Differential effects of the estrogen receptor agonist estradiol on toxicity induced by enzymatically-derived or autoxidation-derived oxysterols in human ARPE-19 cells

Abstract

Purpose/aim of the study: Disturbances in cholesterol metabolism and increased levels of cholesterol oxidation products (oxysterols) in retina may contribute to age-related macular degeneration (AMD). The role of oxysterols or of their target receptors liver X receptors (LXRs) and estrogen receptors (ERs) in the pathogenesis of MD is ill-known. The purpose of this study is to determine the extent to which the oxysterols 27-hydroxycholesterol (27-OHC), 25-hydroxycholesterol (25-OHC) and 7-ketocholesterol (7-KC) affect the transcriptional activity of LXR and ER.

Materials and methods: ARPE-19 cells, untreated or incubated with 27-OHC, 25-OHC or 7-KC for 24 h were harvested. We used Western blot analyses for detecting ERs and LXRs expression, dual luciferase assays for measuring LXRs and ERs transcriptional activity, cytotox-ONE homogeneous membrane integrity assay for measuring cytotoxicity, JC-1 method for measuring mitochondrial membrane potential changes and ELISA for measuring cytokine levels.

Results: Both LXRs and ERs are expressed and are transcriptionally active in ARPE-19 cells. 27-OHC, 25-OHC and 7-KC inhibited ER-mediated transcriptional activity, whereas 27-OHC and 25-OHC increased LXR-mediated transcription. E2 reduced 25-OHC and 27-OHC-induced cytotoxicity, mitochondrial permeability potential decline, and cytokine secretion. The LXR agonist GW3965 or the LXR antagonist 5α-6α-epoxycholesterol-3-sulfate (ECHS) did not offer protection against either 27-OHC and 25-OHC or 7-KC.

Conclusions: Increased levels of oxysterols can decrease ER and increase LXR signaling. ER agonists can offer protection against cytotoxic effects of 27-OHC and 25-OHC, two oxysterols derived by enzymatic reactions. Although they exert similar toxicity, the cellular mechanisms involved in the toxic effects of oxysterols whether derived by enzymatic or autoxidation reactions appear to be different.

FIGURE 1

(A) Western blots showing ERα and ERβ protein levels in cytosolic and nuclear fractions of untreated ARPE-19 cells. (B) Increased ERE-luciferase activity by E2 indicates that estrogen receptors are functional. (C) Western blots showing LXRα and LXRβ protein expression in cytosolic and nuclear fractions of untreated ARPE-19 cells. (D) GW3965 increased LXRE-luciferase activity indicating that liver × receptors are functional. Reporter activity values are expressed as arbitrary units. **p<0.01; ***p<0.001.

FIGURE 2

Cells were treated with vehicle (control), 10 µM 25-OHC, 10 µM 27-OHC or 25 µM 7-KC in the presence or not of 1 µM GW3965 (LXR agonist), 1 µM ECHS (LXR antagonist), 10nM E2 (ER agonist) or 1 µM ICI182780 (ER antagonist), for 24 h. (A) 27-OHC, 25-OHC and 7-KC decreased ERE-luciferase activity. ARPE-19 cells were transfected in parallel with ER reporter, and negative control and cells were subjected to 27-OHC, 25-OHC and 7-KC treatments for 24 h. Oxysterols decreased ERE-luciferase activity (p = 0.001 for 27-OHC, p<0.0001 for 25-OHC, p = 0.016 for 7-KC). (B) ARPE-19 cells were transfected in parallel with LXR reporter, and negative control and cells were subjected to oxysterol treatments for 24 h. 25-OHC and 27-OHC increased LXRE-luciferase activity (p = 0.031, 0.040, respectively). 7-KC appeared to reduce the luciferase activity, but was not statistically significant (p = 0.061). *p<0.05; **p<0.01; ***p<0.001.

FIGURE 3

Cells were treated with vehicle (control), 10 µM 25-OHC, 10 µM 27-OHC or 25 µM 7-KC in the presence or absence of 1 µM GW3965, 1 µM ECHS, 10nM E2 or 1 µM ICI182780, for 24 h. (A) 27-OHC is cytotoxic to cells (p = 0.001), and treatment with the ER agonist E2 reduced the cytotoxicity induced by 27-OHC (p = 0.019). (B) 25-OHC is also cytotoxic to cells (p<0.0001) and treatment with E2 reduced the 25-OHC-induced cytotoxicity (p = 0.012). (C) 7-KC is cytotoxic to cells (p<0.00001) but neither E2 nor ICI182780 significantly reduced cells from 7-KC-induced cytotoxicity (p = 0.114, 0.932, respectively). (D) Treatment with the LXR agonist GW3965 or with the LXR antagonist ECHS did not offer any protection against 27-OHC-induced cytotoxicity. (E) Treatment with GW3965 or ECHS did not offer any protection against 25-OHC-induced cytotoxicity. (F) Treatment of GW3965 or ECHS along with 7-KC did not offer any protection against 7-KC-induced cytotoxicity. *p<0.05; **p<0.01; ***p<0.001 versus control; #p<0.05 versus 27-OHC or 25-OHC.

FIGURE 4

Cells were treated with vehicle (control), 10 µM 25-OHC, 10 µM 27-OHC or 25 µM 7-KC in the presence or absence of 1 µM GW3965, 1 µM ECHS, 10nM E2 or 1 µM ICI182780, for 24 h. JC-1 assay was used for detection of mitochondrial membrane potential. (A) 27-OHC decreased the mitochondrial membrane potential significantly (p = 0.022). The ER agonist E2 restored the mitochondrial membrane potential decline induced by 27-OHC (p = 0.022). (B) 25-OHC caused a decrease in mitochondrial membrane potential (p = 0.025), which is counteracted by E2 (p = 0.040). (C) 7-KC-induced mitochondrial membrane potential decline (p = 0.034) was not significantly counteracted by E2 (p = 0.892) or ICI182780 (p = 0.996). (D–F) Neither the LXR agonist GW3965 nor the LXR antagonist ECHS counteracted the 27-OHC, 25-OHC or 7-KC-induced mitochondrial membrane potential decline. *p<0.05; **p<0.01; ***p<0.001 versus control; #p<0.05 versus 27-OHC or 25-OHC.

FIGURE 5

ELISA was used to measure IL-6 and TNF-α secretion from ARPE-19 cells. (A) IL-6 secretion was significantly increased after 24 h treatment with 27-OHC compared to control cells (p = 0.023). Treatment with E2, GW3965 and ECHS significantly reduced the 27-OHC-induced increase in IL-6 secretion (p = 0.001, p<0.0001, p = 0.001). (B, C) 25-OHC and 7-KC in the presence or absence of E2, ICI, GW3965 or ECHS showed no significant difference in IL-6 secretion when compared with controls. (D) TNF-α secretion was significantly increased after 24 h treatment with 27-OHC compared to control cells (p = 0.008). Treatment with E2, GW3965 and ECHS significantly reduced the 27-OHC-induced increase in TNF-α secretion (p<0.0001, p<0.0001, p = 0.005, respectively). (E, F) 25-OHC and 7-KC in the presence or absence of E2, ICI, GW3965 and ECHS showed no significant difference in TNF-α secretion when comparing with controls. *p<0.05 versus control #p<0.05, ###p<0.001 versus 27-OHC.