606 Translation and growth pathways are directly influenced by autoimmune regulator (Aire) in skin keratinocytes

Abstract

In response to environmental stressors, such as ultraviolet B (UVB) radiation, keratinocytes activate a stress response marked by coordination between signaling pathways associated with proliferation, DNA damage, and apoptosis. Herein, we have identified autoimmune regulator (AIRE), which is classically thought of as a transcriptional regulator, as a key influencer of apoptosis, the DNA damage response (DDR), and surprisingly protein synthesis in stressed skin keratinocytes. Initially, we created keratinocyte cell lines stably and inducibly expressing AIRE (either wild-type or with function blocking mutations) fused to the BirA* biotin ligase and subjected these cells to a proximity based biotinylation screen (BioID) to identify potential AIRE binding partners. Using isobaric labeling (iTRAQ) coupled to tandem mass spectrometry, we identified that the AIRE binding partners most affected by function blocking AIRE mutations included UVB-associated proteins related to apoptosis, DNA damage, and protein translation. To validate these findings, UVB-irradiation (25 mJ/cm2) of CRISPR-Cas9 mediated AIRE knock-out (KO) keratinocytes showed increased apoptotic signaling (BIM, BID, NOXA, FOXO3a, & FasL) and cell death (Cleaved Caspase-3 -8 -9 and Cleaved PARP) compared to wild-type cells. The DDR (P-CHK1, P-CHK2, P-H2B, P-ATM, P-ATR, & γH2AX) was also increased in keratinocytes that lacked AIRE following exposure to UVB (25 mJ/cm2) or peroxide treatment (100 μM H2O2, 30 min). Exposure to UVB radiation concurrently increased protein translation pathways (P-S6K, P-S6, & P-4EBP1) in AIRE KO cells. Using puromycin incorporation (3 μM), we found global protein synthesis was similarly increased in AIRE KO cells following a 2h serum pulse. This influence of AIRE on protein translation indicates a novel non-nuclear function for AIRE. Altogether, our data reveal AIRE to be a coordinate regulator of multiple UVB-induced cell stress response pathways in skin keratinocytes and helps to expand our understanding of AIRE’s function in skin photobiology.