Osteoclasts (Ocs) synthesize and respond to chemokines such as interleukin-8 (IL-8). IL-8 influences osteoclasts by causing membrane contraction, increased motility and inhibition of bone resorptive activity. It is assumed that well organized cytoplasmic adhesion structures connected to th cytoskeleton, called podosomes, affect changes in osteoclastic motility. Podosomes are formed by a conglomerate of proteins, including talin, cortactin , tensin and F-actin. In these studies, morphological, biochemical and molecular events relating to IL-8-induced dissociation of podosomes will be monitored.
Aim 1 proposes immunofluorescent confocal microscopy as a means to characterize the disruption of podosomes.
In Aim 2, the changes in the levels of transcription and expression of certain cytoskeletal proteins induced by IL-8 will be assessed with northern and western blotting as well as RNase Protection Assays (RPAs). Experiments in Aim 3 will assess protein phosphorylation mediated by the regulatory protein, c-Src, using an antiphosphotyrosine monoclonal antibody and western immunoblot methods.
Aim 4 will correlate specific IL-8-regulated signaling pathways leading to alterations in cytoskeletal proteins and podosome structure. Here, the involvement of signaling molecules and pathways such as PKA, PKC, MAP kinase, Ca2+, and superoxide will be assessed by inclusion of specific inhibitors The proposed studies will help elucidate the role of IL-8 in modulating osteoclastic activity during normal as well as pathological bone remodeling.
