Aggregation of Integrins and RhoA Activation Are Required for Thy-1-induced Morphological Changes in Astrocytes

Abstract

Thy-1, a cell adhesion molecule abundantly expressed in mammalian neurons, binds to a ␤ 3 -containing integrin on astrocytes and thereby stimulates the assembly of focal adhesions and stress fibers. Such events lead to morphological changes in astrocytes that resemble those occurring upon injury in the brain. Extracellular matrix proteins, typical integrin ligands, bind to integrins and promote receptor clustering as well as signal transduction events that involve small G proteins and cytoskeletal changes. Here we investigated the possibility that the cell surface protein Thy-1, when interacting with a ␤ 3 -containing integrin on astrocytes, could trigger signaling events similar to those generated by extracellular matrix proteins. DI-TNC 1 astrocytes were stimulated with Thy-1-Fc immobilized on beads, and increased RhoA activity was confirmed using an affinity precipitation assay. The effect of various inhibitors on the cellular response was also studied. The presence of Y-27632, an inhibitor of Rho kinase (p160ROCK), a key downstream effector of RhoA, significantly reduced focal adhesion and stress fiber formation induced by Thy-1. Similar effects were obtained when astrocytes were treated with C3 transferase, an inhibitor of RhoA. Alternatively, astrocytes were transfected with an expression vector encoding fusion proteins of enhanced green fluorescent protein with either the Rho-binding domain of Rhotekin, which blocks RhoA function, or the dominantnegative N19RhoA mutant. In both cases, Thy-1-induced focal adhesion formation was inhibited. Furthermore, we observed that RhoA activity after stimulation with soluble Thy-1-Fc molecule was augmented upon further cross-linking using protein ASepharose beads. The same was shown by crosslinking ␤ 3 -containing integrin with anti-␤ 3 antibodies. Together, these results indicate that Thy-1-mediated astrocyte stimulation depended on ␤ 3 integrin clustering and the resulting increase in RhoA activity.Focal adhesions (FAs) 1 mediate strong adhesion to the substrate and anchor actin stress fibers (SFs) through a supramolecular complex that consists of many different cytoskeletal as well as signaling molecules (reviewed in Refs. 1 and 2). The unique features of FAs suggest that they are employed as mechanosensors to inform cells about the immediate environment. As such, FAs are implicated in transmembrane crosstalk between extracellular matrix (ECM) proteins and intracellular signal transduction pathways via integrin receptors (reviewed in Refs. 3 and 4).The interaction between ECM components and integrins leads to reorganization of the cytoskeleton and regulation of cell migration. Such events have been most extensively studied in fibroblasts (reviewed in Ref. 5) and linked there to activation of small GTPases belonging to the Rho family of monomeric 20 -30-kDa GTP-binding proteins. Like other members of the Ras superfamily, Rho acts as a molecular switch, cycling between an active GTP-bound state and an inactive, GDP-bound state (reviewed in Refs. 6 and 7). The best-cha...

Lipocalin-2 Is an Autocrine Mediator of Reactive Astrocytosis

“…Previously, it was demonstrated that RhoA negatively regulated astrocyte stellation (Suidan et al, 1997;Abe and Misawa, 2003;Chen et al, 2006;Burgos et al, 2007), migration (Höltje et al, 2005), and the IL-1␤-induced reactive astroglial phenotype (John et al, 2004). RhoA activation was, however, required for Thy-1-induced morphological changes in astrocytes that resembled those in brain injuries (Avalos et al, 2004). lcn2, in the current study, stimulated the growth of astrocyte processes and locomotive activity by activating Rho and ROCK pathway.…”

“…The Rho family of small G-proteins has been frequently involved in structural changes of astrocytes (Suidan et al, 1997;Ramakers and Moolenaar, 1998;Avalos et al, 2004;John et al, 2004;Höltje et al, 2005;Chen et al, 2006;Osmani et al, 2006;Borán and García, 2007) and other cell types (Takai et al, 2001;Etienne-Manneville and Hall, 2002;Heo and Meyer, 2003;Hall, 2005). Previously, it was demonstrated that RhoA negatively regulated astrocyte stellation (Suidan et al, 1997;Abe and Misawa, 2003;Chen et al, 2006;Burgos et al, 2007), migration (Höltje et al, 2005), and the IL-1␤-induced reactive astroglial phenotype (John et al, 2004).…”

VEGF–integrin interplay controls tumor growth and vascularization

“…Ligand binding to integrins promotes integrin clustering and subsequent association with cytoplasmic signaling proteins (19,22) to mediate outside-in signaling. Therefore, we next examined the ability of ␣v␤3 WT and S752P integrins to cluster and determined the role of ␣v␤3 clustering in VEGF expression modulation.…”

“…Cells were lysed (18) and clarified, and 500 g of protein was immunoprecipitated with an antibody to ␤3 integrin. In other experiments, cells were stimulated to promote integrin clustering and homogenized in sample buffer (19). Shc protein was analyzed by immunoblotting with antibodies against phospho-Shc (Y317) (Cell Signaling Technology, Beverly, MA) and total Shc (Upstate Biotechnology, Lake Placid, NY).…”

Chemomechanical mapping of ligand–receptor binding kinetics on cells

“…Both the receptor location and the kinetics of ligand binding are important to the understanding of receptor-driven functions within cells, but few experimental approaches provide simultaneous access to spatial, temporal, and intermolecular force dynamics in individual, whole cells (1). Such quantification is crucial to understanding how cells within or among subpopulations may respond differentially to the same ligand [e.g., drug responsivity (2) and differentiation (3)] and how ligand binding can depend on clustering of multiple molecules [e.g., synapse formation (4)] or cytoskeletal association [e.g., focal adhesion formation (5)]. Several impressive experimental approaches including flow cytometry, immunocytochemical staining, Förster resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) are based on optical signals that require either fluorophore-labeling or genetic modification of cell surface proteins (1).…”