RESUMO
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.
Assuntos
Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Glutationa/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Western Blotting , Catalase/genética , Catalase/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Imunofluorescência , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Masculino , Necrose , Oxidantes/farmacologia , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND & AIMS: Heterozygous germline mutations in PRKCSH cause autosomal dominant polycystic liver disease (PCLD), but it is not clear how they lead to cyst formation. We investigated whether mutations in cyst epithelial cells and corresponding loss of the PRKCSH gene product (hepatocystin) contributed to cyst development. METHODS: Liver cyst material was collected through laparoscopic cyst fenestration from 8 patients with PCLD who had a heterozygous germline mutation in PRKCSH. Tissue sections from 71 cysts (2-14 per patient) were obtained for hepatocystin staining and mutation analysis. Cyst epithelium was acquired using laser microdissection; DNA was isolated and analyzed for loss of heterozygosity (LOH) and somatic mutations using restriction analysis and sequencing. Common single nucleotide polymorphisms (SNPs) in a 70-kilobase region surrounding the germline mutation were used to determine variations in the genomic region with LOH. RESULTS: The wild-type allele of PRKCSH was lost (LOH) in 76% of cysts (54/71). Hepatocystin was not detected in cyst epithelia with LOH, whereas heterozygous cysts still expressed hepatocystin. The variation observed in the LOH region analysis indicates that cysts develop independently. We also detected somatic mutations in PRKCSH in 17% (2/12) of the cysts without LOH. Trans-heterozygous mutations in SEC63 were not observed. CONCLUSIONS: Among patients with PCLD who have a heterozygous germline mutation in PRKCSH, we found secondary, somatic mutations (second hits) in more than 76% of the liver cyst epithelia. PCLD is recessive at the cellular level, and loss of functional PRKCSH is an important step in cystogenesis.
Assuntos
Cistos/genética , Glucosidases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Hepatopatias/genética , Perda de Heterozigosidade , Mutação/genética , Adulto , Proteínas de Ligação ao Cálcio , Cistos/fisiopatologia , Análise Mutacional de DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Hepatopatias/fisiopatologia , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
UNLABELLED: 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-beta-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.
Assuntos
Microdomínios da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Peroxissomos/fisiologia , Fenômenos Biológicos , Colesterol/fisiologia , Células Hep G2 , HumanosRESUMO
UNLABELLED: 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-alpha 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.
Assuntos
Caveolina 1/metabolismo , Hepatócitos/metabolismo , Peroxissomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Aciltransferases/metabolismo , Animais , Fenofibrato/análogos & derivados , Fenofibrato/farmacologia , Masculino , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Peroxinas , Peroxissomos/efeitos dos fármacos , Ratos , Ratos Wistar , Frações Subcelulares/metabolismoRESUMO
UNLABELLED: 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 (D(4)CA). Over time, media and cells were collected and the levels of D(4)CA, D(4)-tauro-CA (D(4)TCA), and D(4)-glyco-CA (D(4)GCA) were quantified by liquid chromatography-tandem mass spectrometry (LC/MS/MS). Subcellular accumulation of D(4)-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 D(4)CA to D(4)TCA and D(4)GCA. The relative amounts of D(4)TCA and D(4)GCA produced were dependent on the presence of glycine or taurine in the medium. Treatment of D(4)CA-exposed hepatocytes with 30-150 µg/mL digitonin led to the complete release of D(4)CA, D(4)GCA, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (cytosolic marker). Full release of D(4)TCA, catalase, and BAAT was only observed at 500 µg/mL digitonin, indicating the presence of D(4)TCA in membrane-enclosed organelles. D(4)TCA was detected in fractions of purified peroxisomes, which did not contain D(4)CA and D(4)GCA. 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.
Assuntos
Aciltransferases/metabolismo , Ácidos e Sais Biliares/metabolismo , Hepatócitos/metabolismo , Peroxissomos/metabolismo , Taurina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Colatos/metabolismo , Cromatografia Líquida , Digitonina/farmacologia , Masculino , Peroxissomos/efeitos dos fármacos , Ratos , Ratos Wistar , Espectrometria de Massas em TandemRESUMO
Autosomal dominant polycystic liver disease (PCLD) is a rare progressive disorder characterized by an increased liver volume due to many (>20) fluid-filled cysts of biliary origin. Disease causing mutations in PRKCSH or SEC63 are found in approximately 25% of the PCLD patients. Both gene products function in the endoplasmic reticulum, however, the molecular mechanism behind cyst formation remains to be elucidated. As part of the translocon complex, SEC63 plays a role in protein import into the ER and is implicated in the export of unfolded proteins to the cytoplasm during ER-associated degradation (ERAD). PRKCSH codes for the beta-subunit of glucosidase II (hepatocystin), which cleaves two glucose residues of Glc(3)Man(9)GlcNAc(2) N-glycans on proteins. Hepatocystin is thereby directly involved in the protein folding process by regulating protein binding to calnexin/calreticulin in the ER. A separate group of genetic diseases affecting protein N-glycosylation in the ER is formed by the congenital disorders of glycosylation (CDG). In distinct subtypes of this autosomal recessive multisystem disease specific liver symptoms have been reported that overlap with PCLD. Recent research revealed novel insights in PCLD disease pathology such as the absence of hepatocystin from cyst epithelia indicating a two-hit model for PCLD cystogenesis. This opens the way to speculate about a recessive mechanism for PCLD pathophysiology and shared molecular pathways between CDG and PCLD. In this review we will discuss the clinical-genetic features of PCLD and CDG as well as their biochemical pathways with the aim to identify novel directions of research into cystogenesis.
Assuntos
Cistos/congênito , Hepatopatias/congênito , Proteínas de Ligação ao Cálcio , Cistos/metabolismo , Glucosidases/metabolismo , Glicosilação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Hepatopatias/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Proteínas de Ligação a RNARESUMO
UNLABELLED: 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 (CCl(4))-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 CCl(4)-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.
Assuntos
Fígado/citologia , Fígado/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/classificação , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/fisiologia , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Hepatócitos/metabolismo , Humanos , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Fígado/efeitos dos fármacos , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/patologia , Masculino , Estresse Oxidativo/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/farmacologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
INTRODUCTION: ESM-1 plays a role in the regulation of angiogenesis and is released by activated endothelial cells. OBJECTIVE: To test the hypothesized that ESM-1 is increased in preeclampsia (PE). METHODS: Plasma samples from high risk pregnancies divided in 23 healthy (CON), 11 severe early-onset PE (SE) and 7 severe late-onset PE (SL) pregnancies were collected at regular intervals between week 12 and birth. ESM-1 was measured by ELISA. RESULTS: (see figure) Between GA week 24 and birth, ESM-1 concentrations were significantly increased in both early and late preeclampsia compared to controls (Mann Whitney, p<0.05). Surprisingly, the concentration of ESM-1 also differed between the three groups at weeks 12 and 16. The ESM-1 concentration of healthy pregnancies (mean±SEM:1857±861pg/ml) and those that developed severe late-onset PE (1298±371pg/ml) are comparable, but in those pregnancies that develop severe early-onset PE, the concentration (410±355pg/ml) is significantly lower as compared with healthy pregnancies. CONCLUSIONS: ESM-1 concentrations are increased during early and late severe preeclampsia, which may be due to endothelial cell activation in these conditions. Interestingly, since ESM-1 is decreased at 12-16 weeks in patients that later on develop early onset severe PE, it might be a prognostic marker which can determine the risk of women to develop severe early-onset PE already as early as 12 to 16 weeks of gestation.
RESUMO
BACKGROUND: In humans, N-acetyl L-aspartate (NAA) has not been detected in other tissues than the brain. The physiological function of NAA is yet undefined. Recently, it has been suggested that NAA may function as a molecular water pump, responsible for the removal of large amounts of water from the human brain. Ovarian tumors typically present as large cystic masses with considerable fluid accumulation. METHODOLOGY AND PRINCIPAL FINDINGS: Using Gas Chromatography-Mass Spectrometry, we demonstrated that NAA was present in a high micromolar concentration in oCF of epithelial ovarian tumors (EOTs) of serous histology, sometimes in the same range as found in the extracellular space of the human brain. In contrast, oCF of EOTs with a mucinous, endometrioid and clear cell histological subtype contained a low micromolar concentration of NAA. Serous EOTs have a cellular differentiation pattern which resembles the lining of the fallopian tube and differs from the other histological subtypes. The NAA concentration in two samples of fluid accumulation in the fallopian tube (hydrosalpinx) was in the same ranges as NAA found in oCF of serous EOTs. The NAA concentration in oCF of patients with serous EOTs was mostly 10 to 50 fold higher than their normal serum NAA concentration, whereas in patients with other EOT subtypes, serum and cyst fluid NAA concentration was comparable. CONCLUSIONS AND SIGNIFICANCE: The high concentration of NAA in cyst fluid of serous EOTs and low serum concentrations of NAA in these patients, suggest a local production of NAA in serous EOTs. Our findings provide the first identification of NAA concentrations high enough to suggest local production outside the human brain. Our findings contribute to the ongoing research understanding the physiological function of NAA in the human body.