We investigated whether the fibrogenic actions of acetaldehyde, the immediate oxidation product of ethanol, are mediated via Wingless (WNT) and/or β-catenin pathways in human hepatic stellate cells (HSC). First, we show that both β-catenin small inhibitory RNA and a dominant negative-MYC expression vector markedly down-regulated the expressions of fibrogenic genes in freshly isolated HSC. We further show that acetaldehyde up-regulated platelet-derived growth factor receptor beta mRNA and protein expressions ranging from 4.0- to 7.2-fold (P<0.001). Acetaldehyde induced MYC and collagen type-1 alpha-2 mRNA and protein expressions were WNT independent because DKK1, an antagonist of the canonical WNT/β-catenin pathway, completely failed to block these inductions. Acetaldehyde increased phospho-glycogen synthase kinase-3 beta (GSK3B) protein by 31% (P<0.01), whereas phospho-β-catenin protein decreased by 50% (P ≤ 0.01). Significantly, in contrast to 43% (P<0.01) inhibition of β-catenin nuclear translocation in nucleoredoxin (NXN)-overexpressed HSC, acetaldehyde profoundly stimulated β-catenin nuclear translocation by 51%, (P<0.01). Acetaldehyde also increased the cellular reactive oxygen species level 2-fold (P<0.001) with a concomitant 2-fold (P<0.001) increase in 4-hydroxynonenal adducts. Conversely, there was a 44% decrease (P<0.001) in glutathione levels with a concomitant 76% (P<0.001) decrease in the level of NXN/ disheveled (DVL) complex. Based on these findings, we conclude that actions of acetaldehyde are mediated by a mechanism that inactivates NXN by releasing DVL, leading to the inactivation of GSK3B, and thereby blocks β-catenin phosphorylation and degradation. Thus, the stabilized β-catenin translocates to the nucleus where it up-regulates the fibrogenic pathway genes. This novel mechanism of action of acetaldehyde has the potential for therapeutic interventions in liver fibrosis induced by alcohol.
Keywords: 2′,7′- dichorofluorescein diacetate; 4-HNE; 4-hydroxynonenal; 5,5′-dithiobis(2-nitrobenzoic acid); ACH; ACTA1; Acetaldehyde; COL1A2; DCFDA; DMEM; DTNB; DVL; Dulbecco's modified Eagle's medium; FBS; GAPDH; GSH; GSK3B; HSC; Hepatic stellate cells; NXN; Nucleoredoxin; Oxidative stress; PDGFRB; PVDF; ROS; SDS-PAGE; TCF/LEFT; WNT; Wingless; acetaldehyde; alpha-smooth muscle actin; cell factor/lymphocyte enhancer factor; collagen type-1 alpha-2; disheveled; fetal bovine serum; glutathione; glyceraldehyde-3-phosphate dehydrogenase; glycogen synthase kinase-3 beta; hepatic stellate cells; nucleoredoxin; platelet-derived growth factor receptor beta; polyvinylidene difluoride; reactive oxygen species; sodium dodecyl sulfate polyacrylamide gel electrophoresis; β-Catenin.
Published by Elsevier Inc.