Survivin signaling is regulated through nuclear factor-kappa B pathway during glycochenodeoxycholate-induced
Kewei Wang1, John J Brems, Richard L Gamelli
1Department of Pediatrics and Surgery/Section of Pediatric Surgery, Rush, University Medical Center, Chicago, IL 60612, USA. kewei_wang@rush.edu
Unlabelled:
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.
Insights
Low-dose glycochenodeoxycholate (GCDC) triggers a biphasic hepatocyte apoptosis response, mediated by Survivin and nuclear factor-kappaB (NF-κB) signaling. This biphasic pattern is dose-dependent and linked to antiapoptotic gene expression.
Area of Science:
- Hepatology
- Molecular Biology
- Cell Death Research
Background:
- Primary hepatocytes undergo apoptosis when exposed to glycochenodeoxycholate (GCDC).
- The precise signaling mechanisms driving GCDC-induced hepatocyte apoptosis are not fully understood.
- Investigating antiapoptotic gene roles is crucial for understanding GCDC-induced cell death.
Purpose of the Study:
- To elucidate the signaling pathways involved in GCDC-induced hepatocyte apoptosis.
- To investigate the role of antiapoptotic genes, specifically Survivin, in this process.
- To determine if GCDC-induced apoptosis exhibits a dose-dependent biphasic phenomenon.
Main Methods:
- Primary rat hepatocytes were cultured and treated with varying concentrations and time intervals of GCDC.
- Apoptosis was assessed using TUNEL assays, DNA fragmentation, and caspase activity measurements.
- Gene and protein expression (Survivin, NF-κB) were analyzed via RT-PCR, qRT-PCR, Western blotting, EMSA, and ChIP assays.
Main Results:
- GCDC induced a biphasic hepatocyte apoptosis response specifically at a 50μM dosage, absent at higher concentrations (200μM).
- Survivin, an antiapoptotic gene, exhibited a biphasic expression pattern correlating with GCDC dosage.
- Activation of nuclear factor-kappaB (NF-κB) paralleled Survivin up-regulation, and its inhibition abolished the biphasic response.
Conclusions:
- Low-dose GCDC induces a biphasic apoptosis response in hepatocytes.
- This biphasic response is regulated by Survivin expression.
- The NF-κB signaling pathway mediates Survivin's role in GCDC-induced hepatocyte apoptosis.
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