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Joyce Tombran-Tink, Yanling Liu, Anzor Grivitishvilli; Elevated Glucose Alters Cytoskeleton-tight Junction Dynamics In RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5937.
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© ARVO (1962-2015); The Authors (2016-present)
We investigated the effects of glucose on cytoskeleton and tight junction control in the retinal pigment epithelial cells to understand how glucose shifts influence morphology, barrier function, and adhesion properties of these cells.
Human ARPE19 cells were grown in long term cultures supplemented with varying concentrations of glucose between 5.5-30 mM in medium containing 10% FBS. Cultures were maintained for > 45 days in glucose and osmotically balanced conditions with fresh medium replaced every 4 days. Semi confluent cells were harvested after long-term glucose exposure and processed for transcriptional and translational analyses of cytoskeletal and tight junctions proteins and for electron microscopy.
Morphologically, RPE cells were larger with increasing glucose concentrations. At the electron microscopic level, complex dense arrays of cytoskeletal structures and large vacuoles filled with glycogen granules were visible. Cytoskeletal structures were often organized as tightly packed circular bundles in addition to the parallel cytoskeleton network seen in the cells. Increases in protein and mRNA expression of cytokeratin, tubulin, and actin filaments were also evident. Regulation of three tight junction proteins was examined in the long-term high glucose treated cells. Occludin, and its associated scaffolding protein, ZO1 showed decreased transcription and translational levels while tricellulin, a protein found in vertically oriented TJ strands at tricellular junctions, was elevated with increasing concentrations of glucose. An increase in the phosphorylation of pERK accompanied these gene expression changes.
Elevated glucose alters tight junction and cytoskeleton mechanics in the RPE cells and may do so through the activation of the MAPK pathway. Our studies suggest that fluctuations in glucose levels may contribute to disturbances in cell size and shape, polarized cell phenotype, barrier function, trafficking of glucose transporters and other intracellular transport, and cell adhesion dynamics in the RPE cells. These structural and biochemical shifts in the RPE may have feed forward undercurrents in pathological developments of diabetic retinopathy.
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