title: Triamcinolone acetonide destabilizes VEGF mRNA in Müller cells under continuous cobalt stimulation.
abstract: OBJECTIVE
  To identify the molecular mechanism of steroid-induced downregulation of vascular endothelial growth factor (VEGF) synthesis in Müller cells.
  
  
  METHODS
  Confluent cultures of human Müller cells (MIO-M1) were treated with 100 microM CoCl(2), 1 microg/mL triamcinolone acetonide (TA), or both. VEGF secretion was measured with respect to time by ELISA. VEGF mRNA quantity and stability were analyzed by reverse transcriptase-polymerase chain reaction. The activity of hypoxia-inducible factor (HIF)-1 was measured by the relative binding of HIF-1 protein to the hypoxia response element (HRE), by gel shift and ELISA. The HIF-1alpha protein level was determined with Western blot.
  
  
  RESULTS
  TA decreased VEGF secretion by at least 50% in the presence of continuous cobalt stimulus. VEGF mRNA decreased 50- to 100-fold 6 hours after treatment with TA and cobalt compared with cobalt alone. VEGF mRNA stability was decreased in cobalt-stimulated, TA-treated cells compared with cobalt alone in cells synchronized by exposure to actinomycin D. HIF-1alpha protein level was sustained for the entire 24-hour treatment period and partitioned into nuclear, not cytosolic, fractions. HIF-1 activity was decreased by 20% to 30% in the presence of TA and cobalt compared with cobalt alone.
  
  
  CONCLUSIONS
  TA may decrease VEGF synthesis by nongenomic destabilization of VEGF mRNA in cobalt-stimulated Müller cells. There was little effect on the total HIF-1alpha protein level, HIF-1 partitioning, and HIF-1 activity.
authors.affiliation: Cole Eye Institute, Cleveland Clinic Foundation, OH 44195, USA. searsj@ccf.org
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Sears
authors.forename: George
authors.initials: G
authors.lastname: Hoppe
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology*; D003609:Dactinomycin / Q000494:pharmacology; D015536:Down-Regulation; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014222:Triamcinolone Acetonide / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D004338:Drug Combinations; D005938:Glucocorticoids; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D019384:Nucleic Acid Synthesis Inhibitors; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D003609:Dactinomycin; D003035:Cobalt; C018021:cobaltous chloride; D014222:Triamcinolone Acetonide