Katsiougiannis S, Tenta R, Skopouli F N
Department of Nutrition and Dietetics, Harokopio University, Athens, Greece.
Dental Research Institute, UCLA School of Dentistry, Los Angeles, CA, USA.
Clin Exp Immunol. 2015 Aug;181(2):244-52. doi: 10.1111/cei.12638.
The aim of this study was to examine the levels of endoplasmic reticulum (ER) stress in minor salivary glands, to investigate the interplay between ER stress-induced autophagy and apoptosis in human salivary gland (HSG) cells and to test the effect of ER stress-induced apoptosis on the cellular redistribution of the two major Sjögren's syndrome (SS) autoantigens Ro/Sjögren's syndrome-related antigen A (SSA) and La/Sjögren's syndrome-related antigen B (SSB). Minor salivary gland biopsies from SS patients and sicca controls were examined by immunohistochemistry for the expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/BiP) as an indicator of unfolded protein response (UPR). HSG cells were treated with thapsigargin (TG) and cell viability, autophagy and apoptosis were assessed. Immunoblot was applied to detect the conversion of LC3I to LC3II and the protein levels of GRP78/BiP and X-box binding protein-1 (XBP-1). Apoptosis was evaluated by a single-stranded DNA enzyme-linked immunosorbent assay (ELISA). Ro/SSA and La/SSB localization was visualized using immunofluorescence. GRP78/BiP was expressed by acinar and ductal epithelial cells in salivary glands of patients and sicca controls. TG treatment induced autophagy, as indicated by enhanced protein expression of LC3II. The protein levels of UPR marker XBP-1 were increased after TG treatment, while GRP78/BiP levels were decreased. TG treatment resulted in induction of HSG apoptosis. Ro/SSA and La/SSB autoantigens were localized predominantly to the cytoplasm in resting cells, while they were redistributed to cell membrane and blebs in the apoptotic cells. In conclusion, ER stress is activated in minor salivary gland epithelial cells from SS patients and controls. ER stress-induced apoptosis in HSG cells leads to cell surface and apoptotic blebs relocalization of Ro/SSA and La/SSB autoantigens.
本研究旨在检测小唾液腺中内质网（ER）应激水平，探究ER应激诱导的自噬与人类唾液腺（HSG）细胞凋亡之间的相互作用，并测试ER应激诱导的凋亡对两种主要干燥综合征（SS）自身抗原Ro/干燥综合征相关抗原A（SSA）和La/干燥综合征相关抗原B（SSB）细胞再分布的影响。通过免疫组织化学检测SS患者和干燥对照组小唾液腺活检组织中78 kDa葡萄糖调节蛋白/结合免疫球蛋白蛋白（GRP78/BiP）的表达，作为未折叠蛋白反应（UPR）的指标。用毒胡萝卜素（TG）处理HSG细胞，并评估细胞活力、自噬和凋亡情况。应用免疫印迹法检测LC3I向LC3II的转化以及GRP78/BiP和X盒结合蛋白-1（XBP-1）的蛋白水平。通过单链DNA酶联免疫吸附测定（ELISA）评估凋亡情况。使用免疫荧光观察Ro/SSA和La/SSB的定位。GRP78/BiP在患者和干燥对照组唾液腺的腺泡和导管上皮细胞中表达。TG处理诱导了自噬，表现为LC3II蛋白表达增强。TG处理后UPR标志物XBP-1的蛋白水平升高，而GRP78/BiP水平降低。TG处理导致HSG细胞凋亡。Ro/SSA和La/SSB自身抗原在静息细胞中主要定位于细胞质，而在凋亡细胞中重新分布到细胞膜和小泡。总之，SS患者和对照组的小唾液腺上皮细胞中ER应激被激活。ER应激诱导的HSG细胞凋亡导致Ro/SSA和La/SSB自身抗原重新定位到细胞表面和凋亡小泡。
