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Chen Liang, Jing Z Cui, Luba Kojic, William Jia, Joanne A Matsubara; Expression of A Disintegrin and Metalloproteinase 17 (ADAM 17) in RPE cell culture is associated with apoptosis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6557. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
ADAM17 has been reported to be an indispensible regulator of almost every cellular event from proliferation to death. However, the role of ADAM17 in eye diseases is unknown. The purpose of this study is to investigate the expression and the function of ADAM17 in RPE cells in vitro and in postmortem eyes of AMD and non-AMD donors.
ARPE19 cells were stimulated with IL-1β, H2O2 or TNF-α to trigger ADAM17 activation for 24 hrs. Cells were then lysed, and samples extracted for total, cytoplasmic or nuclear protein. ADAM17 protein expression was assessed by western blot. Nuclear localization of ADAM17 was further evaluated by immunocytochemistry on culture slides. Apoptosis in stimulated ARPE19 cells was studied using a pan-caspase inhibitor, flow cytometry and annexin V/PI labeling. Results were analyzed by one-way ANOVA, p < 0.05.
Total protein analysis revealed three forms of ADAM17: proform (130kD), mature form (100kD) and cytoplasmic tail form (40kD). Significant fold increase of the mature form was observed for IL-1β (1.8±0.45), H2O2 (1.5±0.04), TNF-α (2.0±0.28) stimulation; no significant changes were observed in the cytoplasmic tail form in any of the stimulation regimes. However, the cytoplasmic tail form was significantly increased in the nuclear protein material in all treatment groups (1.9 -4.0 fold). Cell fluorescence staining further confirmed ADAM17 signal in RPE nuclei. Nuclear cytoplasmic tail form was reduced with pretreatment of pan-caspase inhibitor, which blocks caspase-dependent apoptosis. Corroborative immunohistochemistry data were observed from sections of human AMD eyes, in which increased ADAM17 levels were found in the AMD eyes (N=13) and in the nuclear compartment when compared to age-matched controls (N=7).
This is the first study to demonstrate a nuclear localization of ADAM17 in RPE cell under stress in vitro and its role in RPE apoptosis. Additionally, the presence of ADAM17 in RPE nuclei of postmortem eyes is suggestive of a possible association with AMD disease processes.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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