EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation
EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation
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EGFR 信号传导通过调节 Rab5a 激活增强巨噬细胞中 TLR4 细胞表面表达和功能
DOI：
10.1007/s13238-019-00668-8
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发表时间：
2019-11
期刊：
影响因子：
21.1
通讯作者：
Fan Jie
中科院分区：
文献类型：
--
作者：
Tang Jing;Zhou Bowei;Scott Melanie J.;Chen Linsong;Lai Dengming;Fan Erica K.;Li Yuehua;Wu Qiang;Billiar Timothy R.;Wilson Mark A.;Wang Ping;Fan Jie
Toll 样受体 4 (TLR4) 是感应细菌脂多糖 (LPS) 的关键受体，也是 Toll 样受体家族中研究最多的成员 (Kawai 和 Akira，2007；Kayagaki 等，2013；Klein 等，2015)。细胞表面 TLR4 表达由从高尔基体到细胞膜的受体运输与细胞表面受体内化到内体区室之间的平衡决定（Saltoh，2009）。在骨髓源性巨噬细胞（BMDM）中，我们观察到LPS诱导的BMDM表面EGFR磷酸化，并且这种磷酸化通过PD168393或TAPI-1预处理而被抑制（图S1）。接下来，我们测量了 LPS 处理后细胞表面 TLR4 表达的动态变化。 LPS处理后6、12和24小时，与对照相比，BMDM表面的TLR4表达分别增加～2、～6和～9倍。然而，EGFR 抑制剂 PD168393 在所有时间点均抑制 LPS 介导的 TLR4 细胞表面表达增加（图 1 A 和 1 B）。这些改变也在小鼠巨噬细胞系 RAW264 中得到证实。 7个细胞（图S2）。然后，在 LPS 前 30 分钟，对 C57BL/6 小鼠腹膜内 (ip) 注射 LPS (10 mg/kg)，有或没有 EGFR 抑制剂厄洛替尼 (100 mg/kg BW，强饲给药) 预处理。 LPS处理后24小时，收集腹腔巨噬细胞。 LPS 诱导巨噬细胞表面 TLR4 表达增加 5 倍，厄洛替尼预处理抑制 LPS 响应的 TLR4 细胞表面表达（图 1 C 和 1 D）。然后，用LPS处理来自EGFR-/-小鼠的BMDM细胞24小时，与WT BMDM细胞相比，LPS处理未能诱导EGFR-/- BMDM中TLR4的细胞表面表达增加（图1E和1F）。体内也显示出类似的结果（图1G和1H），其中与WT小鼠相比，来自EGFR−/−小鼠的腹膜巨噬细胞上的TLR4表面表达在LPS攻击后24小时没有增加。这些发现表明 EGFR 磷酸化对于 LPS 诱导的巨噬细胞中 TLR4 细胞表面表达的上调至关重要。 EGFR 磷酸化抑制剂 PD168393 有效抑制 LPS 诱导的 TLR4 磷酸化（图 1 I）。此外，LPS诱导的TLR4磷酸化在EGFR−/− BMDM中显着降低（图1J）。我们将TLR4 674和688 Tyr磷酸化位点突变为Ala。然后用MD2、CD14、EGFR和TLR4或TLR4突变体转染HEK293细胞。 LPS处理不能导致TLR4突变组中EGFR的磷酸化（图1K和1L）。我们还测量了 TLR4 磷酸化对 TLR4 细胞膜表达的影响。 LPS处理24小时后，与表达WT-TLR4的细胞相比，在TLR4突变体表达细胞中LPS诱导的TLR4细胞表面表达显着降低（图1M和1N）。 LPS处理后30分钟，LPS还诱导BMDM中TLR4和EGFR的共定位，并且这被EGFR磷酸化抑制剂PD168393抑制(图1O)。此外，这种TLR4和EGFR响应LPS的共定位也依赖于TLR4的磷酸化（图S3）。然而，在对照、LPS或LPS加PD168393预处理中，EGFR不与TLR4共免疫沉淀（图1P）。我们进一步发现，LPS 在 6、12 和 24 小时显着增加 EGFR，但不增加 TLR4、mRNA 和总蛋白表达，并且这种情况被 PD168393 预处理抑制（图 S4A-D）。大部分 TLR4 受体储存在亚细胞区室中，例如高尔基体和内体 (Husebye et al., 2006)。自从 EGFR ...
Toll-like receptor 4 (TLR4) is a key receptor sensing bacterial lipopolysaccharide (LPS), and is the most investigated member of the Toll-like receptor family (Kawai and Akira, 2007; Kayagaki et al., 2013; Klein et al., 2015). Cell surface TLR4 expression is determined by the balance between receptor trafficking from the Golgi apparatus to the cell membrane, and internalization of the cell surface receptor into endosomal compartments (Saltoh, 2009). In bone marrow-derived macrophages (BMDM), we observed LPS-induced EGFR phosphorylation on the surface of BMDM, and this was inhibited by pretreatment with PD168393 or TAPI-1 (Fig. S1). Next, we measured dynamic changes in cell surface TLR4 expression after LPS treatment. At 6, 12, and 24 h after LPS treatment, TLR4 expression on the surface of BMDM was increased∼ 2-,∼ 6-, and∼ 9-fold, respectively, as compared with controls. EGFR inhibitor PD168393, however, inhibited LPS-mediated increases in cell surface expression of TLR4 at all time points (Fig. 1 A and 1 B). These alterations were also confirmed in a mouse macrophage cell line, RAW264. 7 cells (Fig. S2). Then, C57BL/6 mice were injected intraperitoneally (ip) with LPS (10 mg/kg) with or without pretreatment of EGFR inhibitor, erlotinib (100 mg/kg BW, gavage administration) at 30 min prior to LPS. At 24 h after LPS treatment, peritoneal macrophages were collected. LPS induced 5-fold increases in TLR4 expression on the macrophage surface, and erlotinib pretreatment inhibited TLR4 cell surface expression in response to LPS (Fig. 1 C and 1 D). Then, BMDM cells from EGFR−/− mice were treated with LPS for 24 h and LPS treatment failed to induce increased cell surface expression of TLR4 in EGFR−/− BMDM compared with WT BMDM cells (Fig. 1 E and 1 F). Similar findings were shown in vivo (Fig. 1 G and 1 H), where in contrast to WT mice, TLR4 surface expression on peritoneal macrophages from EGFR−/− mice was not increased at 24 h after LPS challenge. These findings indicate that EGFR phosphorylation is essential for LPS-induced upregulation of TLR4 cell surface expression in macrophages. EGFR phosphorylation inhibitor, PD168393, effectively suppressed LPS-induced TLR4 phosphorylation (Fig. 1 I). In addition, LPS-induced TLR4 phosphorylation was dramatically decreased in EGFR−/− BMDM (Fig. 1 J). We mutated TLR4 674 and 688 Tyr phosphorylation site into Ala. Then HEK293 cells were transfected with MD2, CD14, EGFR and TLR4 or TLR4 mutant. LPS treatment could not lead to the phosphorylation of EGFR in TLR4 mutant group (Fig. 1 K and 1 L). We also measured the effect of TLR4 phosphorylation on TLR4 cell membrane expression. 24 h after LPS treatment LPS-induced cell surface expression of TLR4 was markedly decreased in TLR4 mutant-expressing cells compared with cells expressing WT-TLR4 (Fig. 1 M and 1 N). LPS also induced co-localization of TLR4 and EGFR in BMDM at 30min after LPS treatment, and this was suppressed by EGFR phosphorylation inhibitor PD168393 (Fig. 1 O). In addition, this kind of co-localization between TLR4 and EGFR in response to LPS also depended on the phosphorylation of TLR4 (Fig. S3). However, EGFR did not co-immunoprecipitate with TLR4 in control, LPS, or LPS plus PD168393 pretreatment (Fig. 1 P). We further found that LPS significantly increased EGFR, but not TLR4, mRNA and total protein expression at 6, 12 and 24 h, and this was inhibited by PD168393 pretreatment (Fig. S4A–D). A large proportion of TLR4 receptors are stored in subcellular compartments, such as the Golgi apparatus and endosomes (Husebye et al., 2006). Since EGFR …
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影响因子：
12.4
作者：
T. Kawai;S. Akira
通讯作者：
T. Kawai;S. Akira
影响因子：
2.8
作者：
S. Saitoh
通讯作者：
S. Saitoh
影响因子：
4.8
作者：
Chen, Yen-Jen;Hsieh, Ming-Yu;Leu, Tzeng-Horng
通讯作者：
Leu, Tzeng-Horng
影响因子：
4.5
作者：
Klein, Dionne C. G.;Skjesol, Astrid;Husebye, Harald
通讯作者：
Husebye, Harald
影响因子：
12.4
作者：
Li, Z.;Scott, M. J.;Fan, J.
通讯作者：
Fan, J.