Erythropoietin (EPO) is the hormone that stimulates erythropoiesis via activation of its receptor (EPOR) present in erythroid progenitor cells. In adults, more than 90% of EPO is produced in the kidney by a subset of peritubular interstitial fibroblasts. The key regulatory mechanism of EPO expression is hypoxia via activation of the Hypoxia-inducible factor (HIF) pathway. Anemia stimulates EPO production by inducing local hypoxia. Both EPO and EPOR are also expressed in a variety of non-hematopoietic tissues, which implies that EPO may have functions that go beyond regulation of erythropoiesis. For example, a role for EPO in the control of bone mass has been proposed, though it is still highly controversial whether EPO does, indeed, modulate bone development and/or homeostasis, and whether these putative EPO effects on bone are due, at least in part, to activation of EPOR in cells of the osteoblast lineage. We recently showed: 1) cells of the osteoblast lineage can produce and secrete functional EPO; 2) increased activity of HIF-2 in osteoblastic cells stimulates expression of osteoblastic EPO and causes polycythemia; 3) inactivation of HIF-2 in the same cells reduces expression of osteoblastic EPO and decreases number of bone marrow erythroid progenitors. Taken together, these findings indicate that osteoblastic EPO is likely to be physiologically relevant. Along these lines, we also provided evidence that EPOR is expressed in osteoblastic cells (preliminary data). In light of all these novel findings, we are no proposing to explore the physiologic roles of osteoblastic EPO and osteoblastic EPOR. For this purpose, in AIM I we will analyze bones lacking EPO in cells of the osteoblast lineage. In addition, in the same AIM, we will determine whether levels of expression of osteoblastic EPO can be modulated by acute anemia, in order to establish whether anemia is a physiological stimulus for EPO expression in osteoblastic cells. In AIM II, we will study bones lacking EPOR in cells of the osteoblast lineage. Mutant mice lacking EPOR in bone will also be treated with pharmacological doses of exogenous EPO in order to determine whether the actions of systemic EPO on bone are mediated, at least in part, by osteoblastic EPOR. The successful accomplishment of the proposed experiments could lead to the identification of osteoblastic EPO and/or osteoblastic EPOR as novel regulators of bone development and/or homeostasis.