title: Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells.
abstract: Glucocorticoid-induced leucine zipper (GILZ) is involved in anti-inflammatory activities in several animal models and in various cell types. In this study, we explored the role of GILZ in rat retinal vascular endothelial cells.
  
  
  
  Glucocorticoid-induced leucine zipper overexpression or silencing was established using GILZ overexpressing recombinant lentivirus (OE-GILZ-rLV) or short-hairpin RNA targeting GILZ recombinant lentivirus (shRNA-GILZ-rLV), respectively, in rat primary retinal microvascular endothelial cells (RMECs) and intact retina. Seventy-two hours after transfection, RMECs were stimulated with 1000 ng/mL lipopolysaccharide (LPS), 20 μM isoliensinine (an alkaloid derived from the embryos of Nelumbo nucifera, could enhance the dephosphorylation of p65 at Ser536), or PBS for another 24 hours. Western blotting and immunofluorescence were performed to measure protein expression. The concentrations of intercellular adhesion molecule (ICAM)-1 and monocyte chemoattractant protein (MCP)-1 in the RMEC culture media were measured by ELISA.
  
  
  
  Lipopolysaccharide downregulated GILZ expression in RMECs in a time- and dose-dependent manner, and the decrease in GILZ expression was accompanied by increased ICAM-1 and MCP-1 expression. Glucocorticoid-induced leucine zipper overexpression decreased LPS-induced ICAM-1 and MCP-1 expression, whereas GILZ silencing significantly attenuated the production of both cytokines. Glucocorticoid-induced leucine zipper overexpression also inhibited LPS-induced nuclear factor-κB p65 nuclear translocation in RMECs that was mediated by enhanced p65 dephosphorylation. The dephosphorylation of NF-κB p65 further downregulated ICAM-1 and MCP-1 expression in RMECs.
  
  
  
  Glucocorticoid-induced leucine zipper overexpression inhibited NF-κB p65 nuclear translocation by enhancing p65 dephosphorylation. Exogenous GILZ regulated ICAM-1 and MCP-1 expression, which was probably mediated by enhanced p65 dephosphorylation.
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China.
authors.forename: Ruiping
authors.initials: R
authors.lastname: Gu
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China.
authors.forename: Boya
authors.initials: B
authors.lastname: Lei
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Jiang
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China 2Shanghai Key Laboratory of Visual Impairment and Restoration, Fudan University, Shanghai, China.
authors.forename: Gezhi
authors.initials: G
authors.lastname: Xu
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005938:Glucocorticoids / Q000494:pharmacology*; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D016350:Leucine Zippers; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D012164:Retinal Diseases / Q000378:metabolism / Q000473:pathology; D051996:Transcription Factor RelA / Q000378:metabolism*
chemical_list: C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D005938:Glucocorticoids; D051996:Transcription Factor RelA; D018799:Intercellular Adhesion Molecule-1