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Yan Wang, Heming Li, Shihu Zang, Fanfan Li, Yingying Chen, Xiao Zhang, Zongming Song, Qing Peng, Feng Gu; Photoreceptor Cell–Derived CAPN5 Regulates Retinal Pigment Epithelium Cell Proliferation Through Direct Regulation of SLIT2 Cleavage. Invest. Ophthalmol. Vis. Sci. 2018;59(5):1810-1821. doi: https://doi.org/10.1167/iovs.17-22689.
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© ARVO (1962-2015); The Authors (2016-present)
To identify the causative gene and investigate the corresponding mechanisms for an autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) family.
Clinical examination and genetic analysis were performed in a Chinese ADNIV family. To dissect the molecular consequence, we used gene targeting to knock-in a patient's specific mutation in the mouse genome. Immunostaining and immunoprecipitation were harnessed to analyze the colocalization and interaction of CAPN5 with SLIT2 in photoreceptors. The purified SLIT2-N, SLIT2-C fragments, and the conditioned medium from 661W cells with the overexpression of CAPN5 were treated on ARPE-19 cells. The viability of ARPE-19 cells was determined by MTT assays. The activation of protein kinase A (PKA) was analyzed by immunofluorescence and Western blotting in 661W and ARPE-19 cells as well as in frozen retina tissue from wildtype (WT) and knock-in mice.
We identified a novel CAPN5 mutation (p.R289W) in a Chinese family and generated the knock-in CAPN5R289W mouse. This mutation caused abnormal proliferative RPE in both humans and mice. CAPN5 directly cleaved WT SLIT2 in vitro, but not the mutant SLIT2 (p.R1113I). CAPN5 interacted with the SLIT2 in mouse retinal photoreceptors (661W cells) and increased cleavage and secretion of the SLIT2 fragments (SLIT2-N and SLIT2-C). Conditioned medium induced higher levels of secreted SLIT2 fragments, which promoted PKA activation and promoted proliferation of ARPE-19 cells.
The novel CAPN5 mutation (p.R289W) is responsible for the present ADNIV family. The mutant CAPN5 stimulated secretion and cleavage of SLIT2 fragments that may act as a bystander to regulate abnormal RPE cell proliferation for ADNIV.
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