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

Abstract

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 A 2 (PLA 2 ) and cyclooxygenase (COX) pathway during differentiation, with particular emphasis on paracellular permeability. PLA 2 activity, AA release, COX-2 expression, prostaglandin E 2 (PGE 2 ) 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 PLA 2 activity and COX-2 expression and, consequently, a decrease in AA release and PGE 2 synthesis in parallel with a reduction in paracellular permeability. Moreover, the addition of PGE 2 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 PLA 2 , COX-2 expression, and subsequent PGE 2 release are important for the maintenance of paracellular permeability in differentiated Caco-2 cells.-Martín-Venegas, R., S. RoigPérez, R. Ferrer, and J. J. Moreno. Arachidonic acid cascade and epithelial barrier function during Caco-2 cell differentiation.

Role of eicosanoids on intestinal epithelial homeostasis

“…2). Thus, we observed that intestinal epithelial differentiation induces a decrease in both iPLA 2 activity and COX-2 expression and, consequently, a decrease in AA release and PGE 2 synthesis in parallel with a reduction in paracellular permeability and consequently the development of a barrier function that can be disrupted by the exogenous addition of PGE 2 [21]. Recently, we observed that this modulation of the epithelial barrier function is mediated by the interaction with PGE 2 receptors EP 1 and EP 4 .…”

“…These events could also be necessary for the completion of epithelial cell differentiation, or at least for the development of the epithelial barrier function that is characteristic of differentiated epithelium [21]. Defining the molecular mechanisms of action of PGs on cell growth is an area of intense research.…”

PGE2 promotes Ca2+-mediated epithelial barrier disruption through EP1 and EP4 receptors in Caco-2 cell monolayers

“…Caco-2 cells were kindly provided by Dr. David Thwaites at the School of Cell and Molecular Biosciences, University of Newcastle-upon-Tyne (United Kingdom), and were cultured as previously described (30). The cells (passages 107-121) were routinely grown in plastic flasks at a density of 5 ϫ 10 4 cells/cm 2 and cultured in DMEM containing 4.5 g/l D-glucose and 2 mM L-glutamine, supplemented with 1% (vol/vol) nonessential amino acids, 10% (vol/vol) heat-inactivated FBS, 100 U/ml penicillin, and 100 g/ml streptomycin at 37°C in a modified atmosphere of 5% CO 2 in air.…”

“…The medium was replaced every 3 days and on the day before the experiment. Experiments were performed in cultures 19 -21 days after seeding when cells are differentiated (30).…”

“…PP was estimated from unidirectional apical-to-basal D-mannitol fluxes and transepithelial electrical resistance (TER) in cells maintained on filters, as described elsewhere (30). After 3-h incubation with PGE2 or EP1-EP4 agonists and antagonists in the apical and basolateral compartments, monolayers were washed with modified Krebs buffer (room temperature), which had 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.0 mM MgSO4, 0.3 mM NaH2PO4, 10 mM D-glucose, and 10 mM HEPES/Tris (pH 7.4).…”