Human Retinal Pigment Epithelial ARPE-19 Research Articles

Novel YAP1 Activator, Identified by Transcription-Based Functional Screen, Limits Multiple Myeloma Growth.

Yes-associated protein 1 (YAP1) interacts with numerous transcription factors, including TEA-domain family proteins (TEAD) and p73. YAP1 is negatively regulated by the tumor suppressor Hippo pathway. In human cancers, the deregulation of the Hippo pathway and YAP1 gene amplification lead to the activation of YAP1, which induces epithelial-mesenchymal transition (EMT) and drug resistance. YAP1 inhibitors are expected to be useful in cancer therapy. On the other hand, in certain cancers, YAP1 upregulates p73-dependent gene transcription and behaves as a tumor suppressor. Moreover, as YAP1 regulates self-renewal and differentiation of tissue stem cells and plays an important role in tissue homeostasis, YAP1 activators may contribute to the regenerative medicine. With this in our mind, we screened for YAP1 activators by using human retinal pigment epithelial ARPE-19 cells expressing the TEAD-responsive fluorescence reporter under the coexpression of YAP1. From an extensive chemical compound library (n = 18,606) 47 candidate YAP1 activators were identified. These compounds were characterized to determine whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 activator was effective against the human multiple myeloma IM-9 cells and chronic myeloid leukemia K562 cells.Implications: YAP1 activation limits growth, induces apoptosis, and may be useful at suppressing hematological cancers. Mol Cancer Res; 16(2); 197-211. ©2017 AACR.

Correction: Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter.

[This corrects the article DOI: 10.1371/journal.pone.0149255.].

High mobility group B1 up-regulates angiogenic and fibrogenic factors in human retinal pigment epithelial ARPE-19 cells

Hypoxia-induced retinal neovascularization plays a central role in the pathogenesis of diabetic retinopathy. This study aimed to investigate whether hypoxia leads to the release of nuclear high mobility group box 1 (HMGB1) peptides from cultured retinal pigment epithelial ARPE-19 cells, to determine the effect of HMGB1 on angiogenic cytokine production and elucidate the involved signaling pathways. A chemical hypoxia mimetic agent, cobalt chloride, induced SIRT1 downregulation, HMGB1 nucleocytoplasmic relocation and extracellular release from ARPE-19 cells, implicating its autocrine function. Resveratrol treatment significantly reduced secretion of HMGB1 from ARPE-19 cells exposed to hypoxia. Cell proliferation and cell cycle analyses demonstrated that exogenous HMGB1 caused significant growth suppression and G1 cell cycle arrest in ARPE-19 cells. Morphological observations showed that HMGB1 enhanced adhesion, but suppressed migration of ARPE-19 cells. More intriguingly, HMGB1 up-regulated expression of angiofibrogenic factors in ARPE-19 cells, including VEGF, bFGF, TGF-β2, and CTGF. Signal profiling characterization indicated that HMGB1 triggered hyperphosphorylation of Akt, p38 MAPK, and NF-κB, but not that of ERK, JNK, and Smad2, whereas inhibition of PI3K, MAPK, or NF-κB significantly attenuated the HMGB1-driven cytokine overproduction in ARPE-19 cells. Functional neutralization with anti-TLR4 and -RAGE antibodies confirmed that both receptors were involved in the cytokine overproduction. In conclusion, chemically-mimicked hypoxia induced nucleocytoplasmic relocation and release of HMGB1 peptides, which in turn up-regulated the production of angiofibrogenic factors in RPE cells, thereby contributing to the pathogenesis of hypoxia-associated diabetic retinopathies. Conversely, blockades of intraocular HMGB1 bioavailability or signal activation may prevent angiofibrogenesis in development of diabetic retinopathy.

Differential autophagic effects of vital dyes in retinal pigment epithelial ARPE-19 and photoreceptor 661W cells

Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage.

Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal

Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells.

Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter.

Microbiota of the large intestine synthesizes considerable amount of vitamin B1 (thiamine) in the form of thiamine pyrophosphate (TPP). We have recently demonstrated the existence of an efficient and specific carrier-mediated uptake process for TPP in human colonocytes, identified the TPP transporter (TPPT) involved (product of the SLC44A4 gene), and shown that expression of TPPT along the gastrointestinal (GI) tract is restricted to the colon. Our aim in this study was to determine the molecular basis of the colon-specific expression of TPPT focusing on a possible epigenetic mechanism. Our results showed that the CpG island predicted in the SLC44A4 promoter is non-methylated in the human colonic epithelial NCM460 cells, but is hyper-methylated in the human duodenal epithelial HuTu80 cells (as well as in the human retinal pigment epithelial ARPE19 cells). In the mouse (where TPPT expression in the GI tract is also restricted to the colon), the CpG island predicted in the Slc44a4 promoter is non-methylated in both the jejunum and colon, thus arguing against possible contribution of DNA methylation in the colon-specific expression of TPPT. A role for histone modifications in the tissue-specific pattern of Slc44a4 expression, however, was suggested by the findings that in mouse colon, histone H3 in the 5’-regulatory region of Slc44a4 is tri-methylated at lysine 4 and acetylated at lysine 9, whereas the tri-methylation at lysine 27 modification was negligible. In contrast, in the mouse jejunum, histone H3 is hyper-trimethylated at lysine 27 (repressor mark). Similarly, possible involvement of miRNA(s) in the tissue-specific expression of TPPT was also suggested by the findings that the 3’-UTR of SLC44A4 is targeted by specific miRNAs/RNA binding proteins in non-colonic, but not in colonic, epithelial cells. These studies show, for the first time, epigenetic mechanisms (histone modifications) play a role in determining the tissue-specific pattern of expression of TPPT in the GI tract.

Docosahexaenoic Acid Protects against High Glucose-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells

Diabetic retinopathy is a leading cause of vision loss and has been correlated with increased oxidative stress. The aim of the present study was to evaluate the protective properties of docosahexaenoic acid (DHA), an omega-3 fatty acid, in human retinal pigment epithelial (RPE) cells exposed to high glucose. Human RPE cell line (ARPE-19) was cultured for 4 days followed by 5 days of exposure to either a normal (5.5 mM) or a high (45 mM) D-glucose concentration in the absence and presence of DHA (100 µM). Reduced form of glutathione (GSH), total antioxidant capacity (TAC), total nitrites, and malodialdehyde (MDA) were assessed. 4-Hydroxynonenal (HNE), another lipid peroxidation product, was determined by immunocytochemistry. Cell viability was assessed by the MTT assay. The results showed that both TAC and GSH content were significantly decreased after the high glucose challenge. The presence of DHA prevented the reduction and maintained the TAC and GSH at the levels found in cells exposed to the normal glucose concentration (5.5 mM). Moreover, the levels of total nitrites and MDA were significantly increased after high glucose exposure compared to cell exposed to 5.5 mM glucose. Again, the presence of DHA prevented the increase and maintained the nitrites and MDA at the levels found in control cells. Notably, the high glucose condition led to a significantly increased number of HNE aggresomes as compared to control cells, and DHA completely prevented this increase. In line with the reduced oxidative stress, DHA treatment also completely prevented the high glucose-induced decrease in RPE cell viability. Taken together, this study demonstrated that DHA protected human retinal pigment epithelial ARPE-19 cells from high glucose-induced oxidative damage and cytotoxicity. The results of this study support a role for oxidative stress in high glucose-induced RPE injury and the potential use of omega-3 fatty acids to protect against diabetic retinopathy.

Computational Model of Ca2+ Wave Propagation in Human Retinal Pigment Epithelial ARPE-19 Cells.

ObjectiveComputational models of calcium (Ca2+) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer.MethodsWe combined six essential Ca2+ signaling components into a model: stretch-sensitive Ca2+ channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked.ResultsOur model was able to explain Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance.ConclusionsThe developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.

Identification of A Novel Inhibitor of 5‐Lipoxygenase

Arachidonate 5‐lipoxygenase (5‐LOX) is a member of the lipoxygenase family of enzymes that plays a key role in arachidonic acid metabolism. Arachidonic acid, upon oxygenation by 5‐LOX, forms 5‐hydroperoxyeicosatetraenoic acid (5‐HPETE), which is further converted into proinflammatory leukotrienes such as LTB4 that can have detrimental biological effects. The PNPLA2 gene codes for PEDF‐R, a lipase involved in cell survival and lipid metabolism, found in the RPE and retina. We have identified human PEDF‐R peptide regions E5b (I193–L232) and P1 (T210‐L249) that efficiently protected human retinal pigment epithelial ARPE‐19 and pig primary RPE cells from oxidative stress. We investigated whether binding and inhibiting 5‐LOX by the PEDF‐R peptides are critical to protect RPE cells during oxidative stress conditions. Peptide affinity chromatography showed that human recombinant 5‐LOX binds to immobilized P1. The binding decreased with increasing NaCl concentrations, implying an ionic nature for the interaction. Moreover, full length PEDF‐R bound 5‐LOX in a dose‐response fashion. Interestingly, PNPLA2 expression decreased with increasing oxidative stress with no effect on 5‐LOX expression. Peptides E5b and P1 bound and inhibited the activity of soybean lipoxygenase‐V (LOX‐V) in vitro. The LOX‐V enzymatic inhibition was attenuated when PEDF‐R peptides were captured specifically with a P1 binding protein, fluorescein‐conjugated PEDF (Fl‐PEDF). Moreover, measurement of the 5‐LOX product LTB4 showed that P1 inhibited the formation of LTB4 in ARPE‐19 cells under oxidative stress. Our results support that PEDF‐R is a novel inhibitor of 5‐LOX and PEDF‐R derived peptides, i.e., P1, have therapeutic potential in diseases in which oxidative stress plays a role, such as age‐related macular degeneration. Supported by NIH‐IRP.

Efficacy of Ethanol Extract ofFructus lyciiand Its Constituents Lutein/Zeaxanthin in Protecting Retinal Pigment Epithelium Cells against Oxidative Stress:In VivoandIn VitroModels of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Oxidative stress plays a large role in the pathogenesis of AMD. The present study was to evaluate the effects of Fructus lycii ethanol extract on AMD in mice and to investigate whether combination of lutein and zeaxanthin, two carotenoid pigments in Fructus lycii, could protect human retinal pigment epithelial ARPE-19 cells treated with hydrogen peroxide (H2O2) in vitro. We found that severe sediment beneath retinal pigment epithelium and thickened Bruch membrane occurred in AMD mice. However, Fructus lycii ethanol extract improved the histopathologic changes and decreased the thickness of Bruch membrane. Furthermore, the gene and protein expression of cathepsin B and cystatin C was upregulated in AMD mice but was eliminated by Fructus lycii ethanol extract. Investigations in vitro showed that ARPE-19 cell proliferation was suppressed by H2O2. However, lutein/zeaxanthin not only stimulated cell proliferation but also abrogated the enhanced expression of MMP-2 and TIMP-1 in H2O2-treated ARPE-19 cells. These data collectively suggested that Fructus lycii ethanol extract and its active components lutein/zeaxanthin had protective effects on AMD in vivo and in vitro, providing novel insights into the beneficial role of Fructus lycii for AMD therapy.

Dietary wolfberry ameliorates retinal structure abnormality through activation of AMP activated protein kinase signaling in the db/db type 2 diabetic mouse

Objective To determine the protective effect of dietary wolfberry on retinal abnormality in the db/db mouse at the early stage of type 2 diabetes. Methods db/db mice at the age of 6 weeks were fed low fat diet with or without supplemental 1 kCal % wolfberry for 8 weeks. Human retinal pigment epithelial ARPE-19 cells were exposed to high glucose to mimick hyperglycemia in vitro. Retinal structure was investigated by light microscopy. Alteration of protein expression was monitored by Western blot. siRNA was used to knockdown AMPKα in ARPE-19 cells. Results Dietary wolfberry restored thickness of whole retina, in particular the photoreceptor layer, and integrity of retinal pigment epithelia (RPE). Addition of wolfberry lowered expression of endoplasmic reticulum stress biomarkers BiP, PERK, ATF6, and caspase-12; restored AMPKα, thioredoxin, Mn SOD, and FOXO3α activities. Zeaxanthin mimicked wolfberry preventive effect on AMPKα activation and normalization of reactive oxygen species in ARPE-19 cells exposed to high glucose challenge. Zeaxanthin preventive effect was abolished by siRNA knockdown of AMPKα. Conclusions Dietary wolfberry restoration of AMPKα led to enhanced expression of thioredoxin and Mn SOD, which, in turn, resulted in mitigation of cellular oxidative stress and subsequent prevention of retinal disorganization in the retina of db/db mice at the early stage of type 2 diabetes. Supported by NIH COBRE Grant P20-RR-017686 (to D.L.)

Dietary Supplements Protect Retinal Pigment Epithelial Cells From Hyperglycemic Damage

ObjectiveTo determine the protective effects of wolfberry on hyperglycemic damage in retinal pigment epithelial cells.MethodsHuman retinal pigment epithelial ARPE‐19 cells were treated with 36 mM glucose for 60 hrs, with or without 1 mg/mL crude wolfberry extracts. PKCgamma activation was determined by enzyme activity assay. Gap junction activity was determined by scrape loading/dye transfer assay. Western blotting was used to determined activation of AMP‐activated protein kinase (AMPK), acetyl‐CoA carboxylase (ACC), and apoptosis‐inducing factor (AIF).ResultsHigh glucose‐activated PKCgamma phosphorylated Connexin 50 (Cx50) and Cx57 on serines, and subsequently inhibited gap junction activity. Wolfberry extracts abolished the effects of high glucose on PKCgamma activation. Application of wolfberry extracts abolished the inhibitory effect of high glucose on AMPK activation, completely restored ACC phosphorylation by AMPK, and inhibited AIF translocation out of mitochondria.ConclusionsHigh glucose‐induced AIF‐dependent apoptosis would be protected by application of wolfberry extracts through restoration of cellular gap junctional communication and rebalance of cellular energy homeostasis.