Jannes Woudenberg

Glutathione and antioxidant enzymes serve complementary roles in protecting activated hepatic stellate cells against hydrogen peroxide-induced cell death

BACKGROUND In chronic liver disease, hepatic stellate cells (HSCs) are activated, highly proliferative and produce excessive amounts of extracellular matrix, leading to liver fibrosis. Elevated levels of toxic reactive oxygen species (ROS) produced during chronic liver injury have been implicated in this activation process. Therefore, activated hepatic stellate cells need to harbor highly effective anti-oxidants to protect against the toxic effects of ROS. AIM To investigate the protective mechanisms of activated HSCs against ROS-induced toxicity. METHODS Culture-activated rat HSCs were exposed to hydrogen peroxide. Necrosis and apoptosis were determined by Sytox Green or acridine orange staining, respectively. The hydrogen peroxide detoxifying enzymes catalase and glutathione-peroxidase (GPx) were inhibited using 3-amino-1,2,4-triazole and mercaptosuccinic acid, respectively. The anti-oxidant glutathione was depleted by L-buthionine-sulfoximine and repleted with the GSH-analogue GSH-monoethylester (GSH-MEE). RESULTS Upon activation, HSCs increase their cellular glutathione content and GPx expression, while MnSOD (both at mRNA and protein level) and catalase (at the protein level, but not at the mRNA level) decreased. Hydrogen peroxide did not induce cell death in activated HSCs. Glutathione depletion increased the sensitivity of HSCs to hydrogen peroxide, resulting in 35% and 75% necrotic cells at 0.2 and 1mmol/L hydrogen peroxide, respectively. The sensitizing effect was abolished by GSH-MEE. Inhibition of catalase or GPx significantly increased hydrogen peroxide-induced apoptosis, which was not reversed by GSH-MEE. CONCLUSION Activated HSCs have increased ROS-detoxifying capacity compared to quiescent HSCs. Glutathione levels increase during HSC activation and protect against ROS-induced necrosis, whereas hydrogen peroxide-detoxifying enzymes protect against apoptotic cell death.

Lipid Rafts Are Essential for Peroxisome Biogenesis in HepG2 Cells

Peroxisomes are particularly abundant in the liver and are involved in bile salt synthesis and fatty acid metabolism. Peroxisomal membrane proteins (PMPs) are required for peroxisome biogenesis [e.g., the interacting peroxisomal biogenesis factors Pex13p and Pex14p] and its metabolic function [e.g., the adenosine triphosphate–binding cassette transporters adrenoleukodystrophy protein (ALDP) and PMP70]. Impaired function of PMPs is the underlying cause of Zellweger syndrome and X‐linked adrenoleukodystrophy. Here we studied for the first time the putative association of PMPs with cholesterol‐enriched lipid rafts and their function in peroxisome biogenesis. Lipid rafts were isolated from Triton X‐100–lysed or Lubrol WX–lysed HepG2 cells and analyzed for the presence of various PMPs by western blotting. Lovastatin and methyl‐β‐cyclodextrin were used to deplete cholesterol and disrupt lipid rafts in HepG2 cells, and this was followed by immunofluorescence microscopy to determine the subcellular location of catalase and PMPs. Cycloheximide was used to inhibit protein synthesis. Green fluorescent protein–tagged fragments of PMP70 and ALDP were analyzed for their lipid raft association. PMP70 and Pex14p were associated with Triton X‐100–resistant rafts, ALDP was associated with Lubrol WX–resistant rafts, and Pex13p was not lipid raft–associated in HepG2 cells. The minimal peroxisomal targeting signals in ALDP and PMP70 were not sufficient for lipid raft association. Cholesterol depletion led to dissociation of PMPs from lipid rafts and impaired sorting of newly synthesized catalase and ALDP but not Pex14p and PMP70. Repletion of cholesterol to these cells efficiently reestablished the peroxisomal sorting of catalase but not ALDP. Conclusion: Human PMPs are differentially associated with lipid rafts independently of the protein homology and/or their functional interaction. Cholesterol is required for peroxisomal lipid raft assembly and peroxisome biogenesis. HEPATOLOGY 2010

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.)

Unconjugated bile salts shuttle through hepatocyte peroxisomes for taurine conjugation

Bile acid‐CoA:amino acid N‐acyltransferase (BAAT) conjugates bile salts to glycine or taurine, which is the final step in bile salt biosynthesis. In addition, BAAT is required for reconjugation of bile salts in the enterohepatic circulation. Recently, we showed that BAAT is a peroxisomal protein, implying shuttling of bile salts through peroxisomes for reconjugation. However, the subcellular location of BAAT remains a topic of debate. The aim of this study was to obtain direct proof for reconjugation of bile salts in peroxisomes. Primary rat hepatocytes were incubated with deuterium‐labeled cholic acid (D4CA). Over time, media and cells were collected and the levels of D4CA, D4‐tauro‐CA (D4TCA), and D4‐glyco‐CA (D4GCA) were quantified by liquid chromatography‐tandem mass spectrometry (LC/MS/MS). Subcellular accumulation of D4‐labeled bile salts was analyzed by digitonin permeabilization assays and subcellular fractionation experiments. Within 24 hours, cultured rat hepatocytes efficiently (>90%) converted and secreted 100 μM D4CA to D4TCA and D4GCA. The relative amounts of D4TCA and D4GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D4CA‐exposed hepatocytes with 30‐150 μg/mL digitonin led to the complete release of D4CA, D4GCA, and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (cytosolic marker). Full release of D4TCA, catalase, and BAAT was only observed at 500 μg/mL digitonin, indicating the presence of D4TCA in membrane‐enclosed organelles. D4TCA was detected in fractions of purified peroxisomes, which did not contain D4CA and D4GCA. Conclusion: We established a novel assay to study conjugation and intra‐ and transcellular transport of bile salts. Using this assay, we show that cholic acid shuttles through peroxisomes for taurine‐conjugation. (HEPATOLOGY 2010)

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.)