Ras GTPases: integrins' friends or foes?

Integrins are cell-surface receptors that mediate and coordinate cellular responses to the extracellular matrix (ECM). Cellular signalling pathways can regulate cell adhesion by altering the affinity and avidity of integrins for ECM. The Ras family of small G proteins, which includes H-ras, R-ras and Rap, are important elements in cellular signalling pathways that control integrin function.     

Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.     

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.     

Vesicle-associated membrane protein 2 mediates trafficking of α5β1 integrin to the plasma membrane

Integrins are major receptors for cell adhesion to the extracellular matrix (ECM). As transmembrane proteins, the levels of integrins at the plasma membrane or the cell surface are ultimately determined by the balance between two vesicle trafficking events: endocytosis of integrins at the plasma membrane and exocytosis of the vesicles that transport integrins. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, is involved in the trafficking of α5β1 integrin. VAMP2 was present on vesicles containing endocytosed β1 integrin. Small interfering RNA (siRNA) silencing of VAMP2 markedly reduced cell surface α5β1 and inhibited cell adhesion and chemotactic migration to fibronectin, the ECM ligand of α5β1, without altering cell surface expression of α2β1 integrin or α3β1 integrin. By contrast, silencing of VAMP8, another SNARE protein, had no effect on cell surface expression of the integrins or cell adhesion to fibronectin. In addition, VAMP2-mediated trafficking is involved in cell adhesion to collagen but not to laminin. Consistent with disruption of integrin functions in cell proliferation and survival, VAMP2 silencing diminished proliferation and triggered apoptosis. Collectively, these data indicate that VAMP2 mediates the trafficking of α5β1 integrin to the plasma membrane and VAMP2-dependent integrin trafficking is critical in cell adhesion, migration and survival.     

Phospholipase C epsilon suppresses integrin activation

Phospholipase Cepsilon (PLCepsilon) is a newly described effector of the small GTP-binding protein H-Ras. Utilizing H-Ras effector mutants, we show that mutants H-Ras(G12V/E37G) and H-Ras(G12V/D38N) suppressed integrin activation in an ERK-independent manner. H-Ras(G12V/D38N) specifically activated the PLCepsilon effector pathway and suppressed integrin activation. Inhibition of PLCepsilon activation with a kinase-dead PLCepsilon mutant prevented H-Ras(G12V/D38N) from suppressing integrin activation, and low level expression of H-Ras(G12V/D38N) could synergize with wild-type PLCepsilon to suppress integrins. In addition, knockdown of endogenous PLCepsilon with small interfering RNA blocked H-Ras(G12V/D38N)-mediated integrin suppression. Suppressing integrin function with the H-Ras(G12V/D38N) mutant reduced cell adhesion to von Willebrand factor and fibronectin; this reduction in cell adhesion was blocked by coexpression of the kinase-dead PLCepsilon mutant. These results show that H-Ras suppresses integrin affinity via independent Raf and PLCepsilon signaling pathways and demonstrate a new physiological function for PLCepsilon in the regulation of integrin activation.     

Lamellipodia nucleation by filopodia depends on integrin occupancy and downstream Rac1 signaling

Time-lapse video-microscopy unambiguously shows that fibroblast filopodia are the scaffold of lamellipodia nucleation that allows anisotropic cell spreading. This process was dissected into elementary stages by monitoring cell adhesion on micropatterned extracellular matrix arrays of various pitches. Adhesion structures are stabilized by contact with the adhesive plots and subsequently converted into lamellipodia-like extensions starting at the filopodia tips. This mechanism progressively leads to full cell spreading. Stable expression of the dominant-negative Rac1 N17 impairs this change in membrane extension mode and stops cell spreading on matrix arrays. Similar expression of the dominant-negative Cdc42 N17 impairs cell spreading on homogenous and structured substrate, suggesting that filopodia extension is a prerequisite for cell spreading in this model. The differential polarity of the nucleation of lamellipodial structures by filopodia on homogenous and structured surfaces starting from the cell body and of filopodia tip, respectively, suggested that this process is triggered by areas that are in contact with extracellular matrix proteins for longer times. Consistent with this view, wild-type cells cannot spread on microarrays made of function blocking or neutral anti-beta 1 integrin antibodies. However, stable expression of a constitutively active Rac1 mutant rescues the cell ability to spread on these integrin microarrays. Thereby, lamellipodia nucleation by filopodia requires integrin occupancy by matrix substrate and downstream Rac1 signaling.     

Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

Connections between integrins and Rac GTPase pathways control gonad formation and function in C. elegans

The integrins are a family of alphabeta heterodimeric transmembrane receptors that link extracellular matrix (ECM) proteins to the cytoskeleton and orchestrate cell behaviors. It's been suggested that integrins interact with Rho family small GTPases, such as Rho and Rac. We took advantage of a C. elegans nematode line expressing HA-betatail, a beta integrin transgene inhibiting the functions of endogenous integrins, to determine the combined effects of reducing PAT-3 beta integrin and Rac pathway activities. Double mutants of HA-betatail and unc-73, a guanine nucleotide exchange factor GEF for MIG-2/Rac, had body wall and vulval muscle abnormalities. On the other hand, HA-betatail combined with mutant CED-5, another Rac interacting protein, showed ovulation defects and sterility. RNA-mediated interference (RNAi) of pat-3 on Rac mutant backgrounds also affected gonad structure and function. These results show a functional link between integrins and Rac signaling in muscles and gonads. Furthermore, data showing distinct phenotypes of HA-betatail with unc-73 versus ced-5 suggest some tissue-specificity in the usage of Rac signaling pathways.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.     

Outside-in regulation of integrin clustering processes by ECM components per se and their involvement in actin cytoskeleton organization in a colon adenocarcinoma cell line

We investigated in a colon adenocarcinoma cell line, the exclusive role of extracellular matrix (ECM) components in the absence of soluble factors regarding the integrin clustering processes, and their implication in cell adhesion, spreading and organization of the actin cytoskeleton. Caco-2 cells were shown to express at the plasma membrane 11 integrins, some of which (e.g. alpha3beta1, alpha5beta1, alpha6beta1/beta4, alpha8beta1 and alpha(v)beta1/beta5/beta6) were identified for the first time in this cell line. Cell adhesion and spreading processes were governed essentially by lamellipodium, the regulation of which was shown to be induced by two types of integrin clustering processes mediated by ECM proteins alone. During these phenomena, alpha2beta1, alpha(v)beta6 and alpha6beta1 integrins, the Caco-2 cell specific receptors of type IV collagen, fibronectin and laminin, respectively, were clustered in small focal complexes (point contacts), whereas alpha(v)beta5, the vitronectin receptor in this cell line, was aggregated in focal adhesions. The two levels of integrin clustering induced only F-actin cortical web formation organized in thin radial and/or circular filaments. We conclude thus that ECM components per se through their action on integrin clustering are involved in cell adhesion, cortical actin cytoskeleton organization and cell spreading.     

Efficient dendritic cell priming of T lymphocytes depends on the extracellular matrix protein mindin

Rho guanosine triphosphatases (GTPases) regulate multiple aspects of dendritic cell (DC) function, but what regulates the expression of Rho GTPases in DCs is unknown. Here, we show that the extracellular matrix protein mindin regulates the expression of Rho GTPases in DCs. Mindin(-/-) mice displayed defective CD4+ T-cell priming and impaired humoral immune responses to T-dependent antigens. Mindin(-/-) DCs had reduced expression of Rac1/2 and impaired priming capacity owing to inefficient engagement with T lymphocytes. Ectopic Rac1 expression restored the priming capability of Mindin(-/-) DCs. Furthermore, we show that DC adhesion to mindin matrix was blocked by antibodies to alpha4, alpha5 and beta1 integrins. DCs lacking beta1 integrin had reduced adhesion to mindin matrix, decreased expression of Rac1/2 and impaired priming capacity. These results suggest that mindin-integrin interactions play a key role in regulating Rho GTPase expression in DCs and DC priming of T lymphocytes.     

Tenectin is a novel αPS2βPS integrin ligand required for wing morphogenesis and male genital looping in Drosophila

Morphogenesis of the adult structures of holometabolous insects is regulated by ecdysteroids and juvenile hormones and involves cell-cell interactions mediated in part by the cell surface integrin receptors and their extracellular matrix (ECM) ligands. These adhesion molecules and their regulation by hormones are not well characterized. We describe the gene structure of a newly described ECM molecule, tenectin, and demonstrate that it is a hormonally regulated ECM protein required for proper morphogenesis of the adult wing and male genitalia. Tenectin's function as a new ligand of the PS2 integrins is demonstrated by both genetic interactions in the fly and by cell spreading and cell adhesion assays in cultured cells. Its interaction with the PS2 integrins is dependent on RGD and RGD-like motifs. Tenectin's function in looping morphogenesis in the development of the male genitalia led to experiments that demonstrate a role for PS integrins in the execution of left-right asymmetry.     

Inside-out integrin signalling.

Integrins are expressed by virtually all cells and play key roles in a range of cellular processes. Changes in the integrin surface repertoire provide a means of altering the strength and ligand preferences of cell adhesion. Recent research has examined the affinity modulation of integrins, a rapid and versatile mechanism of cell adhesion regulation. Studies with a prototype, alpha IIb beta 3, indicate that intracellular events influence the conformation and ligand-binding affinity of the extracellular domain of integrins. This 'inside-out' signal transduction appears to be mediated through the integrin cytoplasmic domains. In addition, in some cases affinity modulation of integrins may be cell-type specific. The clarification of the mechanisms of integrin affinity modulation should help explain rapid changes in cell adhesion that occur during cell migration, aggregation and the cell cycle.     

A Spatial Model for Integrin Clustering as a Result of Feedback between Integrin Activation and Integrin Binding

Integrins are transmembrane adhesion receptors that bind extracellular matrix (ECM) proteins and signal bidirectionally to regulate cell adhesion and migration. In many cell types, integrins cluster at cell-ECM contacts to create the foundation for adhesion complexes that transfer force between the cell and the ECM. Even though the temporal and spatial regulation of these integrin clusters is essential for cell migration, how cells regulate their formation is currently unknown. It has been shown that integrin cluster formation is independent of actin stress fiber formation, but requires active (high-affinity) integrins, phosphoinositol-4,5-bisphosphate (PIP2), talin, and immobile ECM ligand. Based on these observations, we propose a minimal model for initial formation of integrin clusters, facilitated by localized activation and binding of integrins to ECM ligands as a result of biochemical feedback between integrin binding and integrin activation. By employing a diffusion-reaction framework for modeling these reactions, we show how spatial organization of bound integrins into clusters may be achieved by a local source of active integrins, namely protein complexes formed on the cytoplasmic tails of bound integrins. Further, we show how such a mechanism can turn small local increases in the concentration of active talin or active integrin into integrin clusters via positive feedback. Our results suggest that the formation of integrin clusters by the proposed mechanism depends on the relationships between production and diffusion of integrin-activating species, and that changes to the relative rates of these processes may affect the resulting properties of integrin clusters.     

Activation pathways of alpha4beta1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation

Alpha4beta1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. Alpha4beta1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti-beta1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of 41 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, alpha-CD3 mAb or the chemokine SDF-1alpha followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (alpha4beta1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the alpha4beta1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of alpha4beta1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements.     

Affinity of integrins for damaged extracellular matrix: alpha v beta 3 binds to denatured collagen type I through RGD sites.

Cellular adhesion receptors termed integrins play an important role in the interaction of cells with extracellular matrix (ECM) during wound healing, development and tumorigenesis. During such events, ECM may become modified or damaged which could alter the types of adhesive signals presented to cells. In this study, cell adhesion and affinity chromatography experiments were performed to determine whether different integrins interact with denatured versus native ECM molecules. Human melanoma cells were found to adhere to denatured versus native type I collagen through different integrins. The cells adhere to denatured collagen through the alpha v beta 3 integrin and this interaction is inhibited by an RGD containing peptide but not by a control peptide. In contrast, adhesion to native type I collagen appears to be mediated by several beta 1 integrins and thus, is not inhibited by either alpha v beta 3 antibodies or the RGD peptide. Affinity chromatography reveals a marked increase in the quantity of alpha v beta 3 isolated on denatured collagen versus native collagen-sepharose. These results suggest that RGD sites in type I collagen may be masked and that they become exposed upon denaturation of the molecule. Wounding of extracellular matrix may, thus, expose RGD sites in collagens that facilitate the interaction of cells with damaged extracellular matrix through RGD binding integrins.     

Induction of alpha v beta 3 integrin-mediated attachment to extracellular matrix in beta 1 integrin (CD29)-negative B cell lines.

beta 1 integrin containing complexes have been implicated as the primary adhesion structures in many lymphocyte extracellular matrix (ECM) interactions. However, many B lymphocytes lack surface expression of the beta 1 subunit, implying that this subpopulation of lymphoid cells must employ alternate adhesion structures if they are to maintain an interactive capacity with ECM. An examination of the adherence properties of the beta 1 integrin-negative B cell line JY indicated that these cells exhibit little or no basal adherence to any of the ECM components examined. However, these cells could be induced to adhere to the ECM components fibronectin, laminin, and vitronectin following treatment with PMA. Blocking studies with monoclonal antibodies indicated the alpha v beta 3 integrin complex was involved in the attachment to each of these ligands. However, the adherence to fibronectin displayed a complex pattern of inhibition suggesting the involvement of other ECM receptors. The utilization of the alpha v beta 3 complex was not unique to the JY cell line. Other B cell lines were observed to employ alpha v beta 3, and these lines similarly lacked expression of beta 1 integrin. These results indicate that alpha v beta 3 can act as a lymphoid ECM-adhesion structure which may provide an alternative means for lymphocytes to interact with ECM. Furthermore, these studies provide evidence for the presence of lymphoid-associated alpha v beta 3 integrins with regulatable activity, which contrasts with the constitutive adhesive potential of these complexes when present on other cell types.