Identification of 7-Ketocholesterol-Modulated Pathways and Sterculic Acid Protective Effect in Retinal Pigmented Epithelium Cells by Using Genome-Wide Transcriptomic Analysis

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. AMD is characterized by the formation of lipidic deposits between the retinal pigment epithelium (RPE) and the choroid called drusen. 7-Ketocholesterol (7KCh), an oxidized-cholesterol derivative, is closely related to AMD as it is one of the main molecules accumulated in drusen. 7KCh induces inflammatory and cytotoxic responses in different cell types, and a better knowledge of the signaling pathways involved in its response would provide a new perspective on the molecular mechanisms that lead to the development of AMD. Furthermore, currently used therapies for AMD are not efficient enough. Sterculic acid (SA) attenuates the 7KCh response in RPE cells and is presented as an alternative to improve these therapies. By using genome-wide transcriptomic analysis in monkey RPE cells, we have provided new insight into 7KCh-induced signaling in RPE cells, as well as the protective capacity of SA. 7KCh modulates the expression of several genes associated with lipid metabolism, endoplasmic reticulum stress, inflammation and cell death and induces a complex response in RPE cells. The addition of SA successfully attenuates the deleterious effect of 7KCh and highlights its potential for the treatment of AMD.

Keywords: 7-ketocholesterol; AMD; retina; sterculic acid; transcriptome.

Figure 1

Cell toxicity and inflammation induced by 7KCh and protective effect exerted by SA on mRPE cells. (A) Cell viability determined by MTS assay in mRPE cells treated with increasing concentrations of 7KCh or with (B) 10 μM SA and 12–20 μM 7KCh for 24 h. (C) Quantification by qRT-PCR of IL6, IL8 and VEGFA gene expression levels in mRPE cells treated with 15 μM 7KCh and 10 μM SA for 24 h, normalized with respect to rRNA 18S expression. (D) Secreted levels of IL-6, IL-8 and VEGF-A in mRPE cells exposed to 15 μM 7KCh and 10 μM SA for 48 h and measured by ELISA. The vehicle group in the graphs represents the control (control-vehicle) and the 7KCh (7KCh-vehicle) treatment. Data are represented as mean ± SEM of at least three different experiments. The dashed and dotted lines are a guidance mark of control and 7KCh value, respectively. ANOVA test was used for statistical analysis followed by Tukey (in A) or Sidak (in B–D) post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

TOP GO categories for biological processes in gene expression altered by 15 μM 7KCh in mRPE cells. DEGs between control and 7KCh-treated cells for 24 h were used to perform Gene Ontology (GO) enrichment analysis. The annotated DEGs were classified into different categories according to GO terms by EnrichGO. Functional groups are shown on the y-axis, while the gene ratio of each category is displayed on the x-axis.

Figure 3

Reversion effect induced by 10 μM SA over several representative genes altered by 15 μM 7KCh. Y-axis shows examples of genes modulated by 7KCh, whereas the X-axis represents the change in the expression of these genes in mRPE cells treated with 15 μM 7KCh, 10 μM SA or with the combination of SA and 7KCh with respect to the control and expressed as Log2FC. Positive Log2FC represents upregulated genes, while negative Log2FC represents downregulated genes. SA gene modulation data were obtained from our previous work [39].

Figure 4

Effect of TLR4 inhibition with CLI-095 on 7KCh-induced toxicity and inflammation. (A) Cell viability determined by MTS assay in mRPE cells treated with 15 μM 7KCh and 10 μM CLI-095 for 24 h. (B) Secreted levels of IL-6, IL-8 and VEGF-A in mRPE cells exposed to 15 μM 7KCh and 10 μM CLI-095 for 48 h and measured with ELISA. CLI-095 was added with a pretreatment of 2 h with respect to 7KCh. The vehicle group in the graphs represents the control (control-vehicle) and the 7KCh (7KCh-vehicle) treatment. Data are represented as mean ± SEM of at least three different experiments. The dashed and dotted lines are a guidance mark of control and 7KCh value, respectively. ANOVA test was used for statistical analysis followed by Sidak post hoc test. * p < 0.05.

Figure 5

ROS released in mRPE cells exposed to 12–20 μM 7KCh for 8 h and 24 h. ROS production levels were measured by oxidation of H₂DCFDA probe to DCF, and 10 mM H₂O₂ was used as a positive control. Data are represented as mean ± SEM of at least three different experiments. The dashed line is a guidance of the control value. ANOVA test was used for statistical analysis followed by Tukey post hoc test. ** p < 0.01; **** p < 0.0001.

Figure 6

UPR signaling induced in response to 7KCh in mRPE cells. (A) Western blot estimation of GRP78 levels in mRPE cells exposed to increasing concentrations of 7KCh for 24 h. GRP78 levels were normalized with respect to Actin band quantification. Data are represented as mean ±SEM of at least three different experiments. The dashed line is a guidance of the control value. ANOVA test was used for statistical analysis followed by Tukey post hoc test. (B) XBP1 splicing detection in the RNAseq analysis in mRPE cells treated with 15 μM 7KCh and/or 10 μM SA. Distribution of reads on XBP1 was visualized using the software IGV (Integrated Genome Viewer). Black arrows indicate the position in which the splicing was performed.

Figure 7

Classical MAPK signaling pathway altered by 15 μM 7KCh treatment in mRPE cells and reversion effect exerted by 10 μM SA over this modulation. In red, proteins expressed at higher levels than control. In green, proteins expressed at lower levels than control. Reprinted and modified with permission from Kyoto Encyclopedia of Genes and Genomes (KEGG). Copyright 2013, KEGG, Kyoto, Japan.

Figure 8

Activation of JNK in response to 7KCh in mRPE cells. (A) Western blot estimation of phosphorylated JNK (p-JNK) levels in mRPE cells exposed to increasing concentrations of 7KCh for 24 h. (B) Cell viability determined by MTS assay in mRPE cells treated with 15 μM 7KCh and increasing concentrations of SP600125 (SP) for 24 h. (C) Secreted levels of IL-6, IL-8 and VEGF-A in mRPE cells exposed to 15 μM 7KCh and 40 μM SP600125 for 48 h and measured with ELISA. (D) Western blot estimation of p-JNK levels in mRPE cells exposed to 20 μM 7KCh and 10 μM SA for 6 h, 12 h and 24 h. In Western blot assays, JNK levels, previously normalized with respect to Actin, were used to normalize p-JNK quantification. In MTS assay and ELISA, the JNK inhibitor SP600125 was added with a pretreatment of 2 h with respect to 7KCh. The vehicle group in the graphs represents the control (control-vehicle) and the 7KCh (7KCh-vehicle) treatment. Data are represented as mean ± SEM of at least three different experiments. The dashed and dotted lines are a guidance mark of control and 7KCh value, respectively. ANOVA test was used for statistical analysis followed by Tukey (in A,B,D) or Sidak (in C) post hoc test.* p < 0.05; ** p < 0.01.

Figure 9

Activation of p38 in response to 7KCh in mRPE cells. (A) Western blot estimation of phosphorylated p38 (p-p38) levels in mRPE cells exposed to increasing concentrations of 7KCh for 24 h. (B) Cell viability determined by MTS assay in mRPE cells treated with 15 μM 7KCh and increasing concentrations of SB203580 (SB) for 24 h. (C) Secreted levels of IL-6, IL-8 and VEGF-A in mRPE cells exposed to 15 μM 7KCh and 40 μM SB203580 for 48 h and measured with ELISA. (D) Western blot estimation of p-p38 levels in mRPE cells exposed to 20 μM 7KCh and 10 μM SA for 6 h, 12 h and 24 h. In Western blot assays, p38 levels, previously normalized with respect to Actin, were used to normalize p-p38 quantification. In MTS assay and ELISA, the p38 inhibitor SB203580 was added with a pretreatment of 2 h with respect to 7KCh. The vehicle group in the graphs represents the control (control-vehicle) and the 7KCh (7KCh-vehicle) treatment. Data are represented as mean ± SEM of at least three different experiments. The dashed and dotted lines are a guidance mark of control and 7KCh value, respectively. ANOVA test was used for statistical analysis followed by Tukey (in A,B,D) or Sidak (in C) post hoc test.* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.