Project Summary/Abstract Osteoclasts (OCs), the bone resorbing cells, arise from myeloid OC progenitors (OCPs) and are critical for bone remodeling and homeostasis. OC differentiation and activity are tightly regulated by intrinsic negative feedback loops (autoregulatory) and by paracrine factors secreted by other cells, most notably osteoprotegerin. Intrinsic regulators are critically important for calibrating physiologic OCgenesis, disruption of which leads to uncontrolled pathologic OCgenesis and osteolysis. Using proteomic studies in an independent study, we have recently identified a novel autoregulatory role of IFN stimulated gene-15 (ISG15), a ubiquitin-like small molecule, in OCgenesis. Specifically, we found that RANKL induces expression of ISG15 in OCPs and OCs, which binds to NEMO (a.k.a. IKKg) to down regulates NF-kB signaling. We found that stimulation of OCPs with RANKL induces IFNa/b secretion by OCPs, which engages and activates type 1 IFN receptor (IFNAR) signaling in OCPs themselves to trigger ISG15-dependent local autoregulatory negative feedback loop to limit the extent of OCgenesis. This mechanism appears to depend on STimulator of INterferon Genes (STING), which we find to be expressed in response to RANKL and is essential for IFNa/b, and ISG15 expression, and was validated by data showing that blocking either STING or IFNa/b diminishes RANKL-induced ISG15 levels and exacerbates OCgenesis. Collectively, these observations suggest that endogenous ISG15 inhibits OCgenesis through its classical binding to target proteins, which is facilitated by the sequential action of highly specific E1 (Ube1L), E2 (UbcH8), E3 (Herc6) ligases, a post-translational modification termed ISGylation that maintains cellular homeostasis. However, ISGylation is a reversible mechanism, whereby under inflammatory conditions, free ISG15 is generated in abundance by cells, secreted to the extracellular (EC) space by secretory vesicles, and acts as a cytokine by binding LFA1 receptor. In this regard, we show that inflammatory stimulation of OCPs with the bacterial product LPS inhibits expression of Ube1L, the E1 enzyme, and induces secretion of ample ECISG15. More surprisingly, we found that ISG15KO mice responded poorly to LPS and developed negligible osteolysis compared to robust bone loss by LPS-induced WT counterparts. This event was TNF-dependent, evident by low levels of TNFa in ISG15KO serum compared to copious amounts of TNF in serum of WT mice. Hence, ECISG15 appears to exacerbate OCgenesis and depends on intact endogenous ISG15. Based on these observations, our overarching hypothesis states that ISG15 has bi-modal functions: under physiologic conditions, ISG15 is conjugated to OC signaling proteins to limit OCgenesis and maintain homeostasis, whereas inflammatory conditions facilitate secretion of free ECISG15, which then acts as an inflammatory cytokine to exacerbate OCgenesis and osteolysis. To test this hypothesis, we will: (1) Elucidate the...