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Martin Busch, Susanne Wasmuth, Albrecht Lommatzsch, Daniel Pauleikhoff; ERK1/2 signaling pathway is activated by complement serum in UV-POS pretreated ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):375.
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Retinal pigment epithelial (RPE) cells undergo functional changes upon complement stimulation, which plays a role in the pathogenesis of age-related macular degeneration. The aim of this study was to investigate the effects of human complement serum (HCS) treatment on p44/42 mitogen-activated protein kinase (ERK1/2) activation in ARPE-19 cells pretreated with UV-irradiated photoreceptor outer segments (UV-POS), which was demonstrated to result in an accumulation of intracellular autofluorescent particles as a possible sign of “aging cells”.
ARPE-19 cells were grown in DMEM/F12 medium supplemented with 10 % fetal calf serum (FCS). For experiments, ARPE-19 cells were cultured for one week in 1 % FCS containing medium. Afterwards, cells were pretreated three times with 10 µg/ml UV-POS. Subsequently, cells were thoroughly washed and were stimulated with 5 % HCS or heat-inactivated HCS (HI-HCS) as a control in FCS-free medium for 24 hours. Total protein was analyzed for phosphorylated (activated) and non-phosphorylated ERK1/2 by western blotting. Cell culture supernatants were examined for their content of VEGF, IL-8, and MCP-1 via ELISA.
The stimulation of UV-POS pretreated ARPE-19 cells with HCS increased the amount of phosphorylated ERK1/2 (P-ERK1/2), whereas UV-POS in combination with HI-HCS or HCS alone (without UV-POS pretreatment) had no strong impact on the frequency of P-ERK1/2 when compared to the HI-HCS control. In contrast, the non-phosphorylated form did not differ between all treatment groups. Also, homogeneous expression of the housekeeping gene β-actin could be observed under the various conditions. The ELISA data revealed the trend that the production of VEGF, IL-8, and MCP-1 was increased in response to HCS. This HCS effect was pronounced in UV-POS pretreated ARPE-19 cells.
ERK1/2 signaling pathway demonstrated an increased phosphorylation and therefore activation in UV-POS pretreated ARPE-19 cells in response to complement serum. This was associated with an increased secretion of cytokines. Although possibly superimposed by other signaling pathways, ERK1/2 activation may be involved in the proinflammatory and proangiogenic changes of RPE-cells in response to complement and these effects may be even more distinct, if RPE cells are under metabolic stress.
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