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Sondag, Gregory R., Ph.D., December 2015Biomedical SciencesOSTEOACTIVIN AND CD44 :A NOVEL INTERACTION STIMULATING BONE FORMATION AND INHIBITING BONE RESORPTIONDissertation Advisor: Fayez F. Safadi, Ph.D.Osteoporosis is a growing problem for the elderly population. The bisphosphonate anti-resorptive class of drugs is the most commonly prescribed medication, however, the need for anabolic agents to stimulate bone formation is crucial. Osteoactivin was first discovered in bone using the op osteopetrotic mutant rat model. Our lab was the first to show that Osteoactivin stimulates bone formation in osteoblasts. Although several pieces of literature exist describing the role of Osteoactivin in both osteoblasts and osteoclasts, little is known about the signaling pathway involved in its function. In this study, we used the recombinant Osteoactivin in culture to determine its effects on osteoblast and osteoclast differentiation in vitro. First, we added recombinant Osteoactivin to osteoblast and mesenchymal stem cells in culture and found an enhancement in osteoblast differentiation and function by ALP staining, activity, mineralization and upregulation of gene expression. We found that Osteoactivin binds to and interacts with CD44 to stimulate osteoblast differentiation. Furthermore, we determined that CD44 deficient mice have a skeletal phenotype in vivo and ex vivo that results in less bone mass. Lastly, we found that Osteoactivin mediated enhancement of osteoblast differentiation is mediated through the CD44-ERK signaling pathway.In our next study, we wanted to determine if the lipid raft is important for Osteoactivin mediated signaling in osteoblast. Our results have shown that Osteoactivin co-localizes within caveolin rich lipid rafts. Furthermore, Osteoactivin stimulates clustering of CD44 with Caveolin-1 and disruption of lipid rafts prevents this process. Lastly, we show that disruption of lipid rafts results in an inhibition of Osteoactivin mediated ERK signaling in osteoblasts.In our last study, as opposed to the above role of Osteoactivin in osteoblast formation we wanted to determine the role of recombinant Osteoactivin during osteoclast differentiation and function. Our results reveal that recombinant Osteoactivin inhibits osteoclast differentiation and size in a dose dependent manner. Futhermore, we show that Osteoactivin reduces ERK and AKT phosphorylation in osteoclasts. The Osteoactivin mediated inhibition of osteoclasts was found to involve its interaction with CD44. Lastly, we show that recombinant Osteoactivin inhibits osteoclast migration and recruitment in wild type samples in vivo, and that this effect is abrogated in CD44 deficient mice. Overall, we believe that Osteoactivin and its interaction with CD44 results in the enhancement of osteoblast differentiation and an inhibition of osteoclast resorption. |
