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SYK REGULATES CLATHRIN-MEDIATED ENDOCYTOSIS OF SHIGA TOXIN AND PI3K ACTIVATION IN CACO-2 HUMAN INTESTINAL EPITHELIAL CELLS
X Wang, D Ranev, H Gaisano, C Lingwood, CW Chow
Departments of Medicine and Biochemistry, University of Toronto
Infections with Shiga toxin (Stx), produced by Shigella dysenteriae and enterohemorrhagic Escherichia coli strains are endemic in the developing world and associated with sporadic outbreaks of food-borne gastroenteritis in the developed world, and can lead to serious complications such as hemorrhagic colitis and haemolytic uremic syndrome. Susceptibility to the cytotoxic effects of Stx is apparently related to the level of Gb3 expression, which is high in the renal endothelium.
While the intestinal epithelia are the first point of contact with Stx, they do not express Gb3. It is not clear how Stx is able to traverse the intestinal epithelia to reach the systemic circulation. Recent studies in human intestinal epithelial cells have revealed a Gb3-independent route of Stx internalization. Studies in HeLa epithelial cells have identified Syk tyrosine kinase to mediate Stx-induced phosphorylation of clathrin and clathrin-mediated endocytosis. Syk is known to regulate receptor mediated internalization of diverse membrane receptors. We have recently identified robust Syk expression in intestinal epithelia.
We postulated that in intestinal epithelial cells, Syk is activated by Stx and that it regulates internalization of Stx via clathrin-mediated endocytosis and that it regulates downstream signaling pathways that mediate cell activation.
Using the human intestinal Caco-2 epithelial cell line and purified Stx, we observed recruitment of the normally cytosolic Syk to the plasma membrane upon Stx inoculation. This was accompanied by co-localization with ezrin, a cytoskeletal membrane linker protein that possesses a specific binding domain (the ITAM) for Syk. Syk-ezrin co-localization was accompanied by Syk-clathrin co-localization: these persisted for up to 30 min following conditions permissive for Stx internalization. These confocal imaging studies were confirmed in protein biochemical analyses that revealed co-precipitation of Syk with ezrin and with clathrin, within minutes of Stx inoculation.
Syk is known to regulate multiple downstream signaling pathways in leukocytes, including PI3K. Using Western blot analysis to assess phosphorylation of Akt, an assay of PI3K activation, we found that Akt phosphorylation in response to Stx was Syk dependent, as knock-down of Syk by siRNA significantly abrogated Akt phosphorylation. Syk knock-down also enhanced Stx-induced cleavage of caspase 3, a marker of apoptosis.
Together, our observations revealed that Syk plays a role in clathrin-mediated endocytosis of Stx, and Stx-induced PI3K activation and Stx-induced apoptosis.