Caracterización de regiones promotoras del gen ae2 humano en respuesta a udca y glucocorticoides

Resumen

Characterization of human AE2 gene promoter regions responsive to UDCA and glucocorticoids Fabián Octavio Arenas Ríos Division of Gene Therapy and Hepatology, CIMA-CUN, University of Navarra - School of Medicine Pamplona, Navarra, Spain, 2008 Background and aims. Primary biliary cirrhosis (PBC), is a chronic cholestatic disease associated to autoimmune phenomena PBC patients show alterations in both the biliary bicarbonate secretion and the liver expression of the bicarbonate carrier in the hepatobiliary tract AE2 (SLC4A2). Ursodeoxycholic acid (UDCA), a bile acid which produces a bicarbonate-rich choleresis, is the treatment of choice in PBC. There is, however, a subset of PBC patients with a poor response to UDCA monotherapy, and combination of UDCA with glucocorticoids seems to be beneficial in those cases. To address the mechanism of this benefit, we investigated whether UDCA, alone or in combination with glucocorticoids, could have an effect on AE2 gene expression and analyzed the possible mechanisms involved. Methods. We used human liver cells from hepatocyte and cholangiocyte lineages and treated them with dexamethasone and/or UDCA (as well as with other bile acids). We analyzed the effects of these compounds on: i) the transcriptional expression of AE2 isoforms by measuring the mRNA levels through real-time PCR; ii) the AE2 activity by microfluorimetry; iii) the AE2 alternate promoter activity through luciferase-reporter gene assays; and iv) transcriptional factor interactions on AE2 promoter sequences by using chromatin immunoprecipitation (ChIp) assays. Results. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2, with no changes in the expression of the complete AE2a isoform. Increased alternative expression correlated with Also, UDCA+dexamethasone combination enhanced the AE2 activity in the hepatobiliary cells. Combination effects remained after replacing UDCA with its taurine and glycine conjugates ¿Cbut not with cholic or chenodeoxycholic acids. In vitro and in vivo luciferase-reporter gene assays showed that UDCA+dexamethasone combination upregulates human AE2 alternate overlapping promoter sequences. ChIp assays showed that UDCA+dexamethasone combination induces p300-related interactions between HNF1 and the glucocorticoid receptor on the AE2 alternate promoter. Western blot analysis indicated that HNF1¿Á is absent in normal human cholangiocytes, but its role might be replaced by HNF1¿Â as was demonstrated though transactivation experiments and ChIp assays. Conclusions. These findings in hepatobiliary cells provide evidence for a relevant role of the AE2 alternative expression in the physiology and pathophysiology of the hepatobiliary tract, and reveal the potentiality of naturally occurring compounds with therapeutic properties such as UDCA and glucocorticoids to modulate the alternative expression. Thus, the combination of these compounds improves the AE2-mediated chloride/bicarbonate exchange via upregulation of the AE2 alternate promoter. All these effects are specific for UDCA and its glycine and taurine conjugates. In the case of human intrahepatic cholestatic diseases, activation of this mechanism may result in improved bicarbonate-rich choleresis. Our data provide a rational support for the use of combined therapy with UDCA and glucocorticoids in PBC patients who respond poorly to UDCA alone.