EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation
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EGFR phosphorylation inhibitor, PD168393, effectively suppressed LPS-induced TLR4 phosphorylation (Fig. 1I). In addition, LPS-induced TLR4 phosphorylation was dramatically decreased in EGFR−/− BMDM (Fig. 1J). 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. 1K and 1L). 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. 1M and 1N). 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. 1O). 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. 1P). 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 inhibitor decreased cell surface but not total TLR4 expression in response to LPS, we hypothesized that EGFR phosphorylation contributes to the transportation of TLR4 from the Golgi apparatus to the cell surface. Golgi marker GM130 applied to visualize the spatial relationship between the Golgi apparatus and TLR4 in BMDM. As shown in Fig. 1Q, LPS treatment reduced the co-localization of GM130 and TLR4, and PD168393 pretreatment partially restored the co-localization between Golgi and TLR4. Meanwhile, LPS lost its ability to reduce the co-localization between GM130 and TLR4 in EGFR−/− BMDM (Fig. 1R). These data suggested that TLR4 is transported from Golgi to cell surface following LPS treatment and this is regulated by EGFR phosphorylation.
Epidermal growth factor receptor pathway substrate 8 (EPS8)/related to the N-terminus of tre oncogene (RN-TRE) and growth-factor receptor-bound protein 2 (GRB2)/Ras and Rab interactor 1 (RIN1) have been reported to coordinate the function of Rab5 GEFs and GTPase-activating proteins (GAPs) for the maintenance of normal trafficking of cell membrane receptors (Mendelsohn and Baselga, 2006; Chen et al., 2012). LPS increased EPS8 and GRB2 mRNA and protein expression, and this was significantly inhibited by PD168393 pretreatment (Fig. S5A, S5B and S5E). LPS also increased RN-TRE expression, but this was not suppressed by PD168393 (Fig. S5C and S5D). LPS did not affect the expression of RIN1 (Fig. S5C and S5E). We further demonstrated using coimmunoprecipitation that GRB2 and EPS8 associated with TLR4 at 6 h after LPS treatment (Fig. S5F), and these findings were visualized by immunofluorescence imaging in BMDM cells (Fig. S5G). We found knockdown of any of EPS8/RN-TRE/GRB2/RIN1significantly suppressed cell surface expression of TLR4 at 24 h following LPS treatment (Fig. S5H and S5I), suggesting that EPS8/RN-TRE/GRB2/RIN1 serve as a signaling pathway mediating activation of Rab5a and subsequent upregulation of macrophage surface expression of TLR4 and EGFR in response to LPS.
We measured LPS binding to the cell, detected activation of p38, and ERK1/2 as the downstream signaling of TLR4, and measured cytokine release from the macrophages. Binding of Alexa Fluor®488-conjugated LPS to BMDM was gradually increased over the 24 h after LPS treatment, and PD168393 significantly decreased LPS binding (Fig. S6A and S6B). LPS significantly promoted the phosphorylation of p38 and ERK1/2 at 6, 12 and 24 h after LPS treatment, and these changes in phosphorylation were partially suppressed by PD168393 pretreatment (Fig. S6C). PD168393 also inhibited LPS-induced reactive oxygen species (ROS) production in BMDM at 12 h and 24 h after LPS (Fig. S6D–E). Lastly, we demonstrated that PD168393 markedly inhibited LPS-induced expression of IL-1β, IL-10, IL-6 and TNF-α in both BMDM and RAW264.7 cells at 24 h after LPS treatment (Figs. S6F and S7). Furthermore, knockdown of EPS8, GRB2, or Rab5a in BMDM suppressed LPS-induced cytokine expression at 24 h (Fig. 6G) and phosphorylation of p38 and ERK1/2 (Fig. S6H). In addition, LPS failed to induce phosphorylation of p38 and ERK1/2 in BMDM isolated from EGFR−/− or Rab5a−/− mice (Fig. S6I), and failed to induce the expression of cytokines (Fig. S6J).
RAW264.7 cells and BMDM were treated with LPS for 24 h followed by assessment of cell death. As shown in Fig. S8A–D, LPS increased cell death in RAW264.7 cells and BMDM and this was attenuated with PD168393 pretreatment, which prevents the up-regulation of TLR4 cell surface expression. At 24 h after LPS treatment, macrophages were collected from peritoneal lavage fluid from the mice and macrophages identified. As shown in Fig. S8E and S8F, macrophage death increased from 2.2% to 14.1% in response to LPS, and erlotinib pretreatment significantly prevented LPS-induced macrophage death. Necroptosis and pyroptosis are two major types of cell death known to be induced by LPS (Pilla et al., 2014; Li et al., 2016). At 12 h and 24 h after LPS stimulation DNA fragmentation and caspase-1 activation, as detected by flow cytometry, occurred in BMDM (Fig. S9A and S9C). Morphologically, we observed nuclear condensation and enlarged cell size plus caspase-1 activation (Fig. S9B) by using confocal microscopy at 24 h after LPS treatment. These cellular alterations are known characteristics of pyroptosis (Miao et al., 2011). EGFR phosphorylation inhibitor PD168393 suppressed this macrophage pyroptosis (Fig. S9A–D). In addition, immunoblotting and confocal microscopy showed that LPS induced association of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 in BMDM, a key molecular event that drives cell necroptosis (Fig. S9E and S9F).
This study elucidates a novel mechanism, in which Rab5a plays an important role in promoting macrophage surface expression of TLR4 after LPS stimulation. EGFR phosphorylation leads to the activation of its substrates EPS8 and GRB2, which, in turn, activate ras effector RIN1 and GAP protein RN-TRE. RIN1 and RN-TRE work together to coordinate the equilibrium of GTP and GDP-bound forms of Rab5a to secure the process of receptor internalization. Importantly, receptor internalization is an essential step to promoting increased cell surface expression of TLR4, as well as enhanced inflammation in response to LPS. This study, at least in part, elucidates why EGFR inhibitor is able to attenuate inflammatory responses to LPS and protect endotoxemic animals from death. In addition, during experiments, we included both male and female mice, and thus, the EGFR-mediated enrichment of TLR4 cell surface expression is not sex-dependent.
This work was supported by the National Institutes of Health Grant R01-HL-079669 (J.F. and M.A.W.), National Institutes of Health Grant R01-HL-139547 (J.F. and M.A.W.), National Institutes of Health Grant R01HL076179 (P.W. and J.F.), VA Merit Award 1I01BX002729 (J.F.), VA BLR&D Research Career Scientist Award BX004211 (J.F.), National Natural Science Foundation of China 81671957 (J.T.), Key projects of Guangdong Natural Science Foundation 2018B030311038 (J.T.), Science and Technology Planning Project of Guangdong Province 2016A020215212 (J.T.), and National Institutes of Health Grant R01GM102146 (M.J.S).
J.T., B.Z., L.C., D.L., E.K.F., Y.L. Q.W., planned and did experiments including cell isolation and treatment, confocal microcopy, Western blotting, and flow cytometry; J.T. and B.Z. did animal experiments; J.T., M.J.S., T.R.B., M.A.W., P.W., and J.F. planned the project and conceived the experiments; J.T., M.J.S., and J.F. conceived the data and wrote the manuscript.
Jing Tang, Bowei Zhou, Melanie J. Scott, Linsong Chen, Dengming Lai, Erica K Fan, Yuehua Li, Qiang Wu, Timothy R. Billiar, Mark A. Wilson, Ping Wang and Jie Fan declare no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
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