Gastroenterology

Gastroenterology

Volume 123, Issue 2, August 2002, Pages 599-607
Gastroenterology

Basic–Liver, Pancreas, and Biliary Tract
Organ-specific alterations in RARα:RXRα abundance regulate rat Mrp2 (Abcc2) expression in obstructive cholestasis,☆☆,

Previously published in abstract form (Hepatology 2001;34:367A) and presented at the annual meeting of the American Association for the Study of Liver Diseases.
https://doi.org/10.1053/gast.2002.34758Get rights and content

Abstract

Background & Aims: 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 RARα:RXRα, and that interleukin 1β (IL-1β) repressed promoter activity via this element. We hypothesized that cytokines, which are up-regulated in obstructive cholestasis, would reduce nuclear RARα:RXRα 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 RARα:RXRα protein abundance and activity were assessed by using electrophoretic mobility shift assays (EMSA) and immunoblots. IL-1β abundance was determined by enzyme-linked immunosorbent assay. RARα, RXRα, and Mrp2 RNA levels were determined by using ribonuclease protection assays (RPA). Results: Mrp2 down-regulation and IL-1β up-regulation were observed in liver after BDL. This was temporally associated with down-regulation of liver RARα:RXRα nuclear protein levels and binding to the Mrp2 promoter cis element. Renal RARα:RXRα and Mrp2 expression were preserved under these conditions. IL-1β treatment of primary hepatocytes reduced Mrp2 and RXRα expression. Conclusions: Organ-specific regulation of Mrp2 expression in obstructive cholestasis is associated with cytokine-dependent alterations in RARα:RXRα 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.

GASTROENTEROLOGY 2002;123:599-607

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Materials

Cell culture medium and reagents were obtained from Gibco Life Technologies, Inc. (Gaithersburg, MD). Mouse recombinant tumor necrosis factor α (TNFα) and IL-1β were obtained from R&D Systems (Minneapolis, MN). Chenodeoxycholic acid (CDCA) was obtained from Sigma (St. Louis, MO). The synthetic RXRα ligand, LG364, was provided by Dr. P. Heyman (Ligand Pharmaceuticals, San Diego, CA). RXRα (catalog no. sc-553), RARα (catalog no. sc-551), and SH-PTP1 (catalog no. sc-287) polyclonal antibodies were

Obstructive cholestasis reduces liver, but not kidney, expression of Mrp2

Prior studies from our laboratory and others have shown suppression of Mrp2 RNA and protein expression in the liver in obstructive cholestasis.7, 21, 22, 23 We performed RPA by using total RNA from liver and kidney of rats subjected to BDL for 1, 3, and 14 days to determine whether changes in Mrp2 expression in this model were organ specific. As shown in Figure 1, we confirmed that Mrp2 RNA expression was reduced in liver after BDL, to 66% ± 22% of sham levels by day 3, and to 44% ± 8% of sham

Discussion

The multidrug resistance protein 2 (Mrp2 or Abcc2) is the principal hepatic canalicular multispecific organic anion transporter, and it mediates excretion of bilirubin-diglucuronide and a variety of conjugates of lipophilic substances via bile under normal conditions. Our group and others have previously characterized down-regulation of hepatic Mrp2 RNA and protein expression and function in experimental models of cholestatic liver disease.7, 21, 22, 23 More recently, we and other investigators

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Address requests for reprints to: Lee A. Denson, M.D., Yale Child Health Research Center, Department of Pediatrics, 464 Congress Avenue, P.O. Box 208081, New Haven, Connecticut 06520-8081. e-mail: [email protected]; fax: (203) 737-5972.

☆☆

Supported by National Institutes of Health grant DK02700 (to L.A.D.), DK25636 (to J.L.B.), Charles H. Hood Foundation (to L.A.D.), and Yale Liver Center (DK P30-34989).

The authors acknowledge the excellent technical support of Himmat Bajwa.

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