PROJECT SUMMARY/ABSTRACT Osteopontin (OPN) is involved in the pathogenesis of a wide variety of inflammatory disorders, including infections, autoimmune diseases, cancer, and other diseases accompanied with inflammation. OPN is primarily considered to be a pro-inflammatory molecule and is generally known to induce immune responses. However, recent studies suggested that OPN occasionally functions as an anti-inflammatory molecule, and how OPN switches its role is entirely elusive. It is critical to know how OPN plays the two-sided role, particularly because OPN attracts attentions as a pharmaceutical target and a disease marker in various diseases. To this end, this proposed study seeks to understand the molecular mechanism by which OPN switches immune responses, and will verify the mechanistic hypothesis with in vivo animal disease models using five lines of OPN mutant mice. Our central hypothesis is: Intracellular OPN (iOPN) is a major player to make OPN’s role switch between pro- inflammatory and anti-inflammatory. Here, OPN has two OPN isoforms, secreted (sOPN) and intracellular (iOPN). In the past, we first succeeded to biochemically confirm the presence of iOPN, and revealed the mechanism of iOPN’s generation and its functions. As a pioneer of the iOPN field, we have been continuously discovering new roles of iOPN, as well as those of sOPN. To study OPN in an isoform-specific fashion, it is important to have cells and mouse lines that exclusively express one of two isoforms. We are currently equipped not only with iOPN and sOPN expression vectors, but we will use multiple lines of OPN mutant mice, which make it possible for us to evaluate the isoform-specific role of OPN in in vivo disease models. The objective of this proposed study is to determine how iOPN functions as an anti-inflammatory molecule; and the long-term goal of this project is to elucidate the roles of iOPN and sOPN in inflammation.