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 50μM, whereas it was not found at higher dosages such as 200μM. GCDC stimulated the expression of antiapoptotic Survivin, which also presented a biphasic response. The activation of nuclear factor-kappaB (NF-κB) corresponded with the up-regulation of Survivin. The inhibitor of NF-κB, 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-κB, 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-κB signaling pathway.
Wang
Kewei
K
Department of Pediatrics and Surgery/Section of Pediatric Surgery, Rush, University Medical Center, Chicago, IL 60612, USA. kewei_wang@rush.edu
Brems
John J
JJ
Gamelli
Richard L
RL
Holterman
Ai-Xuan
AX
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D005999:Glycochenodeoxycholic Acid / Q000633:toxicity*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D000077022:Survivin
C516989:Birc5 protein, rat; D008869:Microtubule-Associated Proteins; D016328:NF-kappa B; D000077022:Survivin; D005999:Glycochenodeoxycholic Acid