Contribution of the WNK1 kinase to corneal wound healing using the tissue‐engineered human cornea as an in vitro model

Abstract

Damage to the corneal epithelium triggers important changes in the extracellular matrix (ECM) to which basal human corneal epithelial cells (hCECs) attach. These changes are perceived by integrin receptors that activate different intracellular signalling pathways, ultimately leading to re‐epithelialization of the injured epithelium. In this study, we investigated the impact of pharmacological inhibition of specific signal transduction mediators on corneal wound healing using both monolayers of hCECs and the human tissue‐engineered cornea (hTEC) as an in vitro 3D model. RNA and proteins were isolated from the wounded and unwounded hTECs to conduct gene profiling analyses and protein kinase arrays. The impact of WNK1 inhibition was evaluated on the wounded hTECs as well as on hCECs monolayers using a scratch wound assay. Gene profiling and protein kinase arrays revealed that expression and activity of several mediators from the integrin‐dependent signaling pathways were altered in response to the ECM changes occurring during corneal wound healing. Phosphorylation of the WNK1 kinase turned out to be the most striking activation event going on during this process. The inhibition of WNK1 by WNK463 reduced the rate of corneal wound closure in both the hTEC and hCECs grown in monolayer compared with their respective negative controls. WNK463 also reduced phosphorylation of the WNK1 downstream targets SPAK/OSR1 in wounded hTECs. These in vitro results allowed for a better understanding of the cellular and molecular mechanisms involved in corneal wound healing and identified WNK1 as a kinase important to ensure proper wound healing of the cornea.

Control of Podocyte and Glomerular Capillary Wall Structure and Elasticity by WNK1 Kinase

“…details have emerged only recently. WNK463 inhibits corneal wound healing and reduces filopodium formation in engineered corneas and cultured corneal epithelial cells where WNK1 is the predominant WNK isoform expressed (Desjardins et al, 2019). A pathway including WNK1, SPAK and NKCC1 mediates vascular tone and the pressor response to α 1 adrenergic agonists in WNK1 +/− mice (Bergaya et al, 2012).…”

“…WNK1 participates in fundamental biologic processes including angiogenesis, and mitosis (Xie et al, 2009(Xie et al, , 2013Tu et al, 2011;Gallolu Kankanamalage et al, 2016). WNK1 kinase is also involved in cell migration and adhesion where it controls cytoskeletal structure and migration-associated local cell volume fluctuations as demonstrated in endothelial cells, T cells, corneal epithelial cells and certain cancers, notably glioblastoma cells (Haas et al, 2011;Dbouk et al, 2014;Zhu et al, 2014;Kochl et al, 2016;Desjardins et al, 2019).…”

The Human Tissue-Engineered Cornea (hTEC): Recent Progress

“…In a recent attempt to further characterize the mechanistic details of the signal transduction pathways activated during corneal wound healing, we have shown that phosphorylation-mediated activation of the WNK1 kinase was one particularly important event occurring during hTEC wound healing [ 336 ] ( Figure 7 ). WNK1 is the founding member of a family that comprises four evolutionarily conserved serine–threonine kinases (WNK1, WNK2, WNK3, and WNK4) that share 85% homology over their kinase domains [ 337 ].…”

“…( C ) Cell lysates from the central and external areas of wounded hTECs were analyzed by immunoblotting to confirm the phosphokinase array results for the mediator WNK1. Actin was used as the loading control (Figure adapted from Reference [ 336 ] with the permission of Journal of Tissue Engineering and Regenerative Medicine).…”

In vitro reconstructed 3D corneal tissue models for ocular toxicology and ophthalmic drug development

“…Vesicating agents are reported to cause clinical and histopathological changes related to inflammation and blistering in the eye that are results of alkylation, oxidative stress, lipid peroxidation, and induction of inflammatory responses (Goswami et al 2016). Corneal models have been useful in studying molecular mechanisms of vesication to aid in the development of therapies against oxidative stress and vesicant-induced ocular injuries (Desjardins et al 2019;Kaluzhny et al 2020). Also, ocular surface diseases were addressed using an in vitro 3D stromal model of keratoconus disease (Karamichos et al 2012) and a model of endothelial cell maturation to study endothelial layer differentiation and injury (Hutcheon et al 2019).…”