Imaizumi Tadaatsu, Hayakari Ryo, Watanabe Shojiro, Aizawa Tomomi, Matsumiya Tomoh, Yoshida Hidemi, Tsuruga Kazushi, Kawaguchi Shogo, Tanaka Hiroshi
Department of Vascular Biology, Hirosaki, Japan.
Department of Pediatrics, Hirosaki, Japan.
Kidney Blood Press Res. 2017;42(5):942-950. doi: 10.1159/000485084. Epub 2017 Nov 22.
BACKGROUND/AIMS: Cylindromatosis (CYLD), a deubiquitinase, negatively regulates nuclear factor-κB in various cells. However, its potential roles in glomerular inflammation remain unclear. Because the activation of the Toll-like receptor 3 (TLR3)/type I interferon (IFN) pathways plays a pivotal role in chronic kidney diseases (CKD), we examined the role of CYLD in the TLR3 signaling in cultured human mesangial cells (MCs).
We stimulated CYLD-silenced MCs with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of dsRNA, and studied representative TLR3/IFN-β pathways (i.e., TLR3/IFN-β/retinoic acid-inducible gene-I (RIG-I)/CCL5, and TLR3/IFN-β/melanoma differentiation associated gene 5 (MDA5)/CXCL10 axes) using RT-PCR, western blotting, and ELISA. We also used immunofluorescence staining and microscopy to examine mesangial CYLD expression in biopsied specimens from patients with CKD.
CYLD silencing resulted in an increase of poly IC-induced RIG-I and MDA5 protein levels and increased CCL5 and CXCL10 mRNA and protein expression, but unexpectedly decreased mRNA expressions of RIG-I and MDA5. Interestingly, CYLD silencing did not affect IFN-β or the phosphorylated STAT1 (signal transducers and activator of transcription protein 1). CYLD was highly expressed in biopsied specimens from patients with proliferative lupus nephritis (LN).
CYLD inhibits post-transcriptional regulation of RIG-I and MDA5 expression following TLR3 activation in MCs. CYLD may be involved in the pathogenesis of CKD, especially pathogenesis of LN.
背景/目的：圆柱瘤蛋白（CYLD）是一种去泛素化酶，可在多种细胞中对核因子-κB进行负调控。然而，其在肾小球炎症中的潜在作用仍不清楚。由于Toll样受体3（TLR3）/I型干扰素（IFN）通路的激活在慢性肾脏病（CKD）中起关键作用，我们研究了CYLD在人肾小球系膜细胞（MCs）培养物中TLR3信号传导中的作用。
我们用双链RNA的合成类似物聚肌苷酸-聚胞苷酸（poly IC）刺激CYLD沉默的MCs，并使用逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法和酶联免疫吸附测定（ELISA）研究代表性的TLR3/IFN-β通路（即TLR3/IFN-β/视黄酸诱导基因I（RIG-I）/CCL5和TLR3/IFN-β/黑色素瘤分化相关基因5（MDA5）/CXCL10轴）。我们还使用免疫荧光染色和显微镜检查了CKD患者活检标本中系膜CYLD的表达。
CYLD沉默导致poly IC诱导的RIG-I和MDA5蛋白水平升高，CCL5和CXCL10 mRNA及蛋白表达增加，但出乎意料的是RIG-I和MDA5的mRNA表达降低。有趣的是，CYLD沉默不影响IFN-β或磷酸化信号转导和转录激活因子1（STAT1）。CYLD在增殖性狼疮性肾炎（LN）患者的活检标本中高表达。
CYLD抑制MCs中TLR3激活后RIG-I和MDA5表达的转录后调控。CYLD可能参与CKD的发病机制，尤其是LN的发病机制。
