Visit our Expo - Redox and Inflammation signaling 2012

Session XIV : Transcriptional and translational control Poster XIV, 2
Bile acids modulate p53 transcriptional targeting in primary rat hepatocytes
Joana D. Amaral, Rui E. Castro, Susana Solá, Cecília M.P. Rodrigues
Centro de Patogénese Molecular, Faculty of Pharmacy, University of Lisbon, 1600-083
Lisbon, Portugal. E-mail: jamaral@ff.ul.pt
Ursodeoxycholic acid (UDCA) is a potent inhibitor of classical mitochondrial pathways of
apoptosis. Further, UDCA prevents transforming growth factor beta1 (TGF-beta1)-induced
hepatocyte apoptosis by modulating the E2F-1/p53/Bax pathway. However, the
mechanism(s) by which UDCA regulates gene transcription remains to be clarified. The aim
of this study was to evaluate the modulation of a specific pro-apoptotic transcriptional target
of p53 by several bile acids. Primary rat hepatocytes were cotransfected with plasmid DNA
encoding wild-type or mutant p53 and a reporter gene construct that utilized the bax gene
promoter to drive transcription of chloramphenicol acetyltransferase (CAT). A luciferase
reporter construct was used to assess transfection efficiencies. Hepatocytes were transfected
and simultaneously treated with either vehicle or 100 µM of several bile acids. Twelve hours
later, 1 nM TGF-beta1 was included in the cultures. After an additional 36 h, hepatocytes
were harvested for CAT ELISA and luciferase assays. Total protein extracts were also
prepared and evaluated by immunoblotting. Cultures were scored for apoptotic cells by
Hoechst staining. Electrophoretic mobility shift assays (EMSA) were performed in nuclear
extracts from COS-7 cells, producing high levels of endogenous p53. Our results confirmed
that UDCA abrogated TGF-beta1- and p53-induced hepatocyte apoptosis. DNA-binding
activity of p53 was not altered by the bile acid. Nevertheless, in functional studies,
hepatocytes transfected with wild-type p53 and treated with UDCA showed a marked
decrease in bax transcriptional activation. When the mutant form of p53 was used, UDCA no
longer exerted its protective effect. Similar results were obtained after incubation of cells
with both taurine- and glycine-conjugated analogues of UDCA. However, when cells were
treated with lithocholic, deoxycholic, or chenodeoxycholic acids, the pro-apoptotic gene was
further activated at the transcription level, revealing that bile acids are able to differentially
modulate transcription of p53-driven bax. Taken together, these data suggest that proapoptotic
p53 is a specific molecular target of UDCA, further clarifying the role of bile acids
at modulating apoptosis. (Supported by POCTI/SAU-FCF/62479/04 from FCT, Portugal).
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