The goal of this once-revised competing renewal application is to pursue an ongoing research program identifying the function of c-src which, in the osteoclast, is required for bone resorption and is unique to c-src. The hypothesis that c-src may play a unique role in osteoclast function stems from three observations: First, deletion of the c-src proto-oncogene in the mouse leads to osteopetrosis without other apparent cellular defects. Second, the cellular defect induced by c-src gene deletion is cell autonomous. Third, the osteoclast not only expresses c-src at high levels but also other members of the Src family of non-receptor tyrosine kinases, implying that although present, they cannot compensate for src deletion in osteoclast function. During the first five years of this program, the applicant has made significant progress towards understanding the potential functions of c-src in bone resorption. However, despite the progress made, the mechanism(s) by which src deletion leads to a decrease in bone resorption is not yet definitively characterized. The most significant advances during the previous support period include: 1) the identification of the integrin alphaVbeta3 signaling pathway as the most likely to be affected by the absence of Src in the osteoclast, and 2) the characterization of the tri-molecular complex comprising Pyk2, Src and Cbl as a key molecular element involved in the activation and de-activation of adhesion that is necessary for normal osteoclast mobility. These observations led to the development of a model for podosome assembly and disassembly, which now serves as the central working hypothesis to be tested in the coming years. The central concept that has emerged from these studies was the notion of the crucial importance of podosome assembly and disassembly in the process of osteoclastic bone resorption.
The specific Aims of this revised application are therefore to: 1. Test the various steps of the podosome assembly-disassembly model through specific disruption of each presumed molecular interaction in the Pyk2-Src-Cbl complex, downstream of integrins in vitro. 2. When validated in vitro, generate transgenic mice expressing a selected number of the mutant Pyk2, Src or Cbl constructs targeted to the osteoclast by the TRAP promoter. 3. Further analyze the effects of knockouts and double knockouts of these molecules on osteoclast biology, adhesion, motility and bone resorption in vivo and in vitro. 4. Determine, which other molecules are associating with the Src complex upon adhesion.
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