Our central hypotheses are that estrogen (E) deficiency has both direct and indirect effects on bone, that these two effects cause bone loss by different mechanisms, and that most of the bone loss of aging women can be explained by both effects acting in concert. Postmenopausal women undergo two distinct phases of bone loss - a rapid, transient phase that begins at menopause (due to loss of the direct suppressive effects of E on bone cell function) and a slow, subsequent phase that continues indefinitely (due mainly to loss of E effects on peripheral calcium metabolism leading to secondary hyperparathyroidism (HPT) and, indirectly, to bone loss). Although much has been learned in recent years about the pathophysiology of bone loss in aging women, the most important remaining research need is to define the mechanisms by which these direct (E deficiency only) and indirect (E deficiency plus parathyroid hormone [PTH] excess) skeletal effects produce bone loss. Although there have been extensive studies using in vitro systems and experimental animals, there are very limited data in women. We will apply novel methodological approaches that will allow us to study these mechanism directly in humans. In Aim 1, we will determine unequivocally whether IL-1beta and TNFalpha are essential mediators of the increased bone resorption induced by acute E-withdrawal by intervening with the IL-1beta blocker, anakinra, and the TNFalpha blocker, etanercept. In Aims 2 and 3, we will apply a new method that we have developed to isolate osteoblast and osteoclast precursors from bone marrow, to study their differentiation and function, and to assess their content of mRNA for putative regulatory cytokines and cytokine receptors. These methods utilize 2-color flow cytometry/FACS, intensity of fluorescent probes, and real time RT-PCR. In Aim 2, we will determine if E inhibits differentiation of osteoclast lineage cells through a direct action and, if so, if this occurs by regulating c-Fms or RANK expression or by other mechanisms. Finally, in Aim 3, we will define the molecular mechanisms of the biphasic action of PTH on bone turnover (intermittent administration increases mainly formation whereas continuous increases mainly resorption) and how E modifies them. In addition to helping define the pathophysiology of the two phases of bone loss in postmenopausal women, these data will also help explain molecular mechanisms for the newly approved anabolic regimen of intermittent PTH(1-34) for treating osteoporosis.