31 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Preincubation of cells with anti-3 integrin function-blocking antibody significantly inhibited the induction of lamellipodia by Gal-3. Rac1 GTPase, a member of the family of Rho GTPases, well known for its role in the reorganization of the actin cytoskeleton and formation of lamellipodial extensions. Experiments including knockdown of -1,6-agglutinin (PHA-L, specific for core 1,6GlcNAc-branched N-glycans) reduced cell migration, suggesting that 1,6GlcNAc-branched glycans are involved in the process of cell motility (Przybylo et al., 2008). In recent years, studies aimed at characterization of the mechanisms by which integrin glycans regulate cell migration have revealed that interactions between integrin glycans and carbohydrate-binding proteins, galectins, play an essential role in integrin-dependent cell adhesion and migration (Carcamo et al., 2006; Fischer et al., 2005; Friedrichs et al., 2008; Goetz et al., 2008; Lagana et al., 2006; Levy et al., 2003; Nishi Rabbit polyclonal to ZNF75A et al., 2003; Zhuo et al., 2008). For example, Lagana et al. have shown that galectin-3 (Gal-3; also known as LGALS3) interactions with MGAT5-altered N-glycans at the cell surface of mammary carcinoma cells promote 51 integrin activation and cell motility (Lagana et al., 2006); 41, 51 and 47 integrins have been identified as major Gal-1 glycosylated binding partners involved in immune synapse formation, pre-B-cell-receptor clustering and activation (Rossi et al., 2006); and Gal-8 has been shown Benidipine hydrochloride to form high-affinity interactions with 1 integrins, modulate cell-matrix interactions and promote cell distributing by activating Rho GTPases and PI3K (Diskin et al., 2009; Levy et al., 2001; Levy et al., 2003). Studies in our laboratory have focused on the role of a structurally unique member of the galectin family, Gal-3, in the process of cell migration (Cao et al., 2002). We have exhibited that: (1) migrating epithelia of healing mouse corneas express elevated levels of Gal-3 compared with nonmigrating epithelia of normal corneas; (2) the rate of re-epithelialization of corneal wounds is usually significantly slower in Gal-3-deficient mice compared with wild-type mice; and (3) exogenous Gal-3 stimulates re-epithelialization of corneal wounds in a carbohydrate-dependent manner (Cao et al., 2002). However, the molecular mechanism by which Gal-3 influences re-epithelialization of corneal wounds remains unknown. In the present study, we demonstrate for the first time that Gal-3 promotes formation of lamellipodia by activating 31-integrinCRac1 signaling in epithelial cells and that carbohydrate-mediated conversation between Gal-3 and complex Benidipine hydrochloride N-glycans on 31 integrin is usually involved in Gal-3-induced lamellipodia formation. Results Exogenous Gal-3 promotes cell scattering, lamellipodia formation, and cell motility In an effort to characterize the mechanism by which Gal-3 enhances re-epithelialization of corneal wounds in vivo (Cao et al., 2002), experiments were performed to determine whether Gal-3 promotes Benidipine hydrochloride initiation of migratory phenotype in corneal epithelium. For this, the HCLE cells were incubated in the absence or the presence of Gal-3 and the morphology of the cells, in particular lamellipodia formation, was analyzed after staining Benidipine hydrochloride with TRITC-phalloidin. Lamellipodia are actin-rich, fan-shaped, membrane protrusions at the leading edge of motile cells (Small and Resch, 2005). As early as 30 minutes after exposure to Gal-3, 80% of the cells displayed lamellipodial membrane protrusions as opposed to 5% in control cells (Fig. 1A). The stimulatory effect of Gal-3 on lamellipodia formation was dose dependent (Fig. 1B, inset) and specifically inhibited by a competing sugar, -lactose, but not by an irrelevant disaccharide, sucrose (Fig. 1A), suggesting that this carbohydrate recognition domain name of Gal-3 is usually involved in the formation of lamellipodia in HCLE cells. In time-lapse video microscopy, Gal-3-treated cells showed colony dispersion and a cell scattering effect. As early as 2 moments after activation with Gal-3, cell-cell dissociation was detected (supplementary material Movie 1), the scattering of colonies gradually increased, and by 10 minutes after exposure to Gal-3, formation of lamellipodia and filopodia was obvious in the majority of cells. Furthermore, the cells that experienced dissociated from your colonies were migratory. By contrast, the cells incubated in medium alone (supplementary material Movie 2) or in the presence of Gal-3 with.