Osteoclasts differentiate from the monocyte-macrophage lineage, and, as mononuclear cells, exhibit a variety of markers of the mature osteoclast including amplified vacuolar H+ ATPase expression. Under the influence of regulatory factors, these tb form resorptive osteoclasts. In this application, we propose to examine several aspects of the carefully regulated differentiation of osteoclasts: 1) We will examine the molecular mechanisms controlling H+-ATPase amplification in differentiating cells of the monocyte-macrophage lineage. TAP-1 cells undergoing monocyte to macrophage differentiation show enhanced transcription of a human vacuolar H+ ATPase 56kD subunit """"""""brain"""""""" isoform. The TAP-1 cells will be transfected with promoter-reporter constructs form the 5' flanking region of the gene for the 56kD subunit to investigate the regulatory elements that control this expression; 2) We will determine which proteinases are present in the mammalian osteoclast and which of these is/are responsible for bone matrix degradation: Immunocytochemistry, immunoblots, Northern analysis, and in situ hybridization with antibody and cDNA probes specific for several metalloproteinases and cathepsins will be used to determine which are present in the osteoclast. Extracts from mammalian osteoclasts will be studied in vitro for collagenolytic properties, and inhibitors and immunodepletion will be used to determine which proteinases actually degrade bone. The regulation of expression of crucial proteinases during osteoclast differentiation will be examine; precursor cells into resorptive osteoclasts in vitro will be isolated from conditioned medium by chromatographic protein chemistry methods. The effects of the factor on proteinase expression and secretion and on H+-ATPase gene expression and distribution in osteoclasts will be studied. The enzymatic properties of the H+-ATPase in mammalian osteoclasts generated in vitro will be determined. These studies will advance our understanding of osteoblast-derived differentiation factors, and the cellular and molecular events occurring during osteoclast differentiation and the acquisition of the bone- resorptive state.
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