title: TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle.
abstract: Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca(2+) signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca(2+) entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP-BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca(2+) (EGTA; 1mM) or intracellular Ca(2+) (BAPTA; 5μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca(2+) influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca(2+) and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
authors.forename: Pawan K
authors.initials: PK
authors.lastname: Vohra
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Thompson
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA; Department of Physiology & Biomedical Engineering, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
authors.forename: Venkatachalem
authors.initials: V
authors.lastname: Sathish
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
authors.forename: Alexander
authors.initials: A
authors.lastname: Kiel
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
authors.forename: Calvin
authors.initials: C
authors.lastname: Jerde
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA; Department of Physiology & Biomedical Engineering, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Pabelick
authors.affiliation: Department of Biochemistry and Molecular Biology, University of North Dakota, 264 Centennial Dr, Grand Forks, ND 58202, USA.
authors.forename: Brij B
authors.initials: BB
authors.lastname: Singh
authors.affiliation: Department of Anesthesiology, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA; Department of Physiology & Biomedical Engineering, Mayo Clinic, 200 First St SW, Rochester, MN 55905, USA. Electronic address: Prakash.ys@mayo.edu.
authors.forename: Y S
authors.initials: YS
authors.lastname: Prakash
mesh_terms: D019208:Brain-Derived Neurotrophic Factor / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000473:pathology; D009130:Muscle, Smooth / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012137:Respiratory System / Q000473:pathology*; D050052:TRPC Cation Channels / Q000378:metabolism*
chemical_list: D019208:Brain-Derived Neurotrophic Factor; D011993:Recombinant Fusion Proteins; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D049452:Green Fluorescent Proteins; C579455:BDNF protein, human; D002118:Calcium
keywords: 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; ASM; Asthma; BAL; BAPTA; BDNF; ELISA; GFP; Inflammation; Lung; NGF; NT; Neurotrophin; ROI; SOCE; STIM1; Signaling; TLR; TNFα; TRPC; TrkB; Tropomyosin-related kinase; airway smooth muscle; brain-derived neurotrophic factor; bronchial alveolar lavage; enzyme linked immunosorbent assay; green fluorescent protein; nerve growth factor; neurotrophins; region of interest; siRNA; small interference ribose nucleic acid; store-operated calcium entry; stromal interactive molecule one; toll-like receptor; transient receptor potential channel; tropomyosin-related kinase; tumor necrosis factor-alpha
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