Fringe regulation of Notch signaling in osteoclasts - Project Summary/Abstract Notch signaling both positively and negatively regulates differentiation and function of osteoclasts, and signaling through the different Notch receptors is modulated through glycosylation first by Protein O-fucosyltransferase 1 (POFUT1) and then by members of the Fringe family of N-acetylglucosaminyl transferases. Multiple positions of the Notch extracellular domain are subject to O-fucose extension by Fringes, and different Fringes (LFNG, MFGN, and RFNG) demonstrate differences in both patterns of glycosylation and alteration in ligand-specific receptor activation. Osteoclasts and their precursors express multiple Notch receptors and ligands as well as all three Fringes, and this project will study the combinatorial effect of these Fringes on activation of Notch receptors and resultant osteoclast formation and function. The overall hypothesis for this study is combinations of Fringe-catalyzed glycosylation modulate Notch signaling intensity and varying levels of Notch signaling produce either pro- or anti- osteoclast effects. This will be tested in 2 specific aims. In aim 1, contributions of individual Fringes will be investigated through targeted overexpression and knockdown followed by quantification of Notch1 and Notch2 activation, osteoclast differentiation and activity, and determination of Notch1 and Notch2 receptor glycosylation patterns. In aim 2, the relationship between level of Notch signaling and osteoclast differentiation will be investigated through tuned expression of active Notch1 and Notch2 intracellular domains in pre-osteoclasts followed by high throughput RNA sequencing to determine patterns of Notch-regulated gene expression and resultant level of osteoclast differentiation. Completion of this work will deepen understanding of Notch modulation of osteoclast formation and function specifically, but also contribute to general mechanisms of Notch signaling and the combinatorial effects of Fringe-mediated glycosylation. In the long term, this knowledge may contribute to the development of new bone-preserving therapies based on modulation, rather than total inhibition, of osteoclast activity.
