Additional Supporting Information may be found in the online version of this article. “
“The retinoid X receptor a (RXRα; NR2B1), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid learn more and xenobiotic metabolism and homeostasis. Studies indicated that post-translational modification of RXRα, in particular, inflammation-induced phosphorylation of several sites, can lead to further post-translational modification (e. g., ubiquitination
and SUMOylation), as well as altered RXRa stability and subce l l ular localization. We have found that inflammation-induced reduction in RXRα nuclear quantities involves JNK-dependent phosphorylation at Ser260.Details regarding the fate, induction, localization and function of RXRα where Ser260 is phosphorylated are poorly understood, due to the lack of specific detecting reagents, cell
lines and mouse models. To begin to address these important issues, we developed and characterized an Torin 1 ic50 anti-pRXRα Ser260 antibody (p260 Ab) and verified its specificity and sensitivity with shRNA knockdown, immunoblotting and confocal immunofluorescence assays in both human and mouse models. The phosphorylation of RXRα Ser260 is significantly increased in both nuclear and cytoplasmic compartments of IL-1 β-treated Huh-7 cells and LPS-treated mouse liver, with a novel
finding of a submembrane localization at baseline which is increased in response to inflammation. Moreover, the JNK Elongation factor 2 kinase inhibitor, SP600125, inhibits IL-1 –β-induced upregulation of pRXRα Ser260 in Huh-7 cells, suggesting that JNK is a necessary upstream kinase involved in RXRα Ser260 phosphorylation. Initial explorations with confocal immunofluorescence microscopy and co-immunoprecipitation (Co-IP) assays identified submembrane phospho-RXRα interactions with the submembrane protein β-catenin. Moreover, Co-IP and immunofluorescence assays revealed that inflammation increases the interaction between phospho-RXRα and β-catenin in IL-1 β treated Huh-7 cells and LPS treated mouse livers in both cytoplasmic and subplasma membrane locales extend our knowledge of the potential biological roles played by RXRα species. We conclude that inflammatory stimuli induce JNK-dependent RXRα Serine260 phosphorylation, the interaction between p-catenin and RXRα, and the subcellular redistribution of RXRα, including heretofore novel cytoplasmic and submembrane locales. Disclosures: The following people have nothing to disclose: Hong Tang, Zhining Den, Astrid Kosters, Daniel A. Moore, Saul J.