A2E-induced inflammation and angiogenesis in RPE cells in vitro are modulated by PPAR-α, -β/δ, -γ, and RXR antagonists and by norbixin

Abstract

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9'-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -β/δ and -γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E's deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

Keywords: N-retinylidene-N-retinylethanolamine (A2E); norbixin; peroxisome proliferator-activated receptor (PPAR); retinal pigment epithelium (RPE); retinoic X receptor (RXR).

Figure 1

A2E induces the transactivation of all PPAR isoforms. Effect of increasing concentration of A2E on endogenous PPAR transactivation (A). Effect of A2E (20 μM) on the transactivation of over-expressed PPAR-α (B), PPAR β/δ (C) and PPAR-γ (D). Effect of selective PPAR agonists GW9578 (GW95, 20 μM), GW0742 (GW07, 30 μM) and troglitazone (TGZ, 20 μM) on the respective transactivation of over-expressed PPAR-α (E), PPAR β/δ (F) and PPAR-γ (G). Bars represent mean ± s.e.m. with n = 3–4. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 compared to control (CONT) corresponding to DMSO alone added to the medium. (One-way ANOVA, Dunnett’s post-test).

Figure 2

Norbixin does not transactivate PPARs, but inhibits PPARγ transactivation induced by TGZ. Effect of GW9578, GW0742 and TGZ and of increasing norbixin (NBX) concentrations on over-expressed PPARα (A), PPARβ/δ (B) and PPARγ (C). Effect of GW9578 (20 μM), of GW0742 (30 μM) and of TGZ (20 μM) alone or in competition with NBX (20 μM) on over-expressed PPARα (D), PPARβ/δ (E) and PPARγ (F) transactivation. Bars represent mean ± s.e.m. with n = 3–6. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001 compared to CONT corresponding to DMSO alone added to the medium; ^#p < 0.01 compared to TGZ (One-way ANOVA, Dunnett’s post-test).

Figure 3

Norbixin inhibits the transactivation of PPARs induced by A2E. Effect of A2E (20 μM), NBX (20 μM) and A2E (20 μM) + NBX (20 μM) on endogenous PPAR transactivation (A). Effect of A2E (20 μM), NBX (20 μM) and A2E (20 μM) + NBX (20 μM) on over-expressed PPARα (B), PPARβ/δ (C) and PPARγ (D). Bars represent mean ± s.e.m. with n = 3–6. ^*or ^#p < 0.05, ^###p < 0.001, ^****or ^####p < 0.0001 compared to CONT (DMSO alone) or to A2E, respectively (One-way ANOVA, Dunnett’s post-test).

Figure 4

Norbixin inhibits the transactivation of NF-κB and AP-1 and regulates the expression of inflammatory and angiogenic factors stimulated by A2E in RPE cells in vitro. Effect of A2E (20 μM), NBX (20 μM) and NBX (20 μM) + A2E (20 μM) on NF-κB (A) and AP-1 (B) transactivation. Effect of A2E (30 μM), NBX (20 μM) and NBX (20 μM) + A2E (30 μM) on IL-6 (C), IL-8 (D), IL-18 (E), CCL2 (F), VEGF (G) and MMP9 (H) mRNA expression, and on AKT (I) and ERK (J) protein phosphorylation. Bars represent mean ± s.e.m. with n = 3–9. ^*or ^#p < 0.05, ^**or ^##p < 0.01, ^***p < 0.001, ^****or ^####p < 0.0001 compared to CONT (DMSO alone) or to A2E, respectively (One-way ANOVA, Dunnett’s post-test).

Figure 5

PPAR-α and PPAR-γ antagonists partly reproduce the effects of norbixin on inflammation but not on VEGF expression induced by A2E in RPE cells in vitro. Effect of PPAR-α, -β/δ and -γ selective antagonists MK886 (MK, 1 μM), GSK3787 (GSK, 1 μM) and T007907 (T007, 10 μM) respectively alone or with A2E (20 μM) on PPAR (A), NF-κB (B) and AP-1 (C) transactivation, and on IL-6 (D), IL-8 (E) and VEGF (F) mRNA expression. Bars represent mean ± s.e.m. with n = 3 ^* or ^#p < 0.05, ^** or ^##p < 0.01, ^***p < 0.001, ^**** or ^####p < 0.0001 compared to CONT (DMSO alone) or to A2E, respectively (One-way ANOVA, Dunnett's post-test).

Figure 6

PPAR-α, PPAR- β/δ and PPAR-γ antagonists used in co-treatment partly reproduce the effects of norbixin on inflammation but not on IL-6 expression and PPAR transactivation induced by A2E in RPE cells in vitro. Effect of a mixture of PPAR-α, -β/δ and -γ selective antagonists MK886 (MK, 1 μM), GSK3787 (GSK, 1 μM) and T007907 (T007, 10 μM) alone or with A2E (20 μM) on NF-κB (A), AP-1 (B) transactivation, and on IL-8 (C), VEGFa (D) and IL-6 (E), mRNA expression. Effects on PPAR (F) and RXR (G) transactivation. Bars represent mean ± s.e.m. with n = 3–6 and ^** or ^##p < 0.01, ^***p < 0.001, ^**** or ^####p < 0.0001 compared to CONT (DMSO alone) or to A2E, respectively (One-way ANOVA, Dunnett's post-test).

Figure 7

Norbixin inhibits the transactivation of RXRs induced by A2E. Effect of increasing concentrations of A2E on RXR transactivation (A). Effect of A2E (20 μM) and HX630 (5 μM) on RXR transactivation (B). Effect of A2E (20 μM), NBX (20 μM) and A2E (20 μM) + NBX (20 μM) on RXR transactivation (C). Effect of HX630 (5 μM) alone or in competition with NBX (20 μM) on RXR transactivation (D). Bars represent mean ± s.e.m. with n = 3–5. ^** or ^##p < 0.01, ^***p < 0.001, ^****p < 0.0001 compared to CONT (DMSO alone) or to A2E, respectively (One-way ANOVA, Dunnett's post-test).

Figure 8

Partial inhibition of RXR and PPAR transactivation by the pan-RXR-antagonist HX531 reduces A2E-induced inflammation and angiogenesis. Effect of A2E (20 μM), HX531 (5 μM) alone and HX531 (5 μM) + A2E (20 μM) on RXR (A), PPAR (B), NF-κB (C) and AP-1 (D) transactivation, and on IL-6 (E), IL-8 (F) and VEGF (G) mRNA expression. Bars represent mean ± s.e.m. with n = 3–4. ^*p < 0.05, ^** or ^##p < 0.01, ^###p < 0.001, ^**** or ^####p < 0.0001 compared to CONT (DMSO alone) or to A2E respectively (One-way ANOVA, Dunnett's post-test).