HER2 activation results in β-catenin-dependent changes in pulmonary epithelial permeability

Abstract

The receptor tyrosine kinase human epidermal growth factor receptor-2 (HER2) is known to regulate pulmonary epithelial barrier function; however, the mechanisms behind this effect remain unidentified. We hypothesized that HER2 signaling alters the epithelial barrier through an interaction with the adherens junction (AJ) protein β-catenin, leading to dissolution of the AJ. In quiescent pulmonary epithelial cells, HER2 and β-catenin colocalized along the lateral intercellular junction. HER2 activation by the ligand neuregulin-1 was associated with tyrosine phosphorylation of β-catenin, dissociation of β-catenin from E-cadherin, and decreased E-cadherin-mediated cell adhesion. All effects were blocked with the HER2 inhibitor lapatinib. β-Catenin knockdown using shRNA significantly attenuated neuregulin-1-induced decreases in pulmonary epithelial resistance in vitro. Our data indicate that HER2 interacts with β-catenin, leading to dissolution of the AJ, decreased cell-cell adhesion, and disruption of the pulmonary epithelial barrier.

Keywords: adherens junction; cell adhesion; epithelial cell; human epidermal growth factor receptor-2; neuregulin-1; permeability; receptor tyrosine kinase; β-catenin.

Fig. 1.

Human epidermal growth factor receptor-2 (HER2) is associated with β-catenin in unstimulated pulmonary epithelial cells. Primary human alveolar type I and II (ATI and II), NuLi-1, A549, and H292 (A) and normal human bronchial epithelial (NHBE) cells (B) were grown at an air-fluid interface (ALI) and lysed, and HER2 was immunoprecipitated (IP) followed by Western immunoblotting (IB) performed for β-catenin. IgG served as a negative control. C: NuLi-1 cells were transfected with a HER2 yellow fluorescent protein (YFP) construct, and HER2-β-catenin was assessed by confocal microscopy for HER2 (green) and β-catenin (red). D and E: enlarged images of merged HER2-β-catenin staining (E, yellow). Arrows identify merged HER2-β-catenin staining along the basolateral membrane. n ≥ 3.

Fig. 2.

HER2 activation results in Y-654 phosphorylation of β-catenin. A: Western blot for p-Y-654 β-catenin and total β-catenin from confluent NuLi-1 cells grown at ALI stimulated with neuregulin-1 (NRG-1) (20 nM) with and without lapatinib. B: densitometry of p-Y-654 β-catenin/total β-catenin relative to corresponding control in NuLi-1 cells. C: Western blot for p-HER2 and total HER2 from confluent NuLi-1 cells grown at ALI stimulated with NRG-1 (20 nM) with and without lapatinib. D: Western blot for p-Y-654 β-catenin and total β-catenin from confluent NHBE cells grown at ALI stimulated with NRG-1 (20 nM) with and without lapatinib. E: densitometry of p-Y-654 β-catenin/total β-catenin relative to corresponding control in NHBE cells. F: Western blot for p-HER2 and total HER2 from confluent NHBE cells grown at ALI stimulated with NRG-1 (20 nM) with and without lapatinib. Data are presented as means ± SE. *P < 0.05, experiments, n ≥ 3.

Fig. 3.

HER2 activation is associated with β-catenin-E-cadherin dissociation. A: NuLi-1 cells grown at ALI were stimulated with NRG-1 (20 nM) with and without lapatinib, and E-cadherin immunoprecipitation followed by β-catenin and E-cadherin Western blotting was performed. B: densitometry of E-cadherin/β-catenin association by coimmunoprecipitation in NuLi-1 cells treated with and without lapatinib relative to corresponding control. C: NHBE cells grown at ALI were treated with NRG-1 and lapatinib followed by immunoprecipitation for E-cadherin and Western blotting for E-cadherin and β-catenin. D: densitometry of E-cadherin/β-catenin association by coimmunoprecipitation in NHBE cells treated with and without lapatinib relative to corresponding control. Data are presented as means ± SE. *P < 0.05, n ≥ 3.

Fig. 4.

HER2 activation results in decreased E-cadherin-mediated pulmonary epithelial cell adherence. A: adherence of calcein-acetoxymethyl (AM)-loaded NuLi-1 cells seeded onto rh-E-cadherin-Fc chimera protein-bound plates. NuLi-1 cells were loaded with calcein and seeded onto plates coated with an rh-E-cadherin Fc. Cells were treated with NRG-1 with and without lapatinib and lapatinib alone. Use of an E-cadherin-blocking antibody served as a control to demonstrate binding of cells to the plate through E-cadherin. B: adherence of calcein-AM-loaded NHBE cells in the same fashion as NuLi-1 cells. Data are presented as means ± SE. *P < 0.0001, **P < 0.001, †P < 0.005, n > 3.

Fig. 5.

HER2-mediated changes in epithelial resistance require β-catenin. β-Catenin Western blotting in NuLi-1 cells transfected with a β-catenin shRNA or a nontargeting (NT) shRNA. Normalized resistance in NuLi-1 cells transfected with a β-catenin shRNA and a NT shRNA were grown to confluence at ALI and exposed to NRG-1. Data are presented as means ± SE. *P < 0.05, n ≥ 3.

Fig. 6.

HER2-dependent signaling is ligand specific. NuLi-1 cells transfected with NT HER2 or EGFR shRNA were exposed to TGF (20 ng/ml), and Western blotting was performed for p-Y-654 β-catenin, total β-catenin, p-HER2, total HER2, p-EGFR, total EGFR, and actin. Representative blots are shown. n ≥ 3.