Age-Related Susceptibility to Apoptosis in Human Retinal Pigment Epithelial Cells Is Triggered by Disruption of p53–Mdm2 Association

Abstract

PURPOSE.Relatively little is known about the contribution of p53/Mdm2 pathway in apoptosis of retinal pigment epithelial (RPE) cells or its possible link to dysfunction of aging RPE or to related blinding disorders such as age-related macular degeneration (AMD).METHODS. Age-associated changes in p53 activation were evaluated in primary RPE cultures from human donor eyes of various ages. Apoptosis was evaluated by activation of caspases and DNA fragmentation. Gene-specific small interfering RNA was used to knock down expression of p53. RESULTS.We observed that the basal rate of p53-dependent apoptosis increased in an age-dependent manner in human RPE. The age-dependent increase in apoptosis was linked to alterations in several aspects of the p53 pathway. p53 phosphorylation Ser15 was increased through the stimulation of ATM-Ser1981. p53 acetylation Lys379 was increased through the inhibition of SIRT1/2. These two posttranslational modifications of p53 blocked the sequestration of p53 by Mdm2, thus resulting in an increase in free p53 and of p53 stimulation of apoptosis through increased expression of PUMA (p53 upregulated modulator of apoptosis) and activation of caspase-3. Aged RPE also had reduced expression of antiapoptotic Bcl-2, which contributed to the increase in apoptosis. Of particular interest in these studies was that pharmacologic treatments to block p53 phosphorylation, acetylation, or expression were able to protect RPE cells from apoptosis.CONCLUSIONS. Our studies suggest that aging in the RPE leads to alterations of specific checkpoints in the apoptotic pathway, which may represent important molecular targets for the treatment of RPE-related aging disorders such as AMD. (Invest Ophthalmol Vis Sci. 2012;53:8350-8366) DOI:10.1167/ iovs.12-10495 A major challenge in vision research is the identification of the causative factors that lead to declines in retinal function that will ultimately result in age-related macular degeneration (AMD) for a projected 3 million individuals in the United States with neovascular AMD and/or geographic atrophy by 2020. 1 One promising strategy in understanding this multifactorial disease process is to identify and track agedependent changes in cellular pathways, especially in cells of the retinal pigment epithelium (RPE) because these represent the gatekeepers whose loss of functional integrity has been linked to retinal aging.2-4 RPE cells constitute a major component of the blood-retinal barrier (BRB) and loss of integrity of tight junctions (TJs) and adherens junctions (AJs) in RPE can disrupt photoreceptor homeostasis. 5-9 RPE cells also phagocytosize tips of outer segments normally shed by photoreceptors, generate melanosomes to function as a light and heat sink, provide trophic factors, and recycle visual pigments. Many of the hallmarks of AMD reflect malfunctions of these RPE-related pathways, including photobleaching of melanosomes, accumulation of lipofuscin granules, impairment of outer segment phagocytosis, formation of drusen, and breakdown of the BRB in a...

Quercetin alleviates 4-hydroxynonenal-induced cytotoxicity and inflammation in ARPE-19 cells

“…HNE is known to induce apoptosis, which is also a characteristic of aging RPE cells, a phenomenon which has been implicated in the early pathogenesis of AMD (Bhattacharya et al, 2012;Kook et al, 2008;Sharma et al, 2008). We demonstrated here that quercetin could protect stressed ARPE-19 cells from HNE-induced cytotoxicity and decrease the release of three inflammatory mediators, IL-6, IL-8, and MCP-1 when compared to cells treated with HNE and the equivalent amount of quercetin diluent DMSO.…”

Genomic regulation of senescence and innate immunity signaling in the retinal pigment epithelium

“…We have previously shown that aging in human RPE cells activates p53-mediated apoptosis through increased level and post-translational modification of p53, increased levels of the pro-apoptotic marker PUMA, activation of caspase-3, increased levels of CDKN1A, a known transcriptional target of p53, and reduced levels of antiapoptotic BCL2, all of which lead to an overall increase in apoptosis (Bhattacharya et al 2012, 2011). To investigate age-related changes in p53-mediated senescence and inflammation pathways, we measured the levels of p53 and its target proteins in primary RPE cultures from young and aged donors.…”

“…Importantly, p53 regulates innate immune mediated processes, including increased expression of TLRs, cytokines and chemokines, enhancement and stimulation of IFN signaling, IFN regulatory factor-9, and regulates expression of IFN stimulated genes and IFI family genes, (Gugliesi et al 2005; Kwak et al 2003; Menendez et al 2013, 2011; Munoz-Fontela et al 2008; Shatz et al 2012). Aging in the RPE is associated with increased p53 levels and increased p53-mediated apoptosis (Bhattacharya et al 2012). Thus p53 may play an important role in regulating aging in the RPE and in development of AMD.…”

High-glucose-induced CARM1 expression regulates apoptosis of human retinal pigment epithelial cells via histone 3 arginine 17 dimethylation: Role in diabetic retinopathy

“…In the present study, we showed that blockage of H3R17 asymmetric dimethylation attenuated hyperglycemia-induced RPE cell apoptosis. Indeed, p53 and its target genes induce RPE cells apoptosis [32][33][34]. Hence, one can imagine, perhaps when RPE cells exposes to hyperglycemia, CARM1 and dimethylated H3R17 being recruited to p53 responsive elements of apoptotic target gene promoter regions to induce RPE cells apoptosis.…”