A20/TNFAIP3 Discriminates Tumor Necrosis Factor (TNF)-Induced NF-κB from JNK Pathway Activation in Hepatocytes.

Federico Pinna,Ursula Kummer,Peter Schirmacher,Nicolas Huber,Katharina Beuke,Michaela Bissinger,Sven Sahle,Thomas Longerich,Kai Breuhahn

doi:10.3389/fphys.2017.00610

Abstract

In the liver tumor necrosis factor (TNF)-induced signaling critically regulates the immune response of non-parenchymal cells as well as proliferation and apoptosis of hepatocytes via activation of the NF-κB and JNK pathways. Especially, the induction of negative feedback regulators, such as IκBα and A20 is responsible for the dynamic and time-restricted response of these important pathways. However, the precise mechanisms responsible for different TNF-induced phenotypes under physiological stimulation conditions are not completely understood so far. In addition, it is not known if varying TNF concentrations may differentially affect the desensitization properties of both pathways. By using computational modeling, we first showed that TNF-induced activation and downstream signaling is qualitatively comparable between primary mouse hepatocytes and immortalized hepatocellular carcinoma (HCC) cells. In order to define physiologically relevant TNF levels, which allow for an adjustable and dynamic NF-κB/JNK pathway response in parenchymal liver cells, a range of cytokine concentrations was defined that led to gradual pathway responses in HCC cells (1–5 ng/ml). Repeated stimulations with low (1 ng/ml), medium (2.5 ng/ml) and high (5 ng/ml) TNF amounts demonstrated that JNK signaling was still active at cytokine concentrations, which led to dampened NF-κB signaling illustrating differential pathway responsiveness depending on TNF input dynamics. SiRNA-mediated inhibition of the negative feedback regulator A20 (syn. TNFAIP3) or its overexpression did not significantly affect the NF-κB response. In contrast, A20 silencing increased the JNK response, while its overexpression dampened JNK phosphorylation. In addition, the A20 knockdown sensitized hepatocellular cells to TNF-induced cleavage and activity of the effector caspase-3. In conclusion, a mathematical model-based approach shows that the TNF-induced pathway responses are qualitatively comparable in primary and immortalized mouse hepatocytes. The cytokine amount defines the pathway responsiveness under repeated treatment conditions with NF-κB signaling being dampened ‘earlier’ than JNK. A20 appears to be the molecular switch discriminating between NF-κB and JNK signaling when stimulating with varying physiological cytokine concentrations.

Highlights

The role of the cytokine tumor necrosis factor (TNF) in the liver has been investigated intensively
We recently developed a computational ordinary differential equation (ODE) model for TNF-induced NF-κB activation based on quantitative and time-resolved experimental data derived from primary murine hepatocytes (Pinna et al, 2012)
Analyzing the expression of selected NF-κB pathway constituents revealed that both tested hepatocellular carcinoma (HCC) (Hepa1-6 and Hep56) cell lines expressed higher p65 and IκBα protein amounts than primary hepatocytes (Figure 1A)

Summary

Introduction

The role of the cytokine tumor necrosis factor (TNF) in the liver has been investigated intensively. Biological functions of TNF and subsequent activation of the NF-κB signaling pathway are associated with inflammatory processes, hepatocyte proliferation in response to acute or chronic liver damage, as well as tumorigenesis (DiDonato et al, 2012). The hepatocellular response upon TNF stimulation is based on a sequence of post-translational modifications occurring downstream of the TNF receptor (TNFRI) with several proteins organized in the signalosome (Ruland, 2011). Binding of TNF to TNFRI sequentially activates the mitogen-activated protein kinase kinase kinase 7 (MAP3K7/MEKK7, syn: TAK1) followed by the recruitment of receptor-interacting serine/threonineprotein kinase 1 (RIPK1) and additional scaffold proteins (e.g., TAB, TRAF) to the signalosome. Direct transcriptional NF-κB targets, such as TNF α-induced protein 3 (TNFAIP3, synonym: A20) or nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) are essential for the fast and efficient shut-down of this pathway in terms of a negative feedback regulation (Ruland, 2011)

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