title: Regulatory role of cytosolic phospholipase A2alpha in the induction of CD40 in microglia.
abstract: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction.
  
  
  
  Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction.
  
  
  
  Addition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation.
  
  
  
  Our results show for the first time that cPLA2upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS.
authors.affiliation: Infectious Diseases Laboratory, Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel.
authors.forename: Yafa Fetfet
authors.initials: YF
authors.lastname: Malada-Edelstein
authors.affiliation: Infectious Diseases Laboratory, Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel.
authors.forename: Nurit
authors.initials: N
authors.lastname: Hadad
authors.affiliation: Infectious Diseases Laboratory, Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Beer-Sheva, Israel. ral@bgu.ac.il.
authors.forename: Rachel
authors.initials: R
authors.lastname: Levy
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D019013:CD40 Antigens / Q000096:biosynthesis*; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D054513:Group IV Phospholipases A2 / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D019013:CD40 Antigens; D014409:Tumor Necrosis Factor-alpha; D054513:Group IV Phospholipases A2
keywords: CD40; Cytosolic phospholipase A2α; Interferon gamma; Lipopolysaccharide; Microglia; Nuclear factor-κB