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S‐Adenosylhomocysteine Inhibits NF ‐κ B ‐Mediated Gene Expression in Hepatocytes and Confers Sensitivity to TNF Cytotoxicity
Author(s) -
Watson Walter H.,
Burke Tom J.,
Doll Mark A.,
McClain Craig J.
Publication year - 2014
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/acer.12315
Subject(s) - cytotoxicity , tumor necrosis factor alpha , chemistry , gene , gene expression , microbiology and biotechnology , biology , in vitro , biochemistry , immunology
Background Chronic alcohol exposure results in liver injury that is driven in part by inflammatory cytokines such as tumor necrosis factor‐α ( TNF ). Hepatocytes are normally resistant to the cytotoxic effects of TNF , but they become sensitized to TNF by chronic alcohol exposure. Recently, we reported that the decrease in the ratio of S ‐adenosylmethionine ( SAM ) to S ‐adenosylhomocysteine ( SAH ) that occurs with alcoholic liver injury renders hepatocytes sensitive to TNF cytotoxicity. The purpose of this study was to determine whether inhibition of the transcription factor nuclear factor‐kappaB ( NF ‐κ B ) contributed to TNF ‐induced cell death in hepatocytes with high levels of SAH . Methods Primary human hepatocytes or H ep G 2 cells were pre‐incubated with a combination of adenosine plus homocysteine to increase SAH levels. Following exposure to TNF , viability was determined by the MTT assay, and activation of the NF ‐κ B pathway was assessed by measuring degradation of cytosolic I κ B ‐α, phosphorylation and translocation of NF ‐κ B to the nucleus, and expression of NF ‐κ B ‐dependent genes. TNF ‐induced apoptotic signaling pathways were assessed by monitoring levels of the anti‐apoptotic protein, A 20, and cleavage products of the caspase‐8 substrate, RIP 1. Results NF ‐κ B ‐mediated gene expression was inhibited in cells with high SAH , despite the fact that TNF ‐induced degradation of the cytoplasmic inhibitor I κ B ‐α and accumulation of NF ‐κ B in the nucleus persisted for much longer. In contrast to control cells, the NF ‐κ B that accumulated in the nucleus of cells with high SAH levels was not phosphorylated at serine 536, a modification associated with activation of the transactivation potential of this transcription factor. The inhibition of transactivation by NF ‐κ B resulted in lower mRNA and protein levels of the anti‐apoptotic protein A 20 and increased cleavage of RIP 1. Conclusions High SAH levels inhibited NF ‐κ B ‐mediated gene expression and sensitized primary hepatocytes and H ep G 2 cells to the cytotoxic effects of TNF . It is likely that crosstalk with other transcription factors is perturbed under these conditions, resulting in still other changes in gene expression.