A novel role for p115RhoGEF in regulation of epithelial plasticity.

Epithelial plasticity plays a critical role during physiological processes, such as wound healing and tissue regeneration, and dysregulation of epithelial plasticity can lead to pathological conditions, such as cancer. Cell-cell junctions are a critical feature of epithelial cells and loss of junctions is associated with acquisition of mesenchymal features, such as enhanced protrusion and migration. Although Rho has been implicated in regulation of junctions in epithelial cells, the role of Rho signaling in the regulation of epithelial plasticity has not been understood. We show that members of the RGS RhoGEFs family play a critical role in regulation of epithelial cell-cell junctions in breast epithelial cells. We identify a novel role for p115RhoGEF in regulation of epithelial plasticity. Loss of p115RhoGEF leads to decreased junctional E-cadherin and enhanced protrusiveness and migration. Conversely, overexpression of p115RhoGEF enhanced junctional E-cadherin and inhibited cell protrusion and migration. siRNA screen of 23 Rho effectors showed that members of the Diaphanous-Related Formin (DRF) family are required for p115RhoGEF-mediated changes in epithelial plasticity. Thus, our data indicates a novel role for p115RhoGEF in regulation of epithelial plasticity, which is dependent on Rho-DRF signaling module

Members of diaphanous related formin (DRF) family are required to mediate regulation of epithelial plasticity by p115RhoGEF.

<p>A) p115-OE MCF7 cells were transfected with SMARTpool™ siRNA or individual siRNA oligos targeting <i>DIAPH1</i>. <i>DIAPH1</i>-depleted p115-OE MCF7 cells showed decreased junctional E-cadherin as compared to <i>DIAPH1-</i>expressing p115-OE MCF7 cells. B) p115-OE MDA-MB-231 cells were treated with SMARTpool™ siRNA or individual siRNA oligos targeting <i>DIAPH1</i>. <i>DIAPH1</i> knockdown blocked the change in morphology seen in cells that overexpressed p115RhoGEF. C) Quantitation of E-cadherin ratio in p115-OE MCF7 cells showed a significant decrease in upon <i>DIAPH1</i> knockdown as compared to cells that were treated with control siRNA (mean ± SE). 90 cell-cell junctions were analyzed from 3 experiments. D) Quantitation of morphology in terms of circularity in p115-OE MDA-MB-231 cells showed a significant change upon <i>DIAPH1</i> knockdown as compared to cells that were treated with control siRNA (mean ± SE). 75 cells were analyzed from 3 experiments E) Representative immunoblots showing knockdown of <i>DIAPH1</i> upon treatment with <i>DIAPH1</i> oligo#1, <i>DIAPH1</i> oligo#2 and <i>DIAPH1</i> SMARTpool™ siRNA in MCF7 or MDA-MB-231 cells that overexpressed p115RhoGEF. Scale bars represent 10 µm.</p

Expression levels of p115RhoGEF affect cell migration.

<p>A) Scratch wound assay showed a moderate but significant increase in migration of p115RhoGEF-depleted MCF7 cells as compared to cells treated with control siRNA. Phase contrast images show boundaries of wounds in cell monolayer at the end of migration assay (24 hrs) while the boundary of the wounds at the beginning of the assay (0 hrs) are indicated by superimposed white lines in order to reduce the number of images shown. For both control and p115RhoGEF-depleted cells, three separate locations were imaged at 0 h and 24 h after making the scratch and the difference in wound area was determined using ImageJ software. White arrow indicates direction of cell migration. Migration was measured by measuring the difference in area covered by the cell monolayer at 0 h and 24 h. Cells treated with p115RhoGEF siRNA showed a significant increase in migration as compared to control siRNA treated cells (n = 3). B) Scratch wound assay in MCF10A cells showed a significant increase in migration of p115RhoGEF-depleted MCF10A cells as compared to cells treated with control siRNA (n = 3). C) Scratch wound assay showed a significant decrease in migration of MCF7 cells that overexpressed p115RhoGEF as compared to control cells that expressed GFP alone. D) Scratch wound assay showed a modest but significant decrease in migration of MCF10A cells that overexpressed p115RhoGEF as compared to control cells. E) Results for MDA-MB-231 single cell migration assay. Control (GFP) and p115RhoGEF-overexpressing (p115 OE) MDA-MB-231 cells were plated on MatTek plates and images were collected every 30 min for 6 h. During image capture, cells were maintained at 37°C and 5% CO2 using a Live Cell™ chamber. Individual cell positions in sequential images were determined using the cell tracking tool in Slidebook software and x-y co-ordinates with starting points adjusted to (0,0) were plotted. Migration of 5 cells was measured in each experiment and data was pooled from three individual experiments. p115-OE MDA-MB-231 cells displayed decreased migration as compared to control cells that expressed GFP alone. Quantitation of average total displacement which is a measure of the total distance travelled by the migrating cell showed a significant decrease in cells that overexpressed p115RhoGEF as compared to control cells that expressed GFP alone.</p

Overexpression of p115RhoGEF enhances junctional E-cadherin and blocks protrusions.

<p>A) MCF7 cells that overexpressed p115RhoGEF were stained for E-cadherin and actin. p115RhoGEF overexpressors showed an increase in junctional E-cadherin as well as increase in junctional actin as compared to control cells. B) Overexpression of p115RhoGEF in MCF10A cells showed enhanced junctional E-cadherin and a more subtle effect on junctional actin as compared to control cells that overexpressed GFP alone. C) Actin staining of MDA-MB-231 cells that overexpressed p115RhoGEF showed loss of protrusions and a change in morphology with a clear ring of actin at the cell periphery. D) Quantitation of the ratio of junctional over cytoplasmic E-cadherin in MCF7 and MCF10A cells showed a significant increase in junctional E-cadherin in cells that overexpressed p115RhoGEF (mean ± SE) as compared to control. A total of 45 cell-cell junctions for both MCF7 and MCF10A from 3 separate experiments were analyzed. p-value was calculated using 2 tailed t-test. E) Representative immunoblot showing overexpression of p115RhoGEF (p115 OE) as compared to endogenous levels in control cells in each of the three cell lines. Scale bars represent 10 µm.</p