The production of interleukin-6 (IL-6) by bone and marrow stromal cells is inhibited by l7beta-estradiol; and estrogen loss (ovariectomy) causes an IL-6-mediated upregulation of osteoclastogenesis in mice. In addition, human IL-11, a newly discovered cytokine which seems to be produced exclusively by bone marrow stromal cells, exhibits osteoclastogenic and bone resorptive properties. Based on this evidence, the following hypothesis will be tested: Loss of estrogen in women, similar to the case with mice, unleashes the production and/or action of cytokines, such as IL-6, in the marrow microenvironment which are, in turn, responsible for an upregulation of hematopoietic progenitors of osteoclasts and increased osteoclast development. To do this, bone marrow aspirates will be obtained during and three months following ovariectomy from 30 premenopausal women and 20 women undergoing hysterectomy without ovariectomy. In addition, 50 ml of venous blood and 30 ml of urine will be obtained prior to the operation and at one, two, and three months after the operation. Using low density cells from the bone marrow aspirate, ex vivo cultures will be established and the formation of colony forming units for granulocyte-erythrocyte- megakaryocyte-monocyte (CFU-GEHM) and the colony forming units for granulocyte-monocyte (CFU-GM), as well as the production of interleukin-6 and interleukin-11 in supernatants and the formation of TRAPase-positive cells that bind 125I-calcitonin in the presence of either 1,25(OH)2D3, PTH, IL-1, IL-11, or the combination of l,25(OH)2D3 and IL-11 will be determined. In addition, the concentration of estradiol, calcium, phosphorus, osteocalcin, l,25(OH)2D3, PTH, and a complete blood count, as well as the concentration of creatinine, hydroxyproline, and pyridinium crosslinks will be measured in the blood and urine samples. Each of the cellular parameters determined in the bone marrow cultures will be examined for possible correlations with the estrogen status, and the markers of bone remodeling. In addition to the clinical protocol and as a secondary goal of the proposal, aliquots of the bone marrow cells from ovariectomized women will be cultured on smooth cortical bone slices and utilized for the systematic exploration of experimental conditions for the formation of bone excavating (pit forming) human osteoclasts in vitro. It is expected that these studies will advance knowledge regarding the mechanism of the osteopenia associated with estrogen loss from the experimental animal models to the human situation.
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