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Nur Musfirah Mahmud, Umar Sharif, Paul Kay, Tengku Ain Fathlun Tengku Kamalden, Yit C. Yang, Simon P Harding, Luminita Paraoan; Modulation of cathepsin L expression and NF-kB signalling pathway effectors by advanced glycation end-products in retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1726.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated that an accumulation of advanced glycation end-products (AGEs) in the Bruch’s membrane (BrM) can alter retinal pigment epithelium (RPE) lysosomal activity by changing expression of key effectors and inhibitors. Altered activity of lysosomal cysteine proteases, the cathepsins, can in turn impact signalling via the NF-kB pathway. The aim of this study therefore was to analyse the effects of AGEs on specific lysosomal cathepsins, and the endogenous levels of effectors of the NF-kB signalling pathway in RPE.
ARPE-19 cells were cultured on AGE-containing BrM mimics in vitro for 7-14 days. Intracellular processing of the cysteine proteases cathepsins B, L and S were assessed by qPCR and immunoblotting, while their intracellular activity was assessed using fluorescence-based cleavage assays. Expression of NF-kB (p65) and its main regulatory protein, IκBα, was assessed by qPCR and immunoblotting. Statistical analysis was performed using the independent T-test.
Levels of the active form of cathepsin L were significantly decreased following AGE exposure (33%, p=<0.027). This decrease was also manifested as a decrease (36%, p=0.02) in its intracellular activity. Cathepsin S active form decreased by 74% (p=0.004), yet this change had no effect on its activity. Transcript expression of cathepsins L and S was unaltered, suggesting effects on protein processing rather than at the transcriptional level. Expression, processing and activity of cathepsin B were unaltered. Under the same conditions, NF-κB p65 and Ser536P p65 protein levels were reduced by 37% (p=0.0003) and 52% (p=0.03) respectively. In addition, IκBα expression was significantly down regulated (31%, p=0.02).
AGE accumulation in the BrM, a common consequence of the ageing process, affects the expression and activity of the key lysosomal effector, cathepsin L. Following exposure to AGEs, the overall endogenous levels of key effectors of the NF-kB signalling pathway are also decreased. These results, together with data from previous studies, support the hypothesis that these processes are related. Altered NF-kB-driven transcription affects expression and secretion of pro- and anti- inflammatory cytokines by the RPE, potentially contributing to drusen biogenesis and AMD pathogenesis.
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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