N-(4-hydroxyphenyl)retinamide induces apoptosis in human retinal pigment epithelial cells: Retinoic acid receptors regulate apoptosis, reactive oxygen species generation, and the expression of heme oxygenase-1 and Gadd153
N-(4-hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid (RA) derivative and a potential cancer preventive agent, is known to exert its chemotherapeutic effects in cancer cells through induction of apoptosis. Earlier work from our laboratory has shown that relatively low concentrations of 4HPR induce neuronal differentiation of cultured human retinal pigment epithelial (ARPE-19) cells (Chen et al., 2003, J Neurochem 84:972-981). However, at higher concentrations of 4HPR, these cells showed morphological changes including cell shrinkage and cell death. Here we demonstrate that ARPE-19 cells treated with 4HPR exhibit a dose- and time-dependent induction of apoptosis as evidenced by morphological changes, mono- and oligonucleosome generation, and increased activity of caspases 2 and 3. The 4HPR-induced apoptosis as well as the activation of caspases 2 and 3 were blocked by both retinoic acid receptors (RAR) pan-antagonists, AGN193109 and AGN194310, and by an RARalpha-specific antagonist AGN194301. 4HPR treatment also increased reactive oxygen species (ROS) generation in ARPE-19 cells in a time-dependent manner as determined from the oxidation of 2',7'-dichlorofluorescin. In addition, the increase in the expression of heme oxygenase-1 (HO-1), a stress response protein, and the growth arrest and DNA damage-inducible transcription factor 153 (Gadd153) in response to the ROS generation were also blocked by these receptor antagonists. Pyrrolidine dithiocarbamate (PDTC), a free-radical scavenger, inhibited 4HPR-induced ROS generation, the expression of its downstream mediator, Gadd153, and apoptosis in the pretreated cells. Therefore, our results, clearly demonstrate that 4HPR induces apoptosis in ARPE-19 cells and that RARs mediate this process by regulating ROS generation as well as the expression of Gadd153 and HO-1.
"The ARPE-19 human retinal pigment epithelial cell line was obtained from ATCC (Manassas, VA). The cells (passages 23 through 26) were grown in Dulbecco’s modified Eagle’s medium containing nutrient mixture F12, 50/50 mix (Cellgro, Herndon, VA) supplemented with 5% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 μg/ml), as described previously . "
[Show abstract][Hide abstract] ABSTRACT: The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines.
Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting.
Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1β, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic.
Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1β). The induction of miR-146a showed a dependency on IL-1β, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.
"The miR-9 expression in ARPE-19 cells in response to 4HPR treatment was further investigated. Earlier work from our laboratory has shown that 4HPR-induced apoptosis of ARPE-19 cells is preceded by reactive oxygen species (ROS) generation . The increase in HMOX1 expression is a consequence of oxidative stress. "
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are important regulators of many cellular functions due to their ability to target mRNAs for degradation or translational inhibition. Previous studies have reported that the expression of microRNA-9 (miR-9) is regulated by retinoic acid and reactive oxygen species (ROS). We have previously shown that N-(4-hydroxyphenyl)-retinamide (4HPR), a retinoic acid derivative, induces ROS generation and apoptosis in cultured human retinal pigment epithelial (RPE) cells, known as ARPE-19 cells. The aim of the present study was to investigate the expression of miR-9 in ARPE-19 cells in response to 4HPR treatment, and to identify other miRNAs normally expressed in these cells.
ARPE-19 cells in culture were treated with 4HPR, the total RNA fractions were isolated, and the expression of various miRNAs and mRNAs was analyzed using real-time PCR. The miRNA expression profile of ARPE-19 cells was analyzed using microarray hybridization.
Treatment of ARPE-19 cells with 4HPR resulted in apoptosis characterized by the increased expression of HMOX1 and GADD153 genes. A twofold increase in the expression of miR-9 was also observed during this response. Potential binding sites for the transcription factors encoded by CEBPA and CEBPB genes were found to be present in the putative promoter regions of all three genes encoding miR-9. 4HPR-induced miR-9 expression was associated with parallel increases in the expression of these transcription factor genes. 5-Aza-2'-deoxycytidine, a methyl transferase inhibitor, also increased the expression of miR-9 in ARPE-19 cells. Microarray hybridization analysis identified let-7b, let-7a, miR-125b, miR-24, miR-320, miR-23b, let-7e, and let-7d as the most abundant miRNAs normally expressed in ARPE-19 cells. These miRNAs are known to regulate cell growth, differentiation or development. The 4HPR treatment increased the expression of miR-16, miR-26b, miR-23a, and miR-15b in ARPE-19 cells, although these increases were modest when compared to the increase in the expression of miR-9.
Our studies demonstrate that miR-9 is expressed in the RPE cell line ARPE-19, and its expression is increased by a retinoic acid derivative and by an inhibitor of promoter hypermethylation. Several miRNAs with inherent ability to regulate cell growth, differentiation and development are also normally expressed in ARPE-19 cells. Thus, miR-9 and other miRNAs could be important in maintaining RPE cell function.
[Show abstract][Hide abstract] ABSTRACT: Death receptors are important modulators of the extrinsic apoptotic pathway. Activating certain death receptors such as death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (i.e., DR4 and DR5) selectively kills cancer cells via induction of apoptosis while sparing normal cells. Thus, soluble recombinant TRAIL and agonistic antibodies to DR4 or DR5 have progressed to phase I and phase II clinical trials. Many cancer therapeutic drugs including chemotherapeutic agents have been shown to induce the expression or redistribution at the cell surface of death receptors including TRAIL death receptors. In addition, chemotherapeutic agents have also been shown to enhance induction of apoptosis by TRAIL or agonistic antibodies or overcome cell resistance to TRAIL or agonistic antibodies. Targeted induction of apoptosis by activation of the death receptor-mediated extrinsic apoptotic pathway should be an ideal therapeutic strategy to eliminate cancer cells. Therefore, death receptors, particularly TRAIL death receptors, have emerged as an important cancer therapeutic target. This article will focus on reviewing and discussing the modulation of death receptors by cancer therapeutic agents and its implications in cancer therapy.
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