The RhoGAP protein Deleted in Liver Cancer 3 (DLC3) is essential for adherens junctions integrity

Abstract

Epithelial cell-cell contacts are mediated by E-cadherin interactions, which are regulated by the balanced local activity of Rho GTPases. Despite the known function of Rho at adherens junctions (AJs), little is known about the spatial control of Rho activity at these sites. Here we provide evidence that in breast epithelial cells the Deleted in Liver Cancer 3 (DLC3) protein localizes to AJs and is essential for E-cadherin function. DLC3 is a still poorly characterized RhoA-specific GTPase-activating protein that is frequently downregulated in various types of cancer. We demonstrate that DLC3 depletion leads to mislocalization of E-cadherin and catenins, which was associated with impaired cell aggregation and increased migration. This is explained by aberrant local Rho signaling because ROCK inhibition restored cell-cell contacts in DLC3 knockdown cells. We thus identify DLC3 as a novel negative regulator of junctional Rho and propose that DLC3 loss contributes to carcinogenesis by compromising epithelial integrity.

Figure 1

GFP–DLC3α co-localizes with E-cadherin at cell–cell adhesions and ectopic expression impairs differentiation of MCF10A cells in 3D cultures. (a) MCF7 and (b) MCF10A cells stably expressing wild-type and GAP-inactive (K725E) GFP-tagged DLC3α were stained with antibodies specific for E-cadherin and β-catenin (red), respectively. (c) MCF10A cells stably expressing wild-type and GAP-inactive (K725E) GFP–DLC3α were cultured in matrigel for 6 days and stained with an E-cadherin-specific antibody (red); nuclei were counterstained with TOPRO-3 (blue). (a) The images shown are stacks of several confocal sections and (b, c) single confocal sections. Scale bar, 20 μm.

Figure 2

DLC3 depletion disrupts cell junctions. (a) HEK293T cells were transfected with a GFP–DLC3α expression plasmid along with siRNAs targeting DLC3 (siDLC3-I and siDLC3-II), respectively, or siLacZ. Cells were lysed 48 h post transfection and GFP–DLC3α expression was analyzed by immunoblotting with a GFP-specific antibody (top). The membrane was reprobed with a tubulin-specific antibody (bottom). (b) MCF7 cells were transfected with siLacZ, siDLC3-I and siDLC3-II, respectively. Post transfection (72 h), RNA was extracted and cDNA was analyzed by semi-quantitative RT–PCR using DLC3-specific primers. GADPH was amplified as a loading control. (c) MCF7 cells were transfected with siLacZ, siDLC3-I and siDLC3-II, respectively. Post transfection (72 h), cells were stained with antibodies specific for E-cadherin, β-catenin and p120-catenin. The images shown are stacks of several confocal sections. Scale bar, 20 μm.

Figure 3

DLC3 depletion reduces cell–cell adhesion and increases cell motility. MCF7 cells were transfected with siLacZ, siDLC3-I and siDLC3-II, respectively. (a, b) Post transfection (48 h), cells were replated onto collagen-coated dishes. The next day, confluent cell monolayers were treated with 4 mM EGTA for 15 min. Cells were washed and incubated with fresh medium. In (a), cells were fixed and stained at the indicated times (scale bar, 100 μm), in (b) the normalized mean impedance (cell index) of duplicate wells measured with an xCELLigence device is plotted. (c) Post transfection (48 h), cells were suspended in hanging drops as described in the Materials ans methods section. The next day, spheroids were resuspended by pipetting and photographed. Scale bar, 200 μm. (d) Post transfection (72 h), cells in medium containing 0.5% FCS were plated into Transwell filters coated with collagen on the underside. The bottom chamber contained medium with 10% FCS. After overnight incubation, migrated cells were fixed and stained. Data shown are the mean±s.e.m. of duplicate wells and are representative of 3 independent experiments. Values for siDLC3-I and siDLC3-II versus siLacZ were statistically significant (Student's t-test; ***P<0.001).

Figure 4

ROCK inhibition restores AJs in DLC3-depleted cells. (a, b) MCF7 cells were transfected with siLacZ and siDLC3-II. (a) Post transfection (72 h), cells were stained with a vinculin-specific antibody and AlexaFluor-546 conjugated phalloidin. (b) Two days post transfection, cells were treated overnight with 10 μM Y27632 or DMSO (cont.) before staining with antibodies specific for pMLC (green) and β-catenin (red). Images shown are stacks of several confocal sections. Scale bar, 20 μm.