STIMs and Orai1 regulate cytokine production in spinal astrocytes.
STIMs and Orai1 regulate cytokine production in spinal astrocytes.
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DOI：
10.1186/s12974-016-0594-7
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发表时间：
2016-05-31
影响因子：
9.3
通讯作者：
Hu H
中科院分区：
文献类型：
--
作者：
Gao X;Xia J;Munoz FM;Manners MT;Pan R;Meucci O;Dai Y;Hu H
我们先前的研究表明，钙通道阻滞剂(YM-58483)具有中枢镇痛作用。然而，这种效应的细胞和分子机制仍有待确定。众所周知，神经胶质细胞在中枢敏化中起着重要作用。SOC进入(SOCE)与多种细胞类型有关，包括大脑皮层星形胶质细胞。然而，SOCC家族在星形胶质细胞功能中的作用尚未确定。在此，我们深入研究了SOCCs在脊髓星形胶质细胞中的表达及其功能意义。原代培养的星形胶质细胞取新生(P2-P3)CD1小鼠。实时荧光定量聚合酶链式反应和蛋白质印迹分析分别检测mRNAs和蛋白质的表达。使用钙成像系统测量SOCE。用共聚焦显微镜检测活细胞STIM1易位。采用酶联免疫吸附试验检测细胞因子水平。我们发现SOCC家族在脊髓星形胶质细胞中有表达，并且环匹阿松酸(CPA)耗尽内质网中的钙储存导致大量持续的钙内流，而这种钙内流被SOCC抑制剂所阻断。利用siRNA敲除方法，我们确定STIM1和Orai1是脊髓星形胶质细胞中SOCCs的主要成分。我们还观察了thapsigargin(TG)或CPA诱导的STIM1和Orai1的点状形成。此外，SOCC的激活显著促进了脊髓星形胶质细胞产生肿瘤坏死因子-α和IL-6，而STIM1或Orai1基因被敲除后，这两种作用明显减弱。重要的是，STIM2和OraI1基因的敲除显著减少了脂多糖诱导的肿瘤坏死因子-α和IL-6的产生，而不会改变细胞的存活率。本研究首次提出了STIM1、STIM2和Orai1介导SOCE并参与脊髓星形胶质细胞细胞因子产生的证据。我们的发现为未来评估SOCC在疼痛和其他与星形胶质细胞异常活动相关的中枢神经系统疾病中的作用提供了基础。
Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes. Primary cultured astrocytes were prepared from neonatal (P2–P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay. We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability. This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.