Gao Xinghua, Xia Jingsheng, Munoz Frances M, Manners Melissa T, Pan Rong, Meucci Olimpia, Dai Yue, Hu Huijuan
Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
J Neuroinflammation. 2016 May 31;13(1):126. doi: 10.1186/s12974-016-0594-7.
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.
我们之前的研究表明，一种储存式钙通道（SOCC）抑制剂（YM-58483）具有中枢镇痛作用。然而，这种作用的细胞和分子机制仍有待确定。众所周知，胶质细胞在中枢敏化中起重要作用。SOCC内流（SOCE）已被证明在包括皮质星形胶质细胞在内的多种细胞类型中发挥作用。然而，SOCC家族在星形胶质细胞功能中的作用尚未确定。在此，我们全面研究了SOCC在脊髓星形胶质细胞中的表达及其功能意义。
从新生（P2-P3）CD1小鼠制备原代培养的星形胶质细胞。分别通过实时PCR和蛋白质印迹分析评估mRNA和蛋白质的表达。使用钙成像系统测量SOCE。使用共聚焦显微镜检测活细胞中STIM1的转位。通过酶联免疫吸附测定法测量细胞因子水平。
我们发现SOCC家族在脊髓星形胶质细胞中表达，用环匹阿尼酸（CPA）从内质网耗尽钙储存会导致大量持续的钙内流，这被SOCC抑制剂阻断。使用小干扰RNA敲低方法，我们确定STIM1和Orai1是脊髓星形胶质细胞中SOCC的主要成分。我们还观察到毒胡萝卜素（TG）或CPA诱导的STIM1和Orai1的点状形成。此外，SOCC的激活显著促进脊髓星形胶质细胞中TNF-α和IL-6的产生，而STIM1或Orai1的敲低则大大减弱了这种作用。重要的是，STIM2和Orai1的敲低显著降低了脂多糖诱导的TNF-α和IL-6的产生，而不改变细胞活力。
本研究首次证明STIM1、STIM2和Orai1介导SOCE并参与脊髓星形胶质细胞中的细胞因子产生。我们的发现为未来评估SOCC在疼痛和其他与星形胶质细胞活动异常相关的中枢神经系统疾病中的作用提供了基础。
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