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 ph orbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phor bol ester exposure reduces protein kinase C (PKC) levels in numerous c ell types including T84, as shown here. Under PKC-downregulated condit ions, 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 morphologi cal changes as assessed by thymidine incorporation studies. The effect of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significant ly greater than that of controls. Flux studies localized the permeabil ity defect to the tight junction. PKC-associated alterations in the pe rijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the ob served functional changes. Most interesting was the fact that monolaye r reassembly to the original columnar epithelial phenotype and reestab lishment of barrier function occurred upon normalization of PKC levels . This model of reversible monolayer disassembly will allow investigat ion into the relationship between epithelial structure and function an d examination of factors that govern monolayer formation.