Yang Won Seok, Moon Soo Young, Lee Mee Jeong, Lee Eun Kyoung, Park Su-Kil
Division of Nephrology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
Asan Institute for Life Sciences, Seoul, Republic of Korea.
Cell Physiol Biochem. 2017;43(6):2434-2445. doi: 10.1159/000484396. Epub 2017 Oct 27.
BACKGROUND/AIMS: We investigated how diosgenin, a steroidal sapogenin, has anti-tumor necrosis factor-α (TNF-α) effects in human aortic endothelial cells (HAECs).
Tumor necrosis factor receptor 1 (TNFR1) was assessed by Western blot analysis. Intracellular Ca2+ was measured using Fluo-4 AM. Immunofluorescence staining was performed for a disintegrin and metalloprotease 10 (ADAM10).
Diosgenin (1 ∼ 100 nM) induced ectodomain shedding of TNFR1 within 30 min and attenuated TNF-α-induced intercellular adhesion molecule-1 (ICAM-1) expression. Upon treatment with diosgenin, extracellular Ca2+ entered into the cells via L-type calcium channels, whereas diosgenin-induced ectodomain shedding of TNFR1 was almost completely inhibited by BAPTA-AM (intracellular Ca2+ chelator), verapamil (L-type calcium channel antagonist) and the absence of extracellular Ca2+. Diosgenin caused translocation of ADAM10 to the cell surface, which was mediated by extracellular Ca2+ influx. Depletion of ADAM10 prevented diosgenin-induced ectodomain shedding of TNFR1 and abolished the inhibitory effect of diosgenin on TNF-α-induced ICAM-1 expression. Diosgenin did not induce extracellular Ca2+ influx and ectodomain shedding of TNFR1 in cells depleted of 1,25D3-membrane associated rapid response steroid-binding receptor (1,25D3-MARRS receptor/ERp57).
Diosgenin elicits L-type calcium channel-mediated extracellular Ca2+ influx, and thereby induces ADAM10-mediated ectodomain shedding of TNFR1. This effect of diosgenin was exerted through 1,25D3-MARRS receptor/ERp57.
背景/目的：我们研究了甾体皂苷元薯蓣皂苷元如何在人主动脉内皮细胞（HAECs）中发挥抗肿瘤坏死因子-α（TNF-α）的作用。
通过蛋白质免疫印迹分析评估肿瘤坏死因子受体1（TNFR1）。使用Fluo-4 AM测量细胞内Ca2+。对去整合素和金属蛋白酶10（ADAM10）进行免疫荧光染色。
薯蓣皂苷元（1～100 nM）在30分钟内诱导TNFR1的胞外域脱落，并减弱TNF-α诱导的细胞间黏附分子-1（ICAM-1）表达。用薯蓣皂苷元处理后，细胞外Ca2+通过L型钙通道进入细胞，而BAPTA-AM（细胞内Ca2+螯合剂）、维拉帕米（L型钙通道拮抗剂）和细胞外Ca2+缺失几乎完全抑制了薯蓣皂苷元诱导的TNFR1胞外域脱落。薯蓣皂苷元导致ADAM10转位至细胞表面，这是由细胞外Ca2+内流介导的。ADAM10的缺失阻止了薯蓣皂苷元诱导的TNFR1胞外域脱落，并消除了薯蓣皂苷元对TNF-α诱导的ICAM-1表达的抑制作用。在缺乏1,25二羟维生素D3膜相关快速反应类固醇结合受体（1,25D3-MARRS受体/内质网蛋白57）的细胞中，薯蓣皂苷元未诱导细胞外Ca2+内流和TNFR1的胞外域脱落。
薯蓣皂苷元引发L型钙通道介导的细胞外Ca2+内流，从而诱导ADAM10介导的TNFR1胞外域脱落。薯蓣皂苷元的这种作用是通过1,25D3-MARRS受体/内质网蛋白57发挥的。
