Osteoclasts are multinucleated giant cells specialized for the removal of both the inorganic and organic phases of bone. Despite some recent insights from the effects of the targeted deletion of the c-fos, PU.1 and NF-kappaB transcription factor genes, the mechanisms by which transcription factors control the process of OC differentiation remain unclear. This has prompted us to develop a new method for generating osteoclastogenic cell lines (MOCP-5), isolated novel OC-specific cDNAs (i.e. cathepsin K) and their mouse genomic clones. Cathepsin K, which was recently cloned by the P.I. employing differential cDNA screening, and which plays a central role in normal bone remodeling as well as in pathological processes, is largely if not exclusively limited in its expression to OC and is a useful model protein for studies of osteoclast differentiation. The hypothesis of the proposed studies is that the determination of OC differentiation involves OC-specific transcription factors (OCTFs i.e. lineage-specific factors) which bind cis-regulatory elements of OC-specific genes, and function together with ubiquitous transcription factors (i.e. c-fos, PU.1 and NF-KB), to activate OC- specific genes expression, and hence control OC differentiation. Therefore, the cathepsin K gene critical cis-regulatory element-binding proteins (CCREBPs) should be OCTFs.
In Aim 1, the mouse cathepsin K gene critical cis-regulatory elements (CCRE) will be mapped by mutagenesis of the cathepsin K promoter in vitro (MOCP-5 cells) and in vivo (transgenic mice).
In Aim 2, The CCRE-binding protein(s) (CCREBP) will be characterized by mobility shift analyses and DNase I footprinting. CCREs homologs and function will be sought in other OC-expressed genes.
In Aim 3, the cDNA encoding the CCREBP(s) will be isolated by Southwestern screening, or alternatively, by expression cloning or protein purification. The temporal and spatial expression of CCREBPs will be discerned by northern blot and in situ hybridization. Functional analysis of CCREBPs as putative OCTFs for regulation of OCs differentiation will be determined by co-transfection, forced expression and antisense blockade (Aim 4). The work proposed in this application should not only expand our basic understanding of the molecular mechanisms of OC differentiation, but will also aid our ability to develop therapeutic means of intervention in diseases involving increased bone resorption such as osteoporosis, periodontal disease, and arthritis.
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