Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Glucocorticoids (GC) have been used to treat inflammatory disease for more than six decades, but the broad spectrum of therapeutic effects is accompanied by significant metabolic adverse effects. Recent studies suggest that glucocorticoid-induced leucine zipper (GILZ) mimics the anti-inflammatory effects of GC, including inhibiting the activation of macrophage, B cells, Th1 and Th17 cells, but lacks GC adverse effects. However, little is known about molecular mechanisms of GILZ action that could empower drug discovery.
Expression of GILZ and acetylated GILZ was measured by Western blotting in murine bone marrow-derived macrophages. WT and mutated GILZ plasmids were co-transfected with an NF-κB luciferase reporter into human microvascular endothelial cells (HMECs). IL-6 and MCP-1 were measured by ELISA. NF-κB activity was measured using a NF-κB luciferase reporter assay. Interactions between GILZ and p65/NF-κB were detected by Fluorescence lifetime imaging (FLIM)/ Fluorescence Resonance Energy Transfer (FRET).
Results: GILZ acetylation was detected in macrophages, and TSA, a deacetylase inhibitor, increased GILZ acetylation in a time-dependent manner. WT GILZ was constructed in the pcDNA3.1 expression plasmid and overexpression of GILZ significantly inhibited NF-κB activity and IL-6 and MCP-1 expression. Using an acetylation substrate-binding site alignment approach, we predicted three potential GILZ acetylation sites at K37, K77 and K108. Acetylation of GILZ was lost when K77, but not K37 or K108, was mutated to arginine (K77R). Mutation at K77 aborgated GILZ inhibition of NF-κB activity, and IL-6 and MCP-1 expression. Co-IP and FLIM/FRET demonstrated that GILZ mutation at K77 prevented interaction with p65/NF-κB.
Conclusion: Acetylation of GILZ at K77 is required for its interaction with NF-κB and anti-inflammatory effects. This suggests a potential target for enhancing the anti-inflammatory effects of GILZ as a GC mimic via inducing K77 acetylation.
To cite this abstract in AMA style:Fan H, Wang D, Cheng Q, Harris J, Jones S, Yang YH, Morand E. Targeted Mutations Identify the Active Site of Glucocorticoid-Induced Leucine Zipper (GILZ) [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/targeted-mutations-identify-the-active-site-of-glucocorticoid-induced-leucine-zipper-gilz/. Accessed August 4, 2021.
« Back to 2016 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeted-mutations-identify-the-active-site-of-glucocorticoid-induced-leucine-zipper-gilz/