Abstract
Purpose: The anti-inflammatory activities of protein glucocorticoid-induced leucine zipper (GILZ) have been demonstrated in vivo and in vitro. Here, we examined the potential effect of a synthetic peptide derived from the leucine zipper motif and proline-rich region of GILZ on suppressing inflammatory responses in primary cultured rat Müller cells.Methods: Peptides were selected from amino acids 98–134 of the GILZ protein (GILZ-p). Solid-phase peptide synthesis was used to generate the cell-penetrating peptide TAT, which was bound to the amino terminus of GILZ-p. Primary cultured retinal Müller cells were stimulated with lipopolysaccharide (LPS) alone or in combination with different concentrations of GILZ-p, and the interaction of GILZ-p with nuclear factor (NF)-κB p65 in Müller cells was investigated by western blotting, immunoprecipitation, and immunofluorescence. The expression of the Müller cell gliosis marker glial fibrillary acidic protein (GFAP), functional protein aquaporin (AQP)-4, and the inflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF) α, intercellular adhesion molecule (ICAM)-1, and monocyte chemoattractant protein (MCP)-1 was measured by Western Blotting. The concentration of those cytokines in culture medium was measured by using Enzyme-Linked Immunosorbent Assay.Results: The synthesized GILZ-p, which was water-soluble, entered cells and bound with NF-κB p65, inhibiting p65 nuclear translocation. GILZ-p inhibited the LPS-induced expression of GFAP, IL-1β, TNFα, ICAM-1, and MCP-1 in Müller cells and prevented the LPS-induced downregulation of AQP4.Conclusions: These results indicate that GILZ-p interacted with NF-κB p65 and suppressed p65 nuclear translocation, thereby inhibiting inflammatory cytokine release and Müller cell gliosis.
Highlights
Sight-threatening intraocular inflammation accounts for a significant proportion of visual disability and is involved in many ocular diseases, including those of infectious, non-infectious, and multiple para-inflammatory types (Durrani et al, 2004; Deibel and Cowling, 2013; Forrester et al, 2013; Huerva et al, 2015)
We explored the effect of the synthetic peptide GILZ98−134 derived from the leucine zipper motif and proline-rich region of Glucocorticoid-induced leucine zipper (GILZ) on inflammatory responses induced by lipopolysaccharide (LPS) in primary cultured rat Müller cells
The flow cytometric analyze showed that about 7.92% Müller cells successfully associated with GILZ-p at 15 min after treatment (Supplementary Figure S1)
Summary
Sight-threatening intraocular inflammation accounts for a significant proportion of visual disability and is involved in many ocular diseases, including those of infectious, non-infectious, and multiple para-inflammatory types (Durrani et al, 2004; Deibel and Cowling, 2013; Forrester et al, 2013; Huerva et al, 2015). Glucocorticoid-induced leucine zipper (GILZ), which was first described as a glucocorticoid-induced protein (D’Adamio et al, 1997), belongs to the leucine zipper protein family (Ayroldi and Riccardi, 2009; Pinheiro et al, 2013) It is a dexamethasone-inducible gene that mediates glucocorticoid (GC) actions in a variety of cell types, such as in T cell, B cell, macrophage and endothelium cell. The C-terminal region of GILZ contains eight prolines (P), eight glutamic acid (E) residues, and five PxxP sequences Such sequences were previously shown to mediate protein–protein interactions (Holt and Koffer, 2001). We explored the effect of the synthetic peptide GILZ98−134 derived from the leucine zipper motif and proline-rich region of GILZ on inflammatory responses induced by lipopolysaccharide (LPS) in primary cultured rat Müller cells
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