Activation of the small GTPase Rac is sufficient to disrupt cadherin-dependent cell-cell adhesion in normal human keratinocytes

Abstract

To achieve strong adhesion to their neighbors and sustain stress and tension, epithelial cells develop many different specialized adhesive structures. Breakdown of these structures occurs during tumor progression, with the development of a fibroblastic morphology characteristic of metastatic cells. During Ras transformation, Rac-signaling pathways participate in the disruption of cadherin-dependent adhesion. We show that sustained Rac activation per se is sufficient to disassemble cadherin-mediated contacts in keratinocytes, in a concentration- and time-dependent manner. Cadherin receptors are removed from junctions before integrin receptors, suggesting that pathways activated by Rac can specifically interfere with cadherin function. We mapped an important region for disruption of junctions to the putative second effector domain of the Rac protein. Interestingly, although this region overlaps the domain necessary to induce lamellipodia, we demonstrate that the disassembly of cadherin complexes is a new Rac activity, distinct from Rac-dependent lamellipodia formation. Because Rac activity is also necessary for migration, Rac is a good candidate to coordinately regulate cell-cell and cell-substratum adhesion during tumorigenesis.

Figure 1

Rac activation does not strengthen cadherin-dependent adhesion in keratinocytes. Cells containing mature intercellular junctions were microinjected with constitutively active Rac (L61Rac, 0.5 mg/ml). Activated Rac was injected either alone (a–d), or in combination with activated H-Ras (V12Ras + L61Rac, g and h). Controls show keratinocytes injected with H-Ras alone (V12Ras, e and f). After 2 h of incubation, cells were fixed and stained for E-cadherin (b, d, f, and h). In c and d, keratinocytes were preextracted with Triton X-100 before fixation (see MATERIALS AND METHODS). Microinjected patches of cells are seen in a, c, e, and g. Arrowheads (f and h) show absence of cadherin staining at junctions. Arrows (b and d) point to similar levels of cadherin staining in microinjected cells. Bar, 50 μm.

Figure 2

Rac signaling pathways contribute to the destabilization of cadherin receptors after Ras activation. HaCat cells were microinjected with an expression vector containing dominant-negative Rac (N17Rac, a–c), oncogenic Ras (V12Ras, d–f), or both together (g–i). After 5 h, cells were fixed and double labeled for the myc tag (b, e, and h) and E-cadherin (c, f, and i). Injected patches were identified by coinjection of Dextran-Texas Red (a, d, and g). Arrows (c and i) point to the presence of cadherin staining at junctions; arrowhead (f) shows the absence of cadherin staining between two expressing cells. Bar, 50 μm.

Figure 3

Increased Rac activation perturbs the stability of cadherin receptors in mature junctions. Keratinocytes grown in standard medium (mature cell-cell contacts) were microinjected with different concentrations of constitutively active Rac (L61Rac) as follows: 0.25 mg/ml (a–c), 0.5 mg/ml (d–f), 1.0 mg/ml (g–i), and 2.0 mg/ml (j–l). After a 2-h incubation in the same medium, cell were fixed and double labeled for E-cadherin (b, e, h, and k) and integrins (c, f, i, and l). Injected cells are seen in a, d, g, and j. Arrows (i and l) show integrin staining at junctions; arrowheads (h and k) show absence of cadherin receptors at junctions. Bar, 50 μm.

Figure 4

Increasing concentrations of constitutively active Rac (L61Rac) perturb newly formed cell-cell adhesion and epithelial cell shape. Different concentrations of activated Rac (L61Rac) were microinjected into keratinocytes in the absence of intercellular contacts as follows: 0.25 mg/ml (a and b), 0.5 mg/ml (c and d), 1.0 mg/ml (e and f), and 2.0 mg/ml (g and h). Calcium-dependent cell-cell adhesion was induced for 2 h; cells were then fixed and labeled for E-cadherin, followed by an FITC-conjugated anti-mouse IgG (b, d, f, and h). Injected cells are seen in a, c, e, and g. Bar, 50 μm.

Figure 5

Sustained Rac activation interferes with the stability of newly formed cadherin-dependent contacts. Constitutively active Rac (L61Rac, 0.5 mg/ml) was microinjected into cells grown without contacts, and cadherin-dependent cell-cell adhesion was induced for 1 h (a and b), 2 h (c and d), or 5 h (e and f). Alternatively, L61Rac-pRK5myc expression vector was injected into the nucleus of HaCat cells (g and h), and after a 2-h expression, cell-cell contacts were induced for further 4 h. Cells were fixed and labeled for E-cadherin (b, d, f, and h) as stated in Figure 2; injected patches of cells are visualized in a, c, e, and g. In g, cells are labeled for the myc-tag epitope. Bar, 75 μm for g and h; 50 μm for all the other images.

Figure 6

Mapping the Rac domain important for the disruption of cadherin-dependent adhesion. (a) Comparison of the Rac domains relevant for disruption of cadherin-mediated contacts in keratinocytes and lamella formation in Swiss 3T3 cells (Diekmann et al., 1995; data not shown). Rac protein (□), Rho protein (▪), and the small GTPase functional domains are represented: N-terminal effector domain 1, insert region, and the C-terminal effector domain 2. Chimeras containing portions of the Rac sequence (□) and Rho sequence (▪) are also shown: Rac⁷³Rho (3.35 mg/ml); Rac¹²⁶Rho (0.53 mg/ml); Rac¹⁴³Rho (0.43 mg/ml); Rac¹⁷⁵Rho (2.39 mg/ml) (see text for details). Activated Rac and Rho were tested at 0.5 mg/ml. (b) Recombinant proteins used in this study were separated in SDS-PAGE and visualized by Coomassie blue staining. Molecular weight markers are shown on the left (top to bottom): 36.5, 26, and 20 kDa.

Figure 7

Different chimeric RacRho molecules were injected into keratinocytes grown in the absence of cell-cell contacts, and calcium-dependent adhesion was induced for 4 h (a–f). The following proteins were injected: Rac¹²⁶Rho (a and b); Rac¹⁴³Rho (c, d, g, and h); and Rac¹⁷⁵Rho (e and f). Injected cells (a, c, e, and g) and E-cadherin staining (b, d, and f) are visualized. In g and h, Rac¹⁴³Rho was injected into Swiss 3T3 fibroblasts and cells were stained for filamentous actin (h). Bar, 50 μm.

Figure 8

Second effector domain analysis with respect to disruption of cadherin-dependent adhesion and lamella formation. Different positions within the putative second effector domain were mutated to alanine to generate four mutants in a constitutively active Rac background. Similar results were obtained for all mutants, and representative pictures are shown for the mutants A162 A163 (a, b, e, and f) and A170 A171 (c, d, g, and h). To evaluate disruption of cadherin function in keratinocytes (a–d), cell-cell contacts were induced for 4–5 h after microinjection, and cells were then fixed and stained for E-cadherin (b and d). The same mutants were also analyzed for their ability to induce formation of lamellae/ruffles in Swiss 3T3 cells, after staining with FITC-phalloidin (e–h). Bar, 50 μm.

Figure 9

Characterization of the Rac second effector domain mutants. (a) Quantification of the effects of the Rac mutants on cadherin-dependent adhesion. Patches of keratinocytes microinjected with the different mutants were scored for the presence of perturbed cadherin staining at intercellular junctions and expressed as a percentage of the total number of patches (see MATERIALS AND METHODS). (b) Quantification of the lamella-inducing activity of Rac mutants. Swiss 3T3 cells were injected with the different proteins and scored as a percentage of cells showing ruffles/lamellae. (c) Titration of lamellipodia formation induced by L61Rac, A162 A163, and A170 A171. The same amount of recombinant protein (2, 1, or 0.5 mg/ml) was injected into Swiss 3T3 cells and the percentage of injected cells with ruffles/lamellae were scored. (d) Summary of Rac mutants' ability to perturb cadherin adhesion in keratinocytes or induce lamellipodia in fibroblasts. A diagram representing constitutively active Rac containing the relevant domains (effector domains 1 and 2, insert domain), and the different mutants generated in the second effector domain. Unless otherwise stated, in the microinjection experiments mutants were used at the following concentrations: A147 A148, 0.89 mg/ml; A162 A163, 2.26 mg/ml; A166 A167, 2.91 mg/ml; and A170 A171, 3.57 mg/ml. L61Rac was tested at 0.5 mg/ml in keratinocytes and at 2 mg/ml in Swiss 3T3 cells. ∗p < 0.005; ^@p < 0.01 (Student's t test). Results are the mean of at least three independent experiments; Figure 9c shows the mean of at least two experiments for each concentration of distinct mutants. Error bars represent SD.

Figure 9

Characterization of the Rac second effector domain mutants. (a) Quantification of the effects of the Rac mutants on cadherin-dependent adhesion. Patches of keratinocytes microinjected with the different mutants were scored for the presence of perturbed cadherin staining at intercellular junctions and expressed as a percentage of the total number of patches (see MATERIALS AND METHODS). (b) Quantification of the lamella-inducing activity of Rac mutants. Swiss 3T3 cells were injected with the different proteins and scored as a percentage of cells showing ruffles/lamellae. (c) Titration of lamellipodia formation induced by L61Rac, A162 A163, and A170 A171. The same amount of recombinant protein (2, 1, or 0.5 mg/ml) was injected into Swiss 3T3 cells and the percentage of injected cells with ruffles/lamellae were scored. (d) Summary of Rac mutants' ability to perturb cadherin adhesion in keratinocytes or induce lamellipodia in fibroblasts. A diagram representing constitutively active Rac containing the relevant domains (effector domains 1 and 2, insert domain), and the different mutants generated in the second effector domain. Unless otherwise stated, in the microinjection experiments mutants were used at the following concentrations: A147 A148, 0.89 mg/ml; A162 A163, 2.26 mg/ml; A166 A167, 2.91 mg/ml; and A170 A171, 3.57 mg/ml. L61Rac was tested at 0.5 mg/ml in keratinocytes and at 2 mg/ml in Swiss 3T3 cells. ∗p < 0.005; ^@p < 0.01 (Student's t test). Results are the mean of at least three independent experiments; Figure 9c shows the mean of at least two experiments for each concentration of distinct mutants. Error bars represent SD.

Figure 10

Binding properties of Rac mutants with known Rac targets. (a) In vitro interaction: L61Rac, A162 A163, and A170 A171 were labeled with radioactive GTP and allowed to interact with different Rac-binding proteins: GST-RhoGAP, GST-PAK, GST-ROK-α, GST-POSH, and GST-MLK2. Different amounts of recombinant protein were spotted onto the membranes (10, 5, or 1 μg). GST was used as a negative control; positive (RhoGAP and PAK) and negative controls were used at 10 μg. (b) Yeast two-hybrid interaction by using L61Rac or the second effector domain mutants as baits and the following targets as prey: PAK, MLK2, MLK3, and IQGAP2. Empty vector was used as a negative control. ∗, IQGAP interaction was tested at lower concentration of 3AT (10 mM) because the association with activated Rac was barely detectable with the standard concentration used for the other targets (25 mM). A summary of the yeast two-hybrid results with all the targets tested is shown in Table 1.