GENE EXPRESSION DURING OSTEOCLAST DIFFERENTIATION

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The osteoclast is a unique cell which arises from a hematopoietic precursor it shares with cells of the granulocyte and monocyte lineages. When activated, it becomes polarized, forms a ruffled border and causes release of bone mineral and proteolytic degradation of the bone matrix. The goal of this project is to elucidate changes in expression of specific genes during osteolysis and during differentiation of the precursors. We plan to quantify changes in the expression of two classes of specific genes: a) those directly associated with osteolysis (such as carbonic anhydrase II, the lysosomal protease cathepsin 0, the ruffled border proton pump, and superoxide dismutase) and b) hormonal responses of these to regulatory factors such as calcitonin, vitamin A, phorbol myristate acetate, 1,25 dihydroxyvitamin D-3 and tumor necrosis factor. We will also plan to determine the functional importance of these gene products by manipulating the phenotype of cultured avian osteoclasts. This will be done by the introduction of DNA constructs which express sense or antisense RNA, thereby raising or lowering the levels of specific cellular marker proteins independent of hormonal regulation of the cells. Two model systems will be used for these studies. The human promyelocytic leukemic cell line HL-60 shares phenotypic features with early precursor cells in the osteoclast lineage. This cell line will be used as a representative of an early osteoclast progenitor. As a model for mature osteoclasts, we will use isolated chicken osteoclasts. These cells, unlike mammalian osteoclasts, are available in sufficient quantities to allow application of the techniques of molecular biology.