Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells
Fabián Arenas, … , Jesús Prieto, Juan F. Medina
Fabián Arenas, … , Jesús Prieto, Juan F. Medina
Published January 10, 2008
Citation Information: J Clin Invest. 2008;118(2):695-709. https://doi.org/10.1172/JCI33156.
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Research Article Hepatology

Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells

Abstract

Primary biliary cirrhosis (PBC) is a cholestatic disease associated with autoimmune phenomena and alterations in both biliary bicarbonate excretion and expression of the bicarbonate carrier AE2. The bile acid ursodeoxycholic acid (UCDA) is currently used in treatment of cholestatic liver diseases and is the treatment of choice in PBC; however, a subset of PBC patients respond poorly to UDCA monotherapy. In these patients, a combination of UDCA and glucocorticoid therapy appears to be beneficial. To address the mechanism of this benefit, we analyzed the effects of UDCA and dexamethasone on AE2 gene expression in human liver cells from hepatocyte and cholangiocyte lineages. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2 and enhanced AE2 activity. Similar effects were obtained after replacing UDCA with UDCA conjugates. In in vitro and in vivo reporter assays, we found that a UDCA/dexamethasone combination upregulated human AE2 alternate overlapping promoter sequences from which AE2b1 and AE2b2 are expressed. In chromatin immunoprecipitation assays, we demonstrated that combination UCDA/dexamethasone treatment induced p300-related interactions between HNF1 and glucocorticoid receptor on the AE2 alternate promoter. Our data provide a potential molecular explanation for the beneficial effects of the combination of UDCA and glucocorticoids in PBC patients with inadequate response to UDCA monotherapy.

Authors

Fabián Arenas, Isabel Hervias, Miriam Úriz, Ruth Joplin, Jesús Prieto, Juan F. Medina

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Figure 8

In vitro and in vivo role of GREcore motifs for the effects of UDCA and dexamethasone on AE2 alternate promoter.

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In vitro and in vivo role of GREcore motifs for the effects of UDCA and ...
(A) Luciferase activity of PLC/PRF/5 cells transiently transfected either with wild-type construct I-b1 or with deleted construct I-b1[–358b1/–239b1]Δ (cf. Figure 5) and treated for 24 hours with UDCA and/or dexamethasone. (B) Luciferase activity in PLC/PRF/5 cells transfected either with construct I-b1 or with constructs mutated at the GREcore motif(s) (cf. Figure 5) and treated for 24 hours with UDCA and/or DEX. In both A and B, luciferase activities (normalized with Renilla activity) are given as fold activity relative to the activity in cells transfected with construct I-b1 in the absence of UDCA and DEX; data are mean ± SD; n = 9 each. (C) Representative images of abdominal luminescence in BALB/c mice injected with 30 μg of luciferase constructs mutated at the GREcore motif(s) (cf. Figure 5) and treated with UDCA, DEX, UDCA plus dexamethasone, or just vehicle, as described in Methods. Normalizing values are quotients obtained for each animal after DNA quantifications (through real-time PCR) for the luciferase gene (as transfected gene) and the albumin gene (as endogenous gene) in the liver. (D) Values of abdominal luminescence in mice injected and treated as in C. Luminescence units from each animal had been corrected with corresponding normalizing values and are shown as fold corrected luminescence units relative to those in mice injected with construct I-b1 and with just vehicle (i.e., with no UDCA or DEX administration). Data are mean ± SD; n = 4 each.