Decreased expression of insulin-like growth factor binding protein-5 during N-(4-hydroxyphenyl)retinamide-induced neuronal differentiation of ARPE-19 human retinal pigment epithelial cells: regulation by CCAAT/enhancer-binding protein.

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.

Methionine sulfoxide reductase B2 is highly expressed in the retina and protects retinal pigmented epithelium cells from oxidative damage.

Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial enzyme that converts methionine sulfoxide (R) enantiomer back to methionine. This enzyme is suspected of functioning to protect mitochondrial proteins from oxidative damage. In this study we report that the retina is one of the human tissues with highest levels of MSRB2 mRNA expression. Other tissues with high expression were heart, kidney and skeletal muscle. Overexpression of an MSRB2-GFP fusion protein increased the MSR enzymatic activity three-fold in stably transfected cultured RPE cells. This overexpression augmented the resistance of these cells to the toxicity induced by 7-ketocholesterol, tert-butyl hydroperoxide and all-trans retinoic acid. By contrast, knockdown of MSRB2 by a miRNA in stably transfected cells did not convey increased sensitivity to the oxidative stress. In the monkey retina MSRB2 localized to the ganglion cell layer (GLC), the outer plexiform layer (OPL) and the retinal pigment epithelium (RPE). MSRB2 expression is most pronounced in the OPL of the macula and foveal regions suggesting an association with the cone synaptic mitochondria. Our data suggests that MSRB2 plays an important function in protecting cones from multiple type of oxidative stress and may be critical in preserving central vision.

Retinoic acid regulates the human methionine sulfoxide reductase A (MSRA) gene via two distinct promoters.

MSRAs (methionine sulfoxide reductases A) are enzymes that reverse the effects of oxidative damage by reducing methionine sulfoxide back to methionine and recovering protein function. In this study we demonstrate that the transcriptional regulation of the human MSRA gene is complex and driven by two distinct promoters. Both promoters demonstrate high expression in human brain and kidney tissues. The upstream (promoter 1) regulates the msrA1 transcript that codes for the mitochondrial form of MSRA and is highly active in a broad range of cell lines. The downstream promoter (promoter 2) regulates the msrA2/3 transcripts that code for the cytosolic/nuclear forms of MSRA and is generally less active. Promoter 2 contains a 65 bp putative enhancer region that is very active in the retinal pigment epithelium-derived D407 cell line. Both promoters are partially regulated by all-trans retinoic acid via RARA and other RARs.

Intraretinal lipid transport is dependent on high density lipoprotein-like particles and class B scavenger receptors.

PURPOSE: In our companion paper we demonstrated that circulating lipoproteins enter the retina via the retinal pigment epithelium (RPE) and possibly Müller cells. In order to understand how these lipids are transported within the retina, expression and localization of the main proteins known to be involved in systemic lipid transport was determined. METHODS: Expression of ABCA1, apoA1 (the major HDL protein), SR-BI, SR-BII, CD36, lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblots. Localization was determined by immunohistochemistry using fresh monkey vibrotome sections and imaged by confocal microscopy. RESULTS: ABCA1 and apoA1 were localized to the ganglion cell layer, retinal pigment epithelium (RPE), and rod photoreceptor inner segments. ApoA1 was also observed associated with rod photoreceptor outer segments, presumably localized to the interphotoreceptor matrix (IPM). The scavenger receptors SR-BI and SR-BII localized mainly to the ganglion cell layer and photoreceptor outer segments; in the latter they appear to be associated with microtubules. LCAT and CETP localized mainly to the IPM. CONCLUSIONS: The presence and specific localization of these well-known lipid transport proteins suggest that the retina employs an internal lipid transport mechanism that involves processing and maturation of HDL-like particles.

7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFκB but independently of reactive oxygen species formation.

PURPOSE: 7-Ketocholesterol (7KCh) accumulates in oxidized lipoprotein deposits and is known to be involved in macrophage foam cell formation and atherosclerosis. 7-KCh is present in the primate retina and is associated with oxidized lipoprotein deposits located in the choriocapillaris, Bruch's membrane, and retinal pigment epithelium (RPE). 7-KCh can also be formed in the retina as a consequence of light-induced iron release. The purpose of this study was to examine the signaling pathways involved in the 7KCh-mediated inflammatory response focusing on three cytokines, VEGF, IL-6, and IL-8. METHODS: ARPE-19 cells were treated with 7KCh solubilized in hydroxypropyl-ß-cyclodextrin. Cytokines were quantified by qRT-PCR (mRNA) and ELISA (protein) using commercially available products. NFκB activation was determined by IκBα mRNA induction. RESULTS: Treatment of ARPE-19 cells with 15 µM 7KCh markedly induced the expression of VEGF, IL-6, and IL-8. No increase in NOX-4 expression or ROS formation was detected. 7KCh induced the phosphorylation of ERK1/2 and p38MAPK, and inhibitors to these kinases markedly reduced the cytokine expression but did not affect the IκBα mRNA expression. By contrast, inhibition of PI3K and PKCζ significantly decreased the cytokine and IκBα mRNA expression. Inhibition of the IκB kinase complex essentially ablated all cytokine induction. CONCLUSIONS: 7KCh induces cytokines via three kinase signaling pathways, AKT-PKCζ-NFκB, p38 MAPK, and ERK. The MAPK/ERK pathways seem to preferentially enhance cytokine induction downstream from NFκB activation. The results of this study suggest that 7KCh activates these pathways through interactions in the plasma membrane, but the mechanism(s) remains unknown.