Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.