title: TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements.
abstract: BACKGROUND
  TREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.
  
  
  METHODS
  We determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.
  
  
  RESULTS
  Cytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.
  
  
  CONCLUSIONS
  Although cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells.
authors.affiliation: Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN, United States of America; Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwingshackl
authors.affiliation: Department of Biomedical Engineering, University of Memphis, Memphis, TN, United States of America.
authors.forename: Esra
authors.initials: E
authors.lastname: Roan
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Teng
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, TN, United States of America; Department of Medicine, University of Tennessee Health Science Center, Memphis, TN, United States of America.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Waters
mesh_terms: D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism*; D047630:Depsipeptides / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D018613:Microscopy, Confocal; D015739:Nocodazole / Q000494:pharmacology; D024683:Potassium Channels, Tandem Pore Domain / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D014404:Tubulin / Q000378:metabolism
chemical_list: D000199:Actins; D018932:Chemokine CCL2; D047630:Depsipeptides; D015850:Interleukin-6; D024683:Potassium Channels, Tandem Pore Domain; D014404:Tubulin; C103742:potassium channel protein TREK-1; C057531:jasplakinolide; D015638:Cytochalasin D; D015739:Nocodazole