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Protocadherin-1 binds to SMAD3 and suppresses TGF-beta 1-induced gene transcription

Tellez, G. F., Vandepoele, K., Brouwer, U., Koning, H., Elderman, R. M., Hackett, T-L., Willemse, B. W. M., Holloway, J., Van Roy, F., Koppelman, G. H. & Nawijn, M. C., 1-Oct-2015, In : American Journal of Physiology - Lung Cellular and Molecular Physiology. 309, 7, p. L725-L735 11 p.

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  • Protocadherin-1 binds to SMAD3 and suppresses TGF-beta 1-induced gene transcription

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DOI

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-alpha 1 (TGF-beta 1)-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-beta 1 was measured in BEAS-2B cells transfected with PCDH1 and in 16HBE cells transfected with PCDH1-small-interfering RNA (siRNA). TGF-beta 1-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 over-expressing both proteins, and at endogenous protein levels in 16HBE cells. TGF-beta-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-beta target genes in BEAS-2B cells, whereas knockdown of PCDH1 in human PBECs increased TGF-beta-induced gene expression. In conclusion, we demonstrate that PCDH1 binds to SMAD3 and regulates its activation by TGF-beta 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-beta.

Original languageEnglish
Pages (from-to)L725-L735
Number of pages11
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume309
Issue number7
Publication statusPublished - 1-Oct-2015

    Keywords

  • Mothers against decapentaplegic homolog-3, airway epithelium, functional genetics, asthma susceptibility, transforming growth factor-beta-induced gene expression, EPITHELIAL-MESENCHYMAL TRANSITION, AIRWAY EPITHELIUM, ASTHMA, CELLS, BETA, EXPRESSION, ASSOCIATION, FAMILY, LUNG, PATHOPHYSIOLOGY

ID: 23076818