Print version ISSN 0716-9760
Biol. Res. vol.35 no.2 Santiago 2002
Biol Res 35: 239-246, 2002
Regulation of Rho Family GTPases by Cell-Cell and
Integrins and cadherins are transmembrane adhesion receptors that are necessary for cells to interact with the extracellular matrix or adjacent cells, respectively. Integrins and cadherins initiate signaling pathways that modulate the activity of Rho family GTPases. The Rho proteins Cdc42, Rac1, and RhoA regulate the actin cytoskeleton. Cdc42 and Rac1 are primarily involved in the formation of protrusive structures, while RhoA generates myosin-based contractility. Here we examine the differential regulation of RhoA, Cdc42, and Rac1 by integrin and cadherin signaling. Integrin and cadherin signaling leads to a decrease in RhoA activity and activation of Cdc42 and Rac1. When the normal RhoA suppression is antagonized or RhoA signaling is increased, cells exhibited impaired spreading on the matrix protein fibronectin and decreased cell-cell adhesion. Spreading on fibronectin and the formation of cell-cell adhesions is decreased in cells expressing dominant negative forms of Cdc42 or Rac1. These data demonstrate that integrins and cadherins regulate Rho proteins in a comparable manner and lead us to speculate that these changes in Rho protein activity participate in a feedback mechanism that promotes further cell-matrix or cell-cell interaction, respectively.
Key Terms: Integrins, Cadherins, Adhesion, Rho, Rac, Cdc42
Integrins and cadherins are two distinct families of transmembrane cell adhesion receptors. Integrins allow cells to adhere to the extracellular matrix. Cadherins bind homotypically to cadherins on neighboring cells and are responsible for the development of adherens junctions in epithelial tissues. During the formation of both cell-matrix and cell-cell adhesions, cells undergo morphological changes, one of which is a reorganization of the actin cytoskeleton. The Rho family of small GTPases are key regulators of the actin cytoskeleton. Rho proteins function as molecular switches, relaying biochemical signals from extracellular cues that give rise to intracellular responses. By controlling the activity of numerous downstream effectors, Rho GTPases are able to regulate a range of cellular functions such as endocytosis, adhesion, polarity, spreading, migration, gene expression, and cell growth.
Function of Rho Family GTPases
Because Rho proteins are involved in dynamic cellular processes their activity is tightly regulated (Van Aelst and D'Souza-Schorey, 1997; Kaibuchi et al., 1999). Most Rho proteins cycle between an active GTP-bound state and an inactive GDP-bound state (Van Aelst and D'Souza-Schorey, 1997). These proteins are activated upon association with members of the Dbl family of guanine nucleotide exchange factors (GEFs). GEFs catalyze the release of GDP allowing GTP to spontaneously bind and thereby activate the proteins. In this conformation the GTPases interact with and stimulate the activity of effectors. Active Rho GTPases return to an inactive state upon hydrolysis of GTP to GDP. Although these proteins possess intrinsic GTPase activity, this activity is significantly augmented in the presence of GTPase activating proteins (GAPs). Rho GTPases are also negatively regulated by guanine nucleotide disassociation inhibitors which extract Rho proteins from the plasma membrane and prevent nucleotide release (Olofsson, 1999).
Cdc42, Rac1, and RhoA are the most thoroughly understood members of the Rho family. These proteins play a pivotal role in determining the architecture of the actin cytoskeleton. Cdc42 and Rac1 stimulate the formation of two types of membrane protrusions known as filopodia and lamellipodia (Ridley et al., 1992; Nobes and Hall, 1995). Filopodia are finger-like projections which may act as sensors, probing the extracellular milieu. Lamellipodia are broad sheets rich in polymerizing actin. Both of these protrusive structures allow cells to physically extend into new areas and are thus commonly found along the circumference of spreading cells and at the leading edge of motile cells (Van Aelst and D'Souza-Schorey, 1997).
RhoA activation results in the formation of focal adhesions and contractile bundles of actin and myosin known as stress fibers (Ridley and Hall, 1992). Induction of these structures is dependent on the RhoA effector RhoKinase/ROK/ROCK. Activation of RhoKinase stimulates an increase in myosin-based contractility (Kaibuchi et al., 1999). The resulting augmentation of contractility causes myosin filament formation and the bundling of filamentous actin into stress fibers (Chrzanowska-Wodnicka and Burridge, 1996). The tension exerted by stress fibers is responsible for the maturation of small focal complexes into larger focal adhesions by aggregating integrins and their associated proteins. Focal adhesions have a structural role, as they are sites of strong adhesion, but also serve as centers for many signal transduction pathways initiated by integrins and other transmembrane receptors (Schoenwaelder and Burridge, 1999; Sastry and Burridge, 2000).
Deciphering how signals from integrins and cadherins alter the activity levels of Rho family proteins is essential for understanding the mechanism by which adhesion regulates cell behavior. In this study, we investigated how cell-matrix and cell-cell adhesion regulates Rho proteins. Previous studies have demonstrated that integrin signaling activates Rac1 and Cdc42 and that activation of these proteins is important for normal cell spreading (Price et al., 1998; del Pozo et al., 2000). Here we found that engagement of integrins with the extracellular matrix protein fibronectin triggered a transient decrease in RhoA activity. Inactivation of RhoA by cell-matrix adhesion was mediated by c-Src-dependent activation of p190RhoGAP. This decrease in RhoA activity was found to be important for cell spreading on fibronectin and the prevention of premature stress fiber formation. Signaling from cadherins regulated Rho proteins in a similar manner. Engagement of cadherins inhibited RhoA and activated both Cdc42 and Rac1. Previous reports have found that activation of RhoA (Zhong et al., 1997; Jou and Nelson, 1998) and inhibition of Cdc42 or Rac1 block the formation of cell-cell adhesions (Braga et al., 1997; Hordijk et al., 1997; Kuroda et al., 1997; Takaishi et al., 1997). Together, our results and those of previous studies suggest that integrins and cadherins both trigger RhoA inhibition and activation of Rac1 and Cdc42. Furthermore, we propose that the regulation of Rho GTPases in this particular manner is necessary for normal cell-matrix and cell-cell adhesion downstream of integrin and cadherin signaling, respectively.
Integrin Engagement Suppresses RhoA Activity
Previous studies have demonstrated that integrin-mediated adhesion to the extracellular matrix protein fibronectin activates Cdc42 and Rac1 (Price et al., 1998; del Pozo et al., 2000). In contrast to Cdc42 and Rac1, RhoA is transiently inhibited by adhesion to fibronectin (Ren et al., 1999). We found that, in addition to fibronectin, this inhibition of RhoA can be stimulated by treating cells with RGD (arginine, glycine, aspartate)-containing peptides (Arthur et al., 2000), which mimic the integrin binding domain of fibronectin, or by plating cells on the type III repeats 7-10 of fibronectin (data not shown). Cells lacking the tyrosine kinases c-Src, c-Yes, and c-Fyn (Klinghoffer et al., 1999) failed to inactivate RhoA in response to adhesion on fibronectin (Arthur et al., 2000). This activity is restored upon expression of c-Src, suggesting that c-Src expression is sufficient for RhoA inactivation by integrins. Furthermore, integrin engagement stimulates c-Src-dependent tyrosine phosphorylation and activation of the RhoA-specific inhibitor p190RhoGAP. To address the role of p190RhoGAP in mediating the inactivation of RhoA we stably expressed a dominant negative mutant of this GAP, p190RhoGAPR1283A (Arthur and Burridge, 2001), in Rat1 cells (p190-RA cells). Rat1 cells were also generated that express an empty vector as a negative control (Mock cells). When Mock cells were plated on fibronectin RhoA was rapidly inhibited (Figure 1A). However, when parallel experiments were conducted with p190-RA cells this inactivation was greatly attenuated (Figure 1B). Taken together, these findings suggest that integrin-mediated cell adhesion to the extracellular matrix protein fibronectin inhibits RhoA through a mechanism dependent on p190RhoGAP.
|Fig. 1. Integrin-mediated adhesion to fibronectin inhibits RhoA by activation p190RhoGAP. Mock cells (A) or p190-RA cells (B) were suspended for one hour then plated on fibronectin-coated plates for the indicated periods of time (minutes). Cells were then lysed and active RhoA was precipitated with GST-RBD (Ren et al, 1999). The total levels of RhoA are also shown as loading controls.|
Integrins are also known receptors for cell surface proteins such as the immunoglobulin superfamily members, L1 (Montgomery et al., 1996) and Thy-1 (Leyton et al., 2001; Avalos et al., 2002, this issue). Thus, it would be interesting to see whether integrin signaling initiated by interaction with such proteins activates Rho family members in a similar fashion.
Requirement of RhoA Inactivation in Cell Spreading
To investigate the biological significance of RhoA inactivation in response to cell-matrix adhesion, we analyzed the actin cytoskeleton in the Mock and p190-RA cell lines. After 20 minutes on fibronectin, cells were labeled with Texas Red-conjugated phalloidin to reveal the structure of filamentous actin. Mock cells were well spread but lacked prominent actin bundles (Figure 2A). The inability of Mock cells to form stress fibers at this time point is consistent with the low level of RhoA activity observed after 15-30 minutes of adhesion to fibronectin (Figure 1A). In contrast, the p190-RA cells developed robust stress fibers at this time point (Figure 2B) and exhibited impaired spreading (Arthur and Burridge, 2001). The precocious formation of stress fibers is in accord with the elevated level of RhoA activity observed in Figure 1B. These data suggest that the function of RhoA inactivation by integrin signaling is to prevent premature stress fiber formation and to allow cells to efficiently spread on the extracellular matrix.
Fig. 2. Rho inactivation by integrin-mediated adhesion to fibronectin is required for the prevention of premature stress fiber formation. Mock cells (A) or p190-RA cells (B) were held in suspension for one hour then plated on fibronectin-coated coverslips for twenty minutes. The cells were labeled with Texas-Red phalloidin to analyze the organization of the actin cytoskeleton.
Regulation of RhoA, Rac1, and Cdc42 by Cell-Cell Adhesion
To explore the role of cell-cell adhesion in regulating Rho family GTPases we employed two systems to initiate cadherin signaling. One method takes advantage of the dependence of calcium for cadherin function. Incubation of MDCK cells in low calcium media inhibits E-cadherin binding, resulting in the disassembly of adherens junctions. Restoration of normal calcium levels allows cadherin engagement and cell-cell junction formation. The second system we employed consists of plating CHO cells that are stably expressing C-cadherin (C-Cad cells) on tissue culture plates that are coated with the extracellular domain of C-cadherin (CEC1-5).
The activity levels of Rho proteins were measured after cadherin engagement. Using the calcium switch procedure, an increase in Cdc42 activity was detected within 5 minutes of calcium restoration (Figure 3A). Cdc42 activation was blocked when calcium was restored in the presence of E-cadherin inhibitory antibodies, suggesting that E-cadherin function is crucial for Cdc42 stimulation (Noren et al., 2001). Similarly, plating C-Cad cells on the CEC1-5 increased Rac1 activity levels (Figure 3B). In contrast to Cdc42 and Rac1 activation by cadherin-dependent signaling, cadherin engagement decreased the levels of active RhoA at 30 minutes (Figure 3C) and these continued to drop at later time points (data not shown). Both Rac1 activation and RhoA inhibition failed to occur in CHO cells expressing a truncated mutant of C-cadherin that lacks intracellular sequences (Noren et al., 2001). These findings imply that cytoplasmic interactions mediate the observed changes in RhoA and Rac1 activity. Taken together, our studies demonstrate that cell-cell adhesion mediated by cadherins inhibits RhoA and activates both Cdc42 and Rac1.
| ||Fig. 3. Cadherin-mediated adhesion activates Cdc42 and Rac1 and inhibits RhoA. (A) Cell-cell adhesion was initiated by the addition of calcium containing media to MDCK cells that had been starved of calcium. After the indicated periods of time (minutes) cells were lysed and active Cdc42 was precipitated with GST-Rac1/cdc42 binding domain of PAK as described previously (Noren et al., 2001). (B, C) CHO cells expressing C-cadherin (C-Cad) were suspended for thirty minutes and then plated on tissue culture plates coated with the extracellular domain of C-cadherin (Noren et al., 2001). At the specified time points cells were lysed and active Rac1 (B) and RhoA (C) were precipitated with GST- Rac1/cdc42 binding domain of PAK or GST-RhoA-binding domain of Rhoteckin, respectively. The total levels of the GTPases are shown as loading controls. These data are representative of at least 4 independent experiments.|
Rho Proteins and Cell-Matrix Adhesion
This and previous studies have established that integrin-mediated adhesion to extracellular matrix proteins modulates the activity of Rho family GTPases. Adhesion to matrix proteins such as fibronectin stimulates an increase in Rac1 and Cdc42 activity (Price et al., 1998). Cdc42 and Rac1 promote the formation of protrusive membrane structures such as filopodia and lamellipodia (Ridley et al., 1992; Nobes and Hall, 1995). Accordingly, activation of these Rho proteins in response to adhesion is necessary for efficient cell spreading. Although it is not clear how matrix-adhesion activates Cdc42 and Rac1, recent evidence suggests that members of the VAV subfamily of GEFs may be involved. In hematopoietic cells Vav-1 is thought to mediate signaling pathways downstream of integrins that lead to changes in cell morphology (Cichowski et al., 1996; Yron et al., 1999). Subsequent studies have suggested the VAV-2 is necessary for cell spreading and may be regulated by cell-matrix adhesion in some but not all cells (Liu and Burridge, 2000; Marignani and Carpenter, 2001). Other exchange factors that are candidates for activating Rho proteins in response to adhesion include PIX and DOCK180. PIX and DOCK180 associate with Paxillin and p130CAS, respectively; two proteins that are both regulated by integrin-mediated adhesion and localize to focal adhesions (Petch et al., 1995; Kiyokawa et al., 1998; Turner, 2000).
Regulation of RhoA by integrin-mediated signaling is complex. At longer time points (45-90 minutes) RhoA is activated by adhesion to fibronectin (Ren et al., 1999). The delayed activation may be a result of fibronectin associating with integrins or syndecan-4 (Couchman and Woods, 1999). In this study we have focused on the mechanism and significance of the transient inhibition of RhoA that precedes the activation phase. We have demonstrated that this inactivation of RhoA is mediated by c-Src-dependent tyrosine phosphorylation and activation of p190RhoGAP (Arthur et al., 2000). Activated p190RhoGAP then downregulates the levels of GTP-bound RhoA by augmenting its intrinsic GTPase activity. Subsequent studies have suggested that similar to c-Src and p190RhoGAP, the tyrosine kinase FAK is involved in RhoA inhibition by integrin signaling (Ren et al., 2000). It is presently unclear if FAK participates in signaling from c-Src and p190RhoGAP to RhoA or if it inactivates RhoA through a parallel mechanism. However, given the interdependence of c-Src and FAK (Klinghoffer et al., 1999; Sieg et al., 1999) it is likely that these kinases function cooperatively in RhoA inactivation by cell-matrix adhesion.
Rho Proteins and Cell-Cell Adhesion
In this study we have shown that signaling events mediated by cadherins result in RhoA inhibition and the activation of Cdc42 and Rac1. These findings are in accord with other studies that have examined how Rho proteins are regulated by cell-cell adhesion (Kim et al., 2000; Nakagawa et al., 2001; Noren et al., 2001; Kovacs et al., 2002; Lampugnani et al., 2002). Signaling directly from engaged cadherins was found to be sufficient for signaling to both RhoA and Rac1 (Noren et al., 2001). However, Cdc42 activation was observed in the calcium switch procedure but not in the C-cadherin system (Noren et al., 2001). Perhaps cadherin-mediated cell-cell adhesion initiates juxtacrine signaling by bringing signaling receptors in contact with membrane-bound ligands on neighboring cells.
At this time it is not known how cell-cell adhesion gives rise to RhoA inhibition as well as Cdc42 and Rac1 activation. Since Rho proteins are inactivated by GAPs, we are examining potential RhoA specific GAPs that might be downstream from cadherin signaling. Recent evidence has suggested that PI-3-Kinase activity is increased in response to cell-cell adhesion (Pece et al., 1999) and we and others have found that PI-3-Kinase activity is necessary for Rac1 activation by cadherins (data not shown) (Nakagawa et al., 2001). Furthermore, the activity of the Rac GEF Tiam1 is stimulated by PI-3-Kinase and this GEF localizes to cell-cell contacts (Hordijk et al., 1997; Sander et al., 1998).
Additionally, recent data indicates that Tiam1 translocates to the insoluble fraction in response to cadherin expression (Lampugnani et al., 2002), suggesting that Tiam1 might be involved in relaying signals from cadherins. We are currently examining the potential roles of Tiam1 and other Rho family GEFs in signaling events downstream of cadherins.
Feedback Signaling by Rho Proteins to Cell-Matrix and Cell-Cell Adhesions
Both integrin and cadherin-dependent signaling events give rise to RhoA inhibition as well as Cdc42 and Rac1 activation. Intriguingly, increased RhoA signaling or decreased Rac1 or Cdc42 signaling all inhibit the interaction of cells with the extracellular matrix and neighboring cells. For example, Cdc42 and Rac1 are required for cell spreading on fibronectin (Price et al., 1998; del Pozo et al., 2000). Hyperactivation of RhoA antagonizes cell spreading and attenuates membrane dynamics in adherent cells (Arthur and Burridge, 2001; Cox et al., 2001). Conversely, inhibition of the RhoA effector RhoKinase enhances membrane ruffling (Rottner et al., 1999). Similar to spreading on matrix proteins, Cdc42 and Rac1 are necessary for the formation of cell-cell adhesions (Hordijk et al., 1997; Kuroda et al., 1997; Takaishi et al., 1997; Jou and Nelson, 1998). Furthermore, reduced RhoA activity is necessary for cell-cell junction formation (Braga et al., 1997; Zhong et al., 1997; Jou and Nelson, 1998) while high levels of RhoA activity disrupts junction formation (Zhong et al., 1997; Jou and Nelson, 1998). However, complete inactivation of RhoA blocks cell spreading and cell-cell contact (Zhong et al., 1997), suggesting that some RhoA signaling is required for adhesion. Collectively these data suggest that suppression of RhoA to moderate levels and stimulation of Cdc42 and Rac1 in response to integrin or cadherin signaling participates in promoting the interaction of cells with the extracellular matrix or adjacent cells, respectively.
The authors wish to thank Becky Worthylake for critical reading of this manuscript. This work was supported by grants from NIH (GM 29860 and DE 13079).
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Corresponding author:University of North Carolina. Department of Cell and Developmental Biology. 108 Taylor Hall, CB# 7090, Chapel Hill, NC 27599. e-mail: email@example.com. Telephone: 919-966-5783. Fax: 919-966-1856.
Received: May 14, 2002. In revised form: June 18, 2002. Accepted: June 21, 2002