A Repurposed Drug Screen for Compounds Regulating Aquaporin 5 Stability in Lung Epithelial Cells.

John Villandre,Yanwen Chen,Yuan Liu,Emily Vaiz,Beyza Tuncer,Ferhan Tuncer,Bill B Chen,Dan Camarco,Travis B Lear,Karina Lockwood,Virginia White,John Evankovich

doi:10.3389/fphar.2022.828643

John Villandre, Yanwen Chen + Show 10 more

Open Access

https://doi.org/10.3389/fphar.2022.828643

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Abstract

Aquaporin 5 (AQP5) is expressed in several cell types in the lung and regulates water transport, which contributes to barrier function during injury and the composition of glandular secretions. Reduced AQP5 expression is associated with barrier dysfunction during acute lung injury, and strategies to enhance its expression are associated with favorable phenotypes. Thus, pharmacologically enhancing AQP5 expression could be beneficial. Here, we optimized a high-throughput assay designed to detect AQP5 abundance using a cell line stably expressing bioluminescent-tagged AQP5. We then screened a library of 1153 compounds composed of FDA-approved drugs for their effects on AQP5 abundance. We show compounds Niclosamide, Panobinostat, and Candesartan Celexitil increased AQP5 abundance, and show that Niclosamide has favorable cellular toxicity profiles. We determine that AQP5 levels are regulated in part by ubiquitination and proteasomal degradation in lung epithelial cells, and mechanistically Niclosamide increases AQP5 levels by reducing AQP5 ubiquitination and proteasomal degradation. Functionally, Niclosamide stabilized AQP5 levels in response to hypotonic stress, a stimulus known to reduce AQP5 levels. In complementary assays, Niclosamide increased endogenous AQP5 in both A549 cells and in primary, polarized human bronchial epithelial cells compared to control-treated cells. Further, we measured rapid cell volume changes in A549 cells in response to osmotic stress, an effect controlled by aquaporin channels. Niclosamide-treated A549 cell volume changes occurred more rapidly compared to control-treated cells, suggesting that increased Niclosamide-mediated increases in AQP5 expression affects functional water transport. Taken together, we describe a strategy to identify repurposed compounds for their effect on AQP5 protein abundance. We validated the effects of Niclosamide on endogenous AQP5 levels and in regulating cell-volume changes in response to tonicity changes. Our findings highlight a unique approach to screen for drug effects on protein abundance, and our workflow can be applied broadly to study compound effects on protein abundance in lung epithelial cells.

Highlights

The family of aquaporin proteins (AQP) display tissue and cellspecific expression patterns to regulate water transport across various membranes
Niclosamide increased AQP5, but not AQP4, in human bronchial epithelial (HBE) cells (n 6 wells/condition) (Figures 5E–G). These results suggest that Niclosamide stabilizes AQP5 by preventing its ubiquitination and degradation; further, the effects of Niclosamide to increase AQP5 are observed on endogenous protein in A549 and primary HBE cells
In A549 cells, treatment with Niclosamide resulted in greater AQP5 levels assayed by immunoblot, as well as greater cell volume changes as measured by calcein fluorescence in hypertonic media. These results suggest that increased AQP5 abundance in response to Niclosamide is associated with an increase in functional water transport, providing a link between Niclosamide treatment and the physiologically relevant cellular effect of water transport

Summary

Introduction

The family of aquaporin proteins (AQP) display tissue and cellspecific expression patterns to regulate water transport across various membranes. Aquaporin-1 (AQP1), Aquaporin-4 (AQP4), and Aquaporin-5 (AQP5) are expressed in several cells types (Verkman, 2007; Yadav et al, 2020). Preclinical models of murine acute lung injury show a pattern where AQP5 protein abundance is reduced in the lung following injury (Towne et al, 2000; Hasan et al, 2014; Vassiliou et al, 2017; Zhang J. et al, 2018), and some studies suggest restoring its expression could be protective (Jiang et al, 2015). AQP5 deficiency has been implicated in preclinical models of xerostomia, sialadenitis, and Sjogren’s syndrome, and restoration of aquaporin channels in these tissues is associated with increased saliva secretion (Lai et al, 2016). Given the rationale that increasing AQP5 levels could be beneficial in a number of disease states, we set out to determine whether compounds from a repurposed drug library could affect total AQP5 levels

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