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Originally published In Press as doi:10.1194/jlr.M500564-JLR200 on April 3, 2006

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Journal of Lipid Research, Vol. 47, 1416-1423, July 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation

Raquel Martín-Venegas, Sònia Roig-Pérez, Ruth Ferrer and Juan José Moreno1

Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain

Published, JLR Papers in Press, April 3, 2006.

1 To whom correspondence should be addressed. e-mail: jjmoreno{at}ub.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The small intestinal epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and differentiation. The intestinal epithelium constitutes a permeability barrier regulating the vectorial transport of ions, water, and solutes. Morphological changes during cell differentiation, as well as changes in the activity of brush-border enzymes and the expression of transport proteins, are well established. However, little is known about the arachidonic acid (AA) cascade underlying epithelial cell differentiation or its role in the development of epithelial barrier function. The main purpose of this study was to examine the activity of the high-molecular-weight phospholipases A2 (PLA2) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA2 activity, AA release, COX-2 expression, prostaglandin E2 (PGE2) production, and paracellular permeability were studied in preconfluent, confluent, and differentiated Caco-2 cell cultures. Our results show that Caco-2 differentiation induces a decrease in both calcium-independent PLA2 activity and COX-2 expression and, consequently, a decrease in AA release and PGE2 synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE2 to differentiated cells, at concentrations similar to those detected in nondifferentiated cultures, induces the disruption of epithelial barrier function. These results suggest that AA release by calcium-independent PLA2, COX-2 expression, and subsequent PGE2 release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.

Abbreviations: AA, arachidonic acid; BEL, bromoenol lactone; COX, cyclooxygenase; cPLA2, cytosolic phospholipase A2; iPLA2, Ca2+-independent phospholipase A2; PGE2, prostaglandin E2; PLA2, phospholipase A2; sPLA2, secretory phospholipase A2; TEER, transepithelial electrical resistance

Supplementary key words intestine • tight junctions • phospholipase A2 • cyclooxygenase • prostaglandins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The epithelium of the small intestine is a complex structure formed by numerous cell types involved in many homeostatic functions, such as nutrient digestion and absorption, defense from luminal contents, and peptide secretion (1). This epithelium is a highly dynamic system continuously renewed by a process involving cell proliferation and migration from the stem cell compartment, located at the bottom of the crypts, to programmed cell death and the extrusion of terminally differentiated cells from the tip-villus to the intestinal lumen (2, 3). The entire process of proliferation, differentiation, apoptosis, and extrusion occurs over 3 to 5 days depending on the species (3). Cell differentiation of the intestinal epithelium has been the subject of extensive study. The morphological changes during cell differentiation (4), the growth kinetics of epithelial cells (5), and the changes in brush-border enzyme activities during prenatal and postnatal development (6) are well established. However, little is known about the signals underlying cell differentiation along the crypt-villus axis.

The differentiation process of absorptive cells, the most abundant in the intestinal epithelium, can be modeled in vitro using Caco-2 cell cultures. Although cancerous in origin, these cells undergo a gradual differentiation process that takes place spontaneously once confluence has been reached and that is completed after 21–25 days in culture (7). Consequently, Caco-2 cells acquire morphological and functional polarity and show enzymatic activities, as well as protein and mRNA levels, of brush-border membrane enzymes and transporters highly comparable to those of mature enterocytes (7–9). In this sense, Caco-2 cell cultures provide an important tool for studying and obtaining greater insight into the regulation of human intestinal epithelial cellular processes such as enterocytic differentiation (4, 10).

The intestinal epithelium constitutes a permeability barrier that regulates the vectorial transport of ions, solutes, and water. Epithelial cells adhere to each other via three distinct adhesion systems: tight junctions, adherent junctions, and desmosomes. Of these, tight junctions are the most apical components and determine the paracellular permeability across the intestinal epithelium and the interface (fence) between apical and basolateral membrane domains (11). The existence of a relationship between cell differentiation and tight junction structure and permeability has been demonstrated in vivo in the small intestine; tight junction strand counts are higher in differentiated tip-villus cells than in nondifferentiated crypt cells (12).

Arachidonic acid (AA) mobilization from cellular phospholipids by phospholipase A2 (PLA2) is the rate-limiting step in the AA cascade. PLA2s are a growing family of enzymes with distinct substrate specificity, cofactor requirements, subcellular localization, and cellular function. Thus, mammalian PLA2 enzymes have been broadly classified into three groups: low-molecular-weight Ca2+-dependent secretory phospholipase A2 (sPLA2), high-molecular-weight Ca2+-dependent cytosolic phospholipase A2 (cPLA2), and high-molecular-weight Ca2+-independent phospholipase A2 (iPLA2). After AA release, the fatty acid is oxidized by oxygenases such as cyclooxygenases (COXs), lipoxygenases, or cytochrome P-450 monooxygenases. Thus, AA is the precursor for the further metabolism of many biologically active products, collectively termed eicosanoids (13). This large family of bioactive lipids have pleiotropic actions and modulate diverse physiological responses, including cell growth and differentiation and pathological conditions involving altered cellular proliferation. COX catalyzes the conversion of AA to prostaglandin H2, the immediate precursor of prostanoids. In eukaryotic cells, two COX isoforms have been identified and cloned (14): COX-1 is encoded by a housekeeping gene and is thought to be involved in the maintenance of physiological functions, whereas the expression of COX-2 is induced by various stimuli, including mitogens (15). The overexpression of COX-2 has been associated with a variety of proliferative diseases such as colorectal cancer (16). In this sense, enhanced prostaglandin E2 (PGE2) synthesis has been observed in human colon cancer tissue (17), thus increasing the tumorigenic potential of colonic epithelial cells (18).

Recently, we observed that cPLA2 and iPLA2 were expressed in Caco-2 cells. We also demonstrated that iPLA2 was involved in AA release and the subsequent prostaglandin production induced by serum. Moreover, we reported the first evidence that iPLA2 may be involved in the signaling pathways involved in the control of Caco-2 proliferation (19). The main objective of this study was to examine the activity of high-molecular-weight PLA2, a key enzyme of the AA cascade, during Caco-2 differentiation. Furthermore, we determined the role of PLA2s on Caco-2 differentiation, with particular emphasis on the relationship between PLA2 activities, AA release, COX-2 expression, AA metabolite production, and paracellular permeability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Tissue culture supplies, including Transwells, were obtained from Costar (Cambridge, MA). D-[2-3H]mannitol (specific activity, 30 Ci/mmol), [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]AA) (200–240 Ci/mmol), and phosphatidylcholine-L-{alpha}-1-palmitoyl 2-arachidonyl [arachidonyl-1-14C] (50–60 mCi/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). DMEM, heat-inactivated FBS, penicillin G, streptomycin, and trypsin/EDTA were purchased from BioWhittaker Europe (Verviers, Belgium). Bromoenol lactone (BEL) was obtained from Alexis Corp. (San Diego, CA). Nonessential amino acids, L-glutamine, BSA, ethidium bromide, acridine orange, aprotinin, leupeptin, diethyldithiocarbamic acid, PMSF, DTT, calcium ionophore A23187, PMA, and Igepal CA-630 as well as other chemicals were supplied by Sigma Chemical (St. Louis, MO). Polyclonal antiserum directed against COX-2 was acquired from Cayman Chemicals (Ann Arbor, MI). The enhanced chemiluminescence substrate, Supersignal West Dura Extended Duration Substrate, was provided by Pierce (Rockford, IL). All other reagents were of analytical grade.

Cell culture
Caco-2 cells were kindly provided by Dr. David Thwaites (School of Cell and Molecular Biosciences, University of Newcastle upon Tyne, UK). The cells (passages 107–116) were routinely grown in 75 or 150 cm2 plastic flasks at a density of 5 x 104 cells/cm2 and cultured in DMEM supplemented with 4.5 g/l D-glucose, 1% (v/v) nonessential amino acids, 2 mM L-glutamine, 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a modified atmosphere of 5% CO2 in air. Cells were harvested with trypsin/EDTA and passed to 60 or 100 mm diameter plastic dishes, 12 mm plastic clusters, or 12 mm polycarbonate filters (Transwells) with a pore size of 0.4 µm. For growth on filters, cells were seeded at a density of 4 x 105 cells/cm2. Growth medium was replaced twice per week and the day before the experiment. The experiments were performed in cells maintained for 3, 7, or 21 days in culture (preconfluent, confluent, or differentiated cells, respectively). Cell viability was estimated with ethidium bromide/acridine orange staining, as described by Parks et al. (20).

Transmission electron microscopy
Preconfluent, confluent, and differentiated monolayers grown on filters were prepared for transmission electron microscopy observation as described previously (21). Briefly, the filters were fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 2 h at 4°C and subsequently washed in 0.2 M PBS. The samples were postfixed in OsO4 (1% in 0.2 M PBS), dehydrated in acetone, and further embedded in Spurr's embedding medium (22). Ultrathin sections (60 nm; Ultracut; Reichert-Jung) were stained with uranyl acetate and lead citrate, according to Reynolds (23), and examined with a Philips EM 301 electron microscope operating at 80 kV. Sample processing and observation were carried out at the Serveis Cientificotècnics of the Universitat de Barcelona.

Sucrase activity
Sucrase, a brush-border marker enzyme of cell differentiation, was assayed in accordance with Dahlqvist (24) in homogenates of cells cultured in clusters. The results were expressed as mU/mg protein.

Paracellular permeability
Paracellular permeability was estimated from transepithelial electrical resistance (TEER) and unidirectional apical-to-basal D-mannitol fluxes in cells maintained on filters. TEER was determined as described by Hidalgo, Raub, and Borchardt (8). Monolayers grown on filters were gently washed by sequential transfer through four beakers containing 500 ml of modified Krebs buffer (room temperature) containing (mM) 137 NaCl, 5.4 KCl, 2.8 CaCl2, 1.0 MgSO4, 0.3 NaH2PO4, 10 D-glucose, and 10 HEPES/Tris (pH 7.4). The filters were then placed in culture wells containing 1.5 and 0.7 ml of modified Krebs buffer in the basolateral and apical compartments, respectively, and TEER was determined using a Millicell-ERS voltohmmeter (Millipore, Bedford, MA). Results are expressed as {Omega}/cm2 monolayer surface area. The resistance of the supporting membrane in filters was subtracted from all readings before calculations.

Unidirectional D-mannitol fluxes were determined in accordance with Thwaites et al. (25). After TEER determination, apical medium was replaced by the same volume of modified Krebs buffer containing 0.2 µCi/ml D-[2-3H]mannitol, and the cells were incubated for 5 min at 37°C. At the end of the incubation, basal medium was withdrawn and radioactivity was counted in a scintillation counter (1500 Tri-Carb®; Packard, Downers Grove, IL).

Protein determination
Total protein was measured by the Bradford method (26) using the Bio-Rad protein assay (Hercules, CA) with BSA as a standard.

PGE2 quantification
PGE2 concentration was determined in the culture medium of monolayers maintained in dishes. To stimulate the cells, the cultures were maintained overnight without FBS and then incubated for 3 h with 10% FBS or with 1 µM PMA. At the end of the incubation, 250 µl aliquots of the incubation medium were acidified with 1 ml of formic acid (1%) and PGE2 was extracted in ethyl acetate. After discarding the aqueous phase, the organic phase was evaporated under a stream of N2. The overall recovery for the extraction procedure, established by including [3H]PGE2 (DuPont-New England Nuclear, Boston, MA), was estimated to be 88 ± 2% (mean ± SEM, n = 4). PGE2 levels were determined with a PGE2 monoclonal enzyme immunoassay kit (Cayman Chemicals) according to the manufacturer's protocol. Finally, results are expressed as pg PGE2/mg protein.

Determination of PLA2 activity
PLA2 activity was assayed in cells maintained in clusters and stimulated as described above. At the end of the incubation with FBS or PMA, the medium was replaced by cold lysis buffer (200 mM Tris-HCl, 500 µg/ml aprotinin, 500 µg/ml leupeptin, 500 µg/ml PMSF, 400 µM NaF, and 400 µM Na3VO4) and the cells were scrapped off using a rubber policeman and briefly sonicated. PLA2 activity was determined as described previously (27) using phosphatidylcholine-L-{alpha}-1-palmitoyl 2-arachidonyl [arachidonyl-1-14C] as substrate (28). To discriminate between different PLA2s, 0.5 mM DTT was added to the PLA2 assay mixture to inactivate sPLA2 (29), and BEL was added to inactivate iPLA2 (30). These agents were solubilized in DMSO and diluted in medium. The final concentration of DMSO never exceeded 0.1%.

Incorporation and release of [3H]AA
In cultures maintained in clusters, the medium was removed and replaced by 0.5 ml of DMEM containing 0.1% fatty acid-free BSA and 0.1 µCi of [3H]AA (1 nM). Cells were incubated for 4 h and washed three times with 0.5% BSA-containing medium to remove unincorporated [3H]AA. Cell lipids were then extracted with ethyl acetate (5 ml) acidified with 1% formic acid. The organic phase was evaporated under a N2 stream, and the residue was resuspended in chloroform. Finally, an aliquot was separated by thin-layer chromatography in a solvent system consisting of chloroform-methanol-glacial acetic acid-water (100:30:35:3, v/v/v/v) to determine [3H]AA incorporated into phospholipids.

To determine [3H]AA release, [3H]AA-labeled monolayers were stimulated with FBS, PMA, or A23187 in the presence or absence of BEL (10 µM). The medium was then removed to determine the amount of radioactivity released from the cells. Cells were solubilized with Triton X-100, and the radioactivity was quantified. The release of [3H]AA was expressed as the percentage of total cell-incorporated radioactivity.

Measurement of the relative amounts of [3H]AA metabolized
To determine the amount of [3H]AA metabolized, cultures were incubated with [3H]AA (0.1 µCi) for 2 h, and cell supernatant was extracted with 5 ml of ethyl acetate acidified with 1% formic acid. The organic phase was evaporated and resuspended in 100 µl of chloroform and purified by ascending chromatography on silica thin-layer plates using as a solvent the mixture diethyl ether-hexane-acetic acid (60:40:1, v/v/v), which resolves AA from different oxidation metabolites. Thin-layer plates were exposed to rhodamine B (0.2%) and activated at 100°C. [3H]AA and [3H]AA metabolites fluoresce under ultraviolet light. Finally, the radioactive spots were quantified by scratching and scintillation counting.

Western blot analysis
Cells grown in plastic dishes were washed twice with ice-cold PBS, scraped off into PBS containing 2 mM sodium EDTA, and pelleted. The pellets were then sonicated in PBS containing 4 mM sodium EDTA, 500 µg/ml aprotinin, 500 µg/ml leupeptin, 500 µg/ml PMSF, and 400 µg/ml diethyldithiocarbamic acid and resuspended in lysis buffer containing 200 mmol/l Tris-HCl, 200 mmol/l NaCl, 2% Igepal CA-630, 400 µM NaF, and 200 µM DTT. Immunoblot analysis for COX-2 was performed as follows: 20 µg of protein from cell lysate was separated by 7.5% SDS-PAGE (31) and blotted for 1 h at a constant voltage of 100 V onto a nitrocellulose membrane (Trans-Blot; 0.4 µm pore size; Bio-Rad) using a MiniProtean II system (Bio-Rad). A prestained SDS-PAGE protein standard (Bio-Rad) was used as a molecular weight marker to check transfer efficiency. Membranes were blocked with 5% nonfat milk powder in PBS-0.1% Tween 20 for 1 h. A rabbit polyclonal antiserum directed against COX-2 was applied at a dilution of 1:2,000 for 1 h. The specificity of antibody used was established previously (32). The blots were washed several times in PBS-0.1% Tween 20 and incubated with goat anti-rabbit antibody at a 1:2,000 dilution for 1 h. For ß-actin immunoblotting, stripped membranes were overlaid with monoclonal anti-actin antibody (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by rabbit anti-mouse antibody (1:1,000; Santa Cruz Biotechnology). Antibody binding was visualized by an enhanced chemical luminescence technique using Supersignal West Dura Extended Duration Substrate (Pierce) and Kodak Bio-Max Light-2 film (Rochester, NY).

Data analysis
Results are expressed as means ± SEM. Differences between groups were assessed using either Student's t-test or one-way ANOVA followed by the least significant difference test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PLA2 activity and AA release during Caco-2 cell differentiation
After 21 days in culture, the cells showed a more developed and organized brush border and higher sucrase and TEER values (Fig. 1 ) compared with preconfluent and confluent monolayers, thus attaining a small intestine-like phenotype.


Figure 1
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  		Fig. 1. A: Transmission electron micrographs of Caco-2 cell brush-border membrane from preconfluent (a; 3 days), confluent (b; 7 days), and differentiated (c; 21 days) cultures. B, C: Sucrase activity and transepithelial electrical resistance (TEER) in preconfluent, confluent, and differentiated Caco-2 cell monolayers. Results are expressed as means ± SEM (n = 6). Mean values labeled with different letters are significantly different (P < 0.05).

 
In a previous study, we observed that growing 3T6 fibroblasts release large amounts of prostanoids after stimulation by different means until the cells reach confluence (33). Similarly, preconfluent Caco-2 cultures stimulated with FBS (10%) or PMA (1 µM) produced significant amounts of PGE2, which was reduced significantly after cell-cell interaction at confluence (Fig. 2 ). Interestingly, an additional reduction in PGE2 release was observed in differentiated Caco-2 cell cultures only in response to PMA (Fig. 2). These results suggest that differentiation of Caco-2 cells may be related to changes in the AA cascade. To further investigate this possibility, PLA2 activity was determined in control cells and after FBS or PMA stimulation at different growth/differentiation stages. The results show that total PLA2 activity decreased markedly when Caco-2 cultures reached confluence, with a further decrease observed after differentiation (Table 1 ). In the presence of DTT, an inactivator of the sPLA2 enzyme (34), no modifications were observed in PLA2 activity in any of the culture conditions tested. However, PLA2 activity in the presence of BEL, an irreversible iPLA2 inhibitor (30), showed a decrease that was more pronounced in preconfluent cultures. These data suggest that preconfluent and confluent Caco-2 cultures mainly contain cPLA2 and iPLA2 activities, whereas differentiated cultures mainly contain cPLA2 activity. Therefore, the changes observed can be correlated with a pronounced decrease in iPLA2 activity in confluence, followed by a slight decrease until differentiation. These changes in PLA2 activity appear to be essential for AA mobilization and show good correlation with [3H]AA release (Table 2 ). Thus, [3H]AA release induced by FBS, PMA, or A23187 in preconfluent cultures was higher than in differentiated cultures. Inhibition of iPLA2 with BEL reduced [3H]AA release in preconfluent cultures but had a minor effect on differentiated monolayers. Therefore, iPLA2 activity is involved in AA mobilization in nondifferentiated cultures but does not appear to be involved in differentiated Caco-2 cultures.


Figure 2
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  		Fig. 2. Prostaglandin E2 (PGE2) concentration in the culture medium of preconfluent, confluent, and differentiated Caco-2 cell cultures. The cells were maintained overnight in DMEM without FBS and then stimulated for 3 h with 10% FBS or PMA (1 µM). PGE2 in cell supernatant was determined by enzyme immunoassay. Results are expressed as means ± SEM (n = 6). # Significant difference (P < 0.05) with respect to control conditions. * Significant difference (P < 0.05) with respect to preconfluent conditions. ** Significant difference (P < 0.05) with respect to confluent cultures.

 

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  		TABLE 1. PLA2 activity in preconfluent, confluent, and differentiated Caco-2 cells

 

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  		TABLE 2. [3H]AA release from preconfluent, confluent, and differentiated Caco-2 cells

 
Caco-2 cell differentiation could depend upon the culture conditions, such as cell support. Thus, we compared [3H]AA release using Caco-2 cultures on plastic clusters or polycarbonate filters. Preconfluent, confluent, and differentiated Caco-2 cells cultured on filters released 6.1, 6.9, and 6.3% in control conditions and 19.8, 18.3, and 14.85% in the presence of FBS, respectively. Because these results were similar to [3H]AA release in cell cultures maintained on plastic (Table 2), we suggest that the different cell support used did not affect the variables determined in this study.

On the other hand, the impairment of [3H]AA release in differentiated Caco-2 might be the consequence of a minor [3H]AA incorporation. However, this possibility can be rejected because similar [3H]AA incorporation to Caco-2 phospholipids was observed in the three culture conditions (Table 3 ).


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  		TABLE 3. [3H]AA incorporation and metabolization by preconfluent, confluent, and differentiated Caco-2 cells

 
COX-2 expression and AA metabolism during Caco-2 cell differentiation
The formation and release of prostanoids from Caco-2 cells is a multistep process requiring AA release and the subsequent enzymatic conversion of AA by COXs. To investigate the possibility that COX-2 expression induced by FBS or PMA decreased as cells progressed to confluence and/or differentiation, COX-2 expression was studied in these experimental conditions. Figure 3 shows a significant decrease in COX-2 expression induced by FBS, from preconfluent to confluent cells, and a further decrease to differentiated cells. PMA-stimulated cells only showed a decrease from confluent to differentiated cells, one less pronounced than that detected in FBS-stimulated cells. On the other hand, we added [3H]AA to preconfluent, confluent, and differentiated Caco-2 cells and measured [3H]AA metabolism. The results show a significant decrease of [3H]AA metabolism in confluent cultures and a further impairment in differentiated cells (Table 3). Together, these results suggest that the reduction in COX-2 expression observed in differentiated Caco-2 cells acts as a rate-limiting step in AA metabolism.


Figure 3
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  		Fig. 3. Cyclooxygenase-2 (COX-2) levels in preconfluent (P), confluent (C), and differentiated (D) Caco-2 cell cultures. Monolayers were stimulated for 3 h with 10% FBS or PMA (1 µM) and scraped off to determine COX-2 mass. Results are representative of three Western blots. COX-2 values were normalized to ß-actin expression.

 
Relationship between PGE2 levels and paracellular permeability in Caco-2 cultures
The data suggest that during differentiation of Caco-2 cells, AA release, PLA2 activity, and COX-2 mass are decreased, leading to a decrease of PGE2 synthesis. Taking into account that there were marked differences in PGE2 concentrations in our experimental conditions (differentiated Caco-2 cultures presenting the lowest PGE2 levels), we examined the effects of PGE2-supplemented culture medium on paracellular permeability. The exogenous addition of PGE2 to differentiated cells, at a PGE2/protein ratio detected in confluent or preconfluent cultures, decreased TEER and increased D-mannitol fluxes (Fig. 4A , B). These effects were dose-dependent and maximal after 1–3 h of incubation with this mediator. After 6 h of incubation, TEER recovery was almost complete, whereas D-mannitol fluxes needed a longer period to reach control values. The highest PGE2 concentration tested (1,000 pg/ml), which would be more characteristic of an inflammatory process, induced the highest enhancement of paracellular permeability, which was completely recovered only after 24 h of incubation. Thus, PGE2 appears to be involved in the regulation of paracellular permeability during Caco-2 cell differentiation. These data suggest that Caco-2 differentiation is related to significant changes in the AA cascade that are involved in the development of typical TEER and the paracellular permeability of differentiated epithelium.


Figure 4
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  		Fig. 4. TEER (A) and D-mannitol fluxes (B) in differentiated Caco-2 cells (open squares, control) incubated for 0–24 h with 10 (closed squares), 100 (open circles), or 1,000 (closed triangles) pg/ml PGE2 in the apical and basolateral compartments. Ten and 100 pg/ml correspond to the PGE2 concentrations found in confluent and preconfluent cultures, respectively. Results are expressed as means ± SEM (n = 4). Mean values labeled with different letters, within each incubation period, are significantly different (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As intestinal epithelial cells progress from the crypt-villus unit, they enter a terminal differentiation program that involves, in the case of absorptive cells, the cessation of proliferation and the polarization of morphological and functional features (6). In intestinal Caco-2 cells, this process is completed after 21 days in culture (8). In this study, we observed that Caco-2 cell differentiation is accompanied by marked changes in the AA cascade and that these events can be correlated with the development of epithelial barrier function.

Cell differentiation is characterized by the remodeling of phospholipid fatty acid composition, leading to an increase in polyunsaturated fatty acids, such as AA, and a decrease in monounsaturated and saturated fatty acids. These changes have been described as a general feature of intestinal cell differentiation (35, 36), recently confirmed in Caco-2 cells (37). Apparently, the fact that differentiated Caco-2 cells have more AA esterified to phospholipids than nondifferentiated cells but produce less PGE2 remains something of a paradox. Our findings show that the impairment of PGE2 synthesis during Caco-2 differentiation resulted from profound changes in the AA cascade, such as the decrease in PLA2 activity and COX-2 expression and the consequent impairment of AA metabolism and release to PGE2. Upon differentiation, which occurred 21 days postconfluence, the ability of Caco-2 cells to release [3H]AA from their membranes decreased after stimulation with FBS, PMA, and A23187. The mechanism underlying this effect is not clear, although the results obtained with DTT and BEL indicate that BEL-sensitive iPLA2 activity may be responsible for the impairment of AA release, whereas cPLA2 activity was present in both nondifferentiated and differentiated Caco-2 cultures. In contrast, Bailleux et al. (38) reported that the postconfluence decrease in [3H]AA release depends on cPLA2 activity in the case of Madin-Darby canine kidney type II and Caco-2 cultures.

Thus, our results and previous findings demonstrating that iPLA2 participates in the signaling pathways involved in the control of Caco-2 proliferation (18) suggest that iPLA2 is involved in PGE2 release in proliferative Caco-2 cells, whereas differentiation appears to depress this specific PLA2 activity. Interestingly, the impairment of AA release in differentiated Caco-2 cells was observed in cultures triggered with both pharmacological agents (PMA and the calcium ionophore A23187) and receptor-mediated stimuli such as FBS growth factors. In this situation, AA release appeared as the main rate-limiting step in PGE2 synthesis, which decreased in parallel with AA release in differentiated Caco-2 cells compared with nondifferentiated cells. Thus, our experimental results indicate that cPLA2 is primarily responsible for AA release in differentiated Caco-2 cells, whereas iPLA2 together with cPLA2 may be involved in AA release in nondifferentiated cultures. iPLA2 has been suggested to be involved in phospholipid remodeling, signal transduction, and apoptosis (for review, see 39), although, to our knowledge, this is the first report showing changes in BEL-sensitive iPLA2 activity during epithelial differentiation. As mentioned above, nondifferentiated Caco-2 cells proliferate more rapidly than differentiated cells, whereas proliferating cells contain substantially less AA in their phospholipids than their differentiated counterparts (37). This has been attributed to preferential ß-oxidation of AA (40), although differentiation-associated decreases in PLA2 activity could also be involved.

On the other hand, COX-2 was highly expressed in nondifferentiated cultures and strongly decreased with differentiation, suggesting that this downregulation is involved in Caco-2 cell differentiation. A similar modulating effect of COX-2 has been described for mucociliary differentiation in human tracheobronchial epithelial cells (41) and for adipose tissue differentiation (42). On the basis of our results, we propose that the impairment of iPLA2 activity and COX-2 expression may be involved in the mechanisms that induce the impairment of [3H]AA metabolism and consequently the decrease of PGE2 release during Caco-2 differentiation.

Interactions of Caco-2 cells with the extracellular environment regulate a variety of cellular functions, such as cell differentiation. Furthermore, Caco-2 cells integrate a complex array of signals during this process. Butyrate and vitamin D3 are examples of compounds that affect Caco-2 differentiation through overlapping or complementary pathways (43, 44). Our results suggest that prostaglandins such as PGE2 could also be involved in Caco-2 cell differentiation. Thus, impairment of the AA cascade with a subsequent decrease in PGE2 release could be necessary to complete Caco-2 differentiation, or at least to complete the development of the epithelial barrier function characteristic of differentiated Caco-2 cultures. In this way, Li et al. (45) observed that PGE2 inhibits the expression of differentiation-related genes and regulates chondrocyte maturation.

Maintenance of barrier function in the intestinal epithelium is crucial for normal physiological homeostasis and is a characteristic of differentiated epithelia. For this reason, we were interested to determine whether prostanoids contribute to the regulation of this important function. Paracellular permeability was assessed in differentiated cultures by measuring TEER and D-mannitol fluxes. Our results show that the exogenous addition of PGE2, in a concentration range similar to that found in nondifferentiated cultures, induced a decrease in TEER while conversely promoting permeability to D-mannitol. These results agree with those reported previously by Ma and Pedram (46), who observed that prostaglandins increase endothelial paracellular permeability, and by Resta-Lenert and Barrett (47), who proposed that COX-2 upregulation and prostaglandin release induced by enteroinvasive bacteria can modulate epithelial barrier function in Caco-2 cell cultures.

Increased levels of PGE2 were described in the mucosa of patients with ulcerative colitis and Crohn's disease (48, 49), pathologies characterized by a defect in epithelial barrier function (50). The precise mechanism by which prostanoids modify this function, however, remains unclear. Increasing PGE2 levels in differentiated Caco-2 cultures to the levels found in nondifferentiated Caco-2 cultures is sufficient to lessen the characteristic paracellular permeability noted in differentiated epithelium. To our knowledge, this is the first report showing the contribution of the AA cascade in Caco-2 differentiation, the role of BEL-sensitive iPLA2 and COX-2 expression in AA release, and the role of PGE2 production in the maintenance of the characteristic paracellular permeability occurring in differentiated Caco-2 cell cultures.


   ACKNOWLEDGMENTS
 
This study was supported by Grants BFI2001-3397, BFU2004-04960/BFI, and BFU2005-05899/BFI from the Ministerio de Educación y Ciencia and Grants 2001SGR00134 and 2005SGR0632 from the Generalitat de Catalunya. The authors are grateful for the valuable help of the Serveis Cientificotècnics of the Universitat de Barcelona. R.M-V. and S.R-P. are recipients of Recerca i Docència (Universitat de Barcelona) and Formació d'Investigadors (Generalitat de Catalunya) grants, respectively.

Manuscript received December 27, 2005 and in revised form March 28, 2006.


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