蛋白激酶 Cα 信号通路参与 TNF-α诱导的人肾小球系膜细胞内 IP3R1 的表达。
PKCα signaling pathway involves in TNF-α-induced IP3R1 expression in human mesangial cells.
机构信息
Department of Digestion, Jiujiang First People's Hospital, Jiujiang 332000, China.
出版信息
World J Emerg Med. 2012;3(4):282-6. doi: 10.5847/wjem.j.issn.1920-8642.2012.04.008.
BACKGROUND
This study aimed to explore the effects of TNF-α on the expression of IP3R1 mRNA and protein in human mesangial cells (HMCs), and to elucidate the mechanism of TNF-α relating to IP3R1 expression in the occurrence of hepatorenal syndrome (HRS).
METHODS
HMCs were stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2, 4, 8, and 24 hours). The expression changes of IP3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting. Several inhibitors including D609, U73122, PP1, safingol, rottlerin and non-radioactive protein kinase C (PKC) were used to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs.
RESULTS
The levels of IP3R1 mRNA at 2 hours after TNF-α exposure were significantly enhanced and peaked at 8 hours in HMCs (P<0.01), then descended at 24 hours (P<0.01). The levels of IP3R1 protein at 4 hours after TNF-α exposure were obviously increased and peaked at 24 hours after TNF-α exposure (P<0.01). Compared to the control group, safingol (PKCα inhibitor) and D609 (phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-αinduced expression of IP3R1 mRNA (3.30±0.81 vs. 1.95±0.13, P<0.05; 2.10±0.49, P<0.01) and IP3R1 protein (3.09±0.13 vs. 1.86+0.39, P<0.01; 1.98±0.02, P<0.01). TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay, and the effect was markedly attenuated by pretreatment with D609 or safingol.
CONCLUSION
TNF-α increased the expression of IP3R1 and this was mediated, at least in part, through the PC-PLC/PKCα signaling pathways in HMCs.
背景
本研究旨在探讨 TNF-α 对人肾小球系膜细胞（HMC）中 IP3R1mRNA 和蛋白表达的影响，并阐明 TNF-α 与肝性肾综合征（HRS）中 IP3R1 表达相关的机制。
方法
用肿瘤坏死因子-α（TNF-α）100ng/ml 刺激 HMC 不同时间（2、4、8 和 24 小时）。用实时定量聚合酶链反应和免疫印迹检测 IP3R1mRNA 和蛋白的表达变化。用 D609、U73122、PP1、sa⁃fingol、rottlerin 和非放射性蛋白激酶 C（PKC）等几种抑制剂来检测 TNF-α调节 HMC 中 IP3R1 信号转导的机制。
结果
TNF-α 暴露后 2 小时 IP3R1mRNA 水平明显增强，在 HMC 中 8 小时达到高峰（P<0.01），然后在 24 小时下降（P<0.01）。TNF-α 暴露后 4 小时 IP3R1 蛋白水平明显升高，在 TNF-α 暴露后 24 小时达到高峰（P<0.01）。与对照组相比，sa⁃fingol（PKCα 抑制剂）和 D609（磷脂酰胆碱特异性磷脂酶 C 抑制剂）显著阻断了 TNF-α 诱导的 IP3R1mRNA 表达（3.30±0.81 vs. 1.95±0.13，P<0.05；2.10±0.49，P<0.01）和 IP3R1 蛋白表达（3.09±0.13 vs. 1.86+0.39，P<0.01；1.98±0.02，P<0.01）。非放射性 PKC 测定显示，TNF-α 刺激 8 小时后促进 PKCα 激活，PKCα 磷酸化达到最大值，用 D609 或 sa⁃fingol 预处理可显著减弱该作用。
结论
TNF-α 增加了 IP3R1 的表达，这至少部分是通过 HMC 中的 PC-PLC/PKCα 信号通路介导的。
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