Negative regulation of RPE cell attachment by carbohydrate-dependent cell surface binding of galectin-3 and inhibition of the ERK-MAPK pathway.

Adhesion and spreading of retinal pigment epithelial (RPE) cells on fibronectin-rich extracellular matrices is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). In the present study we explored the capacity of galectin-3, a β-galactoside-binding endogenous lectin, to inhibit early PVR-associated cellular events from a therapeutic perspective. We assessed the relative expression levels of galectin-3 in native RPE and dedifferentiated, cultured RPE. Galectin-3 was constitutively expressed under in vivo and in vitro conditions and was abundant in cultured cells. Treatment of human RPE cells with soluble galectin-3 disclosed no toxicity within control limits up to 250 μg/ml. When added to the medium, galectin-3 dose-dependently inhibited attachment and spreading of the cells on fibronectin by more than 75%. When coated on the plastic surface, galectin-3 alone impaired attachment and spreading of RPE cells, and reduced attachment but not spreading on fibronectin. Galectin-3 bound to the cell surface, and, as determined by the use of the competing sugar β-lactose, galectin-3-mediated effects were dependent on carbohydrate binding. To ascertain the role of the ability of galectin-3 to form pentamers, we proteolytically removed the N-terminal, cross-linking section. The remaining C-terminal carbohydrate-binding domain alone failed to bind to cells and was functionally inactive. These results emphasize the relevance of both properties, i.e., glycan-binding and cross-linking of glycan moieties, for the inhibitory activity of galectin-3. Incubation of mobilized RPE cells with galectin-3 significantly disturbed microfilament assembly and, in correlation with decreased attachment, inhibited ERK phosphorylation. Therefore, galectin-3, acting as a cross-linking lectin on the cell surface, negatively regulates attachment and spreading of RPE cells in vitro. This effect, at least in part, is attributed to an inhibition of the ERK-MAPK pathway, which prevents cytoskeletal rearrangements needed for RPE cell attachment and spreading. Further investigation at this pathway may disclose a promising nouveau perspective for treatment and prophylaxis of early PVR.