title: Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription.
abstract: Genetic studies have identified Protocadherin-1 (PCDH1) and Mothers against decapentaplegic homolog-3 (SMAD3) as susceptibility genes for asthma. PCDH1 is expressed in bronchial epithelial cells and has been found to interact with SMAD3 in yeast two-hybrid (Y2H) overexpression assays. Here, we test whether PCDH1 and SMAD3 interact at endogenous protein levels in bronchial epithelial cells and evaluate the consequences thereof for transforming growth factor-β1 (TGF-β1)-induced gene transcription. We performed Y2H screens and coimmunoprecipitation (co-IP) experiments of PCDH1 and SMAD3 in HEK293T and 16HBE14o(-) (16HBE) cell lines. Activity of a SMAD3-driven luciferase reporter gene in response to TGF-β1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-β1-induced gene expression was quantified in BEAS-2B clones overexpressing PCDH1 and in human primary bronchial epithelial cells (PBECs) transfected with PCDH1-siRNA. We confirm PCDH1 and SMAD3 interactions by Y2H and by co-IP in HEK293T cells overexpressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-β-induced activation of a SMAD3-driven reporter was reduced by exogenous PCDH1 in BEAS2B cells, whereas it was increased by siRNA-mediated knockdown of endogenous PCDH1 in 16HBE cells. Overexpression of PCDH1 suppressed expression of TGF-β target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-β-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-β signaling in bronchial epithelial cells. We propose that PCDH1 and SMAD3 act in a single pathway in asthma susceptibility that affects sensitivity of the airway epithelium to TGF-β.
authors.affiliation: University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Department of Pediatric Pulmonology and Pediatric Allergology, Groningen, The Netherlands; University of Groningen, University Medical Center Groningen, Experimental Pulmonology and Inflammation Research (EXPIRE), Department of Pathology & Medical Biology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom;
authors.forename: Grissel
authors.initials: G
authors.lastname: Faura Tellez
authors.affiliation: Department of Biomedical Molecular Biology, Ghent University & Inflammation Research Center, VIB, Ghent, Belgium;
authors.forename: Karl
authors.initials: K
authors.lastname: Vandepoele
authors.affiliation: University of Groningen, University Medical Center Groningen, Experimental Pulmonology and Inflammation Research (EXPIRE), Department of Pathology & Medical Biology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;
authors.forename: Uilke
authors.initials: U
authors.lastname: Brouwer
authors.affiliation: University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Department of Pediatric Pulmonology and Pediatric Allergology, Groningen, The Netherlands; University of Groningen, University Medical Center Groningen, Experimental Pulmonology and Inflammation Research (EXPIRE), Department of Pathology & Medical Biology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;
authors.forename: Henk
authors.initials: H
authors.lastname: Koning
authors.affiliation: University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Department of Pediatric Pulmonology and Pediatric Allergology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; Centre for Heart Lung Innovation and Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada; and.
authors.forename: Robin M
authors.initials: RM
authors.lastname: Elderman
authors.affiliation: Centre for Heart Lung Innovation and Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, St. Paul's Hospital, Vancouver, British Columbia, Canada; and.
authors.forename: Tillie-Louise
authors.initials: TL
authors.lastname: Hackett
authors.affiliation: University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Department of Pediatric Pulmonology and Pediatric Allergology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;
authors.forename: Brigitte W M
authors.initials: BW
authors.lastname: Willemse
authors.affiliation: Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom; Human Development and Health, Faculty of Medicine, University of Southampton, Southampton, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Holloway
authors.affiliation: Department of Biomedical Molecular Biology, Ghent University & Inflammation Research Center, VIB, Ghent, Belgium;
authors.forename: Frans
authors.initials: F
authors.lastname: Van Roy
authors.affiliation: University of Groningen, University Medical Center Groningen, Beatrix Children's Hospital, Department of Pediatric Pulmonology and Pediatric Allergology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands;
authors.forename: Gerard H
authors.initials: GH
authors.lastname: Koppelman
authors.affiliation: University of Groningen, University Medical Center Groningen, Experimental Pulmonology and Inflammation Research (EXPIRE), Department of Pathology & Medical Biology, Groningen, The Netherlands; GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; m.c.nawijn@umcg.nl.
authors.forename: Martijn C
authors.initials: MC
authors.lastname: Nawijn
mesh_terms: D001249:Asthma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D001980:Bronchi / Q000378:metabolism* / Q000473:pathology; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism* / Q000473:pathology; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D000091363:Protocadherins; D020545:Respiratory Mucosa / Q000378:metabolism* / Q000473:pathology; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D015820:Cadherins; C081683:PCDH1 protein, human; D000091363:Protocadherins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D053773:Transforming Growth Factor beta1
keywords: Mothers against decapentaplegic homolog-3; airway epithelium; asthma susceptibility; functional genetics; transforming growth factor-β-induced gene expression