Yang Won Seok, Kim Jin Ju, Lee Mee Jeong, Lee Eun Kyoung, Park Su-Kil
Division of Nephrology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea.
Asan Institute for Life Sciences, Seoul, Republic of Korea.
Cell Physiol Biochem. 2018;51(4):1632-1644. doi: 10.1159/000495651. Epub 2018 Nov 29.
BACKGROUND/AIMS: High-mobility group box 1 (HMGB1) elicits inflammatory responses through interactions with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). We investigated how RAGE and TLR4 expressions are regulated after HMGB1 stimulation in cultured human aortic endothelial cells (HAECs).
RAGE and TLR4 expressions were analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorogenic substrate.
Upon treatment with HMGB1, both RAGE and TLR4 began to decrease in cell lysate and remained decreased up to 24 h. The decrease in cellular RAGE and TLR4 was accompanied by an increase of N-terminal fragment of RAGE and TLR4 in culture supernatant, indicating ectodomain shedding of the receptors. HMGB1 activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while HMGB1-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. HMGB1-induced ectodomain shedding of RAGE and TLR4 was prevented by siRNA depletion of ADAM17 as well as TAPI-2, an inhibitor of ADAM family, and SB203580. HMGB1 pretreatment abolished p38 MAPK activation in response to 2nd HMGB1 stimulation. In the cells depleted of ADAM17, HMGB1-induced p38 MAPK activation was prolonged. siRNA depletion of RAGE, but not TLR4, suppressed HMGB1-induced p38 MAPK activation.
In response to HMGB1 stimulation, HAECs rapidly undergo ectodomain shedding of RAGE and TLR4, and thereby become insensitive to further HMGB1 stimulation. ADAM17, activated through RAGE-p38 MAPK pathway, is implicated in the ectodomain cleavage of the receptors.
背景/目的：高迁移率族蛋白B1（HMGB1）通过与晚期糖基化终产物受体（RAGE）和Toll样受体4（TLR4）相互作用引发炎症反应。我们研究了在培养的人主动脉内皮细胞（HAECs）中，HMGB1刺激后RAGE和TLR4的表达是如何被调节的。
通过蛋白质免疫印迹分析和免疫荧光染色分析RAGE和TLR4的表达。使用荧光底物测量解整合素和金属蛋白酶17（ADAM17）的活性。
用HMGB1处理后，细胞裂解物中的RAGE和TLR4均开始减少，并持续减少至24小时。细胞中RAGE和TLR4的减少伴随着培养上清液中RAGE和TLR4的N端片段增加，表明受体的胞外域脱落。HMGB1激活p38丝裂原活化蛋白激酶（p38 MAPK）和ADAM17，而p38 MAPK抑制剂SB203580可抑制HMGB1诱导的ADAM17激活。ADAM17的小干扰RNA（siRNA）耗竭以及ADAM家族抑制剂TAPI-2和SB203580可阻止HMGB1诱导的RAGE和TLR4胞外域脱落。HMGB1预处理消除了对第二次HMGB1刺激的p38 MAPK激活。在ADAM17耗竭的细胞中，HMGB1诱导的p38 MAPK激活延长。RAGE的siRNA耗竭而非TLR4的siRNA耗竭可抑制HMGB1诱导的p38 MAPK激活。
响应HMGB1刺激，HAECs迅速经历RAGE和TLR4的胞外域脱落，从而对进一步的HMGB1刺激不敏感。通过RAGE-p38 MAPK途径激活的ADAM17参与受体的胞外域裂解。
