Studies on the molecular interactions of collagen type XVI.
Part I: The role of collagen XVI in pathological disorders
Part II: Extablishment of a retroviral mediated gene silencing model
Molecular interactions of collagen XVI in Crohn’s disease
In Crohn’s disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF), provided by intact tissue is disturbed due to inflammation and cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original extracellular matrix (ECM) leads ISEMF to exit this cycle, however, in fibrosis ISEMF remain. It is known that ISEMF produce and deposit collagen types I, III, IV and V; but synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and the effect of collagen XVI on cell proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall and ISEMF reveal increasing gene and protein expression of collagen XVI with concurrent increasing inflammation. ISEMF display more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This might involve recruitment of α1 integrin, since its cell surface expression on ISEMF is increased in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of α1ß1 integrin into the focal contacts.
Mechanistical studies of collagen XVI by a retroviral mediated knockdown model in murine fibroblasts
Collagen XVI, a member of FACIT collagens (fibril associated collagens with interrupted triple helices) is described as macro-molecule of the ECM. Very little is known about its role in cell-matrix-interactions and in cell signalling. However it has been demonstrated that cells interact with collagen XVI via integrin α1β1 and α2β1. These interactions presumably determine organization of extracellular components and their communication with cells. We have established a retroviral mediated collagen XVI knockdown in NIH3T3 fibroblasts to investigate the role of collagen XVI in cell-matrix interactions. Specific shRNAs against collagen XVI were utilized in parallel to a viral luciferase vector construct as control. After successful transduction, positive cell clones were further selected by antibiotic resistance. Knockdown efficiency was determined on mRNA and protein level and further downstream experiments were performed with respect to adhesion and proliferation. Differential protein expression in knockdown cells was compared to the control by 2D-gelelectrophoresis.
The knockdown resulted in 80-90 % inhibition of collagen XVI gene and protein expression and mass spectrometry revealed several differentially expressed proteins. Collagen XVI inhibited cell lysates showed a lack of macrophage migration inhibitory factor (MIF) and prolylpeptidyl-cis-trans-isomerase A (PPIA). The gene expression of these proteins was slightly up-regulated, however, western blot analysis confirmed 2D-gelelectrophoresis results. Proliferation of knockdown cells was generally reduced and was not influenced by the presence of collagen XVI as cell culture substrate. The cytokine MIF influences migration and proliferation of fibroblasts during wound healing. MIF induces synthesis of collagens in fibroblasts (collagen I, III, IV, V and VI) whereas PPIA contributes to correct protein folding in the cytoplasm of cells. Collagen XVI acts as adapter molecule in organizing suprastructures. Therefore, we assume that the lack of collagen XVI detains fibroblasts in arranging fibrillar stuctures which results in disturbed initial adhesion. The amelioration of cellular adhesion to the available matrix indicates a compensation in integrin expression pattern. Reduced proliferation together with decreased MIF expression hints at changes in the differentiation stage and has to be further elucidated
