Proton pump inhibitor lansoprazole is a nuclear liver X receptor agonist

Abstract

The liver X receptors (LXRalpha and LXRbeta) are transcription factors that control the expression of genes primarily involved in cholesterol metabolism. In the brain, in addition to normal neuronal function, cholesterol metabolism is important for APP proteolytic cleavage, secretase activities, Abeta aggregation and clearance. Particularly significant in this respect is LXR mediated transcriptional control of APOE, which is the only proven risk factor for late onset Alzheimer's disease. Using a transactivation reporter assay for screening pharmacologically active compounds and off patent drugs we identified the proton pump inhibitor Lansoprazole as an LXR agonist. In secondary screens and counter-screening assays, it was confirmed that Lansoprazole directly activates LXR, increases the expression of LXR target genes in brain-derived human cell lines, and increases Abca1 and Apo-E protein levels in primary astrocytes derived from wild type but not LXRalpha/beta double knockout mice. Other PPIs activate LXR as well, but the efficiency of activation depends on their structural similarities to Lansoprazole. The identification of a widely used drug with LXR agonist-like activity opens the possibility for systematic preclinical testing in at least two diseases--Alzheimer's disease and atherosclerosis.

Figure 1. Lansoprazole induces LXR α/β mediated transcriptional activity in a transactivation assay

The assay was performed using Cos7 cells transfected with a Firefly luciferase reporter gene containing 3 copies of LXR response element, an expression plasmid for LXRα (A) or LXRβ (B), and a Renilla luciferase reporter for normalization. LXRα/β dual agonist T0901317 (Cpd T0) with EC50=25nM was used for comparison. A. Lansoprazole (Cpd L) at 5 μM activates LXRα to 80% of the T0 EC50. B. At 10 μM Lansoprazole activates LXRβ to 120% of T0 EC50. Means are fold of veh. *, p<0.05, **, p< 0.01 versus veh

Figure 2. PPI structural analogs increase ABCA1 mRNA expression in H4 human neuroglioma cell line

Cells were treated with 20 μM of Lansoprazole (Cpd L), Pantoprazole (Cpd P), Esomeprazole (Cpd E) and Omeprazole (Cpd O) for 24 h and ABCA1 mRNA expression was analyzed by QPCR. Controls were treated with veh and T0 (25 nM) for the same time. Means are presented as fold of veh. *, p<0.05, ***, p < 0.001 versus veh.

Figure 3. Lansoprazole upregulates ABCA1 expression in human glial-derived cell lines in a concentration-dependent manner

Cells were treated as indicated for 24 h and ABCA1 mRNA expression was analyzed by RT-QPCR. A. H4 neuroglioma cell line. B. U-87 (astrocytoma, APOE3/3). C. CCF (astrocytoma, APOE3/4) D. U-118 (astrocytoma, APOE2/4). Means are presented as fold of vehicle (*, p<0.05; **, p< 0.01; ***, p<0.001)

Figure 4. Lansoprazole upregulates LXR target genes expression in WT but not in LXRα/β-dko primary glial cells

Primary astrocytes derived from WT, LXRα^−/−, LXRβ^−/− and LXRα/β-dko newborn pups were treated with 20 μM Cpd L for 48 h. Control cells were treated with veh or T0 and Abca1 and Apoe mRNA expression were determined by RT-QPCR. A. Increased expression of Abca1 in all cell types but LXRα/β-dko; B. Apoe mRNA is upregulated in WT and LXRβ^−/− astrocytes (*, p< 0.05; **, p< 0.01; ***, p<0.001).

Figure 5. Protein levels of Abca1 and Apo-E are increased in response to Lansoprazole treatment

Primary astrocytes derived from WT newborn pups were treated with 20 μM Cpd L for 48 hrs. Control cells were treated with veh or T0 (1 μM) and Abca1 (A), cell associated Apo-E (B) and secreted Apo-E (C) were determined by western blot as described in the text (*, p< 0.05; **, p< 0.01; ***, p<0.001).

Figure 6. Lansoprazole increases ABCA1 and Apo-E intracellular protein level, and Apo-E secretion from CCF cells expressing APOE3/4 alleles

CCF cells were treated with 20 μM Lansoprazole for 48 h and controls with veh or T0 (1 μM). ABCA1 (A) and Apo- E (B and C) protein level was measured by WB using anti-human Apo-E antibody. Data are results of two experiments in quadruplicate. ABCA1 and Apo-E levels were normalized on β-actin and presented as fold of veh treatment. *, p <0.05, **, p< 0.01 versus vehicle.