Pro-Inflammatory Cytokine Tnf Alpha Induces Endoplasmic Reticulum Stress Through Reactive Oxygen Species Generation In Human Airway Smooth Muscle Cells

Airway inflammation plays a key role in asthma. In various diseases, inflammation leads to an increase in reactive oxygen species (ROS) generation and is associated with accumulation of unfolded proteins leading to endoplasmic reticulum (ER) stress. In response to ER stress, a homeostatic signaling cascade is triggered to restore normal function by halting protein translation and activating protein degradation and expression of molecular chaperones involved in protein folding. Three ER stress protein markers located at the ER membrane are involved in this signaling cascade: protein kinase RNA‐like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol‐requiring enzyme 1 (IRE1α). In particular, phosphorylation of IRE1α catalyzes the alternative splicing of XBP1 mRNA leading to expression of the active XBP1 transcription factor. In this study, we hypothesize that pro‐inflammatory cytokine TNFα induces ROS production triggering ER stress in human airway smooth muscle (hASM). hASM cells were isolated from lung specimen incidental to patient surgery and exposed to TNFα (20 ng/ml) for 1, 3, 6, 12 or 24 h. To measure ROS production, hASM cells were loaded with the general oxidative stress indicator CM‐H2DCFDA or with the mitochondrial superoxide indicator MitoSOX™ Red. The CM‐H2DCFDA and MitoSOX™ Red signals were visualized using a Nikon A1R confocal imaging system. Images were analyzed using ImageJ. Exposure of hASM to tunicamycin (2 μg/ml) was used as a positive control for ER stress. Expression of IRE1α and PERK were determined by Western blot. Phosphorylation of IRE1α was also evaluated by immunoprecipitation and Western blot (Wes Simple Western™ system). The expression of the spliced isoform of the transcription factor XBP1 was examined by quantitative real‐time PCR (RT‐PCR). We found that ROS production increased in hASM within 1 h of TNFα exposure, followed by an increase in IRE1α phosphorylation within 1–3 h. Finally after 3–6 h, there was an increase in alternative splicing of XBP1 mRNA. Together these results show that the pro‐inflammatory cytokine TNFα increases ROS generation in hASM cells and induces an ER stress response that includes an increase in IRE1α phosphorylation and splicing of XBP1. This ER stress response may affect many downstream targets, among which we believe Mfn2 is a major target which affects mitochondrial Ca 2+ buffering, with specific effects on hASM force and/or increased cell proliferation associated with asthma. Support or Funding Information NIH grant HL126451 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .