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MUCOSAL BIOLOGY

Collagen IV regulates Caco-2 migration and ERK activation via ₁₁- and ₂₁-integrin-dependent Src kinase activation

Departments of Surgery, Wayne State University, and John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201-1932

Submitted 16 June 2003 ; accepted in final form 5 November 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. ₁₁- Or ₂₁-integrin blockade with ₁- or ₂-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130^Cas tyrosine phosphorylation, but dominant-negative p130^Cas did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either ₁₁- or ₂₁-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.

extracellular matrix; focal adhesion kinase; cell spreading; cell adhesion

Potential mediators of FAK signaling initiated by collagen IV include the Src family nonreceptor tyrosine kinases, which can associate with the FAK Y397 autophosphorylation site via their SH2 domains (14, 48, 63). In the present work, we characterized the mechanism of ERK activation by collagen IV in Caco-2 cells by using dominant-negative forms of FAK and Src and inhibition of Src by PP2. We characterized the integrins involved in collagen IV adhesion, cell spreading, and signaling in Caco-2 cells through the use of function-blocking integrin antibodies. Finally, we characterized the role of Src kinase in regulating Caco-2 cell spreading and migration on collagen IV by using a dominant-negative form of Src and inhibition of Src by the Src inhibitor PP2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials. DMEM, FBS, Lipofectamine Plus reagent, and monoclonal antibodies to ₃ (clone P1B5)- and ₁ (clone P4C10)-integrin subunits for adhesion blockade studies were obtained from Life Technologies (Gaithersburg, MD). Monoclonal antibodies to ₁ (clone FB12)-, ₂ (clone P1E6)-, ₃ (clone ASC-1)- and ₁ (clone HB1.1)-integrin subunits used for immunoprecipitation and immunoblotting and Western blot stripping reagent were obtained from Chemicon International (Temecula, CA). HA-tag monoclonal antibody 12CA5 and transferrin were obtained from Roche Applied Science (Indianapolis, IN). Trypsin, soybean trypsin inhibitor, collagen IV, poly-L-lysine (PLL; mol wt 70,000–150,000), and horseradish peroxidase conjugated rabbit anti-mouse IgG were obtained from Sigma (St. Louis, MO). FAK, p130^Cas, and RC20 phosphotyrosine monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY) and Upstate Biotechnology (Lake Placid, NY). Phosphospecific polyclonal antibodies to FAK Tyr(P)397 and FAK Tyr(P)576 were obtained from Biosource International (Camirillo, CA). FAK Tyr(P)925 phosphospecific polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or Biosource International and Src polyclonal antibody was obtained from Santa Cruz Biotechnology. Phosphospecific monoclonal and polyclonal antibodies to the activated forms of ERK1 and -2 and polyclonal antibody to ERK protein were obtained from Cell Signaling Technology (Beverly, MA). Myc-tag monoclonal antibody 9E10 and myc-tag polyclonal antibody were obtained, respectively, from Covance (Richmond, CA) and Upstate Biotechnology. Horseradish peroxidase-conjugated donkey anti-rabbit IgG, protein G Sepharose, protein A Sepharose, electrophoresis equipment, transfer apparatus, and Hyperfilm MP were obtained from Pharmacia Biotech (Piscat-away, NJ). PP2 was obtained from Calbiochem (San Diego, CA). Protein molecular weight standards were obtained from Bio-Rad (Hercules, CA). pcDNA 3.1 and pcDNA 3.1 H lacZ were obtained from Invitrogen (La Jolla, CA). Expression vectors with HA-tagged FAK, HA-FAK Y397F, and HA-FAK Y925F were generous gifts from Dr. David Schlaepfer (The Scripps Research Institute, La Jolla, CA). The myc-ERK2 expression vector was a generous gift from Dr. Christopher Marshall (Institute of Cancer Research, London, UK). The dominant-negative Src (K296R/Y527F) expression vector and the control vector pUSE were obtained from Upstate Biotechnology. Expression vectors with HA-tagged p130^Cas, HA-tagged substrate domain deleted-p130^Cas (SD-p130^Cas), and the control vector pSSR were a generous gift from Drs. Tetsuya Nakamoto and Hisamaru Hirai (University of Tokyo, Tokyo, Japan).

Cell culture. The Caco-2 cell line used for this work was a subclone (Caco-2_BBE) of the original Caco-2 cell line that was selected for its ability to differentiate in culture as indicated by formation of an apical brush border and brush-border enzyme expression and has been previously described (42, 43). Passages 45–67 Caco-2 cells were maintained at 37°C with 8% CO₂ in DMEM with (in mM) 4 glutamine, 1 sodium pyruvate, 0.1 MEM nonessential amino acids solution, 10 HEPES (pH 7.4), and 4,500 mg/l D-glucose, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml transferrin, and 3.7 g/l NaHCO₃ and supplemented with 10% FBS.

Coating of cell culture dishes. Cell culture dishes were coated with a saturating concentration (36) of collagen IV (12.5 µg/ml) in precoating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.4). Dishes were coated with PLL as described by the manufacturer. For adhesion to integrin antibodies, dishes were precoated with antibodies to ₁- or ₂-integrin subunits at 10 µg/ml in PBS (in mM: 2.8 NaH₂PO₄, 7.2 Na₂HPO₄, 150 NaCl). For experiments in which integrin subunit antibodies were used to block adhesion or cell spreading, collagen IV-coated dishes were overlaid with 1% heat-inactivated (80°C, 30 min) BSA in PBS for 45 min at 37°C before adhesion to prevent integrin subunit antibodies from binding to culture dishes. For these experiments, 1% BSA was also included in the medium.

Cell lysis. After adhesion, dishes were placed on ice, rinsed with ice-cold Tris-buffered saline (10 mM Tris pH 7.4, 150 mM NaCl), and were then lysed on ice in modified radioimmunoprecipitation buffer (in mM: 50 Tris, pH 7.4, 150 NaCl, 1 EDTA, 1 EGTA, 1 PMSF, 1 Na₃VO₄, 50 NaF, 10 sodium pyrophosphate, and 1% Triton X-100, 10% glycerol, 1% deoxycholic acid, 0.1% SDS, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 1 µM microcystin LR) (Sigma). Lysates were centrifuged at 15,000 g for 10 min at 4°C and supernatants were stored at -80°C. Protein concentrations were determined by the bicin-choninic acid method (Pierce, Rockford IL). For lysates used in integrin immunoprecipitations, divalent cation chelators, SDS, and deoxycholic acid were omitted from the cell lysis buffer and NaF was reduced to 1 mM, and 1 mM MgCl₂ and 1 mM CaCl₂ were added.

Immunoprecipitation and Western blotting. For all experiments described in this article in which immunoprecipitation was required, two rounds of immunoprecipitations were performed and then combined before rinsing with immunoprecipitation buffer (cell lysis buffer without deoxycholic acid or SDS and with 1 mM NaF) to ensure complete immunoprecipitation of protein. Immunoprecipitations of protein-matched samples were performed at 4°C by using protein G Sepharose and myc-tag monoclonal antibody 9E10, protein A Sepharose and HA-tag monoclonal antibody 12CA5, or protein G Sepharose and integrin subunit antibodies. p130^Cas immunoprecipitations were performed with protein A Sepharose, rabbit anti-mouse IgG secondary antibody, and p130^Cas monoclonal antibody. Gel-loading buffer was added to immunoprecipitates after rinsing, and samples were boiled and resolved on SDS-polyacrylamide gels. Blots were detected with enhanced chemiluminescence (ECL) or ECL Plus reagent (Pharmacia Biotech) after transfer of gels to Immobilon P membranes (Millipore; Bedford, MA).

Quantification of adhesion after treatment of Caco-2 cells with integrin subunit antibodies. Subconfluent cells were incubated for 18–24 h with reduced serum medium containing 0.1% FBS. Cells were then harvested by using trypsin-EDTA, and trypsinization was stopped by using soybean trypsin inhibitor. Cells were then rinsed twice with serum-free medium and were resuspended at 1.6 x 10⁵ cells/ml in serum-free medium containing 1% BSA. Integrin subunit antibodies were added at 2 µg/ml and cell suspensions were incubated at 37°C with rotation for 45 min. Cell suspension (1.5 ml) was then added to six-well dishes coated with collagen IV. Collagen IV-coated dishes were blocked with BSA before addition of cell suspension. In all adhesion experiments, Caco-2 cells were allowed to adhere to dishes coated with PLL, collagen IV, or integrin subunit antibodies at 37°C for 20 min, the time point at which maximal ERK activation occurs after adhesion to collagen IV (46), unless indicated otherwise. After adhesion, dishes were rinsed with Tris-buffered saline, fixed with 10% formalin solution (Sigma), and stained with Harris modified hematoxylin (Fisher Scientific, Pittsburgh, PA). Twenty high-powered fields (x200) were counted for each well to determine relative adhesion.

Transfections. Caco-2 cells were transfected with Lipofectamine Plus reagent as described previously (46). After transfection, cells were incubated with normal medium for 20–24 h and then with reduced-serum medium for an additional 18–24 h before experiments. Previous work (64) indicated that 20–25% of cells are transfected by using this procedure. On account of this low transfection efficiency, the effect of dominant-negative forms of Src or FAK on ERK activation was determined in cells cotransfected with 6 µg of the dominant-negative construct DNA (or appropriate vector control) and 1.5 µg of myc-tagged ERK2. For cell-spreading studies, cells were cotransfected with 16 µg dominant-negative Src and 1 µg 3.1 H lacZ (to indicate transfected cells) or with 6 µg p130^Cas or SD-p130^Cas and 1.5 µg 3.1 H lacZ.

Cell spreading assays. Cells grown and harvested as described above were resuspended in serum-free medium with 1% BSA. Cells were allowed to initially adhere for 5 min at 37°C to collagen IV-coated 12-well dishes blocked with BSA. Cells were then extensively rinsed (5–6 times) with cold serum-free media and inspected to make sure that nonadherent cells had been completely removed. Cold serum-free medium containing 1% BSA and integrin subunit antibodies diluted 1:150 (6.7 µg/ml) were then added, and cells were incubated on ice for 30 min. Cells were returned to 37°C for 90 min and fixed with 10% formalin solution. Cells were then stained with Harris modified hematoxylin and counterstained with eosin solution (Sigma).

For measuring the effects of dominant-negative Src and SD-p130^Cas on Caco-2 cell spreading, cells cotransfected with 3.1 H lacZ as described in Transfections were harvested as in adhesion experiments and allowed to spread for 2 h in serum-free medium on collagen IV-coated flasks blocked with 1% BSA. For these experiments, cells were plated at low density (10,000 cells per T25 flask) to avoid false positives resulting from diffusion of stain through gap junctions. Cells were then fixed and stained for lacZ expression. The percentage of spread lacZ positive cells out of total lacZ positive cells was then determined in a blinded count. At least 200 lacZ positive cells were counted for each condition in each experiment.

Cell migration assays. Cells plated on collagen IV-coated petri dishes blocked with BSA were incubated overnight in reduced serum medium. A uniform migrating front was made by gently placing a razor blade on the dish and removing cells with a cotton swab to avoid removing collagen IV. To verify that collagen IV was still present, Caco-2 cell adhesion and spreading was compared in newly coated dishes and in areas of dishes in which cells were removed. Cell spreading and adhesion were similar in denuded areas and newly coated dishes and, in both cases, were prevented by a combination of ₁- and ₂-integrin subunit antibodies (data not shown). Dishes were then blocked again with BSA, and inhibitors were added in serum-free medium. After 18 h at 37°C, cells were rinsed with PBS, fixed in 10% formalin, and then stained with Harris modified hematoxylin and eosin. Migration was quantified by measuring the area of at least five randomly chosen fields for each condition in three or more experiments.

Statistical analysis. Where indicated, results were compared by using the paired Student's t-test and considered statistically significant when P < 0.05. All experiments were done independently at least three times, unless otherwise indicated.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Src kinase inhibition decreases ERK activation. To understand signaling events in Caco-2 cells initiated by the basement membrane matrix protein collagen IV in a system amenable to experimental manipulation, we characterized signaling in serum-starved Caco-2 cells after adhesion to collagen IV in serum-free medium. Our previous work (46) suggested that Src kinases are activated in Caco-2 cells by collagen IV adhesion, because FAK is phosphorylated at the Src phosphorylation site FAK Y576. Previous work (51, 61) in NIH3T3 fibroblasts has suggested that integrin activation of Src family kinases is an early event after fibronectin adhesion. The precise mechanism of Src activation is not completely understood but appears to involve both dephosphorylation of Src Y527 and phosphorylation of Src Y416 and perhaps other tyrosines (reviewed in Ref. 22). Adhesion of Caco-2 cells to collagen IV (Fig. 1A) resulted in a significant increase in Src Y416 phosphorylation compared with adhesion to the control substrate PLL (34.3 ± 10.4% increase, n = 6, P < 0.05). This increase in Src Y416 phosphorylation after collagen IV adhesion is comparable with that observed in other cell types after adhesion to fibronectin (13), which is mediated by different integrins. The Src family kinase inhibitor PP2 (24), which was used at 10 µM throughout the work described in this article, significantly inhibited Src Y416 phosphorylation after adhesion to collagen IV (59.1 ± 9.1% inhibition compared with control cells adherent to collagen IV, n = 6, P < 0.01). Although the Src Tyr(P)416 antibody recognizes other phosphorylated Src family members in addition to Src kinase, the band detected with an Src kinase-specific antibody in blot reprobes corresponded to the Src Tyr(P)416 band. This suggests that the effects of PP2 and dominant-negative Src on signaling events described in this work are most likely exerted through Src kinase, although contributions from other Src family members cannot be ruled out.

View larger version (46K): [in this window] [in a new window]   Fig. 1. Effect of Src kinase inhibition on ERK activation in Caco-2 cells adherent to collagen IV (Col IV). A: lysates of PP2 (10 µM)-treated cells adherent to the control substrate poly-L-lysine (PLL) or collagen IV for 10 min were immunoblotted with an antibody to Src Tyr(P)416. B: lysates of adherent cells treated with PP2 were immunoblotted with a monoclonal active ERK antibody. The blot was then reprobed for total ERK protein. C: cells were cotransfected with myc-ERK2 and either dominant-negative Src or pUSE vector control as described in MATERIALS AND METHODS. Adherent cell lysates were immunoprecipitated for myc-ERK2 by using monoclonal myc-tag antibody as described in MATERIALS AND METHODS and blotted with active ERK polyclonal antibody. In dominant-negative Src-transfected cells, expression of both myc-ERK2 and dominant-negative Src was similar in suspension cells before adhesion and PLL and collagen IV-adherent cells (not shown), indicating reduced myc-ERK2 levels did not result from incomplete immunoprecipitation or an effect of dominant-negative Src on adhesion. D: cells were treated with the MEK inhibitor PD-98059 (20 µM) before adhesion to PLL or collagen IV for 20 min. Lysates were immunoblotted for active ERK then reprobed for ERK protein. One of two identical experiments is shown.

We examined the role of Src kinase in collagen IV-dependent ERK activation by treatment of cells with PP2 and by transfecting cells with dominant-negative Src. We measured ERK activation (Figs. 1, 2, and 5) after 20-min adhesion, which we (46) have previously shown to be the time of the maximal ERK response after Caco-2 cell adhesion to collagen IV. Both PP2 treatment (68.7 ± 3.1% inhibition, n = 3, P < 0.01; Fig. 1B) and dominant-negative Src cotransfected with an myc-tagged ERK2 construct (Fig. 1C) strongly inhibited ERK activation on collagen IV after 20-min adhesion. Whereas expression of myc-ERK2 was lower in cells cotransfected with dominant-negative Src, densitometric analysis of blots in which PLL bands of pUSE and dominant-negative Src transfected cells were exposed to similar intensity indicated that dominant-negative Src inhibited ERK activation by almost 90% (153.4 ± 28.4% increase in ERK activation on collagen IV in pUSE cotransfected cells; 17.0 ± 11.8% increase in ERK activation on collagen IV in dominant-negative Src cotransfected cells, n = 3, P < 0.05). As expected, the mitogen-activated protein ERK kinase (MEK) inhibitor PD-98059 (20 µM) almost completely blocked ERK activation by collagen IV (Fig. 1D).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Role of focal adhesion kinase (FAK) Y925 phosphorylation in collagen IV ERK activation in Caco-2 cells. A: lysates of PP2 (10 µM)-treated cells adherent to PLL or collagen IV for 20 min were immunoblotted with an antibody to FAK Tyr(P)397, FAK Tyr(P)576, or FAK Tyr(P)925. FAK Tyr(P)576 blot was reprobed for FAK protein. Because FAK Tyr(P)925 antibody could not be stripped off with standard techniques, this blot was reprobed with an antibody to p85 to confirm equal protein loading. B: cell lysates were immunoprecipitated with monoclonal p130Cas antibody and immunoblotted for phosphotyrosine. A set of immunoprecipitations prepared in parallel was immunoblotted for p130Cas protein. C: cells were transfected with HA-FAK, HA-FAK Y397F, or HA-FAK Y925F as described in MATERIALS AND METHODS. Adherent cell lysates were immunoprecipitated with HA-tag monoclonal antibody as described in MATERIALS AND METHODS. Immunoprecipitates were then immunoblotted for FAK Tyr(P)925. A separate set of immunoprecipitates were immunoblotted with HA-tag polyclonal antibody to confirm equivalent expression of HA-FAK constructs. One of two identical experiments is shown. D: cells were cotransfected with myc-ERK2 and either FAK Y925F mutant or the pcDNA 3.1 control vector as described in MATERIALS AND METHODS. Adherent cell lysates were immunoprecipitated for myc-ERK2 with myc-tag monoclonal antibody and immunoblotted with active ERK polyclonal antibody. Blots were reprobed with an myc-tag polyclonal antibody to measure myc-ERK2 expression. Total cell lysates were immunoblotted with anti-HA monoclonal antibody to show HA-FAK Y925F expression.

View larger version (54K): [in this window] [in a new window]   Fig. 5. A: lysates of Caco-2 cells adherent for 20 min to dishes coated with PLL, 1-integrin subunit antibody, 2-integrin subunit antibody, or 1-integrin subunit antibody were immunoblotted for the active forms of ERK, FAK Tyr(P)397, FAK Tyr(P)576, or FAK Tyr(P)925. The active ERK and FAK Tyr(P)576 blots were then reprobed for total protein. The FAK Tyr(P)925 blot was reprobed with a polyclonal antibody to p85 to demonstrate equal protein loading, because the FAK Tyr(P) 925 antibody could not be stripped off with standard techniques. One of 3 similar experiments is shown. Note that, unlike experiments described in Figs. 3 and 5B, antibodies to integrin subunits were used to coat dishes rather than block adhesion to collagen IV. B: cells were treated with the IgG1 control antibody or antibodies to the indicated integrin subunits as described in MATERIALS AND METHODS before adhesion to PLL or collagen IV for 20 min. Adherent cell lysates were immunoblotted for FAK Tyr(P)397, FAK Tyr(P)576, FAK Tyr(P)925 or the active form of ERK. The FAK Tyr(P)576 and active ERK immunoblots were then stripped and reprobed for total protein. One of three independent experiments with similar results is shown.

View larger version (35K): [in this window] [in a new window]   Fig. 3. A: effect of integrin subunit antibodies on Caco-2 cell adhesion to collagen IV. Before adhesion to collagen IV, cells were treated with antibodies (2 µg/ml) to the indicated integrin subunits as described in MATERIALS AND METHODS. After 20-min adhesion, nonadherent cells were rinsed off and adherent cells were fixed with a 10% formalin solution and stained with hematoxylin. Adhesion was determined from 3 independent experiments as described in MATERIALS AND METHODS. *P < 0.05 compared with cells pretreated with antibody to the 1-integrin subunit; **P < 0.01 compared with IgG1 control antibody-treated cells. B: quantification of relative expression of 1-integrin heterodimers. Caco-2 cell lysates were immunoprecipitated with antibodies to the indicated integrin subunits as described in MATERIALS AND METHODS and then immunoblotted for the coprecipitating 1-integrin subunit. Quantification relative to total 1-integrin expression was determined from immunoprecipitations of 3 independently prepared lysates.

Caco-2 cell adhesion to collagen IV is mediated by ₁₁- and ₂₁-integrins. Six integrins (₁₁, ₂₁, ₃₁, ₉₁, ₁₀₁, and ₁₁₁) have been observed to bind to collagens, and expression of ₁₁-, ₂₁-, and ₃₁-integrins has been detected in human intestinal epithelial cells in vivo (7). We examined the role of these integrins in adhesion of Caco-2 cells to collagen IV through the use of function-blocking integrin subunit antibodies (Fig. 3A). As expected, a functional antibody to the ₁-integrin subunit almost completely blocked adhesion to collagen IV. A functional antibody to the ₁-integrin subunit inhibited adhesion of Caco-2 cells to collagen IV by 65% compared with cells treated with the IgG1 control antibody. A functional antibody to ₂-integrin subunit had no effect on adhesion by itself but in combination with the ₁-integrin subunit antibody inhibited adhesion to collagen IV by 97%. Treatment of cells with ₂-integrin subunit antibody at 10 µg/ml (5 times the concentration used in Fig. 3A) also failed to inhibit adhesion (data not shown). A functional antibody to the ₃-integrin subunit had no effect by itself or in combination with the other antibodies. This suggests that both ₁₁- and ₂₁-integrins are involved in Caco-2 cell adhesion to collagen IV but that the ₃₁-integrin does not play an important role in this adhesion.

We examined relative expression of the -integrin subunits by immunoprecipitating Caco-2 cell lysates for the ₁-, ₂-, or ₃-integrin subunits then immunoblotting for the coprecipitating ₁-integrin subunit that is tightly associated with the -integrin subunit. This indicated that ₁₁-, ₂₁-, and ₃₁-integrins constituted, respectively, 22.1 ± 6.1, 35.9 ± 3.8, and 4.0 ± 0.5% of the total cellular ₁-integrin heterodimers (Fig. 3B). We also detected low ₃₁-integrin expression in ₃-integrin subunit immunoprecipitates of surface iodinated Caco-2 cells (not shown). The remaining fraction of ₁-integrin heterodimers likely consists mostly of the ₅₁ (fibronectin receptor)- and ₆₁ (laminin receptor)-integrin heterodimers, which were also detected in immunoprecipitates of surface iodinated Caco-2 cells (not shown). The higher relative expression of the ₁₁- and ₂₁-integrins in the undifferentiated Caco-2 cells used for these studies is consistent with in vivo observations of ₁- and ₂- but not ₃-integrin subunit expression in the undifferentiated intestinal epithelial crypt cells (35).

Caco-2 cell spreading on collagen IV is mediated by ₁₁- and ₂₁-integrins. We used the functional antibodies to the ₁- and ₂-integrin subunits to investigate the role of ₁₁- and ₂₁-integrins in Caco-2 cell spreading on collagen IV (Fig. 4, A and B). Treatment with ₁-integrin subunit antibody significantly inhibited cell spreading, as indicated by the percentage of spread cells, compared with IgG1 control antibody treatment. Treatment with antibody to the ₂-integrin subunit significantly inhibited cell spreading compared with treatment with the IgG1 control antibody, although to a lesser extent than treatment with antibody to the ₁-integrin subunit. As for cell adhesion, treatment with the combination of antibodies to the ₁- and ₂-integrin subunits inhibited Caco-2 cell spreading on collagen IV significantly more than treatment with antibody to either integrin subunit alone (Fig. 4, A and B).

View larger version (52K): [in this window] [in a new window]   Fig. 4. Treatment with antibodies to the 1- or 2-integrin subunits inhibits Caco-2 cell spreading on collagen IV. A: cells were allowed to adhere to collagen IV for 5 min. Dishes were rinsed thoroughly to remove nonadherent cells and adherent cells were then treated with control IgG1 antibody or with antibodies to the indicated integrin subunits as described in MATERIALS AND METHODS. After being spread for 90 min at 37°C, cells were fixed with 10% formalin, stained with hematoxylin, and then counterstained with eosin. B: quantification of A on the basis of 3 independent experiments. In each experiment, at least 250 cells were counted for each condition. *P < 0.05 compared with IgG1-treated cells; #P < 0.05 compared with 1- or 2-integrin subunit antibody-treated cells.

Either ₁₁- or ₂₁-integrin can initiate FAK, Src, and ERK activation by collagen IV. To determine whether either ₁₁- or ₂₁-integrin can each initiate FAK, Src, and ERK activation by collagen IV, we allowed Caco-2 cells to adhere to dishes coated with antibodies to either the ₁-, ₂-, or ₁-integrin subunit. Unlike experiments described in Figs. 3A and 5B in which antibody treatment of cells was used to block adhesion to collagen IV, antibodies were used to coat culture dishes (Fig. 5A). Adhesion to either ₁- or ₂-integrin subunit antibody-coated dishes stimulated phosphorylation of the FAK autophosphorylation site Y397, phosphorylation of the Src phosphorylation sites FAK Y576 and FAK Y925, and ERK activation compared with PLL adherent control cells (Fig. 5A). Although stimulation of ERK activation was not as strong after adhesion to ₁-integrin subunit antibody compared with ₂-integrin subunit, densitometric analysis indicated that this activation was significant (57.7 ± 8.4% increase compared with PLL, n = 3, P < 0.05). Caco-2 cells did not adhere to dishes coated with the IgG1 control antibody, preventing this comparison. As expected, on the basis of results from adhesion to -integrin subunit antibodies, adhesion to ₁-integrin subunit antibody also stimulated phosphorylation of FAK Y397, -Y576, and -Y925, and ERK activation. Whereas adhesion to integrin subunit antibodies does not necessarily mimic adhesion to matrix proteins, these results suggest that in Caco-2 cells either ₁₁- or ₂₁-integrin are capable of activating FAK, Src, and ERK.

We also examined the role of each integrin in signaling initiated by collagen IV adhesion by selectively blocking either the ₁₁- or ₂₁-integrin with functional antibodies to the ₁- or ₂-integrin subunit before adhesion. We observed collagen IV stimulation of ERK activation and phosphorylation of FAK Y397, -Y576 and -Y925 after blockade of either ₁₁- or ₂₁-integrin (Fig. 5B), suggesting again that both integrins are capable of initiating FAK, Src, and ERK activation by collagen IV. Because the combination of ₁- and ₂-integrin subunit antibodies inhibited adhesion to collagen IV by 97% (Fig. 3A), the effect of the combined antibodies on signaling from collagen IV could not be assessed in this experiment. A potential concern regarding interpretation of this experiment might be that crosslinking of integrins with antibody in solution initiates signaling. Stimulation of ERK activation and FAK phosphorylation did not occur in cells adherent to PLL (which mediates cell adhesion independently of integrins) treated with antibody to either integrin subunit compared with PLL adherent cells treated with the control antibody IgG1, suggesting that this does not occur in Caco-2 cells under these conditions (Fig. 5B).

Effect of Src inhibition on Caco-2 cell spreading and migration on collagen IV. We investigated the role of Src activation initiated by collagen IV in regulating Caco-2 cell spreading by treatment with PP2 or by coexpressing dominant-negative Src along with a lacZ expression plasmid to indicate transfected cells. Both expression of dominant-negative Src and treatment of cells with PP2 significantly inhibited cell spreading (Fig. 6, A and B). One potential Src-dependent regulator of Caco-2 migration is p130^Cas. This adaptor protein is rapidly tyrosine phosphorylated after collagen IV adhesion (46) and regulates cell spreading and migration in some cell types (12, 27). Whereas p130^Cas tyrosine phosphorylation is strongly inhibited by treatment of cells with PP2 (Fig. 2B), neither overexpression of p130^Cas nor expression of SD-p130^Cas, which strongly inhibits tyrosine phosphorylation of p130^Cas in transfected Caco-2 cells (46), significantly affected cell spreading (Fig. 6C).

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effect of Src inhibition on Caco-2 cell spreading. A: cells were cotransfected with dominant-negative Src along with 3.1 H lacZ as described in MATERIALS AND METHODS. Cells were allowed to spread on collagen IV for 2 h and were then fixed and stained for lacZ expression. The percentage of lacZ-positive cells that had spread was then determined from 3 independent experiments in a blinded count as described in MATERIALS AND METHODS. *P < 0.05 compared with pUSE transfected cells. B: Caco-2 cells were treated with 10 µM PP2 before collagen IV adhesion. After 2-h spreading on collagen IV in the presence of PP2, cells were fixed and stained as described in MATERIALS AND METHODS. Measurements of cell size were based on at least 100 randomly chosen individual cells that were not in contact with other cells for each condition in 3 independent experiments. *P < 0.05 compared with control. C: cells were cotransfected with SSR control vector, wild-type p130Cas, or SD-p130Cas along with 3.1 H lacZ as described in MATERIALS AND METHODS. Cell spreading was determined as described in MATERIALS AND METHODS and A. SD-p130Cas, p130Cas with substrate domain deleted.

We confirmed our observations about the role of Src in cell spreading by measuring the effect of Src inhibition by PP2 in a model of cell migration that corresponds more closely to the in vivo movement of intestinal epithelial cells. PP2 strongly inhibited sheet migration of Caco-2 cells on collagen IV (54.2 ± 5.6% inhibition compared with control, P < 0.05; Fig. 7, A and B). Control experiments indicated a similar increase in cell number between control (41.1 ± 12.3% increase) and PP2 (46.5 ± 15.0% increase)-treated cells under the conditions used for these migration assays (data not shown). Although measurements of cell number are not necessarily a precise indicator of proliferation, this suggests that reduced cell proliferation is not responsible for the effect of PP2 on Caco-2 migration on collagen IV. If the effects of Src on cell migration were mediated via FAK Y925-dependent activation of ERK, it might be predicted that ERK inhibition by PD-98059 would have an effect on cell migration similar to PP2. Although the ERK inhibitor PD-98059 significantly inhibited sheet migration of Caco-2 cells on collagen IV (11.5 ± 3.9% inhibition compared with control, P < 0.05; Fig. 7, A and B), this inhibition was much lower than that observed with the Src inhibitor PP2 (P < 0.05). This suggests that Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.

View larger version (54K): [in this window] [in a new window]   Fig. 7. A: sheet migration of control, PD-98059 (20 µM), and PP2 (10 µM)-treated Caco-2 cells on collagen IV. Line indicates starting point of migration. Migrations were performed as described in MATERIALS AND METHODS. B: quantification of sheet migration of Caco-2 cells on collagen IV. Migration area was determined from 3 or more independent experiments as described in MATERIALS AND METHODS. *P < 0.05 compared with control; #P < 0.05 compared with control or PD-98059-treated cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The differential expression of integrins in intestinal epithelial cells (reviewed in Ref. 35) and basement membrane extracellular matrix proteins (reviewed in Ref. 8) along the intestinal epithelial crypt-villus axis in vivo suggests a potential role for cell-matrix interactions in regulating intestinal epithelial cell differentiation and migration. Additionally, the extracellular matrix influences restitution of mucosal wounds not only as a physical substrate but also by modulating the expression, organization, and activation of the relevant intracellular proteins and by modulating the expression and organization of receptors for soluble factors in the extracellular environment which influence cell migration (reviewed in Refs. 4, 19, 44). Effects of physical forces, such as repetitive deformation on intestinal epithelial cells, also appear to be matrix and integrin mediated (65). Several studies have demonstrated matrix composition changes (1, 11, 52), changes in matrix metalloproteinease expression (6, 25, 57), and increased apoptosis (38, 53) associated with coeliac disease, Crohn's disease, ulcerative colitis, and collagenous colitis, suggesting that perturbation of normal cell-matrix interactions may play an important role in some intestinal pathobiology. Our previous work (64) indicates a role for FAK and ERK activation in regulation of Caco-2 cell migration and that FAK is required for ERK activation by collagen IV (46). We sought to further elucidate the signal transduction pathways initiated in intestinal epithelial cells by collagen IV, a major component of the intestinal epithelial basement membrane in vivo, and to characterize their role in regulating Caco-2 intestinal epithelial cell ERK activation, cell spreading, and cell migration on this matrix protein.

The work described in this article suggests that Src kinase is an important regulator of ERK activation on collagen IV (Fig. 1) and that this activation can be mediated by either ₁₁- or ₂₁-integrin (Fig. 5). These results suggest that the mechanisms of ERK activation for ₁₁- and ₂₁-integrin in the Caco-2 cells studied here are different from those described in other cell types such as fibroblasts and endothelial cells. In wild-type dermal fibroblasts expressing both ₁₁- and ₂₁-integrins, but not in dermal fibroblasts from ₁-integrin subunit-deficient mice, Shc phosphorylation and activation of ERK is observed after adhesion to collagen I or a combination of collagen I and collagen IV (45). Additionally, crosslinking of ₁₁-integrin in MG-63 osteosarcoma cells (60) stimulates ₁₁-integrin association with caveolin and subsequent phosphorylation of Shc. In the Caco-2 cells studied here, collagen IV stimulated ERK activation via ₂₁-integrin (Fig. 5B) and adhesion of Caco-2 cells to ₂-integrin subunit antibody-coated dishes or to collagen IV via ₁₁-integrin-stimulated ERK activation independently of both caveolin, which is not expressed in the Caco-2 cells used in this study, and Shc phosphorylation, which does not occur in Caco-2 cells in response to either collagen I, collagen IV, fibronectin, or laminin-1 adhesion (46).

One proposed pathway for integrin activation of ERK after fibronectin adhesion involves association of Grb2 with Src-phosphorylated FAK Tyr(P)925 after association of Src with FAK Tyr(P)397, based on in vitro studies showing association of Grb2 with FAK Tyr(P)925 (49) and coimmunoprecipitation of Grb2 with transfected wild-type FAK but not FAK Y925 after fibronectin adhesion of HEK-293 cells (50). Although expression of FAK Y397F in HEK-293 cells inhibits fibronectin activation of ERK, expression of FAK Y925F actually increases ERK activation (50), presumably due to additional FAK-dependent pathways proceeding through FAK Tyr(P)397. Thus a direct role for Src phosphorylation of FAK Y925 in matrix activation of ERK has not been demonstrated. In other studies (32, 61), FAK-independent ERK activation by fibronectin in NIH3T3 fibroblasts is observed. Whereas our previous results (46) indicated FAK-dependent activation of ERK by collagen IV adhesion, they did not distinguish whether this activation required only FAK Y397 autophosphorylation or, additionally, required phosphorylation of FAK Y925 by Src. The results in this article provide direct evidence that an important signaling pathway for ERK activation by collagen IV in Caco-2 cells requires Src phosphorylation of FAK Y925 and suggest that FAK Y397 autophosphorylation is required for FAK Y925 phosphorylation by Src after collagen IV adhesion (Figs. 1 and 2). Because both ERK and phosphorylation of FAK Y925 are activated by adhesion to collagen IV when either ₁₁- or ₂₁-integrin is blocked by integrin subunit functional antibodies (Fig. 5B) and after adhesion to ₁- and either ₁- or ₂-integrin subunit antibody-coated dishes (Fig. 5A), our results suggest that adhesion mediated via either ₁₁- or ₂₁-integrin can stimulate ERK activation via FAK Y925 phosphorylation in Caco-2 cells.

The finding that collagen IV adhesion stimulates ERK activation in undifferentiated Caco-2 cells may also have implications for intestinal epithelial cell differentiation in vivo. As noted above, the activated forms of ERK1 and -2 are detected predominantly in the crypt region in which intestinal epithelial cells are undifferentiated and in which the ₁- and ₂-integrin subunits are highly expressed (2). Ding et al. (21) found that ERK activation was inversely correlated with cell differentiation in Caco-2 cells and that treatment with the MEK inhibitor PD-98059 enhanced differentiation of Caco-2 cells in response to sodium butyrate. Determining a potential role for ₁₁- and ₂₁-integrin-initiated ERK activation by collagen IV in regulating intestinal epithelial cell differentiation will be an interesting topic for future studies.

Results of the adhesion blockade studies suggested that ₁₁- or ₂₁-integrin can mediate Caco-2 cell adhesion to collagen IV. These studies also indicated that the ₃₁-integrin was not involved in collagen IV adhesion in Caco-2 cells, although it is possible that failure to observe any role in collagen IV adhesion resulted from the very low expression of this integrin in Caco-2 cells (Fig. 3B). Additionally, the cell-spreading studies (Fig. 4) suggested either ₁₁- or ₂₁-integrin can mediate Caco-2 cell spreading on collagen IV and that either integrin can stimulate FAK, Src, and ERK activation in response to collagen IV adhesion. These findings, along with the similar expression patterns of the ₁₁- and ₂₁-integrins in the human intestinal epithelium in vivo (7), the lower expression of ₃₁-integrin in intestinal crypt cells in vivo (35) and the failure thus far to detect an overt phenotype relating to intestinal epithelial cells in both ₁-integrin subunit null mice (23) and ₂-integrin subunit null mice (15, 26) suggest that the function of ₁₁- and ₂₁-integrins in the human intestinal epithelium may be at least partially redundant. Although dextran sodium sulfate-induced colitis in mice was ameliorated in ₁-integrin subunit-deficient mice and by blockade of ₁₁-integrin with an ₁-integrin subunit-blocking antibody, this appeared to result from inhibition of monocyte rather than intestinal epithelial cell function (30). Whereas mutagenesis studies (28, 29) in the mouse mammary epithelial cell line NMuMG-3 indicate that different signal-transduction pathways may be initiated by the cytoplasmic regions of the ₁- and ₂-integrin subunits, any unique roles for ₁₁- and ₂₁-integrins in intestinal epithelial cells remain to be identified.

In Caco-2 cells, the ₂₁-integrin also acts as a receptor for laminin-5 (39). This previous study also suggested that the ₁₁-integrin in Caco-2 cells might act as a receptor for laminin-1, because ₁-integrin antibody, but not antibodies to the ₂-, ₃-, ₆-, and ₄-integrin subunits, inhibited adhesion to laminin-1. In vivo, however, laminins-1 and -5 (9, 31) are expressed in the villus but not in the crypt region in which ₁₁- and ₂₁-integrins are expressed. Both ₁₁-integrin, in PC12 adrenal pheochromocytoma cells, and ₂₁-integrin, in HT 1080 fibrosarcoma cells (16), have been shown to act as receptors for laminin-2, which is expressed in the basement membrane of the crypt of the adult small intestine (9). It is not known, however, whether these integrins act as receptors for laminin-2 in intestinal epithelial cells. Intestinal epithelial crypt cells may also make contact with collagen I in intestinal epithelial wounds that penetrate to the interstitial matrix below the basement membrane. We have previously observed that collagen I also strongly initiates FAK and ERK activation at similar levels as collagen IV in Caco-2 cells (46), suggesting a possible role for the signaling pathways described here in mucosal healing under these conditions. In vitro studies (55) have indicated that the I domain of the ₂-integrin subunit binds more strongly to fibrillar collagens, including collagen type I, compared with the basement membrane collagen type IV, whereas the I domain of the ₁-integrin subunit has the opposite binding preferences. This would be consistent with our results suggesting a more important role for ₁₁-integrin in mediating adhesion and spreading of Caco-2 cells on collagen IV (Figs. 3 and 4). It will be of interest to determine the relative contributions of the ₁₁- and ₂₁-integrins in adhesion and spreading of Caco-2 cells on collagen type I.

Our results suggested an important role for Src kinase in collagen IV regulation of both Caco-2 cell spreading and migration (Figs. 6 and 7). Although both PP2 and PD-98059 strongly inhibit ERK activation (Fig. 1) and ERK activation by collagen IV requires Src kinase-dependent phosphorylation of FAK Y925 (Fig. 2), Src kinase inhibition by PP2 has a much greater inhibitory effect on Caco-2 migration than ERK inhibition by PD-98059. This suggests that Src kinase regulates Caco-2 cell migration on collagen IV primarily through ERK-independent pathways. Potential downstream mediators of the ERK-independent pathways by which Src kinase regulates Caco-2 cell migration include the adaptor proteins p130^Cas and paxillin, both of which are rapidly tyrosine phosphorylated in Caco-2 cells after collagen IV adhesion (46). Whereas the role of paxillin in ERK activation by collagen IV has not been addressed, expression of dominant-negative p130^Cas did not affect ERK activation by collagen IV in Caco-2 cells (46). Although this would be consistent with a role for p130^Cas in ERK-independent regulation of migration by collagen IV in Caco-2 cells and p130^Cas has been observed to regulate cell spreading and migration on fibronectin in other cell types (12, 27), neither expression of a dominant-negative p130^Cas nor overexpression of wild-type p130^Cas (Fig. 6B) affected cell spreading on collagen IV. Although results from cell spreading studies do not necessarily apply to cell migration, this suggests that p130^Cas may not be an important regulator of Caco-2 cell migration on collagen IV.

The nature of the interaction between FAK and Src also requires clarification. Whereas our results indicate that Src-dependent phosphorylation of FAK Y925 requires FAK Y397 autophosphorylation, they do not address whether FAK is required for Src activation per se. Work in Src-deficient and FAK-deficient fibroblasts suggests a complex relationship between FAK and Src in regulation of integrin-mediated signaling events (13, 58). In Src-deficient fibroblasts transfected with various Src mutants, Src kinase activity is essential for integrin-mediated migration and cell spreading on fibronectin, although association of Src with FAK is not required for cell spreading or for Src phosphorylation of p130^Cas and paxillin in this system. As we observed for Caco-2 cells after adhesion to collagen IV (Fig. 1A), there is only a moderate increase in Src Y416 phosphorylation after cell adhesion to fibronectin in this system (13). Additionally, Src-dependent phosphorylation of p130^Cas is not affected in FAK-null fibroblasts (58). It is of interest that TNF- has also been demonstrated recently to regulate intestinal epithelial cell migration by a Src-dependent mechanism involving FAK (17). This suggests that there may be common features in regulation of intestinal epithelial migration by matrix and other factors present in the intestinal epithelial milieu.

Matrix is only one of many factors that regulate intestinal epithelial cell migration. In addition to TNF-, as noted above, polyamines (37, 59) and several growth factors present in the intestine, including the transforming growth factor- and transforming growth factor- families, insulin-like growth factors, and trefoil factors, among others, have been identified as potential regulators of intestinal epithelial cell migration (reviewed in Ref. 20 and references cited therein). Previous work (5) has shown that epidermal growth factor appears to exert a matrix-specific effect on Caco-2 migration by modulation of integrin expression and organization, and we have also observed that collagen IV enhances the ability of epidermal growth factor to activate ERK in Caco-2 cells (data not shown). Additionally, matrix has been observed to regulate the proliferative response of enterocytes to growth factors (62). Determining how these diverse factors cooperate to regulate cell migration and which play an important role in vivo in both normal and pathobiological conditions will be important subjects for future studies.

In conclusion, we have observed that Caco-2 cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires Src kinase phosphorylation of FAK Y925 and that collagen IV can stimulate FAK, Src, and ERK activity in Caco-2 cells via either ₁₁- or ₂₁-integrin. Additionally, our results suggest that adhesion and cell spreading of Caco-2 cells on collagen IV can be mediated by either ₁₁- or ₂₁-integrin and that Src kinase regulates migration on collagen IV, primarily by ERK-independent pathways. Whereas in vitro cell culture studies should be interpreted with caution, the results described in this article and our previous observations (46, 64) suggest that FAK, Src, and ERK activation by intestinal epithelial basement membrane matrix proteins may play important roles in regulating intestinal epithelial cell migration.

	ACKNOWLEDGMENTS

 
We thank the following people for technical assistance and generous gifts of expression vectors: Dr. David Schlaepfer (The Scripps Research Institute, La Jolla, CA) for HA-FAK, HA-FAK Y397F, and HA-FAK Y925F; Dr. Christopher Marshall (Institute of Cancer Research, London, UK) for myc-tagged ERK2; Drs. Tetsuya Nakamoto and Hisamaru Hirai (University of Tokyo) for expression vectors with HA-tagged p130^Cas, HA-tagged substrate domain deleted-p130^Cas (SD-p130^Cas), and the control vector pSSR; and Denise Besserts for help with microscopy.

GRANTS

This research was supported, in part, by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-60771 and a Veterans Affairs Merit Award (to M. D. Basson).

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. Basson, Chief, Surgical Service (11 Surg), John D. Dingell Veterans Affairs Medical Center, 4646 John R Street, Detroit, MI 48201-1932 (E-mail: marc.basson{at}med.va.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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