592 REDD1/DDIT4 regulates the glucocorticoid receptor function in human keratinocytes

Abstract

Glucocorticoids (Gcs) are widely used for inflammatory skin diseases. However, Gcs are also notorious for adverse effects such as skin atrophy. The glucocorticoid receptor (GR) is a transcription factor mediating Gcs action. Previously, we found that GR target gene REDD1 is causatively involved in atrophic effects of Gcs, and that REDD1 inhibitors could protect skin from steroid hypoplasia. In this work, we searched for the mechanisms underlying REDD1 effects on GR function using HaCaT REDD1 KO keratinocytes generated by CRISPR/Cas9 approach. Transcriptome analysis revealed that induction of GR target genes (FKBP51, SGK1, GILZ, BIRC3, and KLF9) by glucocorticoid fluocinolone acetonide (FA) in REDD1 KO was markedly lower than in Cas9-control cells. Interestingly, induction of mitochondrial GR targets, MT-ND2 and MT-CYTB, by FA was also decreased in REDD1 KO cells compared to control. This suggested the existence of novel feed forward loop in GR activation via REDD1. Next, we assessed whether a) REDD1 KO affected GR expression at RNA/protein levels, and b) modified the major steps in GR activation: GR phosphorylation at activation site Ser211 and GR nuclear import. Surprisingly, the difference between REDD1 KO and control cells in terms of GR expression and activation was rather marginal. We also did not find direct protein-protein interaction between GR and REDD1 using co-immunoprecipitation (co-IP). It is known that GR function is strongly regulated by chaperones including immunophilins FKBP51 and FKBP52. Specifically, FKBP51 inhibits GR activity via altered nuclear translocation and inhibition of interaction between GR and its ligands, and potentially may affect GR-DNA binding. We found that FKBP51 protein levels were increased in REDD1 KO keratinocytes, while FKBP52 levels did not change significantly. The ongoing experiments are focused on the evaluation of FKBP51 interaction with GR in the absence of REDD1 and the effect of FKBP51 knockdown on response of REDD1 KO cells to FA.