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Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells.

ABSTRACT: Purpose:Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor genes in RPE cells is mediated by activator protein-1 (AP-1), and whether c-Fos and c-Jun genes are regulated by extracellular osmolarity. Methods:Hyperosmotic media were made up with the addition of NaCl or sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. DNA binding of AP-1 protein was evaluated with electrophoretic mobility shift assay (EMSA). Results:High NaCl and the addition of sucrose triggered expression of the c-Fos gene, but not of the c-Jun gene. High NaCl also increased the levels of c-Fos and phosphorylated c-Jun proteins and the level of DNA binding of AP-1. Hypoosmolarity decreased the expression of the c-Fos and c-Jun genes. NaCl-induced expression of the c-Fos gene was in part mediated by NFAT5. Autocrine/paracrine activation of fibroblast growth factor and adenosine A1 receptors is involved in mediating NaCl-induced expression of the c-Fos gene. Pharmacological inhibition of the AP-1 activity decreased the NaCl-induced expression of the HIF-1?, NFAT5, VEGF, PlGF, and TGF-?2 genes, and prevented the NaCl-induced secretion of PlGF but not of VEGF. Conclusions:The data indicate that AP-1 is activated in RPE cells in response to extracellular hyperosmolarity and mediates in part via the NaCl-induced expression of VEGF and PlGF, and secretion of PlGF. It is suggested that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in part via activation of AP-1.

SUBMITTER: Kleiner J

PROVIDER: S-EPMC6178337 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells.

Kleiner Jana J Hollborn Margrit M Wiedemann Peter P Bringmann Andreas A

Molecular vision 20181009

<h4>Purpose</h4>Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor ge ...[more]

PMID: 30310263

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Project description:BackgroundThe retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols.MethodsSphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches.FindingsWe provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate that the Hippo signaling pathway is important in iRPESC reprogramming.InterpretationThe Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs. FUND: National Natural Science Foundation of China and the National Institute of Health USA.

Project description:Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-?), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved.In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-? antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-? protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-? or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and ?-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-? and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of ?-catenin, VEGF, and p22phox and coimmunoprecipitation of ?-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-? following pretreatment with ?-catenin transcriptional inhibitors, XAV939 or JW67, or transfection with p22phox siRNA and compared to appropriate controls.Compared to the non-lasered control, TNF-? and VEGF protein were increased in the RPE/choroids in a murine laser-induced CNV model (p<0.05). An intravitreal neutralizing antibody to mouse TNF-? reduced CNV volume, and VEGF protein in the RPE/choroids (p<0.01) and oxidized phospholipids within CNV compared to IgG control (p<0.05). In cultured RPE cells and compared to controls, TNF-? induced ROS generation and increased activation of NOX4, an isoform of NADPH oxidase; both were prevented by pretreatment with the apocynin or VAS2870 or knockdown of p22phox, a subunit of NADPH oxidase. TNF-? treatment increased VEGF expression (p<0.001) and the formation of a transcriptional complex of ?-catenin and T-cell transcriptional factor; both were prevented by pretreatment with apocynin or knockdown of p22phox. Inhibition of ?-catenin by XAV939, but not the nuclear factor kappa B inhibitor, Bay 11-7082, prevented TNF-?-induced VEGF upregulation.Our results support the thinking that TNF-? contributes to CNV by upregulating VEGF production in RPE cells through ROS-dependent activation of ?-catenin signaling. These results provide mechanisms of crosstalk between inflammatory mediator, TNF-?, and ROS in RPE cells.

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Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells.

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Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells.

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