Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, Sapporo, Japan.
Department of Cell Science, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Tissue Barriers. 2023 Jul 3;11(3):2106113. doi: 10.1080/21688370.2022.2106113. Epub 2022 Jul 26.
Lipolysis-stimulated lipoprotein receptor (LSR), a lipid metabolism-related factor localized in tricellular tight junctions (tTJs), plays an important role in maintaining the epithelial barrier. LSR is highly expressed in well-differentiated endometrial endometrioid carcinoma (EEC), and its expression decreases during malignancy. Angubindin-1, a novel LSR ligand peptide, regulates tTJs without cytotoxicity, enhances paracellular permeability, and regulates epithelial barrier via c-Jun N-terminal kinase (JNK)/cofilin. In this study, we investigated the immune-modulatory roles of an anti-LSR antibody in the treatment of EEC in vitro compared to those of angubindin-1. We prepared an antibody against the extracellular N-terminal domain of human LSR (LSR-N-ab) and angubindin-1. EEC cell-line Sawano cells in 2D and 2.5D cultures were treated with 100 μg/ml LSR-N-ab or 2.5 μg/ml angubindin-1 with or without protein tyrosine kinase 2β inhibitor PF431396 (PF43) and JNK inhibitor SP600125 (SP60) at 10 μM. Treatment with LSR-N-ab and angubindin-1 decreased LSR at the membranes of tTJs and the activity of phosphorylated LSR and phosphorylated cofilin in 2D culture. Treatment with LSR-N-ab and angubindin-1 decreased the epithelial barrier measured as TEER values in 2D culture and enhanced the epithelial permeability of FD-4 in 2.5D culture. Treatment with LSR-N-ab, but not angubindin-1, induced apoptosis in 2D culture. Pretreatment with PF43 and SP60 prevented all the changes induced by treatment with LSR-N-ab and angubindin-1. Treatment with LSR-N-ab and angubindin-1 enhanced the cell metabolism measured as the mitochondrial respiration levels in 2D culture. LSR-N-ab and angubindin-1 may be useful for therapy of human EEC via enhanced apoptosis or drug absorption.
脂解刺激脂蛋白受体（LSR）是一种位于三细胞紧密连接（tTJs）中的脂质代谢相关因子，在维持上皮屏障中发挥重要作用。LSR 在分化良好的子宫内膜样癌（EEC）中高度表达，其表达在恶性肿瘤过程中降低。一种新型的 LSR 配体肽 Angubindin-1 调节 tTJs 而没有细胞毒性，增强细胞旁通透性，并通过 c-Jun N 末端激酶（JNK）/丝切蛋白调节上皮屏障。在这项研究中，我们研究了抗 LSR 抗体在体外治疗 EEC 中的免疫调节作用，并与 Angubindin-1 进行了比较。我们制备了针对人 LSR 胞外 N 端结构域的抗体（LSR-N-ab）和 Angubindin-1。在 2D 和 2.5D 培养的 Sawano 细胞系中，用 100μg/ml LSR-N-ab 或 2.5μg/ml Angubindin-1 处理，同时用蛋白酪氨酸激酶 2β抑制剂 PF431396（PF43）和 JNK 抑制剂 SP600125（SP60）处理，浓度均为 10μM。在 2D 培养中，LSR-N-ab 和 Angubindin-1 处理降低了 tTJ 膜上的 LSR 和磷酸化 LSR 及磷酸化丝切蛋白的活性。LSR-N-ab 和 Angubindin-1 处理降低了 2D 培养中上皮屏障的电阻值（TEER 值），并增强了 2.5D 培养中 FD-4 的上皮通透性。LSR-N-ab 处理诱导 2D 培养中的细胞凋亡，但 Angubindin-1 处理则无此作用。用 PF43 和 SP60 预处理可预防 LSR-N-ab 和 Angubindin-1 处理引起的所有变化。LSR-N-ab 和 Angubindin-1 处理增强了 2D 培养中细胞代谢的线粒体呼吸水平。LSR-N-ab 和 Angubindin-1 可能通过增强细胞凋亡或药物吸收而有助于人类 EEC 的治疗。
