Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells

Abstract

Background/Aims: Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal–regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. Methods: Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. Results: Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. Conclusions: Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells.

Extracellular Hsp70 modulates the inflammatory response of cigarette smoke extract in NCI‐H292 cells

“…JNK and p38 inhibitors exhibited a significant antagonistic effect with rhHsp70, and although they did not significantly reduce IL-1 secretion compared to rhHsp70 alone, this may indicate the role of JNK and p38 signalling pathways in the synthesis of this cytokine. These results are consistent with previous literature that showed that eHsp70 can activate MAPK and NF-B signalling pathways depending on the cell type (Somensi et al, 2017;Zhe et al, 2016).…”

Pro‐inflammatory effects of extracellular Hsp70 on NCI‐H292 human bronchial epithelial cell line

“…The effect of extracellular Hsp70 is mediated through cell surface receptors such as c‐type lectin receptors, scavenger receptors and pattern recognition receptors for advanced glycation end products (RAGE) as well as TLR2 and TLR4. It was demonstrated that eHsp70 can stimulate the production of pro‐inflammatory cytokines through activation of NF‐κB and MAPKs pathways when acting as TLR2/4 receptors agonist . Furthermore, signalling cascades triggered by various TLRs are known to interfere with each other, and cross‐talk between different signalling pathways activated by DAMPs or PAMPs may represent a regulatory mechanism that can control a potentially harmful immune response .…”

“…While intracellular Hsp70 exerts a powerful protective and anti‐inflammatory effect, extracellular Hsp70 (eHsp70) is considered as one of the DAMPs and therefore might be able to activate innate and inflammatory responses . eHsp70 can stimulate a pro‐inflammatory effect through binding to several receptors, most notably TLR2 and TLR4, followed by activation of mitogen‐activated protein kinases (MAPKs) and/or nuclear factor κB (NF‐κB) signal transduction pathways, resulting in cytokine production …”

“…8,10,11 eHsp70 can stimulate a pro-inflammatory effect through binding to several receptors, most notably TLR2 and TLR4, followed by activation of mitogen-activated protein kinases (MAPKs) and/or nuclear factor κB (NF-κB) signal transduction pathways, resulting in cytokine production. 8,[12][13][14][15] Hsp70 has been implicated in the pathogenesis of COPD. A case-control study reported that higher level of plasma Hsp70 might be associated with an increased risk of COPD among coal workers.…”

Major Differences in Hypoxia Tolerance and P38 Regulation Among Different Renal Cells

“…Among many signaling molecules, Mitogen-activated protein kinases (MAPKs) are early responders to insufficient oxygen. They are made up of four well-described subgroups, i.e.,the extracellular signal-regulated kinases-1 and -2 (ERK1/2), the c-Jun N-terminal kinases (JNK), p38 MAPKs, and extracellular signal-regulated kinase-5 (ERK5/BMK1) [4-6]. In general, ERK1/2 is relevant to cell proliferation/survival, while p38 is associated with cell apoptosis [7-13].…”