Differentiation restricted endocytosis of cell penetrating peptides in MDCK cells corresponds with activities of Rho-GTPases

Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a gamma-zein related sequence. Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-gamma/TNF-alpha a induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-gamma/TNF-alpha a induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery.