To understand the cellular and molecular mechanisms involved in regulation of physiological and pathological bone resorption, there is a critical need to develop a marker that identifies osteoclasts and permits distinction of these cells from hematopoietic precursors and other multinucleated giant cells (e.g. foreign body giant cells). To fulfill this need, we have cloned cDNAs that encode high affinity receptors for calcitonin (CT), the principal peptide hormone that regulates the functional activity of osteoclasts. The goal of this application is to exploit our knowledge of the structure of the human CT receptor (CTR) and the availability of molecular probes to answer the following questions: I. What are the structural and functional properties of the osteoclast CTR? RNA prepared from human giant cell tumors of bone and the reverse transcriptase/polymerase chain reaction (RT-PCR) and cloning techniques will be utilized to sequence and characterize the structural and functional properties of the human osteoclast-type CTR. 2. What is the phenotypic relationship between bone resorbing cells in physiological and pathological remodeling? Are there osteoclast """"""""subtypes"""""""" without CTRs and what controls their development? In situ hybridization techniques and immunohistochemistry with antibodies to the human CTR will be used to demonstrate the distribution and specific cell-types expressing the CTR. Characterization and comparison of cells in specific bone lesions, e.g. Paget's disease of bone and other forms of granulomatous lesions in which the phenotype of the giant cells and their relationship to osteoclasts is not well understood will be undertaken. Examination of bone samples from individuals with refractoriness to CT will provide insights into the mechanisms by which CTR expression is regulated in these conditions. 3. What are the cellular and molecular mechanisms regulating CTR gene expression during osteoclast differentiation? We propose to clone and characterize the human CTR gene. To define the specific and potentially unique regulatory sequences responsible for expression of the CTR, constructs of the CTR 5' region containing the putative promoter regions of the CTR gene will be linked to a chloramphenicol transferase (CAT) reporter gene and screened for the capacity of specific hormones or cytokines to induce CTR expression using transfection in human mononuclear cell lines. This system can then be used to identify potentially unique factors that have the capacity to regulate CTR gene expression.
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