Galectin-3: Endozytose und Recycling in polaren Epithelzellen sowie seine Rolle im klarzelligen Nierenzellkarzinom

Polar epithelial cells are the prerequisite for a functional organ like the kidney. They build a monolayer on the outer surface of the organ forming a barrier and exchange system between the organ and its environment. The polarized structure of epithelial cells is characterized by apical and basolateral plasma membrane domains. These two membrane domains are separated by tight junctions and differ in their lipid and protein composition. The polarity of the cell is maintained by a directional transport of lipids and proteins to their target membrane. In apical protein transport the sorting pathways can be subdivided by the affinity of the transported protein to lipid rafts into a lipid raft dependent or independent pathway. The separation of the transport routes takes place in a post-Golgi compartment, based on the formation of high molecular clusters mediated by the sorting receptor galectin-3. Galectin-3 is a galactose-binding lectin acting as an apical sorting platform for raft independent glycoproteins. It is not only present intracellularly but can also be transported via an unconventional secretory pathway into the extracellular space. Presumably it can be taken up from the extracellular space into the cell again, but the exact mechanism of endocytosis is still unclear. In the present study a recycling process of endocytosed recombinant human galectin-3 could be observed by biochemical analyses. By using TGN-Exit analyses followed by DRM-isolation it could be shown that galectin-3 is present in the extracellular space 60 min after TGN-block. During its resumption this lectin is obviously associated with lipid rafts. Results of high-resolution microscopy and the use of a lipid raft inhibitor confirmed a lipid raft dependent endocytosis of galectin-3. Furthermore the binding affinity of galectin-3 to sugar residues on the cell surface seems to play a pivotal role in internalization of the lectin. Additionally the association of galectin-3 with lipid rafts and the endocytosis occur in a pHdependent manner. During the recycling process galectin-3 and its ligands pass different endosomal compartments with varying pH. Furthermore in the present work, first indications for the influence of the pH during apical protein transport of the p75 neurotrophin receptor and the glycoprotein gp80 could be collected. At the same time evidence of a pH dependent galectin-3 induced cluster formation was determined. Galectin-3 is involved in many cellular processes like cell cycle regulation, cell-cell adhesion, apoptosis and angiogenesis. Due to these functions it is possible that this lectin participates in tumor progression in renal cells. Confocal fluorescence microscopy of human renal tissue and clear cell renal cell carcinoma tissue were used to obtain new insights in the localization of galectin-3 as well as various apical and basolateral marker proteins. In normal kidney tissue galectin-3 can be observed in single epithelial cells in the distal tubules and collecting ducts while in tumor tissue the localization is diffuse and shifted into the cell Nucleus. The expression of galectin-3 increases in the tumor tissue compared to the normal kidney tissue in 75% of patients with clear cell renal cell carcinoma used in this study. This increase in protein expression during tumor differentiation correlates with the decrease in the expression of the cell-cell adhesion protein CEACAM1. Furthermore, some interaction partners of galectin-3 during tumor progression could be identified by using a galectin-3 affinity column.