Fiona A.J. van den Heuvel

Tissue Distribution of the Death Ligand TRAIL and Its Receptors

Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor distribution, we performed an immunohistochemical (IHC) analysis of the expression of TRAIL-R1, TRAIL-R2, the anti-apoptotic TRAIL receptor TRAIL-R3, and TRAIL in normal human and chimpanzee tissues. In humans, hepatocytes stained positive for TRAIL and TRAIL receptors and bile duct epithelium for TRAIL, TRAIL-R1, and TRAIL-R3. In brains, neurons expressed TRAIL-R1, TRAIL-R2, TRAIL-R3 but no TRAIL. In kidneys, TRAIL-R3 was negative, tubuli contorti expressed TRAIL-R1, TRAIL-R2, and TRAIL, and cells in Henles loop expressed only TRAIL-R2. Heart myocytes showed positivity for all proteins studied. In colon, TRAIL-R1, TRAIL-R2, and TRAIL were present. Germ and Leydig cells were positive for all proteins studied. Endothelium in liver, heart, kidney, and testis lacked TRAIL-R1 and TRAIL-R2. In alveolar septa and bronchial epithelium TRAIL-R2 was expressed, brain vascular endothelium expressed TRAIL-R2 and TRAIL-R3, and in heart vascular endothelium only TRAIL-R3 was present. Only a few differences were observed between human and chimpanzee liver, brain, and kidney. In contrast to human, chimpanzee bile duct epithelium lacked TRAIL, TRAIL-R1, and TRAIL-R3, lung and colon showed no TRAIL or its receptors, TRAIL-R3 was absent in germ and Leydig cells, and vascular endothelium showed only TRAIL-R2 expression in the brain. In conclusion, comparable expression of TRAIL and TRAIL receptors was observed in human and chimpanzee tissues. Lack of liver toxicity in chimpanzees after rhTRAIL administration despite TRAIL-R1 and TRAIL-R2 expression is reassuring for rhTRAIL application in humans. (J Histochem Cytochem 52:821–831, 2004)

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)

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.)

Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis.

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome‐mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild‐type p53 levels and sensitize HPV‐positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF‐related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL‐induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL‐induced apoptosis in a caspase‐dependent and time‐dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6‐mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132‐induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL‐induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X‐linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV‐positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.

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.)

Sinusoidal endothelial cells are damaged and display enhanced autophagy in myelodysplastic syndromes

Terminally differentiated bone marrow cells in low-risk myelodysplasia (MDS) undergo enhanced cell death leading to ineffective haematopoiesis. The cell death type may be cell-specific: studies in MDS erythroblasts have shown enhanced apoptosis but also autophagy, while megakaryocytes undergo a caspase-3 independent non-apoptotic death (Houwerzijl et al, 2009). A strong connection has been shown between the erythroid and endothelial lineage. Erythroid and endothelial cells might originate from a common precursor cell (Lancrin et al, 2009) and in MDS haematopoietic and circulating endothelial cells can carry similar chromosomal abnormalities (Della Porta et al, 2008). These findings raise the question whether endothelial cells in MDS might also be prone to cell death pathways and might have a disrupted architecture. To investigate this question in more detail, particularly in order to quantify vascularisation and study cell ultrastructure, immunohistochemistry and electron microscopy were performed on bone marrow samples of patients with refractory anaemia (RA, n = 3), refractory cytopenias with multilineage dysplasia (RCMD, n = 5), RA with ringed sideroblasts (RARS, n = 3), RCMD with ringed sideroblasts (RCMD-RS, n = 5), RA with excess blasts type 1 (RAEB-1, n = 2), acute myeloid leukaemia (AML, n = 9), and healthy controls (n = 4). Three AML patients had a proven prophase of MDS. According to the International Prognostic Scoring System (IPSS) patients with RA, RARS, RCMD, and RCMDRS (n = 16) were categorized as lower-risk, i.e. low risk (n = 6) and intermediate risk-1 (n = 10). The RAEB-1 patients were high-risk (n = 2). For patient characteristics see Table SI. CD34-immunostaining of MDS bone marrow biopsies demonstrated increased microvessel density (MVD; supporting materials and methods). In both RA/RCMD (n = 8) and RARS/RCMD-RS (n = 5) MVD was significantly increased compared to normal marrow [number of vessels: 15 1 4 4 (mean SD) and 12 1 7 3/power field respectively, versus 3 5 2 9 in healthy controls, P < 0 05]. MVD in high-risk MDS (n = 2) and AML (n = 6) was also significantly increased (18 7 6 6 and 13 1 4 8 respectively, P < 0 05) compared to normal, but not significantly different from low-risk MDS (see Fig 1). Vascular endothelial growth factor (VEGF) staining of endothelial cells was evaluated semiquantitively. Normal bone marrow endothelial cells demonstrated no to weak staining of VEGF, while the VEGF intensity of endothelial cells in RA/RCMD (n = 6), RARS/RCMD-RS (n = 6), and AML (n = 2) was significantly increased and varied between weak to strong indicating that, in line with the increased MVD, VEGF expression by MDS endothelial cells is significantly elevated (see Fig 1). Ultrastructural analysis of endothelial cells was performed on haematons of a subgroup of the lower-risk MDS (RA: n = 3, RCMD: n = 2, RARS: n = 3, RCMD-RS: n = 4) and AML patients (n = 5) and compared to normal bone marrow (n = 3). Haematons are compact particles containing haematopoietic progenitor cells residing within a stromal framework, including adipocytes, mesenchymal cells, macrophages and endothelial cells (Blazsek et al, 2000, and Fig S1). Haematons can be isolated from the spicules from the bone marrow aspirate. In haematons sinusoidal endothelial cells can be observed in an architecturally preserved form. In MDS patients, endothelial cells demonstrated irregular cell membranes with numerous small protrusions and frequent sprouting, compatible with neoangiogenesis (Table SII). In AML there was less sprouting and membrane outlines were smoother, however extensive networks of microvessels were observed. Abnormally shaped endothelial cells without pericytes and with degradation of the basal membrane were observed in both MDS and AML. Extensive cytoplasmic vacuolization was present in MDS endothelial cells, especially in RARS and RCMS-RS. The majority of these vacuoles had double membranes, a characteristic of autophagy. In addition, an increased number of large and dense secondary lysosomes were present in the damaged MDS endothelial cells (Fig 2 and Table SII). Mitochondria were mainly ultrastructurally normal. No features of apoptosis were found in MDS endothelial cells, and this was confirmed by a negative immunostaining for caspase-3 and caspase-8 (data not shown). LC3, a marker of autophagic membranes (see supplementary material and methods), could be demonstrated on membranes and inside the majority of the vacuoles in MDS endothelial cells (Fig 2). In AML, autophagy was also observed in endothelial cells but at a low level, which might be linked to the prophase of MDS in 33% of the AML patients. Thus, enhanced autophagy was not a general phenomenon related to an increase in MVD. It is likely that in both MDS and AML the increased MVD might change bone marrow blood flow, thereby triggering hypoxia leading to upregulation of