Contribution of the WNK1 kinase to corneal wound healing using the tissue-engineered
human cornea as an in vitro model
SHORT RUNNING TITLE: Altered phosphorylation of WNK1 in corneal wound healing
Pascale Desjardinsa,b,c
, Camille Couturea,b,c
, Lucie Germaina,b,c and *Sylvain L. Guérina,b
aCUO-Recherche, Médecine Régénératrice – Centre de recherche du CHU de Québec -
Université Laval, Québec, Canada; and Centre de Recherche en Organogénèse expérimentale
de l’Université Laval/LOEX, bDépartement d’Ophtalmologie and cDépartement de Chirurgie,
Faculté de médecine, Université Laval, Québec, QC, Canada.
* Corresponding author:
Dr. Sylvain L. Guérin, CUO-Recherche/LOEX
Hôpital du Saint-Sacrement, Centre de recherche du CHU de Québec
Québec, QC, Canada
Phone: (418) 682-7565; Fax: (418) 682-8000
E-mail: [email protected]
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SUMMARY
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 signaling 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 tissue￾engineered human cornea (hTECs) 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 to 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.
KEYWORDS
Tissue-engineering, biomaterial, cornea, wound healing, WNK1 kinase, signal transduction
pathway
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1 INTRODUCTION
The eyes are one of the most precious sensory organs of the human body. They provide us
vision, which is essential to perceive and interact with our surrounding environment. The
functionality of the visual system relies on each structure composing the eye. One such
particularly important structure is the cornea. The cornea forms the transparent anterior
segment of the eye and accounts for three-fourths of the refractive power of this organ
(Eghrari, Riazuddin, & Gottsch, 2015). However, because of its superficial anatomical
localization, it is particularly vulnerable to abrasive forces and various traumas. In the USA,
the rate of eye injury exceeds one million each year and it is estimated that approximately
20% of the population will experience eye injury at least once in their lifetime (Ljubimov &
Saghizadeh, 2015). Approximately 75% of all eye injuries affect the cornea and are due to
foreign bodies or abrasive damages. Other causes of corneal wounds include burns, punctures
and viral or bacterial infections (McGwin & Owsley, 2005). In general, successful wound
healing occurs through the renewal of corneal epithelial stem cells, but in some cases,
particularly in the presence of a severe injury, or when the wound is not treated properly or
rapidly, corneal injury may result in permanent visual impairment or lead to corneal blindness
(Daniels, Dart, Tuft, & Khaw, 2001).
To investigate corneal wound healing, several models have been used over the years. Among
others, the ex vivo culture organ model has been used to study healing of the cornea
(Carrington & Boulton, 2005; Foreman, Pancholi, Jarvis-Evans, McLeod, & Boulton, 1996;
Gipson & Anderson, 1980; Janin-Manificat et al., 2012; Zagon, Sassani, & McLaughlin,
2000). However, these studies are limited by the availability of healthy donors and the delay
between donor’s death and the arrival of the tissue in the laboratory. As for in vivo
experiments, they are expensive and difficult to conduct because of the inherent variability
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among animals (Bentley et al., 2001; Buhren et al., 2009; Ferrington et al., 2013; Friedenwald
& Buschke, 1944; Pallikaris, Papatzanaki, Stathi, Frenschock, & Georgiadis, 1990). Cell
monolayers in vitro models are definitively less complex than the native cornea can be of
interest due to their ease and rapidity of use (Ker-Woon, Abd Ghafar, Hui, Mohd Yusof, &
Wan Ngah, 2015; Lu, Wang, Dai, & Lu, 2010; Nelson, Silverman, Lima, & Beckman, 1990;
Rieck, Cholidis, & Hartmann, 2001; Robciuc, Arvola, Jauhiainen, & Holopainen, 2018).
Recent works in the field of tissue engineering have led to the emergence of new three￾dimensional corneal equivalents. Some current approaches rely on the use of decellularized
biological materials, as in the case of amniotic membrane and animal cornea (Ghezzi, Rnjak￾Kovacina, & Kaplan, 2015). Others rely on the use of a variety of natural and synthetic
polymers, such as collagen-chitosan, cross-linked recombinant collagen and polyacrylamide
or polyethylene glycol, in combination with primary derived corneal cells or immortalized
corneal cell lines (Griffith et al., 1999; Reichl, Bednarz, & Muller-Goymann, 2004; Van
Goethem et al., 2006). Using ascorbic acid to promote secretion of extracellular matrix
(ECM) by fibroblasts in a procedure known as the self-assembly approach (Auger, Remy￾Zolghadri, Grenier, & Germain, 2002; Germain L, 2004), we succeeded in the reconstruction
of a human, three-dimensional, tissue-engineered cornea (hTEC) (Carrier et al., 2008;
Germain et al., 1999; Proulx et al., 2010). When mechanically wounded, the hTEC triggers
processes such as cell migration and proliferation, to allow reepithelialization of the damaged
tissue, which is found to be very similar to wound healing of a native cornea (Carrier et al.,
2008; Couture et al., 2016; Zaniolo, Carrier, Guerin, Auger, & Germain, 2013).
During corneal wound healing, many processes such as cell migration, proliferation,
differentiation and adhesion will chronologically take part in the wound healing response.
The first event is the migration of cells adjacent to the wounded area whose function is to seal
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the wound, followed by their proliferation and vertical differentiation in order to produce the
stratified neoepithelium. The last event consists in the reassembly of adhesion structures
(Agrawal & Tsai, 2003; C. Y. Liu & Kao, 2015). Corneal wound healing relies on cell-cell
and cell-matrix interactions, themselves mediated in great part by ECM components, but also
by growth factors and cytokines (Nishida & Tanaka, 1996; Wilson et al., 2001; Zieske,
2001). Upon corneal epithelium injury, important changes occur in the composition of the
ECM to which the basal corneal epithelial cells attach. These alterations are detected by
integrins, a family of membrane-anchored receptors that trigger outside-in signalling between
ECM and the cell (Hynes, 1987). Integrins act by recognizing ECM components as their
ligand and subsequently activating different intracellular signalling pathways (Giancotti &
Ruoslahti, 1999). Although it is now well establish that intracellular signalling by integrins is
a key step in the reepithelialization process of wounded cornea, yet the specific mediators
involved in that mechanism remain unknown.
In the present study, we used both monolayers of human corneal epithelial cells (hCECs) and
three-dimensional human tissue-engineered corneas (hTECs) as in vitro models to investigate
the impact of such ECM changes on the gene expression pattern and the activity of several
intracellular signalling kinases during corneal wound closure. Phosphorylation of the
serin/threonine protein kinase WNK1 (With no- lysine (K) kinase 1) turned out to be the most
striking activation event occurring during wound healing of damaged hTECs.
Pharmacological inhibition of WNK1 by WNK463 prevents activation of its downstream
targets Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress
responsive 1 (OSR1) kinases and considerably impedes the process of corneal wound healing
in our biological models, suggesting that WNK1 plays a particularly important role in order
to ensure proper healing of the cornea.
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2 MATERIALS AND METHODS
This study was conducted in accordance with our institution’s guidelines and the Declaration
of Helsinki. The protocols were approved by the CHU de Québec – Université Laval hospital
and Université Laval Committees for the Protection of Human Subjects.
2.1 Cell culture, production and wounding of the tissue-engineered human corneas
Human corneal epithelial cells (hCECs) were isolated from the limbal area of normal eyes of
44 (hCEC-44), 52 (hCEC-52), 70 (hCEC-70X) and 71 (hCEC-71) year-old donors obtained
from the Banque d’Yeux Nationale of the Centre Universitaire d’Ophtalmologie (CHU de
Québec – Université Laval, Québec, QC, Canada). HCECs were cultured in the presence of a
feeder layer of irradiated murine Swiss-3T3 fibroblasts, frozen and stored in liquid nitrogen
until use for hTEC production, as previously reported (Carrier et al., 2008; Gaudreault et al.,
2003; Germain et al., 1999; Germain L, 2004). In the present study, new batches of hTECs
were produced, wounded and left to recover at the air-liquid interface using the same protocol
as recently described (Couture et al., 2016). Briefly, hCECs were seeded on reconstructed
stroma containing corneal fibroblasts, cultured 7 days in submerged conditions and 7 days at
the air-liquid interface to induce epithelial cell differentiation and vertical stratification. Then,
hTECs were wounded, 14 days after the addition of epithelial cells. When indicated, the
WNK1 inhibitor WNK463 (50nM, 1µM or 10µM; Novartis, Basel, Switzerland) was added
to the culture medium of hTECs immediately after they were wounded with the biopsy
punch. The specific concentrations selected for this pharmacological compound are based on
MTS assays conducted on hCECs grown as a monolayer (see Supplementary Methods and
Supplementary Figure 1). Wound closure was then monitored macroscopically for 6 days and
photographed at 24 h intervals with a Zeiss Imager.Z2 microscope (Zeiss Canada Ltd, North
York, ON, Canada). All experiments were conducted in quadruplicates except when indicated
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otherwise. Epithelial tissue from the central area and the external ring of both wounded and
unwounded (used as negative controls) hTECs were harvested four days post-wounding to
collect both total RNA and proteins required for microarray and Western blot analyses,
respectively, except for the SPAK/OSR1 Western that used total proteins from six days post￾wounding. HCEC-44, hCEC-52 and hCEC-71 were used for the preparation of hTECs used
for gene profiling analyses whereas hCEC-52 and hCEC-71 were used for the wound healing
experiments conducted with the inhibitor WNK463.
2.2 Cell cycle analysis
HCEC-52 and hCEC-70X (3×105 cells) were each plated with irradiated murine 3T3
fibroblasts (7.5×105 cells) in 9,6 cm2 plates in DH medium (Dulbecco-Vogt modification of
Eagle’s medium with Ham’s F12 in a 3 : 1 ratio) supplemented with 5% FetalClone II serum,
5 μg/mL insulin, 0.4 μg/mL hydrocortisone, 10 ng/mL epidermal growth factor, 10−10 M
cholera toxin, 100 IU/ml Penicilin, and 25 µg/ml Gentamycin, and with (1 µM) or without
(control (Ctrl) DMSO) WNK463. When cells reached confluence, hCECs were collected,
fixed with 70% cold ethanol for 30 min, washed with PBS and stored at 4°C until use. Fixed
cells were then treated RNase A (100 μg/ml) and PI (50 μg/ml) at room temperature in the
dark for 30 min. The DNA content of the cells was analyzed using flow cytometry and
acquisition of data for 50 000 events were performed using a BD Accuri™ C6 Plus flow
cytometer (BD Biosciences, San Jose, CA). The distribution of hCECs within each phase of
the cell cycle was analyzed using the BD Accuri™ C6 Plus flow cytometry workstation
software.
2.3 Scratch wound assay
HCEC-52, hCEC-70X and hCEC-71 (2.5×105 cells) were each plated in duplicates with
irradiated murine 3T3 fibroblasts (7.5×105
cells) in 9,6 cm2 plates in complete DH medium.
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When cells reached confluence, a 1 mm large X 35mm long scratch was created in the center
of the plate using a P200 pipet tip (Sarstedt, Nümbrecht, Germany) prior to addition of either
WNK463 (50nM or 1µM; Novartis) or DMSO (negative control) to fresh culture media.
Wound closure was monitored and photographs collected at various time intervals (0, 5, 8, 9,
10, 11, 12, 13, 14 and 15h).
2.4 Measurement of cells growth rate
HCEC-52 and hCEC-70X (2.5×105 cells) were plated with irradiated murine 3T3 fibroblasts
(7.5×105 cells) in 9,6 cm2 tissue-culture plates in complete DH medium. WNK463 (50nM or
1µM; Novartis) or DMSO (negative control) was added to fresh culture media every day.
When cells reached confluence (or growth arrest), they were harvested and counted using a
cell and particle counter (Z2; Beckman Coulter, Mississauga, ON, Canada). Cell counts were
performed at passages (P) P3, P4 and P5. The growth rate per day was determined using the
initial number of cells, final number of cells and the duration of culture in days according the
following formula:
Growth rate =
log((