YAP, ΔNp63, and β-Catenin Signaling Pathways Are Involved in the Modulation of Corneal Epithelial Stem Cell Phenotype Induced by Substrate Stiffness

Abstract

Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche’s distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and β-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/β-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.

Biomechanical Modulation Therapy—A Stem Cell Therapy Without Stem Cells for the Treatment of Severe Ocular Burns

“…52,58 Conversely, the stiffening of the limbus matrix (e.g., due to burn injury or fibrosis) has been shown to rapidly change LESC phenotype in multiple species, 52,59 with YAP activation and nuclear translocation initiating a signaling cascade leading to increased cell activation, migration, and differentiation via suppression of Np63 and Wnt/β-catenin signaling and increased expression of BMP4. 58 Numerous studies indicate that these mechanisms of action not only regulate stem cell response in the cornea and other epithelia 60 but might also provide the link between chronic inflammation and LSCD, 61 metaplasia, 62 and subsequent vision loss. 63,64 Although the course of ocular burns depends on the nature of its agent, most severe cases end up affecting the biomechanical properties of the anterior cornea, including in the limbus.…”

“…Research has shown that the localized use of low doses of collagenase type I is a simple but efficient method to soften collagen-based substrates in vitro by reducing their density and in this way reproduce the compliance of the natural limbus. 52,58 Moreover, LESCs grown on these softer substrates showed increased expression of LESC-characteristic markers and lower YAP expression and activation, which in turn promotes LESC maintenance, proliferation, stratification, and survival. But, most importantly, this enzymatic strategy was then successfully shown to change the mechanical stiffness both in vivo and ex vivo while maintaining overall tissue structure (i.e., without causing tissue melting or ectasia).…”

“…52 Cells residing on collagenase-treated areas better retained an undifferentiated, LESC-like behavior, whereas cells on stiffer, untreated areas assumed a more differentiated phenotype. 52,53,58 This modulation of cell fate was shown to depend on the biomechanical similarity of the substrate with the natural limbus 52,53,70 and not on structural or compositional changes (e.g., interactions with new topographical or biochemical cues exposed by the enzyme treatment).…”

“…In addition, testing in LSCD corneas ex vivo will allow us to ascertain if increased stiffness varies with injury severity and to determine if individual protocol adjustments are required. Finally, we believe that future studies aimed at developing alternative methods for modulating tissue biomechanics or controlling stem cell phenotype via mechanotransduction signaling (e.g., using biochemical or molecular biology tools to regulate BMP4 expression) 58 merit further consideration and can find extended applications in ophthalmology and beyond. 78…”

“… 53 , 54 Together with its co-effector TAZ, YAP is a well-known molecular regulator of stem cell fate 55 , 56 and is crucially involved in downstream signaling promoting LESC maintenance, proliferation, and stratification. 57 , 58 Moreover, YAP inactivation in response to limbus-like matrix compliance has been associated with both direct and indirect upregulation of the LESC markers ΔNp63, Wnt/β-catenin, and ABCG2. 52 , 58 Conversely, the stiffening of the limbus matrix (e.g., due to burn injury or fibrosis) has been shown to rapidly change LESC phenotype in multiple species, 52 , 59 with YAP activation and nuclear translocation initiating a signaling cascade leading to increased cell activation, migration, and differentiation via suppression of ΔNp63 and Wnt/β-catenin signaling and increased expression of BMP4.…”