Heparan Sulfate–Osteoprotegerin Interactions In Bone Remodeling Bone remodeling is a life-long process controlled by the balanced activity of the bone-forming osteoblasts and the bone-resorbing osteoclasts. In many prevalent and costly medical conditions such as osteoporosis, rheumatoid arthritis, periodontitis and cancer, the balance is tipped towards bone resorption as a result of excessive osteoclast activity. The receptor activator of nuclear factor kappa-B ligand (RANKL), produced by osteoblasts as a membrane-associated cytokine, plays a central role in bone remodeling by promoting osteoclastogenesis. To regulate the activity of RANKL, osteoblasts secrete osteoprotegerin (OPG) as a soluble decoy receptor for RANKL. While it is known that the ratio between RANKL and OPG plays a decisive role in bone remodeling, how this ratio is regulated at the cell surface of osteoblasts remains poorly understood. Heparan sulfate (HS), a universal glycosaminoglycan that all mammalian cells express at the cell surface, could regulate RANKL/OPG ratio because it binds OPG with high affinity. Indeed, a couple of studies have shown that HS regulates the bioavailability of OPG in tumor settings. Therefore, our central hypothesis is that HS regulates the RANKL/OPG ratio in bone remodeling. To test this hypothesis, our overall objective here is to elucidate how HS interacts with OPG and how HS–OPG interactions regulate osteoclastogenesis and bone remodeling. Guided by strong preliminary data generated in the lab, our hypothesis will be tested by pursuing the following three specific aims. Aim1. Identify the structural details of HS–OPG interactions. We will employ complementary biophysical methods including hydrogen/deuterium exchange mass spectrometry, small-angle X-ray scattering and X-ray crystallography to understand the structural basis of HS– OPG interactions, OPG dimerization and HS–OPG–RANKL ternary complex. Aim2. Determine the biological significance of HS–OPG–RANKL ternary complex. We will manipulate HS–OPG interactions genetically and biochemically and determine the mechanism by which HS promotes the productivity of OPG–RANKL interactions at the cell surface. Aim 3. Determine the role of HS–OPG interactions in osteoclastogenesis in vivo. By using a novel OPG knock-in mouse strain, we will examine the consequence of altering HS–OPG interactions in bone remodeling under physiological and pathological conditions. Results from the proposed experiments will substantially advance our understanding of the cellular regulation of the OPG–RANKL interactions by elucidating the role of HS in the system. Importantly, these results are expected to have positive translational impact because by identifying how HS regulates the RANKL/OPG ratio in osteoblasts, we may be able to manipulate RANKL/OPG ratio by modulating HS–OPG interactions to control excessive osteoclastogenesis.