RHOGEFS IN EPITHELIA

The role of RhoGEFs in epithelial polarity using a three-dimensional model

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 Obiettivo del progetto (Objective)

'Rho-family GTPases control many aspects of cell behavior through the regulation of multiple signaling pathways. Failure to properly regulate Rho GTPases can have deleterious consequences in human development and disease. A key group of spatial and temporal regulators of Rho-family GTPases are the Rho GEF proteins. Rho GEFs serve to couple diverse regulatory stimuli to Rho GTPase activation. There are a large number of Rho GEFs encoded by the human genome and a systematic study addressing cellular function is needed. With this proposal, I aim to complete an unbiased large-scale functional analysis of all the Rho GEF proteins using a lentiviral-RNAi based strategy to identify Rho-family GEF proteins involved in epithelial cell morphogenesis. To achieve this project I will use the three-dimensional Madin-Darby canine kidney epithelial cell (3D-MDCK) system, which is an excellent model to investigate epithelial morphogenesis in vitro. I will verify RNAi mediated knockdown of expression for candidate GEFs and demonstrate that the phenotypes caused by RNAi are not primarily due to off-target effects. Then, I will characterize the phenotypes of Rho GEF RNAi knockdowns in detail and determine the subcellular localization of the GEF proteins. Finally, I aim to determine the Rho GTPase specificity for Rho GEF candidates. The analysis of Rho GEF function in the MDCK 3D cell culture system might have particular relevance to cancer since their aberrant regulation can lead to malignant transformation and a majority of human tumors are thought to be epithelial in origin. In this proposal, I will carry out a more systematic, genome wide knockdown of all known human Rho GEFs with the goal of identifying and characterizing important regulators of MDCK epithelial cell morphogenesis. I hope that the insights I gain from the analysis of Rho GEF function in MDCK epithelial cells will aid our understanding of development and disease in both the kidney and other epithelial tissues.'

Introduzione (Teaser)

The disrupted functioning of Rho-family proteins can have a negative effect on cellular activity. Resulting alterations in gene expression can lead to tumour growths.

Descrizione progetto (Article)

Rho-family guanosine triphosphatases (GTPases) are small proteins that regulate numerous aspects of intracellular behaviour such as cell proliferation, cell death and gene expression. Cdc42, Rac1 and RhoA are three such proteins whose proper functioning is necessary to avoid disruptions in normal human development and the onset of disease. A key group of Rho-family GTPases are the Rho guanine nucleotide exchange factor (GEF) proteins, a large number of which are encoded by the human genome and play a role in stimulating Rho GTPase activation.

The 'The role of RhoGEFs in epithelial polarity using a three-dimensional model' (RhoGEFs in epithelia) project has set out to perform an unbiased large-scale functional analysis of all the Rho GEF proteins. The end goal is to identify Rho-family GEF proteins involved in the cancerous growth of epithelial cells, as their disrupted regulation can lead to malignant transformation. Rho GEF functioning is being studied in an MDCK (Madin-Darby Canine Kidney Cells, a virus tissue culture) 3D system.

Having generated a library of short hairpin RNAs (shRNAs) targeting Rho-family GEFs, the project partners produced viruses, collecting and filtering them for infection of MDCK cells. The infected cells were examined and plated to allow for cyst formation, and then fixed and stained for further study. The results of this reagent treatment approach were not satisfactory and researchers opted to take a candidate screening approach to knockdown or reduce the gene expression of specific GEFs that are expressed in MDCK cells.

With this new system, Cdc42 specific GEF Intersectin2 (ITSN2) has been shown to be a candidate for regulating the Cdc42 activity that controls the formation of lumen. This is the interior of a tubular structure such as urinary collecting tubes or kidney ducts. Investigation of ITSN2 knockdown cells revealed a reduction in levels of Cdc42-GTP, explaining the aberrant phenotype in development of cancerous cysts.

ITSN2 has also been shown to localise at centrosomes: this is where microtubles are produced, and an excess volume of these are often found in cancer cells. Other study results have so far shown that ITSN2 and Cdc42 are necessary for normal cell division processes.