scholarly journals Characterization of key residues and membrane association domains in retinol dehydrogenase 10

2009 ◽  
Vol 419 (1) ◽  
pp. 113-123 ◽  
Author(s):  
Yusuke Takahashi ◽  
Gennadiy Moiseyev ◽  
Krysten Farjo ◽  
Jian-xing Ma
Keyword(s):  
Enzymatic Activity ◽  
Cultured Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Binding Motif ◽  
Dependent Manner ◽  
Retinol Dehydrogenase ◽  
Oxidoreductase Activity ◽  
Cofactor Binding ◽  
Key Residues

RDH10 (retinol dehydrogenase 10) was originally identified from the retinal pigment epithelium and retinal Müller cells. It has retinoid oxidoreductase activity and is thought to play a role in the retinoid visual cycle. A recent study showed that RDH10 is essential for generating retinoic acid at early embryonic stages. The present study demonstrated that wild-type RDH10 catalysed both oxidation of all-trans-retinol and reduction of all-trans-retinal in a cofactor-dependent manner In vitro. In cultured cells, however, oxidation is the favoured reaction catalysed by RDH10. Substitution of any of the predicted key residues in the catalytic centre conserved in the RDH family abolished the enzymatic activity of RDH10 without affecting its protein level. Unlike other RDH members, however, replacement of Ser197, a key residue for stabilizing the substrate, by glycine and alanine did not abolish the enzymatic activity of RDH10, whereas RDH10 mutants S197C, S197T and S197V completely lost their enzymatic activity. These results suggest that the size of the residue at position 197 is critical for the activity of RDH10. Mutations of the three glycine residues (Gly43, Gly47 and Gly49) in the predicted cofactor-binding motif (Gly-Xaa3-Gly-Xaa-Gly) of RDH10 abolished its enzymatic activity, suggesting that the cofactor-binding motif is essential for its activity. Deletion of the two hydrophobic domains dissociated RDH10 from the membrane and abolished its activity. These studies identified the key residues for the activity of RDH10 and will contribute to the further elucidation of mechanism of this important enzyme.

scholarly journals Acadesine suppresses TNF-α induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244307
Author(s):  
Nikolaos E. Efstathiou ◽  
Giannis A. Moustafa ◽  
Daniel E. Maidana ◽  
Eleni K. Konstantinou ◽  
Shoji Notomi ◽  
...  
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Complement Component ◽  
Adenosine Kinase ◽  
Age Related Macular Degeneration ◽  
Dependent Manner ◽  
Rpe Cells ◽  
Age Related ◽  
Tnf Α ◽  
Complement Component 3

Rationale Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells. Methods ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis. Results Acadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine’s effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it’s action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn’t prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3. Conclusions Our results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

scholarly journals Isolation of Bovine Retinal Pigment Epithelial Cells Using Adhesion to Agarose: Demonstration of Cellular and Regional Heterogeneity

2003 ◽  
Vol 51 (1) ◽  
pp. 121-124 ◽  
Author(s):  
Eleonore Fröhlich ◽  
Elke Maier ◽  
Christian Klessen
Keyword(s):  
Enzymatic Activity ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Histochemical Staining ◽  
Retinal Pigment Epithelial Cells ◽  
Isolated Cells ◽  
Regional Heterogeneity ◽  
Rpe Cells ◽  
A New Technique

The retinal pigment epithelium (RPE) shows cell heterogeneity in morphology and enzymatic activity. Routine isolation procedures for RPE cells may reduce enzymatic activity and prevent the quantification of regional enzymatic differences in vivo. We developed a new technique for the isolation of RPE cells based on adhesion of the cells to agarose. The morphology of the isolated cells resembled that of RPE cells in vivo. The cells were viable in the dye exclusion test and showed a histochemical staining pattern as RPE cells in vivo. With this technique, quantitative regional differences in the enzymatic activities were detected.

scholarly journals Transcriptome profiling of embryonic retinal pigment epithelium reprogramming

2021 ◽  
Author(s):  
Jared A Tangeman ◽  
Agustín Luz-Madrigal ◽  
Sutharzan Sreeskandarajan ◽  
Erika Grajales- Esquivel ◽  
Lin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinal Pigment Epithelium ◽  
Cell Cycle Progression ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Retina Development

AbstractThe plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in an FGF2-dependent manner. To characterize the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from 1) intact RPE, 2) transiently reprogrammed RPE (t-rRPE) 6 hours post-retinectomy, and 3) reprogrammed RPE (rRPE) 6 hours post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for MAPK-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis were used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) regulators, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

scholarly journals Retinoid metabolism in cultured human retinal pigment epithelium

1988 ◽  
Vol 250 (2) ◽  
pp. 459-465 ◽  
Author(s):  
S R Das ◽  
P Gouras
Keyword(s):  
Fatty Acid ◽  
Culture Medium ◽  
Cultured Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Retinyl Palmitate ◽  
Lipid Binding ◽  
Retinoid Metabolism ◽  
Hydrolysis Of

Uptake, esterification and release of all-trans-retinol in primary cultures of human retinal epithelium were studied. Cultured cells were supplemented with 3H-labelled 11,12-all-trans-retinol, using fatty-acid-free albumin as the carrier. This led to incorporation of retinal and the formation of all-trans- and 11-cis-retinyl palmitate. The metabolism of the all-trans ester was monitored in a medium containing various concentrations of foetal-bovine serum (FBS). In 20% (v/v) FBS, the ester was hydrolysed, and all-trans-retinol was released into the culture medium. In the absence of FBS, little ester was hydrolysed and no retinol was found in the medium. Dialysed or heat-inactivated FBS or fatty-acid-free albumin was as effective as FBS in provoking ester hydrolysis and retinol release. The concentration-dependency of this effect on FBS was matched by the corresponding concentrations of albumin alone. A linear relationship was also found between interphotoreceptor retinoid-binding protein and retinoid release. Haemoglobin, which does not bind retinoids, is ineffective in this capacity. It is concluded that lipid-binding substances, mainly albumin, in FBS act as acceptors for retinol and drain the cultured cells of this molecule. The release of the retinol is coupled to the hydrolysis of retinyl esters in the cell, so that there is little or no net hydrolysis of ester if there is no acceptor for retinol in the culture medium. This effect explains why cultured human retinal epithelial cells are depleted of their stores of retinoids when maintained in medium supplemented with FBS.

scholarly journals Glutathione Metabolism and the Novel Role of Mitochondrial GSH in Retinal Degeneration

Antioxidants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 661
Author(s):  
Parameswaran G. Sreekumar ◽  
Deborah A. Ferrington ◽  
Ram Kannan
Keyword(s):  
Cultured Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Critical Role ◽  
Basal Respiration ◽  
Age Related Macular Degeneration ◽  
Glutathione Metabolism ◽  
Cell Organelles ◽  
Atp Production ◽  
Age Related

Glutathione (GSH) is present ubiquitously, and its role as a crucial cellular antioxidant in tissues, including the retina, is well established. GSH’s antioxidant function arises from its ability to scavenge reactive oxygen species or to serve as an essential cofactor for GSH S-transferases and peroxidases. This review summarizes the general functions, retinal distribution, disorders linked to GSH deficiency, and the emerging role for mitochondrial GSH (mGSH) in retinal function. Though synthesized only in the cytosol, the presence of GSH in multiple cell organelles suggests the requirement for its active transport across organellar membranes. The localization and distribution of 2-oxoglutarate carrier (OGC) and dicarboxylate carrier (DIC), two recently characterized mitochondrial carrier proteins in RPE and retina, show that these transporters are highly expressed in human retinal pigment epithelium (RPE) cells and retinal layers, and their expression increases with RPE polarity in cultured cells. Depletion of mGSH levels via inhibition of the two transporters resulted in reduced mitochondrial bioenergetic parameters (basal respiration, ATP production, maximal respiration, and spare respiratory capacity) and increased RPE cell death. These results begin to reveal a critical role for mGSH in maintaining RPE bioenergetics and cell health. Thus, augmentation of mGSH pool under GSH-deficient conditions may be a valuable tool in treating retinal disorders, such as age-related macular degeneration and optic neuropathies, whose pathologies have been associated with mitochondrial dysfunction.

scholarly journals Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina.

1993 ◽  
Vol 121 (2) ◽  
pp. 335-343 ◽  
Author(s):  
D Gundersen ◽  
S K Powell ◽  
E Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Cultured Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Neural Retina ◽  
Apical Surface ◽  
Neural Cell Adhesion ◽  
Adhesion Role

The retinal pigment epithelium (RPE) is unique among epithelia in that its apical surface does not face a lumen, but, instead, is specialized for interaction with the neural retina. The molecules involved in the interaction of the RPE with the neural retina are not known. We show here that the neural cell adhesion molecule (N-CAM) is found both on the apical surface of RPE in situ and on the outer segments of photoreceptors, fulfilling an important requisite for an adhesion role between both structures. Strikingly, culture of RPE results in rapid redistribution of N-CAM to the basolateral surface. This is not due to an isoform shift, since the N-CAM expressed by cultured cells (140 kD) is the same as that expressed by RPE in vivo. Rather, the reversed polarity of N-CAM appears to result from the disruption of the contact between the RPE and the photoreceptors of the neural retina. We suggest that N-CAM in RPE and photoreceptors participate in these interactions.

scholarly journals Transcriptome Profiling of Embryonic Retinal Pigment Epithelium Reprogramming

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 840
Author(s):  
Jared A. Tangeman ◽  
Agustín Luz-Madrigal ◽  
Sutharzan Sreeskandarajan ◽  
Erika Grajales-Esquivel ◽  
Lin Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinal Pigment Epithelium ◽  
Cell Cycle Progression ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Retina Development

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

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