Abstract
Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/β-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues.
Highlights
Dysfunction and death of retinal pigment epithelium (RPE) constitute the final common pathway in age-related macular degeneration, the leading cause of legal blindness among the elderly, as well as retinitis pigmentosa [2] and Stargardt’s macular dystrophy [3]
These results collectively indicated that contact inhibition unlocked by EGTA with epidermal growth factor (EGF)+FGF-2 was accompanied by loss of the normal RPE phenotype and gain of a mesenchymal phenotype suggestive of epithelial-mesenchymal transition (EMT)
The results showed that the T cell factor/lymphoid enhancer factor (TCF/LEF) promoter activity was low in cells treated with phosphate-buffered saline (PBS) but elevated 15-fold in cells treated with EGTA+EGF+FGF-2, and such an elevated activity was abolished by XAV939
Summary
Dysfunction and death of retinal pigment epithelium (RPE) constitute the final common pathway in age-related macular degeneration, the leading cause of legal blindness among the elderly (reviewed by Binder et al [1]), as well as retinitis pigmentosa [2] and Stargardt’s macular dystrophy [3]. EMT occurs in a number of pathological diseases involving RPE (reviewed by Saika et al [14]), such as proliferative vitreoretinopathy, in which RPE undergoes EMT to become fibroblastic and contractile cells, leading to tractional retinal detachment and blindness (reviewed by Nagasaki et al [15] and Pastor et al [16]). The detrimental outcome of proliferative vitreoretinopathy is attributed to proliferation of abnormal RPE [15]. It remains unclear whether EMT is inevitable upon loss of contact inhibition or whether different growth factors might have different impacts on proliferation, even if EMT is elicited
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