(Verbatim from the Applicant): This application focuses on two novel autocrine/paracrine factors, which we have identified, that are produced by both murine and human OCLs: AXII, a stimulator of OCL formation, and OIP-2, an inhibitor of OCL formation. It is our hypothesis that these factors play a critical role in the physiologic regulation of OCL formation. Thus, understanding their mechanism of action will provide important insights into the control of osteoclastogenesis. To determine how these factors regulate OCL formation, we will pursue the following Specific Aims: (la) Determine the mechanism of action of AXII on OCI formation. AXII stimulates OCL formation through its capacity to induce production of high local levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) by CD4+ T-cells and marrow stromal cells. We will confirm that AXII can induce CFU-GM colony growth and test if AXII also acts directly on OCL precursors. In addition, we will assess the effects of AXII on RANK ligand and osteoprotegerin (OPG) expression. Since both T-cells and marrow stromal cells bind AXII and secrete GM-CSF, we will determine the relative contribution of T-cells by assessing OCL formation in marrow cultures depleted of total T-cells or CD4+ T-cells or adding CD4+ 1-cells to highly purified human OCL precursors treated with AXII. (lb) Identify and clone the AXII receptor we recently identified on marrow stromal cells. The receptor will be characterized in terms of its binding affinity and specificity for AXII and then will be cloned from a human marrow stromal cell library. (2a) Determine the mechanism responsible for the inhibitory action of OIP-2 on OCL formation. OIP-2 is identical to the enzyme legumain and appears to act directly on OCL precursors to inhibit OCL formation. We will confirm that OIP-2 inhibits OCL formation by highly purified OCL precursors, and test if OIP-2 affects RANKL, VDR, RANK or OPG expression in normal marrow cultures. (2b) Map the domains of OIP-2 responsible for its OCL inhibitory activity. We will confirm that the C-terminal domain of OIP-2 mediates its inhibitory effects on OCLs, assess if enzyme activity is required for OIP-2 activity, confirm that OCL precursors bind OIP-2, and clone and characterize the OIP-2 receptor.
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