Calpain Mediates Integrin-induced Signaling at a Point Upstream of Rho Family Members

Abstract

Integrin-induced adhesion leads to cytoskeletal reorganizations, cell migration, spreading, proliferation, and differentiation. The details of the signaling events that induce these changes in cell behavior are not well understood but they appear to involve activation of Rho family members which activate signaling molecules such as tyrosine kinases, serine/threonine kinases, and lipid kinases. The result is the formation of focal complexes, focal adhesions, and bundles and networks of actin filaments that allow the cell to spread. The present study shows that -calpain is active in adherent cells, that it cleaves proteins known to be present in focal complexes and focal adhesions, and that overexpression of -calpain increased the cleavage of these proteins, induced an overspread morphology and induced an increased number of stress fibers and focal adhesions. Inhibition of calpain with membrane permeable inhibitors or by expression of a dominant negative form of -calpain resulted in an inability of cells to spread or to form focal adhesions, actin filament networks, or stress fibers. Cells expressing constitutively active Rac1 could still form focal complexes and actin filament networks (but not focal adhesions or stress fibers) in the presence of calpain inhibitors; cells expressing constitutively active RhoA could form focal adhesions and stress fibers. Taken together, these data indicate that calpain plays an important role in regulating the formation of focal adhesions and Rac-and Rho-induced cytoskeletal reorganizations and that it does so by acting at sites upstream of both Rac1 and RhoA.Adhesion of cells on integrin substrates results in cell spreading, migration, differentiation, and proliferation and is essential for events such as inflammation, platelet clot formation, development, and wound healing (1-3). Recent evidence has shown that one of the early events following integrin-ligand interactions is the clustering of integrins into small complexes with signaling molecules (4, 5). The subsequent activation of Cdc42 and Rac1 induces the polymerization of new actin filaments that organize into bundles and submembranous networks, thus, causing the extension of filopodia and lamellipodia (4 -8). The dynamic breakdown and formation of new focal complexes in the extending lamellipodia allows the cells to spread. RhoA then becomes activated and induces the formation of larger complexes of integrins, cytoskeletal proteins, and signaling molecules known as focal adhesions. RhoA also induces myosin to interact with actin filaments, causing the filaments to assemble into bundles known as stress fibers that terminate at the focal adhesions and allow tension to be generated on ligand-occupied integrin in the spreading cells (9 -12).By analogy to activation of Ras proteins by growth factor receptors, it is likely that Rho family members are activated by exchange factors that are recruited to sites of ligand-occupied integrin (13-19). However, little is known about the mechanisms inducing recruitment or activation of ...

Inhibition of Calpain Is a Component of Nitric Oxide-Induced Down-Regulation of Human Mast Cell Adhesion

“…Inhibition of calpain has been shown to result in disassembly of stress fibers, loss of focal complexes, and focal adhesions in endothelial cells. In this system, calpain appears to mediate integrin-induced signaling at sites upstream of both Rac1 and RhoA (45). Furthermore, inhibition of calpain activity in T cells decreases the ability of the cells to adhere and spread on FN (46).…”

Myoblast migration is prevented by a calpain‐dependent accumulation of MARCKS

“…However, in most of these studies, both µ-and m-calpain activities were inhibited, using calpastatin overexpression, general pharmacological inhibitors or by targeting the gene encoding the common regulatory subunit (Huttenlocher et al, 1997;Potter et al, 1998;Croce et al, 1999;Dourdin et al, 1999). Other authors have focused only on µ-calpain particularly by means of a dominant negative µ-calpain or by using a cell type that presents a negligible level of m-calpain (Kulkarni et al, 1999;Azam et al, 2001). For the first time, we have employed a strategy that targets separately, or concomitantly, the two isoenzymes, thus shedding light on the importance of m-calpain during myoblast migration.…”

“…Besides, treatments with specific calpain inhibitors or null mutation of capn4 in non-muscular cells induce a decrease of migration (Huttenlocher et al, 1997;Dourdin et al, 2001). Few studies suggested that inhibited calpain activity is associated to an increase of cell attachment (Huttenlocher et al, 1997) while other works indicated a drop in cell adhesion and a defect in focal adhesion formation (Kulkarni et al, 1999;Dedieu et al, 2003(b)). All these data suggest that ubiquitous calpains could participate to the regulation of cell migration but in different ways.…”

Proteomics analysis of H-RAS-mediated oncogenic transformation in a genetically defined human ovarian cancer model

“…For example, calpain is implicated as a downstream effector of the G q signaling pathway for inhibition of Wnt/b-catenin-regulated cell proliferation (Li and Iyengar, 2002). Calpain has also been reported to be important for ERK/MAP kinase activation associated with epidermal growth factor receptor-mediated fibroblast motility (Glading et al, 2000) and for integrin-induced signaling upstream of Rho GTPases (Kulkarni et al, 1999). In addition, calpain is involved in caspase-independent apoptosis in ovarian and breast cancers (Bao et al, 2002).…”