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Yan Fan; p53-independent Role of MDM2 in Suppressing NF-κB-mediated Inflammation in Retinal Pigment Epithelium Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1059.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous studies indicated that Nutlin-3, a MDM2 inhibitor inhibits pathologic retinal endothelial cell proliferation through a p53-dependent mechanism, but the p53-independent functions and effect on retinal pigment epithelial (RPE) cell viability have not been adequately elucidated. LPS induced RPE inflammation via NF-κB is a typical model for endotoxin-induced uveitis. Our current study suggests that Nutlin-3, in a non-toxic, low dosage, significantly inhibited LPS-induced NF-κB activation, independent of p53 function. Here, we sought to investigate the underlying mechanism of the non-canonical, p53-indendepent function of Nutlin-3, as well as the role of MDM2 in ocular inflammation. We hypothesized that MDM2 inhibitors possess an anti-inflammatory function benefiting patients suffering from retinal inflammatory diseases.
For the in vitro experiments, the ARPE-19-NF-κB-luciferase reporter cell line was used to record NF-κB activity during LPS and Nutlin-3 treatment. Cell viability and proliferation was assessed using a cell death ELISA and MTT assays. The downstream pro-inflammatory response of NF-kB was tested by q-PCR or ELISA. NF-κB-GFP and p53 knockout mice were used for the in vivo experiments. After LPS intraperitoneal and Nutlin-3 intravitreal injection, ocular inflammation was monitored via H&E staining, focal and full-field ERG, and SD-OCT. The eyes were harvested and subjected for western blotting, q-PCR, immune staining, and ELISA for assessing the anti-inflammatory effect of Nutlin-3.
ARPE-19 NF-kB-luciferase cells were pre-treated with the indicated amounts of Nutlin-3 for 30 minutes to 7 hours, followed by stimulating with LPS (10 ng/ml) for various time intervals. LPS alone dramatically induced NF- κB luciferase reporter gene activity, and Nutlin-3 pre-treatment significantly attenuated LPS-induced response (p< 0.05). Inhibition of LPS-induced NF-κB by Nutlin-3 is in both dose- and time-dependent manner. More interestingly, this occured in a p53-independent manner.
Our data suggests that Nutlin-3 has a novel p53-indepenent function in RPE cells by inhibiting LPS mediated NF-κB RPE inflammation. MDM2 inhibitors may have a therapeutic effect in treating patients with uveitis and other retinal inflammatory diseases.
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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