Kristina Mayer

• Extracellular cathepsins are involved in physiological and in pathological conditions, in which they may cause disturbances of normal balances of enzymatic activity. To determine whether cathepsins contribute to the onset of inflammatory responses leading to post-operative ileus (POI), we used a rat model of standardized small intestine trauma. In non-damaged tissue, cathepsins were located in endocytic vesicles of enterocytes. After trauma, a localized release of cathepsin B was observed along with degradation of components of the ECM. Hence, extracellular cathepsin activity might lead to an impairment of mucosal barrier function, making cathepsin B a potential candidate for promotion of inflammatory responses after trauma. To better characterize molecular mechanisms underlying POI, an in-vitro model system for mechanical manipulation of enterocytes was developed. The established compressive loading system allows to reproducibly induce damage, which is characterized by necrotic cell death leading to release of cathepsins. Released cathepsins B and D were proteolytically active as soluble enzymes in conditioned media of damaged cells, whereas cathepsin L re-associated with the cell surfaces. Hence, released cathepsins may serve proteolytic functions as soluble or cell surface-immobilized enzymes. To elucidate the underlying secretion mechanisms in damaged tissues, we took advantage of an intestine specific gene-expression driven by the A33-antigen-promotor which contains regulatory elements including CDX binding sites. The activity of the intestine-specific transcription factor Cdx1 is pivotal for an intestinal expression pattern of the A33 antigen gene. We cloned the A33 antigen-promoter into a cathepsin B-EGFP vector. Co-transfection with Cdx1 enabled A33-antigen promoter-controlled expression of cathepsin B-EGFP in CHO cells. The long-term objective of these in vitro experiments is to apply the results in order to establish a transgenic mouse model.