The reduction of bone mass with loss of ovarian function and the sensitive reversal of this phenomenon with estrogen replacement are well documented. Yet, the precise mechanisms by which estrogens exert their potent antiresorptive effects on bone remain unknown. Preliminary studies leading to this application indicate that: 1) estradiol inhibits the production of interleukin-6 (IL-6) induced by cytokines and systemic hormones in bone marrow stromal and osteoblastic cell lines from animals and humans, as well as in primary cultures of calvaria cells; and that estrogen withdrawal from primary bone cultures causes a surge of IL-6 production; 2) tumor necrosis factor (TNF) is produced by bone cells and serves as an amplifier of IL-1-, PTH-, and 1,25(OH)2D3-induced stimulation of IL-6 production as well as a stimulator of osteoclast development in the calvaria cultures; and estradiol as well as an anti-IL-6 neutralizing antibody, suppress this effect, while exogenous IL-6 promotes osteoclast development; 3) trabecular bone loss induced by ovariectomy in the mouse is associated with increased osteoclast development in ex-vivo bone marrow cell cultures and is reversed by estrogen replacement. Based on this evidence, it is hypothesized that the bone loss associated with estrogen withdrawal might be due, at least in part, to the removal of inhibitory effects of estrogens on the production and/or action of cytokines such as, IL-6 and TNF, which act in a paracrine fashion to regulate osteoclast formation. To advance this hypothesis, the molecular mechanism of the estrogen-induced inhibition of IL-6 production will be investigated with nuclear run-on assays, IL-6 promoter/CAT transfection experiments, and mRNA stability assays. Further, the interactions between estrogens (and their withdrawal) with PTH, 1,25(OH)2D3,IL-1, and TNF in the regulation of IL-6 production, as well as the effects of estrogens on the production of other bone relevant cytokines such as leukemia inhibitory factor (LIF) and macrophage-colony stimulating factor (M-CSF) will be investigated. In addition, the link between estrogen-regulated IL-6 production and osteoclast development in calvaria and bone marrow cultures will be explored. Finally, the link between estrogen deficiency, cytokine production, and bone loss in the ovariectomized mouse model, will be sought by examining whether in-vivo administration of neutralizing anti-IL-6 (and other anti-cytokine) antibodies can reverse the ovariectomy-induced bone loss. This project should provide novel insight into the pathophysiology of postmenopausal osteoporosis and perhaps suggest new therapeutic approaches for its management.
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