PKCδ silencing alleviates saturated fatty acid induced ER stress by enhancing SERCA activity
PKCδ silencing alleviates saturated fatty acid induced ER stress by enhancing SERCA activity
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DOI：
10.1042/bsr20170869
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发表时间：
2017-12-22
影响因子：
4
通讯作者：
Wen, Liangzhi
中科院分区：
文献类型：
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作者：
Lai, Shujie;Li, Yan;Wen, Liangzhi
蛋白激酶C δ（PKC δ）在非酒精性脂肪性肝病（NAFLD）中起重要作用，但其机制尚不清楚。本研究探讨PKCd在NAFLD发生发展中的作用，并研究PKCd、钙稳态和内质网（ER）应激（ERS）之间的关系。采用软脂酸（PA）诱导L02细胞脂肪变性模型。使用油红O染色和甘油三酯（TG）检测试剂盒评价脂质蓄积。siRNA下调PKCd表达。RT-PCR和Western blotting检测ERS标记的表达。用共聚焦显微镜记录Ca 2+内流的荧光。用超微ATP酶试剂盒测定肌钙蛋白酶（SERCA）活性。PA处理诱导L02细胞脂质沉积，破坏ER结构，并以时间依赖的方式增加PKCd的激活。此外，PA处理显著增加ERS标记物、IG重链结合蛋白（Bip）和CCAAT增强子结合蛋白（CHOP）的同源蛋白的表达。PKCd沉默下调Bip和CHOP表达，表明成功缓解ERS。与阴性对照相比，PKCd siRNA处理的L02细胞中由PA刺激诱导的增加的钙储存显著降低。此外，PA引起的SERCA活性降低在PKCd siRNA转染的细胞中恢复。据我们所知，这是第一个报告表明，抑制PKCd通过增强SERCA活性和稳定钙稳态来抑制ERS。
Protein kinase C delta (PKC delta) plays an important role in nonalcoholic fatty liver disease (NAFLD), however, the mechanism remains unknown. The present study explored the role of PKCd in NAFLD development and investigated the relationships between PKCd, calcium homeostasis, and endoplasmic reticulum (ER) stress (ERS). Hepatic steatosis cell model was induced by palmitic acid (PA) in L02 cells. Lipid accretion was evaluated using Oil Red O staining and a triglyceride (TG) detection kit. PKCd was down-regulated by siRNA. RT-PCR and Western blotting were used to detect the expression of ERS markers. The fluorescence of Ca2+ influx was recorded using confocal microscopy. Sarco-ER Ca2+-ATPase (SERCA) activity was measured by ultramicro-ATP enzyme test kit. PA treatment induced lipid accretion in L02 cells, destroyed the ER structure, and increased PKCd activation in a time-dependent manner. Further, PA treatment significantly increased the expression of ERS markers, Ig heavy chain binding protein (Bip), and homologous proteins of CCAAT-enhancer binding proteins (CHOP). PKCd silencing down-regulated Bip and CHOP expression, indicating a successful alleviation of ERS. The increased calcium storage induced by PA stimulation was significantly decreased in L02 cells treated with PKCd siRNA compared with the negative control. Moreover, diminished SERCA activity caused by PA was recovered in PKCd siRNA transfected cells. To the best of our knowledge, this is the first report demonstrating that the inhibition of PKCd alleviates ERS by enhancing SERCA activity and stabilizing calcium homeostasis.