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Ocoxin is a nutritional supplement that has been shown to exert antioxidant and immunomodulatory responses in patients with chronic hepatitis C. The present work aimed to determine the effects of Ocoxin on activated hepatic stellate cells (HSC), the cell type mainly responsible for collagen deposition in the fibrotic liver. Ocoxin was found to reduce the survival of a cell line of immortalized non-tumoral rat HSC in a dose-response fashion and to diminish collagen type I levels. This latter effect was observed even at doses not affecting cell survival, pointing to an antifibrogenic action for the supplement. The decrease in viability exerted by Ocoxin on HSC correlated with an increase in histone-associated fragments in the cytoplasm and with increased activity of caspase-3, indicating the induction of apoptosis. To determine the molecular mechanisms mediating Ocoxin-induced apoptosis, the activation of members of the MAPK family was analyzed. Incubation of HSC with Ocoxin caused a transient and dramatic enhancement on ERK, JNK, and p38 MAPK phosphorylation levels. Using specific inhibitors for these enzymes, p38 MAPK was identified as a key mediator of the apoptotic effect of Ocoxin on HSC., (© 2022. The Author(s).)

Background: Cholangiocarcinoma (CCA) is still a deadly tumour. Histological and molecular aspects of thioacetamide (TAA)-induced intrahepatic CCA (iCCA) in rats mimic those of human iCCA. Carcinogenic changes and therapeutic vulnerabilities in CCA may be captured by molecular investigations in bile, where we performed bile proteomic and metabolomic analyses that help discovery yet unknown pathways relevant to human iCCA., Methods: Cholangiocarcinogenesis was induced in rats (TAA) and mice (Jnk Δhepa  + CCl 4  + DEN model). We performed proteomic and metabolomic analyses in bile from control and CCA-bearing rats. Differential expression was validated in rat and human CCAs. Mechanisms were addressed in human CCA cells, including Huh28-KRAS G12D cells. Cell signaling, growth, gene regulation and [U- 13 C]-D-glucose-serine fluxomics analyses were performed. In vivo studies were performed in the clinically-relevant iCCA mouse model., Results: Pathways related to inflammation, oxidative stress and glucose metabolism were identified by proteomic analysis. Oxidative stress and high amounts of the oncogenesis-supporting amino acids serine and glycine were discovered by metabolomic studies. Most relevant hits were confirmed in rat and human CCAs (TCGA). Activation of interleukin-6 (IL6) and epidermal growth factor receptor (EGFR) pathways, and key genes in cancer-related glucose metabolic reprogramming, were validated in TAA-CCAs. In TAA-CCAs, G9a, an epigenetic pro-tumorigenic writer, was also increased. We show that EGFR signaling and mutant KRAS G12D can both activate IL6 production in CCA cells. Furthermore, phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in serine-glycine pathway, was upregulated in human iCCA correlating with G9a expression. In a G9a activity-dependent manner, KRAS G12D promoted PHGDH expression, glucose flow towards serine synthesis, and increased CCA cell viability. KRAS G12D CAA cells were more sensitive to PHGDH and G9a inhibition than controls. In mouse iCCA, G9a pharmacological targeting reduced PHGDH expression., Conclusions: In CCA, we identified new pro-tumorigenic mechanisms: Activation of EGFR signaling or KRAS mutation drives IL6 expression in tumour cells; Glucose metabolism reprogramming in iCCA includes activation of the serine-glycine pathway; Mutant KRAS drives PHGDH expression in a G9a-dependent manner; PHGDH and G9a emerge as therapeutic targets in iCCA., (© 2022. The Author(s).)

Inborn errors of metabolism (IEM) encompass a group of monogenic diseases affecting both pediatric and adult populations and currently lack effective treatments. Some IEM such as familial hypercholesterolemia or X-linked protoporphyria are caused by gain of function mutations, while others are characterized by an impaired protein function, causing a metabolic pathway blockage. Pathophysiology classification includes intoxication, storage and energy-related metabolic disorders. Factors specific to each disease trigger acute metabolic decompensations. IEM require prompt and effective care, since therapeutic delay has been associated with the development of fatal events including severe metabolic acidosis, hyperammonemia, cerebral edema, and death. Rapid expression of therapeutic proteins can be achieved hours after the administration of messenger RNAs (mRNA), representing an etiological solution for acute decompensations. mRNA-based therapy relies on modified RNAs with enhanced stability and translatability into therapeutic proteins. The proteins produced in the ribosomes can be targeted to specific intracellular compartments, the cell membrane, or be secreted. Non-immunogenic lipid nanoparticle formulations have been optimized to prevent RNA degradation and to allow safe repetitive administrations depending on the disease physiopathology and clinical status of the patients, thus, mRNA could be also an effective chronic treatment for IEM. Given that the liver plays a key role in most of metabolic pathways or can be used as bioreactor for excretable proteins, this review focuses on the preclinical and clinical evidence that supports the implementation of mRNA technology as a promising personalized strategy for liver metabolic disorders such as acute intermittent porphyria, ornithine transcarbamylase deficiency or glycogen storage disease., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 ( Jnk1/2 ) knockout mice. Floxed JNK1/2 ( Jnk f/f ) and Jnk ∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines ( Tnf , Tgfβ1 ) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk ∆hepa compared with Jnk f/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk ∆hepa animals compared with Jnk f/f littermates. Finally, thioacetamide (TAA) administration to Jnk ∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl 4 -treated Jnk ∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA.

The endocytosis of ligand-bound receptors and their eventual recycling to the plasma membrane (PM) are processes that have an influence on signalling activity and therefore on many cell functions, including migration and proliferation. Like other tyrosine kinase receptors (TKR), the insulin receptor (INSR) has been shown to be endocytosed by clathrin-dependent and -independent mechanisms. Once at the early endosome (EE), the sorting of the receptor, either to the late endosome (LE) for degradation or back to the PM through slow or fast recycling pathways, will determine the intensity and duration of insulin effects. Both the endocytic and the endosomic pathways are regulated by many proteins, the Arf and Rab families of small GTPases being some of the most relevant. Here, we argue for a specific role for the slow recycling route, whilst we review the main molecular mechanisms involved in INSR endocytosis, sorting and recycling, as well as their possible role in cell functions.

Background and Aims: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors., Approach and Results: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl 4 treatment (Jnk Δhepa + DEN + CCl 4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in Jnk Δhepa + DEN + CCl 4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status., Conclusions: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies., (© 2020 by the American Association for the Study of Liver Diseases.)

Objective: Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis., Design: G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-β1 (TGFβ1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl 4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology., Results: G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFβ1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFβ1 fibrogenic responses in hHSC. TGFβ1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity., Conclusions: Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign ( n = 36) and malignant conditions, CCA ( n = 36) or PDAC ( n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy ( 1 H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids ( n = 10) and proteins ( n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy.

AGAP2 (Arf GAP with GTP-binding protein-like domain, Ankyrin repeat and PH domain 2) isoform 2 is a protein that belongs to the Arf GAP (GTPase activating protein) protein family. These proteins act as GTPase switches for Arfs, which are Ras superfamily members, being therefore involved in signaling regulation. Arf GAP proteins have been shown to participate in several cellular functions including membrane trafficking and actin cytoskeleton remodeling. AGAP2 is a multi-tasking Arf GAP that also presents GTPase activity and is involved in several signaling pathways related with apoptosis, cell survival, migration, and receptor trafficking. The increase of AGAP2 levels is associated with pathologies as cancer and fibrosis. Transforming growth factor beta-1 (TGF-β1) is the most potent pro-fibrotic cytokine identified to date, currently accepted as the principal mediator of the fibrotic response in liver, lung, and kidney. Recent literature has described that the expression of AGAP2 modulates some of the pro-fibrotic effects described for TGF-β1 in the liver. The present review is focused on the interrelated molecular effects between AGAP2 and TGFβ1 expression, presenting AGAP2 as a new player in the signaling of this pro-fibrotic cytokine, thereby contributing to the progression of hepatic fibrosis., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Liver damage induces hepatic stellate cells (HSC) activation, characterised by a fibrogenic, proliferative and migratory phenotype. Activated HSC are mainly regulated by transforming growth factor β 1 (TGFβ1), which increases the production of extracellular matrix proteins (e.g. collagen-I) promoting the progression of hepatic fibrosis. AGAP2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 2) is a GTPase/GTP-activating protein involved in the actin remodelling system and receptor recycling. In the present work the role of AGAP2 in human HSC in response to TGFβ1 was investigated. LX-2 HSC were transfected with AGAP2 siRNA and treated with TGFβ1. AGAP2 knockdown prevented to some extent the proliferative and migratory TGFβ1-induced capacities of LX-2 cells. An array focused on human fibrosis revealed that AGAP2 knockdown partially prevented TGFβ1-mediated gene expression of the fibrogenic genes ACTA2, COL1A2, EDN1, INHBE, LOX, PDGFB, TGFΒ12, while favored the expression of CXCR4, IL1A, MMP1, MMP3 and MMP9 genes. Furthermore, TGFβ1 induced AGAP2 promoter activation and its protein expression in LX-2. Moreover, AGAP2 protein levels were significantly increased in liver samples from rats with thioacetamide-induced fibrosis. In addition, AGAP2 silencing affected TGFβ1-receptor 2 (TGFR2) trafficking in U2OS cells, blocking its effective recycling to the membrane. AGAP2 silencing in LX-2 cells prevented the TGFβ1-induced increase of collagen-I protein levels, while its overexpression enhanced collagen-I protein expression in the presence or absence of the cytokine. AGAP2 overexpression also increased focal adhesion kinase (FAK) phosphorylated levels in LX-2 cells. FAK and MEK1 inhibitors prevented the increase of collagen-I expression caused by TGFβ1 in LX-2 overexpressing AGAP2. In summary, the present work shows for the first time, that AGAP2 is a potential new target involved in TGFβ1 signalling, contributing to the progression of hepatic fibrosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 are implicated in the progression of liver fibrosis. However, the role of Nox5 is not yet known, mainly due to the lack of this enzyme in rat and mouse genomes. Here we describe the expression and functional relevance of Nox5 in the human cell line of hepatic stellate cells (HSC) LX-2. Under basal conditions, three long (Nox5-L: Nox5α, -β, and -δ) and a short (Nox5-S or Nox5ε) splice variants were detected, which were silenced with specific siRNAs for Nox5. The most abundant isoform was Nox5-S, accounting for more than 90% of Nox5 protein. Overexpression of Nox5β generated reactive oxygen species (ROS) in the presence of calcium, as judged by the production of hydrogen peroxide, L-012 luminescence and cytochrome c reduction. Nox5ε did not generated ROS under these conditions, and a reduced ROS production was observed when co-expressed with Nox5β. In contrast, dihydroethidium oxidation was increased by Nox5β or Nox5ε, suggesting that Nox5ε induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5β and Nox5ε stimulated the proliferation of LX-2 cells and the collagen type I levels, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-β and angiotensin II upregulated Nox5 expression, which was reduced in cells pre-incubated with catalase. Further studies silencing Nox5 in TGF-β-treated cells resulted in a reduction of collagen levels via p38 MAPK. Collectively, these results show for the first time that Nox5 can play a relevant role in the proliferation and fibrosis on human HSC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Unfolded protein response (UPR) triggered as a consequence of ER stress has been shown to be involved in the development of different pathologies, including fibrotic disorders. In the present paper we explore the role played by UPR on a key fibrogenic parameter in the liver: collagen type I levels in activated hepatic stellate cells (HSC). Using Brefeldin A (BFA) as an ER stress inducer we found that UPR correlated with enhanced mRNA and protein levels of collagen type I in a cell line of immortalized non-tumoral rat HSC. Analysis of the three branches of UPR revealed the activation of IRE1α, PERK and ATF6 in response to BFA, although PERK activation was shown not to be involved in the fibrogenic action of BFA. BFA also activated p38 MAPK in an IRE1α-dependent way and the p38 MAPK inhibitor SB203580 prevented the increase in collagen type I mRNA and protein levels caused by BFA, suggesting the involvement of this kinase on this effect. Analysis of Smad activation showed that phosphorylated nuclear levels of Smad2 and 3 were increased in response to BFA treatment. Inhibition of Smad3 phosphorylation by SIS3 prevented the enhancement of collagen type I levels caused by BFA. Pretreatment with IRE1α and p38 MAPK inhibitors also prevented the increased p-Smad3 accumulation in the nucleus, suggesting an IRE1α-p38 MAPK-Smad pathway to be responsible for the fibrogenic action of BFA on HSC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (H(2)DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H(2)DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H(2)DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H(2)DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques., (Copyright © 2015. Published by Elsevier Inc.)

The turnover of extracellular matrix (ECM) components can generate signals that regulate several cellular functions such as proliferation, differentiation, and apoptosis. During liver injury, matrix metalloproteases (MMPs) production is enhanced and increased levels of peptides derived from extracellular matrix proteins can be generated. Synthetic peptides with sequences present in extracellular matrix proteins were previously found to induce both stimulating and apoptotic effects on several cell types including the inflammatory cells monocytes/macrophages. Therefore, in inflammatory liver diseases, locally accumulated peptides could be also important in regulating hepatic fibrosis by inducing apoptosis of hepatic stellate cells (HSC), the primary cellular source of extracellular matrix components. Here, we describe the apoptotic effect of fibronectin peptides on the cell line of human hepatic stellate cells LX-2 based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion, and accumulation of Bax protein. We also found that these peptides trigger the activation of Src kinase, which in turn mediated the increase of JNK and p38 activities. By the use of specific inhibitors we demonstrated the involvement of Src, JNK, and p38 in apoptosis induced by fibronectin peptides on HSC. Moreover, fibronectin peptides increased iNOS expression in human HSC, and specific inhibition of iNOS significantly reduced the sustained activity of JNK and the programmed cell death caused by these peptides. Finally, the possible regulatory effect of fibronectin peptides in liver fibrosis was further supported by the ability of these peptides to induce metalloprotease-9 (MMP-9) expression in human monocytes., (© 2014 Wiley Periodicals, Inc., A Wiley Company.)

General control nonderepresible 2 (GCN2) is a highly conserved cytosolic kinase that modulates a complex response for coping with the stress owing to lack of amino acids. GCN2 has been recently shown to be involved in the regulation of metabolic balance and lipid degradation rate in the liver. We hypothesized that GCN2 could have a role in in hepatic fibrogenesis and in the response to acute or chronic liver injury. Activation of GCN2 in primary or immortalized human hepatic stellate cells by incubation with medium lacking the essential amino acid histidine correlated with decreased levels of collagen type I protein and mRNA, suggesting an antifibrogenic effect of GCN2. In vivo studies with Gcn2 knock-out mice (Gcn2(-/-)) showed increased susceptibility to both acute or chronic liver damage induced by CCl(4), as shown by higher alanine aminotransferase and aspartate aminotransferase activities, increased necrosis and higher inflammatory infiltrates compared with wild-type mice (WT). Chronic CCl(4) treatment increased deposition of interstitial collagen type I more in Gcn2(-/-) mice than in WT mice. Col1a1 and col1a2 mRNA levels also increased in CCl(4)-treated Gcn2(-/-) mice compared with WT mice. These results suggest that GCN2 is a key regulator of the fibrogenic response to liver injury.

Inflammatory conditions are characterized by continuous overproduction of nitric oxide (NO) that can contribute to cell survival but also to cell demise by affecting apoptosis. These facts are important in regulation of hepatic fibrogenesis during exposure to inflammatory stress, since elevated NO may pose the risk of cells with a pro-fibrogenic phenotype giving rise to a sustained proliferation leading to chronic fibrosis. Since nitration of tyrosine residues occurs in a range of diseases involving inflammation, we tested the hypothesis that nitration of specific proteins could result in apoptosis of hepatic stellate cells (HSC), the primary cellular source of matrix components in liver diseases. We found the peroxynitrite generator SIN-1 to promote apoptosis in human and rat HSC, based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax protein. We also showed that SIN-1-induced apoptosis of HSC was due to protein nitration. Among the tyrosine-nitrated proteins, tyrosine kinase Lyn was identified. SIN-1 triggered a signaling pathway through Src kinase Lyn activation that resulted in increased activity of the tyrosine kinase Syk. The involvement of these signaling molecules in the apoptotic process induced by SIN-1 as well as the mechanism by which they are activated was confirmed by using specific inhibitors. In summary, NO, via protein-nitration, could play an important role in controlling liver fibrosis resolution by regulation of HSC apoptosis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In eukaryotic cells amino acid deprivation triggers a response aimed to ensure cell survival in stress conditions. In the present work we analyzed the effects of amino acid deprivation on intracellular levels of reactive oxygen species (ROS) of hepatic stellate cells (HSC), a key cell type in the development of liver fibrosis. Histidine deprivation caused in the human immortalized HSC cell line LX-2 a fast decrease of intracellular ROS levels that was also observed in HSC incubated either with leucine-free or amino acid-free medium, but not with glucose-free medium. Phosphorylation of GCN2 kinase and its substrate eIF2alpha was induced by histidine deprivation. Reversion studies and activation of GCN2 by tRNA and the proteasome inhibitor MG-132 showed a correlation between GCN2 phosphorylation and diminished ROS levels. However, a lack of correlation between eIF2alpha phosphorylation and ROS levels was found using salubrinal, an inhibitor of eIF2alpha phosphorylation, suggesting a role for GCN2 unrelated to its activity as eIF2alpha kinase. LX-2 cells treated with histidine-free medium presented reduced SOD activity that could account for the decrease on ROS levels. Histidine deprivation as well as activation of GCN2 by treatment with tRNA, caused an increase in LX-2 cell viability, suggesting amino acid restriction to present a protective effect in HSC which is mediated by GCN2 activation., (Copyright 2010 S. Karger AG, Basel.)

The sustained overproduction of nitric oxide (NO) observed in inflammatory conditions can contribute to cell demise by affecting apoptosis. Nitration of tyrosine residues occurs in a range of diseases involving macrophage activation. Since NO induces apoptosis in monocytes/macrophages, we tested the hypothesis that nitration of specific proteins could result in apoptotic cell death. The peroxynitrite generator SIN-1 promoted apoptosis in monocytes based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax and p53 proteins. We also found that the signaling pathway triggered by SIN-1 was initiated through tyrosine kinase and Rac activation and resulted in increased JNK and p38 activities. Among the tyrosine-nitrated proteins, Rac and Lyn were identified. Using specific inhibitors for different signaling and effector molecules involved in the apoptotic process we demonstrate that NO, via protein-nitration, could play an important role in controlling the inflammatory response by regulation of monocyte homeostasis.

The amino acid leucine causes an increase of collagen alpha1(I) synthesis in hepatic stellate cells through the activation of translational regulatory mechanisms and PI3K/Akt/mTOR and ERK signaling pathways. The aim of the present study was to evaluate the role played by reactive oxygen species on these effects. Intracellular reactive oxygen species levels were increased in hepatic stellate cells incubated with leucine 5 mM at early time points, and this effect was abolished by pretreatment with the antioxidant glutathione. Preincubation with glutathione also prevented 4E-BP1, eIF4E and Mnk-1 phosphorylation induced by leucine, as well as enhancement of procollagen alpha1(I) protein levels. Inhibitors for MEK-1 (PD98059), PI3K (wortmannin) or mTOR (rapamycin) did not affect leucine-induced reactive oxygen species production. However, preincubation with glutathione prevented ERK, Akt and mTOR phosphorylation caused by treatment with leucine. The mitochondrial electron chain inhibitor rotenone and the NADPH oxidase inhibitor apocynin prevented reactive oxygen species production caused by leucine. Leucine also induced an increased phosphorylation of IR/IGF-R that was abolished by pretreatment with either rotenone or apocynin. Therefore, leucine exerts on hepatic stellate cells a prooxidant action through NADPH oxidase and mitochondrial Reactive oxygen species production and these effects mediate the activation of IR/IGF-IR and signaling pathways, finally leading to changes in translational regulation of collagen synthesis.

TNF-alpha has been shown to inhibit procollagen alpha1(I) expression in hepatic stellate cells (HSC), although the molecular mechanisms involved have not been fully established. In the present work, we studied the possible role played by oxidative stress and NFkappaB on the antifibrogenic action of TNF-alpha on a cell line of rat HSC. Treatment of HSC with TNF-alpha did not affect either intracellular levels of reactive oxygen species or lipid peroxidation, but caused a decrease on reduced glutathione (GSH) levels. Restoration of intracellular GSH by incubation with exogenous GSH prevented the inhibition of procollagen alpha1(I) levels caused by TNF-alpha. The effect of GSH was not mimicked by antioxidants like deferoxamine, tempol or trolox. Activation of NFkappaB by TNF-alpha was also abolished by preincubation of HSC with GSH, but not by deferoxamine, tempol or trolox. These results point to GSH depletion as a mediator of TNF-alpha action in HSC.

The essential amino acid leucine has been described to specifically activate signaling pathways leading to the activation of the translational machinery and the increase of total protein synthesis. Regulation of type I collagen production by hepatic stellate cells (HSC) is a multistep process involving transcriptional and post-transcriptional mechanisms. In the present work we studied the effect of leucine on translation regulation of collagen alpha1(I) production in HSC and the signaling pathways involved. Treatment of HSC with 5 mM leucine did not alter half-life or steady state levels of procollagen alpha1(I) mRNA, but caused an increase in procollagen alpha1(I) protein that correlated with changes of components involved in translational regulation, like enhanced 4E-BP1, Mnk-1, and eIF4E phosphorylation. Leucine also induced mTOR, ERK, and Akt phosphorylation in HSC, without affecting p38 and JNK activation. Pre-treatment of HSC with PD098059, wortmannin, or rapamycin prevented the profibrogenic action of leucine due to the inhibition of different molecular mechanisms. These results suggest leucine is a profibrogenic agent for HSC, activating signaling pathways that lead to an enhancement of collagen alpha1(I) production through translational regulation., (Copyright 2006 Wiley-Liss, Inc.)

S-adenosylmethionine (AdoMet) and 5'-methylthioadenosine (MTA) exert a protective action on apoptosis induced by okadaic acid in primary rat hepatocytes but not in human transformed HuH7 cells. In the present work, we analyzed the role played by the JNK/activator protein (AP)-1 pathway in this differential effect. Okadaic acid induced the phosphorylation of JNK and c-Jun and the binding activity of AP-1 in primary hepatocytes, and pretreatment with either AdoMet or MTA prevented those effects. In HuH7 cells, pretreatment with either AdoMet or MTA did not affect JNK and c-Jun activation or AP-1 binding induced by okadaic acid. In both cell types, p38 was activated by okadaic acid, but neither AdoMet nor MTA presented a significant effect on its activity. Therefore, the differential effect of both AdoMet and MTA on the JNK/AP-1 pathway could explain their antiapoptotic effect on primary hepatocytes and the lack of protection they show against okadaic acid-induced apoptosis in hepatoma cells.

Synthetic peptides with sequences present in extracellular matrix protein fibronectin have been described to stimulate human monocytes. We describe now that one of these peptides, FN6, induces apoptotic effects on monocytes and we investigate the molecular mechanisms involved in the regulation of this response. Incubation of monocytes with FN6 induces the activation of the small GTPase Rac. In turn, Rac mediates the increase of both JNK and p38 activities in a sustained fashion, as well as the phosphorylation levels of their respective substrates c-Jun and ATF-2. FN6 also stimulates caspases -9 and -3 and the delayed proteolysis of its substrates PARP and D4-GDI. In addition, initiator caspases-1 and -5 were activated by FN6 treatment of monocytes but, in contrast to that observed for caspases-9 and -3, this effect was not dependent on JNK or p38 activities. These kinases also mediated the increase of Bax levels, but only in some conditions Bcl-2 depletion caused by the peptide. Moreover, whereas initially only caspase-1 is involved in caspase-3 activation, later on caspase-9 seems also to participate. Therefore, we demonstrate that FN6 stimulation allows multiple, JNK and p38-dependent and -independent interacting signals to regulate the apoptotic response in human monocytes.

Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein-3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell-cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase-3 activation, Bcl-2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes.

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA), is a derivative of amphetamine with hepatotoxic effects that has been shown to induce apoptosis of cultured liver cells. In the present work, we studied the role played by oxidative stress in the apoptotic response caused by MDMA on a cell line of hepatic stellate cells (HSC). MDMA-treatment provoked oxidative stress determined as reactive oxygen species (ROS) accumulation and decrease of intracellular reduced glutathione levels. Pre-treatment with the antioxidant pyrrolidine dithiocarbamate blocked ROS production but did not prevent MDMA-induced apoptosis of HSC. The pro-oxidant menadione induced in HSC ROS production and apoptosis that were prevented by pyrrolidine dithiocarbamate, showing HSC to be susceptible to oxidative stress-induced apoptosis. Addition of exogenous GSH or its precursor NAC potentiated the apoptotic action of MDMA but blocked apoptosis induced by menadione. Pre-treatment of HSC with the cytochrome P450 inhibitor quinine diminished the extent of apoptosis caused by MDMA, suggesting the involvement of a metabolic derivative of MDMA on its apoptotic effect. Nuclear factor NF-kappaB was activated by MDMA in a oxidative stress independent fashion and played a protective role in the apoptotic response, since inhibition of NF-kappaB by treatment with parthenolide or by viral infection with a dominant-negative form of NIK (Ad5dnNIK) resulted in an increase of MDMA-induced cell death. In summary, MDMA-induced apoptosis of HSC is accompanied, but not caused by oxidative stress; a metabolic derivative of the drug is responsible for the apoptotic effect of MDMA, which is partially blocked by NF-kappaB activation.

"Ecstasy" (3,4-methylenedioxymethamphetamine, MDMA) has been shown to be hepatotoxic for human users, but molecular mechanisms involved in this effect remained poorly understood. MDMA-induced cell damage is related to programmed cell death in serotonergic and dopaminergic neurons. However, until now there has been no evidence of apoptosis induced by MDMA in liver cells. Here we demonstrate that exposure to MDMA caused apoptosis of freshly isolated rat hepatocytes and of a cell line of hepatic stellate cells (HSC), as shown by chromatin condensation of the nuclei and accumulation of oligonucleosomal fragments in the cytoplasm. In both cell types, apoptosis correlated with decreased levels of bcl-x(L), release of cytochrome c from the mitochondria and activation of caspase 3. In HSC, but not in hepatocytes, MDMA induced poly(ADP-ribose)polymerase (PARP) proteolysis. These results suggest that apoptosis of liver cells could be involved in the hepatotoxicity of MDMA.

The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

It has been suggested that left ventricular fibrosis in spontaneously hypertensive rats (SHR) is the result of both exaggerated collagen synthesis and insufficient collagen degradation. We have shown previously that chronic treatment with the angiotensin II type 1 receptor antagonist losartan results in diminished synthesis of collagen type I molecules and reversal of myocardial fibrosis in SHR. This study was designed to investigate whether losartan also affects the extracellular degradation of collagen type I fibers in the left ventricle of SHR. The study was performed in 30-week-old normotensive Wistar-Kyoto rats (WKY), untreated SHR, and SHR treated with orally administered losartan (20 mg/kg per day) for 14 weeks before they were killed. Ventricular collagenase activity was determined by degradation of [(14)C]collagen with tissue extracts. Ventricular expression of tissue inhibitor of metalloproteinases 1 (TIMP-1) mRNA was analyzed by Northern blot. A histomorphometric study of the left ventricle was performed in all rats. Compared with WKY, SHR exhibited left ventricular hypertrophy, increased (P<0.05) blood pressure, left ventricular collagen volume fraction and TIMP-1 mRNA, and diminished (P<0.05) collagenase activity. After the treatment period, blood pressure was higher (P<0.05) in losartan-treated SHR than in WKY, and no significant differences were noted in the remaining parameters between the 2 strains of rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy, diminished (P<0.05) blood pressure, left ventricular collagen volume fraction and TIMP-1 mRNA, and increased (P<0.05) collagenase activity. These results suggest that the transcription of the TIMP-1 gene is upregulated in the hypertrophied and fibrotic left ventricle of adult SHR. Upregulation of TIMP-1 may account for diminished collagenase activity in the myocardium of those rats. Chronic angiotensin II type 1 receptor blockade with losartan resulted in inhibition of TIMP-1 expression and stimulation of collagenase activity in the left ventricle of SHR. It is proposed that angiotensin II may facilitate myocardial fibrosis in SHR by depressing the collagenase-mediated extracellular degradation of collagen fibers.

Tumor necrosis factor alpha (TNF-alpha) is one of the key cytokines of the acute phase response and of many inflammatory processes. This cytokine has several antifibrogenic actions and down-regulates the expression of the type I collagen genes and induces the expression of metalloproteinases. Because TNF-alpha directly antagonizes some fibrogenic actions of transforming growth factor beta(1) (TGF-beta(1)), we considered it important to map the cis-acting regulatory element of the alpha1(I) collagen (col1a1) promoter involved in TNF-alpha responsiveness in hepatic stellate cells (HSC), to investigate the transcription factors that bind to it, and to establish possible mechanisms by which TNF-alpha down-regulates its expression. In this article, we show the presence of a functional TNF-alpha-responsive element (TaRE) in the -378 to -345 region of the col1a1 promoter. This element colocalizes with a previously reported TGF-beta(1)-responsive element. We further demonstrate that TNF-alpha induces nuclear translocation and binding of transcriptional complexes containing p20C/EBPbeta, p35C/EBPbeta, and C/EBPdelta to this sequence of the promoter. Transient overexpression of C/EBPdelta or p20C/EBPbeta, the natural dominant negative form of C/EBPbeta in HSC, down-regulated activity of a CAT reporter vector driven by -412 to +110 of the col1a1 promoter. Taken together, these data suggest that the -378 to -340 region of the col1a1 promoter is the site of convergence of different stimuli that ultimately modulate col1a1 gene transcription.

3,4-Methylenedioxymethamphetamine, MDMA ("Ecstasy"), has been previously shown to produce cell necrosis and fibrosis in the liver. Our aim was to study the effect of MDMA on the type I collagen production by a cell line of hepatic stellate cells (HSC), the cell type mainly responsible for collagen synthesis in the liver. We demonstrated that MDMA increases alpha1(I) procollagen mRNA levels and that this increase correlates with glutathione depletion and enhanced hydrogen peroxide production by HSC. Pre-treatment with either glutathione monoethyl ester or deferoxamine prevents the MDMA-induced alpha1(I) procollagen mRNA expression, indicating oxidative stress to be a mediator of this effect. Lipid peroxidation was not detected in MDMA-treated cells and therefore does not seem to be involved in the pro-fibrogenic action of MDMA on HSC., (Copyright 1999 Academic Press.)

Oxidative stress plays a key role in liver fibrosis. Both inflammatory cells and activated Kupffer cells produce H2O2, an oxidant involved in the activation of hepatic stellate cells (HSC). Increased production of reactive oxygen intermediates (ROIs) in fibrotic livers is associated in part with the up-regulation of transforming growth factor beta (TGF-beta), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-beta induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF-beta-elicited alpha1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-beta induces the accumulation of H2O2, and that this oxidant is, in turn, directly involved in up-regulating the expression of the col1a1 gene. While the addition of H2O2 to HSC induced the expression of alpha1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF-beta-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped a cis-acting element (-370 to -344) essential for TGF-beta responsiveness. We further showed that TGF-beta induced the activation and binding of a C/EBPbeta-containing transcriptional complex to this sequence, an effect that was also mimicked by the addition of H2O2. Taken together, these data demonstrate a direct connection between TGF-beta-mediated accumulation of H2O2 and the up-regulation of col1a1 gene in HSC.

Background: In response to nonspecific inflammatory stimuli, circulating monocytes produce several cytokines that regulate the expression of liver acute phase protein genes. Patients with alcoholic liver disease have several manifestations of the acute phase response, including elevated serum levels of interleukin (IL)-1 (IL-1), IL-6 and tumor necrosis factor-alpha (TNF-alpha). However, the role of the acute phase response on liver fibrogenesis has not been explored., Experimental Design: In this communication we report experiments performed to investigate whether turpentine, an acute phase response inducer in rats has any effect on alpha 1 (I) procollagen gene expression in the liver. We also investigated which of the cytokines is responsible for the turpentine effect and whether IL-6 and TNF-alpha had an effect on alpha 1 (I) procollagen mRNA expression by liver fat-storing cells (FSC)., Results: We show that alpha 1 (I) procollagen mRNA is increased in livers of turpentine-treated rats, and that an antibody to IL-6 as well as colchicine inhibit this effect. We also show that rIL-6 induces the expression of alpha 1 (I) procollagen mRNA in cultured FSC but not in hepatocytes. We demonstrated that the IL-6 effect is a transcriptional event that requires "de novo" protein synthesis. In addition to its effect on collagen gene expression, rIL-6 also stimulates expression of transforming growth factor-beta and fibronectin mRNAs. TNF-alpha inhibits alpha 1 (I) procollagen expression in FSC by 24 to 48 hours. However, TNF-alpha induces a transient expression of alpha 1 (I) procollagen mRNA by 2 to 3 hours. This increase is preceded by the induction of IL-6 mRNA., Conclusions: We conclude that IL-6 produced during the acute phase response, alone or in conjunction with other cytokines, could play an important role in liver fibrogenesis by inducing the expression of collagen, fibronectin, and transforming growth factor-beta mRNAs in FSC.

The Ca²( +) -dependent ATPase activity of spinach chloroplast coupling factor 1 (CF₁) is activated by treatment with dithiothreitol (DDT). If excess of this reagent is eliminated by gel filtration, an Eadie-Hofstee biphasic plot is obtained. These results are consistent with the existence of two active forms of the enzyme governed by the redox state. We have observed that SDS-polyacrylamide gel electrophoresis pattern is affected by the pretreatment of the samples under those two different conditions. Spontaneous activation of the samples, due to a limited proteolytic process, has also been detected. In this case the electrophoretic pattern was also affected. The protease implied in this process could be a cystein protease co-isolated with CF₁. These observations suggest that limited proteolysis, as well as redox-induced changes, are involved in the physiological regulation of the enzyme.

The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.