title: All-trans retinoic acid induces COX-2 and prostaglandin E2 synthesis in SH-SY5Y human neuroblastoma cells: involvement of retinoic acid receptors and extracellular-regulated kinase 1/2.
abstract: BACKGROUND
  Our recent results show that all-trans retinoic acid (ATRA), an active metabolite of vitamin A, induces COX-dependent hyperalgesia and allodynia in rats. This effect was mediated by retinoic acid receptors (RARs) and was associated with increased COX-2 expression in the spinal cord. Since ATRA also up-regulated COX-2 expression in SH-SY5Y human neuroblastoma cells, the current study was undertaken to analyze in these cells the mechanism through which ATRA increases COX activity.
  
  
  METHODS
  Cultured SH-SY5Y neuroblastoma cells were treated with ATRA. COX expression and kinase activity were analyzed by western blot. Transcriptional mechanisms were analyzed by RT-PCR and promoter assays. Pharmacological inhibitors of kinase activity and pan-antagonists of RAR or RXR were used to assess the relevance of these signaling pathways. Production of prostaglandin E2 (PGE2) was quantified by enzyme immunoabsorbent assay. Statistical significance between individual groups was tested using the non-parametric unpaired Mann-Whitney U test.
  
  
  RESULTS
  ATRA induced a significant increase of COX-2 expression in a dose- and time-dependent manner in SH-SY5Y human neuroblastoma cells, while COX-1 expression remained unchanged. Morphological features of differentiation were not observed in ATRA-treated cells. Up-regulation of COX-2 protein expression was followed by increased production of PGE2. ATRA also up-regulated COX-2 mRNA expression and increased the activity of a human COX-2 promoter construct. We next explored the participation of RARs and mitogen-activated peptide kinases (MAPK). Pre-incubation of SH-SY5Y human neuroblastoma cells with either RAR-pan-antagonist LE540 or MAP kinase kinase 1 (MEK-1) inhibitor PD98059 resulted in the abolition of ATRA-induced COX-2 promoter activity, COX-2 protein expression and PGE2 production whereas the retinoid X receptor pan-antagonist HX531, the p38 MAPK inhibitor SB203580 or the c-Jun kinase inhibitor SP600125 did not have any effect. The increase in RAR-beta expression and extracellular-regulated kinase 1/2(ERK1/2) phosphorylation in ATRA-incubated cells suggested that RARs and ERK1/2 were in fact activated by ATRA in SH-SY5Y human neuroblastoma cells.
  
  
  CONCLUSIONS
  These results highlight the importance of RAR-dependent and kinase-dependent mechanisms for ATRA-induced COX-2 expression and activity.
authors.affiliation: Facultad de Medicina, Departamento de Fisiología, Campus Universitario, Universidad de Alcalá, Madrid, Spain. matilde.alique@uah.es <matilde.alique@uah.es>
authors.forename: Matilde
authors.initials: M
authors.lastname: Alique
authors.forename: Juan F
authors.initials: JF
authors.lastname: Herrero
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Lucio-Cazana
mesh_terms: D045744:Cell Line, Tumor; D051546:Cyclooxygenase 2 / Q000096:biosynthesis*; D015232:Dinoprostone / Q000096:biosynthesis* / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000502:physiology*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D018168:Receptors, Retinoic Acid / Q000819:agonists / Q000502:physiology*; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: D047428:Protein Kinase Inhibitors; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D015232:Dinoprostone