Fig. 6.
Type II transmembrane serine protease Hepsin mediates epithelial integrity and BM defects as well as transformation in Lkb1-deficient 3D acini. (A) Loss of Lkb1 (Lkb1lox/lox and W-Myc;Lkb1lox/lox) in the 3D acini enhances immunostaining of Hepsin and leads to its redistribution from cell borders to cytosol. (Scale bar: 20 μm.) (B) Overexpression of Hepsin in 3D MMEC acini (3-d culture) results in Hepsin redistribution and asymmetrical, distorted acinar morphology as well as filling of the luminal space. E-cadherin visualizes the cell borders in figures. (Scale bar: 20 μm.) (C) Overexpression of Hepsin in 3D MMEC acini induces disruption of apicobasal polarity and deterioration of BM as indicated by mislocalization of ZO-1 and uneven staining of nidogen. (Scale bar: 20 μm.) (D) shRNA silencing of Hepsin rescues loss of Lkb1-induced epithelial phenotypes. The graph shows mean and SD values from three independent experiments. (E) shRNA silencing of Hepsin rescues loss of Lkb1-induced BM defect. (Scale bar: 20 μm.) (F) Phase-contrast images of day 10 W-Myc;Lkb1lox/lox MMEC acini transduced with pLKO.1 shHepsin demonstrate partial reversion of the branching/aggregation (transformation) phenotype. (Scale bar: 100 μm.) (G) Mean and SD of branching/aggregating acini quantitated from three independent experiments. (H) shRNA silencing of Hepsin rescues BM defect in acini with Myc and Lkb1 loss double lesion. (Scale bar: 20 μm.) (I) Loss of Lkb1 induces concomitant loss of desmoplakin and Hepsin from desmosomal junctions. (Left) Colocalization of desmoplakin and Hepsin in cell-cell borders. (Center and Right) Ablation of desmoplakin and Hepsin form cell-cell borders in AdCre-infected Lkb1lox/lox and W-Myc;Lkb1lox/lox MMECs grown in 2D cell culture. (Scale bar: 20 μm.) (J) Dissociation of Hepsin and desmoplakin from cell-cell junctions on calcium depletion. For the calcium switch assay, MMECs were treated with EGTA for 30 min, after which EGTA was removed and the cells were incubated for an additional 2 h. (Scale bar: 20 μm.)