Department of Medicine, Division of Pulmonary, Critical Care, Sleep and Allergy, University of Illinois at Chicago, Chicago, IL 60612, USA.
Cells. 2023 Oct 5;12(19):2405. doi: 10.3390/cells12192405.
GADD45a is a gene we previously reported as a mediator of responses to acute lung injury. GADD45a-/- mice express decreased Akt and increased Akt ubiquitination due to the reduced expression of UCHL1 (ubiquitin c-terminal hydrolase L1), a deubiquitinating enzyme, while GADD45a-/- mice have increased their susceptibility to radiation-induced lung injury (RILI). Separately, we have reported a role for sphingolipids in RILI, evidenced by the increased RILI susceptibility of SphK1-/- (sphingosine kinase 1) mice. A mechanistic link between UCHL1 and sphingolipid signaling in RILI is suggested by the known polyubiquitination of SphK1. Thus, we hypothesized that the regulation of SphK1 ubiquitination by UCHL1 mediates RILI. Initially, human lung endothelial cells (EC) subjected to radiation demonstrated a significant upregulation of UCHL1 and SphK1. The ubiquitination of EC SphK1 after radiation was confirmed via the immunoprecipitation of SphK1 and Western blotting for ubiquitin. Further, EC transfected with siRNA specifically for UCHL1 or pretreated with LDN-5744, as a UCHL1 inhibitor, prior to radiation were noted to have decreased ubiquitinated SphK1 in both conditions. Further, the inhibition of UCHL1 attenuated sphingolipid-mediated EC barrier enhancement was measured by transendothelial electrical resistance. Finally, LDN pretreatment significantly augmented murine RILI severity. Our data support the fact that the regulation of SphK1 expression after radiation is mediated by UCHL1. The modulation of UCHL1 affecting sphingolipid signaling may represent a novel RILI therapeutic strategy.
GADD45a 是我们之前报道的一种介导急性肺损伤反应的基因。由于去泛素化酶 UCHL1（泛素 C 端水解酶 L1）的表达减少，GADD45a-/- 小鼠的 Akt 表达降低，Akt 泛素化增加，而 GADD45a-/- 小鼠对辐射诱导的肺损伤（RILI）的易感性增加。另外，我们已经报道了鞘脂在 RILI 中的作用，这一点可以从 SphK1-/-（鞘氨醇激酶 1）小鼠对 RILI 的易感性增加得到证明。UCHL1 和 SphK1 在 RILI 中的鞘脂信号之间的机制联系是通过 SphK1 的已知多泛素化来提示的。因此，我们假设 UCHL1 对 SphK1 泛素化的调节介导了 RILI。最初，受到辐射的人肺内皮细胞（EC）表现出 UCHL1 和 SphK1 的显著上调。通过免疫沉淀 SphK1 和 Western blot 检测泛素，证实了 EC SphK1 在辐射后的泛素化。此外，在辐射前用针对 UCHL1 的 siRNA 转染 EC 或用 LDN-5744（UCHL1 抑制剂）预处理的 EC，在两种情况下，SphK1 的泛素化都减少了。进一步，UCHL1 的抑制作用减弱了 SphK1 介导的内皮细胞屏障增强作用，通过跨内皮电阻来测量。最后，LDN 预处理显著增加了小鼠 RILI 的严重程度。我们的数据支持这样一个事实，即辐射后 SphK1 的表达受到 UCHL1 的调节。UCHL1 对 SphK1 表达的调节可能代表了一种新的 RILI 治疗策略。
