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

Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cell s 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 tr ansmembrane protein to be localized to the tight junction of both epithelia l 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 mi croscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicate d that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remaine d unchanged for at least 2 hours in cells treated with 10(-7) M TPA compare d to their corresponding control cells. As the phosphorylation state of occ ludin is thought to be important in both tight junction assembly and regula tion, the effect of phorbol ester treatment on the phosphorylation of occlu din was investigated. Surprisingly, activation of protein kinase C,vith 10- 7 M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithe lial electrical resistance. This dephosphorylation of occludin, occuring af ter 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 premeab ility, then protein kinase C is apparently further upstream in the signalin g pathway regulating epithelial barrier function, with a downstream serine/ threonine phosphatase acting upon occludin.