title: IL-13 desensitizes β2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism.
abstract: BACKGROUND
  β2-Adrenergic receptor (β2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of β2-agonists is well studied, type 2 inflammation could also affect β2AR function.
  
  
  OBJECTIVE
  We sought to evaluate the effect of the type 2 cytokine IL-13 on β2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2).
  
  
  METHODS
  HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and β2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2.
  
  
  RESULTS
  Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in β2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients.
  
  
  CONCLUSIONS
  IL-13 treatment of HAECs leads to β2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) β2ARs, suggesting that the beneficial effects of β2-agonists could be blunted in patients with type 2 associated asthma.
authors.affiliation: University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa; Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Palermo, Italy.
authors.forename: Giusy D
authors.initials: GD
authors.lastname: Albano
authors.affiliation: University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa. Electronic address: zhaoj2@upmc.edu.
authors.forename: Jinming
authors.initials: J
authors.lastname: Zhao
authors.affiliation: University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Emily B
authors.initials: EB
authors.lastname: Etling
authors.affiliation: Center for Research on Health Care Data Center, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Seo Young
authors.initials: SY
authors.lastname: Park
authors.affiliation: University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Haizhen
authors.initials: H
authors.lastname: Hu
authors.affiliation: University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: John B
authors.initials: JB
authors.lastname: Trudeau
authors.affiliation: Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Palermo, Italy.
authors.forename: Mirella
authors.initials: M
authors.lastname: Profita
authors.affiliation: University of Pittsburgh Asthma Institute at UPMC, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pa.
authors.forename: Sally E
authors.initials: SE
authors.lastname: Wenzel
mesh_terms: D000328:Adult; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001249:Asthma / Q000175:diagnosis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D016022:Case-Control Studies; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D018793:Interleukin-13 / Q000378:metabolism* / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D049488:Phosphatidylethanolamine Binding Protein / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*
chemical_list: D018793:Interleukin-13; C511482:PEBP1 protein, human; D049488:Phosphatidylethanolamine Binding Protein; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP; D001093:Arachidonate 15-Lipoxygenase; D054769:G-Protein-Coupled Receptor Kinase 2; D007545:Isoproterenol
keywords: 15-lipoxygenase 1; G protein receptor kinase 2; IL-13; asthma; cAMP; phosphatidylethanolamine binding protein 1; β(2)-adrenergic receptor
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