EARLY RESPONSE PATTERNS IN DIFFERENTIATING OSTEOBLASTS

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Because of its high incidence and devastating socio-economic consequences, osteoporosis is an important disorder of the aging population. The disease causes 1.5 million fractures, including 250,000 hip fractures, annually in the United States. Following hip fracture, 50% of the survivors cannot walk independently again and 30-50% require long-term nursing care. The costs of osteoporosis are estimated at $7 to $10 billion annually in the USA. To treat and to prevent this disorder the basic physiological mechanisms regulating bone cell biology must be understood. Eicosanoids (e.g., prostaglandins, leukotrienes, etc.) are key components of a target cell's early responses to various stimulators including growth factors, cytokines, and hormones. While eicosanoid metabolism has been studied in several osteoblastic cell systems and in bone organ cultures, gaps in our knowledge concerning their local production remain. Although eicosanoid production is an early response to stimulation (altered production is evident within minutes) most investigators have measured (only) PGE2 release hours, or days, after treatment. These data may not reflect the dynamic responses of this signal transduction pathway in stimulated osteoblastic cells. Moreover, selecting a priori which eicosanoid to measure increases the probability that major responses in other unexamined members of the eicosanoid family will be missed. This is a critical issue since different eicosanoids often exert opposing biological effects. Presently, even the classes of eicosanoids produced by human osteoblastic cells is unknown. therefore, eicosanoid metabolism will be studied in normal adult human osteoblast-like (hOB) cells derived from trabecular bone explants. The use of hOB cells as the model system for the human osteoblast avoids potential complications introduced in trans-species studies and the abnormal cell responses commonly observed in spontaneous transformants. The hOB cells are partially differentiated and steroid responsive. the impact of 1,25(OH)2D3-induced differentiation on hOB cell eicosanoid metabolism will be examined. Eicosanoid release following treatment with the osteotropic agents estrogen, TGFbeta, PTH, interleukin 1 and IGF-I will be studied and compared in naive and 1,25(OH)2D3-treated hOB cells. These studies will focus on eicosanoid production in short-term incubations (120 minutes or less). Most analyses will initially use HPLC to examine the array of eicosanoids produced by the hOB cells. Follow-up studies will employ specific antisera for quantitative purposes. This work will define the characteristics of hOB cell eicosanoid metabolism, and determine the impact of differentiation on the production of these bioactive lipids. The regulation of hOB cell eicosanoid metabolism by the osteotropic agents will be studied both before and during induced cell differentiation. The molecular mechanism of any differentiation-associated changes in eicosanoid production will be probed at the level of hOB cell phospholipid metabolism. The proposed investigations may influence the design and use of pharmacologic interventions into the progress f osteoporosis and other metabolic bone diseases.