Organ-specific alterations in RAR alpha:RXR alpha abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis.
OBJECTIVE
Obstructive cholestasis is associated with adaptive changes in expression of hepatocyte transport proteins. These include a significant reduction in hepatic expression of Mrp2 (Abcc2), the principal canalicular multispecific organic anion transporter. Renal Mrp2 expression is preserved under these conditions. We have recently reported that the rat Mrp2 promoter is activated by RAR alpha:RXR alpha, and that interleukin 1 beta (IL-1 beta) repressed promoter activity via this element. We hypothesized that cytokines, which are up-regulated in obstructive cholestasis, would reduce nuclear RAR alpha:RXR alpha levels, and that this would be associated with suppression of hepatic Mrp2 expression.


METHODS
Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham surgery, and liver and kidney RNA and protein were isolated. Primary rat hepatocytes were treated with bile acids, retinoids, or cytokines, and RNA and protein were isolated. Mrp2 and RAR alpha:RXR alpha protein abundance and activity were assessed by using electrophoretic mobility shift assays (EMSA) and immunoblots. IL-1 beta abundance was determined by enzyme-linked immunosorbent assay. RAR alpha, RXR alpha, and Mrp2 RNA levels were determined by using ribonuclease protection assays (RPA).


RESULTS
Mrp2 down-regulation and IL-1 beta up-regulation were observed in liver after BDL. This was temporally associated with down-regulation of liver RAR alpha:RXR alpha nuclear protein levels and binding to the Mrp2 promoter cis element. Renal RAR alpha:RXR alpha and Mrp2 expression were preserved under these conditions. IL-1 beta treatment of primary hepatocytes reduced Mrp2 and RXR alpha expression.


CONCLUSIONS
Organ-specific regulation of Mrp2 expression in obstructive cholestasis is associated with cytokine-dependent alterations in RAR alpha:RXR alpha nuclear receptors. Preservation of renal Mrp2 expression may permit urinary excretion of toxic organic anions and xenobiotics under conditions in which biliary excretion is impaired.
Denson
Lee A
LA
Department of Pediatrics and Yale Child Health Research Center, Yale University School of Medicine, New Haven, Connecticut 06520-8081, USA. lee.denson@yale.edu
Bohan
Alan
A
Held
Matthew A
MA
Boyer
James L
JL
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics*; D002779:Cholestasis / Q000378:metabolism*; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000378:metabolism; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000235:genetics*; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics*
D018528:ATP-Binding Cassette Transporters; C423013:Abcc2 protein, rat; D002352:Carrier Proteins; D007375:Interleukin-1; D009687:Nuclear Proteins; C000607073:Rara protein, rat; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors