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1.
Biochim Biophys Acta ; 1863(8): 2115-23, 2016 08.
Article in English | MEDLINE | ID: mdl-27155082

ABSTRACT

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.


Subject(s)
Brefeldin A/pharmacology , Collagen Type I/biosynthesis , Endoplasmic Reticulum Stress/drug effects , Endoribonucleases/physiology , Hepatic Stellate Cells/drug effects , MAP Kinase Signaling System/drug effects , Multienzyme Complexes/physiology , Protein Serine-Threonine Kinases/physiology , Smad3 Protein/physiology , Unfolded Protein Response/drug effects , p38 Mitogen-Activated Protein Kinases/physiology , Animals , Cell Line , Collagen Type I/genetics , Endoplasmic Reticulum Stress/physiology , Endoribonucleases/antagonists & inhibitors , Fibrosis , Gene Expression Regulation/drug effects , Hepatic Stellate Cells/metabolism , Imidazoles/pharmacology , Multienzyme Complexes/antagonists & inhibitors , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Unfolded Protein Response/physiology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
2.
Free Radic Biol Med ; 84: 263-278, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25841776

ABSTRACT

Excess of saturated free fatty acids, such as palmitic acid (PA), in hepatocytes has been implicated in nonalcoholic fatty liver disease. α-Lipoic acid (LA) is an antioxidant that protects against oxidative stress conditions. We have investigated the effects of LA in the early activation of oxidative and endoplasmic reticulum stress, lipid accumulation, and Nrf2-mediated antioxidant defenses in hepatocytes treated with PA or in rats fed a high-fat diet. In primary human hepatocytes, a lipotoxic concentration of PA triggered endoplasmic reticulum stress, induced the apoptotic transcription factor CHOP, and increased the percentage of apoptotic cells. Cotreatment with LA prevented these effects. Similar results were found in mouse hepatocytes in which LA attenuated PA-mediated activation of caspase 3 and reduced lipid accumulation by decreasing PA uptake and increasing fatty acid oxidation and lipophagy, thereby preventing lipoapoptosis. Moreover, LA augmented the proliferation capacity of hepatocytes after PA challenge. Antioxidant effects of LA ameliorated reactive oxygen species production and endoplasmic reticulum stress and protected against mitochondrial apoptosis in hepatocytes treated with PA. Cotreatment with PA and LA induced an early nuclear translocation of Nrf2 and activated antioxidant enzymes, whereas reduction of Nrf2 by siRNA abolished the benefit of LA on PA-induced lipoapoptosis. Importantly, posttreatment with LA reversed the established damage induced by PA in hepatocytes, as well as preventing obesity-induced oxidative stress and lipoapoptosis in rat liver. In conclusion, our work has revealed that in hepatocytes, Nrf2 is an essential early player in the rescue of oxidative stress by LA leading to protection against PA-mediated lipoapoptosis.


Subject(s)
Antioxidants/pharmacology , Apoptosis , Hepatocytes/physiology , NF-E2-Related Factor 2/physiology , Thioctic Acid/pharmacology , Active Transport, Cell Nucleus , Animals , Antioxidant Response Elements , Cells, Cultured , Diet, High-Fat/adverse effects , Humans , Male , Membrane Potential, Mitochondrial , Non-alcoholic Fatty Liver Disease/metabolism , Oxidative Stress , Palmitic Acid/pharmacology , Rats, Wistar , Reactive Oxygen Species/metabolism
3.
J Cell Physiol ; 230(3): 546-53, 2015 Mar.
Article in English | MEDLINE | ID: mdl-24976518

ABSTRACT

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.


Subject(s)
Extracellular Matrix Proteins/metabolism , Fibronectins/metabolism , Liver Cirrhosis/metabolism , Peptides/metabolism , Apoptosis/genetics , Caspase 3/metabolism , Cell Differentiation/genetics , Cell Line , Cell Proliferation/genetics , DNA Fragmentation , Fibronectins/genetics , Gene Expression Regulation , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Humans , Liver Cirrhosis/pathology , Matrix Metalloproteinase 9/metabolism , bcl-2-Associated X Protein/metabolism
4.
Exp Dermatol ; 22(2): 125-30, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23362871

ABSTRACT

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis produced as a consequence of desmoglein (Dsg) and non-Dsg autoantibodies binding to several targeting molecules localized on the membrane of keratinocytes. Nitric oxide (NO) may exert a pathogenic function in several immunological processes. We have previously demonstrated that neural nitric oxide synthase (nNOS) plays part in PV acantholysis. Also, our group has described a relevant role for HER [human epidermal growth factor receptor (EGFR) related] isoforms and several kinases such as Src (Rous sarcoma), mammalian target of rapamycin (mTOR) and focal adhesion kinase (FAK), as well as caspases in PV development. Using a passive transfer mouse model of PV, we aimed to investigate the relationship between the increase in nNOS and EGFR, Src, mTOR and FAK kinase upregulation observed in PV lesions. Our results revealed a new function for nNOS, which contributes to EGFR-mediated PV acantholysis through the upregulation of Src, mTOR and FAK. In addition, we found that nNOS participates actively in PV at least in part by increasing caspase-9 and caspase-3 activities. These findings underline the important issue that in PV acantholysis, caspase activation is a nNOS-linked process downstream of Src, mTOR and FAK kinase upregulation.


Subject(s)
Acantholysis/enzymology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Nitric Oxide Synthase Type I/metabolism , Pemphigus/enzymology , TOR Serine-Threonine Kinases/metabolism , src-Family Kinases/metabolism , Animals , Biopsy , Caspase 3/metabolism , Caspase 9/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Humans , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred C57BL , Time Factors
5.
Exp Dermatol ; 21(4): 254-9, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22320676

ABSTRACT

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis and by autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mammalian target of rapamycin (mTOR), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate whether upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor (FI), the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FI before PV-IgG injection prevented the changes in both Bax and Bcl-2 expression and caspase-9 and caspase-3 activities induced by PV-IgG. Finally, FI reduced the expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel role of phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Pemphigus/prevention & control , Acantholysis/prevention & control , Animals , Animals, Newborn , Blister/prevention & control , Caspase Inhibitors , Disease Models, Animal , ErbB Receptors/antagonists & inhibitors , Focal Adhesion Protein-Tyrosine Kinases/immunology , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Immunization, Passive , Immunoglobulin G/administration & dosage , Mice , Mice, Inbred C57BL , Models, Biological , Pemphigus/enzymology , Pemphigus/immunology , Phosphorylation , Protein Kinase Inhibitors/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Tyrosine/chemistry , src-Family Kinases/antagonists & inhibitors
6.
Biochem Pharmacol ; 81(3): 451-8, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-21056031

ABSTRACT

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.


Subject(s)
Apoptosis , Hepatic Stellate Cells/metabolism , Molsidomine/analogs & derivatives , Nitric Oxide/metabolism , Peroxynitrous Acid/metabolism , Animals , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line , DNA Fragmentation , Humans , Inflammation/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Liver Cirrhosis/metabolism , Molsidomine/metabolism , Molsidomine/pharmacology , Protein-Tyrosine Kinases/metabolism , Rats , Signal Transduction , Syk Kinase , Tyrosine/metabolism , src-Family Kinases/metabolism
7.
Apoptosis ; 13(11): 1356-67, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18819005

ABSTRACT

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.


Subject(s)
Apoptosis , Monocytes/metabolism , Nitric Oxide/metabolism , Caspase 3/metabolism , Caspase 9/metabolism , DNA Fragmentation , Humans , Inflammation , Leukocytes, Mononuclear/metabolism , MAP Kinase Kinase 4/metabolism , MAP Kinase Signaling System , Macrophages/metabolism , Models, Biological , Nitrogen/chemistry , Signal Transduction
8.
Biochim Biophys Acta ; 1773(11): 1681-8, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17707924

ABSTRACT

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.


Subject(s)
Collagen Type I/biosynthesis , Hepatocytes/cytology , Hepatocytes/drug effects , Leucine/pharmacology , Protein Biosynthesis/drug effects , Reactive Oxygen Species/pharmacology , Acetophenones/pharmacology , Animals , Carrier Proteins/metabolism , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Glutathione/metabolism , Hepatocytes/enzymology , Intracellular Signaling Peptides and Proteins , Models, Biological , Oxidative Stress/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Phosphorylation/drug effects , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats , Reactive Oxygen Species/metabolism , Receptor, Insulin/metabolism , Rotenone/pharmacology , Signal Transduction/drug effects , Superoxides/metabolism , TOR Serine-Threonine Kinases
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