Brush border alterations in the pathogenesis of intestinal diseases

The intestine is the location of diseases of enormous social and economic impact, like the inflammatory bowel disease (affecting about 1 person in every 500 in the UK) and the colon carcinoma (the second most frequent malignancy in developed countries). In addition numerous infectious agents use the intestinal tract to gain access into the body. The intestine therefore fulfils an important mechanical and immunological barrier function. Alterations of this barrier count among the diverse factors that predispose to the development of conditions that affect the intestine. Understanding how the intestinal barrier develops and maintains its integrity is key to prevent those diseases. An important component of the intestinal barrier are the epithelial cells, which constitute the largest cell population of the intestine and bear a so called brush border. The brush border is a complex anatomical structure whose assembly is still not well understood. We will use a cell line with characteristics of intestinal epithelial cells to investigate the consequences of altering the components of the brush border. By doing so we will learn about the assembly of the intestinal barrier and the consequences of its disruption for the development of inflammatory bowel disease and other conditions.

Alterations in the cytoskeleton of the apical region of the intestinal epithelial cells are emerging as predisposing factors to intestinal pathology. Such is the case of mutations in keratins, which are suspected to result in disrupted cell polarity and increased mechanical fragility, or absence of villin, which results in increased apoptosis in a mouse model of induced colitis. Our work with an I-plastin deficient mouse shows that this cytoskeleton protein plays a key role in the organisation of the intestinal brush border (BB), as its absence results in mechanical fragility and increased sensitivity to induced colitis. This suggests that I-plastin deficiency may function as a predisposing factor to the development of inflammatory bowel disease through a mechanism that involves, in part, malfunction of the keratin network.
In order to complement and extend our studies we will resort to the intestinal epithelial cell line CaCo2-BBe and apply lentiviral vector technology for RNA silencing and ectopic expression of one or more BB components (more specifically plastins, villin, espin and keratins 8 and 19). We will use immunofluorescence confocal microscopy to investigate alterations in the distribution of major proteins of the apical pole at several stages during development of polarity. Electron microscopy techniques will be applied to investigate the microvilli (organization, density and morphology), the terminal web and the junctional complex. Biochemical studies will address whether any of the BB proteins becomes overexpressed or underregulated as a compensatory mechanism to manipulation of a particular BB component. Finally, the functional integrity of the epithelial monolayer (transepithelial electrical resistance, targeting of hydrolases and functioning of transporters) will be investigated in response to alterations in the composition of the BB.
Our aim is to investigate the consequences of these manipulations on the composition, morphology and functionality of the BB. We will learn about the contribution of cytoskeleton components to the assembly and maintenance of the architecture of the intestinal BB and the consequences of disrupting the BB for the pathogenesis of IBD and other intestinal diseases.