Molecular and Systems Pharmacology PhD Program, Emory University, Atlanta, Georgia, USA.
Immunology and Molecular Pathogenesis PhD Program, Emory University, Atlanta, Georgia, USA.
Physiol Rep. 2021 Sep;9(17):e15023. doi: 10.14814/phy2.15023.
Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508-CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib-1, which also decreased CFTR function. The SMase-mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5' adenosine monophosphate-activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non-CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508-CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid-SMase protein than nHBEs. Lastly, we determined that airway-like neutrophils, which are increased in the CF lung, secrete acid-SMase. Identifying the mechanism of SMase-mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid-SMase from cell types relevant to CF.
囊性纤维化跨膜电导调节因子（CFTR）功能丧失导致囊性纤维化（CF）。在肺部，这表现为免疫细胞浸润和细菌感染，导致组织破坏。以前的工作已经确定，急性细菌鞘氨醇酶（SMase）会降低无 CF 个体（nHBE）和 CF 个体（cfHBE，纯合 ΔF508-CFTR 突变）的支气管上皮细胞中的 CFTR 功能。本研究重点探讨了这种效应的机制。SMase 增加了二氢神经酰胺的丰度，使用 ceranib-1 阻断神经酰胺酶可以模拟这种结果，这也降低了 CFTR 功能。SMase 介导的抑制机制不涉及细胞 CFTR 丰度的减少或 CFTR 从顶端表面的去除，也不涉及 5' 腺苷单磷酸激活蛋白激酶的激活。为了确定这些神经鞘脂失衡的病理相关性，我们评估了 cfHBE 和 cfHNEs（鼻）与非 CF 对照相比的神经鞘脂谱。CF 细胞中的神经鞘磷脂、神经酰胺和二氢神经酰胺大量增加。用 VX445+VX661 校正 ΔF508-CFTR 转运减少了一些神经鞘磷脂和所有神经酰胺，但加剧了二氢神经酰胺的增加。用 CFTR 增强剂 VX770 进一步处理并没有影响这些变化，这表明错误折叠的 CFTR 得到了充分的拯救。我们还发现 cfHBE 表达的酸性-SMase 蛋白比 nHBE 多。最后，我们确定气道样中性粒细胞在 CF 肺中增加，分泌酸性-SMase。鉴于 CF 细胞中神经鞘脂的失衡以及与 CF 相关的细胞类型中酸性-SMase 的分泌，确定 SMase 介导的 CFTR 抑制机制将非常重要。
