No abstract

Transferrin receptor (TfR)-mediated endocytosis and transcytosis in enterocyte-like Caco-2 cells was investigated in order to elucidate the transport mechanism of orally administered Tf-fusion proteins. Cellular uptake and pulse chase studies were performed in Caco-2, MCF-7 and bladder carcinoma (5637) cells using 125I-labeled Tf (125I-Tf). Co-localization studies of Rab 11 and FITC-Tf endocytosed at either the apical or basolateral membrane were performed in polarized Caco-2 cells grown on Transwells, using confocal laser scanning microscopy (LSM510, Zeiss). Unlike in MCF-7 or 5637 cells, where rapid recycling of Tf was observed, a significant amount of endocytosed 125I-Tf accumulated in Caco-2 cells. This accumulation was especially noticeable with the internalization of 125I-Tf from the apical membrane of polarized Caco-2 cells. Confocal microscopy studies showed that apically, but not basolaterally, endocytosed FITC-Tf was delivered to a Rab11-positive compartment. Our results suggest that a significant amount of apically endocytosed Tf in intestinal epithelial cells is transported to a Rab11-positive compartment, possibly a late endosomal and slow recycling compartment. The Rab11-positive compartment may control the release of apically internalized Tf for either slow recycling to apical membrane or processing to transcytotic compartments.

The potential application of transferrin receptors as delivery vehicles for transport of macromolecular drugs across intestinal epithelial cells is limited by several factors, including the low level of transferrin receptor-mediated transcytosis, particularly in the apical-to-basolateral direction. The GTPase inhibitor, AG10 (tyrphostin A8), has been shown previously to increase the apicalto-basolateral transcytosis of transferrin in Caco-2 cells. However, the mechanism of the increased transcytosis has not been established. In this report, the effect of AG10 on the trafficking of endocytosed transferrin among different endosomal compartments as well as the involvement of Rab11 in the intracellular trafficking of transferrin was investigated. Confocal microscopy studies showed a high level of colocalization of FITC-transferrin with Rab5 and Rab11 in Caco-2 cells pulsed at 16°C and 37°C, which indicated the presence of apically endocytosed FITCtransferrin in early endosomes and apical recycling endosomes at 16°C and 37°C, respectively. The effect of AG10 on the accumulation of transferrin within different endosomal compartment was studied, and an increase in the transcytosis and recycling of internalized 125 I-labeled transferrin, as well as a decrease in cell-associated 125 I-labeled transferrin, was observed in AG10-treated Caco-2 cells pulsed at 37°C for 30 min and chased for 30 min. Moreover, confocal microscopy showed that FITC-transferrin exhibited an increased level of colocalization with Rab11, but not with Rab5, in the presence of AG10. These results suggest an effect of AG10 on the later steps of transferrin receptor trafficking, which are involved in subsequent recycling, and possibly transcytosis, of endocytosed transferrin in Caco-2 cells.