scholarly journals MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 791-802 ◽  
Author(s):  
R L Shattuck ◽  
L D Wood ◽  
G J Jaffe ◽  
A Richmond
Keyword(s):  
Northern Blot ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Fold Increase ◽  
Tnf Alpha ◽  
Mrna Levels ◽  
Nf Kappa B ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Alpha Beta

We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift analyses indicate that IL-1 and TNF alpha induce NF-kappa B complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappa B complexes may result in the inability of the cytokines IL-1 and TNF alpha to activate transcription of the MGSA/GRO genes. IL-1 and TNF alpha posttranscriptionally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells, IL-1 increases the half-life of MGSA/GRO alpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNF alpha transcriptionally and posttranscriptionally regulate MGSA/GRO alpha, -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, the major effect of IL-1 and TNF alpha is on mRNA stability.

MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells

1994 ◽  
Vol 14 (1) ◽  
pp. 791-802
Author(s):  
R L Shattuck ◽  
L D Wood ◽  
G J Jaffe ◽  
A Richmond
Keyword(s):  
Northern Blot ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Fold Increase ◽  
Tnf Alpha ◽  
Mrna Levels ◽  
Nf Kappa B ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Alpha Beta

We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift analyses indicate that IL-1 and TNF alpha induce NF-kappa B complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappa B complexes may result in the inability of the cytokines IL-1 and TNF alpha to activate transcription of the MGSA/GRO genes. IL-1 and TNF alpha posttranscriptionally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells, IL-1 increases the half-life of MGSA/GRO alpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNF alpha transcriptionally and posttranscriptionally regulate MGSA/GRO alpha, -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, the major effect of IL-1 and TNF alpha is on mRNA stability.

scholarly journals Multidrug-Resistant Pseudomonas aeruginosa Evokes Differential Inflammatory Responses in Human Microglial and Retinal Pigment Epithelial Cells

2020 ◽  
Vol 8 (5) ◽  
pp. 735
Author(s):  
Poonam Naik ◽  
Sukhvinder Singh ◽  
Sushma Vishwakarma ◽  
Inderjeet Kaur ◽  
Vivek Pravin Dave ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Retinal Pigment Epithelial ◽  
Inflammatory Responses ◽  
Retinal Pigment ◽  
Multidrug Resistant ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Infected Cells

Increasing incidences of multidrug-resistant (MDR) pathogens causing endophthalmitis threaten our ability to treat this condition, and the modulation of inflammatory responses by MDR bacteria is not known. In this study, using human microglia and retinal pigment epithelial (RPE) cells, we compare the inflammatory responses of sensitive (S-PA) and multidrug-resistant (MDR-PA) clinical isolates of Pseudomonas aeruginosa. Infected cells were subjected to qPCR analysis, enzyme-linked immunosorbent assay (ELISA), and immunostaining to assess the expression of inflammatory mediators. Both microglia and RPE cells, challenged with S-PA and MDR-PA, induced a time-dependent expression of inflammatory cytokines. Significant differences were observed in expression levels of Toll-like receptors (TLR) TLR4, TLR5, and TLR9 in microglia cells challenged with MDR-PA vs. S-PA. Similarly, mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α, Interferon (IFN)-γ, and matrix metalloproteinase (MMP)-9 were also higher in MDR-PA-infected cells. At protein levels, upregulation was observed for IL-10 (p = 0.004), IL-8 (p = 0.0006), IL-1β (p = 0.02), and Granulocyte-macrophage colony-stimulating factor (GM-CSF) (p = 0.0006) in cells infected MDR-PA versus S-PA in both microglia and RPE cells; however, the response was delayed in RPE cells. Heatmap and STRING analysis highlighted the existence of a cross-talk between the inflammatory and cytokine-mediated signaling pathways. Our study highlights a differential inflammatory response evoked by MDR vs. sensitive pathogens in retinal cells during endophthalmitis.

scholarly journals Sulforaphane Enhances the Ability of Human Retinal Pigment Epithelial Cell against Oxidative Stress, and Its Effect on Gene Expression Profile Evaluated by Microarray Analysis

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Liang Ye ◽  
Ting Yu ◽  
Yanqun Li ◽  
Bingni Chen ◽  
Jinshun Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Microarray Analysis ◽  
Retinal Pigment Epithelial ◽  
Nuclear Translocation ◽  
Retinal Pigment ◽  
Regulation Of Gene Expression ◽  
Age Related Macular Degeneration ◽  
Rpe Cells ◽  
Pigment Epithelial

To gain further insights into the molecular basis of Sulforaphane (SF) mediated retinal pigment epithelial (RPE) 19 cell against oxidative stress, we investigated the effects of SF on the regulation of gene expression on a global scale and tested whether SF can endow RPE cells with the ability to resist apoptosis. The data revealed that after exposure to H2O2, RPE 19 cell viability was increased in the cells pretreated with SF compared to the cell not treated with SF. Microarray analysis revealed significant changes in the expression of 69 genes in RPE 19 cells after 6 hours of SF treatment. Based on the functional relevance, eight of the SF-responsive genes, that belong to antioxidant redox system, and inflammatory responsive factors were validated. The up-regulating translation of thioredoxin-1 (Trx1) and the nuclear translocation of Nuclear factor-like2 (Nrf2) were demonstrated by immunoblot analysis in SF treated RPE cells. Our data indicate that SF increases the ability of RPE 19 cell against oxidative stress through up-regulating antioxidative enzymes and down-regulating inflammatory mediators and chemokines. The results suggest that the antioxidant, SF, may be a valuable supplement for preventing and retarding the development of Age Related Macular Degeneration.

scholarly journals GEP100/ARF6 regulates VEGFR2 signaling to facilitate high-glucose-induced epithelial-mesenchymal transition and cell permeability in retinal pigment epithelial cells

2019 ◽  
Vol 316 (6) ◽  
pp. C782-C791 ◽  
Author(s):  
Zhi-Peng You ◽  
Shan-Shan Chen ◽  
Zhong-Yi Yang ◽  
Shu-Rong Li ◽  
Fan Xiong ◽  
...  
Keyword(s):  
Cell Migration ◽  
High Glucose ◽  
Retinal Pigment Epithelial ◽  
Epithelial Mesenchymal Transition ◽  
Retinal Pigment ◽  
Cell Permeability ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Vegfr2 Signaling

Cell permeability and epithelial-mesenchymal transition (EMT) were found to be enhanced in diabetic retinopathy, and the aim of this study was to investigate the underlying mechanism. ARPE-19 cell line or primary retinal pigment epithelial (RPE) cells were cultured under high or normal glucose conditions. Specific shRNAs were employed to knock down ADP-ribosylation factor 6 (ARF6), GEP100, or VEGF receptor 2 (VEGFR2) in ARPE-19 or primary RPE cells. Cell migration ability was measured using Transwell assay. Western blotting was used to measure indicated protein levels. RPE cells treated with high glucose showed increased cell migration, paracellular permeability, EMT, and expression of VEGF. Knockdown of VEGFR2 inhibited the high-glucose-induced effects on RPE cells via inactivation of ARF6 and MAPK pathways. Knockdown ARF6 or GEP100 led to inhibition of high-glucose-induced effects via inactivation of VEGFR2 pathway. Knockdown of ARF6, but not GEP100, decreased high-glucose-induced internalization of VEGFR2. High-glucose enhances EMT and cell permeability of RPE cells through activation of VEGFR2 and ARF6/GEP100 pathways, which form a positive feedback loop to maximize the activation of VEGF/VEGFR2 signaling.

Hesperidin Prevents High Glucose-Induced Damage of Retinal Pigment Epithelial Cells

Planta Medica ◽  
2018 ◽  
Vol 84 (14) ◽  
pp. 1030-1037 ◽  
Author(s):  
Wayne Liu ◽  
Shorong-Shii Liou ◽  
Tang-Yao Hong ◽  
I-Min Liu
Keyword(s):  
High Glucose ◽  
Retinal Pigment Epithelial ◽  
Colorimetric Assay ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Antioxidant Enzyme Activities ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Rpe Cells ◽  
Pigment Epithelial

AbstractThe present study aimed to determine whether hesperidin, a plant-based active flavanone found in citrus fruits, can prevent high glucose-induced retinal pigment epithelial (RPE) cell impairment. Cultured human RPE cells (ARPE-19) were exposed to a normal glucose concentration (5.5 mM) for 4 d and then soaked in either normal (5.5 mM) or high (33.3 mM) concentrations of D-glucose with or without different concentrations of hesperidin (10, 20, or 40 µM) for another 48 h. The survival rates of the cells were measured using a 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay. With the help of a fluorescent probe, the intracellular production of reactive oxygen species (ROS) was evaluated. Colorimetric assay kits were used to assess the antioxidant enzyme activities, and western blotting was used to measure the expression of apoptosis-related protein. Hesperidin was effective in inhibiting high glucose-induced ROS production, preventing loss of cell viability, and promoting the endogenous antioxidant defense components, including glutathione peroxidase, superoxide dismutase, catalase, and glutathione, in a concentration-dependent manner. Furthermore, high glucose triggered cell apoptosis via the upregulation of caspase-9/3, enhancement of cytochrome c release into the cytosol, and subsequent interruption of the Bax/Bcl-2 balance. These detrimental effects were ameliorated by hesperidin in a concentration-dependent manner. We conclude that through the scavenging of ROS and modulation of the mitochondria-mediated apoptotic pathway, hesperidin may protect RPE cells from high glucose-induced injury and thus may be a candidate in preventing the visual impairment caused by diabetic retinopathy.

scholarly journals Differential induction of gene expression by basic fibroblast growth factor and neuroD in cultured retinal pigment epithelial cells

2000 ◽  
Vol 17 (2) ◽  
pp. 157-164 ◽  
Author(s):  
RUN-TAO YAN ◽  
SHU-ZHEN WANG
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Fibroblast Growth ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Two Factors

Embryonic chick retinal pigment epithelial (RPE) cells can undergo transdifferentiation upon appropriate stimulation. For example, basic fibroblast growth factor (bFGF) induces intact RPE tissue younger than embryonic day 4.5 (E4.5) to transdifferentiate into a neural retina. NeuroD, a gene encoding a basic helix-loop–helix transcription factor, triggers de novo production of cells that resemble young photoreceptor cells morphologically and express general neuron markers (HNK-1/N-CAM and MAP2) and a photoreceptor-specific marker (visinin) from cell cultures of dissociated E6 RPE (Yan & Wang, 1998). The present study examined whether bFGF will lead to the same transdifferentiation phenomenon as neuroD when applied to dissociated, cultured E6 RPE cells, and whether interplay exists between the two factors under the culture conditions. Dissociated E6 RPE cells were cultured in the presence or absence of bFGF, and with or without the addition of retrovirus expressing neuroD. Gene expression was analyzed with immunocytochemistry and in situ hybridization. Unlike neuroD, bFGF did not induce the expression of visinin, or HNK-1/N-CAM and MAP2. However, bFGF elicited the expression of RA4 immunogenicity; yet, many of these RA4-positive cells lacked a neuronal morphology. Addition of bFGF to neuroD-expressing cultures did not alter the number of visinin-expressing cells; misexpression of neuroD in bFGF-treated cultures did not change the number of RA4-positive cells, suggesting the absence of interference or synergistic interaction between the two factors. Our data indicated that bFGF and neuroD induced the expression of different genes in cultured RPE cells.

scholarly journals S-Allylmercapro-N-Acetylcysteine Attenuates the Oxidation-Induced Lens Opacification and Retinal Pigment Epithelial Cell Death In Vitro

Antioxidants ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 25 ◽  
Author(s):  
Naphtali Savion ◽  
Samia Dahamshi ◽  
Milana Morein ◽  
Shlomo Kotev-Emeth
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment Epithelial Cell ◽  
Retinal Pigment ◽  
Lens Opacity ◽  
Glutathione Level ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Lens Opacification

The capacity of S-Allylmercapto-N-acetylcysteine (ASSNAC) to protect human retinal pigment epithelial (RPE) cells (line ARPE-19) and porcine lenses from oxidative stress was studied. Confluent ARPE-19 cultures were incubated with ASSNAC or N-acetyl-cysteine (NAC) followed by exposure to oxidants and glutathione level and cell survival were determined. Porcine lenses were incubated with ASSNAC and then exposed to H2O2 followed by lens opacity measurement and determination of glutathione (reduced (GSH) and oxidized (GSSG)) in isolated lens adhering epithelial cells (lens capsule) and fiber cells consisting the lens cortex and nucleus (lens core). In ARPE-19 cultures, ASSNAC (0.2 mM; 24 h) increased glutathione level by 2–2.5-fold with significantly higher increase in GSH compared to NAC treated cultures. Similarly, ex-vivo exposure of lenses to ASSNAC (1 mM) significantly reduced the GSSG level and prevented H2O2 (0.5 mM)-induced lens opacification. These results demonstrate that ASSNAC up-regulates glutathione level in RPE cells and protects them from oxidative stress-induced cell death as well as protects lenses from oxidative stress-induced opacity. Further validation of these results in animal models may suggest a potential use for ASSNAC as a protective therapy in retinal degenerative diseases as well as in attenuation of oxidative stress-induced lens opacity.

Experimental eye enlargement in mature animals changes the retinal pigment epithelium

1999 ◽  
Vol 16 (4) ◽  
pp. 619-628 ◽  
Author(s):  
ALISON M. HARMAN ◽  
ROBERT HOSKINS ◽  
LYN D. BEAZLEY
Keyword(s):  
Retinal Pigment Epithelium ◽  
Retinal Pigment Epithelial ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Expansion Process ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Multinucleated Cells ◽  
Eye Opening ◽  
Comparable Size

Form deprivation has been shown to result in myopia in a number of species such that the eye enlarges if one eye is permanently closed at the time of eye opening. In the quokka wallaby, the eye grows slowly throughout life. After form deprivation, the eye enlarges by 1–1.5 years of age to the size of that in a 4–6-year-old animal and the number of multinucleated retinal pigment epithelial (RPE) cells in the enlarged retina remains much lower than would be expected in eyes of comparable size. Here we have repeated the experiment but examined animals at 4 years of age. The sutured eye grew significantly larger than did its partner. Numbers of RPE cells were comparable between sutured and partner eyes but were lower than in normal animals of similar age. Reductions in RPE cell density were greater in nasal than in dorsal or ventral retina and were not seen in temporal retina. The distribution of multinucleated cells was quite different in the sutured and open eyes. As in normal eyes, partner eyes had most multinucleated cells in ventral retina, while in the sutured eyes such cells were located mainly in the far periphery. In conclusion, the RPE is significantly changed by the eye enlargement process. However, it is not known whether this change results from an active part played by the RPE in the retinal expansion process or whether the changes are simply a result of a passive increase in area of the RPE.

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