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TNF? selectively activates the IRE1?/XBP1 endoplasmic reticulum stress pathway in human airway smooth muscle cells.

ABSTRACT: Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNF? mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNF? triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mitofusin 2 (Mfn2). In hASM cells isolated from lung specimens incidental to patient surgery, dose- and time-dependent effects of TNF? exposure were assessed. Exposure of hASM to tunicamycin was used as a positive control. Tempol (500 ?M) was used as superoxide scavenger. Activation of three ER stress pathways were evaluated by Western blotting: 1) autophosphorylation of inositol-requiring enzyme1 (IRE1?) leading to splicing of X-box binding protein 1 (XBP1); 2) autophosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) leading to phosphorylation of eukaryotic initiation factor 2?; and 3) translocation and cleavage of activating transcription factor 6 (ATF6). We found that exposure of hASM cells to tunicamycin activated all three ER stress pathways. In contrast, TNF? selectively activated the IRE1?/XBP1 pathway in a dose- and time-dependent fashion. Our results indicate that TNF? does not activate the PERK and ATF6 pathways. Exposure of hASM cells to TNF? also decreased Mfn2 protein expression. Concurrent exposure to TNF? and tempol reversed the effect of TNF? on IRE1? phosphorylation and Mfn2 protein expression. Selective activation of the IRE1?/XBP1 pathway in hASM cells after exposure to TNF? may reflect a unique homeostatic role of this pathway in the inflammatory response of hASM cells.

SUBMITTER: Yap J

PROVIDER: S-EPMC7099431 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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TNFα selectively activates the IRE1α/XBP1 endoplasmic reticulum stress pathway in human airway smooth muscle cells.

Yap John J Chen Xujiao X Delmotte Philippe P Sieck Gary C GC

American journal of physiology. Lung cellular and molecular physiology 20200115 3

Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNFα mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNFα triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mit ...[more]

PMID: 31940218

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Project description:Accumulation of damaged or misfolded proteins resulted from oxidative protein modification induces endoplasmic reticulum (ER) stress by activating the pathways of unfolded protein response. In pathologic hemolytic conditions, extracellular free hemoglobin is submitted to rapid oxidation causing heme release. Resident cells of atherosclerotic lesions, after intraplaque hemorrhage, are exposed to heme leading to oxidative injury. Therefore, we raised the question whether heme can also provoke ER stress. Smooth muscle cells are one of the key players of atherogenesis; thus, human aortic smooth muscle cells (HAoSMCs) were selected as a model cell to reveal the possible link between heme and ER stress. Using immunoblotting, quantitative polymerase chain reaction and immunocytochemistry, we quantitated the markers of ER stress. These were: phosphorylated eIF2α, Activating transcription factor-4 (ATF4), DNA-damage-inducible transcript 3 (also known as C/EBP homology protein, termed CHOP), X-box binding protein-1 (XBP1), Activating transcription factor-6 (ATF6), GRP78 (glucose-regulated protein, 78kDa) and heme responsive genes heme oxygenase-1 and ferritin. In addition, immunohistochemistry was performed on human carotid artery specimens from patients who had undergone carotid endarterectomy. We demonstrate that heme increases the phosphorylation of eiF2α in HAoSMCs and the expression of ATF4. Heme also enhances the splicing of XBP1 and the proteolytic cleavage of ATF6. Consequently, there is up-regulation of target genes increasing both mRNA and protein levels of CHOP and GRP78. However, TGFβ and collagen type I decreased. When the heme binding proteins, alpha-1-microglobulin (A1M) and hemopexin (Hpx) are present in cell media, the ER stress provoked by heme is inhibited. ER stress pathways are also retarded by the antioxidant N-acetyl cysteine (NAC) indicating that reactive oxygen species are involved in heme-induced ER stress. Consistent with these findings, elevated expression of the ER stress marker GRP78 and CHOP were observed in smooth muscle cells of complicated lesions with hemorrhage compared to either atheromas or healthy arteries. In conclusion, heme triggers ER stress in a time- and dose-dependent manner in HAoSMCs. A1M and Hpx as well as NAC effectively hamper heme-induced ER stress, supporting their use as a potential therapeutic approach to reverse such a deleterious effects of heme toxicity.

Dataset Information

TNF? selectively activates the IRE1?/XBP1 endoplasmic reticulum stress pathway in human airway smooth muscle cells.

Publications

TNFα selectively activates the IRE1α/XBP1 endoplasmic reticulum stress pathway in human airway smooth muscle cells.

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