The Interaction Of Racl Protein Activation With Endothelial Cell Permeability And Cytoskeleton Reorganization

Background and object: Rho GTPases are members of the Ras superfamily of monomeric 20~30 kDa GTP-binding proteins. Each of these GTPases act as a molecular switch, cycling between an active GTP-bound, and an inactive GDP-bound state. In the GTP-bound form, they are able to interact with target molecules to initiate a down-stream response, while an intrinsic GTPase activity returns the proteins to the GDP-bound state, to complete the cycle and terminate signal transduction. Activated Rho GTPases interact with cellular target proteins or effectors to trigger a wide variety of cellular responses, including the reorganization of the actin cytoskeleton and changes in gene transcription.During the occurrion of MOF and SIRS, the increase of endothelial permeability is controlled by a variety of the cell factors such as TNF and LPS, derived from blood or surrounding tissues in response to inflammatory mediators. However, researches from a number of laboratories over the past years had revealed that most of the cell factors and inflammatory mediators induced endothelial cell impaired via the changes of cellular actin cytoskeleton, contraction or retraction of cells and the resultant formation of interendothelials gaps. Recently, some studies on the interaction in reorganization of actin cytoskeleton with the gap of interendothelial cell formation identified that the Rho GTPases play crucial roles in increasing of endothelial permeability induced by inflammatory mediators. However, Rac1 as one of the most extensively characterized members of small GTPase, there are few examples that have been implicated in actin reorganization. So in this study, we will discuss how it regulated the cytoskeleton reorganization and endothelial permeability change.Methods: The gene fragment of p21 binding domain (hPBD) which may bind the Rac1/GTP and cdc42/GTP was amplified by RT-PCR and cloned into the pGEX-2T prokaryotic expression vector. The expression of GST-hPBD fusion protein was induced by IPTG from E.coli BL21 and purificated using Glutathione Sepharose 4B affinity chromatography. The activated Rac1 protein in ECV304 was detected with GST-hPBD by pull down assays. The expression of activated Rac1 were investigated in endothelial cells activated with TNF and LPS stimulation by pull down assays. The changes of cell actin cytoskeleton were stained with Rhodamine-Phalloidin on slide, and endothelia permeability was detected by horseradish peroxidase in transwell model.Results: we constructed the pGEX-2T/hPBD prokaryotic expression vector successfully, made its expression efficiently into E.coli BL21, and obtained 75% of the GST-hPBD fusion protein purified. The activated Rac1 protein was detected in ECV304 cells by the GST-hPBD protein. The expression of activated Rac1 protein increased with the stimulation of TNF and LPS obviously in the endothelial cells with time elongate, as well as the increase of endothelial permeability. It was also found that the cells shape was changed , the cellular ratio of length to width was increased, the interendothelial gaps were formed and basement membrane were impared compared to normal cells. As the same times, F-actin microfilaments in cells were reorganized , the formation of stress fibre were induced and the polarity of cells dispeared by fluore-scence microscopy. Conclusion: In our studies, a useful methods were provided to detecte the expression of activated Rac1 protein. Although there is no a directed evidence, Rac1 proteins were activated by inflammatory factors in parallel to endothelial permeability increasion and cytoskeleton reorganization showed that Rac1 protein may be act on the important roles in the regulation of endothelial cell premeability.