POS0100 ACTIVATION OF THE CELLULAR INTEGRATED STRESS RESPONSE IN LABIAL SALIVARY GLANDS FROM SJÖGREN’S SYNDROME PATIENTS.

Abstract

BackgroundSjögren’s syndrome (SS) is a chronic autoimmune disease characterized by inflammation of the exocrine glands and severe symptoms of eye and mouth dryness. Disorders in the saliva secretion process have been associated with oxidative and endoplasmic reticulum (ER) stress in combination with inflammatory responses. The integrated stress response (ISR) is a mechanism that allows cells to modify their gene expression program to restore homeostasis and promote their survival against various extrinsic and intrinsic stress signals, such as hypoxia, nutrient deprivation, viral infections, inflammatory factors (cytokines, chemokines, inflammasomes), and accumulation of unfolded proteins in the ER, among others (1). The ISR is regulated by four kinases: PERK, PKR, HRI and GCN2, that dimerize and autophosphorylate to become active and each one responds to different stress stimuli. The signaling pathways that are activated in response to stress factors stimulate the phosphorylation of eIF2α, which causes a transient inhibition of global protein synthesis and induction of synthesis of some specific genes like ATF4 and NRF2. ATF4 induces the transcription of genes involved in metabolism and nutrient uptake, redox status, and regulation of apoptosis. Dephosphorylation of eIF2α is the ISR termination signal to restore protein synthesis and is mediated by the PP1 complex, which recruits the catalytic subunit PP1c and one of its two regulatory subunits: GADD34 or CREP.ObjectivesTo evaluate the presence and functional state (phosphorylation) of the ISR sensing kinases: PERK, PKR, HRI and GCN2; the levels of eIF2α /p-eIF2α and the key ISR transcription factors ATF4 and NRF2, as well as subunits of the complex involved in the ISR termination: PP1c, GADD34 and CREP in labial salivary glands (LSG) of SS-patients.MethodsBiopsies of LSG from 12 SS-patients and 11 control subjects were studied. The levels of mRNA, protein and phospho (p)-protein of the ISR components were determined by RT-qPCR and Western blotting.ResultsOur results show increased levels of p-PERK/PERK ratio (11/11), PKR (7/11), p-PKR (7/11), p-PKR/PKR ratio (7/11), eIF2α (5/11), p-eIF2α (5/11) and ATF4 (11/11) in LSG from SS-patients compared to control subjects. No significant changes were found in mRNA levels of HRI, GCN2, and GADD34 between LSG from SS-patients and control subjects. Decreased protein levels of HRI (8/12), p-GCN2 (6/11), eIF2α (6/11), p-eIF2α (6/11), NRF2 (11/12), and p-NRF2 (12/12) were found in LSG showing scarce parenchyma and high fibrosis and fat infiltration. On the other hand, PP1c and CREP showed decreased mRNA and protein levels in all SS-patients LSG. Interestingly, Ro autoantibodies and focus score were negatively correlated with PP1c and NRF2 mRNA and protein levels whereas positively correlated with PKR mRNA levels.ConclusionThe overexpression and activation of some ISR kinases together with the decrease in the PP1c/CREP phosphatase complex suggests a continuous activation of ISR, resulting in p-eIF2α to remain activated in LSG from SS-patients. This would explain the high protein levels of ATF4 and of target genes involved in the antioxidant response in LSG from SS patients suggesting that ISR activation plays a key role in pro-survival response to cellular stress.