Rho: theme and variations

AbstractIn addition to their roles in organizing the actin cytoskeleton, members of the Rho family of GTP-binding proteins have recently been implicated in a plethora of other functions, including the activation of kinase cascades and transcription factors, and the control of endocytosis and secretion. Alongside this expansion in proposed functions has been the identification of multiple target proteins that interact directly with Rho, Rac or Cdc42. Molecular connections are now being made along the signalling pathways activated by members of the Rho family

Life at the Leading Edge

Cell migration requires sustained forward movement of the plasma membrane at the cell's front or “leading edge.” To date, researchers have uncovered four distinct ways of extending the membrane at the leading edge. In lamellipodia and filopodia, actin polymerization directly pushes the plasma membrane forward, whereas in invadopodia, actin polymerization couples with the extracellular delivery of matrix-degrading metalloproteases to clear a path for cells through the extracellular matrix. Membrane blebs drive the plasma membrane forward using a combination of actomyosin-based contractility and reversible detachment of the membrane from the cortical actin cytoskeleton. Each protrusion type requires the coordination of a wide spectrum of signaling molecules and regulators of cytoskeletal dynamics. In addition, these different protrusion methods likely act in concert to move cells through complex environments in vivo

p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, beta-catenin and alpha-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, beta-catenin and alpha-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, beta-catenin and alpha-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, beta-catenin or alpha-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion

CD73 represses pro-inflammatory responses in human endothelial cells

<p>Abstract</p> <p>Background</p> <p>CD73 is a 5'-ectonucleotidase that produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors to induce cellular responses. CD73 has been reported to regulate expression of pro-inflammatory molecules in mouse endothelium. Our aim is to determine the function of CD73 in human endothelial cells.</p> <p>Methods</p> <p>We used RNAi to deplete CD73 levels in human umbilical cord endothelial cells (HUVECs).</p> <p>Results</p> <p>CD73 depletion resulted in a strong reduction in adenosine production, indicating that CD73 is the major source of extracellular adenosine in HUVECs. We find that CD73 depletion induces a similar response to pro-inflammatory stimuli such as the cytokine TNF-α. In CD73-depleted cells, surface levels of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin increase. This correlates with increased translocation of the transcription factor NF-kB to the nucleus, which is known to regulate ICAM-1, VCAM-1 and E-selectin expression in response to TNF-α. Adhesion of monocytic cells to endothelial cells is enhanced. In addition, CD73-depleted cells become elongated, have higher levels of stress fibres and increased endothelial permeability, resembling known responses to TNF-α.</p> <p>Conclusions</p> <p>These results indicate that CD73 normally suppresses pro-inflammatory responses in human endothelial cells.</p

Shear stress–induced endothelial cell polarization is mediated by Rho and Rac but not Cdc42 or PI 3-kinases

Shear stress induces endothelial polarization and migration in the direction of flow accompanied by extensive remodeling of the actin cytoskeleton. The GTPases RhoA, Rac1, and Cdc42 are known to regulate cell shape changes through effects on the cytoskeleton and cell adhesion. We show here that all three GTPases become rapidly activated by shear stress, and that each is important for different aspects of the endothelial response. RhoA was activated within 5 min after stimulation with shear stress and led to cell rounding via Rho-kinase. Subsequently, the cells respread and elongated within the direction of shear stress as RhoA activity returned to baseline and Rac1 and Cdc42 reached peak activation. Cell elongation required Rac1 and Cdc42 but not phosphatidylinositide 3-kinases. Cdc42 and PI3Ks were not required to establish shear stress–induced polarity although they contributed to optimal migration speed. Instead, Rho and Rac1 regulated directionality of cell movement. Inhibition of Rho or Rho-kinase did not affect the cell speed but significantly increased cell displacement. Our results show that endothelial cells reorient in response to shear stress by a two-step process involving Rho-induced depolarization, followed by Rho/Rac-mediated polarization and migration in the direction of flow