Beta 1 integrins signal lipid second messengers required during cell adhesion.

Clustering of integrin receptors during cell adhesion stimulates signal transduction across the cell membrane. Second messengers are generated, activating cytosolic proteins and causing cytoskeletal assembly and rearrangement. HeLa cell adhesion to a collagen substrate has been shown to initiate an arachidonic acid-mediated signaling pathway, leading to the activation of protein kinase C (PKC) and cell spreading. To determine the role of integrin receptors in triggering this signaling pathway, monoclonal antibodies to beta 1 integrins were used to either cluster integrins on the cell surface or to provide an integrin-dependent substrate for cell adhesion. Using this approach, we have defined a pathway required for cell spreading that can be initiated by the ligation of integrins and leads to the activation of PKC. Specifically, our results indicate that clustering beta 1 integrins results in the activation of phospholipase A2 leading to the production of arachidonic acid and the activation of PKC.     

Avidity modulation activates adhesion under flow and requires cooperativity among adhesion receptors

An early step in activation of leukocyte adhesion is a release of integrins from cytoskeletal constraints on their diffusion, leading to rearrangement and, consequently, increased avidity. Static adhesion assays using purified ligand as a substrate have demonstrated that very low doses of cytochalasin D disconnect beta2-integrins from their cytoskeletal links, allowing rearrangement and activating adhesion. The adhesion process in blood vessels is poorly simulated by these assays, however, for two reasons: leukocyte adhesion to endothelium 1), occurs in the presence of blood flow and 2), involves the simultaneous interactions of multiple sets of adhesion molecules. We investigated the effect of cytochalasin D, at concentrations that increase integrin diffusion but do not alter leukocyte shape and surface features, on adhesion of leukocytes to endothelial cells under flow. Cytochalasin D increased the number of rolling cells, the number of firmly adherent cells, and the duration of both rolling and firm adhesion. These effects required endothelial cell expression of ICAM-1, the ligand for leukocyte beta2-integrins. The beta2-integrin-ICAM-1 interaction alone was not sufficient, however. Experiments using purified substrates demonstrated that avidity effects on activation of adhesion under flow require functional cooperativity between integrins and other adhesion receptors.     

Dynamitin controls Beta 2 integrin avidity by modulating cytoskeletal constraint on integrin molecules

Dynamitin, a subunit of the microtubule-dependent motor complex, was implicated in cell adhesion by binding to MacMARCKS (Macrophage-enriched myristoylated alanine-rice C kinase substrate). However, how dynamitin is involved in cell adhesion is unclear despite the fact that both MacMARCKS and microtubules regulate beta(2) integrin activation. We report that dynamitin regulates beta(2) integrin avidity toward iC3b by modulating the lateral mobility of beta(2) integrin molecules. Using the single particle tracking method, we found that integrin molecular mobility in cells expressing the fusion protein CFP (cyan fluorescent protein)-dynamitin or CFP-MB (the MacMARCKS binding domain peptide of dynamitin) increased 6-fold over the control cells, suggesting that disturbing dynamitin function dramatically altered the cytoskeletal constraint on beta(2) integrin molecules. Further mechanistic studies revealed that overexpression of dynamitin stimulated the phosphorylation of endogenous MacMARCKS protein, which lead to the enhanced tyrosine phosphorylation of paxillin. This effect of dynamitin correlates with the observation that higher concentration of PKC inhibitor is required to block beta(2) integrin mobility in dynamitin-expressing cells. Although dynamitin acts at the point of MacMARCKS phosphorylation, it is upstream of RhoA, because its effect was blocked by RhoA inhibitor. Thus, we conclude that dynamitin is a part of the cytoskeletal constraint that locks beta(2) integrin in the inactive form.     

Microtubule-dependent regulation of Rho GTPases during internalisation of Yersinia pseudotuberculosis

Internalisation of the human pathogen Yersinia pseudotuberculosis via interaction of bacterial invasin with host beta1 integrins depends on the actin cytoskeleton and involves Src family kinases, focal adhesion kinase, p130Crk-associated substrate, proline-rich tyrosine kinase 2, Rac, Arp 2/3 complex and WASP family members. We show here that Rho GTPases are regulated by the microtubule system during bacterial uptake. Interfering with microtubule organisation using nocodazole or paclitaxel suppressed uptake by HeLa cells. The nocodazole effect on microtubule depolymerisation was partially inhibited through overexpression of Rac, Cdc42, RhoG or RhoA and completely prevented by expression of Vav2. This suggests that microtubules influence Rho GTPases during invasin-mediated phagocytosis and in the absence of functional microtubules Vav2 can mimic their effect on one, or more, of the Rho family GTPases. Lastly, overexpression of p50 dynamitin partially inhibited bacterial uptake and this effect was also blocked by co-expression of Vav2, thus further implicating this guanine nucleotide exchange factor in activating Rho GTPases for internalisation during loss of microtubule function.     

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.     

Competition for talin results in trans-dominant inhibition of integrin activation

The ability of integrin adhesion receptors to undergo rapid changes in affinity for their extracellular ligands (integrin activation) is essential for the development and function of multicellular animals and is dependent on interactions between the integrin beta subunit-cytoplasmic tail and the cytoskeletal protein talin. Cross-talk among different integrins and between integrins and other receptors impacts many cellular processes including adhesion, spreading, migration, clot retraction, proliferation, and differentiation. One form of integrin cross-talk, transdominant inhibition of integrin activation, occurs when ligand binding to one integrin inhibits the activation of a second integrin. This may be relevant clinically in a number of settings such as during platelet adhesion, leukocyte trans-migration, and angiogenesis. Here we report that competition for talin underlies the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i). beta tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii). trans-dominant inhibition can be reversed by overexpression of integrin binding and activating fragments of talin, and (iii). expression of another non-integrin talin-binding protein, phosphatidylinositol phosphate kinase type Igamma-90, also inhibits integrin activation. Thus, the sequestration of talin by the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.     

Activation-enhanced alpha(IIb)beta(3)-integrin-cytoskeleton interactions outside of focal contacts require the alpha-subunit

Integrins link the cell's cytoskeleton to the extracellular matrix, as well as to receptors on other cells. These links occur not only at focal contacts but also at smaller integrin-containing protein complexes outside of focal contacts. We previously demonstrated the importance of focal contact-independent integrin-cytoskeleton interactions of beta(2) integrins: activation of adhesion resulted from a release of integrins from cytoskeletal constraints. To determine whether changes in integrin-cytoskeleton interactions were related to activation of the integrin, we used single particle tracking to examine focal contact-independent cytoskeletal associations of alpha(IIb)beta(3)-integrin, in which activation results in a large conformational change. Direct activation of alpha(IIb)beta(3) by mutation did not mimic activation of lymphocytes with phorbol ester, because it enhanced integrin-cytoskeleton interactions, whereas activation of lymphocytes decreased them. Using additional integrin mutants, we found that both alpha- and beta-cytoplasmic domains were required for these links. This suggests that 1) both beta(2)- and beta(3)-integrins interact with the cytoskeleton outside of focal contacts; 2) activation of a cell and activation of an integrin are distinct processes, and both can affect integrin-cytoskeleton interactions; and 3) the role of the alpha-subunit in integrin-cytoskeleton interactions in at least some circumstances is more direct than generally supposed.     

Chemical modulation of VLA integrin affinity in human breast cancer cells

The fact that disruption of integrin-extracellular matrix contacts leads to cell death, has converted cell adhesion into a potential target for the control of invasive cancer. In this work, we studied the functional consequences of the interference with the activity of the very late activation antigen (VLA) family of integrins in human breast cancer cell lines of distinct malignancy. The alpha2beta1-mediated adhesion reduced the entry of highly malignant, hormone-independent breast cancer cells into apoptosis. Adhesion of breast cancer cells through the VLA integrins alpha2beta1 and alpha5beta1 was significantly reduced by an apoptosis-inducing natural triterpenoid, dehydrothyrsiferol (DT), when studied on low amounts of extracellular matrix. This effect was dose-dependent, not related to cell toxicity and not shared with apoptosis-inducing standard chemotherapeutics, such as doxorubicin and taxol. The compound did not affect either the cell surface expression level of VLA integrins or cell distribution of vinculin and actin during cell spreading. In addition, neither phosphorylation of the focal adhesion kinase pp125FAK on Tyr397 nor the protein kinase B (Akt/PKB) on Ser473 was significantly altered by DT. The integrin activation level, assessed by binding of soluble collagen to the alpha2beta1 integrin, was reduced upon cell treatment with DT. Importantly, the TS2/16, an anti-beta1 activating monoclonal antibody was able to rescue DT-treated cells from apoptosis. Since the activation state of integrins is increasingly recognized as an essential factor in metastasis formation, findings presented herein reveal that the chemical regulation of integrin affinity may be a potential therapeutic strategy in cancer therapy.     

Adhesion of neutrophils to fibronectin: role of the cd66 antigens

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.     

Beta3 tyrosine phosphorylation and alphavbeta3-mediated adhesion are required for Vav1 association and Rho activation in leukocytes

Integrin alpha(v)beta(3)-mediated adhesion of hematopoietic cells to vitronectin results in activation of the Rho GTPases. Mutation of beta(3) tyrosine residue 747, previously shown to disrupt cell adhesion, results in sustained activation of Cdc42 and diminished Rac and Rho activity. We investigated the role of the hematopoietically restricted guanine nucleotide exchange factor Vav1 in alpha(v)beta(3)-mediated adhesion. We find that Vav1, a guanine nucleotide exchange factor for Rac and Rho, associates with alpha(v)beta(3) upon cell adhesion to vitronectin and that this association requires beta(3) tyrosine phosphorylation. Expression of exogenous Vav1 demonstrates that Y160F, but not wild type or the Vav1Y174F mutant, inhibits Rac and Rho activation during alpha(v)beta(3)-mediated cell adhesion to vitronectin. Cells expressing Vav1Y160F exhibit a sustained Cdc42 activation similar to nonphosphorylatable beta(3) mutants. In addition, cytoskeletal reorganization and cell adhesion are severely suppressed in Vav1Y160F-transfected cells, and Vav1Y160F fails to associate with beta(3) integrins. Furthermore, Vav1 itself is selectively phosphorylated upon tyrosine 160 after alpha(v)beta(3)-mediated adhesion, and the association between Vav1 and beta(3) occurs in specific response to adhesion to substrate. These studies describe a phosphorylation-dependent association between beta(3) integrin and Vav1 which is essential for cell progression to a Rho-dominant phenotype during cell adhesion.     

Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment

Fluid shear stress is a critical determinant of vascular remodeling and atherogenesis. Both integrins and the small GTPase Rho are implicated in endothelial cell responses to shear but the mechanisms are poorly understood. We now show that shear stress rapidly stimulates conformational activation of integrin alpha(v)beta3 in bovine aortic endothelial cells, followed by an increase in its binding to extracellular cell matrix (ECM) proteins. The shear-induced new integrin binding to ECM induces a transient inactivation of Rho similar to that seen when suspended cells are plated on ECM proteins. This transient inhibition is necessary for cytoskeletal alignment in the direction of flow. The results therefore define the role of integrins and Rho in a pathway leading to endothelial cell adaptation to flow.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.     

Evidence that beta3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from the focal complexes and focal adhesions that form as Rac and RhoA become active

Interaction of integrins with the extracellular matrix leads to transmission of signals, cytoskeletal reorganizations, and changes in cell behavior. While many signaling molecules are known to be activated within Rac-induced focal complexes or Rho-induced focal adhesions, the way in which integrin-mediated adhesion leads to activation of Rac and Rho is not known. In the present study, we identified clusters of integrin that formed upstream of Rac activation. These clusters contained a Rac-binding protein(s) and appeared to be involved in Rac activation. The integrin clusters contained calpain and calpain-cleaved beta3 integrin, while the focal complexes and focal adhesions that formed once Rac and Rho were activated did not. Moreover, the integrin clusters were dependent on calpain for their formation. In contrast, while Rac- and Rho-GTPases were dependent on calpain for their activation, formation of focal complexes and focal adhesions by constitutively active Rac or Rho, respectively, occurred even when calpain inhibitors were present. Taken together, these data are consistent with a model in which integrin-induced Rac activation requires the formation of integrin clusters. The clusters form in a calpain-dependent manner, contain calpain, calpain-cleaved integrin, and a Rac binding protein(s). Once Rac is activated, other integrin signaling complexes are formed by a calpain-independent mechanism(s).     

Fcgamma-receptors induce Mac-1 (CD11b/CD18) mobilization and accumulation in the phagocytic cup for optimal phagocytosis

Functional interactions between Fcgamma-receptors (FcgammaR) and the beta2 integrin Mac-1 (CD11b/CD18) have been described, but the molecular basis of this relationship remains unclear. Although the glycosylphosphatidylinositol-linked receptor FcgammaRIIIB of human neutrophils is constitutively associated with Mac-1, we found no evidence for direct physical association between Mac-1 and the FcgammaR of mouse macrophages, which are transmembrane proteins. Nevertheless, Mac-1 accumulated in the phagocytic cup following engagement of FcgammaR by IgG-opsonized particles. Blocking the CD18 chains of beta2 integrins by using specific antibodies reduced Mac-1 accumulation in the cup. These antibodies or the addition of the recombinant CD11b I-domain inhibited the ingestion of IgG-opsonized particles. FcgammaR cross-linking stimulated cell adhesion to surfaces coated with Mac-1 ligands and in addition enabled macrophages to bind C3bi-opsonized particles, indicating that FcgammaR-derived signals induce activation of Mac-1. Measurements of fluorescence recovery after photobleaching revealed that whereas most (>80%) of Mac-1 is immobile in resting cells, stimulation of FcgammaR markedly increases the mobile fraction of the integrin. Activation of Mac-1 by FcgammaR required the activity of Src family tyrosine kinases, phosphatidylinositol 3-kinase and phospholipase C, with the release of diacylglycerol and stimulation of protein kinase C. Because elevated cytosolic Ca2+ was not required, we suggest that novel protein kinase C isoforms are involved in Mac-1 activation. These results suggest that FcgammaR stimulation promotes Mac-1 clustering into high avidity complexes in phagocytic cups by releasing the integrin from cytoskeletal constraints and enhancing its lateral diffusion. FcgammaR can enhance host defense by activating Mac-1 (and possibly other integrins), having a synergistic effect on pathogen engulfment and promoting the adherence of phagocytes at sites of infection.     

The N-terminal domains of talin cooperate with the phosphotyrosine binding-like domain to activate beta1 and beta3 integrins

The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the beta3 integrin cytoplasmic tail is required for beta3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate beta3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for beta1 activation, and talin F3 binds beta1 and beta3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate beta1 integrins. beta1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate beta1 activation. This effect is not due to increased affinity for the integrin beta tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of beta3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated beta3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of beta1 and beta3 integrins.     

PKB mediates c-erbB2-induced epithelial beta1 integrin conformational inactivation through Rho-independent F-actin rearrangements

Signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to inhibit the adhesive function of the collagen receptor integrin alpha(2)beta(1) in human mammary epithelial cells. This anti-adhesive effect is mediated by the MAP ERK kinase 1/2 (MEK1/2) and protein kinase B (PKB) pathways. Here, we show that both pathways mediate suppression of matrix adhesion by causing the extracellular domain of the beta(1) integrin subunit to adopt an inactive conformation. The conformational switch was also dependent on rapid and extensive actin depolymerisation. While neither activation nor inhibition of the Rho GTPase affected this rearrangement, Rho was found to be activated by c-erbB2 and to be necessary for conformation-dependent integrin inactivation and, apparently by a different mechanism, a delayed re-formation of stress fibers which did not restore integrin function. Interestingly, the initial actin depolymerisation as well as its effects on integrin function was shown to be mediated by PKB. These results demonstrate how oncogenic growth factor signalling inhibits matrix adhesion by multiple pathways converging on integrin conformation and how Rho signalling can profoundly influence integrin activation in a cytoskeleton-independent manner.     

Activation of beta1 integrins induces cell-cell adhesion

Integrins are highly regulated receptors that can function in both cell-substrate and cell-cell adhesion. We have found that the activating anti-beta1 mAb, 12G10, can specifically and rapidly induce both cell-substrate and cell-cell adhesion of HT-1080 human fibrosarcoma and other cell types. Binding of mAb 12G10 induced clustering of cell-surface integrins, and the preferential localization of beta1 integrins expressing the 12G10 epitope at cell-cell adhesion sites. Fab fragments of mAb 12G10 induced HT-1080 cell-cell adhesion as effectively as did intact antibodies, suggesting that integrin clustering was not due to direct antibody crosslinking. Latrunculin B, an inhibitor of F-actin polymerization, inhibited cell-cell adhesion but not the clustering of integrins. Results from a novel, two-color cell-cell adhesion assay suggested that nonactivated cells can bind to activated cells and that integrin activation-induced HT-1080 cell-cell adhesion minimally requires the interaction of activated alpha2beta1 with nonactivated alpha3beta1. These findings suggest that HT-1080 cell-cell adhesion induced by integrin activation require a signaling process involving integrin clustering and the subsequent organization of the cytoskeleton. Integrin activation could therefore play a key role in cell-cell adhesion.     

Alpha 4 beta 1 integrin/VCAM-1 interaction activates alpha L beta 2 integrin-mediated adhesion to ICAM-1 in human T cells

Modulation of integrin affinity and/or avidity provides a regulatory mechanism by which leukocyte adhesion to endothelium is strengthened or weakened at different stages of emigration. In this study, we demonstrate that binding of high-affinity alpha 4 beta 1 integrins to VCAM-1 strengthens alpha L beta 2 integrin-mediated adhesion. The strength of adhesion of Jurkat cells, a human leukemia T cell line, or MnCl2-treated peripheral blood T cells to immobilized chimeric human VCAM-1/Fc, ICAM-1/Fc, or both was quantified using parallel plate flow chamber leukocyte detachment assays in which shear stress was increased incrementally (0.5-30 dynes/cm2). The strength of adhesion to VCAM-1 plus ICAM-1, or to a 40-kDa fragment of fibronectin containing the CS-1 exon plus ICAM-1, was greater than the sum of adhesion to each molecule alone. Treatment of Jurkat or blood T cells with soluble cross-linked VCAM-1/Fc or HP2/1, a mAb to alpha 4, significantly increased adhesion to ICAM-1. These treatments induced clustering of alpha L beta 2 integrins, but not the high-affinity beta 2 integrin epitope recognized by mAb 24. Up-regulated adhesion to ICAM-1 was abolished by cytochalasin D, an inhibitor of cytoskeletal rearrangement. Taken together, our data suggest that the binding of VCAM-1 or fibronectin to alpha 4 beta 1 integrins initiates a signaling pathway that increases beta 2 integrin avidity but not affinity. A role for the cytoskeleton is implicated in this process.     

Integrin regulation by RhoA in thymocytes

The guanine nucleotide-binding protein Rho has essential functions in T cell development and is important for the survival and proliferation of T cell progenitors in the thymus. To explore the mechanisms used by RhoA to control thymocyte biology, the role of this GTPase in the regulation of integrin-mediated cell adhesion was examined. The data show that RhoA activation is sufficient to stimulate beta(1) and beta(2) integrin-mediated adhesion in murine thymocytes. RhoA is also needed for integrin activation in vivo as loss of Rho function impaired the ability of thymocytes to adhere to the extracellular matrix protein VCAM-1 and prevented integrin activation induced by the GTPases Rac-1 and Rap1A in vivo. The regulated activity of integrins is needed for cell motility and in the present study it was seen that RhoA activity is critical for integrin-mediated thymocyte migration to chemokines in vitro. Thus, RhoA has a critical role in regulating cell adhesion and migration during T cell development.     

Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading

Integrin-induced cell adhesion results in transmission of signals that induce cytoskeletal reorganizations and resulting changes in cell behavior. The cytoskeletal reorganizations are regulated by transient activation and inactivation of Rho GTPases. Previously, we identified mu-calpain as an enzyme that is activated by signaling across beta1 and beta3 integrins. We showed that it mediates cytoskeletal reorganizations in bovine aortic endothelial (BAE) and Chinese hamster ovary (CHO) cells and does so by acting upstream of Rac1 activation. Here we show that mu-calpain is also involved in inactivating RhoA during integrin-induced signaling. Cleavage of RhoA was detectable in BAE cells plated on an integrin substrate; it did not occur in cells plated on poly-l-lysine. Cleavage was inhibited by calpain inhibitors. In vitro, mu-calpain cleaved RhoA generating a fragment of the same size as in intact cells. The cleavage site was identified, an HA-tagged construct expressing calpain-cleaved RhoA generated, and the construct expressed in BAE and CHO cells. Calpain-cleaved RhoA inhibited integrin-induced stress fiber assembly and decreased cell spreading. Together, our data show that calpain cleaves RhoA and generates a form that inhibits integrin-induced stress fiber assembly and cell spreading.