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 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.

Expression and Function of the Lipocalin-2 (24p3/NGAL) Receptor in Rodent and Human Intestinal Epithelia

“…Moreover, transcytosis of Tf has been demonstrated in Caco-2 cells [11,17,45]. We have previously demonstrated that MT and Tf are ligands of r24p3-R/hNGAL-R [24], therefore, we investigated whether hNGAL-R-mediated uptake of PC 3 , MT and Tf is associated with transcytosis of these proteins in colon-like Caco-2 BBE cells.…”

“…So far, in vivo studies have provided limited evidence for expression of the megalin/cubilin/amnionless receptor complex in the terminal ileum [13,14] (reviewed in 15, which plays a role in apical endocytosis of he intrinsic factor-vitamin B12 complex and possibly Tf [16]. Moreover, the Tf receptor may mediate apical uptake and transcytosis of Tf though, in contrast to in vitro studies [11,17,18], evidence for apical expression of the Tf receptor in vivo is sparse [18]. Here we focussed on the possibility that endo-/transcytosis of proteins, such as Tf, but also MT and PC 3 , may also be mediated by the r24p3-R/hNGAL-R in the intestine.…”

“…Overall, A546-Tf transcytosis was more efficient than that of A488-PC 3 or A546-MT (Figure 6G). This specific difference could be due to the presence of apical Tf-R in Caco-2 cells [11,17] that may additionally contribute to efficient RME and transcytosis. Furthermore, the magnitude of hNGAL-R-dependent A546-MT uptake was higher than A488-PC 3 or A546-Tf (Figure 6B, CD and 6F), suggesting that A546-MT is preferentially taken up by the hNGAL-R endocytosis pathway in colon-like Caco-2 BBE cells and/or that other pathways for RME of A546-MT have a lower affinity to this ligand.…”

“…In this intestinal segment it is thought to mediate apical internalization of specific proteins, such as the intrinsic factor-vitamin B12 complex [15] or transferrin (Tf) [16]. In addition, apical Tf uptake and transcytosis has been linked to a Tf receptor in vitro [11,17,18]. …”

Targeting receptor-mediated endocytotic pathways with nanoparticles: Rationale and advances

“…Tf (and likely the TfR itself), has been demonstrated to undergo transcytosis in gastrointestinal epithelium [63] as well as across the blood-brain barrier [64], and this transcytotic pathway has been exploited for the trans-epithelial and trans-endothelial delivery of therapeutic agents across these barriers.…”