title: Regulation of phagocytosis and cytokine secretion by store-operated calcium entry in primary isolated murine microglia.
abstract: Microglia are immune effector cells in the central nervous system that participate in tissue repair, inflammatory responses, and neuronal degeneration. The most important signaling factor in the differentiation of immune-active cells after stimulation is the sustained high calcium concentration in the cytosol, which is called store-operated calcium entry (SOCE). Recently, the molecular identity of the store-operated channel (SOC) has revealed that Orai1, Orai2, Orai3, Stim1, and Stim2 constitute the most of SOC. In this study, we demonstrate that Orai1- and Stim1-mediated SOC regulated the phagocytic activity and cytokine release of primary isolated murine microglia. RT-PCR analysis revealed that primary cultured microglia from neonatal ICR mouse brains had Orai1, Orai2, Orai3, and Stim1. To elucidate the role of SOCE in the immune functions of microglia, pharmacological inhibitors or knockdown with Orai1 or Stim1 siRNA was applied, and UDP-induced phagocytic activity and LPS-induced cytokine secretion activity were compared. The pharmacological inhibition and siRNA effect was verified by measuring thapsigargin (TG)-, ATP-, or UDP-activated SOCE Ca2+ influx and proper siRNA-mediated knockdown was verified by western blot analysis. UDP-induced phagocytic activity was inhibited by pharmacological inhibitors of SOCE, such as SKF96365 or 2-APB, and knockdown of Orai1 and Stim1. Cytokine secretion of TNF-α and IL-6 by LPS treatment was also inhibited by SKF96365 and knockdown of Orai1 and Stim1. Meanwhile, LPS stimulation-induced NF-κB activation was not altered, but NFAT1 activity was attenuated with Stim1 knockdown. These results indicate that SOCE, which was composed of Orais and Stim1, regulates UDP-induced phagocytosis and LPS-stimulated cytokine secretion in microglia.
authors.affiliation: Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
authors.forename: Dae Keon
authors.initials: DK
authors.lastname: Heo
authors.affiliation: Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
authors.forename: Hye Min
authors.initials: HM
authors.lastname: Lim
authors.affiliation: Department of Physiology, Dongguk University College of Medicine, Gyeongju 780-714, Republic of Korea; Channelopathy Research Center, Dongguk University College of Medicine, Goyang 410-773, Republic of Korea.
authors.forename: Joo Hyun
authors.initials: JH
authors.lastname: Nam
authors.affiliation: Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.
authors.forename: Min Goo
authors.initials: MG
authors.lastname: Lee
authors.affiliation: Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea. Electronic address: jooyoungkim@yuhs.ac.
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Kim
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism; D000071740:ORAI1 Protein; D010587:Phagocytosis / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014530:Uridine Diphosphate / Q000494:pharmacology
chemical_list: D015220:Calcium Channels; D016207:Cytokines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; D034741:RNA, Small Interfering; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; D014530:Uridine Diphosphate; D019284:Thapsigargin; D014357:Trypsin; D002118:Calcium
keywords: Cytokine secretion; Microglia; Orai1 (CRACM1 or TMEM142A); Phagocytosis; Stim1; Store-operated calcium entry (SOCE)