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Am J Physiol Gastrointest Liver Physiol 294: G906-G913, 2008. First published January 31, 2008; doi:10.1152/ajpgi.00043.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Bile acids modulate tight junction structure and barrier function of Caco-2 monolayers via EGFR activation

Francesco Raimondi,1 Pasquale Santoro,1 Maria Vittoria Barone,2 Serena Pappacoda,1 Maria Luisa Barretta,1 Merlin Nanayakkara,2 Carmela Apicella,1 Letizia Capasso,1 and Roberto Paludetto1

1Division of Neonatology, Department of Pediatrics, and 2Department of Pediatrics and European Laboratory for the Investigation of Food-Induced Diseases, "Federico II" University, Naples, Italy

Submitted 24 January 2007 ; accepted in final form 30 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intestinal and systemic illnesses have been linked to increased gut permeability. Bile acids, whose luminal profile can be altered in human disease, modulate intestinal paracellular permeability. We investigated the mechanism by which selected bile acids increase gut permeability using a validated in vitro model. Human intestinal Caco-2 cells were grown in monolayers and challenged with a panel of bile acids. Transepithelial electrical resistance and luminal-to-basolateral fluxes of 10-kDa Cascade blue-conjugated dextran were used to monitor paracellular permeability. Immunoprecipitation and immunoblot analyses were employed to investigate the intracellular pathway. Redistribution of tight junction proteins was studied by confocal laser microscopy. Micromolar concentrations of cholic acid, deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased dextran flux in a reversible fashion. Coincubation of 50 µM CDCA or DCA with EGF, anti-EGF monoclonal antibody, or specific src inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) abolished the effect. A concentration of 50 µM of either CDCA or DCA also induced EGF receptor phosphorylation, occludin dephosphorylation, and occludin redistribution at the tight junction level in the same time frame and in a reversible fashion. We conclude that selected bile acids modulate intestinal permeability via EGF receptor autophosphorylation, occludin dephosphorylation, and rearrangement at the tight junction level. The effect is mediated by the src family kinases and is abolished by EGF treatment. These data also support the role of bile acids in the genesis of necrotizing enterocolitis and the protective effect of EGF treatment.

epithelial growth factor receptor; epidermal growth factor; intestinal permeability

TIGHT JUNCTIONS (TJs) are the main structures limiting molecular diffusion across the intestinal epithelium. Failure of barrier function may result from increased paracellular permeability and can lead to passage of antigens or microbes; this has been linked to the pathogenesis of different illnesses, such as sepsis or food allergy. In particular, necrotizing enterocolitis (NEC), the most frequent and feared gastrointestinal emergency in premature newborns, although multifactorial in origin, has been related to both intestinal barrier dysfunction and to bile acid (BA) metabolism (9, 14, 15). A consistent body of evidence suggests that BAs are able to interact with the cell's plasma membrane, leading to ligand-independent activation of epithelial growth factor receptor (EGFR) and intracellular signaling (3, 22, 30). Previous papers have also suggested a role for BAs in modulation of transepithelial permeability (1). Because EGFR may play a role in TJ rearrangement (4), we have hypothesized that BAs, EGFR, TJ rearrangement, and increased paracellular permeability may indeed be connected.

In this study, using the in vitro model of Caco-2 cells grown in monolayers, we provide evidence of a role for BAs in modulating intestinal paracellular permeability by the rearrangement of occludin, a structural protein of TJs (16). We demonstrate that the effect is EGFR dependent and is prevented by EGF treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Cell culture chemicals were obtained from GIBCO-Life Technologies (Milano, Italy). Cascade blue-dextran (10 kDa) was purchased from Invitrogen (Carlsbad, CA). Unconjugated bile acids, atropine, and Texas red-phalloidin were purchased from Sigma (St. Louis, MO). Trypan blue and all other reagents of analytic grade were also purchased from Sigma.

Proteins were determined by the protein assay from Bio-Rad Laboratories (Munich, Germany). ECL-Plus was from Amersham Biosciences UK. Protein inhibitor cocktail tablets were from Roche Diagnostics (Mannheim, Germany).

Antibodies. Antibodies used were anti-occludin (H279; rabbit polyclonal), anti-EGFR (1005; rabbit polyclonal; used for Western blots), clone 528 (mouse monoclonal; used for both inhibition experiments and immunoprecipitation), anti-phosphotyrosine (PY-99, mouse monoclonal), anti-ERK1/2 (mouse monoclonal), and protein A/G PLUS-agarose. All of the above antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Mouse anti-occludin-FITC was from Zymed Laboratories (San Francisco, CA). Secondary antibodies (both anti-rabbit and anti-mouse) were from Amersham Biosciences, UK. Anti-src (mouse monoclonal), anti-phospho-src (rabbit monoclonal), anti-ErbB-2 (rabbit polyclonal), and anti-phospho-ErbB-2 (rabbit polyclonal) were from Cell Signaling Technology (Danvers, MA). The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002, the src family kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2), and the MEK inhibitor PD-98059 were from Alexis Biochemicals (Vinci, Italy).

Cell culture. Human Caco-2 intestinal cells were purchased from the "Istituto Zooprofilattico della Lombardia e dell'Emilia" (Brescia, Italy). Cells were grown as previously described (20, 21) in DMEM containing 25 mmol/l glucose and supplemented with 10% FBS, 1% nonessential amino acids, 2 mmol/l L-glutamine, 1% penicillin-streptomycin, and 1% sodium pyruvate. Cells were maintained in a humidified atmosphere (95%) of 5% CO2 in air at 37°C. Single cell suspensions were obtained from 70 to 80% confluent cultures by incubation with 0.05% trypsin and then seeded at 105 cells/cm2 onto 13- or 25-mm glass coverslips, detachable polycarbonate microporous cell culture inserts (Snapwells, 12-mm diameter, 0.4-mm pore size; Costar, Cambridge, MA), and 24-well plates (multiwell; Becton & Dickinson, Franklin Lakes, NJ) according to experimental needs. Because vectorial electrolyte transport requires cells to grow in a polarized fashion with structured intercellular TJs, Caco-2 cells need to be cultured for at least 21 days before experiments (~11–13 days after confluence). Before experiments, cells were starved overnight in a low-serum (0.5%) medium. Conventional medium was replaced by phenol red- and serum-free medium to avoid interferences with fluorescent tracer's measurements. The apical side of human intestinal cell line Caco-2 monolayers was challenged with chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), cholic acid (CA), and deoxycholic acid (DCA).

Cell viability assays. BA cytotoxicity was investigated using two-well-validated assays. Lactate dehydrogenase release and the Trypan blue exclusion tests were performed as previously described (19).

Transepithelial electrical resistance experiments. Transepithelial electrical resistance (TEER) over a 24-h period was measured as previously described (19). Briefly, after we removed the Snapwells from the incubator and allowed a 10-min acclimatizing period under a sterile hood, we placed the entire Snapwell into a resistance chamber (Snap-Endhom; World Precision Instruments) connected to a voltmeter (Millipore, Billerica, MA). A 1.0-cm2 planar electrode was used for all measurements. The screw cap allowed the planar electrode to remain at the same distance from the monolayers in repeated measurements. Monolayers with a basal reading between 350 and 450 {Omega}/cm2 were used for this study.

Paracellular permeability assays. The apical sides of Snapwell plates were preincubated with 50 µM of a selected BA for 10 min to allow TJ rearrangement. Then 2 mg/ml of Cascade blue-Dextran (10 kDa) was added. After 30 min, basolateral compartment medium was replaced with fresh medium and collected, and the amount of diffused marker was measured with a Perkin-Elmer (Wellesley, MA) 2000 fluorescence spectrophotometer (excitation wavelength = 365 nm, emission wavelength = 440 nm). After 120 min, the basolateral compartment medium was collected again and analyzed as described above.

Assays with inhibitors. The addition of 50 µM BAs was preceded by a 20-min preincubation with either 1) the anti-EGFR blocking antibody (2 µg/ml; clone 528), 2) the selective inhibitor of src family kinases (10 µM PP-2; prepared as 10 mM stock solution and stored at –20°C), 3) the MAPK inhibitor PD-098059 (50 µM; prepared as 25 mM stock solution in DMSO and stored at –20°C), 4) the PI3-kinase inhibitor LY-294002 (25 µM, prepared as a 1,000x stock solution), or 5) atropine, a muscarinic receptor inverse agonist (0.1 µM). EGF (1.5, 15, and 80 nM) was added together with the appropriate BA.

Immunoprecipitation and immunoblot analyses. After 10, 20, and 120 min of incubation with the appropriate BA, monolayers were washed twice and scraped with ice-cold PBS without calcium and magnesium. All subsequent steps were performed at 4°C. EGFR was extracted by lysis of the cells on a rotating shaker for 30 min in Tris·HCl-Triton X-100 buffer (50 mM Tris·HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100, 50 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 1 mM Na3VO4, with protease inhibitors cocktail). Occludin and Src were extracted by P-40 (NP-40) extraction buffer (25 mM HEPES, 150 mM NaCl, 4 mM EDTA, 25 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 1% NP-40, with protease inhibitors cocktail). Suspensions were centrifuged again for 30 min, and the pellet was discarded. The supernatants were then incubated overnight on a rotating shaker with either anti-EGFR antibody (Santa Cruz, clone 528, 1.5 µg/mg), anti-occludin (3 µg/mg protein), or anti-src (mouse; 1:100). After the addition of protein A/G PLUS-agarose (120 min on a rotating shaker), samples were centrifuged (1 min at 14,000 rpm), and the pellet was washed three times with the appropriate ice-cold lysis buffer, resuspended in sample buffer, and boiled for 5 min. After protein A/G PLUS-agarose was discarded by centrifugation, samples were run on a 10% (occludin and src immunoprecipitates) or a 7.5% (EGFR and ErbB-2 immunoprecipitates) SDS-PAGE, and proteins were transferred onto a nitrocellulose membrane. Blocking was performed for 2 h with 5% non-fat dried milk, and membranes were incubated in 3% BSA-Tris-buffered saline-Tween 20 (TBST) with anti-phosphotyrosine (1:1,000) or anti-phospho-src (rabbit, 1:1,000). Membranes were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibody (45 min, 1:6,000 or 45 min, 1:2,000 for anti-phosphotyrosine and anti-phospho-src, respectively) in 3% non-fat dried milk-TBST and detected with ECL-Plus according to the manufacturer's instructions. Blots were stripped (100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris·HCl, pH 7.6) for 30 min at 60°C and then washed in TBST before they were reprobed with anti-EGFR (1:1,000; rabbit, Clone 1500), anti-occludin (1:500), or anti-src (rabbit, 1:1,000) overnight in 3% non-fat dried milk on an orbital shaker. Membranes were washed again in TBST and incubated with horseradish peroxidase-conjugated secondary antibody (45 min, 1:5,000 or 45 min, 1:2,000, respectively, for anti-occludin and anti-src) in 3% non-fat dried milk-TBST and detected with ECL-Plus.

ERK1/2 activation. Differentiated Caco-2 monolayers were treated with 50 µM CDCA. After 10, 20, and 120 min, cells were washed twice and scraped with ice-cold PBS without calcium and magnesium. All subsequent steps were performed at 4°C. Lysis was performed for 30 min on a rotating shaker in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0,25% sodium deoxycholate, 1% NP-40, with the protease inhibitors cocktail). Suspensions were centrifuged again for 30 min, and the pellet was discarded. Samples were resuspended in sample buffer, boiled for 5 min, and then loaded onto a 10% SDS-PAGE gel. Proteins were transferred onto a nitrocellulose membrane. Blocking was performed for 2 h with 5% BSA-TBST, and membranes were incubated with the primary antibodies (1:1,000) for 2 h followed by incubation with the appropriate secondary antibody for 45 min at room temperature. ECL-Plus detection of the immunoreactive bands was performed according to the manufacturer's instructions.

Confocal microscopy. Caco-2 cells were grown on glass coverslips, challenged with 50 µM CDCA for 20 or 120 min, and then rinsed three times with 0.01 M PBS (pH 7.4). Cells were fixed in 3% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked in 1% FBS-PBS for 30 min at room temperature, and then rinsed again in PBS. Staining with monoclonal fluorescent FITC anti-human occludin antibody (1:50; Invitrogen) was performed overnight at 4°C in a dark, humid chamber. Staining with Texas red-phalloidin (1:1,000) was performed for 45 min at room temperature. Monolayers were washed three times for 10 min in 0.01 M PBS (pH 7.4), mounted, and then examined with a confocal fluorescence microscope (LSM 510; Zeiss, Leipzig, Germany).

Statistical analysis. Results are presented as means ± SD. The data were analyzed by using a commercial software package (GraphPad Prism; GraphPad Software, San Diego, CA). All data sets passed the normality test; one-way ANOVA, together with Tukey's post hoc test, was used to compare different groups of data. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Selected BAs induce TEER decrement and increase paracellular permeability. As previously reported (23), none of the BAs tested is toxic at concentrations of 50–100 µM. A nonsignificant increase of lactate dehydrogenase release and of the number of Trypan blue-positive cells was observed at 200 µM for all studied BAs, except UDCA. Concentrations above 400 µM were toxic for the cells (not shown). As shown in Fig. 1A, 50 µM CDCA, DCA, and CA, but not UDCA, induced a transient and statistically significant (P < 0.05; n = 5) TEER decrement after 20 min that reverted within 120 min. No further changes were observed up to 24 h.


Figure 1
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  		Fig. 1. Selected bile acids (BAs) induce transepithelial electrical resistance (TEER) decrement and increase paracellular permeability. A: time course of 50 µM luminally applied BAs. Ursodeoxycholic acid (UDCA) was not effective, but chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and cholic acid (CA) induced a transient TEER decrement, which peaked at 20 min. Within 120 min, TEER returned to baseline. Data are presented as percent decrease of TEER; n = 10. *P < 0.05 vs. control. B: spectrophotometric analysis of dextran fluxes (2 mg/ml; applied as described in MATERIALS AND METHODS). CDCA, DCA, and CA induced a paracellular passage of 10-kDa Cascade blue-conjugated dextran. The effect occurred within 30 min of stimulation and returned to baseline within 120 min. n = 10. 20' and 120', 20 and 120 min, respectively. *P < 0.05 vs. control (CTR).

 
Because TEER decrement is only an indirect method to study TJ efficiency, we tested CDCA, DCA, and CA for their ability to induce a paracellular flux of macromolecules, using a 10-kDa fluorescent dextran. Figure 1B shows that all tested BAs, with the exclusion of UDCA, were able to promote the apical-to-basolateral passage of dextran. Similarly to the TEER decrement, paracellular permeability increases within 30 min of stimulation and returns to control levels within 120 min.

BAs decrease TEER and increase paracellular permeability via EGFR activation. CDCA and DCA, the most effective BAs tested in the previous set of experiments, were used in further studies. Both the anti-EGFR blocking antibody (anti-EGFR 528) and 15 nM EGF inhibited BA-induced TEER decrement (Fig. 2A) and the passage of Cascade blue-dextran from the apical-to-basolateral side of the chamber (Fig. 2B). EGF (1.5 nM) was not effective; however, 80 nM EGF was as effective as 15 nM EGF (not shown).


Figure 2
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  		Fig. 2. BAs decrease TEER and increase paracellular permeability via epithelial growth factor receptor (EGFR) activation. A: both anti-EGFR blocking antibody (clone 528, 2 µg/ml) and 15 nM EGF blocked CDCA and DCA (50 µM)-induced TEER decrement (at 20 min). Results were normalized to the TEER value of the control; n = 5. *P < 0.05 vs. control (CTRL). B: paracellular fluxes of dextran (2 mg/ml), measured as described in Paracellular permeability assay were abolished in the presence of both anti-EGFR (clone 528, 2 µg/ml) and 15 nM EGF.

 
BAs induce EGFR phosphorylation and occludin dephosphorylation in a similar fashion. As shown in Fig. 3A, the addition of CDCA increased EGFR phosphorylation, with a peak at 20 min. After 120 min, the phosphorylation level returned to a level similar to that of control. Although results with DCA were similar to those with CDCA, UDCA induced negligible EGFR phosphorylation (Fig. 3B).


Figure 3
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  		Fig. 3. BAs induce EGFR phosphorylation. Immunoblots of Caco-2 cells lysates are with anti-phosphotyrosine antibodies (p-Tyr) or anti-EGFR antibody (Tot), immunoprecipitated with anti-EGFR monoclonal antibody (clone 528). A: time course of CDCA (50 µM)-induced EGFR activation. Total EGFR (170 kDa) was revealed with a polyclonal antibody after stripping the blots. Activated EGFR was revealed with anti-phosphotyrosine monoclonal antibody. EGFR phosphorylation peaked at 20 min and returned to baseline within 120 min. B: total EGFR and EGFR activation at 20 min induced by DCA, CDCA, and UDCA (50 µM). Only CDCA and DCA elicited an increment of EGFR phosphorylation, whereas UDCA had no effect. Results are representative of 3 separate experiments, showing similar results. Numbers at bottom of blots report densitometric data and represent the increase of tyrosine phosphorylation vs. controls and are normalized for the total protein amount.

 
Occludin, which is required in a phosphorylated state at the TJ level (6), became dephosphorylated after monolayers were treated with CDCA (Fig. 4A). The effect was transient and occurred with a time frame similar to the TEER decrement, the increase of paracellular permeability, and the EGFR phosphorylation. Again, DCA stimulation of Caco-2 monolayers gave results similar to those for CDCA, whereas UDCA stimulation was not effective (Fig. 4B).


Figure 4
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  		Fig. 4. BAs induce occludin dephosphorylation. Immunoblots of Caco-2 cells lysates are with anti-phosphotyrosine antibodies (p-Tyr) or anti occludin (Tot), immunoprecipitated with anti-occludin monoclonal antibody. A: time course of occludin phosphorylation induced by 50 µM CDCA treatment. Total occludin was revealed after stripping the membranes, and phosphorylated occludin was revealed with anti-phosphotyrosine monoclonal antibody. CDCA elicited a transient dephosphorylation of occludin at 20 min. After 120 min, occludin returned to levels similar to control. B: at 20 min, DCA and CDCA (50 µM) induced a similar decrement of occludin phosphorylation, whereas UDCA (50 µM) was similar to control. Results are representative of 3 separate experiments showing similar results. C: both anti-EGFR and EGF inhibited the dephosphorylation of occludin induced by CDCA. The stimulation of Caco-2 monolayers with EGF alone or anti-EGFR had no effect on occludin phosphorylation. Numbers at bottom of blots report densitometric data and represent the increase of tyrosine phosphorylation vs. the control and are normalized for the total protein amount. Results are representative of 3 separate experiments showing similar results. D: densitometric analysis of 3 similar experiments in the form of a bar chart comparing the effects of CDCA alone, EGF alone, and their combination on EGFR phosphorylation.

 
Immunoblot of Caco-2 cell lysates with anti-phosphotyrosine antibodies or anti-occludin immunoprecipitated with anti-occludin monoclonal antibody demonstrated that both anti-EGFR blocking antibody and EGF inhibited the dephosphorylation of occludin induced by 50 µM CDCA (Fig. 4C). The stimulation of Caco-2 monolayers with EGF alone or anti-EGFR did not induce occludin dephosphorylation. These results are in agreement with both the TEER decrement and the increased paracellular permeability shown in Fig. 2.

The time course of EGFR activation in response to both EGF and EGF + CDCA is shown in Fig. 5. EGF induced a sustained and continuous increase in phosphorylation up to 120 min (Fig. 5A). Costimulation with both CDCA and EGF caused a mixed phosphorylation pattern, with a peak at 20 min and a still sustained phosphorylation after 120 min. Figure 5B shows the densitometric analysis of three separate experiments and compares the effects of CDCA and EGF alone and in combination on EGFR phosphorylation.


Figure 5
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  		Fig. 5. BAs and EGF trigger EGFR in a different fashion. A: time course of EGFR activation in response to EGF (15 nM) and EGF + CDCA (50 µM). Immunoblot of Caco-2 lysates immunoprecipitated with anti-EGFR monoclonal antibody is shown. Activated EGFR (p-Tyr) was revealed with anti-phosphotyrosine antibodies; EGFR (Tot) was revealed with anti-EGFR polyclonal antibody. Numbers at bottom of blots report densitometric data and represent the increase of tyrosine phosphorylation vs. the control and are normalized for the total amount of the protein. B: densitometric analysis of 3 separate experiments comparing the effect of CDCA, EGF, and their combination on EGFR phosphorylation. Phosphorylation is expressed as fold increase over the corresponding untreated sample. Results are means ± SD.

 
BA-induced activation of EGFR decreases TEER via the activation of src family kinases. Rearrangement of TJs can occur via several biochemical pathways, including MAPK (4), src (5), and PI3-kinase (25). Using selective inhibitors, we showed that, although pretreatment with PP-2, a src family kinases inhibitor, was able to abolish BA-induced TEER decrement, the inhibitors of both ERK1/2 (PD-98059) and PI3-kinase (LY-294002) had no effect (Fig. 6A). Unrelated to a permeability effect, we report that CDCA is able to induce a transient phosphorylation of ERK1/2 after 20 min on Caco-2 monolayers (not shown).


Figure 6
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  		Fig. 6. BAs decrease TEER and induce occludin dephosphorylation via the activation of Src family kinases. A: pretreatment of monolayers with the selective inhibitors of MAPK (PD-098059; 50 µM) and phosphatidylinositol 3-kinase (PI3-kinase; LY-294002; 25 µM) did not block TEER decrement. The selective inhibitor of src family kinases 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP-2) (10 µM) prevented TEER decrement; n = 5. *P < 0.05. B: blots for phosphorylated src (p-src) of cell lysates stimulated with 50 µM CDCA and immunoprecipitated for occludin revealed an increase of p-src recovered with occludin. Numbers at bottom of blots report densitometric data and represent the increase of tyrosine phosphorylation vs. the control and are normalized for the total protein amount.

 
Blots for phosphorylated src of cell lysates stimulated with 50 µM CDCA and immunoprecipitated for occludin revealed an increase of phosphorylated src recovered with occludin (Fig. 6B). Blots for total src gave similar results (not shown), indicating that, on CDCA stimulation, occludin binds a greater amount of activated src.

In separate experiments, we investigated the possible role of muscarinic receptors in BA-induced TEER decrement with the use of 0.1 µM atropine, a muscarinic receptor inverse agonist. Results, shown in Fig. 7, exclude a possible involvement of such receptors in BA activation of EGFR.


Figure 7
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  		Fig. 7. The muscarinic receptor is not involved in BA-induced activation of EGFR. Pretreatment of monolayers with 0.1 µM atropine did not affect BA-induced TEER decrement (n = 3). P < 0.05.

 
BAs induce a rearrangement of occludin at the TJ level. Confocal microscopy allows scanning thin planes of a sample along the z optical axis by controlled micrometer increments. Figure 8 shows the results of treatment of Caco-2 monolayers with 50 µM CDCA. As shown in Fig. 8A, the scanning from top to bottom of an untreated Caco-2 monolayer demonstrates a distinct, continuous line of fluorescence spanning through a limited number of sections (3 or 4), describing an homogenous occludin ring along the entire perimeter of the cells. Figure 8B shows a monolayer treated for 20 min with 50 µM CDCA. Most planes have a patchy organization of intracellular occludin, and its staining is not limited to some sections; however, it can be observed in almost all of the optical sections, suggesting that a significant occludin redistribution had occurred. Figure 8C shows the redistribution of occludin along the z-axis occurring after stimulation with 50 µM CDCA.


Figure 8
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  		Fig. 8. BAs induce a rearrangement of occludin at the tight junction level. Optical sections were along the z-axis (1 µm). A: untreated Caco-2 cells show a linear distribution of occludin along the cell perimeter restricted to sections 3 and 4. B: in monolayers incubated for 20 min with 50 µM CDCA, no organized structure is detected and fluorescence is scattered along all sections (x40 objective). DCA gave similar results (not shown). Results are representative of 5 separate experiments showing similar results. C: confocal fluorescence localization of occludin along the z-axis.

 
Results of a double-staining approach with anti-FITC-occludin antibody and Texas red-phalloidin are shown in Fig. 9. Figure 9 shows images of Caco-2 monolayers after 50 µM CDCA treatment and coincubation with EGF, anti-EGFR antibody, and PP-2. As shown and in agreement with results reported in Figs. 2, 4D, and 6A, 50 µM CDCA-induced occludin and actin rearrangement is inhibited by EGF, anti-EGFR antibody, and PP-2.


Figure 9
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  		Fig. 9. Effect of EGF, EGFR antibody, and PP-2 on occludin rearrangement at the TJ level. Treated Caco-2 monolayers were stained with both anti-FITC-occludin antibody and Texas red-phalloidin. The rearrangement of occludin and actin induced by the incubation of monolayer with 50 µM CDCA is inhibited by EGF, anti-EGFR, and PP-2.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
BAs play an important role in normal digestive processes, and they also have been used as therapeutic agents during recent decades (2, 15). The biological effects depend on the chemical structure of the individual BA and its concentration. In the millimolar range, CDCA and DCA may induce cell damage because of membrane solubilization and may promote the loss of cholesterol and phospholipids (13). At lower concentrations, BAs affect host defense (29), cause hydroperoxide generation, and impair the function of the electron transport chain in isolated rat liver mitochondria (26, 27). They may also function as immunosuppressive agents (17) and are able to induce apoptosis and carcinogenesis. DCA, in particular, stimulates the proliferation of colonic epithelium (11). BAs may also activate EGFR in human cholangiocyte cell line (30) and induce intracellular signaling in HCT-116 cells (22). Remarkably, the CDCA epimer UDCA does not exhibit any of these effects.

The standard concentration of 50 µM used in our experiments is relevant to most clinical situations. In fact, although the normal serum concentration of each specific unconjugated bile acid ranges between 0.2 and 1 µM, it may increase up to 150 µM in chronic cholestatic disease (24). Moreover, tissue concentrations of ~250 µM are common in cholestasis (12, 17).

The mechanisms underlying the multifaceted effects of bile acids are under intense investigations. Recently, via an in vivo rat model (9), BAs have been linked to intestinal epithelial barrier dysfunction and pathogenesis of NEC. It has also been reported to have a protective effect for EGF treatment in an experimental NEC model (9, 15).

In this study, we provide evidence that treatment with DCA and CDCA, but not UDCA, can induce EGFR phosphorylation in Caco-2 monolayers, resulting in an increased paracellular permeability via occludin dephosphorylation and cytoskeletal rearrangement at the TJ level.

EGFR is an intriguing molecule involved in many fundamental biological processes, such as cell proliferation, differentiation, tissue healing, cell cycle progression, and cancer. EGFR is a member of the ErbB family of receptor tyrosine kinases, which consist of an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular tyrosine kinase domain. Phosphorylation of the tyrosine kinase portion leads to receptor autophosphorylation and consequent homodimerization or heterodimerization with receptors of the same family and finally to protein activation and signaling. EGFR downstream effectors include ERK1/2, the PI3-kinase pathway, the src family of kinases, and the JAK and STAT pathways.

The link between BAs, EGFR, and increased paracellular permeability is provided by the blocking effect of anti-EGFR antibodies. It is not entirely clear whether this inhibition is ligand dependent because it is not known, to the best of our knowledge, whether binding of the 528 blocking antibody to the receptor is also able to inhibit the receptor autophosphorylation or whether it may have some effect on receptor mobility in the membrane, influencing the receptor dimerization and/or autophosphorylation, all critical events to distinguish between a ligand-dependent or -independent EGFR activation.

We also show that EGF and BAs trigger EGFR in a different fashion: BAs-induced activation is rapid, peaks at 20 min, and returns to baseline within 120 min (Fig. 3), whereas EGF-induced phosphorylation shows a sustained phosphorylation of the receptor up to 120 min (Fig. 5). When the receptor was stimulated with both EGF and CDCA, densitometry showed a mixed pattern of phosphorylation with a peak at 20 min and a still sustained phosphorylation at 120 min (Fig. 5). These results demonstrate that EGFR activation can be modulated in intensity and duration of activation depending on the nature of the stimulus. We speculate that this signal modulation may influence the cellular response. In other terms, EGFR may trigger different pathways depending on the modality of the activation, namely, either the BAs induced transient activation involved in permeability effects or the sustained EGF-mediated activation, initiating multiple cascades. The addition of EGF, the natural ligand, may stabilize the formation of dimers, inducing a prolonged signaling and in turn a different response. However, the mechanism by which EGF prevents the effects of BAs requires further experiments; e.g., we cannot exclude that EGF may have additional modes of action, such as an influence on receptor internalization.

We attempted to address the issue of how BAs could be linked to EGFR phosphorylation. Because EGFR may form heterodimers with other members of the ErbB family and because ErbB-2 is the preferred heterodimerization partner (8), we investigated the possibility that BAs could induce EGFR-ErbB-2 heterodimers. This hypothesis was abandoned when no band compatible with the ErbB-2 receptor was found in our EGFR coimmunoprecipitation experiments (not shown). We also considered the possibility that BAs induced signaling via muscarinic receptors and transactivation of EGFR, as described in others cell types (7, 28). We provide direct evidence against this hypothesis. In fact, atropine, a specific muscarinic receptor inverse agonist was not able to abolish TEER decrement (Fig. 7). The precise mode of interaction of BAs, EGF, EGFR, and its phosphorylation requires further studies and may well need physics and chemistry expertise's that are presently beyond our reach.

However, we have shed some light into the biochemical events coupling BA-induced EGFR activation to paracellular permeability. MAPK, src, and PI3-kinase are potential targets of EGFR, and they have been described to modulate TJs in a variety of cells types. In particular, MAPK may be transiently activated by BAs (4). The involvement of MAPK and PI3-kinase pathways was excluded by inhibition experiments (Fig. 6A). We found that only the specific inhibitor of the src family kinases, PP-2, was able to inhibit BA-induced TEER decrement. Previous papers described a role of nonreceptor tyrosine kinases in occludin phosphorylation/dephosphorylation and disassembly/reassembly at the TJ level, but conflicting data have been generated (6, 10, 25). Our data show that activated src coimmunoprecipitates with occludin and that the amount found in the immunoprecipitate varies in a time-dependent fashion, with a peak at 20 min after CDCA stimulation, suggesting that activated src plays a main role in TJ rearrangement (Fig. 6B).

The effect of BAs on TJ rearrangement was also studied by confocal microscopy. It has been described that TJs are connected to the actin cytoskeleton by zonula occludens proteins and cingulin. Other authors reported morphological changes of both actin and occludin during TJ rearrangement (16, 18). Our data clearly show that CDCA induces a marked redistribution of intracellular occludin. Untreated Caco-2 cells show a linear distribution of occludin along the cell perimeter. In monolayers incubated with 50 µM CDCA, no organized structure was detected, and fluorescence was distributed in a scattered and clustered fashion along all sections (Fig. 8). Accordingly, double staining with occludin and actin shows a regular immunofluorescence pattern in control samples, whereas in samples treated with CDCA the regular fluorescence pattern along the cell membrane is missing for both actin and occludin, and actin stress fibers can also be observed (Fig. 9). Treatment with EGF, EGFR, and PP-2 reversed the immunofluorescence pattern to the control level (Fig. 9).

In conclusion, EGFR is a crucial intermediate of BA modulation of Caco-2 TJs. BA-induced increase of paracellular permeability can be abolished by preventing EGFR activation with a specific inhibitor or with the engagement of the receptor by the proper ligand, indicating that either EGFR full activation or its complete inhibition blocks the BA effect on intestinal epithelial permeability.

Our in vitro data provide a useful paradigm to the recent in vivo observations that suggest a role for BAs in the genesis of NEC; at the same time, they offer a biological mechanism on the potential of EGF in the prevention and therapy of the most frequent gastrointestinal emergency in neonates (9, 15).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Santoro, Università degli Studi di Napoli "Federico II," Dipartimento di Pediatria, Divisione di Neonatologia, Via S. Pansini, 5 Napoli, Italy (e-mail: pasantor{at}unina.it)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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