Glucocorticoid-Induced Leucine Zipper Suppresses ICAM-1 and MCP-1 Expression by Dephosphorylation of NF-κB p65 in Retinal Endothelial Cells

Abstract

PURPOSE. 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.METHODS. 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 lM 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. RESULTS.Lipopolysaccharide downregulated GILZ expression in RMECs in a time-and dosedependent 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-jB p65 nuclear translocation in RMECs that was mediated by enhanced p65 dephosphorylation. The dephosphorylation of NF-jB p65 further downregulated ICAM-1 and MCP-1 expression in RMECs.CONCLUSIONS. Glucocorticoid-induced leucine zipper overexpression inhibited NF-jB p65 nuclear translocation by enhancing p65 dephosphorylation. Exogenous GILZ regulated ICAM-1 and MCP-1 expression, which was probably mediated by enhanced p65 dephosphorylation.

Glucocorticoid-Induced Leucine Zipper Protects the Retina From Light-Induced Retinal Degeneration by Inducing Bcl-xL in Rats

“…9 GILZ inhibited the nuclear translocation of NF-jB p65 by promoting its dephosphorylation in rat primary retinal microvascular endothelial cells, leading to the inhibition of proinflammatory cytokine release. 10 In addition to its anti-inflammatory effects, GILZ regulates numerous signal transduction pathways involved in cell proliferation and survival. Previously published data indicate that the cell survival regulatory functions of GILZ differ according to cell type.…”

Synthetic Glucocorticoid-Induced Leucine Zipper Peptide Inhibits Lipopolysaccharide-Induced Ocular Inflammation in Rats

“…The biological activity of a full-length GILZ fusion protein has previously been described [19][20][21][22]. Our previous studies have found that full-length GILZ suppressed ICAM-1 and MCP-1 expression by dephosphorylating NF-κB p65 in retinal endothelial cells in vitro [23] and inhibited LPSinduced retinal inflammation in rats in vivo [10]. The full-length GILZ protein contains 134 amino acids, and its C-terminal region (amino acids 98-134) contains 8 proline (P) residues, 8 glutamic acid (E) residues, and 5 PxxP sequences.…”

“…In the present study, we showed that synthetic GILZ-p successfully inhibited LPS-induced retinal Müller cell gliosis, but we did not identify the mechanism underlying this effect. One hypothesis is that GILZ-p inhibits the nuclear translocation of NF-κB and thus downregulates the release of inflammatory cytokines [23]. Another hypothesis is that GILZ-p suppresses the process by which Müller cells become scattered and ameboid, thus reducing the production of inflammatory factors from the scattered ameboid cells.…”

Lymphotoxin α fine-tunes T cell clonal deletion by regulating thymic entry of antigen-presenting cells

“…We identified two putative NF-κB binding sites for c-Rel and p65, by in silico analysis, in the Ccl12 promoter region (Supplementary Table 1 ), suggesting that this gene could be also regulated by the classical NF-κB pathway. The level of p65 phosphorylation at serine 536 (ser536), which is associated with the upregulation of CCL2 45 , 46 , was unaltered in Ltα −/− mTEC lo (Fig. 5d ).…”