S-Adenosylhomocysteine Inhibits NF-κB-Mediated Gene Expression in Hepatocytes and Confers Sensitivity to TNF Cytotoxicity
Article first published online: 13 NOV 2013
Copyright © 2013 by the Research Society on Alcoholism
Alcoholism: Clinical and Experimental Research
Volume 38, Issue 4, pages 889–896, April 2014
How to Cite
Watson, W. H., Burke, T. J., Doll, M. A. and McClain, C. J. (2014), S-Adenosylhomocysteine Inhibits NF-κB-Mediated Gene Expression in Hepatocytes and Confers Sensitivity to TNF Cytotoxicity. Alcoholism: Clinical and Experimental Research, 38: 889–896. doi: 10.1111/acer.12315
- Issue published online: 9 APR 2014
- Article first published online: 13 NOV 2013
- Manuscript Accepted: 30 SEP 2013
- Manuscript Received: 13 FEB 2013
- NIH Grants. Grant Numbers: R21 ES021311, R01 AA018869, P01 AA017103, R01 AA018016, R37 AA010762, P30 AA019360, RC2AA019385, R01 AA015970, R01 DK071765
- Department of Veterans Affairs
- Tumor Necrosis Factor-alpha;
- Alcoholic Liver Disease;
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-κB) contributed to TNF-induced cell death in hepatocytes with high levels of SAH.
Primary human hepatocytes or HepG2 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-κB pathway was assessed by measuring degradation of cytosolic IκB-α, phosphorylation and translocation of NF-κB to the nucleus, and expression of NF-κB-dependent genes. TNF-induced apoptotic signaling pathways were assessed by monitoring levels of the anti-apoptotic protein, A20, and cleavage products of the caspase-8 substrate, RIP1.
NF-κB-mediated gene expression was inhibited in cells with high SAH, despite the fact that TNF-induced degradation of the cytoplasmic inhibitor IκB-α and accumulation of NF-κB in the nucleus persisted for much longer. In contrast to control cells, the NF-κB 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-κB resulted in lower mRNA and protein levels of the anti-apoptotic protein A20 and increased cleavage of RIP1.
High SAH levels inhibited NF-κB-mediated gene expression and sensitized primary hepatocytes and HepG2 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.