Activation of c-Src: a hub for exogenous pro-oxidant-mediated activation of Toll-like receptor 4 signaling

Abstract

To study the role of c-Src kinase in pro-oxidant-induced stimulation of Toll-like receptor 4 (TLR4), we used lipopolysaccharide from Escherichia coli K12 (LPS-EK) and monophosphoryl lipid A, as TLR4-specific agonists and positive controls, and SIN-1 and potassium peroxychromate as pro-oxidant sources. We used the HEK-Blue mTLR4 cell line, which is stably transfected with mouse TLR4 and expresses optimized SEAP reporter under the control of a promoter inducible by NF-κB transcription factor. The level of SEAP released due to TLR4 stimulation was a measure of NF-κB activation. Treatment with either the pro-oxidants or LPS-EK increased SEAP release and TNF-α production in these cells. These treatments also increased intracellular reactive oxygen species accumulation, with an enhanced production of nitric oxide and TBARS to confirm oxidant stress in these cells. Pretreatment with c-Src kinase inhibitors, PP2 and Ca-pY, which act by different mechanisms, decreased these parameters. Pretreatment with SSG, a c-Src activator, enhanced the effects promoted by LPS-EK and pro-oxidants and rescued cells from the PP2- and Ca-pY-induced effects. Curiously, pro-oxidants, but not TLR4 agonist, increased the ratio of TNF-α to IL-10 released, suggesting that pro-oxidants can initiate and maintain an imbalance of TNF-α production over IL-10. To different degrees, both pro-oxidants and TLR4 agonist increased formation of c-Src complexes with TLR4 and IκB-α as coimmunoprecipitates. Both pro-oxidants and TLR4 agonist increased c-Src phosphorylation of the Tyr42 residue in IκB-α, but the pro-oxidant-induced effect was more robust and much longer lasting. Taken together, these studies provide a mechanism whereby c-Src assumes a central role in pro-oxidant-induced NF-κB activation in TLR4 signaling. Pro-oxidant-induced activation of TLR4 through c-Src/NF-κB/IκB-α coupling provides a basis for a molecular dissection of the initiation and maintenance of sterile inflammation that may serve as a "pathophysiologic primer" for many diseases.

Keywords: Free radicals; IκB-α; NF-κB; Pathophysiologic primer; Pro-oxidant; Sterile inflammation; Toll-like receptor 4; c-Src.

Fig. 1. Effect of c-Src inhibitors on secreted embryonic alkaline phosphatase (SEAP) release in mono-phosphoryl lipid A (MPLA)- and pro-oxidants (PPC and SIN-1)-induced HEK-Blue mTLR4 activation

Cells were pre-incubated with various inhibitors for 30 min followed by overnight (~ 16 h) incubation with MPLA, PPC, or SIN-1 in the continued presence of the inhibitors. SEAP release in the conditioned medium was determined using Quanti-Blue, and the absorbance was read at 650 nm. The color-coded column in each Panel (A to C) represents treatment with MPLA, PPC or SIN-1 alone, respectively. The data represent 5 independent experiments conducted in duplicate. ⁺p ≤ 0.05, ^*p ≤ 0.01 and ^#p ≤ 0.001

Fig. 2. Effect of TAK-1 inhibitors on secreted embryonic alkaline phosphatase (SEAP) release in mono-phosphoryl lipid A (MPLA)- and pro-oxidants (PPC and SIN-1)-induced HEK-Blue-mTLR4 activation

Cells were pre-incubated with various inhibitors for 30 min followed by overnight incubation with MPLA, PPC, or SIN-1 in the continued presence of the inhibitors. SEAP release in the conditioned medium was determined using Quanti-Blue, and the absorbance was read at 650 nm. The color-coded column in each Panel (A to C) represents treatment with MPLA, PPC or SIN-1 alone, respectively. The data represent 5 independent experiments. ⁺p ≤ 0.05, and ^#p ≤ 0.001.

Fig. 3. A dose-dependent effect of SIN-1 on secreted embryonic alkaline phosphatase (SEAP) release

Cells were treated with different concentrations of SIN-1 ranging from 1.0 to 500 μM for 16 h. SEAP release in the conditioned medium was determined using Quanti-Blue, and the absorbance was read at 650 nm. (A) Fold changes in SEAP release after treatment with different doses of SIN-1 in HEK-Blue mTLR4 cells and (B) Fold changes after treatment of HEK-Blue null-1v cells with the same concentrations of SIN-1 as in (A). Data represent the mean of four (4) independent experiments carried out in duplicate. *p = 0.05; #p ≤ 0.01

Fig. 4

(A) The effect of LPS-EK, PPC and SIN-1 on secreted embryonic alkaline phosphatase (SEAP) release in HEK-Blue null 1-v cells. Cells were pre-treated with c-Src inhibitors for 30 min followed by incubation overnight with MPLA, PPC, or SIN-1 in the continued presence of the inhibitors. SEAP release in the conditioned medium was determined using Quanti-Blue, and the absorbance was read at 650 nm. The color-coded column in each panel represents treatment with MPLA (Panel A), PPC (Panel B) or SIN-1 (Panel C) alone, respectively. The data represent 5 independent experiments. (B) Effect of TLR4 agonists and PPC on accumulation of intracellular reactive oxygen species (ROS) in HEK-null 1-v cells. Cells were incubated overnight with MPLA, LPS-EK, or PPC. Then, cells were incubated with CellRox® and NucBlue live cell stain for 30 min at 37 °C followed by subsequent PBS washes and fixation with 4 % paraformaldehyde for 15 min. After subsequent washing with PBS, images were acquired using fluorescence microscope. Scale bar = 50 μm.

Fig. 5. Comparative effects of LPS-EK and PPC on c-Src activation in HEK-Blue mTLR4 cells

Cells were pre-incubated with various inhibitors for 30 min followed by stimulation with either LPS-EK (A) or PPC (B) for 20 min in continued presence of the inhibitors. c-Src activation in the cells were determined with FACE™ c-Src kit according to the manufacturer’s instructions. The data are expressed as % ratios of [pTyr418] c-Src (activated) to total c-Src present with respect to the effect of agonist treatment alone. Data represent 3 independent experiments carried out in duplicate with ⁺p ≤ 0.05, ^*p ≤ 0.01 and ^#p ≤ 0.001.

Fig. 6. Immunofluorescence representation of levels of intracellular ROS produced following stimulation of HEK-Blue mTLR4 cells with LPS EK or PPC alone and/or in combination with different inhibitors

Cells were pre-incubated with various inhibitors for 30 min followed by stimulation with LPS-EK (A) or PPC (B) overnight in continued presence of the inhibitors. Cells were then incubated with CellRox® and NucBlue live cell stain for 30 min at 37 °C. Cells were washed in PBS and fixed with 4 % paraformaldehyde for 15 min. After subsequent washes with PBS, images were acquired using fluorescence microscope. (Figs. 6A & 6B) are merged representative pictures of the immunofluorescence data with scale bar 50 μm. (Figs. 6C & 6D) are semi-quantitative histograms of (Figs. 6A & 6B) generated using ImageJ™ software. The data represent 4 independent experiments. pY = Caffeic-pYEEIE; Eb = Ebselen; ⁺p ≤ 0.05; p ≤ 0.01; ^#p ≤ 0.001.

Fig. 7. Effect of c-Src inhibitors used alone and in combination on Nitric oxide production in HEK-Blue mTLR4 cells stimulated with LPS-EK or PPC

Cells were pre-incubated with Ca-pY or Eb for 30 min or SSG for 2 h, followed by stimulation with LPS-EK (A) or PPC (B) overnight in the continued presence of the inhibitors. Culture media was separated from cells, cleared by centrifugation and used to quantify total nitric oxide levels as nitrite/nitrate according to the manufacture’s instructions. The data represent 3 independent experiments conducted in duplicates. Ca-pY = Caffeic-pYEEIE; Eb = Ebselen; SSG = sodium stibogluconate; ⁺p = 0.05.

Fig. 8. Effect of c-Src inhibitors used alone and in combination on lipid peroxides produced in HEK-Blue-mTLR4 cells stimulated with LPS-EK and PPC

Cells were pre-incubated with Ca-pY or Eb for 30 min or SSG for 2 h, followed by stimulation with LPS-EK (A) or PPC (B) overnight in the continued presence of the inhibitors. Cells from which the supernatants were used to quantify total nitric oxide were scraped, lysed and prepared for use to quantify malondialdehyde (MDA) as thiobarbituric acid reacting substances (TBARs) according to the manufacturer’s instructions. The data represent 3 independent experiments carried out in duplicates. Ca-pY = Caffeic-pYEEIE; Eb = Ebselen; SSG = sodium stibogluconate. ⁺p ≤ 0.01.

Fig. 9. Effect of c-Src inhibition on the production of TNF-α and IL-10 following stimulation of HEK-Blue mTLR4 cells with LPS-EK or PPC

Cells were pre-incubated with inhibitors for 30 min followed by stimulation with LPS-EK (A & C) or PPC (B & D) overnight in continued presence of the inhibitors. TNF-α and IL-10 levels in the conditioned medium were determined using their respective ELISA kits according to the manufacturer’s instructions. The ratios of TNF-α to IL-10 following treatments with LPS-EK (E) and PPC (F) were quantified. The data represent 4 independent experiments conducted in duplicate. ⁺p ≤ 0.05, ^*p ≤ 0.01 and ^#p ≤ 0.001 (A & C) TNF-α and IL-10 production in LPS-EK- stimulated cells and (B & D) TNF-α and IL-10 production in PPC-stimulated cells.

Fig. 10. Immunoprecipitation complexes formed by c-Src interactions with TLR4 or with IκB-α in the NFκB activation pathway

Cells were stimulated with MPLA, LPS-EK or PPC (A & B) for 4 h followed by immunoprecipitation with TLR4 (A) or with IκB-α (B). In both cases, coimmunoprecipitates were detected by Western blot with polyclonal anti-c-Src. Experiments were repeated three times with essentially the same results.

Fig. 11. Effect of LPS-EK and PPC on [pTyr42] IκB-α formation and IκB-α degradation in HEK-Blue mTLR4 cells

Cells were stimulated with LPS-EK (A) or PPC (B) for 0, 30, 60, 90, 120 and 180 min. I-κB-α (Y42) formation against IκB-α degradation was determined by Western blots. The time-course graphs (C & D) represent the optical density (OD) ratios of IκB-α (Y-42) and IκB-α immunoblot signals from (A & B) normalized to those of β-actin from the same test groups. The data represent 3 independent experiments with ⁺p ≤ 0.05, ^*p ≤ 0.01 and ^#p ≤ 0.001 compared to time-matched corresponding treatments. For the immunofluorescence data (E & F), cells were incubated with FITC-conjugated secondary antibody and NucBlue live cell stain corresponding to treatments with LPS-EK (A) and PPC (B). Images were acquired using fluorescence microscope (scale bar = 50 μm).

Fig. 12. A simplified schematic representation of a putative mechanism for exogenous prooxidant-induced TLR4 signaling in c-Src-mediated NFκB activation

TLR4 is stimulated in response to exogenous oxidants resulting in the activation of c-Src, which interacts with TLR4. The formation of TLR4/c-Src complex leads to the recruitment of different cytosolic adaptor proteins such as myeloid differentiation 88 protein (MyD88), toll-interleukin 1 receptor adaptor protein (TIRAP), TNF receptor-associated factor 6 (TRAF6), etc. resulting in c-Src/NFκB/IκBα coupled activation. This would result in c-Src phosphorylation of the Tyr42 residue in IκBα kinase to form [pTyr42] IκBα that delays ubiquitination and degradation by proteosome that leads to activation of NFκB for a longer time. Following release of the complex, p50/p65 eventually translocates to the nucleus to induce the expression of new genes.