Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both ␤1 and ␤3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment. Furthermore, these inhibitors alter the fate of integrin receptors at the rear of the cell during migration. A Chinese hamster ovary cell line expressing low levels of calpain I also shows reduced migration rates with similar morphological changes, further implicating calpain in this process. Taken together, the data suggest that calpain inhibition modulates cell migration by stabilizing cytoskeletal linkages and decreasing the rate of retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may therefore be a potential therapeutic approach to control pathological cell migration such as tumor metastasis.Cell migration requires a dynamic interaction between a cell, its substratum, and the actin cytoskeleton. Integrin receptors, which are ␣␤ heterodimers present on the cell surface, play an important role during cell migration by mediating these interactions and transmitting forces between the extracellular matrix and the actin cytoskeleton (1, 2). The mechanisms by which these linkages are regulated and released at the cell's rear during migration are not well understood. Previous studies implicate calcium transients in adhesive release in neutrophils migrating on both fibronectin and vitronectin. However, calcineurin mediates the calcium-dependent release of adhesions at the cell's rear in neutrophils migrating on vitronectin but not on fibronectin (3, 4). The specificity of the calcineurin effect for vitronectin and the ␣v␤3 integrin suggests that other calciumdependent mechanisms are also likely to contribute to detachment during migration.The calcium-dependent protease calpain is an attractive candidate to be a calcium-responsive regulator of cell migration. It localizes to focal adhesions and cleaves many focal adhesionrelated proteins including integrin receptors, focal adhesion kinase, and talin (5-8). Calpain is a cysteine protease with two characterized isoforms, calpain I (-calpain) and II (m-calpain). Both contain an 80-kDa catalytic subunit and a 30-kDa regulatory domain. Activation requires calcium concentrations in the micromolar range and millimolar range for calpain I and II, respectively (9, 10). The increases in calcium seen in migrating cells appear to be within the range to support activation of calpain (3). In this study we use genetic and inhibitor studies to show that inhibition of the calcium-dependent protease calpain reduces both ␤1 and ␤3 integrin-mediated migration. EXPERIMENTAL PROCEDURES Cells and Reagents-CHO1 KI cells ...

Proteases having a neutral pH optimum and an absolute requirement for calcium ion are found in virtually all mammalian cells. Association of calcium-dependent proteases and a specific inhibitor protein with biological membranes seems to be an important regulatory feature of this proteolytic system, and it is likely that membranes are preferred sites for calcium-dependent protease action. Several recent hypotheses for the physiological function of calcium-dependent proteolysis are consistent with a membrane-associated protease action. Calcium-dependent proteases may participate in cell membrane fusion: the proteolysis of membrane proteins, which is required for the efficient fusion of erythrocytes, may be catalyzed by these enzymes. There is also evidence for the involvement of calcium-dependent proteolysis in postsynaptic membrane remodeling in the hippocampus after long-term potentiation. Although the relationship of the proteolysis to synaptic function is not known, it could have important physiological or pathophysiological consequences. Finally, it has recently been suggested that calcium-dependent proteolysis may be a physiologically significant mechanism for activating membrane-associated protein kinase C after exposure of some cell types to phorbol esters or other mitogens. Further pursuit of these hypotheses may reveal a novel role for intracellular calcium-regulated proteolysis in membrane-associated cell functions.

Mammalian cells require extracellular calcium ion to undergo rapid plasma membrane repair seconds after mechanical damage. Utilizing transformed fibroblasts from calpain small subunit knock-out (Capns1 ؊/؊ ) mouse embryos, we now show that the heterodimeric, typical subclass of calpains is required for calcium-mediated survival after plasma membrane damage caused by scraping a cell monolayer. Survival of scrape-damaged Capns1 ؊/؊ cells was unaffected by calcium in the scraping medium, whereas more Capns1 ؉/؉ cells survived when calcium was present. Calcium-mediated survival was increased when Capns1 ؊/؊ cells were scraped in the presence of purified m-or -calpain. Survival rates of scraped Capns1 ؉/؉ , HFL-1, or Chinese hamster ovary cells were decreased by the calpain inhibitor, calpeptin, or the highly specific calpain inhibitor protein, calpastatin. Capns1 ؊/؊ cells failed to reseal following laser-induced membrane disruption, demonstrating that their decreased survival after scraping resulted, at least in part, from failed membrane repair. Proteomic and immunologic analyses demonstrated that the known calpain substrates talin and vimentin were exposed at the cell surface and processed by calpain following cell scraping. Autoproteolytic activation of calpain at the scrape site was evident at the earliest time point analyzed and appeared to precede proteolysis of talin and vimentin. The results indicate that conventional calpains are required for calcium-facilitated survival after plasma membrane damage and may act by localized remodeling of the cortical cytoskeleton at the injury site.

Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular dystrophy (DMD) patients has been reported to result in altered calcium homeostasis. Previous studies have shown a correlative relationship between calcium-dependent protease (calpain) activity in dystrophic muscle and muscle necrosis, but have not tested whether calpain activation precedes cell death or is a consequence of it. To test a causal relationship between calpain activation and muscle cell death in dystrophin deficiency, mdx mice were generated that overexpress a calpastatin transgene in muscle. Calpastatin (CS) is a specific, endogenous inhibitor of m- and micro -calpains that does not inhibit calpain 3 (p94). CS overexpression on a C57/BL 10 background produced no phenotype. Transgenic (Tg) mice crossed with mdx mice were tested for pathological indicators of necrosis, regeneration and membrane damage. Two lines of mice were examined, with different levels of CS overexpression. Both lines of Tg/mdx mice showed reductions in muscle necrosis at 4 weeks of age. These mice had fewer as well as smaller lesions. In addition, one line of mice had significantly less regeneration, indicating a reduction in previous necrosis. The extent of improvement correlated with the level of CS protein expression. Membrane damage, as assessed by procion orange and creatine kinase assays, was unchanged, supporting the idea that calpains act downstream of the primary muscle defect. These data suggest that calpains play an active role in necrotic processes in dystrophic muscle and that inhibition of calpains might provide a good therapeutic option for treatment of DMD.

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