Department of Signal Transduction, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
Department of Signal Transduction, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
J Lipid Res. 2021;62:100121. doi: 10.1016/j.jlr.2021.100121. Epub 2021 Sep 22.
Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB, 6t-LTB, LTC, LTB, and 6t-LTB. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.
白细胞三烯 (LTs) 和鞘脂是参与多种细胞信号转导事件和多种疾病发展的关键脂质介质，如导致哮喘的支气管高反应性。这些脂质介质产生的酶促反应涉及 5-脂氧合酶 (5-LO) 介导的花生四烯酸转化为 LTs 和丝氨酸棕榈酰转移酶 (SPT) 介导的鞘脂从头合成。先前的研究表明内质网膜蛋白 ORM1 样蛋白 3 (ORMDL3) 抑制 SPT 的活性和随后的鞘脂合成。然而，ORMDL3 在 LTs 合成中的作用尚不清楚。在这项研究中，我们使用从 ORMDL3 KO 或对照小鼠分离的腹腔衍生肥大细胞，检查了它们的钙动员、脱颗粒、NF-κB 抑制剂-α磷酸化和 TNF-α 产生。我们发现，与 WT 细胞相比，ORMDL3 KO 的腹腔衍生肥大细胞对抗原的反应性增加。详细的脂质分析表明，与 WT 细胞相比，ORMDL3 缺失细胞不仅产生了更多的鞘脂，而且还产生了 LT 信号介质 LTB、6t-LTB、LTC、LTB 和 6t-LTB。ORMDL3 和 5-LO 代谢途径之间的串扰得到了支持，发现内源性 ORMDL3 和 5-LO 定位于人类肥大细胞中相似的内质网域，并且 ORMDL3 与 5-LO 物理相互作用。进一步的实验表明，5-LO 还与 SPT 的长链 1 和长链 2 亚基相互作用。与这些发现一致，5-LO 敲低增加了神经酰胺水平，而 SPTLC1 的沉默降低了花生四烯酸代谢为 LTs 的水平，达到 5-LO 敲低的水平。这些结果表明 LT 和鞘脂代谢途径之间存在功能串扰，导致脂质信号介质的产生。
