Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance - PubMed (original) (raw)

Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance

Andrei E Medvedev et al. J Biol Chem. 2007.

Abstract

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse DeltaTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IkappaB-alpha degradation, and activation of NF-kappaB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-kappaB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.

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Figures

Fig. 1

Fig. 1. The P712H mutation in the TIR domain of mouse HA-ΔTLR4 abolishes TLR4 signaling and tyrosine phosphorylation

HEK293T cells were transiently transfected with expression vectors encoding HA-tagged ΔTLR4 WT, P712H mutant, or a control vector pCDNA3. After 48 h, total RNA was isolated to examine IL-8 mRNA levels by real-time PCR (A), cell-free supernatants were collected to determine IL-8 production (B), and cell extracts prepared for Western analysis of IRAK-1, IκB-α, p38 phosphorylation, and tubulin expression (C). TLR4 species were also immunoprecipitated with α-HA Ab and subjected to immunoblotting with α-HA Ab (total TLR4 expression) and α-phosphotyrosine Ab (TLR4 phosphorylation) (D). Results of a representative experiment (n=3) are shown.

Fig. 2

Fig. 2. The effect of the PGV714−716 mutation in the TIR domain of human CD4-TLR4 on cell activation and TLR4 tyrosine phosphorylation

CD4-TLR4 WT or PGVP714−716AAA mutant (designated P714A) was overexpressed in HEK293T cells, and transfection with pCDNA3 was used as a control. After 48 h, IL-8 gene expression was examined by real-time PCR (A), IL-8 production was determined in supernatants by ELISA (B), and Western analysis of IRAK-1 phosphorylation, IκB-α degradation, and p38 phosphorylation was performed in cellular extracts. Tubulin expression was assessed as a loading control (C). CD4-TLR4 was immunoprecipitated with α-CD4 Ab and analyzed by immunoblotting with α-Flag Ab (total TLR4 expression) and α-phosphotyrosine Ab (TLR4 phosphorylation) (D). Data from a representative experiment (n=4) are shown.

Fig. 3

Fig. 3. Tyrosine-alanine substitutions in the TIR domain impair TLR4-induced signaling

(A) and (B): HEK293T cells were transiently transfected with expression vectors encoding either CD4-TLR4 WT, CD4-TLR4 P714H mutant, or CD4-TLR4 species expressing Y674A and Y680A mutations. As a control, pCDNA3 was transfected. (A): NF-κB (pELAM-lucifearse) and RANTES (pGL3-RANTES-lucifearse) reporters were co-expressed along with pCMV-β-galactosidase reporter, cells were recovered for 24h, and luciferase vs. β-galactosidase activities were measured. *p<0.005, **p<0.05 reflect statistically significant differences in reporter activation between CD4-TLR4 WT and the indicated mutants. (B): TLR4 species were immunoprecipitated with α-CD4 Ab and subjected to immunoblotting with α-Flag Ab (total TLR4 expression) and α-phosphotyrosine Ab (TLR4 phosphorylation). (C) and (D): Full-length WT, P714H, and Y674A TLR4 species were overexpressed in HEK293T cells along with CD14 and MD-2, in control cultures, pcDNA3 transfection was carried out. (C): NF-κB (pELAM-lucifearse) and pCMV-β-galactosidase reporters were co-transfected, and cells were stimulated with medium or 100 ng/ml LPS for 24 h. Luciferase vs. β-galactosidase activities were measured in cell extracts. (D): After recovery for 24 h, YFP-TLR4 proteins were immunoprecipitated with α-GFP Ab, and total TLR4 expression was examined by immunoblotting with α-GFP Ab. (B) and (D): Cell lysates were subjected to Western analysis of JNK and p38 phosphorylation and IκB-α degradation. β-actin immunoblots show a loading control. The middle band in the JNK immunoblot represents an unspecific band. Results of a representative experiment (n=5) are presented.

Fig. 4

Fig. 4. The effect of the Y674A and Y680A mutations on constitutive (CD4-TLR) and LPS-inducible (full-length YFP-TLR4) interactions of TLR4 with MyD88 and IRAK-1 species

WT, Y674A and Y680A variants of CD4-TLR4 were overexpressed in HEK293T cells with (A) or without (B) YFP-MyD88. CD4-TLR4 species were immunoprecipitated with α-Flag-agarose, and TLR4 interactions with YFP-MyD88 (A) or IRAK-1 (B) were examined using α-GFP or α-IRAK-1 Abs. Total CD4-TLR4 expression was measured by immunoblot analysis of TLR4 immune complexes with α-Flag-HRP, and YFP-MyD88 expression was estimated in immunoprecipitated YFP-MyD88 using α-GFP Ab. (C) and (D): WT, Y674A YFP-TLR4 variants were co-expressed in HEK293T cells along with CD14 and MD-2. LPS stimulation was carried out as indicated (C) or for 5 min (D). YFP-TLR4s or endogenous MyD88 were immunoprecipitated, and TLR4, endogenous MyD88 and IRAK-1 expression was analyzed by immunoblotting with α-GFP, α-MyD88, and α-IRAK-1 Abs, respectively. Results of a representative experiment (n=4) are shown.

Fig. 5

Fig. 5. TLR2 and TLR4 agonists induce tyrosine phosphorylation of respective TLRs

HEK293T cells were transiently transfected with pCDNA3-huCD14, pEFBOS-HA-huMD-2, along with either pCMV-1-Flag-huTLR2 (A,B) or pCDNA3-huTLR4 (C). Cells were recovered for 24 h, followed by stimulation with TLR2 (STF and Pam3Cys, A and B) and TLR4 (LPS, C) agonists. TLR2 and TLR4 were immunoprecipitated from cell extracts with α-Flag and α-TLR4 H80 Ab, respectively, and subjected to immunoblot analysis for total TLR2 (α-Flag Ab) or TLR4 (H80) expression, as well as TLR tyrosine phosphorylation (α-phosphotyrosine Ab PY20). Shown are data of one out of three experiments.

Fig. 6

Fig. 6. The effect of protein tyrosine kinase and Src kinase inhibitors on TLR4 phosphorylation and signal transduction

HEK293/TLR4/MD-2 stable transfectants were pretreated for 45 min with genistein (100 μM), herbimycin A (1 μM), PP1 and PP2 (10 μM each), followed by stimulation with LPS for 15 min (A) or 6 h (B). In NF-κB reporter assays (B), NF-κB (pELAM-luciferase) and β-galactosidase (pCMV-β-galactosidase) reporters were co-transfected. (A): TLR4 tyrosine phosphorylation was analyzed in TLR4 immune complexes and total cell lysates vs. TLR4 total expression using α-phosphotyrosine (PY20) and α-TLR4 Abs. LPS-inducible phosphorylation of p38 was measured with α-phosphop38 Ab, and equal protein loading was controlled by running β-actin immunoblot. (B). NF-κB reporter activation was examined in cell extracts by analysis of luciferase vs. β-galactosidase activities. Shown are results of a representative (n=4) experiments.

Fig. 7

Fig. 7. Endotoxin tolerance induction inhibits TLR4 tyrosine phosphorylation in HEK293/TLR4/MD-2 stable transfectants but does not affect TLR4-MD-2 interactions

Cells were pretreated with for 20 h medium or 10 ng/ml LPS, followed by washing and restimulation with 100 ng/ml LPS as shown. (A): RNA was isolated, reverse-transcribed, and analyzed by real-time PCR for IL-8 gene expression. *p<0.05. (B) TLR4 proteins were immunoprecipitated from cell extracts with α-TLR4 Ab H80 (B) or α-phosphotyrosine Ab PY20 (C). TLR4 immune complexes (B) were analyzed with α-phosphotyrosine Ab PY20 (TLR4 phosphorylation), α-TLR4 Ab H80 (TLR4 total expression), or α-Flag Ab to estimate amounts of Flag-MD-2 associated with TLR4. Immunoprecipitated phosphotyrosine proteins (C) were subjected to immunoblot analysis using α-TLR4 Ab H80. Total cellular extracts were analyzed by immunoblotting with α-TLR4 antiserum to measure total TLR4 protein expression (C). Results of a representative experiment (n=4) are depicted.

Fig. 8

Fig. 8. Suppressed LPS-inducible tyrosine phosphorylation of TLR4 in endotoxin-tolerant human monocytes

Human monocytes were pretreated for 20 h with medium or 10 ng/ml LPS, washed and restimulated with 100 ng/ml LPS over time courses (A and C), or for 1h (IFN-β mRNA induction) and 3 h (TNF-α gene expression, B). *p<0.005; **p<0.05. Cellular extracts were examined by immunoblotting for IκB-α degradation and p38 phosphorylation (A). B: RNA was isolated and subjected to reverse transcription and real-time PCR analysis of TNF-α and IFN-β mRNA levels. (C) Cellular extracts were subjected to immunoprecipitation with α-TLR4 Ab H80 followed by immunoblotting with α-phosphotyrosine Ab PY20 (TLR4 tyrosine phosphorylation). Total TLR4 expression was analyzed by immunoblotting with α-TLR4 antiserum. Results of a representative experiment (N =7) are shown.

Fig. 9

Fig. 9. LPS-induced recruitment of Lyn to TLR4 is suppressed in LPS-tolerant HEK/TLR4/MD-2 cells

Flag-Lyn was overexpressed in HEK293/TLR4/MD-2 stable transfectants, cells pretreated for 20 h with medium or 10 ng/ml LPS, followed by LPS stimulation as shown. Recruitment of Flag-Lyn to TLR4 was examined in TLR4 immunoprecipitates by immunoblotting with α-Flag-HRP or in Flag-Lyn immunoprecipitates immunoblotted with α-TLR4 antibody. Total expression of Flag-Lyn was measured in Lyn immune complexes with α-Flag-HRP, and total TLR4 expression was determined by immunoblotting of cellular extracts with α-TLR4 antibody. β-actin immunoblot was used to control for protein loading. The results of a representative experiment (n=3) are presented.

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