Pro-inflammatory cytokines enhance ERAD and ATF6α pathway activity in salivary glands of Sjogren's syndrome patients

Salivary gland (SG) acinar-cells are susceptible to endoplasmic reticulum (ER) stress related to their secretory activity and the complexity of synthesized secretory products. SGs of Sji5gren's syndrome patients (SS)-patients show signs of inflammation and altered proteostasis, associated with low IRE1 alpha/ XBP-1 pathway activity without avert increases in apoptosis. Acinar-cells may avoid apoptosis by activation of the ATF6 alpha pathway and ER-associated protein degradation (ERAD). The aim of this study was to evaluate the role of pro-inflammatory cytokines in ATF6a pathway/ERAD activation and cell viability in labial salivary glands (LSG) of SS-patients. In biopsies from SS-patients increased ATF6a signaling pathway activity, as evidenced by generation of the ATF6f cleavage fragment, and increased expression of ERAD machinery components, such as EDEM1, p97, SEL1L, gp78, UBE2J1, UBE2G2, HERP and DERLIN1, were observed compared to controls. Alternatively, for pro-(active-caspase-3) and anti-apoptotic (cIAP2) markers no significant difference between the two experimental groups was detected. Increased presence of ATF6f and ERAD molecules correlated significantly with increased expression of pro inflammatory cytokines. These observations were corroborated in vitro in 3D-acini treated with TNF-alpha and/or IFN-gamma, where an increase in the expression and activation of the ATF6a sensor and ERAD machinery components was detected under ER stress conditions, while changes in cell viability and caspase-3 activation were not observed. Cytokine stimulation protected cells from death when co-incubated with an ERAD machinery inhibitor. Alternatively, when cytokines were eliminated from the medium prior to ERAD inhibition, cell death increased, suggesting that the presence of pro-inflammatory cytokines in the medium is essential to maintain cell viability. In conclusion, the ATF6a pathway and the ERAD machinery are active in LSG of SS-patients. Both were also activated by TNF-a and IFN-gamma in vitro in 3D-acini and aided in preventing apoptosis. IFN-gamma levels were elevated in SS-patients and UPR responses triggered in vitro by this cytokine closely matched those observed in LSG from SS-patients, suggesting that cytokines may induce ER stress. (C) 2016 Elsevier Ltd. All rights reserved.