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Beth M. Elledge, Cameron L. Atkinson, Weilue He, Srinivasa R. Sripathi, Wan Jin Jahng; Melatonin Modulates Prohibitin and β-Tubulin in Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3340.
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Retinal pigment epithelium (RPE) plays an imperative role in normal retinal function; it protects photoreceptors from light and phagocytizes rod and cone outer segments during disc shedding. Studies have shown that melatonin plays a role in retinal health and may be beneficial in protection of RPE cells from oxidative stress. Aside from the antioxidant and oxygen-free radical scavenging properties of melatonin, we are interested in its effect on cell cycle and cytoskeletal structure.
In vitro, Human RPE cell line ARPE-19 was grown to 70% confluence and then treated with 100µM DMSO-dissolved melatonin and 100µM H2O2 in DMEM for half an hour, after which the cells were allowed to express for 12 hours in untreated medium. Cells were probed with prohibitin (PHB) or β-tubulin antibody and prepared for immunocytochemistry (ICC) using AlexaFluor488 secondary antibody. Mitochondria and nucleus were fluorescently labeled with MitoTracker Orange and DAPI respectively. Cell morphology was tracked at initial, 30 minutes, and 12 hour time points. Quantitative PHB and β-tubulin levels were measured by Western blot and densitometric analysis using whole cell lysate.
Western blot analysis shows a 3-fold up-regulation of PHB with melatonin treatment versus control. A comparable level of PHB is detected when ARPE-19 cells are subjected to melatonin and H2O2 treatment together. β-tubulin levels are up-regulated under melatonin to a lesser extent. Initial cell shape of ARPE-19 is epitheloid; however, after 30 minute treatment with melatonin the cells undergo a morphological change to a fusiform shape with spindle outgrowths. Cells return to epitheliod shape after 12 hours in untreated medium. ICC of melatonin treated cells illustrates an increase in and dissimilar distribution of PHB and β-tubulin compared to non-melatonin-treated cells.
Melatonin up-regulates PHB concentration and alters cytoskeleton structure in RPE. Mitochondrial arrangement is also changed by melatonin treatment.
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