Regulation of NF-kB by the ZO-1/ZONAB pathway

Epithelia are continuous layers of cells that delineate our tissues and organs. The integrity of epithelia is important for our organs to function normally and to protect us from our environment. For example, breaches in epithelial layers such as the skin or in the lining of the intestine can lead to serious infections. Individual epithelial cells interact with each other via molecular complexes that mediate adhesion but also function as sensors that transmit information about the environment, such as the presence or absence of neighbouring cells, to the cell interior. These sensors are important for the regulation of cell behaviour as they tell cells when to divide and when to stop to grow. If an epithelium is damaged, for example, cells divide and migrate to repair holes until they reach their neighbours and can form new adhesive complexes. Therefore, defects in these sensory mechanisms lead to severe diseases such as cancer. Many inflammatory diseases affect the functional properties of these adhesive molecular complexes. These diseases can be caused by bacteria and viruses, as well as allergens or by our own body defence system and often directly involve components of adhesive complexes. For example, a bacterium that causes gastric cancer injects molecules into epithelial cells that stimulate dissociation of adhesive complexes. Such infections also cause a cellular inflammatory response that helps our body to deal with them. However, these cellular responses can get out of control and cause damage. Here we focus on a new molecular mechanism by which adhesive complexes in epithelial cells can regulate the cellular inflammatory response. We propose to study the molecular mechanism and to determine how it is affected in two models of human disease. One model is based on the mentioned bacterium that causes gastric cancer and the other on cells isolated from colon and prostate cancers. Understanding how adhesive complexes guide the inflammatory response will help us to design new therapies for the above-mentioned diseases.

Epithelial cells adhere to each other via junctional complexes, enabling them to form cellular barriers that separate different tissues and body compartments. Tight junctions are one of these intercellular junctions. They restrict paracellular permeability and are hence required for barrier formation. Tight junctions also contribute to the regulation of epithelial proliferation, differentiation and polarisation via different types of signalling mechanisms. Dissociation of tight junctions results in epithelial barrier breakdown and is associated with many diseases including chronic inflammations and cancer. Such diseases often stimulate proinflammatory signalling by activating NF-?B. Whether tight-junction-associated signalling mechanisms directly contribute to the regulation of proinflammatory signalling is not known. In previous work, we have discovered a tight-junction-associated signalling mechanism that is based on the junctional adaptor ZO-1 and the Y-box transcription factor ZONAB. By forming a junction-associated complex, ZO-1 inhibits nuclear accumulation of ZONAB/DbpA and thereby regulates gene expression and cell proliferation. As there are many pathogens that disrupt tight junctions, we asked whether this protein plays a role in NF-?B activation. Our unpublished results based on functional and biochemical assays now suggest that the ZO-1/ZONAB pathway interacts with NF-?B pathway components. Based on these observations, we hypothesize that ZO-1 and ZONAB regulate proinflammatory signalling by interacting with cellular machinery that controls NF-?B activation, and that this mechanism is functionally important under conditions that trigger the dissociation of intercellular junctions such as microbial infections or cancer. The purpose of this application is to identify the molecular mechanisms by which the ZO-1/ZONAB pathway participates in the regulation of NF-?B activation and whether these mechanisms are of pathological importance. In aim 1, we propose an experimental strategy based on biochemical, functional and morphological assays to determine how and where ZO-1 and ZONAB interact with specific NF-?B activating pathways, and, in aim 2, we plan to use two disease models, H. pylori infection and carcinoma-derived cell lines, to test the functional relevance of the identified mechanisms for the regulation of NF-?B. An additional objective will be to identify possible strategies to exploit this regulatory mechanism for future therapeutic approaches The expected results will be important for the understanding of the role of epithelial junctions in the regulation NF-?B signalling pathways and how these molecular mechanisms contribute to pathological conditions.