Survivin signaling is regulated through nuclear factor-kappa B pathway during glycochenodeoxycholate-induced hepatocyte apoptosis

Hepatocytes in primary culture undergo apoptosis upon exposure to glycochenodeoxycholate (GCDC). The signaling mechanisms of GCDC-induced apoptosis remain unclear. To investigate the role of antiapoptotic genes, we compared apoptotic response in primary hepatocytes following GCDC treatment. The hepatocytes from adult Sprague-Dawley rats were cultured in collagen-coated dishes and treated with GCDC in varying concentrations, or the same concentration at different time intervals. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, DNA fragmentation assay, and caspase assays. Expression of apoptosis-related genes and proteins was evaluated by RT-PCR, quantitative real-time PCR (qRT-PCR), and Western blotting, respectively. The DNA-binding property of a nuclear protein was assessed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. An interesting result was that GCDC caused hepatocyte apoptosis to display a biphasic phenomenon at a dosage of 50 mu M, whereas it was not found at higher dosages such as 200 mu M. GCDC stimulated the expression of antiapoptotic Survivin, which also presented a biphasic response. The activation of nuclear factor-kappaB (NF-kappa B) corresponded with the up-regulation of Survivin. The inhibitor of NF-kappa B, BAY 11-7082, suppressed the expression of Survivin and simultaneously eliminated the biphasic response. The expression of Survivin was transcriptionally mediated by the activation of NF-kappa B, as shown by EMSA and ChIP assay. Conclusions: These results demonstrated that a low dosage of GCDC induced the hepatocyte apoptosis to exhibit the biphasic response, which was regulated by the expression of Survivin through NF-kappa B signaling pathway. (C) 2010 Elsevier B.V. All rights reserved.