Cosin-Roger Jesus, Spalinger Marianne R, Ruiz Pedro A, Stanzel Claudia, Terhalle Anne, Wolfram Lutz, Melhem Hassan, Atrott Kirstin, Lang Silvia, Frey-Wagner Isabelle, Fried Michael, Scharl Michael, Hausmann Martin, Rogler Gerhard
Department of Gastroenterology and Hepatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
PLoS One. 2018 Feb 15;13(2):e0193003. doi: 10.1371/journal.pone.0193003. eCollection 2018.
Gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. Its lack in intestinal macrophages of Crohn's disease (CD) patients is correlated with loss of tolerance against the host gut flora. Gp96 has been stablished to be an essential chaperone for Toll-like receptors (TLRs). We studied the impact of gp96-knockdown on TLR-function in macrophages. TLR2 and TLR4 expression was only decreased but not abolished when gp96 was knocked-down in cell lines, whereas in a monocyte/macrophage specific knock-out mouse model (LysMCre) TLR4 was abolished, while TLR2 was still present. Lipopolysaccharide (LPS)-induced NF-κB activation was still observed in the absence of gp96, and gp96-deficient macrophages were able to up-regulate surface TLR4 upon LPS treatment, suggesting that there is another chaperone involved in the folding of TLR4 upon stress responses. Moreover, LPS-dependent pro-inflammatory cytokines were still expressed, although to a lesser extent in the absence of gp96, which reinforces the fact that gp96 is involved in regulating signaling cascades downstream of TLR4 are impaired upon loss of gp96. In addition, we have also found a reduced phosphorylation of ERK and p38 kinases and an impaired response upon CSF1R activation in gp96 deficient macrophages. Our findings indicate that the loss of gp96 not only impairs TLR4 signaling, but is also associated with a diminished phosphorylation of ERK and mitogen-activated stress kinases resulting in an impaired signalling through several receptors, including CSF1R.
Gp96是多种蛋白质底物的内质网伴侣蛋白。克罗恩病（CD）患者肠道巨噬细胞中缺乏Gp96与对宿主肠道菌群的耐受性丧失相关。Gp96已被确定为Toll样受体（TLR）的一种必需伴侣蛋白。我们研究了巨噬细胞中gp96基因敲低对TLR功能的影响。在细胞系中敲低gp96时，TLR2和TLR4的表达仅降低但未被消除，而在单核细胞/巨噬细胞特异性敲除小鼠模型（LysMCre）中，TLR4被消除，而TLR2仍然存在。在缺乏gp96的情况下，仍可观察到脂多糖（LPS）诱导的NF-κB激活，并且gp96缺陷型巨噬细胞在LPS处理后能够上调表面TLR4，这表明在应激反应时，还有另一种伴侣蛋白参与TLR4的折叠。此外，LPS依赖性促炎细胞因子仍有表达，尽管在缺乏gp96时表达程度较低，这进一步证明gp96参与调节TLR4下游的信号级联反应，在gp96缺失时这些信号级联反应受损。此外，我们还发现gp96缺陷型巨噬细胞中ERK和p38激酶的磷酸化减少，并且在CSF1R激活时反应受损。我们的研究结果表明，gp96的缺失不仅损害TLR4信号传导，还与ERK和丝裂原活化应激激酶的磷酸化减少有关，导致通过包括CSF1R在内的几种受体的信号传导受损。
