PROJECT SUMMARY/ABSTRACT This R21 proposal describes a two-year research plan that will facilitate research program dedicated to determining the role of macropinocytosis in systemic sclerosis (SSc, scleroderma) and SSc-associated interstitial lung disease (ILD). SSc is an autoimmune disorder, which is manifested by skin fibrosis, vasculopathy and the fibrosis of internal organs. The prevalence of SSc is estimated to range from 50 to 300 per million across the world, with one of the highest prevalence rates observed in the United States at 240 per million. Because of its clinical heterogeneity, SSc is a challenging disease to manage which results in the highest disease mortality among the rheumatologic diseases. ILD is the most common lung complication of SSc and is associated with increased mortality. ILD occurs in up to 80% of patients with SSc, with 25–30% developing a severe progressive form of SSc-ILD leading to eventual respiratory failure and death. Little is known about the molecular mechanisms involved in the pathogenesis of SSc and SSc-ILD. This project will focus on elucidating the key roles of macropinocytosis in skin and lung fibrogenesis, as well as its therapeutic targeting. Macropinocytosis is an actin-dependent but clathrin-independent endocytic process that mediates the nonselective internalization of extracellular contents, such as proteins, cell debris, or viruses. Macropinocytosis has been shown to be involved in cell survival, migration and invasion by providing nutrients (e.g., free amino acids and polypeptides) from extracellular environment. However, its role in scleroderma and organ fibrogenesis is unknown. Our preliminary findings suggest that inhibition of macropinocytosis attenuates dermal myofibroblast differentiation and pulmonary fibrosis in mice. Furthermore, we found that vacuolar protein sorting 34 (Vps34), a sole member of class III phosphoinositide-3-kinase (PI3K), is involved in macropinocytosis and fibroblast activation. Based on published and our preliminary findings, we hypothesize that increased macropinocytosis promotes the development of dermal and lung fibrosis in SSc. Thus, its inhibition exerts anti-fibrotic effects in the animal model of skin fibrosis by reducing profibrotic responses in activated fibroblasts. We also hypothesize that Vps34 is essential for macropinocytosis, which in turn confers to fibroblast activation. We will test our hypotheses by addressing the following Specific Aims: Specific Aim #1: To demonstrate that macropinocytosis inhibition attenuates dermal and lung fibrosis in mice by inhibiting fibroblast to myofibroblast differentiation, ECM production, and migration. Specific Aim #2: To demonstrate that Vps34 is a key protein in macropinosome formation and contributes to profibrotic responses of dermal and lung fibroblasts.