Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets

Abstract

Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10(−7) M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10(−7) M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10(−7) M TPA compared to their corresponding control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10(−7) M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occurring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction permeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.