Marco E. Bianchi
Francesco Blasi
Daniele D'Ambrosio
Manuel Fresno
Antonio Lanzavecchia
Federico Major Jr.
Paola Panina-Bordignon
Ruggero Pardi
›› Anne Ridley
Federica Sallusto
Francisco Sanchez-Madrid
Marcus Thelen
Bart Vanhaesebroeck
Marketa Zvelebil
Anne Ridley

Training Module

Direct Supervisor: Anne Ridley, PhD
Location: LICR-UCL
Descriptive title of research activity: Role of Rnd proteins in endothelial cell inflammatory responses

Overall goals: Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) are members of the Rho family of GTPases, which are key coordinators of cell adhesion and migration. The goal of this research is to determine how Rnd proteins and the RhoE partner ROCK I in endothelial cells contribute to leukocyte adhesion and transendothelial migration.

Rationale and significance: RhoA and the RhoA-activated kinases ROCKI/II stimulate actin stress fibre formation, increased contractility and loss of cell-cell junction integrity in endothelial cells4, and RhoA regulates leukocyte adhesion to HUVECs5. Rnd1, Rnd2 and RhoE act antagonistically to RhoA and induce loss of stress fibres in fibroblasts and epithelial cells, and RhoE inhibits cell proliferation1,2, but the response of endothelial cells to Rnd proteins has not been tested. RhoE is highly expressed in HUVECs, and Rnd1 expression is induced by the pro-inflammatory cytokine IL1. We therefore expect that RhoE and Rnd1, and possibly Rnd2, will play important roles in regulating the endothelial cytoskeleton and cell-cell adhesion during inflammation. Interestingly, RhoE enhances the tight junction assembly in epithelial cells. Rnd proteins are unusual Rho family members in that they are not regulated by GTP hydrolysis. Instead, RhoE is regulated by ROCKI-induced phosphorylation, which increases its stability and reduces its membrane association1,3. We propose that RhoE acts to restrict ROCKI activation in response to extracellular stimuli.

Description of work and methodology: The roles of the three Rnd proteins will be investigated primarily in HUVECs, since we have extensive experience in working with these cells4,5. In addition, we will test whether endothelial cells isolated from inflammed tissues (e.g. arthritic joints) have altered levels of Rnd proteins. These samples are available from Dr J-P Girard (MAIN). Finally, we will investigate whether mouse endothelial cells (cardiac, lung) have a similar or different pattern of Rnd expression to human cells, in order to know whether the mouse is an appropriate model for studying Rnd function in endothelial cells in vivo (e.g. by using knockout mice, which are not yet available but could be derived in the future). The levels of RhoE protein and ROCKI-phosphorylated RhoE will be investigated by western blotting with antibodies we have described1,3, and for Rnd1 and Rnd2 using antibodies we are currently developing. The localization of Rnd proteins within cells will be investigated by immunofluorescence for endogenous proteins, and changes in localization following receptor stimulation will be monitored in real time using GFP-Rnd fusions expressed at low levels. Rnd protein expression will be increased using adenoviral vectors to deliver the genes into endothelial cells. Rnd protein expression will be reduced using RNAi. The effects of altered Rnd protein expression on cell-cell junctions will be investigated by immunofluorescence with antibodies against a range of cell-cell junction markers (e.g. occludin, ZO-1, JAMs, VE-cadherin, -catenin)4. Changes to the actin cytoskeleton will be monitored using fluorescent phalloidin4. Responses of endothelial cells to inflammatory stimuli, including TNF, IL1 and Interferonwill be tested, measuring alterations to junction integrity and the cytoskeleton, as well as upregulation of specific receptors involved in leukocyte adhesion (e.g. ICAM-1, VCAM-1, E-selectin). Effects of altered Rnd expression on leukocyte adhesion to endothelial cells and TEM under static conditions will be investigated by timelapse microscopy and confocal microscopy. Rolling and TEM under flow conditions will be tested in collaboration with Dr Ronen Alon’s group, who have extensive experience in these methodologies (MAIN).