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Project

In vitro studies into the downstream regulatory mechanism whereby LRP4 and sclerostin together regulate Wnt siganling activity.

Previous genetic and functional studies have shown that the canonical Wnt signaling pathway is an important pathway in the regulation of bone formation. The pathway is activated by the binding of Wnt ligands to the receptor complex formed by a Frizzled receptor and an LRP5/6 co-receptor. Furthermore, it is well known that sclerostin is an important inhibitor of the pathway that prevents Wnt ligands from binding to LRP/6 and activating the pathway. Although it is clear that sclerostin can also binds to LRP4 and that this interaction is important for the inhibitory actions of sclerostin, the exact downstream mechanism of this interaction is unclear. In literature, it is reported that LRP4 has an endocytosis signal in its intracellular domain and in addition, several years ago one study demonstrated in murine mesenchymal stem cells that sclerostin is internalized via clathrin dependent endocytosis. Consequently, the aim of this study is to investigate the role of LRP4 in the endocytosis of sclerostin. To do this, primary osteoblasts isolated from the long bones of wild type and Lrp4 mutant mice are treated with fluorescent tagged sclerostin and visualized using fluorescent microscopy. In addition, using different LRP4/5/6 mutant constructs, we aim to further investigate the role of the different receptors in the internalization process. Mutant and wild type constructs will be overexpressed in wild type primary osteoblasts and internalization of fluorescent tagged sclerostin will be evaluated using fluorescent microscopy and western blot. The results of these studies will increase the insights in the downstream mechanisms of the inhibitory action of sclerostin and LRP4. Furthermore, we will evaluate the role of LRP5/6 in this process. Overall we believe that this study will increase the knowledge on the regulation of the canonical Wnt signaling pathway by sclerostin and LRP4 which is important since sclerostin is an important therapeutic target for the treatment of osteoporosis and other bone diseases.
Date:1 Apr 2019 →  30 Mar 2020
Keywords:WNT PATHWAY, OSTEOBLAST, BONE FORMATION, ENDOCYTOSIS
Disciplines:Cell signalling, Genetics