The long-term goal of the proposed research is to understand molecular mechanisms of bone resorption, a process that requires osteoclast activation and is altered in multiple bone disorders such as osteopetrosis and osteoporosis. Osteoclastic activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, formation of the sealing zone and polarized ruffled membrane, and directional secretion of acids and lysosomal enzymes in the resorbing surface. Osteoclasts attach to the extracellular matrix at podosomes. Podosomes are dot-like aggregations of actin that cluster in a ring around the cell periphery. They are similar to focal adhesions in fibroblasts. Changes in podosome assembly and disassembly allow osteoclast adhesion, migration, and bone resorption. Integrin-mediated signaling plays a key role in these processes. Integrin engagement triggers activation of proline-rich tyrosine kinase 2 (PYK2), an """"""""adapter-like"""""""" tyrosine kinase that is related to focal adhesion kinase (FAK). PYK2 is highly expressed in osteoclasts and localized at podosomes and tight sealing zones in resorbing osteoclasts. PYK2 knockdown by antisense or knockout leads to defects on podosome formation and bone resorption, indicating that PYK2 plays a critical role in regulating actin cytoskeletal organization in osteoclasts. However, exactly how PYK2 regulates osteoclast function remains unclear. In this proposal, we will focus on how PYK2 regulates podosome assembly in osteoclasts. Using focal adhesions in fibroblasts as a model system, we found that overexpression of PYK2 induced """"""""podosome-like"""""""" focal adhesions. We have studied mechanisms of PYK2 induced cytoskeletal reorganization and found that gelsolin, an actin binding protein that is important for actin filament formation in osteoclasts, is regulated by PYK2. Based on our preliminary results, we hypothesize that PYK2 functions in osteoclasts by regulating gelsolin. To test this hypothesis, we will: 1. Investigate the function of the PYK2-gelsolin interaction in osteoclasts.2. Determine whether gelsolin is a PYK2 substrate in osteoclasts.3. Determine whether and how PYK2 regulates the gelsolin-PIP2 binding Results of the proposed studies will provide insight into PYK2 signaling pathways in regulating osteoclastic functions.
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