F. Pinna, S. Sahle, K. Beuke, M. B. Bissinger, S. Tuncay, L. D'Alessandro, R. Gauges, J. Timmer, Andreas Raue, U. Klingmüller, P. Schirmacher, U. Kummer, K. Breuhahn
- The cytokine tumor necrosis factor (TNF)-α is a key factor in the priming phase of liver regeneration since it activates the NF-κB signalling pathway, which primes hepatocyte for proliferation. In order to understand how TNFα-dependend NF-κB activation contributes to a cellular response in parenchymal liver cells, we aimed to generate a hepatocyte-specific mathematical model based on quantitative and time-resolved data of NF-κB pathway constituents after TNFα administration.
For model parametrization, freshly isolated primary murine hepatocytes were treated with TNFα (10ng/ml) and protein levels (p65, IκBα), phosphorylation (phospho-p65 [Ser536], phospho-IκBα), and transcript levels of NF-κB target genes (e.g., IκBα) were quantitatively defined for up to 4 hours. In addition, cycloheximide treatment of hepatocytes revealed high stability of p65, while IκBα showed 40% degradation within 6 hours. Together with an additional nuclear phosphorylation step of p65 (e.g., at Ser276), theseThe cytokine tumor necrosis factor (TNF)-α is a key factor in the priming phase of liver regeneration since it activates the NF-κB signalling pathway, which primes hepatocyte for proliferation. In order to understand how TNFα-dependend NF-κB activation contributes to a cellular response in parenchymal liver cells, we aimed to generate a hepatocyte-specific mathematical model based on quantitative and time-resolved data of NF-κB pathway constituents after TNFα administration.
For model parametrization, freshly isolated primary murine hepatocytes were treated with TNFα (10ng/ml) and protein levels (p65, IκBα), phosphorylation (phospho-p65 [Ser536], phospho-IκBα), and transcript levels of NF-κB target genes (e.g., IκBα) were quantitatively defined for up to 4 hours. In addition, cycloheximide treatment of hepatocytes revealed high stability of p65, while IκBα showed 40% degradation within 6 hours. Together with an additional nuclear phosphorylation step of p65 (e.g., at Ser276), these data were used to generate an extended TNFα/NF-κB model, which was able to quantitatively fit all experimental data. Model validated was done using p65:IκBα co-immunoprecipitation analysis to define complexed p65 levels over time. Lastly, we computationally analyzed the impact of protein turnover on signal amplitude and integrated signal response (signal strength). The system highly depends on p65 translation and degradation as well as on IκBα transcription and mRNA degradation, while changed total p65 levels didn't significantly affect the system.
Although, NF-κB signalling has been studied in immortalized cells, no computational modelling approaches for hepatocytes or primary cells have been published, yet. Here we present an extended mathematical model for TNFα/NF-κB signalling in primary hepatocytes, suggesting the relevance of p65:IκBα complex formation and protein turnover of NF-κB pathway constituents for the dynamic behaviour of the system in processes such as liver regeneration.…
