Person:
Cubero Palero, Francisco Javier

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First Name
Francisco Javier
Last Name
Cubero Palero
URI
https://hdl.handle.net/20.500.14352/82491
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Medicina
Department
Inmunología, Oftalmología y ORL
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2008 - 200912010 - 20121
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Author: Nieto, Natalia × Subject: 577 ×

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    Ethanol and Arachidonic Acid Synergize to Activate Kupffer Cells and Modulate the Fibrogenic Response via Tumor Necrosis Factor , Reduced Glutathione, and Transforming Growth Factor beta–Dependent Mechanisms
    (Hepatology., 2008) Cubero Palero, Francisco Javier; Nieto, Natalia
    Because of the contribution of ethanol and polyunsaturated fatty acids (PUFAs) to alcoholic liver disease, we investigated whether chronic ethanol administration and arachidonic acid (AA) could synergistically mediate Kupffer cell (KC) activation and modulate the stellate cell (HSC) fibrogenic response. Results: (1) the effects of ethanol and AA on KC and HSC were as follows: Cell proliferation, lipid peroxidation, H(2)O(2), O(2).(-), nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity, and tumor necrosis factor alpha (TNF-alpha) were higher in KC(ethanol) than in KC(control), and were enhanced by AA; HSC(ethanol) proliferated faster, increased collagen, and showed higher GSH than HSC(control), with modest effects by AA. (2) AA effects on the control co-culture: We previously reported the ability of KC to induce a pro-fibrogenic response in HSC via reactive oxygen species (ROS)-dependent mechanisms; we now show that AA further increases cell proliferation and collagen in the control co-culture. The latter was prevented by vitamin E (an antioxidant) and by diphenyleneiodonium (a NADPH oxidase inhibitor). (3) Ethanol effects on the co-cultures: Co-culture with KC(control) or KC(ethanol) induced HSC(control) and HSC(ethanol) proliferation; however, the pro-fibrogenic response in HSC(ethanol) was suppressed because of up-regulation of TNF-alpha and GSH, which was prevented by a TNF-alpha neutralizing antibody (Ab) and by L-buthionine-sulfoximine, a GSH-depleting agent. (4) Ethanol plus AA effects on the co-cultures: AA lowered TNF-alpha in the HSC(control) co-cultures, allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSC(ethanol) co-cultures by counteracting the up-regulation of TNF-alpha and GSH with a significant increase in GSSG and in pro-fibrogenic transforming growth factor beta (TGF-beta). Conclusion: These results unveil synergism between ethanol and AA to the mechanism whereby KC mediate ECM remodeling and suggest that even if chronic ethanol consumption sensitizes HSC to up-regulate anti-fibrogenic signals, their effects are blunted by a second "hit" such as AA.
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    Oxidative stress modulates KLF6Full and its splice variants
    (Alcoholism: Clinical and Experimental Research, 2012) Urtasun, Raquel; Cubero Palero, Francisco Javier; Nieto, Natalia
    Abstract Background: Induction of reactive oxygen species (ROS) is a central mechanism in alcohol hepatotoxicity. Krüppel-like factor 6 (KLF6), a transcription factor and a tumor-suppressor gene, is an early-responsive gene to injury; however, the effect of ROS and alcohol on KLF6 induction is unknown. The aim of this study is to investigate the contribution of 2 sources of ROS, cytochrome P450 2E1 (CYP2E1), NAD(P)H quinone oxidoreductase (NQO1), and alcohol on the modulation of KLF6(Full) expression, splicing to KLF6_V1 and KLF6_V2, and the effect on TNFα, a downstream target. Methods and results: Endogenous ROS production in CYP2E1-expressing HepG2 cells induced mRNA and protein expression of KLF6(Full) and its splice variants compared to control cells. Incubation with pro-oxidants such as arachidonic acid (AA), β-naphtoflavone, and H(2) O(2) further enhanced KLF6(Full) and its splice variants. The AA effects on KLF6(Full) and its splice forms were blocked by vitamin E-which prevents lipid peroxidation-and by diallylsulfide-a CYP2E1 inhibitor. Menadione and paraquat, 2 pro-oxidants metabolized via NQO1, induced KLF6(Full) mRNA in a thiol-dependent manner. Antioxidants and an NQO1 inhibitor suppressed the menadione-dependent increase in KLF6(Full) and its splice variants mRNA. Furthermore, primary hepatocytes and livers from chronic alcohol-fed rats, with elevated lipid peroxidation, H(2) O(2) and CYP2E1 but with low GSH, showed a ~2-fold increase in KLF6(Full) mRNA compared to controls. Inhibition of p38 phosphorylation further up-regulated the CYP2E1 and the AA effects on KLF6(Full) mRNA, whereas inhibition JNK and ERK1/2 phosphorylation decreased both. KLF6_V1 but not KLF6(Full) ablation markedly increased TNFα levels in macrophages; thus, TNFα emerges as a downstream target of KLF6_V1. Conclusions: The novel effect of ROS on modulating KLF6(Full) expression and its splice variants could play a relevant role in liver injury and in TNFα regulation.