Purchase this article with an account.
Anna K. Pilat, Anja Herrnreiter, Christine Skumatz, Janice M. Burke, Tadeusz J. Sarna; Oxidative Stress Increases HO-1 in ARPE-19 Cells but Melanosomes Suppress the Increase when Light is the Stressor. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4776.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
ARPE-19 cells containing phagocytized melanosomes were previously shown to survive hydrogen peroxide-induced oxidative stress better than cells containing control latex beads. Here we asked whether the protective mechanism(s) involves differential expression of antioxidant enzymes in cells containing pigment granules by quantifying enzyme levels after treating cells with hydrogen peroxide or blue light.
ARPE-19 cells were loaded by phagocytosis with porcine RPE melanosomes or black latex beads (control particles). Heme oxygenase-1 (HO-1), HO-2, glutathione peroxidase (GPx), and catalase were quantified by western blot analysis before and after treatment with sub-lethal hydrogen peroxide or blue light (400-450nm). The stress was confirmed as sub-lethal by cell survival analysis using real-time quantification of propidium iodide fluorescence.
Phagocytosis itself produced transient changes in protein levels of some antioxidant enzymes, but steady state levels (7 days post phagocytosis) did not differ in cells containing melanosomes versus beads. Sub-lethal stress, induced by either hydrogen peroxide or light, had no effect on catalase, GPx or HO-2 in either particle-free or particle-loaded cells. In contrast, HO-1 protein was upregulated by treatment with both hydrogen peroxide and light. Particle content did not affect the HO-1 increase induced by hydrogen peroxide, but the increase induced by blue light irradiation was partially blocked in cells containing black beads and blocked even more in cells containing melanosomes.
Upregulation of HO-1 may help protect ARPE-19 cells from stress but it does not explain the differential susceptibility of cells to hydrogen peroxide that we previously observed for cells containing melanosomes versus beads (which did not differ in the HO-1 response). Melanosomes are believed to protect against light but, counterintuitively, the granules blocked the HO-1 increase after light stress. Blockage was partly due to optical screening (since black beads also blocked), but other properties of the pigment granule (perhaps melanin-mediated binding of iron, which regulates HO-1 expression) also contributed. The results indicate a role for HO-1 in the RPE stress response and a multifaceted role for melanosomes in regulating stress susceptibility.
This PDF is available to Subscribers Only