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Yi-Jen Hsueh, Hung-Chi Chen, Sung-En Wu, Tze-Kai Wang, Jan-Kan Chen, David Hui-Kang Ma; Nuclear YAP-promoted proliferation can be induced by lysophosphatidic acid via the PI3K and ROCK pathways in contact-inhibited human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1142. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Silence of p120-catenin has shown promise in inducing proliferation in human corneal endothelial cells (HCECs), but there is concern regarding gene recombination in potential clinical applications. We aimed to develop ex vivo expansion of HCECs using natural compounds, and we hypothesized that lysophosphatidic acid (LPA) can unlock the mitotic block in contact-inhibited HCECs via enhancing nuclear translocation of yes-associated protein (YAP).
Collagenase-isolated HCEC aggregates from stripped Descemet’s membrane were cultured to 14 days, and treated by 100 nM control or YAP-1 siRNA. Cells were further treated with 20 μM LPA and 10 μM BrdU . Likewise, post-confluent b4g12 cells (HCEC cell line) cultured to 4 days under the similar condition. Immunofluorescent staining was performed to demonstrate YAP-1, ZO-1, Na/K-ATPase, SMA and BrdU labeling. B4g12 cells was pretreated with LY294002 (PI3K inhibitor, 20 μM), PD98059 (ERK1/2 inhibitor, 20 μM), SB203580 (p38 inhibitor, 20 μM) or Y27632 (ROCK inhibitor, 20 μM) for pathway assay. Western blot analysis was applied to evidence the regulation of proliferation by targeting p27/p21 and cyclin D1.
Firstly, we verified that exogenous YAP could induce cell proliferation in contact-inhibited HCEC monolayers and post-confluent B4G12 cells. In B4G12 cells, enhanced cyclin D1 expression, reduced p27KIP1/p21CIP1 levels and the G1/S transition were detected upon transfection with YAP. Secondly, we confirmed that LPA induced nuclear expression of YAP and promoted cell proliferation. Moreover, PI3K and ROCK, but not ERK or p38, were required for LPA-induced YAP nuclear translocation. Finally, cells treated with LPA or transfected with YAP remained hexagonal in shape, in addition to unchanged expression of ZO-1, Na/K-ATPase, and SMA, suggestive of a preserved phenotype, without endothelial-mesenchymal transition (EnMT).
Our findings indicate an innovative strategy for ex vivo cultivation of HCECs for transplantation and cell therapy.
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