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

Abstract

Summary To study the role of c-Src kinase in prooxidant-induced stimulation of TLR4, we used LPS-EK and MPLA as TLR4 specific agonists and positive controls, and SIN-1 and PPC as prooxidant sources. We used HEK-Blue mTLR4 cell line that is stably transfected with mouse TLR4 and that 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 prooxidants or LPS-EK increased SEAP release and TNF-α production in these cells. These treatments also increased intracellular ROS 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 prooxidants, and rescued cells from PP2- and Ca-pY-induced effects. Curiously, prooxidants but not TLR4 agonist increased the ratio of TNFα to IL-10 released suggesting that prooxidants can initiate and maintain an imbalance of TNFα production over IL-10. To different degrees, both prooxidant and TLR4 agonist increased formation of c-Src complexes with TLR4 and IκB-α as coimmunoprecipitates. Both prooxidant and TLR4 agonist increased c-Src phosphorylation of Tyr-42 residue in IκB-α, but prooxidant-induced effect was more robust and much longer lasting. Taken together, these studies provide a mechanism whereby c-Src assumes a central role in prooxidant-induced NF-κB activation in TLR4 signaling. Prooxidant-induced activation of TLR4 through c-Src/NFκB/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.

Toll-like receptor 4 signaling: A common pathway for interactions between prooxidants and extracellular disulfide high mobility group box 1 (HMGB1) protein-coupled activation

“…Mechanistically, this is of significance because it confirms that HMGB1 can be released from cells either actively in a regulated process, or passively during necrosis [41, 42]. Our previous work showed that over 94 % of cells remained viable after treatment with PPC (5 μM) and SIN-1 (1.0 mM) as used in the present experiments [32]. These results suggest that prooxidant-induced HMGB1 release from HEK-Blue mTLR4 cells is not caused by cell death, but is likely due to an active process regulated by the levels of iROS.…”

“…We had previously shown in vivo and in vitro that PPC could activate NF-κB mainly through a TLR4-dependent pathway, which was attenuated by prior treatment with antioxidants [23,32]. Our initial experimental evidence for the involvement of prooxidants in promoting HMGB1-induced NF-κB activation is based on SEAP and TNF-α release, and inhibition of their release with TLR4 neutralizing pAb and HMGB1 (BoxA) fragment, a specific antagonist of HMGB1.…”

“…Following subsequent washes with PBS, images were acquired using fluorescence microscope (Axiovert 200M; Zeiss) at excitation and emission wavelengths (λs) of ~644/655 nm for CellROX ® Deep Red reagent and 405/410-550 nm for NucBlue ® . Finally, acquired images were merged and composite histograms were obtained and analyzed [32] with ImageJ (V1.44, http://rsbweb.nih.gov/ij/) software.…”

“…Extracellularly released HMGB1 due to injury can potentially activate TLR4 in an autocrine or paracrine fashion. TLR4 is stimulated in response to exogenous oxidants resulting in the activation of c-Src, which interacts with TLR4 [32]. Increased formation of TLR4/c-Src complex leads to enhanced recruitment of different cytosolic adaptor proteins including toll-interleukin 1 receptor adaptor protein (TIRAP) that results in c-Src/NFκB/IκBα coupled activation.…”

In vitro and in vivo anti-inflammatory activity of Phyllanthus acidus methanolic extract

“…Genistein, a broad-spectrum inhibitor of tyrosine kinases, has been shown to display strong antiinflammatory activity (Kim et al, 2005). Moreover, suppression of Syk or Src by knockdown or conditional knockout strategies resulted in macrophages that failed to express various proinflammatory cytokines Karki et al, 2014;Miller et al, 2012). Although the exact mechanism by which Src and Syk activate NF-κB is not fully understood, these data strongly indicate that Src and Syk could play critical roles in TLR-mediated inflammatory responses and could also be the pharmacological targets of Pa-ME.…”