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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.

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 CCl4 treatment (JnkΔhepa + DEN + CCl4 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 JnkΔhepa + DEN + CCl4 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.

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.

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.

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 andidentifyingbiomarkersforthedifferentialdiagnosisofbiliarystrictures. Comprehensiveanalyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-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. Keywords: human bile; cholangiocarcinoma; pancreatic adenocarcinoma; lipidomics; proteomics; machine-learning &nbsp

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 (1H-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., This research was funded by: Instituto de Salud Carlos III (ISCIII) co-financed by Fondo Europeo de Desarrollo Regional (FEDER) Una manera de hacer Europa, grant numbers: PI16/01126 (M.A.A.), PI19/00819 (M.J.M. and J.J.G.M.), PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129 (J.M.B.); Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation), grant name: Rare Cancers 2017 (J.M.U., M.L.M., J.M.B., M.J.M., R.I.R.M., M.G.F.-B., C.B., M.A.A.); Gobierno de Navarra Salud, grant number 58/17 (J.M.U., M.A.A.); La Caixa Foundation, grant name: HEPACARE (C.B., M.A.A.); AMMF The Cholangiocarcinoma Charity, UK, grant number: 2018/117 (F.J.C. and M.A.A.); PSC Partners US, PSC Supports UK, grant number 06119JB (J.M.B.); Horizon 2020 (H2020) ESCALON project, grant number H2020-SC1-BHC-2018–2020 (J.M.B.); BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia, grant numbers BIO15/CA/016/BD (J.M.B.) and BIO15/CA/011 (M.A.A.). Department of Health of the Basque Country, grant number 2017111010 (J.M.B.). La Caixa Foundation, grant number: LCF/PR/HP17/52190004 (M.L.M.), Mineco-Feder, grant number SAF2017-87301-R (M.L.M.), Fundación BBVA grant name: Ayudas a Equipos de Investigación Científica Umbrella 2018 (M.L.M.). MCIU, grant number: Severo Ochoa Excellence Accreditation SEV-2016-0644 (M.L.M.). Part of the equipment used in this work was co-funded by the Generalitat Valenciana and European Regional Development Fund (FEDER) funds (PO FEDER of Comunitat Valenciana 2014–2020). Gobierno de Navarra fellowship to L.C. (Leticia Colyn); AECC post-doctoral fellowship to M.A.; Ramón y Cajal Program contracts RYC-2014-15242 and RYC2018-024475-1 to F.J.C. and M.G.F.-B., respectively. The generous support from: Fundación Eugenio Rodríguez Pascual, Fundación Echébano, Fundación Mario Losantos, Fundación M Torres and Mr. Eduardo Avila are acknowledged. The CNB-CSIC Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0001 (F.J.C.). Comunidad de Madrid Grant B2017/BMD-3817 (F.J.C.).

ObjectiveHepatic 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.DesignG9a 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 CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology.ResultsG9a 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.ConclusionsDual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

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.

Epigenetic modifications such as DNA and histone methylation functionally cooperate in fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n = 268), peritumoral tissues (n = 154), and HCC cell lines (n = 32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cell growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1, and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. Conclusion: Combined targeting of G9a/DNMT1 with compounds such as CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC.

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 Nox5e) 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. Nox5e 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 Nox5e, suggesting that Nox5e induced intracellular oxidative stress by an unknown mechanism. Functional studies showed that both Nox5β and Nox5e 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.

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.

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.

NADPH oxidase (Nox) variants Nox1, Nox2 and Nox4 have been implicated in the progression of liver fibrosis. However, the role of Nox5 is unknown, 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, these cells expressed a long (Nox5L) and a short (Nox5S) variant which were silenced with specific siRNAs for Nox5. Overexpression of Nox5L generated ROS in the presence of calcium, as judged by the production of extracellular hydrogen peroxide, L-012 luminescence and cytochrome c reduction, while Nox5S did not generated ROS under these conditions. In contrast, dihydroethidium oxidation was increased when either Nox5L or Nox5S were overexpressed. Functional studies revealed that both Nox5L and Nox5S stimulated the proliferation of LX-2 cells and the synthesis of type I collagen, while Nox5 siRNAs inhibited these effects. Interestingly, TGF-beta and angiotensin II induced Nox5, and silencing Nox5 reduced collagen production stimulated by TGF-beta. Collectively, these results suggest for the first time that Nox5 can play a relevant role in HSC proliferation and fibrogenesis.

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.

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.

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.

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.

S-adenosylmethionine (AdoMet) is an essential compound in cellular transmethylation reactions and a precursor of polyamine and glutathione synthesis in the liver. In liver injury, the synthesis of AdoMet is impaired and its availability limited. AdoMet administration attenuates experimental liver damage, improves survival of alcoholic patients with cirrhosis, and prevents experimental hepatocarcinogenesis. Apoptosis contributes to different liver injuries, many of which are protected by AdoMet. The mechanism of AdoMet's hepatoprotective and chemopreventive effects are largely unknown. The effect of AdoMet on okadaic acid (OA)-induced apoptosis was evaluated using primary cultures of rat hepatocytes and human hepatoma cell lines. AdoMet protected rat hepatocytes from OA-induced apoptosis dose dependently. It attenuated mitochondrial cytochrome c release, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. These effects were independent from AdoMet-dependent glutathione synthesis, and mimicked by 5'-methylthioadenosine (MTA), which is derived from AdoMet. Interestingly, AdoMet and MTA did not protect HuH7 cells from OA-induced apoptosis; conversely both compounds behaved as proapoptotic agents. AdoMet's proapoptotic effect was dose dependent and observed also in HepG2 cells. In conclusion, AdoMet exerts opposing effects on apoptosis in normal versus transformed hepatocytes that could be mediated through its conversion to MTA. These effects may participate in the hepatoprotective and chemopreventive properties of this safe and well-tolerated drug.

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.

Abstract —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 P P P P

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 α1(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 α1(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.

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 β (TGF-β), and this cytokine enhances collagen production by cultured HSC. However, the possible link between oxidative stress and the molecular mechanisms by which TGF-β induces collagen gene expression in HSC remains to be elucidated. To address this question, we investigated whether H2O2 is a mediator of TGF-β-elicited α1(I) collagen gene (col1a1) up-regulation. We demonstrated that TGF-β 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 α1(I) procollagen mRNA, catalase, an H2O2 enzyme scavenger, abrogated TGF-β-mediated col1a1 gene up-regulation. We transfected HSC with chimeric plasmids driven by different segments of the mouse col1a1 promoter and mapped acis-acting element (−370 to −344) essential for TGF-β responsiveness. We further showed that TGF-β induced the activation and binding of a C/EBPβ-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-β-mediated accumulation of H2O2 and the up-regulation of col1a1gene in HSC.

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.

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.

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.

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.

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.

OBJECTIVE: Previous studies have shown that as well as left ventricular hypertrophy, myocardial fibrosis develops early in rats with spontaneous hypertension (SHR). The present study was designed to investigate whether chronic treatment with the angiotensin II type 1 (AT1) receptor antagonist losartan modifies collagen type I metabolism and reverses left ventricular fibrosis in young SHR with left ventricular hypertrophy. DESIGN: The study was performed in 30-week-old normotensive Wistar-Kyoto (WKY) rats, untreated SHR and SHR treated with losartan (20 mg/mg per day, orally) for 14 weeks before they were killed. METHODS: Ventricular pro-alpha 1 (I) collagen messenger RNA was analyzed by Northern blot. Serum levels of the carboxy-terminal propeptide of procollagen type I (PIP) and the pyridoline cross-linked telopeptide domain of collagen type I (CITP) were determined by specific radioimmunoassays as markers of collagen type I synthesis and degradation, respectively. Collagen volume fraction was determined in the left ventricle by quantitative morphometry. RESULTS: Compared with WKY rats, SHR exhibited increased (P < 0.05) mean arterial pressure, pro-alpha 1 (I) collagen messenger RNA, PIP and left ventricular collagen volume fraction, and similar CITP values. After the treatment period, mean arterial pressure was higher (P < 0.05) in losartan-treated SHR than in WKY rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy and diminished (P < 0.05) values of mean arterial pressure, PIP and left ventricular collagen volume fraction. No changes in pro-alpha 1 (I) collagen messenger RNA and CITP values were observed with treatment in SHR. No significant differences in the left ventricular collagen volume fraction were observed between treated SHR with normal blood pressure and treated SHR with abnormally high blood pressure at the end of the treatment period. CONCLUSIONS: These results suggest that chronic AT1 blockade with losartan decreases the post-transcriptional synthesis of fibril-forming collagen type I molecules in young SHR. This effect may be involved in the ability of this drug to reverse left ventricular fibrosis in young rats with genetic hypertension. Apart from its antihypertensive action, other mechanisms may mediate the antifibrotic effect of losartan in this animal model.

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.

“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.