Marinò M, Chiovato L, Lisi S, Pinchera A, McCluskey R T
Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy.
Eur J Endocrinol. 2001 Oct;145(4):477-83. doi: 10.1530/eje.0.1450477.
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.
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.
We studied the effect of the microbial product wortmannin, a specific inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells.
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.
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.
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.
磷酸肌醇3激酶（PI3-K）参与包括细胞内运输在内的各种涉及信号传导的细胞过程。PI3-K已被证明在跨培养的肾细胞和未分化的甲状腺细胞的受体介导和非受体介导的转胞吞作用中均发挥作用。
研究PI3-K在甲状腺球蛋白（Tg）跨分化的培养Fisher大鼠甲状腺细胞（FRTL-5细胞）转胞吞作用中的作用，这一过程已知由低密度脂蛋白受体家族成员巨膜蛋白介导。
我们研究了微生物产物渥曼青霉素（一种PI3-K的特异性抑制剂）对Tg跨FRTL-5细胞转胞吞作用的影响。
使用在双室装置上室的滤器上紧密培养的FRTL-5细胞进行转胞吞实验，巨膜蛋白仅在上层细胞表面表达。将Tg添加到上室，细胞在37℃孵育。在从下室收集的液体中测量转胞吞的Tg。为了研究PI3-K的作用，细胞先用渥曼青霉素预孵育。
用渥曼青霉素预孵育FRTL-5细胞不影响Tg的结合和摄取，但导致Tg转胞吞作用显著增加（增加40%-75%，取决于渥曼青霉素的浓度），这表明PI3-K对Tg转胞吞作用具有抑制作用。在使用抗巨膜蛋白单克隆抗体减少Tg转胞吞作用的实验中，用渥曼青霉素预孵育并没有使Tg转胞吞作用从其降低的水平增加，这表明PI3-K参与巨膜蛋白介导的途径。渥曼青霉素不影响FRTL-5细胞从外源添加的Tg中释放三碘甲状腺原氨酸的程度，这用作溶酶体途径中Tg降解的指标，表明PI3-K对转胞吞作用的影响发生在Tg从溶酶体途径转向之后。
PI3-K对培养的甲状腺细胞中巨膜蛋白介导的Tg转胞吞作用具有抑制作用。PI3-K的作用发生在分选后水平，即在Tg绕过溶酶体途径之后。
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