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. 1997 Nov;8(11):2329-44.
doi: 10.1091/mbc.8.11.2329.

Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells

C Zhong  1 , M S Kinch, K Burridge
Affiliations
Free PMC article

Rho-stimulated contractility contributes to the fibroblastic phenotype of Ras-transformed epithelial cells

C Zhong et al. Mol Biol Cell. 1997 Nov.
Free PMC article

Abstract

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell-cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.

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Figures

Figure 1
Figure 1
Ras-transformed MCF10A cells reveal increased contractility. Normal (A) and Ras-transformed (B) MCF10A cells were cultured on flexible silicone rubber substrates and visualized by phase microscopy. The Ras-transformed cells exerted sufficient tension on the underlying rubber to generate wrinkles (B). Bar, 200 μm. (C) The levels of myosin light chain phosphorylation in the normal and Ras-transformed cells were compared.
Figure 2
Figure 2
Rho activity contributes to the increased contractility of Ras-transformed MCF10A cells. Ras-transformed MCF10A cells were cultured on silicone rubber substrates at high density. A–D are images of successive time points from a video recording of cells microinjected with the Rho-inhibitor C3. Cells before injection are seen in A. B shows the cells at the time of injection. Cells were injected with C3 on the right side of the field and are indicated with arrowheads. Within 4.5 min after injection (C), many of the wrinkles in the rubber have decreased. This decrease in wrinkling is more pronounced at about 9 min after injection (D).
Figure 3
Figure 3
BDM and ML-7 are potent inhibitors of contractility in Ras-transformed MCF10A cells. Ras-transformed MCF10A cells plated on silicone rubber substrates were either untreated (A) or treated with 20 mM BDM (B) or 25 μM ML-7 (C) for 1 h. The effects of both inhibitors were reversible. (D) Cells were exposed to 20 mM BDM for 1 h and then treated with normal medium for another 1 h. Bar, 200 μm.
Figure 4
Figure 4
Inhibition of contractility abolishes focal adhesions in Ras-transformed MCF10A cells. Normal MCF10A cells (A and B) or MCF10A cells transformed (T) by oncogenic H-Ras (C–H) were untreated (C and D) or treated either with 20 mM BDM (E and F) or 25 μM ML-7 (G and H) for 2 h. Cells were fixed and stained with vinculin (A, C, E, and G) or paxillin (B, D, F, and H). (A, E, and G) Arrowheads indicate vinculin that is in adherens junctions. (C and D) Arrowheads indicate vinculin or paxillin in prominent focal adhesions within confluent regions of the culture. After treatment with contractility inhibitors, the number and size of focal adhesions in Ras-transformed MCF10A cells within confluent areas are diminished. Bar, 20 μm.
Figure 5
Figure 5
Inhibition of contractility restores more normal cell–cell adherens junctions and cytoskeletal organization to Ras-transformed MCF10A cells. Normal MCF10A cells (A and B) or MCF10A cells transformed (T) by oncogenic H-Ras (C–H) were untreated (C and D) or treated either with 20 mM BDM (E and F) or 25 μM ML-7 (G and H) for 2 h. Cells were stained for E-cadherin after permeabilization (A, C, E, and G) and stained for actin (B, D, F, and H). Arrowheads in A, E, and G mark prominent adherens junctions. In contrast the arrowhead in C indicates a region of cell–cell contact in the untreated Ras-transformed cells exhibiting little E-cadherin staining. The arrowhead in D indicates actin organized in stress fibers, which are absent from the cells in B, F, and H. Bar, 20 μm.
Figure 6
Figure 6
BDM and ML-7 drive E-cadherin into the detergent-insoluble fraction of Ras-transformed MCF10A cells. Monolayers of normal MCF10A (N), Ras-transformed MCF10A (T), Ras-transformed cells treated with either 20 mM BDM (BDM) or 25 μM ML-7 (ML-7) for 2 h were sequentially extracted with 0.5% digitonin and modified RIPA buffer. Total amount of protein in each sample was equalized before immunoprecipitation. All the fractions were immunoprecipitated with anti-E-cadherin antibody and immunoblotted for E-cadherin.
Figure 7
Figure 7
Inhibition of Rho activity partially reverses the transformed phenotype of Ras-transformed MCF10A cells. Ras-transformed MCF10A cells were microinjected with coumarin-BSA (A and B) or coumarin-BSA and 160 μg/ml C3 (C and D). Twenty-five minutes after microinjection, the cells were fixed and stained for vinculin (A and C) or visualized for coumarin-BSA to reveal injected cells (B and D). Single examples of injected cells are indicated with arrows. The arrowhead in C indicates vinculin at the margin of a cell (in focal complexes). (E) Ras-transformed MCF10A cells were microinjected with 160 μg/ml C3 and propidium iodide to label the nuclei of injected cells. One hour later, cells were permeabilized, fixed, and stained with anti-E-cadherin antibody. Arrows indicate three injected cells. Note the lack of E-cadherin staining between the injected cells. Bar, 20 μm.
Figure 8
Figure 8
Blocking Rho function by 19N-RhoA prevents focal adhesion formation but does not promote cell–cell adherens junction formation. Ras-transformed MCF10A cells were microinjected into the nucleus with plasmid pGreen Lantern either alone (A and B) or with a plasmid encoding 19N-RhoA (C and D). Twenty-four hours later, cells were fixed, permeabilized, and stained with antibody against vinculin (A and C). Injected cells were visualized by expression of green fluorescent protein (B and D). Arrows indicate microinjected cells. Bar, 20 μm.
Figure 9
Figure 9
Introduction of constitutively active Rho into normal MCF10A cells is not sufficient to promote the formation of stress fibers. Normal MCF10A cells were microinjected with either GST (A and B) or with 14V Rho-GST at 2.5 mg/ml (C and D) and returned to the incubator for 7 h, followed by staining for GST (B and D) and actin (A and C). Arrows indicate microinjected cells. Bar, 20 μm.
Figure 10
Figure 10
Rho is required for the maintenance and formation of cell–cell adherens junctions in normal MCF10A cells. To examine the effect of C3 on existing adherens junctions, normal MCF10A cells plated for 2 d were either injected with control buffer (A) or 100 μg/ml C3 (B). To visualize the injected cells, the injection buffer contained propidium iodide to label the nuclei. After 30 min of incubation, cells were permeabilized first, then fixed, and stained with E-cadherin. To examine the effect of C3 on the formation of adherens junctions, cells were injected with buffer (C) or 100 μg/ml C3 (D), followed by addition of medium containing 4 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid for 30 min to disrupt the adherens junctions. Ca2+ was then restored by changing the medium back to normal for another 45 min. Cells were then permeabilized, fixed, and stained for E-cadherin. Bar, 20 μm.
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