Hydrogen peroxide: A link between acetaldehyde-elicited α1(i) collagen gene up-regulation and oxidative stress in mouse hepatic stellate cells

Abstract

Ethanol induces liver fibrosis by several means that include, among others, the direct fibrogenic actions of acetaldehyde and the induction of an oxidative stress response. However, the mechanisms responsible for these activities, and the possible connections between oxidative stress and acetaldehyde-induced fibrosis are not well understood. In this communication we investigated the molecular mechanisms whereby acetaldehyde induces mouse ␣1(I) procollagen (col1a1) gene expression in cultured hepatic stellate cells. Transfection assays using reporter plasmids driven by different segments of the col1a1 promoter localized an acetaldehyde-responsive element (AcRE) between nucleotides ؊370 and ؊345. We also show that acetaldehyde enhances binding of a CCAAT/enhancer binding protein-␤ (C/EBP␤)-containing complex to this element, and that this effect is due, at least in part, to an increase in the concentration of nuclear p35C/EBP␤ protein. Although this element overlaps to a previously described transforming growth factor ␤1 (TGF-␤1)-responsive element, the stimulatory effect of acetaldehyde is not mediated through this cytokine, because addition of neutralizing anti-TGF-␤1 antibodies does not prevent acetaldehyde-elicited col1a1 up-regulation. On the other hand, this effect is blocked by the addition of catalase, an H 2 O 2 scavenger. Moreover, this ethanol metabolite stimulates production of H 2 O 2 in stellate cells. Thus, these results suggest that acetaldehyde-induced col1a1 up-regulation is mediated, at least in part, through H 2 O 2 . Altogether, these data suggest that the ؊370 to ؊344

Tumor necrosis factorα down-regulates expression of theα1(I) collagen gene in rat hepatic stellate cells through a p20C/EBPβ- and C/EBPδ-dependent mechanism

“…To delineate the cis-acting regulatory elements required for TNF-␣-mediated col1a1 down-regulation, we performed transient transfection assays using chimeric plasmids driven by different segments of the col1a1 promoter. 15,19 As shown in Fig. 2, expression of the Ϫ412col1a1CAT and Ϫ378col1a1CAT constructs was inhibited by 39.4% Ϯ 13.1% and 42.2% Ϯ 8.7%, respectively (P Ͻ .05) in TNF-␣-treated HSC.…”

“…This TaRE is located between nucleotides Ϫ378 and Ϫ345 of the col1a1 promoter and colocalizes with a TGF-␤ 1 -and acetaldehyde-responsive element described previously. 15,19 Several lines of evidence established the location of the TaRE in the col1a1 promoter and of some of the transcription factors that bind to it: 1) Functional studies revealed that deleting the Ϫ378 to Ϫ345 promoter region of the col1a1 gene renders a reporter vector unresponsive to TNF-␣ down-regulation. 2) EMSA studies revealed that TNF-␣ treatment induces binding of three distinct transcriptional complexes, TN1, TN2a, and TN2b, to the Ϫ412 to Ϫ345 region of the col1a1 promoter.…”

“…Reporter plasmids used in this study, driven by the sequences Ϫ412 to ϩ110 (Ϫ412col1a1CAT), Ϫ378 to ϩ110 (Ϫ378col1a1CAT), Ϫ345 to ϩ110 (Ϫ345col1a1CAT), and Ϫ220 to ϩ110 (Ϫ220col1a1CAT) of the mouse col1a1 gene have been described previously. 15,19 Integrity and orientation of these constructs were verified by DNA sequencing. HSC were cotransfected with 15 µg of each construct plus 3 µg of a luciferase control plasmid, pGL3-Basic (Promega, Madison, WI), using the calcium phosphate method.…”

Zinc supplementation attenuates ethanol- and acetaldehyde-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS) production and by influencing intracellular signaling

“…Of particular interest is the interaction between TGF- and ROS formation. In cultured HSCs, TGF- increases the production of hydrogen peroxide [32], which in turn induces the expression of 1(I) procolagen mRNA [33,34]. The direct profibrogenic effect of hydrogen peroxide has also been observed in co-cultures of HSCs with HepG2 cells overexpressing CYP2E1 [35].…”

“…At concentrations that have been detected in hepatic venous blood during alcohol consumption, acetaldehyde stimulates type I collagen synthesis and gene transcription in cultured rat and human HSCs through protein kinase C (PKC) activation [10]. Acetaldehyde has also been shown to increase NFB (p65) and its binding to the  2 (I) collagen promotor [11] by a mechanism dependent on the accumulation of H 2 O 2 [12,13,14]. CYP2E1 is an important source of reactive oxygen species (ROS) in alcohol-induced injury and fibrosis, generating superoxide (O 2 -) and hydrogen peroxide (H 2 O 2 ).…”