TLR3-mediated apoptosis and activation of phosphorylated Akt in the salivary gland epithelial cells of primary Sjogren's syndrome patients
TLR3-mediated apoptosis and activation of phosphorylated Akt in the salivary gland epithelial cells of primary Sjogren's syndrome patients
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DOI：
10.1007/s00296-012-2381-9
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发表时间：
2013-02-01
影响因子：
4
通讯作者：
Kawakami, Atsushi
中科院分区：
文献类型：
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作者：
Nakamura, Hideki;Horai, Yoshiro;Kawakami, Atsushi
本研究旨在探讨先天性免疫是否参与了原发性干燥综合征（pSS）患者原代培养的唾液腺上皮细胞（SGECs）的凋亡。使用TLR 3配体poly（I：C）进行SGEC凋亡的诱导。磷酸化Akt（pAkt）和切割的caspase 3的活化通过Western印迹或免疫荧光测定。在用每种配体刺激24小时后，在培养的SGEC中观察到TLR 2和TLR 3与pAkt的表达。与肽聚糖或脂多糖刺激相比，聚（I：C）诱导显着的核碎裂，通过Hoechst染色（p = 0.0098）。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色pSS患者和正常受试者的SGEC证实细胞凋亡。通过添加PI 3 K抑制剂LY 294002观察到TUNEL阳性细胞的显著增加。Poly（I：C）磷酸化应激激活蛋白激酶/Jun-terminal激酶和p44/42 MAP激酶以及Akt。此外，LY 294002还抑制了多聚（I：C）诱导的半胱天冬酶3在SGEC中的裂解。使用从正常受试者获得的SGEC也获得了类似的结果。结果首次证明TLR 3通过PI 3 K-Akt信号通路诱导SGECs凋亡。
This study aimed at ascertain whether innate immunity is involved in the apoptosis of primary cultured salivary gland epithelial cells (SGECs) in primary Sjogren's syndrome (pSS). Induction of apoptosis of SGECs was performed using a TLR3 ligand, poly (I:C). Activation of phosphorylated-Akt (pAkt) and cleaved-caspase 3 was determined by Western blotting or immunofluorescence. Expression of TLR2 and TLR3 with pAkt was observed in cultured SGECs after 24-h stimulation with each ligand. Compared with stimulation with the peptidoglycan or lipopolysaccharide, that with poly (I:C) induced significant nuclear fragmentation, as determined by Hoechst staining (p = 0.0098). Apoptosis was confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining of SGECs from pSS patients and a normal subject. A significant increase in TUNEL-positive cells was observed by the addition of a PI3K inhibitor, LY294002. Poly (I:C) phosphorylated stress-activated protein kinase/Jun-terminal kinase and p44/42 MAP kinase as well as Akt. Furthermore, poly (I:C)-induced caspase 3 cleavage in SGECs was also inhibited by LY294002. Similar results were obtained using SGECs obtained from a normal subject. The results demonstrated for the first time that TLR3 induces the apoptotic cell death of SGECs via the PI3K-Akt signaling pathway.