, and 10 mM Na2P4O7).

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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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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Fig. 1.   Comparison of proliferation and differentiation of Caco-2 cells after differential density seeding. Caco-2 cells were seeded into 35 × 10-mm and 100 × 20-mm dishes with precoating collagen I to generate static cell and migrating populations using differential density cell seeding as described in MATERIALS AND METHODS. The solid line and dotted line represent static cells and migrating cells, respectively. A: cell number was assessed at 24, 48, 72, and 96 h after initial seeding by trypsinizing to a single cell suspension and counting with an automated cell counter. The ratios of cell number at initial seeding to cells at each time point were equivalent in both static and migrating cells (n = 4, P > 0.05). B: 2 and 4 days after seeding, Caco-2 cells were lysed in Dulbecco's modified PBS containing 0.5% Triton X-100; 20 µg of total cell lysates were used to test alkaline phosphatase (AKP) and dipeptidyl dipeptidase (DPDD) as described in MATERIALS AND METHODS. Means ± SE of 4 independent experiments are shown. Both AKP and DPDD were at similar levels in static and migrating preparations at 2 and 4 days after initial seeding (n = 4, P > 0.05).

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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Fig. 2.   Membrane/cytoskeletal focal adhesion kinase (FAK) protein (A) and FAK tyrosine phosphorylation (B) in static (S) and migrating (M) Caco-2 cells. Cell lysates (400 µg) enriched for the cytoskeletal fraction were immunoprecipitated with monoclonal anti-FAK coupled to protein A-Sepharose beads. Immunoprecipitates were washed and resolved by 7.5% SDS-PAGE and transferred to nitrocellulose. Densitometric analysis of at least 5 similar experiments is shown, with typical blots in insets. After normalizing each experiment's data to static cells grown on tissue culture plastic, data were expressed as percentage of static cells on tissue culture plastic, shown as means ± SE. A: blots were detected with anti-FAK, visualized using enhanced chemiluminescence (ECL), and densitometrically analyzed. Compared with static cells on same matrix protein, membrane/cytoskeletal FAK decreased substantially in cells on collagen I (Coll), on laminin (Ln), and on fibronectin (Fn) in a matrix-dependent manner (n = 5, P < 0.01, laminin vs. collagen I and fibronectin) but increased on tissue culture plastic (Tp; n = 5, P < 0.01). B: same nitrocellulose membranes immunodetected for total membrane/cytoskeletal FAK protein were stripped and reprobed with horseradish peroxidase-conjugated phosphotyrosine antibody (CR20), visualized using ECL, and analyzed densitometrically. Phosphorylated FAK decreased substantially in cells on collagen I, on laminin, and on fibronectin, but a significant increase was observed in cells migrating on tissue culture plastic compared with static cells. * P < 0.05 between S and M in the same matrix protein; # P < 0.05 collagen I vs. laminin, fibronectin, and tissue culture plastic.



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Fig. 3.   Cytosolic FAK protein (A) and FAK tyrosine phosphorylation (B) in static and migrating Caco-2 cells. Cytosolic FAK protein and phosphorylated FAK were assessed by a method similar to that described for Fig. 2. Densitometric analysis of at least 5 similar experiments is shown, and typical blots are shown in insets. After normalizing each experiment's data to static cells grown on tissue culture plastic, data were expressed as percentage of static cells on tissue culture plastic, shown as means ± SE. A: cytosolic FAK decreased significantly in cells on collagen I, on laminin, and on fibronectin in matrix-independent manner compared with static nonmigrating cells on same matrix protein (# P < 0.05 compared with static tissue culture plastic; n = 5), whereas cells migrating on tissue culture plastic exhibited a substantial increase (* P < 0.05 compared with static cells on each matrix; n = 5). B: cytosolic FAK tyrosine phosphorylation also decreased substantially in cells migrating on matrix proteins but increased in cells migrating on tissue culture plastic compared with static cells. * P < 0.05 between S and M in the same matrix protein.

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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Fig. 4.   Membrane/cytoskeletal paxillin expression (A) and phosphorylation (B) in static and migrating Caco-2 cells. Cell lysates (200 µg) enriched for a membrane/cytoskeletal fraction were immunoprecipitated with monoclonal anti-paxillin coupled to protein A-Sepharose beads. Immunoprecipitates were washed, resolved by 7.5% SDS-PAGE, and transferred to nitrocellulose. Densitometric analysis of at least 5 similar experiments is shown, and typical blots are shown in insets. After normalizing each experiment's data to static cells grown on tissue culture plastic, data were expressed as percentage of static cells on tissue culture plastic, shown as means ± SE. A: blots were detected with anti-paxillin, visualized using ECL, and analyzed densitometrically. Paxillin decreased significantly in cells migrating on matrix proteins studied compared with static nonmigrating cells on same matrix protein, whereas cells migrating on tissue culture plastic demonstrated an increase (n = 5, P < 0.05). B: after stripping the same nitrocellulose membranes immunodetected for total paxillin, blots were reprobed with horseradish peroxidase-conjugated phosphotyrosine antibody (CR20), visualized using ECL, and analyzed densitometrically. Tyrosine-phosphorylated paxillin decreased substantially in cells migrating on matrix proteins studied matrix dependently (n = 5, P < 0.01, laminin vs. collagen I and fibronectin) compared with static cells on same matrix protein. No decrease was observed on tissue culture plastic. * P < 0.05 between S and M in the same matrix.

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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Fig. 5.   Subcellular localization of FAK in static and migrating Caco-2 cells. Caco-2 cells were seeded at densities of 13,000/cm2 and 750/cm2 into 4-well culture slides with and without collagen I precoating to generate static and migrating cells on collagen and tissue culture plastic. After 4 days, cells were stained for FAK using monoclonal anti-FAK and Texas red-conjugated secondary antibody (anti-mouse IgG) as described in MATERIALS AND METHODS. A: FAK distribution in cells migrating on collagen I. Arrows indicate the lamellipodial edge of one cell, which does not exhibit FAK staining. B: FAK distribution in nonmigrating (static) cells on collagen I. Arrow represents a typical FAK staining pattern at cell-cell contacts. C: immunolocalization of FAK in cells migrating on tissue culture plastic. Arrow indicates FAK localized at the lamellipodial edge of one cell. D: immunolocalization of FAK in static cells on tissue culture plastic. Arrow represents a typical FAK staining pattern at cell-cell contacts. Bar = 200 µm.

Paxillin organization in motile and confluent cells on collagen I and tissue culture plastic is shown in Fig. 6. In Fig. 6, panels A and B represent cells on collagen I, but similar results were obtained in cells on fibronectin and laminin (data not shown). In contrast to FAK staining, discrete paxillin staining was observed at lamellipodial edges in cells migrating across matrix proteins in addition to staining at areas of cell-cell contact and in a perinuclear ER-Golgi distribution (Fig. 6A). Interestingly, although the overall cellular paxillin staining might have been less intense in migrating cells compared with static cells on collagen I (Fig. 6B), we noted the most intense paxillin staining at the lamellipodial edge of migrating cells. In cells cultured on tissue culture plastic, paxillin staining was also distributed along the lamellipodia and areas of cell-cell contact. However, stains for paxillin in cells migrating on tissue culture plastic (Fig. 6C) seemed to exhibit more intracellular staining compared with lamellipodial edge staining compared with cells migrating on matrix (Fig. 6A), in which a relative paucity of staining was observed in the lamellipodium behind the edge. In addition, the lamellipodial paxillin staining in cells migrating on plastic (Fig. 6C) appeared more intense than lamellipodial paxillin staining in cells migrating across matrix proteins. As for cells on collagen I, paxillin staining appeared less organized in confluent cells on plastic than in motile cells on plastic (Fig. 6D).


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Fig. 6.   Subcellular localization of paxillin in static and migrating Caco-2 cells. Similar procedures used for FAK staining were applied to stain for paxillin with monoclonal anti-paxillin. A: paxillin distribution in cells migrating on collagen I. Arrows show paxillin reorganization at the lamellipodial edge. B: paxillin distribution in nonmigrating (static) cells on collagen I. C: immunolocalization of paxillin in cells migrating on tissue culture plastic. Arrows show intense paxillin staining within the cell behind the lamellipodial edge. D: immunolocalization of paxillin in static cells on tissue culture plastic. Bar = 200 µm.

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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Fig. 7.   p38 Activation in static and migrating Caco-2 cells. Caco-2 cells were cultured in serum-free media for 24 h before harvesting. Whole cell lysates (20 µg) were resolved by 10% SDS-PAGE and transferred to nitrocellulose. Blots were detected with phosphospecific anti-p38 antibody, visualized using ECL, and analyzed densitometrically. After stripping each phosphorylated p38 blot and reprobing for total p38 protein, we normalized the phosphorylated p38 data against total p38 protein data to correct for any difficulty with equivalent protein loading in each lane. Densitometric analysis of at least 5 similar experiments is shown, and typical phosphorylated and total p38 blots are shown in the inset. Activation of p38 mitogen-activated protein kinase (MAPK) increased substantially in cells migrating on the matrix proteins studied compared with static cells on the same matrix proteins but decreased in cells migrating on tissue culture plastic. * P < 0.05 between S and M in the same matrix.

Phosphorylated p38 was substantially increased in cells migrating across matrix proteins compared with static cells on these same substrates. Cells migrating on tissue culture plastic exhibited the reverse result (Fig. 7). After stripping each phosphorylated p38 blot and reprobing for total p38 protein, we normalized the phosphorylated p38 data against total p38 protein data to correct for any differences in protein loading in each lane. According to these data, phosphorylated p38 in migrating cells on collagen I was 74.2 ± 11.1% of p38 phosphorylation levels in static cells on plastic, whereas phosphorylated p38 in static cells on collagen I was only 47.0 ± 9.2% of p38 phosphorylation levels in static cells on plastic (n = 5, P < 0.001 migrating cells vs. static cells). Similarly, phosphorylated p38 was also increased in motile cells on laminin (105.3 ± 12.1% of static plastic control levels vs. 69.7 ± 6.7% of static plastic control cells, P < 0.05 migrating cells vs. static cells) and fibronectin (113.4 ± 14.8% of static plastic control levels vs. 67.2 ± 11.5% of static plastic control cells, P < 0.05 migrating cells vs. static cells) compared with static cells. However, p38 was decreased by 40.5 ± 7.6% (n = 6, P < 0.05) in cells migrating on tissue culture plastic compared with static cells on tissue culture plastic.

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).


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Fig. 8.   Extracellular signal-regulated kinase (ERK) activation in static and migrating Caco-2 cells. Activation of ERK was examined as described for p38 in Fig. 7. Densitometric analysis of at least 5 similar experiments is shown, and a typical blot is shown in the inset. Activation of ERK1 (A) and ERK2 (B) decreased significantly in cells migrating on matrix proteins studied compared with static cells on same matrix protein. Cells migrating on tissue culture plastic exhibited alterations of ERK1 and -2 similar to cells migrating on matrix proteins. * P < 0.05 between S and M in the same matrix.

In contrast to observations for FAK, paxillin, and p38, cells migrating on tissue culture plastic exhibited changes in ERK1 and ERK2 parallel in direction to those observed in cells migrating across the matrix proteins studied. However, the magnitude of the motility-associated changes in active ERK1 and ERK2 on tissue culture plastic (33.4 ± 7.8% and 29.4 ± 5.2% respectively, n = 6, P < 0.05) was substantially less than those observed upon migration across matrix proteins (P < 0.05 compared with collagen I, laminin, and fibronectin).

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).


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Fig. 9.   Inhibition of Caco-2 migration by FRNK transfection. Caco-2 cells were transiently transfected with hemaglutinin (HA)-tagged FRNK and wild-type FAK, and migration was assayed in a monolayer cell wounding model as described in MATERIALS AND METHODS. The vertical axis represents the ratio of HA-stained cells (expressing transfected FAK or FRNK) to unstained (untransfected) cells in at least 10 random high-power fields along the wound in each dish. FAK/B represents the ratio of HA-FAK-staining cells to unstained cells in the confluent monolayer behind the migrating front. FAK/F represents the ratio of HA-FAK-staining cells to unstained cells in the migrating front. FRNK/B represents the ratio of HA-FRNK-staining cells to unstained cells in the confluent monolayer behind the migrating front. FRNK/F represents the ratio of HA-FRNK-staining cells to unstained cells in the migrating front. Data were pooled from 4 separate and similar studies. * P < 0.002.

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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Fig. 10.   Effect of MAPK-ERK kinase (MEK) and p38 inhibitors on Caco-2 migration. Caco-2 cells were treated with MEK inhibitor PD-98059 and the p38 inhibitor SB-203580 at 5 µM and 10 µM concentrations in a proliferation-blocked monolayer cell wounding model. The number of cells in the wound defect area of at least 10 random high-power fields along the wound was counted 48 h after treatment and expressed as a percentage of the cells migrating into the wound defect area in identically wounded control dishes treated only with a DMSO vehicle control. PD-5 and PD-10 represent PD-98059 concentrations of 5 µM and 10 µM, respectively, and SB-5 and SB-10 represent SB-203580 concentrations of 5 µM and 10 µM respectively. Data was pooled from 4 separate and similar studies. * P < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-alpha 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.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R29-DK-47051 (M. D. Basson).


    FOOTNOTES

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 .


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 278(6):G952-G966