Tyrphostin A8 stimulates a novel trafficking pathway of apically endocytosed transferrin through Rab11-enriched compartments in Caco-2 cells

Abstract

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 apical-to-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 degrees C and 37 degrees C, which indicated the presence of apically endocytosed FITC-transferrin in early endosomes and apical recycling endosomes at 16 degrees C and 37 degrees 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 degrees 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.