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Extracellular matrix (ECM) plays an active and complex role in regulating cellular behaviors, including proliferation and adhesion. This study aimed at delineating the adhesion-induced signaling profiles in cultured human retinal pigment epithelium (RPE) cells and investigating the antiadhesion effect of antiproliferative drugs in this context. RPE R-50 cells grown on various ECM molecules, such as type I and IV collagens, fibronectin, and laminin, were used for adhesion assay and for examining the phosphorylation profiles of signaling mediators including Akt, extracellular signal-regulated kinase (ERK) 1/2, and integrin-linked kinase (ILK) using Western blotting. The cells receiving antiproliferative drug treatment at subtoxic doses were used to evaluate their antiadhesive and suppressive effects on kinase activities. ECM coating enhanced adhesion and spreading of RPE cells significantly. The cellular attachment onto ECM-coated surfaces differentially induced Akt, ERK1/2, and ILK phosphorylation, and concomitantly increased p53 phosphorylation and cyclin D1 expression, but decreased Bcl-2/Bax ratios. Treatment with antiproliferative agents, including 5-fluorouracil, mitomycin C, and daunomycin, at subtoxic doses suppressed the ability of RPE cells to adhere to ECM substratum significantly. This suppression was in part mediated through reduction of integrin β1 and β3 expressions and interfering Akt-ILK signaling activity. Mechanistically, blockade of PI3K/Akt signaling resulted in the suppressed adhesion of RPE cells to ECM. These findings support the hypothesis that, in addition to their antimitogenic effect, antiproliferative agents also exhibit suppressive effect on the adhesiveness of cultured RPE cells. Moreover, inhibitors of the PI3K/Akt signaling mediator can potentially be used as therapeutic agents for proliferative vitreoretinopathy., (Copyright © 2013. Published by Elsevier B.V.)

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. [ABSTRACT FROM AUTHOR]

Methylglyoxal (MGO), a glycolytic metabolite, induces oxidative injury and apoptotic cell death that play a pathogenetic role in age-related macular degeneration (AMD). This study examined the impact of MGO on cell proliferation and autophagy flux in retinal pigment epithelium (RPE) ARPE-19 cells and elucidated the underlying mechanism. Short-term MGO exposure suppressed cell proliferation without induction of apoptotic cell death, increased production of reactive oxygen species, and potentiated H 2 O 2 -exhibited cytotoxicity in ARPE-19 cells. Conversely, pretreatment with N-acetylcysteine, a ROS scavenger, and aminoguanidine, an MGO blocker, prevented MGO-induced growth retardation. MGO significantly enhanced autophagy flux and increased intracellular accumulation of autophagosomes, which was functionally confirmed by addition of autophagy enhancer or inhibitors. Signaling kinetic observation indicated that MGO remarkably triggered phosphorylation of Akt, ERK1/2, p38 MAPK, and JNK1/2. Blockade of kinase activity demonstrated that the hyperphosphorylation of Akt, ERK1/2, JNK, and p38 MAPK were all involved in the MGO-enhanced autophagy and growth-arresting effect in ARPE-19 cells. Moreover, pretreatment with autophagic flux inhibitors including 3-methyladenine, bafilomycin A, and chloroquine effectively ameliorated MGO- but not H 2 O 2 -mediated ARPE-19 cytotoxicity. In conclusion, modulation of autophagy flux activity by using autophagic or kinase inhibitors may be an applicable modality to treat AMD. [ABSTRACT FROM AUTHOR]

This study investigated the regulatory role of microRNA 100 (miR-100) in hydrogen peroxide (H2O2)-induced apoptosis of human retinal pigment epithelial ARPE-19 cells. H2O2 induced oxidative cell death of cultured ARPE-19 cells was measured by cytotoxicity assay. qRT-PCR was used to quantify cytosolic and extracellular contents of miR-100. Kinase and miR-100 inhibition treatments were applied to determine the regulatory signaling pathways involved in cell death regulation. H2O2 dose-dependently reduced viability of ARPE-19 cells and simultaneously upregulated miR-100 levels in both cytosolic and extracellular compartments. Western blotting detection indicated that H2O2 elicited hyperphosphorylation of PI3K/Akt, ERK1/2, JNK, p38 MAPK, and p65 NF-κB. Further kinase inhibition experiments demonstrated that PI3K, p38 MAPK, and NF-κB activities were involved in oxidative-stress-induced miR-100 upregulation in ARPE-19 cells, while blockade of PI3K, JNK, and NF-κB signaling significantly attenuated the oxidative cell death. Intriguingly, MiR-100 antagomir treatment exerted a cytoprotective effect against the H2O2-induced oxidative cell death through attenuating the oxidation-induced AMPK hyperphosphorylation, restoring cellular mTOR and p62/SQSTM1 levels and upregulating heme oxygenase-1 expression. These findings support that miR-100 at least in part mediates H2O2-induced cell death of ARPE-19 cells and can be regarded as a preventive and therapeutic target for retinal degenerative disease. [ABSTRACT FROM AUTHOR]