Activation of the ERK1/2-MAPK Signaling Pathway by Complement Serum in UV-POS-Pretreated ARPE-19 Cells

Abstract

Background: Retinal pigment epithelial (RPE) cells undergo functional changes upon complement stimulation, which play a role in the pathogenesis of age-related macular degeneration (AMD). These effects are in part enhanced by pretreating ARPE-19 cells with UV-irradiated photoreceptor outer segments (UV-POS) in vitro. The aim of this study was to investigate the effects of human complement serum (HCS) treatment on p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2 [ERK1/2]) activation in ARPE-19 cells pretreated with UV-POS. Methods: UV-POS-pretreated ARPE-19 cells were stimulated with 5% HCS or heat-inactivated HCS (HI-HCS) as a control. Pro tein expression of phosphorylated (activated) ERK1/2, total ERK1/2, Bax, and Bcl-2 was analyzed by Western blotting. Cell culture supernatants were analyzed for IL-6, IL-8, MCP-1, and VEGF by enzyme-linked immunosorbent assay (ELISA). Furthermore, extra- and intracellular reactive oxygen species (ROS) were determined. Results: The amount of phosphorylated ERK1/2 was increased in UV-POS-pretreated ARPE-19 cells, especially in combination with HCS stimulation, compared to non-pretreated ARPE-19 cells incubated with HCS alone or HI-HCS. The same observation was made for Bax and Bcl-2 expression. Furthermore, an increase in extra- and intracellular ROS was detected in UV-POS-pretreated ARPE-19 cells. The ELISA data showed that the production of IL-6, IL-8, and MCP-1 tended to increase in response to HCS in both UV-POS-pretreated and non-pretreated ARPE-19 cells. Conclusions: Our data imply that ERK1/2 activation in ARPE-19 cells may represent a response mechanism to cellular and oxidative stress, associated with apoptosis-regulating factors such as Bax and Bcl-2, which might play a role in AMD, while ERK1/2 seems not to represent the crucial signaling pathway mediating the functional changes in RPE cells in response to complement stimulation.

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy

“…This results in elevated ROS, increased Bax and Bcl-2 expression, alongside secretion of IL-6, IL-8 and MCP-1 [33]. OxPOS-induced upregulation of VEGF and the aforementioned cytokines appears to be enhanced by the presence of human complement serum [32,33]. An earlier study using ARPE-19 cells also reported the induction of angiogenic cytokines IL-8 and MCP-1 alongside increased ROS following exposure to OxPOS [62].…”

“…Porcine photoreceptor outer segments (POS) were isolated as described previously [ 26 , 27 , 31 ]. In order to modify POS, a proportion of isolated POS were exposed to a 254 nm wavelength beam for 3 h which delivered 120,000 µJ [ 32 , 33 ]. Use of a commercially available UV cross-linking device allowed a standardized approach to generate oxidatively modified POS (OxPOS), which was subsequently visualized in vitro by transmission electron microscopy (TEM).…”

“…Exposure of ARPE-19 cultures to OxPOS leads to upregulation of ERK1/2, a mitogen-activated protein kinase and an upstream activator of the inflammasome-associated transcription factor NF-κB. This results in elevated ROS, increased Bax and Bcl-2 expression, alongside secretion of IL-6, IL-8 and MCP-1 [33]. OxPOS-induced upregulation of VEGF and the aforementioned cytokines appears to be enhanced by the presence of human complement serum [32,33].…”

Preeclampsia-Associated Alteration of DNA Methylation in Fetal Endothelial Progenitor Cells

“…Another affected pathway was ERK5 signaling. Typically, it is activated by external stimuli such as serum, growth factors, and cellular stressors including hypoxia and altered osmotic pressure (Busch et al, 2018). Simplified, activation results in increased gene expression and cell survival.…”

APOE2 promotes the development and progression of subretinal neovascularization in age-related macular degeneration via MAPKs signaling pathway

“…Besides, activated ERK1/2 and p38 MAPK have pro-inflammatory and proangiogenic properties. Studies have shown that MAPKs pathway promotes the upregulation of VEGF related factors, which leads to nvAMD ( Nagai et al, 2009 , Kyosseva, 2016 , Busch et al, 2018 ). The accumulation of IL-1β, IL-6 and TNF-α reduces after the addition of inhibitors of MAPKs which confirms MAPK is an upstream activator of inflammation-related NF-κB pathway ( Vyas et al, 2014 , Zhang et al, 2019 ).…”