Ethanol and fish oil induce NFκB transactivation of the collagen α2(I) promoter through lipid peroxidation–driven activation of the PKC-PI3K-Akt pathway

Abstract

To analyze whether fish oil, as a source of polyunsaturated fatty acids from the n-3 series, could synergize with ethanol to promote collagen I upregulation in vivo, collagen alpha2(I) promoter-betaGal (COL1A2-betaGal) transgenic mice were fed a diet enriched in fish oil in the presence of ethanol (ethanol group) or dextrose (control group). Ethanol-fed mice showed mild steatosis, increased alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonsterified fatty acids, and plasma alcohol levels along with elevated cytochrome P450 2E1 activity, lipid peroxidation end products, and low glutathione (GSH) levels, which suggested enhanced oxidant stress and liver injury. Increased transactivation of the COL1A2 promoter assessed by betaGal activity was shown in vivo and by transfection with deletion constructs for the collagen alpha1(I) promoter (COL1A1) and COL1A2 promoters in vitro. Transcriptional regulation of both COL1A1 and COL1A2 promoters was validated by nuclear in vitro transcription run-on, northern blot analysis, and quantitative polymerase chain reaction, which was followed by the subsequent upregulation of collagen I protein with no changes in matrix metalloproteinase 13 (MMP 13). To further analyze the potential mechanism for collagen I upregulation, an in vitro coculture model was designed with primary stellate cells seeded on the bottom plate of a Boyden chamber and the rest of the liver cells plated on a cell culture insert, and fish oil or fish oil plus ethanol were added. The combination of fish oil plus ethanol increased nuclear factor kappaB binding to the COL1A2 promoter both in vivo and in the cocultures and also resulted in increased phosphorylation of protein kinase C, activation of PI3 kinase, and phosphorylation of Akt. The in vitro addition of vitamin E prevented such activation and collagen I increase. Furthermore, inhibitors of all 3 kinases blocked the increase in collagen I and NFkappaB binding to the COL1A2 promoter; the latter was also prevented by vitamin E. These results suggest that fish oil (mainly n-3 polyunsaturated fatty acids [PUFAs]) can synergize with ethanol to induce collagen I, transactivating the COL1A2 promoter through a lipid peroxidation-PKC-PI3K-Akt-NFkappaB-driven mechanism in the absence of overt steatosis and inflammation.