Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 1;309(7):L725-35. doi: 10.1152/ajplung.00346.2014. Epub 2015 Jul 24.

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.

Keywords: Mothers against decapentaplegic homolog-3; airway epithelium; asthma susceptibility; functional genetics; transforming growth factor-β-induced gene expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Asthma / genetics
  • Asthma / metabolism
  • Asthma / pathology
  • Bronchi / metabolism*
  • Bronchi / pathology
  • Cadherins / genetics
  • Cadherins / metabolism*
  • Epithelial Cells / metabolism*
  • Epithelial Cells / pathology
  • HEK293 Cells
  • Humans
  • Protein Binding
  • Respiratory Mucosa / metabolism*
  • Respiratory Mucosa / pathology
  • Smad3 Protein / genetics
  • Smad3 Protein / metabolism*
  • Transcription, Genetic*
  • Transforming Growth Factor beta1 / genetics
  • Transforming Growth Factor beta1 / metabolism*
  • Two-Hybrid System Techniques

Substances

  • Cadherins
  • PCDH1 protein, human
  • SMAD3 protein, human
  • Smad3 Protein
  • TGFB1 protein, human
  • Transforming Growth Factor beta1