Over the past several years, new data have accumulated on the lineage and hormonal responsivity of osteoclasts but, with few exceptions, the biochemical mechanisms underlying these phenomena remain poorly understood. The latter gap in information is particularly apparent at the molecular biological level where no literature exists on the isolation, characterization and/or regulation of those genes which define the osteoclast RNA and in identifying several genes (e.g., carbonic anhydrase whose activity appears elevated in osteoclasts relative to related marrow and marrow-derived cells, e.g., macrophages and macrophage multinucleated giant cells. In the present renewal, three major aims are identified that, if attained, should provide significant insight into the gene regulatory mechanisms operational in osteoclasts. The first is to further enlarge the """"""""panel"""""""" of cDNA probes necessary to explore the regulation of gene expression. Such panel enlargement will be achieved using two strategies; in one, previously cloned cDNAs from non-osteoclastic cells or tissues (e.g., human tartrate resistant acid phosphatase) will be used to identify their homologs in chicken osteoclast cDNA libraries; in the other, cDNA clones responding to, as yet, unrecognized osteoclast development/function-related genes will be isolated from osteoclast cDNA libraries by comparative and antibody screening using cDNA probes and monoclonal reagents developed for genes prominently expressed using cDNA probes and monoclonal reagents developed for genes prominently expressed in osteoclasts and related cells.
The second aim i s to determine the pattern of gene expression (i.e., changes in specific mRNA abundance) in differentiating osteoclasts and in osteoclasts responding to select effector substances.
The final aim i s to initiate analysis of the mechanisms underlying the changes in gene expression with a particular focus on transcriptional regulation.
This aim will be achieved using the nuclear runoff technique and the transfection of promoter-reporter gene constructs into osteoclasts and related multinucleated giant cells.
