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

Abstract

Background: Phosphoinositide 3-kinase (PI3-K) is implicated in various cellular processes involving signaling, including intracellular traf®cking. PI3-K has been shown to play a part in both receptorand 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. Design: We studied the effect of the microbial product wortmannin, a speci®c 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 ®lters 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 8C. Transcytosed Tg was measured in¯uids 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.

Insulin uptake across the luminal membrane of the rat proximal tubule in vivo and in vitro

“…In L2 cells, which express high levels of megalin, β2gpI binds to megalin and RAP is known to inhibit its uptake ( 16 ). Insulin internalization is also mediated by megalin, but RAP has no effect on insulin uptake in L2 cells ( 13 ). To study the effect of RAP on insulin and β2gpI endocytosis in WKPT cells, we monitored the uptake of Cy2-insulin or Cy2-β2gpI in the presence of various concentrations of RAP ( Fig.…”

“…Retinol binding protein (RBP) uptake in vivo is also mediated almost entirely by megalin, as suggested by the absence of RBP-containing granules in the PT of megalin knockout mice ( 7 ). In immortalized rat renal proximal tubule (IRPT) cells, RBP transcytosis is also mediated by megalin, but RBP transport to the lysosome and subsequent degradation (55% of internalized RBP) are independent of megalin ( 13 ). Therefore, in the IRPT model, other megalin-independent mechanisms of endocytosis must account for RBP degradation: other non-megalin receptors, or fluid-phase pinocytosis, has been suggested.…”

“…Orlando et al ( 23 ), using chemical cross-linking of 125 I-labeled insulin to a renal microvillar membrane preparation, identified megalin as the binding protein, which required a 1,000-fold excess of unlabeled insulin to compete for binding; it was also competed with by glucagon. In contrast, RAP (receptor-associated protein), which inhibits binding of other endocytosed ligands to megalin, such as retinol binding protein (RBP) ( 13 ), transthyretin ( 27 ), and β 2 -glycoprotein (β2gpI) ( 16 ), did not inhibit binding. In the same study ( 23 ), uptake of FITC-labeled insulin in L2 yolk sac-derived cells (which are rich in megalin) was inhibited by a 50-fold excess of unlabeled insulin and by anti-megalin IgG, but again not by RAP.…”

Proliferative Effects of Bovine and Porcine Thyroglobulins on Thyroid Epithelial Cells

“…in thyroid cells TSH activates this pathway by elevating the level of cAmP in the cells through the activation of adenylyl cyclase, a downstream effecter of the TSh receptor [28,29]. marino et al [30] also demonstrated that PI3k exerted an inhibitory effect on megalin-mediated Tg transcytosis across the cultured FRTL-5 cells. It has been postulated that Tg transcytosis plays an important role in the regulation of thyroid function [31].…”

Vitamin D and Haematopoiesis

“…Megalin and cubilin transport vitamin D, complexed to its serum binding protein. Receptor endocytosis can generate rapid intracellular signals via the Rab5/PI3-kinase pathway [91]. However and from knockout studies, we have excluded the involvement of megalin and cubilin as to the differentiating potency and selectivity of our analogues of 1,25D3 [92].…”