Claudin-18–mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis
Beiyun Zhou, … , Edward D. Crandall, Zea Borok
Beiyun Zhou, … , Edward D. Crandall, Zea Borok
Published February 5, 2018
Citation Information: J Clin Invest. 2018;128(3):970-984. https://doi.org/10.1172/JCI90429.
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Research Article Pulmonology

Claudin-18–mediated YAP activity regulates lung stem and progenitor cell homeostasis and tumorigenesis

Abstract

Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18–/– AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18–/– mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Authors

Beiyun Zhou, Per Flodby, Jiao Luo, Dan R. Castillo, Yixin Liu, Fa-Xing Yu, Alicia McConnell, Bino Varghese, Guanglei Li, Nyam-Osor Chimge, Mitsuhiro Sunohara, Michael N. Koss, Wafaa Elatre, Peter Conti, Janice M. Liebler, Chenchen Yang, Crystal N. Marconett, Ite A. Laird-Offringa, Parviz Minoo, Kunliang Guan, Barry R. Stripp, Edward D. Crandall, Zea Borok

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Figure 3

Activation of YAP signaling in whole lung and alveolar epithelial type II (AT2) cells in Cldn18–/– mice.

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Activation of YAP signaling in whole lung and alveolar epithelial type I...
Representative immunofluorescence (IF) image shows increased nuclear YAP (A) and decreased cytoplasmic p-YAP (B) in lungs of adult Cldn18–/– mice (age ~2 months). n ≥ 3 mice of each genotype. DAPI (blue) is the nuclear counterstain. Scale bars: 20 μm. (C) Representative immunohistochemistry of lung sections at E18 shows increased nuclear YAP (brown) in Cldn18–/–mice. n = 3 for WT, 2 for Cldn18–/– mice. Hematoxylin is the nuclear counterstain (blue). Scale bars: 50 μm. (D) Western blot shows increased YAP and decreased p-YAP in Cldn18–/– AT2 cells (age ~2 months). Tubulin and β-actin are loading controls. n = 2 mice of each genotype. (E) Representative Phos-tag Western blot shows increased dephosphorylated YAP (arrow) in Cldn18–/– AT2 cells (age ~2 months). n = 3 independent experiments. (F) qRT-PCR shows increased expression of YAP target genes in Cldn18–/– versus WT lungs. n = 5 independent experiments. Z test. *P < 0.05. (G) Representative image shows increased expression of YAP target cyclin D1 (brown) in Cldn18–/– (age 2–9 months) mouse lung. Hematoxylin (blue) is the nuclear counterstain. n = 3 mice of each genotype. Scale bars: 50 μm. Western analysis (H) and quantification (I) of whole-lung samples demonstrate higher levels of cyclin D1 protein in Cldn18–/– compared with WT lungs. n = 4 mice of each genotype. Z test. *P < 0.05. (J) Representative image shows increased nuclear YAP (green) and decreased cytoplasmic p-YAP (red) in Cldn18–/– AT2 cells in 3D culture. PI (red) and DAPI (blue) are nuclear counterstains. Scale bars: 50 μm. n = 3 independent experiments. Bar graphs represent the mean ± SEM for F and I.