ABSTRACT Radiation-induced lung injury (RILI) is a common complication in patients administered thoracic radiotherapy that is associated with significant morbidity and mortality and for which targeted therapies currently do not exist. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We subsequently confirmed sphingolipid involvement in murine RILI by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Moreover, mice with a targeted deletion of SphK1 (SphK1-/-) or with reduced expression of S1P receptors (S1PR1+/-, S1PR2-/-, and S1PR3-/-) exhibited marked RILI susceptibility and we observed RILI protection conferred by the S1P analog, (S)-FTY720-phosphonate (Tys). As these findings support an important role for S1P signaling in RILI we hypothesize specific molecular mechanisms involved in sphingolipid-mediated responses to radiation may serve as novel therapeutic targets. This hypothesis serves as the basis for the current proposal which is comprised of three specific aims. One potential molecule of interest in is TRPM2 (transient receptor potential melastatin-2), an oxidant sensitive, non-selective, cation channel expressed in the lung endothelium that is known to regulate endothelial cell (EC) permeability and cellular responses to radiation injury. As our preliminary data implicate TRPM2 as a mediator of both lung EC barrier regulation by S1P and murine RILI susceptibility, Specific Aim #1 will examine the role of TRPM2 in sphingolipid-mediated lung responses to radiation and its potential viability as a therapeutic target in RILI. Separately, we have also identified differential RILI responses mediated by the deubiquitinating enzyme, UCHL1 (ubiquitin carboxyl-terminal esterase L1) and we have confirmed SphK1 ubiquitination regulated by UCHL1 as well UCHL1 regulation by TRPM2. In Specific Aim #2 we will extend these findings to fully define the role of UCHL1 in the regulation of sphingolipids affected by radiation in vivo and in vitro. Finally, in Specific Aim #3 we will study epigenetic events modulated by sphingolipids in response to radiation and characterize specific genes of interest differentially regulated in this context, an entirely novel area of investigation supported by our preliminary data confirming radiation-induced SphK phosphorylation associated with histone acetylation in human lung EC. Collectively, these studies will yield new insights into mechanisms of sphingolipid signaling, mediators of sphingolipid regulation and downstream epigenetic modifications mediated by sphingolipids in response to radiation and our findings may ultimately identify more precise and effective RILI therapeutic strategies.