Dissection TNFα/NF-κB signalling in murine hepatocytes–a model for the priming phase of liver regeneration

During liver regeneration, initiation and termination of hepatocyte proliferation is tightly regulated by a defined subset of secreted factors. The cytokine tumor necrosis factor (TNFα) is specifically secreted by Kupffer cells very early in the priming phase of regeneration and promotes DNA synthesis and cell cycle progression of quiescent hepatocytes by activation of the NF-κB pathway. However, little is known about the dynamic behaviour, structure of feedback mechanisms, and termination of this pathway in hepatocytes. In order to define the molecular mechanisms regulating early TNFα-dependent steps in liver regeneration, we stimulated isolated primary murine hepatocytes with TNFα (10ng/ml) and analyzed the time-resolved expression and activation/phosphorylation of pathway activators (IKKβ), transcription factors (p65/RelA), and feedback components (IkBα and A20) by quantitative western immunoblotting (9 time-points in 12 independent experiments). In addition, expression kinetics of typical NF-κB target transcripts (A20, IkBα) were measured by quantitative real-time PCR. Experimental data at the cell population level revealed rapid and strong pathway activation 10 minutes after TNFα administration, followed by increased expression of negative feedback regulators. In addition to the initial peak, a second activation of was detectable after 40 minutes. In order to define highly dynamic interplay of TNFα/NF-κB pathway constituents in hepatocytes, we developed a (ODE)-based mathematical model. First results of the parameter estimation showed that this model can successfully capture the initial dynamic phase of the pathway. Here we present a simple mathematical model for TNFα/NF-κB signalling specific to murine hepatocytes. Integration of further parameters critically involved in the initiation and termination of regenerative processes, will unravel emergent properties contributing to regenerative capacity of hepatocytes and organ size control.

Literatur: Turner DA., Physiological levels of TNF-alpha stimulation induce stochastic dynamics of NF-kB responses in single living cells. J Cell Sci 2010 Aug 15;123(Pt 16):2834-43, 2010 U. Klingmuller at. al. Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathway- IEE Proc.-Syst. Biol., Vol. 153, No. 6, November 2006 Hoops S., Sahle S., Gauges R., Lee C., Pahle J., Simus N., Singhal M., Xu L., Mendes P. and Kummer U. COPASI — a COmplex PAthway SImulator. Bioinformatics 22, 3067-74., 2006 Funahashi, A., Tanimura, N., Morohashi, M., and Kitano, H., CellDesigner: a process diagram editor for gene-regulatory and biochemical networks, BIOSILICO, 1:159-162, 2003

Liver Regeneration - NF-kB signalling - ODE model - Primary murine heptocytes