Sphingolipids as Novel Therapeutic Targets in Radiation Lung Injury

摘要 放射性肺损伤（RILI）是胸部放疗患者常见的并发症 与显著的发病率和死亡率相关，并且目前靶向治疗不能 存在.虽然分子病因学知之甚少，我们以前的特点是小鼠模型， RILI，其中肺屏障完整性的改变作为潜在的重要病理生物学事件浮出水面， 全基因组肺mRNA水平鉴定了鞘脂代谢途径基因的失调。我们 随后证实鞘脂参与小鼠RILI的辐射诱导的肺 鞘氨醇激酶（SphK）亚型1和2的表达，以及神经酰胺与 血浆、支气管肺泡灌洗液中的1-磷酸鞘氨醇（S1 P）和二氢-S1 P（DHS 1 P）水平，以及 肺组织此外，SphK 1靶向缺失（SphK 1-/-）或S1 P表达降低的小鼠， 受体（S1 PR 1 +/-、S1 PR 2-/-和S1 PR 3-/-）表现出明显的RILI易感性， 由S1 P类似物（S）-FTY 720-膦酸酯（Tys）赋予的保护。由于这些发现支持了 S1 P信号转导在RILI中的重要作用，我们假设参与RILI的特定分子机制， 鞘脂介导的对辐射的反应可以作为新的治疗靶点。这一假设服务于 作为目前提案的基础，该提案包括三个具体目标。一个潜在的 感兴趣是TRPM 2（瞬时受体电位melastatin-2），一种氧化剂敏感的非选择性阳离子 肺内皮细胞中表达的一种已知调节内皮细胞（EC）通透性的通道， 细胞对辐射损伤的反应由于我们的初步数据暗示TRPM 2作为双肺EC的介导剂， 通过S1 P和鼠RILI易感性的屏障调节，特异性目标#1将检查TRPM 2在 鞘脂介导的肺对放射的反应及其作为RILI治疗靶点的潜在可行性。 另外，我们还鉴定了由去泛素化酶介导的不同RILI反应， UCHL 1（泛素羧基末端酯酶L1），我们已经证实SphK 1泛素化受 UCHL 1以及TRPM 2对UCHL 1的调节。在具体目标#2中，我们将扩展这些发现，以充分定义 UCHL 1在体内和体外辐射影响鞘脂调节中的作用。最后，具体 目标#3我们将研究鞘脂调节的表观遗传事件对辐射的反应，并表征 在这种情况下，感兴趣的特定基因差异调节，一个全新的研究领域 我们的初步数据证实了辐射诱导的SphK磷酸化与组蛋白相关， 人肺EC乙酰化。总的来说，这些研究将产生新的见解， 鞘脂信号传导、鞘脂调节的介质和下游表观遗传修饰介导 我们的研究结果可能最终会更精确和有效地确定 RILI治疗策略。

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.