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

• Published in: Cellular physiology and biochemistry Vol. 42; no. 6; pp. 2507 - 2522
• Main Authors: Somensi, Nauana, Brum, Pedro Ozorio, de Miranda Ramos, Vitor, Gasparotto, Juciano, Zanotto-Filho, Alfeu, Rostirolla, Diana Carolina, da Silva Morrone, Maurilio, Moreira, José Claudio Fonseca, Pens Gelain, Daniel
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland S. Karger AG 01-10-2017; Cell Physiol Biochem Press GmbH & Co KG
• Subjects: A549 Cells; Antibiotics; Antigens; Asthma; Cancer therapies; Chemokines; Cytokines; Cytokines - genetics; Cytokines - metabolism; Disease; eHSP70; ERK; Heat shock proteins; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - metabolism; HSP70 Heat-Shock Proteins - pharmacology; Humans; Immunoblotting; Immunoprecipitation; Inflammation; JNK Mitogen-Activated Protein Kinases - metabolism; Kinases; Ligands; Lung cancer; Lung Neoplasms - metabolism; Lung Neoplasms - pathology; Metastasis; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; NF-kappa B - genetics; NF-kappa B - metabolism; NF-κB; Original Paper; Phosphorylation; RAGE; Real-Time Polymerase Chain Reaction; Receptor for Advanced Glycation End Products - antagonists & inhibitors; Receptor for Advanced Glycation End Products - genetics; Receptor for Advanced Glycation End Products - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; RNA Interference; RNA, Small Interfering - metabolism; Rodents; Signal transduction; Signal Transduction - drug effects; Transcriptional Activation; Tumorigenesis; U937 Cells; Brazil; NF-κB; Inflammation; RAGE; Lung cancer; eHSP70; ERK
• ISSN: 1015-8987; 1421-9778
• DOI: 10.1159/000480213

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.