The Na+, K+-ATPase β1 subunit regulates epithelial tight junctions via MRCKα
Haiqing Bai, … , Jennifer L. Young, David A. Dean
Haiqing Bai, … , Jennifer L. Young, David A. Dean
Published January 28, 2021
Citation Information: JCI Insight. 2021;6(4):e134881. https://doi.org/10.1172/jci.insight.134881.
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Research Article Cell biology Pulmonology

The Na+, K+-ATPase β1 subunit regulates epithelial tight junctions via MRCKα

Abstract

An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well understood. Here, we show that overexpression of the NKA β1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport–independent mechanism. Using IP and mass spectrometry, we identified a number of unknown protein interactions of the β1 subunit, including a top candidate, myotonic dystrophy kinase–related cdc42-binding kinase α (MRCKα), which is a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA β1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with acute respiratory distress syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a potentially novel mechanism by which NKA regulates epithelial tight junctions and have identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.

Authors

Haiqing Bai, Rui Zhou, Michael Barravecchia, Rosemary Norman, Alan Friedman, Deborah Yu, Xin Lin, Jennifer L. Young, David A. Dean

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Figure 2

Overexpression of the β1 subunit increases alveolar type I barrier function.

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Overexpression of the β1 subunit increases alveolar type I barrier funct...
(A) Overexpression of the β1 subunit in HEK293T cells increased occludin expression at the plasma membrane. Caveolin-1 was used as membrane loading control. (B) Densitometry of gels in A, with analysis by Student’s t test, **P < 0.01. (C) ATII cells were cotransfected with 4 mg/mL pCMV-Tet3G plasmid and 16 mg/mL pTet3G-human β1 plasmid day 1 after isolation. Cells were then plated on fibronectin-coated coverslips. Doxycycline (1 μg/mL) was added 48 hours later. Representative immunofluorescence staining of ATI cells shows that doxycycline-induced expression of the β1 subunit induces more mature tight junctions, as indicated by occludin (green) and zo-1 (red) staining. Images represent 3 independent experiments. Scale bar: 20 mm. (D) ATII cells were cotransfected as in C, but cells were plated on fibronectin-coated 12-well transwell plates. Twenty-four hours later at day 2, 1 μg/mL of doxycycline (dox) was added to induce β1 gene expression. TEER was measured every 24 hours. ANOVA followed by Bonferroni’s post hoc test was used for statistical analysis, *P < 0.05, **P < 0.01. (E) After TEER measurement at day 4, permeability to 3 kD Texas Red–dextran and 40 kD FITC-dextran was measured for a duration of 2 hours. Data are presented as mean ± SD. ANOVA followed by Bonferroni’s post hoc test was used for statistical analysis, **P < 0.01.