Applicant

Name: Prof. Dr. Andrea Vortkamp

email: andrea.vortkamp@uni-due.de


Interaction of heparan sulfate and integrin signaling in maintaining the articular cartilage

Heparan sulfate (HS) carrying proteoglycans bind to many signaling factors and other secreted proteins. Recent investigations strongly indicate that the degree and level of HS sulfation determine the specificity of these interactions, thereby regulating distribution, activity and receptor binding of the proteins. Unpublished experiments of our group demonstrate that distinct modifications (loss of Ndst1 dependent sulfation) of the HS structure have a protective effect on the articular cartilage during aging and in an induced osteoarthritis (OA) model. Here we plan to investigate the importance of the HS structure on cartilage homeostasis during aging and in induced OA models by analyzing mice carrying different HS modifications (loss of 2-O sulfation and epimerization) (Aim 1). Like integrin signaling, the HS carrying syndecans act as mechanotransducers in chondrocytes. We will thus investigate how chondrocytes carrying an altered HS structure respond to mechanical stress (dynamic compression) using the Flexacell compression system established in the Angele (SP7) group. To receive insight into how the altered HS pattern is translated into a cellular response we will generate RNA expression profiles of wild type and mutant femoral heads and of wild type and mutant chondrocytes exposed to defined mechanical loading conditions (Aim 2). The expression profiles will be compared to those obtained for the ß1 and α10 integrin mutants generated by the Aszodi group to identify common and distinct target genes.

Preliminary experiments also indicated that complete loss of HS leads to a severe reorganization of the extracellular matrix (ECM) and to increased levels of phosphorylated focal adhesion kinase (pFAK) indicating disturbed integrin signaling. We will use immunofluorescence and atomic force microscopy (Aszodi, SP1) to investigate if specific alterations of the HS sulfation pattern lead to distinct modifications in the expression of structural ECM components and of the mechanical quality of the ECM (Aim 3). As integrin signaling is the main regulator of cell matrix interactions we will use immunofluorescence, affinity chromatography (aim 4) and cell culture experiments (aim 5) to decipher how HS affects integrin signaling to organize the ECM structure and if the sulfation pattern is critical for this interaction. With the experiments outlined above we expect to gain first insight into the mechanisms by which HS and integrin signaling are linked, which integrins are regulated and if the HS structure has an impact on the interaction. Based on the outcome we will cross the most informative sulfation mutant to the respective integrin mutant to investigate the epistatic interaction and its importance for the maintenance of the articular cartilage in vivo (Aim 5).