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TNFα Induces Endoplasmic Reticulum Stress in Human Airway Smooth Muscle Cells
Author(s) -
Yap John Qian,
Delmotte Philippe,
Sieck Gary C
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.726.4
Subject(s) - endoplasmic reticulum , xbp1 , unfolded protein response , microbiology and biotechnology , mfn2 , inflammation , mitochondrial fission , chemistry , biology , mitochondrial fusion , mitochondrion , immunology , biochemistry , rna , rna splicing , mitochondrial dna , gene
Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL‐13) enhance cytosolic Ca 2+ concentration ([Ca 2+ ] cyto ) triggering airway smooth muscle (ASM) hyperreactivity (enhanced contractile state). Airway inflammation also induces ASM proliferation and remodeling (enhanced ASM synthetic state). In various diseases inflammation induces sarcoplasmic/endoplasmic reticulum (SR/ER) stress, which decreases SR/ER‐mitochondria interactions, with downstream effects on Ca 2+ regulation. In vascular smooth muscle cells, SR/ER stress promotes smooth muscle contraction and proliferation by disrupting SR/ER‐mitochondrial coupling. We hypothesized that inflammation induces SR/ER stress in human ASM, thereby altering expression of mitochondrial fission/fusion proteins (dynamin‐related protein ‐ Drp1 and mitofusin ‐ Mfn2) with downstream impact on ASM Ca 2+ regulation and contractility as well as proliferation. Human ASM cells were isolated from lung specimen incidental to patient surgery and exposed to TNFα or IL‐13 for 24h. Expression of SR/ER stress markers including PERK (protein kinase‐like endoplasmic reticulum kinase), Bip (Binding immunoglobulin protein also known as 78 kDa glucose‐regulated protein), PDI (protein disulfide isomerase), and mitochondrial fission/fusion proteins Drp1 and Mfn2 were evaluated by Western blot. The expression of the spliced isoform of the transcription factor XBP1 was investigated by quantitative real‐time PCR (RT‐PCR). For mitochondrial morphology (fission/fusion), ASM cells were loaded with Mitotracker Green and images analyzed using ImageJ and Matlab to quantify mitochondrial Form Factor and Aspect Ratio. The effect of inflammation induced SR/ER stress on human ASM proliferation was evaluated using CyQUANT assay. We found that after 24 h exposure to TNFα and IL‐13: 1) expression of SR/ER stress protein markers (PERK, BiP and XBP1) increased; 2) Drp1 expression increased and Mfn2 expression decreased; 3) mitochondrial fragmentation increased; 4) [Ca 2+ ] cyt and [Ca 2+ ] mito responses to ACh stimulation were uncoupled; 5) net [Ca 2+ ] cyt increased; 6) contractility increased; and 7) ASM cell proliferation increased. Together these results suggest that inflammation induces SR/ER stress that leads to changes in Mfn2 and Drp1 expression, which alter mitochondrial dynamics and Ca 2+ regulation resulting in ASM hyperreactivity and remodeling. Support or Funding Information NIH grant HL126451 (GCS).