Impaired IRE1 alpha/XBP-1 pathway associated to DNA methylation might contribute to salivary gland dysfunction in Sjogren's syndrome patients

Abstract

Objectives. Labial salivary glands (LSGs) of SS patients show alterations related to endoplasmic reticulum stress. Glandular dysfunction could be partly the consequence of an altered inositol-requiring enzyme 1 alpha (IRE1 alpha)/X box-binding protein 1 (XBP-1) signalling pathway of the unfolded protein response, which then regulates genes involved in biogenesis of the secretory machinery. This study aimed to determine the expression, promoter methylation and localization of the IRE1 alpha/XBP-1 pathway components in LSGs of SS patients and also their expression induced by IFN-gamma in vitro. Methods. IRE1 alpha, XBP-1 and glucose-regulated protein 78 (GRP78) mRNA and protein levels were measured by qPCR and western blot, respectively, in LSGs of SS patients (n = 47) and control subjects (n = 37). Methylation of promoters was evaluated by methylation-sensitive high resolution melting, localization was analysed by immunofluorescence and induction of the IRE1 alpha/XBP-1 pathway components by IFN-gamma was evaluated in 3D acini. Results. A significant decrease of IRE1 alpha, XBP-1u, XBP-1s, total XBP-1 and GRP78 mRNAs was observed in LSGs of SS patients, which was correlated with increased methylation levels of their respective promoters, and consistently the protein levels for IRE1 alpha, XBP-1s and GRP78 were observed to decrease. IFN-gamma decreased the mRNA and protein levels of XBP-1s, IRE1 alpha and GRP78, and increased methylation of their promoters. Significant correlations were also found between IRE1 alpha/XBP-1 pathway components and clinical parameters. Conclusion. Decreased mRNA levels for IRE1 alpha, XBP-1 and GRP78 can be partially explained by hypermethylation of their promoters and is consistent with chronic endoplasmic reticulum stress, which may explain the glandular dysfunction observed in LSGs of SS patients. Additionally, glandular stress signals, including IFN-gamma, could modulate the expression of the IRE1 alpha/XBP-1 pathway components.

Objectives. Labial salivary glands (LSGs) of SS patients show alterations related to endoplasmic reticulum stress. Glandular dysfunction could be partly the consequence of an altered inositol-requiring enzyme 1 alpha (IRE1 alpha)/X box-binding protein 1 (XBP-1) signalling pathway of the unfolded protein response, which then regulates genes involved in biogenesis of the secretory machinery. This study aimed to determine the expression, promoter methylation and localization of the IRE1 alpha/XBP-1 pathway components in LSGs of SS patients and also their expression induced by IFN-gamma in vitro. Methods. IRE1 alpha, XBP-1 and glucose-regulated protein 78 (GRP78) mRNA and protein levels were measured by qPCR and western blot, respectively, in LSGs of SS patients (n = 47) and control subjects (n = 37). Methylation of promoters was evaluated by methylation-sensitive high resolution melting, localization was analysed by immunofluorescence and induction of the IRE1 alpha/XBP-1 pathway components by IFN-gamma was evaluated in 3D acini. Results. A significant decrease of IRE1 alpha, XBP-1u, XBP-1s, total XBP-1 and GRP78 mRNAs was observed in LSGs of SS patients, which was correlated with increased methylation levels of their respective promoters, and consistently the protein levels for IRE1 alpha, XBP-1s and GRP78 were observed to decrease. IFN-gamma decreased the mRNA and protein levels of XBP-1s, IRE1 alpha and GRP78, and increased methylation of their promoters. Significant correlations were also found between IRE1 alpha/XBP-1 pathway components and clinical parameters. Conclusion. Decreased mRNA levels for IRE1 alpha, XBP-1 and GRP78 can be partially explained by hypermethylation of their promoters and is consistent with chronic endoplasmic reticulum stress, which may explain the glandular dysfunction observed in LSGs of SS patients. Additionally, glandular stress signals, including IFN-gamma, could modulate the expression of the IRE1 alpha/XBP-1 pathway components.