Chalcomoracin prevents vitreous-induced activation of AKT and migration of retinal pigment epithelial cells.
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ABSTRACT:
SUBMITTER: Han H
PROVIDER: S-EPMC8500972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kβ, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.
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Project description:Mutations in the lysosomal membrane protein CLN3 cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL). Activation of the lysosomal ion channel TRPML1 has previously been shown to be beneficial in several neurodegenerative disease models. Here, we tested whether TRPML1 activation rescues disease-associated phenotypes in CLN3-deficient retinal pigment epithelial (ARPE-19 CLN3-KO) cells. ARPE-19 CLN3-KO cells accumulate LAMP1 positive organelles and show lysosomal storage of mitochondrial ATPase subunit C (SubC), globotriaosylceramide (Gb3), and glycerophosphodiesters (GPDs), whereas lysosomal bis(monoacylglycero)phosphate (BMP/LBPA) lipid levels were significantly decreased. Activation of TRPML1 reduced lysosomal storage of Gb3 and SubC but failed to restore BMP levels in CLN3-KO cells. TRPML1-mediated decrease of storage was TFEB-independent, and we identified TRPML1-mediated enhanced lysosomal exocytosis as a likely mechanism for clearing storage including GPDs. Therefore, ARPE-19 CLN3-KO cells represent a human cell model for CLN3 disease showing many of the described core lysosomal deficits, some of which can be improved using TRPML1 agonists.
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Project description:The Notch signaling is an evolutionarily conserved cell-cell communication pathway that plays critical roles in the proliferation, survival, apoptosis, and fate determination of mammalian cells. Retinal pigment epithelial (RPE) cells are responsible for supporting the function of the neural retina and maintaining vision. This study investigated the function of Notch signaling in RPE cells. We found that the members of the Notch signaling pathway components were differentially expressed in RPE cells. Furthermore, blockage of Notch signaling inhibited the migration and proliferation of RPE cells and reduced the expression levels of certain Notch signaling target genes, including HES1, MYC, HEY2, and SOX9. Our data reveal a critical role of Notch signaling in RPE cells, suggesting that targeting Notch signaling may provide a novel approach for the treatment of ophthalmic diseases related to RPE cells.
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Project description:Proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) are characterized by the development of epi-retinal membranes which may exert a tractional force on retina. A lot of inflammatory growth factors may disturb the local ocular cells such as retinal pigment epithelial (RPE) cells, causing them to migrate and proliferate in the vitreous cavity and ultimately forming the PVR membrane. In this study, the signal pathways mediating cell migration of RPE induced by growth factors were investigated. Hepatocyte growth factor (HGF), epidermal growth factor (EGF) or heparin-binding epidermal growth factor (HB-EGF) induced a greater extent of migration of RPE50 and ARPE19 cells, compared with other growth factors. According to inhibitor studies, migration of RPE cells induced by each growth factor was mediated by protein kinase C (PKC) and ERK (MAPK). Moreover, HGF coupled with EGF or HB-EGF had synergistic effects on cell migration and enhanced activation of PKC and ERK, which were attributed to cross activation of growth factor receptors by heterogeneous ligands. Furthermore, using the shRNA technique, PKC? was found to be the most important PKC isozyme involved. Finally, vitreous fluids from PVR and PDR patients with high concentration of HGF may induce RPE cell migration in PKC?- and ERK- dependent manner. In conclusion, migration of RPE cells can be synergistically induced by HGF coupled with HB-EGF or EGF, which were mediated by enhanced PKC? activation and ERK phosphorylation.
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Project description:Purpose:Oxidative stress in retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD). Resveratrol exerts a range of protective biologic effects, but its mechanism(s) are not well understood. The aim of this study was to investigate how resveratrol could affect biologic pathways in oxidatively stressed RPE cells. Methods:Cultured human RPE cells were treated with hydroquinone (HQ) in the presence or absence of resveratrol. Cell viability was determined with WST-1 reagent and trypan blue exclusion. Mitochondrial function was measured with the XFe24 Extracellular Flux Analyzer. Expression of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit was evaluated by qPCR. Endoplasmic reticulum stress protein expression was measured by Western blot. Potential reactions between HQ and resveratrol were investigated using high-performance liquid chromatography mass spectrometry with resveratrol and additional oxidants for comparison. Results:RPE cells treated with the combination of resveratrol and HQ had significantly increased cell viability and improved mitochondrial function when compared with HQ-treated cells alone. Resveratrol in combination with HQ significantly upregulated HO-1 mRNA expression above that of HQ-treated cells alone. Resveratrol in combination with HQ upregulated C/EBP homologous protein and spliced X-box binding protein 1. Additionally, new compounds were formed from resveratrol and HQ coincubation. Conclusions:Resveratrol can ameliorate HQ-induced toxicity in RPE cells through improved mitochondrial bioenergetics, upregulated antioxidant genes, stimulated unfolded protein response, and direct oxidant interaction. This study provides insight into pathways through which resveratrol can protect RPE cells from oxidative damage, a factor thought to contribute to AMD pathogenesis.
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Project description:PurposeTo investigate the effects of inflammatory factors and oxidative stress on cell survival of the human retinal pigment epithelial (RPE) cell line, ARPE-19.MethodsConfluent RPE cells were treated with peripheral blood mononuclear cells-conditioned medium (PCM), H2O2, NaIO3, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, or combinations of these. Cell viability was determined by viability assays and by light microscopy. Effector molecules of cell death were investigated by immunofluorescence microscopy and flow cytometry. Microarrays were performed to screen for differential expression of anti-oxidative enzymes, and protein expression was validated by immunoblotting.ResultsViability of RPE cells was reduced by exposure to inflammatory agents (PCM, IFNγ+/-TNFα) or to oxidative agents (H2O2 or NaIO3). Unexpectedly, cells treated with either H2O2 or NaIO3 were partially protected from cell death by the addition of PCM. This protection was conferred, at least in part, by IFNγ and TNFα. Cell death induced by H2O2 or NaIO3 was preceded by mitochondrial dysfunction and by p62 upregulation, both of which were attenuated by PCM and/or by IFNγ+TNFα. RPE cells co-cultured with activated T cells, or treated with cytokines showed increased expression of anti-oxidative genes, with upregulation of superoxide dismutase 2 protein following PCM treatment.ConclusionOxidative stress-induced cell death was reduced by concomitant inflammatory stress. This is likely due to the cytokine-mediated induction of the anti-oxidative stress response, upregulating protective anti-oxidant pathway(s). These findings suggest caution for the clinical use of anti-inflammatory agents in the management of immune-associated eye diseases such as age-related macular degeneration.
| S-EPMC3660526 | biostudies-literature
Project description:Diabetic retinopathy (DR) is one of the most common causes of blindness globally. Proliferative DR (PDR), an advanced stage of DR, is characterized by the formation of fibrotic membranes at the vitreoretinal interface. The proliferation, migration, and secretion of extracellular matrix molecules in retinal pigment epithelial (RPE) cells contribute to the formation of fibrotic membranes in PDR. Gremlin has been reported to be upregulated in response to elevated glucose levels in the retina of diabetic rat and bovine pericytes. However, the role of gremlin in PDR remains unclear. In the present study, the vitreous concentrations of gremlin were significantly higher in the PDR (67.79 ± 33.96) group than in the control (45.31 ± 12.31) group, and high glucose levels induced the expression of gremlin in RPE cells. The elevated expression of extracellular matrix molecules, such as fibronectin and collagen IV, was significantly reduced by gremlin siRNA in human RPE cells under high-glucose conditions. Thus, gremlin may play a vital role in the development of PDR.
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Project description:The late stage of dry age-related macular degeneration (AMD), or geographic atrophy (GA), is characterized by extensive retinal pigment epithelial (RPE) cell death, and a cure is not available currently. We have recently demonstrated that RPE cells die from necrosis in response to oxidative stress, providing a potential novel mechanism for RPE death in AMD. In this study, we screened U.S. Food and Drug Administration-approved natural compounds and identified gossypol acetic acid (GAA) as a potent inhibitor of oxidative stress-induced RPE cell death. GAA induces antioxidative response and inhibits accumulation of excessive reactive oxygen species in cells, through which it prevents the activation of intrinsic necrotic pathway in response to oxidative stress. Sestrin2 (SESN2) is found to mediate GAA function in antioxidative response and RPE survival upon oxidative stress. Moreover, Forkhead box O3 transcription factor (FoxO3) is further found to be required for GAA-mediated SESN2 expression and RPE survival. Mechanistically, GAA promotes FoxO3 nuclear translocation and binding to the SESN2 enhancer, which in turn increases its transcriptional activity. Taken together, we have identified GAA as a potent inhibitor of oxidative stress-induced RPE necrosis by regulating the FoxO3/SESN2 pathway. This study may have significant implications in the therapeutics of age-related diseases, especially GA.
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Project description:Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly, and oxidative damage to retinal pigment epithelial (RPE) cells plays a major role in the pathogenesis of AMD. Exposure to high levels of atmospheric particulate matter (PM) with an aerodynamic diameter of <2.5 μm (PM2.5) causes respiratory injury, primarily due to oxidative stress. Recently, a large community-based cohort study in the UK reported a positive correlation between PM2.5 exposure and AMD. Sulforaphane (SFN), a natural isothiocyanate found in cruciferous vegetables, has known antioxidant effects. However, the protective effects of SNF in the eye, especially in the context of AMD, have not been evaluated. In the present study, we evaluated the effect of SFN against PM2.5-induced toxicity in human RPE cells (ARPE-19) and elucidated the molecular mechanism of action. Exposure to PM2.5 decreased cell viability in ARPE-19 cells in a time- and dose-dependent manner, potentially due to elevated intracellular reactive oxygen species (ROS). SFN treatment increased ARPE-19 cell viability and decreased PM2.5-induced oxidative stress in a dose-dependent manner. PM2.5-induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), a cell survival factor, was recovered by SFN. PM2.5 treatment decreased the enzymatic activities of the antioxidant enzymes including superoxide dismutase and catalase, which were restored by SFN treatment. Taken together, these findings suggest that SFN effectively alleviates PM2.5-induced oxidative damage in human ARPE-19 cells via its antioxidant effects, and that SFN can potentially be used as a therapeutic agent for AMD, particularly in cases related to PM2.5 exposure.
| S-EPMC8247919 | biostudies-literature
Project description:PurposeTo study the effect of subtoxic levels of hydrogen peroxide (H(2)O(2)) on the expression and release of interleukin-6 (IL-6) by cultured retinal pigment epithelial (RPE) cells and to explore the relevant signal pathways.MethodsCultured human RPE cells were stimulated with various subtoxic concentrations of H(2)O(2) for different periods. Conditioned medium and cells were collected. IL-6 in the medium and IL-6 mRNA in the collected cells were measured using an IL-6 enzyme-linked immunosorbent assay kit and reverse transcriptase polymerase chain reaction, respectively. Nuclear factor-kappaB (NF-κB) in nuclear extracts and phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinases (JNK) in cells cultured with and without H(2)O(2) were measured by NF-κB and MAPK enzyme-linked immunosorbent assay kits. Inhibitors of p38 (SB203580), ERK (UO1026), JNK (SP600125), and NF-κB (BAY11-7082) were added to the cultures before the addition of H(2)O(2) to test their effects(.)ResultsSubtoxic levels of H(2)O(2) (100 µM and less) increased the IL-6 mRNA level and the release of IL-6 protein by the cultured human RPE cells in a dose- and time-dependent manner. This was accompanied by an increase of NF-κB in nuclear extracts and phosphorylated p38 MAPK, ERK, and JNK in cell lysates, particularly in the p38 and NF-κB. The NF-κB inhibitor decreased the H(2)O(2)-induced expression of IL-6. The p38 inhibitor, but not the ERK or JNK inhibitor, completely abolished H(2)O(2)-induced expression of IL-6 by RPE cells. The p38 inhibitor also abolished the increase of NF-κB in nuclear extracts in cells treated with H(2)O(2).ConclusionsH(2)O(2) stimulated the production of IL-6, a key factor in the modulation of immune responses, inflammatory processes, and the occurrence of autoimmune diseases, which recently has been documented to be increased in age-related macular degeneration (AMD). This may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in AMD and may provide a novel target for the treatment of AMD.
| S-EPMC2956668 | biostudies-literature