title: CPI-17 silencing-reduced responsiveness in control and TNF-alpha-treated human bronchi.
abstract: Under pathophysiologic conditions, the modulation of Ca2+ sensitivity and reactivity of bronchial smooth muscle is controlled by protein kinase C-dependent phosphorylation of the newly described protein, CPI-17. The goal of the present study was to assess the key role of this regulatory protein in airway hyperresponsiveness (AHR) using control and TNF-alpha-treated human bronchi as well as a specific siRNA duplex against human CPI-17 transcripts. Validity of a mixed transfection strategy was assessed using the reversible permeabilization method to introduce X-TremeGene (X-TG)-siRNA complexes in an overreactive model of human bronchi treated with TNF. Data demonstrate that X-TG-siRNA complexes targeted against CPI-17 transcripts resulted in a reduction in mRNA and specific protein expression in human bronchial tissues. This approach revealed that overall reactivity of bronchial smooth muscle to methacholine was reduced, while their relaxing responses to beta2-agonist were increased, when compared with responses triggered in control TNF-alpha-treated bronchi. Quantification analysis showed that Ca2+ -sensitivity in both untreated and TNF-alpha-treated bronchi were largely reduced upon transfection with human CPI-17 siRNA-X-TremeGene complexes, while Western blot analysis corroborated the decrease in CPI-17 and MLC phosphorylation levels in pretreated human bronchi. Identical results were obtained upon treatment with an antiinflammatory eicosanoid, 14,15-EET, known to inhibit CPI-17 phosphorylation. Together these results are consistent with a key molecular role for CPI-17 in AHR, in the absence of bronchial wall remodeling.
authors.affiliation: Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
authors.forename: Caroline
authors.initials: C
authors.lastname: Morin
authors.forename: Marco
authors.initials: M
authors.lastname: Sirois
authors.forename: Vincent
authors.initials: V
authors.lastname: Echave
authors.forename: Eric
authors.initials: E
authors.lastname: Rousseau