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.

The Role of Canalicular ABC Transporters in Cholestasis

“…UDCA has, in addition, various other beneficial effects, such as increasing the hydrophilicity of the circulating bile acid pool, cytoprotection against bile acids and cytokines, immune modulation, and anti-inflammatory effects (reviewed by Poupon, 2012). In PBC patients, UDCA combined with budesonide (but not UDCA or budesonide alone) restored the activity of cholangiocyte anion exchanger 2, which mitigated the impaired choleresis in these patients (Arenas et al, 2008). UDCA also induced the antimicrobial peptide cathelicidin in PBC patients, presumably via VDR activation (D'Aldebert et al, 2009).…”

Molecular physiology and genetics of Na+-independent SLC4 anion exchangers

“…Since siRNAmediated suppression of Ae2 in rat cholangiocytes decreased secretin-and taurocholate-stimulated apical HCO 3 -secretion (Banales et al, 2006), decreased Ae2-mediated bile secretion secondary to inflammatory and cytokine-mediated damage may contribute to primary biliary cirrhosis in mouse and human (Arenas et al, 2008 (Salas et al, 2008). Ae2 expression in lymphoid cells (Alper et al, 1988) may thus be of immunological consequence.…”

“…The combination of UDC and glucocorticoids increases transcription from the overlapping intron 2 promoters driving expression of the liver-enriched Ae2 variants Ae2b1 and AE2b2, resulting in increased Ae2 polypeptide and Cl -/HCO 3 -exchange activity. Increased transcription results from enhanced p300-related interaction of HNF-1 and glucocorticoid receptor at promoterbinding sites (Arenas et al, 2008). The synonymous polymorphism of AE2 linked to ursodeoxycholate responsiveness in primary biliary cirrhosis may be part of a haplotype that includes polymorphisms within the AE2b1/2 promoter, or intronic polymorphisms governing mRNA stability.…”

1120 BICARBONATE SECRETION OF MOUSE CHOLANGIOCYTES INVOLVES Na+–HCO3− COTRANSPORT IN ADDITION TO Na+-INDEPENDENT Cl−/HCO3− EXCHANGE

“…13 Autocrine/paracrine stimulation of apical purinergic receptors by ATP is then followed by increased intracellular Ca 2ϩ , activation of apical Ca 2ϩ -dependent Cl Ϫ channels, and efflux of Cl Ϫ , which is likewise exchanged with HCO 3 Ϫ through AE2. Bicarbonate-enriching processes elicited by secretin might also involve CFTR-independent effects of cAMP on the AE2-mediated Cl Ϫ /HCO 3 Ϫ exchange, as observed in vitro in human cholangiocytes in the presence of CFTR inhibitors 3 and in hepatocyte-derived cells 4 that lack CFTR. Acetylcholine (ACh) may further assist biliary HCO 3 Ϫ secretion through Ca 2ϩ -dependent Cl Ϫ efflux from cholangiocytes and by potentiating the effect of secretin on both intracellular cAMP levels and Cl Ϫ /HCO 3 Ϫ exchange in a calcineurin-dependent manner.…”

“…2 In human cholangiocytes, however, basolateral influx of HCO 3 Ϫ occurs mainly through Na ϩ -dependent Cl Ϫ /HCO 3 Ϫ exchange. 3 In both human 4 and rat 5 cholangiocytes, the secretion of HCO 3 Ϫ to bile occurs through a Na ϩ -independent electroneutral Cl Ϫ /HCO 3 Ϫ exchange mediated by the anion exchanger (AE) 2 (AE2/SLC4A2), which is an acid loader of the SLC4/AE family generally involved in intracellular pH (pH i ) regulation. 6 Secretin-stimulated bicarbonate-rich hydrocholeresis has been postulated to involve the synchronized action of AE2 and two additional flux proteins, the cyclic adenosine monophosphate (cAMP)-responsive Cl Ϫ channel cystic fibrosis transmembrane conductance regulator (CFTR) and the water channel aquaporin-1.…”