Alcohol Promotes Exosome Biogenesis and Release via Modulating Rabs and miR-192 Expression in Human Hepatocytes

Exosomes are membrane vesicles released by various cell types into the extracellular space under different conditions including alcohol exposure. Exosomes are involved in intercellular communication and as mediators of various diseases. Alcohol use causes oxidative stress that promotes exosome secretion. Here, we elucidated the effects of alcohol on exosome biogenesis and secretion using human hepatocytes. We found that alcohol treatment induces the expression of genes involved in various steps of exosome formation. Expression of Rab proteins such as Rab1a, Rab5c, Rab6, Rab10, Rab11, Rab27a and Rab35 were increased at the mRNA level in primary human hepatocytes after alcohol treatment. Rab5, Rab6 and Rab11 showed significant induction in the livers of patients with alcohol-associated liver disease. Further, alcohol treatment also led to the induction of syntenin, vesicle-associated membrane proteins (VAMPs), and syntaxin that all play various roles in exosome biogenesis and secretion. VAMP3, VAMP5, VAPb, and syntaxin16 mRNA transcripts were increased in alcohol treated cells and in the livers of alcohol-associated liver disease (ALD) patients. Induction in these genes was associated with increases in exosome secretion in alcohol treated hepatocytes. We found that hepatocyte enriched miR-192 and miR-122 levels were significantly decreased in alcohol treated hepatocytes whereas their levels were increased in the cell-free supernatant. The primary transcripts of miR-192 and miR-122 were reduced in alcohol treated hepatocytes, suggesting alcohol partially affects these miRNAs at the transcriptional level. We found that miR-192 has putative binding sites for genes involved in exosome secretion. Inhibition of miR-192 in human hepatoma cells caused a significant increase in Rab27a, Rab35, syntaxin7 and syntaxin16 and a concurrent increase in exosome secretion, suggesting miR-192 regulates exosomes release in hepatocytes. Collectively, our results reveal that alcohol modulates Rabs, VAMPs and syntaxins directly and partly via miR-192 to induce exosome machinery and release.

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Green tea catechin enhances cholesterol 7α-hydroxylase gene expression in HepG2 cells

British Journal Of Nutrition ◽

10.1017/s0007114507864816 ◽

2008 ◽

Vol 99 (6) ◽

pp. 1182-1185 ◽

Cited By ~ 28

Author(s):

Mak-Soon Lee ◽

Ju-Yeon Park ◽

Hedley Freake ◽

In-Sook Kwun ◽

Yangha Kim

Keyword(s):

Green Tea ◽

Promoter Activity ◽

Human Subjects ◽

Mrna Level ◽

Transcriptional Level ◽

Dependent Manner ◽

Human Hepatoma Cells ◽

Epicatechin Gallate ◽

Tea Catechins ◽

Green Tea Catechins

Green tea catechins are known to have hypocholesterolaemic effects in animals and human subjects. In the present study, we investigated the effects of green tea catechins on the mRNA level and promoter activity of hepatic cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in the conversion of cholesterol to bile acids, in human hepatoma cells. Real-time PCR assays showed that different catechins, ( − )-epicatechin gallate (ECG), ( − )-epigallocatechin-3-gallate (EGCG), ( − )-epigallocatechin (EGC) and ( − )-epicatechin (EC), up regulated the CYP7A1 mRNA level by 5·5-, 4·2-, 2·9- and 1·9-fold, respectively, compared with the control. The − 1312/+358 bp of the CYP7A1 promoter was subcloned into the pGL3 basic vector that includes luciferase as a reporter gene. ECG or EGCG significantly increased CYP7A1 promoter activity by 6·0- or 4·0-fold, respectively, compared with the control. Also, EGCG stimulated CYP7A1 at both mRNA level and promoter activity in a dose-dependent manner. These results suggest that the expression of the CYP7A1 gene may be directly regulated by green tea catechins at the transcriptional level.

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BCL2 protein expression parallels its mRNA level in normal and malignant B cells

Blood ◽

10.1182/blood-2004-01-0243 ◽

2004 ◽

Vol 104 (9) ◽

pp. 2936-2939 ◽

Cited By ~ 17

Author(s):

Yulei Shen ◽

Javeed Iqbal ◽

James Z. Huang ◽

Guimei Zhou ◽

Wing C. Chan

Keyword(s):

B Cells ◽

Protein Expression ◽

B Cell ◽

Posttranscriptional Regulation ◽

Cell Lymphoma ◽

Dominant Role ◽

Mrna Level ◽

B Cell Lymphoma ◽

Transcriptional Level ◽

Bcl2 Protein

Abstract The regulation of B-cell lymphoma 2 (BCL2) protein expression in germinal center (GC) B cells has been controversial. Previous reports have indicated posttranscriptional regulation plays a dominant role. However, a number of recent studies contradicted these reports. Using real-time polymerase chain reaction (PCR) and Standardized Reverse Transcriptase-PCR (StaRT-PCR), we measured the level of mRNA expression in GC, mantle zone (MNZ), and marginal zone (MGZ) cells from laser capture microdissection. Both quantitative RT-PCR measurements of microdissected GC cells from tonsils showed that GC cells had low expression of BCL2 transcripts commensurate with the low protein expression level. These results are in agreement with microarray studies on fluorescence-activated cell sorter (FACS)-sorted cells and microdissected GC cells. We also examined BCL2 mRNA and protein expression on a series of 30 cases of diffuse large B-cell lymphoma (DLBCL) and found, in general, a good correlation. The results suggested that BCL2 protein expression is regulated at the transcriptional level in normal B cells and in the neoplastic cells in most B-cell lymphoproliferative disorders.

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The Spherical Nucleic Acids mRNA Detection Paradox

ScienceOpen Research ◽

10.14293/s2199-1006.1.sor-chem.az1mju.v1 ◽

2016 ◽

Cited By ~ 4

Author(s):

Marie Held ◽

Raphael Levy

Keyword(s):

Gold Nanoparticles ◽

Mrna Level ◽

Membrane Vesicles ◽

Apparent Paradox ◽

Intracellular Vesicle ◽

Intracellular Sensing ◽

The 1950S ◽

Spherical Nucleic Acids ◽

New Applications ◽

Endocytic Compartments

<p>From the 1950s onwards, our understanding of the formation and intracellular trafficking of membrane vesicles was informed by experiments in which cells were exposed to gold nanoparticles and their uptake and localisation, studied by electron microscopy.  In the last decade, building on progress in the synthesis of gold nanoparticles and their controlled functionalisation with a large variety of biomolecules (DNA, peptides, polysaccharides), new applications have been proposed, including the imaging and sensing of intracellular events. Yet, as already demonstrated in the 1950s, uptake of nanoparticles results in confinement within an intracellular vesicle which in principle should preclude sensing of cytosolic events. To study this apparent paradox, we focus on a commercially available nanoparticle probe that detects mRNA through the release of a fluorescently-labelled oligonucleotide (unquenching the fluorescence) in the presence of the target mRNA. Using electron, fluorescence and photothermal microscopy, we show that the probes remain in endocytic compartments and that they do not report on mRNA level. We suggest that the validation of any nanoparticle-based probes for intracellular sensing should include a quantitative and thorough demonstration that the probes can reach the cytosolic compartment.</p>

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Synthesis of Factor VIII in Human Hepatocytes in Culture

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646977 ◽

1988 ◽

Vol 60 (03) ◽

pp. 387-391 ◽

Cited By ~ 8

Author(s):

J Ingerslev ◽

B Sloth Christiansen ◽

L Heickendorff ◽

C Munck Petersen

Keyword(s):

Factor Viii ◽

Light Chain ◽

Gradient Centrifugation ◽

Human Fibroblasts ◽

Human Hepatocytes ◽

Human Hepatocyte ◽

Blood Monocytes ◽

Hep G2 ◽

Human Hepatoma Cells ◽

Sds Page

SummaryAlthough several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78–79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all of these cell cultures, vWf : Ag was indetectable or present as trace. Our results suggest that the human hepatocyte is a production site of FVIII.

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Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis

Blood ◽

10.1182/blood.v88.7.2578.bloodjournal8872578 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2578-2584 ◽

Cited By ~ 81

Author(s):

K Cohen-Solal ◽

JL Villeval ◽

M Titeux ◽

S Lok ◽

W Vainchenker ◽

...

Keyword(s):

Splice Variants ◽

Mrna Level ◽

Constitutive Expression ◽

Experimental Models ◽

Northern Analysis ◽

Transcriptional Level ◽

Severe Thrombocytopenia ◽

Wild Type ◽

Mrna Isoforms ◽

Liver And Kidney

Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.

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Gene expression of lipid storage-related enzymes in adipose tissue of the genetically obese Zucker rat. Co-ordinated increase in transcriptional activity and potentiation by hyperinsulinaemia

Biochemical Journal ◽

10.1042/bj2810607 ◽

1992 ◽

Vol 281 (3) ◽

pp. 607-611 ◽

Cited By ~ 23

Author(s):

I Dugail ◽

A Quignard-Boulangé ◽

X Le Liepvre ◽

B Ardouin ◽

M Lavau

Keyword(s):

Adipose Tissue ◽

Mrna Level ◽

Lipid Storage ◽

Zucker Rats ◽

Transcriptional Level ◽

Transcription Rate ◽

Mrna Levels ◽

Zucker Rat ◽

Obese Zucker Rat ◽

Obese Rats

The genetically obese Zucker rat displays excessive fat storage capacity which is due to a tissue-specific increase in the activities of a number of lipid storage-related enzymes in adipose tissue. The aim of this study was to investigate the molecular mechanism responsible for this phenomenon. Lean (Fa/fa) and obese (fa/fa) Zucker rats were studied during the early stages of adipose tissue overdevelopment, both before (at 16 days of age) and after (at 30 days of age) the emergence of hyperinsulinaemia, in order to delineate the effects of the fatty genotype independently of those of hyperinsulinaemia. Lipoprotein lipase (LPL), glycerophosphate dehydrogenase (GPDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malic enzyme (ME) mRNA levels in the adipose tissue of lean and obese rats were assessed by Northern blot analysis, and the relative transcription rates of the corresponding genes were compared in the two genotypes by a nuclear run-on assay. In normoinsulinaemic 16-day-old pre-obese rats, mRNA levels were increased over control values (LPL, 5-fold; ME, 2-fold; GAPDH, 3-fold), in close correlation with genotype-mediated differences in enzyme activities. Stimulation of the transcription rates of the ME and GAPDH genes was observed in obese rats, which could fully account for differences in steady-state mRNA levels. At this age, GPDH activity, mRNA level and transcription rate were similar in the two genotypes. In hyperinsulinaemic 30-day-old obese rats, a 6-7-fold increase in both mRNA and the transcription rate of GPDH emerged, together with an amplification of the genotype-mediated differences observed in younger animals (GAPDH, 6-fold; ME, 7.9-fold; LPL, 10-fold). These results demonstrate that the obese genotype exerts a co-ordinated control on the expression of these genes in adipose tissue, mainly at the transcriptional level. This genotype effect is greatly amplified by the development of hyperinsulinaemia.

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Different responses of primary normal human hepatocytes and human hepatoma cells toward cyanobacterial hepatotoxin microcystin-LR

Toxicon ◽

10.1016/j.toxicon.2015.08.025 ◽

2015 ◽

Vol 105 ◽

pp. 4-9 ◽

Cited By ~ 8

Author(s):

Tsuyoshi Ikehara ◽

Junichi Nakashima ◽

Shihoko Nakashima ◽

Takeshi Yasumoto

Keyword(s):

Hepatoma Cells ◽

Human Hepatocytes ◽

Human Hepatoma ◽

Human Hepatoma Cells ◽

Normal Human

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AKR1D1 is a novel regulator of metabolic phenotype in human hepatocytes and is dysregulated in non-alcoholic fatty liver disease

Metabolism ◽

10.1016/j.metabol.2019.153947 ◽

2019 ◽

Vol 99 ◽

pp. 67-80 ◽

Cited By ~ 11

Author(s):

Nikolaos Nikolaou ◽

Laura L. Gathercole ◽

Lea Marchand ◽

Sara Althari ◽

Niall J. Dempster ◽

...

Keyword(s):

Liver Disease ◽

Fatty Liver ◽

Fatty Liver Disease ◽

Human Hepatocytes ◽

Metabolic Phenotype ◽

Alcoholic Fatty Liver ◽

Alcoholic Fatty Liver Disease

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Effect of 5-Fluorouracil on the Regulation of Zebrafish Thymidylate Synthase Gene Expression

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.641-642.732 ◽

2013 ◽

Vol 641-642 ◽

pp. 732-735

Author(s):

Chun Xia Song ◽

Qian Zhang ◽

Yu Liang Xiao

Keyword(s):

Gene Expression ◽

Thymidylate Synthase ◽

De Novo ◽

Mrna Level ◽

Translation System ◽

Transcriptional Level ◽

Important Target ◽

Reticulocyte Lysate ◽

First Time ◽

Translational Level

Thymidylate synthase (TS) is an important target in cancer therapy, which is a folate-dependent enyme, catalyzing the de novo synthesis of dUMP. In this report, the effect of 5-flurorouracil (5-FU) on the regulation of TS gene expression was estimated in zebrafish. The results showed 5-FU could significantly increase the TS expression in zebrafish embryos. However, TS mRNA level were remained unchanged. To determine the effect of 5-FU and 5-FdUMP on translation of TS mRNA, a rabbit reticulocyte lysate translation system was used. Addition of 5-FU, not inhibited the translation of TS mRNA. While addition of 5-FdUMP, completely repressed the translation of TS mRNA. Therefore, induced expression of thymidylate synthase by 5-FU in zebrafish occurred in translational level, not in transcriptional level. The findings demonstrated that zebrafish TS protein was able to bind to its own cogate mRNA and the 5-FU regulated TS in the translational level. This is the first time to confirm that the regulation of TS is affected by TS and its cognant mRNA interaction in the whole animal level.

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Syk Plays a Critical Role in the Expression and Activation of IRAK1 in LPS-Treated Macrophages

Mediators of Inflammation ◽

10.1155/2017/1506248 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Jae Gwang Park ◽

Young-Jin Son ◽

Byong Chul Yoo ◽

Woo Seok Yang ◽

Ji Hye Kim ◽

...

Keyword(s):

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Interleukin 1 ◽

Mrna Level ◽

Critical Role ◽

Primary Response ◽

Transcriptional Level ◽

Mrna Levels ◽

Treatment Conditions ◽

Lps Treatment

To address how interleukin-1 receptor-associated kinase 1 (IRAK1) is controlled by other enzymes activated by toll-like receptor (TLR) 4, we investigated the possibility that spleen tyrosine kinase (Syk), a protein tyrosine kinase that is activated at an earlier stage during TLR4 activation, plays a central role in regulating the functional activation of IRAK1. Indeed, we found that overexpression of myeloid differentiation primary response gene 88 (MyD88), an adaptor molecule that drives TLR signaling, induced IRAK1 expression and that piceatannol, a Syk inhibitor, successfully suppressed the MyD88-dependent upregulation of IRAK1 under LPS treatment conditions. Interestingly, in Syk-knockout RAW264.7 cells, IRAK1 activity was almost completely blocked after LPS treatment, while providing a Syk-recovery gene to the knockout cells successfully restored IRAK1 expression. According to our measurements of IRAK1 mRNA levels, the transcriptional upregulation of IRAK1 was induced by LPS treatment between 4 and 60 min, and this can be suppressed in Syk knockout cells, providing an effect similar that that seen under piceatannol treatment. The overexpression of Syk reverses this effect and leads to a significantly higher IRAK1 mRNA level. Collectively, our results strongly suggest that Syk plays a critical role in regulating both the activity and transcriptional level of IRAK1.

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