Annual Meeting of the North American Society for Pediatric Gastroenterology and Nutrition; Orlando, October 22-24, 1998
HUMAN MDR3 mRNA EXPRESSION IS UPREGULATED IN CHOLESTATIC LIVER DISEASE
The class III P-glycoproteins (Pgp3) transport phospholipid across the canalicular membrane of the hepatocyte. Deficiency of human Pgp3 results in progressive familial intrahepatic cholestasis type 3 and is due to mutations in the human MDR3 gene. Our aim was to examine the expression of the hepatic MDR3 gene in pediatric liver disease and to study its in-vitro regulation in the Hep G2 cell line. Methods: Total RNA was isolated from human tissue samples obtained at orthotopic liver transplantation and Northern blot analysis performed after hybridization of the blots with an MDR3 specific cDNA probe. Deletion constructs containing fragments of the MDR3 promoter and a luciferase reporter gene were used in transient transfection experiments to examine regulation of MDR3 gene expression by bile acids. Luciferase activity was determined after transfected Hep G2 cells were incubated in the presence of bile acids for 48 hours. Results: By Northern blot analysis we found that MDR3 mRNA expression was increased in livers from patients with extrahepatic biliary atresia as compared to normal livers obtained from unused donor segments. In these livers, MDR3 became the predominantly expressed isoform in comparison to normal livers in which MDR1 was more highly expressed. Given that MDR3 was upregulated in cholestatic livers, we examined the effect of bile acids on MDR3 expression in Hep G2 cells. By Northern blot analysis, we found that MDR3 mRNA increased in response to the more hydrophobic bile acids. To determine whether upregulation of MDR3 occurred as a result of increased promoter activity, transient transfection experiments were performed using the MDR3 promoter constructs. We found that bile acids such as cholic acid, taurocholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid had no effect on luciferase activity indicating that these bile acids were unable to transcriptionally activate the MDR3 promoter. Conclusion: Although MDR3 mRNA expression is upregulated in livers from patients with cholestatic liver disease and in the Hep G2 cell line in response to bile acids, it appears that this increase involves a mechanism other than transcriptional activation of the MDR3 promoter.
PLENARY SESSION III© 1998 Lippincott Williams & Wilkins, Inc.