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Takato Kagami, Naoki Okumura, Makiko Nakahara, Noriko Koizumi; Involvement of the Nectin-afadin and N-cadherin-catenin systems in the formation of adherence junctions in corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1447.
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© ARVO (1962-2015); The Authors (2016-present)
The apical junction, comprising adherence junctions (AJ) and tight junctions (TJ), acts as a paracellular barrier, ensures cell polarity, and serves as a scaffold for signaling. Cadherin-catenin and nectin-afadin complexes form AJ in various cell types. Though barrier function of corneal endothelium is essential, as is pump function, apical junction, especially that of AJ, has yet to be well elucidated. The purpose of this study was to elucidate the AJ in corneal endothelium.
The expression of N-, E-, P-, R-, VE-, K-, and OB-cadherins, β-catenin, nectin-1, -2, -3, and -4, and afadin was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in corneal endothelium obtained from a human donor cornea. Distribution of N-cadherin, β-catenin, nectin, afadin, and ZO-1 were examined by immunofluorescent staining using the donor cornea. Immunoprecipitation was performed to examine the binding between N-cadherin and β-catenin. siRNA was used to knock down (KD) N-cadherin, afadin, or ZO-1 in cultured monkey corneal endothelial cells (MCECs).
RT-PCR showed that N-, P-, VE-, and OB-cadherin were expressed in the human corneal endothelium. N-cadherin and β-catenin were colocalized at the plasma membrane. Immunoprecipitation showed that N-cadherin and β-catenin were bound together in the MCECs. The expression of nectin-1, -2, -3, and -4, and afadin in the human corneal endothelium was confirmed at the mRNA level. Immunofluorescent staining showed that nectin and afadin were localized at the plasma membrane of corneal endothelium. A confocal-microscopy Z-stack image showed that nectin, afadin, and N-cadherin were co-localized at the basal side, while ZO-1 was localized at the apical side. KD of afadin in the MCECs did not alter the distribution of N-cadherin and ZO-1, and KD of ZO-1 did not alter that of N-cadherin and afadin. In contrast, ND of N-cadherin disrupted the distribution of afadin and ZO-1.
Our findings revealed that AJ of corneal endothelium are formed by N-cadherin-catenin and nectin-afadin systems, and that N-cadherin plays an essential role for maintaining the apical junction of corneal endothelium, at least after the AJ and TJ are once formed.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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