Ectodomain Shedding of RAGE and TLR4 as a Negative Feedback Regulation in High-Mobility Group Box 1-Activated Aortic Endothelial Cells

Abstract

Background/Aims: High-mobility group box 1 (HMGB1) elicits inflammatory responses through interactions with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). We investigated how RAGE and TLR4 expressions are regulated after HMGB1 stimulation in cultured human aortic endothelial cells (HAECs). Methods: RAGE and TLR4 expressions were analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorogenic substrate. Results: Upon treatment with HMGB1, both RAGE and TLR4 began to decrease in cell lysate and remained decreased up to 24 h. The decrease in cellular RAGE and TLR4 was accompanied by an increase of N-terminal fragment of RAGE and TLR4 in culture supernatant, indicating ectodomain shedding of the receptors. HMGB1 activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while HMGB1-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. HMGB1-induced ectodomain shedding of RAGE and TLR4 was prevented by siRNA depletion of ADAM17 as well as TAPI-2, an inhibitor of ADAM family, and SB203580. HMGB1 pretreatment abolished p38 MAPK activation in response to 2nd HMGB1 stimulation. In the cells depleted of ADAM17, HMGB1-induced p38 MAPK activation was prolonged. siRNA depletion of RAGE, but not TLR4, suppressed HMGB1-induced p38 MAPK activation. Conclusion: In response to HMGB1 stimulation, HAECs rapidly undergo ectodomain shedding of RAGE and TLR4, and thereby become insensitive to further HMGB1 stimulation. ADAM17, activated through RAGE-p38 MAPK pathway, is implicated in the ectodomain cleavage of the receptors.

Systemic T Cell Exhaustion Dynamics Is Linked to Early High Mobility Group Box Protein 1 (HMGB1) Driven Hyper-Inflammation in a Polytrauma Rat Model

“…This study is the first indication of diminished levels of surface expression of RAGE and TLR4 on T cells in response to overwhelming levels of HMGB1 in vivo. In an in vitro study, the authors stimulated human aortic endothelial cells (HAECs) with a high concentration of HMGB1 and demonstrated that as time progressed, HAECs rapidly underwent ectodomain shedding of RAGE and TLR4 in response to HMGB1 and was concentration and time-dependent, which caused the cells to become insensitive to further HMGB1 stimulation [ 19 ]. Similarly, in our study, the diminished expression levels of RAGE and TLR4 on T cells in PT could be due to receptor ectodomain shedding and T cell exhaustion/anergy because of the early and untimely activation of T cells by HMGB1.…”

“…However, in extremity trauma, such undertakings by the host’s immune system fail to support adaptive immunological and physiological conditions, leading to deleterious outcomes, thereby requiring therapeutic interventions to modulate mediators to maintain proper functioning. An in vitro study using aortic endothelial cells demonstrated that saturated levels of extracellular HMGB1 lead to cellular exhaustion, causing a negative feedback regulation and ectodomain shedding of RAGE and TLR4 receptors while rendering cells unresponsive to further stimuli [ 19 ]. However, these findings lack in vivo validation in a dysregulated inflammatory state.…”

Differential expression of soluble receptor for advanced glycation end-products (sRAGE) in mice susceptible or resistant to chronic colitis

“…Changes we observed in the levels of RAGE expression suggest that it is the epithelial cell response to T. muris infection that informs subsequent susceptibility to chronic inflammation. The reduction in the amount of RAGE present at the cell membrane we saw by flow cytometry and by immunohistochemistry immediately following T. muris infection could be caused by either internalisation of activated RAGE-ligand complexes or ADAM10-mediated shedding to produce sRAGE [21, 22]. Splice variants of Rage may also result in a truncated RAGE molecule or a modified and actively secreted decoy receptor [23, 24].…”

The role of the MAD2-TLR4-MyD88 axis in paclitaxel resistance in ovarian cancer

“…Secondly, TLR4 shedding can occur as a result of oxidative stress [45] and during cellular senescence reactive oxygen species (ROS) levels are known to be dramatically increased [27]. Indeed, TLR4 shedding may even represent a feedback mechanism to blunt hyper-reactive TLR4-ligand signalling which may occur due to HMGB1 release which also occurs during senescence [31,46]. HMGB1 was examined as part of this study however its expression was not found to be altered post knockdown, which may be as a result of its function as a secreted cytokine.…”