Artikel
Activation of Erk1/2 signaling pathway by complement serum in UV-POS pretreated ARPE-19 cells
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Autoren
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Martin Busch
- Münster
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S. Wasmuth
- Münster
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G. Spital
- Münster
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A. Lommatzsch
- Münster
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D. Pauleikhoff
- Münster
| Veröffentlicht: | 1. Oktober 2015 |
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Gliederung
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Background: RPE cells undergo functional changes upon complement stimulation, which play a role in the pathogenesis of AMD and are enhanced by pre-treatment with UV-irradiated photoreceptor outer segments (UV-POS) in vitro. Here, we investigated the effects of human complement serum (HCS) on p44/42 mitogen-activated protein kinase (ERK1/2) activation in ARPE-19 cells pretreated with UV-POS.
Methods: ARPE-19 cells were pretreated three times with 10 µg/ml UV-POS. Subsequently, cells were stimulated with 5 % HCS or heat-inactivated HCS (HI-HCS) as a control for 24 hours. Phosphorylated (activated) and non-phosphorylated ERK1/2 and Bcl-2 expression were analyzed by western blotting. Cell culture supernatants (SN) were analyzed for the concentration of IL-6, IL-8, MCP-1, and VEGF via ELISA and the content of reactive oxygen species (ROS) was determined photometrically based on the reaction with nitroblue tetrazolium salt.
Results: The amount of phosphorylated Erk1/2 was increased in UV-POS pretreated cells and especially after stimulation with HCS when compared to non-pretreated cells incubated with HCS alone or HI-HCS. This was paralleled by the Bcl-2 expression. In contrast, the expression of non-phosphorylated Erk1/2 and the beta-actin control did not differ between all treatment groups. Furthermore, a raise of ROS in SN of UV-POS pretreated cells was detected. ELISA data revealed an elevated production of IL-6, IL-8, MCP-1, and VEGF in response to HCS in both UV-POS pretreated and non-pretreated cells.
Conclusion: Erk1/2 activation in ARPE-19 cells was induced by UV-POS pretreatment and further enhanced by HCS stimulation. The effect of pronounced cytokine production could be attributed to HCS alone. Thus, we conclude that Erk1/2 does not represent the crucial signaling pathway mediating the functional changes of RPE cells in response to complement stimulation. Erk1/2 activation in ARPE-19 cells may represent a protective mechanism in response to cellular and oxidative stress inducing survival factors as Bcl-2.
