Mechanisms for integrating adhesive signals RhoGTPases

RhoGTPases are central to cell signaling pathways both upstream and downstream of cadherin and integrin adhesions (Huveneers and Danen, 2009; Watanabe et al., 2009), making them prime candidates for mediating integration of adhesion-dependent signals. They are known to modulate a wide range of cellular behaviors including cytoskeletal organization, cell polarity, cell proliferation, and the formation and maturation of adhesive junctions (Etienne-Manneville and Hall, 2002). RhoGTPases have a primary role in regulating the assembly of integrin-based focal adhesions (Hall, 1998). Similarly, the assembly of cadherin-based adherens junctions requires the activity of Rho, Rac and Cdc42 (Van Aelst and Symons, 2002). Rho GTPases must be tightly regulated in the cell given that a certain degree of Rho activity is required for cell–cell adhesion but increased levels of Rho activity disrupt cadherin adhesions (Zhong et al., 1997).

Adhesion through classical cadherins results in increased Rac1 activity upon engagement but inhibits RhoA activity over the course of a few hours (Noren et al., 2001). The effects of cadherin adhesion on RhoGTPases are known to require the cytoplasmic domain of cadherin, but the mechanism of signal transduction remains to be established (Noren et al., 2001; Watanabe et al., 2009). A number of proteins bind the cytoplasmic tails of cadherins and some of these have been shown to regulate RhoGTPase activity. p120 catenin binds to the juxtamembrane region of the cadherin tail and, when overexpressed, can decrease Rho activity while increasing Rac and Cdc42 activity (Braga and Yap, 2005). Another study suggested that p120 catenin interacts with p190 Rho GTPase activating protein (RhoGAP), locally inhibiting Rho in response to Rac activation (Wildenberg et al., 2006). p190 RhoGAP is activated by integrin adhesion and is thought to be a convergence point for signaling by integrins and syndecans (Bass et al., 2008). In epithelia and endothelia, in which both cell–cell and cell–matrix adhesions are required, p190 RhoGAP is likely to be a point of convergent signaling between integrins and cadherins.

Rho is transiently activated by the formation of new integrin adhesions (Ren et al., 1999; Bhadriraju et al., 2007), and this activation can have consequences for the adhesive functions of cadherins. There are several examples of RhoGTPases that act as signaling intermediaries between integrins and cadherins. The activities of Rho and Rac downstream of integrin signaling are thought to regulate the formation of adherens junctions in epithelial cells (Playford et al., 2008). Integrin signaling in colon cancer cells promotes cell–cell junction formation through activation of phosphatidylinositol 3-kinase (PI 3-kinase) and Rac1B (Chartier et al., 2006). In addition to these input–output pathways, RhoGTPases are also implicated in convergent signaling. For example, both integrin and cadherin adhesions have been shown to enhance cell proliferation by promoting the expression of cyclin D1 in a redundant manner through Rac (Fig. 2) (Fournier et al., 2008).

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Fig. 2.

Molecular mechanisms for integration of adhesive signals. Activation of Rac by both cadherins (dark blue) and integrins (light green) upregulates proliferation in an additive manner through an increase in the expression of cyclin D1 (orange arrows). Cyclic cell strain induces cadherin-dependent activation of Rac and cyclin D1, but it has not yet been established how sensitive integrin-mediated promotion of proliferation is to cyclic strain. Cadherins and integrins antagonistically influence the activity of Rho GTPase and thus of Rho (purple arrows). Active Rho has dramatically different effects on cell adhesion depending on the downstream effector it binds. Rho signaling through Dia produces reorganization of actin in a manner that strengthens cell–cell adhesion, whereas signaling through ROCK enhances actin contractility, which in turn promotes cell–cell and cell–matrix adhesion. However, a high level of Rho- and ROCK-mediated actin contractility is antagonistic to cell–cell junctions. Fer kinase is activated by cadherin and integrin adhesions, and activated Fer phosphorylates (P) the actin-organizing protein cortactin (blue arrows). Reorganization of the actin cytoskeleton by cortactin can enhance either cell–cell adhesion or migration. Shear stress across endothelial cells leads to inflammation (green arrows). Shear stress induces the assembly of a VEGFR–VE-cadherin–Shc complex (VEGFR in purple) and the phosphorylation of Shc, which then leads to activation of ERK1/2 and, subsequently, to inflammation. Phosphorylated Shc associates with integrins in a cadherin dependent manner and activates the NF-κB pathway, also resulting in inflammation. Solid lines represent direct interactions or effects, dashed lines indicate an indirect or unknown mechanism. The extracellular matrix is shown beneath the cells in blue and the actin cytoskeleton is represented by the pink lines.

Rho activation is a node in the convergent signaling network initiated by cadherins and integrins that can exert varied effects on adhesions depending on the downstream effectors involved (Fig. 2). Rho signaling through diaphamous Dia reorganizes the actin cytoskeleton to stabilize adherens junctions, whereas Rho-kinase (ROCK) is thought to disrupt cell–cell junctions by activating actomyosin contractility to excess (Sahai and Marshall, 2002). RhoA-mediated activation of the effector ROCK is regulated by cell–matrix adhesion, cell shape and cytoskeletal tension. In fact, tension generated by cell spreading is required for activation of ROCK by RhoA (Bhadriraju et al., 2007). This suggests the existence of a positive feedback loop, in which tension generated by cell–matrix adhesion stimulates activation of ROCK, which in turn enhances the formation and maturation of integrin-based adhesions. Regulation of cytoskeletal tension is also crucial for the accumulation of E-cadherin at cell–cell contacts. ROCK regulates the activity of the motor protein nonmuscle myosin II downstream of initial E-cadherin ligation in order to stabilize newly formed cell–cell junctions (Shewan et al., 2005). Cdc42 limits Rho signaling to achieve the crucial tension levels that are required to maintain cell–cell junctions, preventing excess tension that would probably lead to the dissociation of these adhesions (Warner and Longmore, 2009).

Owing to the complex spatiotemporal regulation of RhoGTPases, it is difficult to separate their roles in the initial establishment of adhesions from those in the signaling events downstream of cadherin and integrin engagement. Furthermore, because these proteins are involved in so many pathways, localization and timing become extremely important in dictating cellular responses to a given stimulus. Rac1 is localized to sites of cell–cell contact where it might have a role in mediating rapid changes in actin organization concurrent with the formation of new cell–cell junctions (Ehrlich et al., 2002). More recent work has shown that localization of both Rac and Rho to cell–cell contacts is essential for the formation and expansion of cadherin adhesions. The spatiotemporal localization of both the GTPases and their effectors suggests specific roles in adhesion formation, with Rac facilitating actin remodeling and Rho stimulating actomyosin contractility (Yamada and Nelson, 2007). The downstream effects of RhoGTPase activation can differ dramatically depending on the site of activation. Rac is required for epithelial wound healing and promotes cell migration when activated at cell–matrix contact sites, but promotes cell–cell adhesion and formation of adherens junctions when activated at cell–cell junctions (Van Aelst and Symons, 2002; Liu et al., 2010). Network complexity increases when we consider the functions of other GTPases, such as the Ras family member Rap1, which has been implicated in regulation of integrin activity downstream of cadherins (see Box 2). Clearly, signaling through GTPases is a much-used mechanism for intracellular communication, and is influenced by both cell–cell and cell–ECM adhesions. Only recently were suitable tools developed that enable GTPase activities to be visualized in time and space, thus revealing specific activation events that mediate interactions between adhesions (Yamada and Nelson, 2007; Wang et al., 2010).

Tyrosine kinases

A number of tyrosine kinases are localized to cell–cell and cell–matrix adhesions (Giannone and Sheetz, 2006; McLachlan et al., 2007) where they function as prominent nodes in the adhesive network. Activation of Src family kinases frequently accompanies the formation of both cell–cell and cell–ECM adhesions. Src is recruited and activated upon E-cadherin ligation and this provides a positive feedback loop that signals through PI 3-kinase to promote the stability of cell–cell contacts (McLachlan et al., 2007). However, Src levels at cell–cell adhesions must be tightly regulated, because constitutively active Src disrupts cell–cell contacts and alters cell morphology (Behrens et al., 1993). Integrin ligation also leads to Src activation and this has divergent downstream consequences, often involving RhoGTPases (Huveneers and Danen, 2009). In epithelial cells, constitutively active Src at sites of integrin–matrix adhesion leads to peripheral accumulation of activated myosin, which is disruptive to cell–cell junctions (Avizienyte et al., 2004). However, moderate Src activation and regulation of actomyosin contractility by ROCK or myosin light-chain kinase (MLCK) are necessary for the integrin-mediated strengthening of E-cadherin adhesions (Martinez-Rico et al., 2010). Although cytoskeletal tension is important for the formation of both cell–cell and cell–matrix adhesions, excessive tension might serve to rip junctions apart or induce changes in protein conformation that lead to junctional instability.

The focal adhesion kinase (Fak, also known as PTK2) is another non-receptor tyrosine kinase, which as a primary signaling partner of Src is implicated in many of the same signaling pathways (Playford and Schaller, 2004). Unlike Src, however, Fak contains a focal adhesion targeting (FAT) sequence, consistent with its role as a downstream effector of integrin adhesion and signaling. For example, transforming growth factor-β (TGF-β) inhibits epithelial-to-mesenchymal transition (EMT) in at least some colon cancer cells, and stimulates increased expression of ECM leading to integrin engagement and activation of Fak (Wang et al., 2004). Fak then promotes E-cadherin expression and cell cohesion (Wang et al., 2004), but the mechanism by which this occurs remains unclear. Nonetheless, this is consistent with Fak knockdown studies, which report stimulation of EMT and inhibition of cadherin adhesion (Yano et al., 2004). Because Fak is normally associated with integrin adhesions there is much speculation about its role in cell–cell junctions. Regulation of RhoGTPases is one possible mechanism of action because Fak can inhibit the activity of Rho, and constitutively active Rho mutants can phenocopy Fak loss-of-function (Playford et al., 2008). Interestingly, Fak has also been shown to localize to cell–cell contacts, although the significance – if any – of its localization at cell–cell adhesions is unclear (Crawford et al., 2003; Playford et al., 2008).

The non-receptor tyrosine kinase Fer is also reported to be involved in communication between cell–cell and cell–ECM adhesive contacts (Arregui et al., 2000). Fer can be activated upon engagement of either cadherins or integrins (El Sayegh et al., 2005; Sangrar et al., 2007). The association of Fer with the actin-organizing protein cortactin might help coordinate cellular response to various adhesive inputs. Cortactin is required for cell spreading on fibronectin, and the activation of cortactin by Fer promotes cell motility (Illes et al., 2006; Sangrar et al., 2007). Phosphorylation of cortactin by Src and/or Fer downstream of E-cadherin ligation enhances cell–cell adhesion (El Sayegh et al., 2005; Ren et al., 2009). This suggests that Fer signaling feeds back to cell–cell and cell–matrix adhesions by promoting cortactin-dependent reorganization of actin (Fig. 2).

Several growth factor receptor tyrosine kinases are reported to regulate cadherin and integrin adhesive functions. For instance, vascular endothelial growth factor (VEGF) signalling through the VEGF receptor 2 (VEGFR2, also known as KDR) both increases integrin-dependent migration and decreases stability of vascular endothelial (VE)–cadherin adhesions (Carmeliet et al., 1999; Byzova et al., 2000). Receptor tyrosine kinases can also enable lateral molecular associations between different types of adhesion. IGF1R forms a ternary complex with E-cadherin and αv-integrins at cell–cell contacts. Binding of the ligand IGF1 causes the relocalization of αv-integrin to focal contacts and an increase in cell migration (Canonici et al., 2008). Thus, IGF1R might sequester integrins at cell–cell contacts and inhibit migration in the absence of growth factor signaling.

Phosphatases

Given the central importance of kinases to many adhesion-dependent cell signaling pathways, it is crucial to also consider the role of phosphatases in the regulation of adhesive networks. There are a number of protein tyrosine phosphatases (PTPs) that associate with cell–cell and cell–ECM adhesions – such as PTP1B, which localizes to the cytoplasmic tail of cadherins and also to focal adhesions (Stoker, 2005; Burridge et al., 2006; Sallee et al., 2006). The catalytic activity of PTP1B is required for dephosphorylation of β-catenin and association of N-cadherin with the actin cytoskeleton, both of which are crucial for the stability of cell–cell junctions (Sallee et al., 2006). PTP1B is also involved in signaling from integrin adhesions, probably through dephosphorylation of the inhibitory tyrosine residue of Src, which leads to activation of Fak (Arregui et al., 1998). Outgrowth of neurites depends on both cell–cell and cell–matrix interactions, and PTP1B has been shown to be required for this process, probably also through regulation of Src activity (Pathre et al., 2001). PTPμ is another member of the PTP family that is necessary for maintaining the integrity of cell–cell adhesions. In addition to its role in stabilizing cell–cell junctions through dephosphorylation of junctional components, PTPμ might also recruit regulatory proteins to sites of cell–cell adhesion (Sallee et al., 2006). Expression of PTPμ in kidney epithelial cells is dependent on a complex between α3β1-integrin and tetraspanin that associates with E-cadherin on the lateral membranes of cells. An interesting feature of this lateral complex coupling is that a specific subset of unligated integrins appears to associate with and promote stability of cell–cell adhesions, and that these are distinct from the subset of integrins involved in matrix adhesion (Chattopadhyay et al., 2003).

Scaffolding and adaptor proteins

The consequences of cadherin and integrin engagement can vary greatly depending upon the specific intracellular environment at the site of signal initiation. Scaffolding and adaptor proteins are crucial elements of adhesion-dependent signaling cascades. They facilitate the localization of key downstream effectors, thereby increasing the probability of interactions between activated signaling components. Receptor for activated C kinase-1 (RACK1, also known as GNB2L1) is a scaffolding protein that binds the cytoplasmic tails of integrins and interacts with intracellular signaling components such as protein kinase C (PKC) (Besson et al., 2002). RACK1 is a crucial component of the E-cadherin and α3β1-integrin–tetraspanin lateral signaling complex described above (Chattopadhyay et al., 2003). Binding to PTPμ might recruit RACK1 to cell–cell adhesions, serving to localize activated PKC or other effectors (Mourton et al., 2001). PKC has numerous roles in promoting cell–cell and cell–matrix adhesions (Larsson, 2006). Localization of active PKC by RACK1 might be key to specifying the types of adhesion that are affected by PKC signaling.

In general, adaptor proteins link signaling components but lack intrinsic enzymatic or kinase activities. Motifs such as the phosphotyrosine-binding Src homology 2 (SH2) domain allow adaptors to bring signaling proteins together and propagate signaling cascades initiated by a diverse array of stimuli. The SH2 domain-containing (Shc) adaptor protein forms a complex with growth factor receptor-bound protein 2 (GRB2) and the Son of Sevenless (SOS) family of guanine nucleotide exchange factors to activate Ras and, subsequently, mitogen-activated protein (MAP) kinases, the pathway of which is activated through many inputs including integrin signaling and mechanical force (Ravichandran, 2001). Shc has recently been shown to associate with both cell–cell and cell–matrix adhesions in response to fluid flow across vascular endothelial cells (Liu et al., 2008). Phosphorylated Shc is recruited to cell–cell junctions at the onset of fluid flow, particularly in areas of tissue where fluid-flow shear stresses are high and result in inflammation. Previous work demonstrated the assembly of a VEGFR2–VE-cadherin complex in response to shear stress and it is now apparent that Shc interacts with components of this complex (Shay-Salit et al., 2002; Liu et al., 2008). Shc is also recruited to integrin adhesions in response to fluid flow, a process that requires the presence of VE-cadherin. Phosphorylation of the MAPK family members ERK1/2 and activation of NF-κB are increased by fluid flow in a Shc-dependent manner. Interestingly the activation of NF-κB, but not the activation of ERK1/2, is dependent on ECM composition (Liu et al., 2008). These data suggest that Shc is a central mediator of fluid flow-induced signaling events that include cadherin-dependent activation of signaling at cell–matrix junctions (Fig. 2).

There are several other classes of protein that can act as adaptors even though they are not traditionally thought of as such. One example is the catenin family of proteins, of which some are involved in both cell–cell adhesion and cell signaling. Catenins are crucial for connecting cadherins to the cytoskeleton, although not necessarily through a direct physical link (Nelson, 2008). Catenins are required for the initiation of many signaling pathways downstream of cadherin adhesion, including those that regulate RhoGTPase activities, actomyosin organization and contractility, and stability of cell–cell junctions (Perez-Moreno and Fuchs, 2006). Plakoglobin (Pkg, also known as JUP or γ-catenin), associates with both desmosomes and adherens junctions where it helps maintain tissue integrity through interactions with α-catenin or desmoplakin (Zhurinsky et al., 2000; Acehan et al., 2008). By serving as a link between cadherin and either α-catenin or desmoplakin, Pkg connects cadherins to actin or intermediate filament networks, respectively. Pkg is reported to suppress motility of cell sheets and single keratinocytes on collagen (Yin et al., 2005). Although the mechanism for regulation of cell motility by Pkg is not clear, there is some evidence that Pkg works through long-range mechanisms to promote fibronectin expression and inhibit Src function (Yin et al., 2005; Todorovic et al., 2010). Pkg is a known target of Src kinase activity, thus it is possible that the presence of Pkg at cell–cell junctions sequesters Src away from cell–matrix adhesions where it typically promotes cell migration (Webb et al., 2004; Lee et al., 2010).

Migration

In cancer metastasis and EMT, integrin and cadherin adhesions display an antagonistic relationship that determines whether cells maintain associations with their neighbors or uncouple and initiate migration (Avizienyte et al., 2002; Guarino, 2007). For example, in ovarian carcinoma cells, downregulation of E-cadherin through matrix metalloproteinase (MMP)-mediated proteolysis is initiated by integrin ligation to collagen (Symowicz et al., 2007). This decreased cadherin-mediated cohesion allows the cells to disperse and migrate independently.

Although this scenario might best reflect the behavior of metastatic cancer cells and other instances of EMT, it is important to consider that normal, intact tissues also undergo migratory processes. In collective cell migration, substrate traction is finely balanced with cell cohesion to maintain organization of the migratory tissue (Fig. 3A). In fact, cadherin adhesions in normal tissues provide instructive signals that regulate the polarity of cells and, as a result, the direction of migration. As cells come in contact with one another, cadherin adhesions are formed, Cdc42 signaling is activated, and the centrosome reorients anteriorly relative to the cell nucleus (Desai et al., 2009; Dupin et al., 2009), indicating that a broad repolarization has occurred throughout the cell. Protrusive activity is suppressed locally near cadherin adhesions, and increased on opposing sides of the cell at sites of integrin–ECM adhesion (Desai et al., 2009; Borghi et al., 2010). Even highly migratory tissues, such as some cranial neural crest, maintain cell–cell adhesions while they move, although these adhesions are dynamic and cell contacts transient. N-cadherin adhesions in neural crest cells are required for the subcellular localization of activated RhoGTPases in response to chemoattractants. Without N-cadherin the cells fail to migrate directionally or collectively (Theveneau et al., 2010). Expression of N-cadherin at the cell surface is regulated locally through signaling pathways that are initiated by β1- and β3-integrins (Monier-Gavelle and Duband, 1997). In border cells of Drosophila melanogaster, asymmetric protrusions initiated by localized Rac activation in a single border cell can alter migratory direction of an entire cluster of border cells (Wang et al., 2010). Cohesion of border cells is stabilized by both β-integrin and Rac signaling through the MAPK Jun N-terminal kinase (JNK), and these signaling proteins are required for normal collective cell migration (Llense and Martin-Blanco, 2008; Wang et al., 2010).

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Fig. 3.

Mechanical inputs modulate adhesive networks in complex tissue systems. (A) Collective migration involves traction forces through integrin–matrix adhesions to drive the tissue forward (green arrows). Extracellular matrix substratum is in purple. The tissue advances against intercellular tissue tension (pink arrows) that is mediated by cadherins (pink rectangles). This intercellular tension is anisotropically distributed in the tissue and is greater within the tissue than at the leading edge. (B) Convergence and extension movements in gastrulation involve coordinated regulation of different adhesion types. Bidirectional protrusive activity (green arrows) mediated by PCP signaling and integrin–matrix adhesions enables sliding of cells past each other, which is a cadherin-dependent process (cadherin adhesions are shown as pink rectangles). Intercalation behavior creates force in anterior and posterior directions, thereby driving axial elongation. (C) In vivo, assembly of matrix (purple lines) can be regulated by force transduced through cadherin adhesions (pink rectangles). In multicellular tissues, cells pull on one another (pink arrows) through cadherin adhesions to increase local mechanical tension. It has been proposed that integrins translocate from sites of cell–cell contact across the free surface of the cell (green arrows) and promote matrix assembly. (D) Endothelial cells in vivo are simultaneously exposed to fluid-flow shear stress, cyclic strain and hydrostatic compression (large open arrows), each representing a mechanical stress that applies force to cadherin and integrin adhesions (filled arrows, color matched to the applicable force). Mechanical forces on cadherins and integrins initiate input–output and convergent type signaling across the adhesive network to regulate cell morphology and physiological responses such as cell proliferation.

Migration of whole tissues is a recurring feature of development and morphogenesis, and wound healing. Collectively migrating tissues use a mechanism of distributed traction, whereby both leading edge cells and those that follow polarize and migrate in a directional manner (Davidson et al., 2002; Farooqui and Fenteany, 2005). Substrate traction forces and intercellular tissue tension are developed as an intact tissue translocates. Trepat and co-workers reported that, although leading cells generate the highest substrate traction, intercellular tension increases progressively as a function of distance from the leading edge (Trepat et al., 2009). Moreover, tension on cadherin adhesions recruits scaffolding proteins, such as vinculin and α-catenin, which mediate cytoskeletal reinforcement of the adhesions and the resultant strain-stiffening response (Chu et al., 2004; le Duc et al., 2010; Yonemura et al., 2010). However, an important question remains when tying these observations together: does tension on cadherin adhesions have ramifications for protrusive activity and polarity of the cell? We speculate that, because cadherins are responsive to mechanical force, changes in the physical linkage of cadherins to cytoskeletal networks and the association with scaffolding proteins have an important role in the polarization of migratory cells and tissues.

Early embryonic morphogenesis

Several examples of adhesive networks can be illustrated by using early-embryonic models of tissue morphogenesis. During convergent extension, mesodermal cells mediolaterally intercalate to form the notochord and drive axial elongation of the embryo (Fig. 3B). The cells are not able to complete this process in the absence of fibronectin, when α5β1-integrin is inhibited, or when cadherin adhesion is altered (Marsden and DeSimone, 2003; Davidson et al., 2006). Moreover, adhesion and signaling of α5β1-integrin can modulate cadherin adhesion, which is required for cell intercalation and cell-sorting behaviors (Marsden and DeSimone, 2003), although the mechanism of signaling between these adhesions remains unclear. Communication between adhesion receptors in this system is also bidirectional. Tension on cadherin adhesions induces integrin-dependent assembly of fibronectin fibrils (Fig. 3C) (Dzamba et al., 2009). Fibronectin fibril assembly is, in turn, required for normal morphogenetic movements in early embryogenesis, such as epiboly and mesendodermal migration (Rozario et al., 2009). Activation of the planar cell polarity pathway (PCP) – a non-canonical Wnt signaling cascade – is required for convergence and extension movements, and also promotes the assembly of the fibronectin matrix by regulating cadherin adhesion and tissue tension (Dzamba et al., 2009). Thus, PCP signaling is probably an important signaling pathway that links cadherin and integrin functions in the early embryo.

We thank our colleagues Bette Dzamba, Tania Rozario and Shuo Wei for reading and commenting on the manuscript. The authors were supported by USPHS grants F32-GM83542; to G.F.W., T32-GM08136; to M.A.B., and R01-HD26402/GM094793 to D.W.D. Deposited in PMC for release after 12 months.