EGFR signaling augments TLR4 cell surface expression and function in macrophages via regulation of Rab5a activation

摘要

EGFR activation promotes TLR4 phosphorylation and cell surface expression of TLR4 in response to LPS. (A and B) BMDM were treated with LPS (1 μg/mL) for 6, 12, or 24 h in the presence or absence of pretreatment of PD or TAPI-1. (A) Flow cytometry analysis of cell surface TLR4 intensity in BMDM. (B) Flow cytometry analysis of cell surface TLR4 intensity in BMDM. (C and D) WT (C57BL/6) mice were treated with LPS (10 mg/kg, i.p.). In some groups, mice were pretreated with erlotinib (100 mg/kg, gavage administration) at 30 min prior to LPS i.p. Peritoneal lavage fluids were collected at 24 h after LPS treatment and peritoneal macrophages were identified with F4/80. TLR4 intensity on the surface of peritoneal macrophage was analyzed by flow cytometry. (E and F) BMDM isolated from WT and mice were treated with LPS (1 μg/mL) for 1 h followed by flow cytometry analysis of cell surface TLR4 intensity. (G and H) WT (C57BL/6) and mice were treated with LPS (10 mg/kg, i.p.) for 24 h. Peritoneal lavage fluids were collected, and peritoneal macrophages were identified with F4/80. TLR4 intensity on the surface of peritoneal macrophage was analyzed by flow cytometry. (I) Western blot analysis of phosphor-TLR4 in BMDM treated with LPS (1 μg/mL) for 30 min with or without PD168393 (PD, 10 μmol/L) pretreatment for 30 min. (J) Western blot analysis of phosphor-TLR4 in BMDM treated with LPS (1 μg/mL) for 30 min. (K–N) HEK293 cells were transfected with , , , , or mutant for 48 h, with treatment of LPS (1 μg/mL) for 30 min or 24 h. (K) Diagram of the TLR4 phosphorylation site mutated plasmid. (L) Western blot analysis of the phosphor-TLR4 and phosphor-EGFR in transfected HEK293 treated with LPS for 30 min. (M and N) Flow cytometry analysis of cell surface TLR4 intensity in transfected HEK293 treated with LPS for 24 h. (O and P) BMDM were treated with LPS (1 μg/mL) for 30 min with or without PD168393 (PD) pretreatment for 30 min. (O) Immune-staining of TLR4 and EGFR in BMDM. (P) Co-immunoprecipitation of TLR4 with EGFR in BMDM. (Q) Immune-staining of TLR4 and GM130 in BMDM treated with LPS (1 μg/mL) for 24 h with or without PD168393 pretreatment for 30 min. (R) Immune-staining of TLR4 and GM130 in BMDM treated with LPS (1 μg/mL) for 24 h. All images and flow cytometric plots are the representatives from at least 4 experiments. The graphs depict mean ± SD of four to six experiments or mice. * < 0.05 as compared with control group; † < 0.05 as compared with the time-matched LPS alone group