Bone resorptive cytokines, including interleukin 1 (IL-1) and tumor necrosis factor (TNF), have been implicated in the bone destruction associated with periodontitis, arthritis, osteoporosis and the hypercalcemia of malignancy. These mediators concomitantly inhibit bone formation through negative effects regulation of matrix protein synthesis by osteoblasts. This combination of activities 'uncouples' bone resorption and formation, thereby leading to a net loss of bone mass. The goal of this application is to elucidate the molecular mechanisms by which these mediators regulate bone remodeling, via effects on gene expression in bone cells.
In Aims 1 and 2, we will identify and evaluate the functional role of IL-1-induced resorption-related genes which are expressed in osteoblasts and osteoclasts. These genes will be identified by differential hybridization from a cDNA library derived from resorbing rat bone, and will be initially characterized by DNA sequencing. Both previously-described and novel genes will be obtained. Bone cells expressing these genes will be identified by in situ hybridization and/or immunolocalization. Functional relevance will be established by antisense deoxyoligonucleotide blockade of bone resorption in vitro.
In Aims 3 and 4, mechanisms of regulation of bone formation by cytokines will be studied at the molecular level, using osteocalcin as a model protein. The TNF-responsive element (TNF-RE) in the osteocalcin promoter will be mapped using deletion and oligonucleotide-directed mutants. The interaction of the TNF-RE with DNA-binding induced regulatory protein(s) will be characterized in gel retardation and DNase I footprint analyses. Regulatory protein(s) will be characterized. Homologs of the osteocalcin TNF-RE will be sought in other bone matrix protein promoters. Elucidation of the mechanisms involved in cytokine-induced uncoupling will permit the rational design of therapeutic agents which interfere with bone destruction in infectious and inflammatory diseases.
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