Mariska Geuken

High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP‐binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up‐regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up‐regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductules. Copyright

Decreased P-glycoprotein (P-gp/MDR1) expression in inflamed human intestinal epithelium is independent of PXR protein levels

Background Altered P‐glycoprotein expression (P‐gp/MDR1) and/or function may contribute to the pathogenesis of gastrointestinal inflammatory disorders. Low intestinal mRNA levels of the pregnane X receptor (PXR) have been linked to low MDR1 mRNA levels in patients with ulcerative colitis (UC). Here we compared intestinal MDR1 mRNA and protein expression in uninflamed and inflamed intestinal epithelium (IE) of patients with gastrointestinal inflammatory disorders to healthy controls. Methods Intestinal mucosal biopsies were obtained from patients with Crohns disease (CD, n = 20), UC (n = 10), diverticulitis (n = 3), collagenous colitis (n = 3), and healthy controls (n = 10). MDR1, iNOS, MRP1, CYP3A4, and PXR expression was determined using real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR), Western blotting, and/or immunohistochemistry. Furthermore, MDR1 expression was determined in human intestinal biopsies and the human colon carcinoma cell line DLD‐1 after exposure to cytokines (TNF‐&agr;, IFN‐&ggr;, and/or IL‐1&bgr;). Results MDR1 mRNA levels in uninflamed colon of UC patients were comparable to healthy controls, while they were slightly decreased in ileum and slightly increased in colon of CD patients. MDR1 expression, however, was strongly decreased in inflamed IE of CD, UC, collagenous colitis, and diverticulitis patients. A cytokine‐dependent decrease of MDR1 expression was observed in human intestinal biopsies, but not in DLD‐1 cells. Remarkably, PXR protein levels were equal in uninflamed and inflamed tissue of CD and UC patients despite low PXR mRNA levels in inflamed tissue. Conclusions MDR1 expression is strongly decreased in inflamed IE of patients with gastrointestinal disorders and this is independent of PXR protein levels. Low MDR1 levels may aggravate intestinal inflammation. (Inflamm Bowel Dis 2007)

A progressive familial intrahepatic cholestasis type 2 mutation causes an unstable, temperature-sensitive bile salt export pump

BACKGROUND/AIMS Progressive familial intrahepatic cholestasis type 2 (PFIC-2) patients have a defect in the hepatocanalicular bile salt secretion. The disease is caused by mutations in the bile salt export pump (BSEP). Ten different missense mutations have been described. In this study, we analysed the effect of the D482G PFIC-2 mutation on BSEP function. METHODS Adenosine triphosphatase (ATPase) and taurocholate transport assays were performed with full-length mouse Bsep (mBsep) with and without the D482G mutation. The effect on expression and subcellular sorting was studied in HepG2 cells, stably expressing enhanced green fluorescent protein (EGFP)-tagged mBsep proteins. RESULTS The D482G mutation did not significantly affect the taurocholate transport activity of mBsep, even though the bile salt-inducible ATPase activity of the mutant protein was slightly reduced. Protein expression and canalicular sorting were strongly affected by the D482G mutation. Mutant EGFP-mBsep protein was only partly glycosylated and detected in both the canalicular membrane and the cytoplasm. At 30 degrees C, the mutant mRNA and protein levels were strongly increased, and the protein was predominantly glycosylated and efficiently targeted to the canalicular membrane. CONCLUSIONS These data suggest that PFIC-2 patients with the D482G mutation express a functional, but highly unstable, temperature-sensitive bile salt export pump.

Human and rat bile acid–CoA:amino acid N‐acyltransferase are liver‐specific peroxisomal enzymes: Implications for intracellular bile salt transport

Bile acid–coenzyme A:amino acid N‐acyltransferase (BAAT) is the sole enzyme responsible for conjugation of primary and secondary bile acids to taurine and glycine. Previous studies indicate a peroxisomal location of BAAT in peroxisomes with variable amounts up to 95% detected in cytosolic fractions. The absence or presence of a cytosolic pool of BAAT has important implications for the intracellular transport of unconjugated/deconjugated bile salts. We used immunofluorescence microscopy and digitonin permeabilization assays to determine the subcellular location of endogenous BAAT in primary human and rat hepatocytes. In addition, green fluorescent protein (GFP)–tagged rat Baat (rBaat) and human BAAT (hBAAT) were transiently expressed in primary rat hepatocytes and human fibroblasts. Catalase and recombinant GFP‐SKL and DsRed‐SKL were used as peroxisomal markers. Endogenous hBAAT and rBaat were found to specifically localize to peroxisomes in human and rat hepatocytes, respectively. No significant cytosolic fraction was detected for either protein. GFP‐tagged hBAAT and rBaat were efficiently sorted to peroxisomes of primary rat hepatocytes. Significant amounts of GFP‐tagged hBAAT or rBaat were detected in the cytosol only when coexpressed with DsRed‐SKL, suggesting that hBAAT/rBaat and DsRed‐SKL compete for the same peroxisomal import machinery. When expressed in fibroblasts, GFP‐tagged hBAAT localized to the cytosol, confirming earlier observations. Conclusion: hBAAT and rBaat are peroxisomal enzymes present in undetectable amounts in the cytosol. Unconjugated or deconjugated bile salts returning to the liver need to shuttle through the peroxisome before reentering the enterohepatic circulation. (HEPATOLOGY 2007;45:340–348.)

ATP binding cassette transporter gene expression in rat liver progenitor cells

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. Methods: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF ϕ 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF ϕ 13 cells by flow cytometry. Results: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF ϕ 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF ϕ 13 cells. Conclusion: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity.

Caveolin-1 is enriched in the peroxisomal membrane of rat hepatocytes.

Caveolae are a subtype of cholesterol‐enriched lipid microdomains/rafts that are routinely detected as vesicles pinching off from the plasma membrane. Caveolin‐1 is an essential component of caveolae. Hepatic caveolin‐1 plays an important role in liver regeneration and lipid metabolism. Expression of caveolin‐1 in hepatocytes is relatively low, and it has been suggested to also reside at other subcellular locations than the plasma membrane. Recently, we found that the peroxisomal membrane contains lipid microdomains. Like caveolin‐1, hepatic peroxisomes are involved in lipid metabolism. Here, we analyzed the subcellular location of caveolin‐1 in rat hepatocytes. The subcellular location of rat hepatocyte caveolin‐1 was analyzed by cell fractionation procedures, immunofluorescence, and immuno‐electron microscopy. Green fluorescent protein (GFP)‐tagged caveolin‐1 was expressed in rat hepatocytes. Lipid rafts were characterized after Triton X‐100 or Lubrol WX extraction of purified peroxisomes. Fenofibric acid–dependent regulation of caveolin‐1 was analyzed. Peroxisome biogenesis was studied in rat hepatocytes after RNA interference–mediated silencing of caveolin‐1 and caveolin‐1 knockout mice. Cell fractionation and microscopic analyses reveal that caveolin‐1 colocalizes with peroxisomal marker proteins (catalase, the 70 kDa peroxisomal membrane protein PMP70, the adrenoleukodystrophy protein ALDP, Pex14p, and the bile acid–coenzyme A:amino acid N‐acyltransferase BAAT) in rat hepatocytes. Artificially expressed GFP–caveolin‐1 accumulated in catalase‐positive organelles. Peroxisomal caveolin‐1 is associated with detergent‐resistant microdomains. Caveolin‐1 expression is strongly repressed by the peroxisome proliferator‐activated receptor‐α agonist fenofibric acid. Targeting of peroxisomal matrix proteins and peroxisome number and shape were not altered in rat hepatocytes with 70%‐80% reduced caveolin‐1 levels and in livers of caveolin‐1 knockout mice. Conclusion: Caveolin‐1 is enriched in peroxisomes of hepatocytes. Caveolin‐1 is not required for peroxisome biogenesis, but this unique subcellular location may determine its important role in hepatocyte proliferation and lipid metabolism. (HEPATOLOGY 2010.)

Up-regulation and cytoprotective role of epithelial multidrug resistance-associated protein 1 in inflammatory bowel disease

MRP1 (multidrug resistance-associated protein 1) is well known for its role in providing multidrug resistance to cancer cells. In addition, MRP1 has been associated with both pro- and anti-inflammatory functions in nonmalignant cells. The pro-inflammatory function is evident from the fact that MRP1 is a high affinity transporter for cysteinyl-leukotriene C4 (LTC4), a lipid mediator of inflammation. It remains unexplained, however, why the absence of Mrp1 leads to increased intestinal epithelial damage in mice treated with dextran-sodium sulfate, a model for inflammatory bowel disease (IBD). We found that MRP1 expression is induced in the inflamed intestine of IBD patients, e.g. Crohn disease and ulcerative colitis. Increased MRP1 expression was detected at the basolateral membrane of intestinal epithelial cells. To study a putative role for MRP1 in protecting epithelial cells against inflammatory cues, we manipulated MRP1 levels in human epithelial DLD-1 cells and exposed these cells to cytokines and anti-Fas. Inhibition of MRP1 (by MK571 or RNA interference) resulted in increased cytokine- and anti-Fas-induced apoptosis of DLD-1 cells. Opposite effects, e.g. protection of DLD-1 cells against cytokine- and anti-Fas-induced apoptosis, were observed after recombinant MRP1 overexpression. Inhibition of LTC4 synthesis reduced anti-Fas-induced apoptosis when MRP1 function was blocked, suggesting that LTC4 is the pro-apoptotic compound exported by epithelial MRP1 during inflammation. These data show that MRP1 protects intestinal epithelial cells against inflammation-induced apoptotic cell death and provides a functional role for MRP1 in the inflamed intestinal epithelium of IBD patients.

Multidrug resistance–associated proteins are crucial for the viability of activated rat hepatic stellate cells

Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP‐binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance–associated protein (Mrp)‐type and multidrug resistance protein (Mdr)–type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC‐833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase‐3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl4)–induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte‐specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl4‐treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. Conclusion: Activated HSCs contain relatively high levels of Mrp1. Mrp‐type transporters are required for the viability of activated HSCs. Mrp‐dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases. (HEPATOLOGY 2008.)