IL-6-induced acetylation of E2F1 aggravates oxidative damage of retinal pigment epithelial cell line

Highlights

• High deacetylation of E2F1 depends on Sirt1 in oxidative stress-adapted RPE cells.

• Deacetylated E2F1 promotes the pentose phosphate pathway through activating the E2F1/HMGA1/G6PD axis.

• IL-6 induces the phosphorylation of Sirt1 and inhibits its deacetylase activity by activating PI3K/AKT/mTOR signaling.

• IL-6-induced acetylation of E2F1 impairs the antioxidant capacity of RPE cells.

Abstract

Oxidative damage in retinal pigment epithelial cells (RPE) is considered to be a crucial pathogenesis of age-related macular degeneration (AMD). Although dysregulation of the DNA repair system has been found in RPE cells of AMD patients, the detailed molecular mechanisms of this dysregulation and their relationship with the intraocular microenvironment of AMD patients remain unclear. Here, we established an RPE model of H₂O₂-induced oxidative stress and found that Sirtuin 1 (Sirt1)-mediated deacetylation of E2F transcription factor 1 (E2F1) was required for oxidation resistance in RPE cells. Moreover, E2F1 induced the expression of the chromatin-binding protein, high mobility group AT-Hook 1 (HMGA1), which promoted the transcription of glucose 6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, to increase NADPH level for antioxidant defense. Interrupting the E2F1/HMGA1/G6PD regulatory axis increased reactive oxygen species (ROS) levels, DNA damage, and apoptosis in RPE cells under oxidative stress. Notably, interleukin 6 (IL-6), an inflammatory cytokine that is known to be upregulated in the intraocular fluid of AMD patients, induced phosphorylation (S47) of Sirt1 by activating PI3K/AKT/mTOR signaling, thereby inhibiting Sirt1 activity and increasing the acetylation of E2F1. Specific inhibitors of PI3K/AKT/mTOR signaling decreased DNA damage and ROS while increasing NADPH in RPE cells. Collectively, our findings demonstrate that IL-6-induced acetylation of E2F1 impairs the antioxidant capacity of RPE cells by disturbing the pentose phosphate pathway, which elucidates a relationship between the intraocular microenvironment and RPE oxidative damage in AMD and provides a possible therapeutic target for AMD.

Introduction

Age-related macular degeneration (AMD) is a major cause of irreversible central vision loss and blindness, which accounts for 8.7% of blindness cases worldwide. The number of AMD cases is expected to reach 200 million by 2020 and rise to about 300 million by 2040 (Jonas, 2014; Wong et al., 2014). AMD is a complex degenerative disease in which macular injury is the primary cause of permanent vision loss. Oxidative stress, protein aggregation, inflammatory responses, and genetic factors play critical roles in AMD pathology (Kauppinen et al., 2016). However, the mechanisms of AMD pathogenesis remain poorly understood, and more efficacious AMD treatments are needed.

The retinal pigment epithelium (RPE), between the choroid and neuroretinal layers, has a variety of important functions including providing nutrition for photoreceptors, phagocytosis of photoreceptor-membrane discs, and formation of the external blood-retinal barrier. However, light exposure, high oxygen pressure, and photoreceptor-segment metabolism contribute to the formation of the oxidative environment in RPE cells, which inevitably leads to oxidative damage. Under physiological conditions, abundant antioxidant molecules and efficient repair systems in RPE cells antagonize the negative effects of the highly oxidized RPE environment. In contrast, reduced antioxidant capacity and abnormal repair processes for reducing oxidative damage eventually induce cellular death of RPE cells and photoreceptors in AMD patients (Jarrett et al., 2008; Tokarz et al., 2013). A previous study showed that DNA oxidative damage is closely related to the occurrence and development of AMD (Hyttinen et al., 2017). Therefore, it is of great scientific significance and clinical value to further explore the mechanisms of DNA oxidative damage in the RPE.

E2F1 is a transcription factor with a wide range of biological functions, which regulates the expression of genes related to DNA replication, damage repair, the cell cycle, and apoptosis (DeGregori and Johnson, 2006). Different post-translational modifications of E2F1 protein determine its different biological functions (Putzer and Engelmann, 2013). Our previous work in cancer cells suggests that E2F1 hypoacetylation maintains continued activation of DNA repair signaling by inducing the expression of checkpoint kinase 1 (CHK1) (Fang et al., 2018). As a key transcription factor in DNA repair, E2F1 directly regulates the expression of multiple DNA repair genes, such as ATM, BRCA1, Msh2, Msh6, PCNA, and RRM2 (Fang et al., 2015; Udayakumar et al., 2010). However, the role of E2F1 in DNA oxidative damage repair has not been reported in RPE cells.

As one of the main pathological features of AMD, chronic inflammation is closely associated with the progression of AMD (Knickelbein et al., 2015). The levels of various inflammatory factors in the sera and intraocular fluids of AMD patients are significantly increased compared with those in healthy controls (Mimura et al., 2018; Subhi et al., 2019). Among these factors, interleukin 6 (IL-6) levels are predictive of AMD progression and constitute one of the known biomarkers of disease activity in AMD (Klein et al., 2014). At present, studies on IL-6 have mainly focused on the clinical characteristics of AMD patients, whereas the pathogenic mechanisms of IL-6 have been rarely reported.

In the present study, we demonstrated that Sirt1-mediated deacetylation of E2F1 upregulated G6PD levels by inducing HMGA1 expression. Continuous expression of G6PD increased signaling of the pentose phosphate pathway via the production of the antioxidant molecule, NADPH. However, IL-6 inhibited the activity of Sirt1 by activating PI3K/AKT/mTOR signaling, consequently increasing DNA damage and apoptosis in RPE cells under oxidative stress.