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 cellular processes involving signaling, including intracellular trafficking. PI3-K has been shown to play a part in both receptor- and non-receptor-mediated transcytosis across cultured kidney cells and undifferentiated thyroid cells.


OBJECTIVE
To investigate the role of PI3-K in transcytosis of thyroglobulin (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cells) - a process known to be mediated by megalin, a member of the low-density lipoprotein receptor family.


METHODS
We studied the effect of the microbial product wortmannin, a specific inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells.


METHODS
Transcytosis experiments were performed using FRTL-5 cells cultured as tight layers on filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper cell surface. Tg was added to the upper chamber and cells were incubated at 37 degrees C. Transcytosed 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 binding and uptake, but resulted in a considerable increase in Tg transcytosis (by 40-75%, depending on the concentration of wortmannin), suggesting that PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcytosis, pre-incubation with wortmannin did not increase Tg transcytosis from its reduced levels, indicating that PI3-K is involved in the megalin-mediated pathway. Wortmannin did not affect the extent of release of tri-iodothyronine 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 pathway.


CONCLUSIONS
PI3-K exerts an inhibitory role on megalin-mediated Tg transcytosis across cultured thyroid cells. PI3-K action takes place at a post-sorting level, after Tg bypassing of the lysosomal pathway.
Marinò
M
M
Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy. m.marino@endoc.med.unipi.it
Chiovato
L
L
Lisi
S
S
Pinchera
A
A
McCluskey
R T
RT
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000502:physiology*; D008247:Lysosomes / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051381:Rats; D013954:Thyroglobulin / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology / Q000378:metabolism*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D013954:Thyroglobulin; D000077191:Wortmannin