Examining Novel Factors that Influence Contact-dependent Osteoblast and Osteoclast Differentiation

A dynamic equilibrium between bone destruction by osteoclasts and bone formation by osteoblasts is responsible for the maintenance of bone integrity, mineral homeostasis and protection from bone-related disease. As such, the focus of this work was to examine novel factors contributing to the contact-dependent differentiation of osteoblasts and osteoclasts. Osteoblast differentiation and maturation is stimulated by multiple external factors, two of which are ascorbic acid (AA) and bone morphogenetic protein-2 (BMP-2). Utilizing MC3T3-E1 cells and primary murine osteoblasts, we identified a novel role for EB1, a microtubule plus-end binding protein, during osteoblast differentiation. AA-stimulation strongly induced EB1 expression and EB1 knockdown significantly impaired the osteoblast differentiation program in both AA- and BMP-2-induced osteoblasts. Furthermore, we identified that EB1 function was important for the global stability of -catenin, a major signaling molecule in osteoblasts. Lastly, the influence of E-cadherin, a cell-cell adhesion and recognition molecule, was investigated in AA-stimulated osteoblasts. Up-regulation of Cdh1 (E-cadherin) paralleled that of Catnb (beta-catenin), and E-cadherin blocking antibody treatment dampened osteoblast-specific gene expression. E-cadherin is also expressed in monocyte/macrophage cells, which are precursors to osteoclasts. Receptor activator of nuclear factor-ÎşB ligand (RANKL)-stimulated osteoclast differentiation involves a period of precursor expansion followed by multiple fusion events to generate a multinucleated osteoclast. Interestingly, our results indicated that E-cadherin participated in early precursor interaction/recognition rather than during periods of osteoclast fusion. In both RAW 264.7 cells and primary murine macrophages, E-cadherin expression and surface localization was highest during early osteoclast differentiation. Utilizing E-cadherin blocking antibodies prior to the onset of fusion delayed osteoclast-specific gene expression and significantly impaired multinucleated osteoclast formation. Long-term imaging revealed that blocking E-cadherin function prolonged the proliferative phase of the precursor population while concomitantly decreasing the proportion of migrating precursors; the lamellipodium and polarized membrane extensions were identified as principal sites of fusion, establishing migration as a requirement for osteoclast differentiation. This work characterizes EB1 and E-cadherin function during osteoblast differentiation and the E-cadherin-mediated transition from proliferative to migratory activities during osteoclast differentiation. Taken together, both studies highlight the value of exploring the early intra- and intercellular events that direct osteoblast and osteoclast differentiation.Ph.D