Enhanced wound healing of tissue-engineered human corneas through altered phosphorylation of the CREB and AKT signal transduction pathways

Couture, Camille; Desjardins, Pascale; Zaniolo, Karine; Germain, Lucie; Guérin, Sylvain L.

doi:10.1016/j.actbio.2018.04.021

Cited by 20 publications

(43 citation statements)

References 66 publications

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“…Akt isoforms and STAT5 phosphorylation profiles observed in this study are also in line with one of our group’s recent publication on an in vitro reconstructed corneal epithelium wound healing model. Couture et al found that STAT5a and STAT5b were less phosphorylated in post-wound regenerating neo-epithelium cells as opposed to less actively proliferating cells of unwounded regions of the model [31]. In that same study, and in accordance with our results, p53 and Akt isoforms 1 to 3 displayed increased phosphorylation statuses in neo-epithelium cells when compared to those of unwounded regions [31].…”

Section: Resultssupporting

confidence: 91%

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Ghio

Cantin-Warren

Guignard

et al. 2018

IJMS

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Human keratinocyte culture has provided the means to treat burns, wounds and skin pathologies. To date, to efficiently culture keratinocytes, cells are cultured on an irradiated feeder layer (iFL), either comprising human (iHFL) or murine (i3T3FL) fibroblasts, and the culture medium is supplemented with a cyclic adenosine monophosphate (cAMP) accumulation inducing agent such as isoproterenol (ISO) or cholera toxin (CT). Previous studies have characterized how the feeder layer type and the cAMP inducer type influence epithelial cells’ phenotype independently from one another, but it is still unknown if an optimal combination of feeder layer and cAMP inducer types exists. We used sophisticated statistical models to search for a synergetic effect of feeder layer and cAMP inducer types on human keratinocytes’ proliferative potential. Our data suggests that, when culturing human keratinocytes, using iHFL over i3T3FL increases population doublings and colony-forming efficiency through signaling pathways involving Ak mouse strain thymoma (Akt, also known as protein kinase B) isoforms 1 to 3, signal transducer and activator of transcription 5 (STAT5), p53, and adenosine monophosphate activated protein kinase α1 (AMPKα1). Both tested cAMP inducers ISO and CT yielded comparable outcomes. However, no significant synergy between feeder layer and cAMP inducer types was detected. We conclude that, to promote human keratinocyte growth in the early passages of culture, co-culturing them with a human feeder layer is preferable to a murine feeder layer.

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Section: Resultssupporting

confidence: 91%

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Ghio

Cantin-Warren

Guignard

et al. 2018

IJMS

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“…(b) Unlike when grown as a monolayer, hCECs that are grown on the hTECs are lying on a complex ECM whose many constituting components likely activate signal transduction pathways (such as the MAPK pathway) that share mediators also used by the WNK1 pathway as well. We recently demonstrated that addition of both the CREB inhibitor C646 and the Akt agonist SC79 to wounded hTECs considerably increased wound closure irrespective of whether it is deposited beneath (Couture et al, ) or over (unpublished results) the reconstructed tissue. It is therefore unlikely that addition of WNK463 underneath the hTEC accounts for the difference observed, which therefore rather favour an impact from the ECM.…”

Section: Discussionmentioning

confidence: 91%

“…We recently demonstrated that hypophosphorylation of CREB and activation of Akt was particularly important to ensure proper closure of wounded hTECs in vitro (Couture et al, ). However, in the present study, we showed that besides these two signal transduction mediators, corneal wound healing also induces a strong phosphorylation of the WNK1 kinase, a mediator that can be activated via multiple routes such as the JAK/STAT, MAPK, or the PI3K/Akt pathways (Figure a).…”

Section: Discussionmentioning

confidence: 99%

“…(1-μM WNK463) could easily be rescued by removing the inhibitor from the culture medium (1µMø of WNK463), the cell's growth rate increasing from 0.03 to 0.37, a value very similar to that measured for control hCECs (0.42). In addition, replacing the WNK463-containing medium with fresh DH medium also restored the ability of the colonies of hCECs to expand again normally after 5 (total of 15 culture days) or 8 days (total of 18 culture days) under inhibitor-free condition We recently demonstrated that hypophosphorylation of CREB and activation of Akt was particularly important to ensure proper closure of wounded hTECs in vitro (Couture et al, 2018). However, in the present study, we showed that besides these two signal transduction mediators, corneal wound healing also induces a strong phosphorylation of the WNK1 kinase, a mediator that can be activated via multiple routes such as the JAK/STAT, MAPK, or the PI3K/Akt pathways (Figure 2a).…”

Section: Inhibition Of Wnk1 Alters the Proliferative Properties Of mentioning

confidence: 96%

“…Impact of hTEC wound healing on the expression of mediators from the MAPK and PI3K/Akt pathwaysWe recently exploited gene profiling on microarray to examine the impact of wound healing on the expression of the major signal transduction mediators using, as a model, our hTECs. Alterations were observed in a number of mediators primarily from the MAPK and PI3K/Akt pathways(Couture, Desjardins, Zaniolo, Germain, & Guerin, 2018). However, our analysis only compared the pattern of genes expressed in the central re-epithelializing wound with the external area surrounding the initial wound in the hTECs.…”

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confidence: 99%

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

Desjardins

Couture

Germain

et al. 2019

J Tissue Eng Regen Med

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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.

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The WNK1 kinase regulates the stability of transcription factors during wound healing of human corneal epithelial cells

Desjardins

Le‐Bel

Ghio

et al. 2022

Journal Cellular Physiology

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Due to its superficial anatomical localization, the cornea is continuously subjected to injuries. Damages to the corneal epithelium trigger important changes in the composition of the extracellular matrix to which the basal human corneal epithelial cells (hCECs) attach. These changes are perceived by membrane-bound integrins and ultimately lead to re-epithelialization of the injured epithelium through intracellular signalin. Among the many downstream targets of the integrin-activated signaling pathways, WNK1 is the kinase whose activity is the most strongly increased during corneal wound healing. We previously demonstrated that pharmacological inhibition of WNK1 prevents proper closure of wounded human tissue-engineered cornea in vitro. In the present study, we investigated the molecular mechanisms by which WNK1 contributes to corneal wound healing. By exploiting transcription factors microarrays, electrophoretic mobility-shift assay, and gene profiling analyses, we demonstrated that the DNA binding properties and expression of numerous transcription factors (TFs), including the well-known, ubiquitous TFs specific protein 1 (Sp1) and activator protein 1 (AP1), were reduced in hCECs upon WNK1 inhibition by WNK463. This process appears to be mediated at least in part by alteration in both the ubiquitination and glycosylation status of these TFs. These changes in TFs activity and expression impacted the transcription of several genes, including that encoding the α5 integrin subunit, a well-known target of both Sp1 and AP1. Gene profiling revealed that only a moderate number of genes in hCECs had their level of expression significantly altered in response to WNK463 exposition. Interestingly, analysis of the microarray data for these deregulated genes using the ingenuity pathway analysis software predicted that hCECs would stop migrating and proliferating but differentiate more when they are grown in the presence of the WNK1 inhibitor. These results demonstrate that WNK1 plays a critical function by orienting hCECs into the appropriate biological response during the process of corneal wound healing.

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Enhanced wound healing of tissue-engineered human corneas through altered phosphorylation of the CREB and AKT signal transduction pathways

Cited by 20 publications

References 66 publications

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

Are the Effects of the Cholera Toxin and Isoproterenol on Human Keratinocytes’ Proliferative Potential Dependent on Whether They Are Co-Cultured with Human or Murine Fibroblast Feeder Layers?

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

The WNK1 kinase regulates the stability of transcription factors during wound healing of human corneal epithelial cells

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