舞蹈病-棘红细胞增多症患者成纤维细胞中钙库操纵性钙内流的锂敏感性及细胞存活情况

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

作者信息

Pelzl Lisann, Elsir Bhaeldin, Sahu Itishri, Bissinger Rosi, Singh Yogesh, Sukkar Basma, Honisch Sabina, Schoels Ludger, Jemaà Mohamed, Lang Elisabeth, Storch Alexander, Hermann Andreas, Stournaras Christos, Lang Florian

机构信息

Department of Medicine III, Tuebingen, Germany.

Department of Neurology and Hertie Institute for Clinical Brain Research, Tuebingen, Germany.

出版信息

Cell Physiol Biochem. 2017;42(5):2066-2077. doi: 10.1159/000479901. Epub 2017 Aug 11.

DOI:10.1159/000479901

PMID:28803243

Abstract

BACKGROUND

The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment.

METHODS

Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry.

RESULTS

SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB.

CONCLUSIONS

In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

摘要

背景

广泛表达的蛋白质 chorein 促进磷酸肌醇 3 激酶（PI3K）途径的激活，从而支持细胞存活。编码 chorein 的基因 VPS13A（液泡蛋白分选相关蛋白 13A）的功能丧失突变会导致舞蹈病 - 棘红细胞增多症（ChAc），这是一种伴有红细胞变形的神经退行性疾病。在小鼠中，chorein 的基因敲除会导致神经元凋亡增加。PI3K 依赖性信号上调 Orai1，Orai1 是一种形成孔道的通道蛋白，可实现储存性钙内流（SOCE）。已表明 Orai1 表达增加和 SOCE 可赋予肿瘤细胞存活能力。锂可上调 SOCE。本研究探讨了 ChAc 成纤维细胞中 SOCE 和/或凋亡是否发生改变，以及是否可通过锂处理进行调节。

方法

从 ChAc 患者和年龄匹配的健康志愿者中分离出成纤维细胞。通过 Fura - 2 荧光估计细胞质钙活性（[Ca2 +]i），在用肌浆内质网钙 - ATP 酶（SERCA）抑制剂毒胡萝卜素（1 μM）耗尽钙储存后重新添加钙后，通过[Ca2 +]i 的增加来估计 SOCE，并通过流式细胞术中 Annexin - V/碘化丙啶染色定量凋亡。

结果

ChAc 成纤维细胞中的 SOCE 明显小于对照成纤维细胞。锂（2 mM，24 小时）显著增加 SOCE，而 Orai1 阻滞剂 2 - 氨基乙氧基二苯硼酸盐（2 - APB，50 μM，24 小时）显著降低 SOCE。ChAc 成纤维细胞中的 Annexin - V 结合和碘化丙啶染色明显高于对照成纤维细胞。在 ChAc 成纤维细胞中，锂处理可显著降低 Annexin - V 结合和碘化丙啶染色，2 - APB 可显著增加，并且在存在 2 - APB 的情况下锂几乎无作用。