title: STIMs and Orai1 regulate cytokine production in spinal astrocytes.
abstract: 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.
authors.affiliation: 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.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Gao
authors.affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Xia
authors.affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
authors.forename: Frances M
authors.initials: FM
authors.lastname: Munoz
authors.affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
authors.forename: Melissa T
authors.initials: MT
authors.lastname: Manners
authors.affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Pan
authors.affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA.
authors.forename: Olimpia
authors.initials: O
authors.lastname: Meucci
authors.affiliation: Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Dai
authors.affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, 19102, USA. hhu@drexelmed.edu.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Hu
mesh_terms: D000813:Anilides / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D005260:Female; D051379:Mice; D000071740:ORAI1 Protein / Q000037:antagonists & inhibitors / Q000502:physiology*; D011247:Pregnancy; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism*; D000071737:Stromal Interaction Molecule 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D000071738:Stromal Interaction Molecule 2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D013830:Thiadiazoles / Q000494:pharmacology
chemical_list: C476308:4-methyl-4'-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)-1,2,3-thiadiazole-5-carboxanilide; D000813:Anilides; D016207:Cytokines; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; C100757:Stim1 protein, mouse; C525024:Stim2 protein, mouse; D000071737:Stromal Interaction Molecule 1; D000071738:Stromal Interaction Molecule 2; D013830:Thiadiazoles
keywords: Astrocytes; Cytokine; Orai1; STIM1; Store-operated calcium channels; The spinal cord