Estrogen replacement therapy has a proven protective effect in the prevention of bone loss in post menopausal osteoporosis however, the mechanism of this beneficial effect is unknown. Recently, several groups have identified estrogen receptors (ER) in cells of an osteoblast lineage. However, it has been difficult to show a direct effect of estrogen on bone cells in vitro, and in those that have responded, the biological effects have been minimal. In addition, other investigators were unable to confirm these findings suggesting that estrogen responsiveness may be heterogeneous and confined to certain subpopulations of osteoblasts. This interpretation may also be the result of a limited number of cells expressing the ER, which hampers a detailed analysis of the biological and biochemical mechanisms involved in estrogen activity. Such studies would be greatly facilitated by access to osteoblastic cells that uniformly express the ER and show measurable biological responses to the cognate hormone, 17beta-estradiol. Our working hypothesis is that ER levels and ER functionality are controlling factors in osteoblast differentiated activities. These activities include producing an extracellular matrix (ECM), activating existing osteoclasts, stimulating the recruitment of osteoclast precursors, and reacting to physiological signals. In this proposal, we have designed a two-part study to determine the role of ER in osteoblasts. In Part I, an expression plasmid containing the human ER is used to stably transfect osteoblastic cell lines thus producing osteoblasts that overexpress the ER. In Part II, human osteoblast cells are isolated from donated bone specimens from patients undergoing elective hip replacement surgery, in order to evaluate their ER levels and ER function. These in vitro analyses of ER and estrogenic responses in hyper-responsive osteoblasts will determine which estrogen-modulated genes are responsible for the beneficial actions of estrogen on bone.
The specific aims are: 1. To establish and characterize stably transfected osteoblast cell line with varying levels of ER. 2. To measure the synthesis of specific components of the differentiated phenotype in osteoblasts over-expressing ER. The structural components to be studied are bone sialoprotein, osteopontin, biglycan and decorin. The regulatory component to be analyzed is bone-specific alkaline phosphatase. The cytokines and growth factors to be analyzed will IL-6 and TGF-beta. 3. To determine the level of ER and measure its functionality in osteoblasts derived from surgically-obtained samples. The level of ER will be determined with a specific immunoassay and ER function will be assessed by co-transfection with an estrogen-response- element (ERE)-containing plasmid harboring a reporter vector (CAT). 4. To transiently transfect the human ER into enriched human osteoblast cultures and measure the phenotypic gene responses of structural, regulatory and cytokine/growth factor components. It has been difficult to demonstrate the direct action of estrogen in normal human bone cells in culture. Such studies would be facilitated by the access to osteoblasts that uniformly express the ER and show biological responses to estrogen. This proposal focuses on and exploits the ability to transfect human osteoblasts with the ER gene at various levels, and investigates the biological functions of the ER in those cells. These clarification's of estrogen's actions on bone will enable a fully reasoned approach to improving existing estrogen replacement therapy in osteoporotic patients.
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