Sustained Induction of ERK, Protein Kinase B, and p70 S6 Kinase Regulates Cell Spreading and Formation of F-actin Microspikes Upon Ligation of Integrins by Galectin-8, a Mammalian Lectin

Abstract

Galectin-8, a member of the galectin family of mammalian lectins, is a secreted protein that promotes cell adhesion and migration upon binding to a subset of integrins through sugar-protein interactions. Ligation of integrins by galectin-8 triggers a distinct pattern of cytoskeletal organization, including formation of F-actin-containing microspikes. This is associated with activation of integrin-mediated signaling cascades (ERK and phosphatidylinositol 3 kinase (PI3K)) that are much more robust and are of longer duration than those induced upon cell adhesion to fibronectin. Indeed, formation of microspikes is enhanced 40% in cells that overexpress protein kinase B, the downstream effector of PI3K. Inhibition of PI3K activity induced by wortmannin partially inhibits cell adhesion and spreading while largely inhibiting microspike formation in cells adherent to galectin-8. Furthermore, the inhibitory effects of wortmannin are markedly accentuated in cells overexpressing PKB or p70S6K (CHO PKB and CHO p70S6K cells), whose adhesion and spreading on galectin-8 (but not on fibronectin) is inhibited ϳ25-35% in the presence of wortmannin. The above results suggest that galectin-8 is an extracellular matrix protein that triggers a unique repertoire of integrin-mediated signals, which leads to a distinctive cytoskeletal organization and microspike formation. They further suggest that downstream effectors of PI3K, including PKB and p70 S6 kinase, in part mediate cell adhesion, spreading, and microspike formation induced by galectin-8. Extracellular matrix (ECM)1 proteins have important functions in providing structural integrity to tissues and in presenting proper environmental cues for cell adhesion, migration, growth, and differentiation (1-5). These functions rely on spatio-temporal expression of adhesive as well as anti-adhesive components of the ECM proteins (6). ECM proteins like fibronectin (7-9), collagen (10), and laminin (11) are best characterized, though other types of proteins, including mammalian lectins, also function as modulators of cell adhesion. Selectins mediate cell-cell interactions (12) through calciumdependent recognition of sialylated glycans (13, 14), whereas galectins, animal lectins that specifically bind ␤-galactoside residues (15), were implicated as modulators of cell-matrix interactions. Although lacking a signal peptide and found mainly in the cytosol, galectins are externalized by an atypical secretory mechanism (16) to regulate cell growth, cell transformation, embryogenesis, and apoptosis (reviewed in Refs. 17 and 18). In accordance with their proposed functions, galectins enhance or inhibit cell-matrix interactions (reviewed in Ref. 19).Cellular adhesion to extracellular matrix proteins is mediated by a diverse class of cell surface ␣␤ heterodimeric receptors known as integrins (2,20,21). In addition to mediating cell adhesion, integrins induce multiple signal transduction pathways that regulate cytoskeletal rearrangements, cell spreading, migration, differentiation, survival, and cell...

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“…36 Alternatively, growth factor receptor-independent activation of signalling intermediates may result from interactions between integrins and extracellular matrix components. 37,38 In control-transfected DU145 cells, IGF stimulation induced phosphorylation of Akt, ERKs, and p38 (Fig 4a). At 48 hours after IGF1R siRNA transfection, DU145 cells showed significant attenuation of IGF-induced Akt and ERK phosphorylation, comparable to results we previously reported in breast cancer cells, 23 although there was no suppression of the p38 response to IGF-I.…”

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“…In the case of human smooth muscle cells Gal-1 binding did not cross-link ␤1 integrin but nonetheless led to a transient increase in tyrosine phosphorylation of two cytoskeleton-associated proteins and to modulation of cell attachment, possibly due to an effect of Gal-1 on ␤1 conformation (13). Cytoskeletal reorganization also occurs following interaction of the tandem repeat-type lectin galectin-8 with ␣3␤1 and ␣6␤1 integrins on Chinese hamster ovary cells or human endothelial cells (38,39). So far, however, the functional relevance of integrin binding for Gal-1-dependent growth-regulatory effects has not been conclusively demonstrated.…”

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“…Second, although vinculin and paxillin are associated with large focal contacts in cells adherent to fibronectin, the number and size of vinculin-and paxillin-containing focal contacts is reduced in cells attached to galectin-8 (23). The differences in actin-microfilament organization are not accompanied by differences in microtubules organization, and a similar microtubular network develops when cell adhere to galectin-8 or fibronectin (24).…”

“…Second, and more important, the differences in cytoskeletal organization, which take place when cells adhere to different matrices, seem to play a critical role. Hence, whereas cells adherent to fibronectin develop an elaborated network of actin bundles associated with well developed focal contacts, cells attached to galectin-8 are characterized by the formation of cortical actin, elaborate F-actin microspikes, and a poorly organized network of actin microfilaments, which is associated with small focal contacts distributed mainly at the cell periphery (23,24). Many of these adhesion sites contain lower amounts of vinculin or paxillin (23).…”