New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine, 800 Washington St, Boston, MA, 02111, USA.
New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine, 800 Washington St, Boston, MA, 02111, USA; Program in Genetics, Molecular & Cellular Biology, Tufts Graduate School of Biomedical Sciences, Tufts University, 136 Harrison Ave, Boston, MA, USA; Program in Pharmacology & Drug Development, Tufts Graduate School of Biomedical Sciences, 136 Harrison Ave, Tufts University, Boston, MA, USA.
Exp Eye Res. 2024 Oct;247:110029. doi: 10.1016/j.exer.2024.110029. Epub 2024 Aug 8.
Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca. Store-operated Ca entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca release from internal sources. The inefficacy of inhibitors of ER Ca channels suggested that this compartment was not the source of the Ca surge caused by tBHP exposure. However, using a Ca-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca homeostasis, with inhibition of mitochondrial Ca release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.
钙稳态失调会引发一系列病理事件，导致组织损伤和细胞死亡。Dynasore 是一种小分子，通过靶向经典的动力蛋白来抑制内吞作用。在之前的一项研究中，我们表明 dynasore 可以通过恢复细胞内钙（Ca）稳态来保护人角膜上皮细胞免受叔丁基过氧化物（tBHP）暴露引起的损伤。在这里，我们报告了一项后续研究的结果，旨在确定损伤 Ca 的来源。储存操纵的 Ca 内流（SOCE）是一种从细胞外环境中恢复细胞内钙库的细胞机制。我们发现 dynasore 可有效阻断用 thapsigargin（TG）处理的细胞中的 SOCE，TG 是一种抑制 Ca 泵入内质网（ER）的小分子。然而，与 dynasore 不同，SOCE 抑制剂 YM-58483 并不干扰 tBHP 暴露引起的细胞溶质 Ca 过载。我们还发现 dynasore 可有效阻断内部来源的 Ca 释放。ER Ca 通道抑制剂无效表明该隔室不是 tBHP 暴露引起的 Ca 激增的来源。然而，使用靶向线粒体的 Ca 测量细胞器被困蛋白指示剂（CEPIA）报告器，我们发现 dynasore 可以阻断 tBHP 暴露引起的线粒体 Ca 释放。我们的结果表明 dynasore 对细胞内 Ca 稳态有多种影响，抑制线粒体 Ca 释放在保护角膜上皮细胞免受 tBHP 暴露引起的氧化应激方面发挥关键作用。
