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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.
Molecular vision 01/2013; 19:737-750. · 2.20 Impact Factor
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ABSTRACT: Insulin-like growth factor (IGF)-binding protein-5 (IGFBP5), an important member of the IGF axis involved in regulating cell growth and differentiation, acts by modulating IGF signaling and also by IGF-independent mechanisms. We identified IGFBP5 by microarray analysis as a gene differentially regulated during N-(4-hydroxyphenyl)retinamide (4HPR)-induced neuronal differentiation of human retinal pigment epithelial (RPE) cells. IGFBP5 is expressed in human RPE cells, and its expression, mRNA as well as protein, is greatly decreased during the 4HPR-induced neuronal differentiation. Exogenous IGFBP5 does not block the neuronal differentiation indicating that IGFBP5 down-regulation may not be a prerequisite for the neuronal differentiation. IGFBP5 down-regulation, similar to neuronal differentiation, is mediated by the MAPK pathway since U0126, an inhibitor of MEK1/2, effectively blocked it. The overexpression of transcription factor CCAAT/enhancer binding protein-beta (C/EBPbeta) inhibited the 4HPR-induced down-regulation of IGFBP5 expression and the neuronal differentiation of RPE cells. Interestingly, the binding of C/EBPbeta to the IGFBP5 promoter was decreased by the 4HPR treatment as indicated by gel shift and chromatin immunoprecipitation analyses. Further, the deletion of C/EBP response element from IGFBP5 promoter markedly decreased the basal promoter activity and abolished its responsiveness to 4HPR treatment in reporter assays, suggesting that the expression of IGFBP5 is regulated by C/EBP. Thus, our results clearly demonstrate that the IGFBP5 expression is down-regulated during 4HPR-induced neuronal differentiation of human RPE cells through a MAPK signal transduction pathway involving C/EBPbeta.
Journal of Cellular Physiology 09/2010; 224(3):827-36. · 3.87 Impact Factor
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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.
Molecular vision 01/2010; 16:1475-86. · 2.20 Impact Factor
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ABSTRACT: NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P(270)KKRKAP(276)) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.
Biochemical and Biophysical Research Communications 08/2006; 345(4):1333-41. · 2.48 Impact Factor
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ABSTRACT: Recently it has been shown that the transport as well as clearance of retinol from isolated rod photoreceptors requires an extracellular factor. Interphotoreceptor retinoid-binding protein (IRBP) is a component of the interphotoreceptor matrix (IPM) and is known to bind visual cycle retinoids. Serum albumin and serum retinol-binding protein (sRBP), proteins capable of binding retinoids, have also been reported to be components of the IPM. It is of interest to know the components present in the IPM that are capable of binding visual cycle retinoids and that also facilitate rhodopsin regeneration. The purpose of this study was to determine the localization of serum albumin, sRBP, and IRBP in bovine retina using immunofluorescence analysis.
Fresh bovine eyes, obtained from a local abattoir, were fixed immediately after enucleation. Tissue sections (100 microm) were incubated with primary antibodies to bovine serum albumin (BSA), sRBP, and IRBP. Sections were washed then incubated 4 h with 4'-6-Diamidino-2-phenylindole (DAPI), Alexa Fluor(R) 488 goat antimouse, and Alexa Fluor 568 goat antirabbit secondary antibodies. Sections were analyzed using a laser scanning confocal microscope equipped with Nomarski optics. Western immunoblot analysis of bovine retinal tissues and protein standards was performed using the primary antibodies to BSA, sRBP, and IRBP to show specificity to their respective antigens.
Immunoblot analysis showed that monoclonal anti-BSA was highly specific for BSA detecting only a single band at about 67 kDa. Antihuman sRBP and antibovine IRBP were also highly specific, recognizing a single band at about 25 and about 133 kDa, respectively. No immunopositive bands were observed in bovine neural retinal when probed with the anti-sRBP antibody; however, a single immunoreactive band at about 67 and about 133 kDa was detected in bovine neural retina by the anti-BSA and IRBP antibodies, respectively. Immunofluorescence analysis showed labeling for IRBP throughout the IPM. IRBP labeling was especially associated with the outer segments of photoreceptors and also with the apical surface of the retinal pigment epithelium. Immunofluorescence labeling for serum albumin was associated only with the lumen of retinal and choroidal blood vessels. Staining for both serum albumin and sRBP in the IPM was negative.
Immunofluorescence analysis of fresh bovine eyes using antibodies to BSA and sRBP clearly shows that serum albumin and sRBP are not components of bovine IPM. IRBP, on the other hand, is localized to the IPM where it is available for the binding and transport of visual cycle retinoids. From these data we conclude that serum albumin and sRBP are not factors that could participate in the binding as well as transport of visual cycle retinoids in the IPM of bovine retina.
Molecular vision 02/2006; 12:1632-9. · 2.20 Impact Factor
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ABSTRACT: ARPE-19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N-(4-hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE-19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide-induced neuronal differentiation was dose and time dependent, started within 1-2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde-binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two-dimensional gel followed by matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated that heat-shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.
Journal of Neurochemistry 04/2003; 84(5):972-81. · 4.06 Impact Factor
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ABSTRACT: Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all-trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480-590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration.
Photochemistry and Photobiology 82(3):741-5. · 2.41 Impact Factor
