Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level

Background: Phosphoinositide 3-kinase (PI3-K) is implicated in various cell ular processes involving signaling, including intracellular trafficking. PI 3-K has been shown to play a part in both receptor- and non-receptor-mediat ed transcytosis across cultured kidney cells and undifferentiated thyroid c ells. Objective: To investigate the role of PI3-K in transcytosis of thyroglobuli n (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cell s)-a process known to be mediated by megalin, a member of the low-density l ipoprotein receptor family. Design: We studied the effect of the microbial product wortmannin, a specif ic inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells. Methods: Transcytosis experiments were performed using FRTL-5 cells culture d as tight layers on filters in the upper chamber of dual chambered devices , with megalin expression exclusively on the upper cell surface. Tg was add ed to the upper chamber and cells were incubated at 37 degreesC. Transcytos ed Tg was measured in fluids collected from the lower chamber. To study the role of PI3-K, cells were pre-incubated with wortmannin. Results: Pre-incubation of FRTL-5 cells with wortmannin did not affect Tg b inding and uptake, but resulted in a considerable increase in Tg transcytos is (by 40-75%, depending on the concentration of wortmannin), suggesting th at PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcyto sis, pre-incubation with wortmannin did not increase Tg transcytosis from i ts reduced levels, indicating that PI3-K is involved in the megalin-mediate d pathway. Wortmannin did not affect the extent of release of tri-iodothyro nine from exogenously added Tg by FRTL-5 cells, which was used as a measure of Tg degradation in the lysosomal pathway, indicating that the effect of PI3-K on transcytosis occurs after diversion of Tg from the lysosomal pathw ay. Conclusions: PI3-K exerts an inhibitory role on megalin-mediated Tg transcy tosis across cultured thyroid cells. PI3-K action takes place at a post-sor ting level, after Tg bypassing of the lysosomal pathway.