ON/OFF and Beyond - A Boolean Model of Apoptosis

Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-α, UV-B irradiation, interleukin-1β and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways.


NF-κB-DNA binding in response to FasL treatment in Jurkat T cells
Jurkat T cells were treated with 25ng/ml FasL for the indicated times and equal amounts of nuclear protein were subjected to EMSA. NF-κB-DNA binding is measured as PSL and expressed as fold increase of untreated cells. Means of two independent experiments with sd are shown.

IκB-α Western Blot in hepatocytes and Jurkat T cells
Hepatocytes were stimulated with TNF-α 25ng/ml, IL-1β 20ng/ml, FasL 50ng/ml, UV irradiation 300J/m² or 600 J/m² and Jurkat Tcells with FasL 50ng/ml, respectively and total extracts were subjected to western blotting targeting IκB-α and after stripping actin as a loading control. Note that cells treated with UV radiation 600 J/m² reveal higher IκB-α levels then the model predicted. After UV radiation cells had to be cultivated further before preparing total extracts so that initial NF-κB activity already induced protein newsynthesis of its negative feedback regulator IκB-α. NF-κB is still active in this state which is shown by EMSA in Fig.2.

IκB-α 39kDa
Actin 45kDa 4 cFLIP and cIAP2 mRNA levels in Jurkat T cells Jurkat T cells were treated with FasL 50ng/ml for 2h, total RNA was isolated and cIAP2 and cFLIP mRNA levels were determined by qRT-PCR. Means of two independent experiments with sd are shown.

XIAP Western Blot in hepatocytes and Jurkat T cells
Hepatocytes were stimulated with TNF-α 25ng/ml, IL-1β 20ng/ml, FasL 50ng/ml, UV irradiation 300J/m² or 600 J/m² and Jurkat T cells with FasL 50ng/ml, respectively, for the indicated times. Total extracts were subjected to western blotting targeting XIAP and after stripping actin as a loading control.

Hepatocytes
Jurkats T cells

Caspase-3 activity in response to FasL and UV irradiation in Jurkat T cells
Jurkats T cells were treated with 25ng/ml TNF-α, 50ng/ml FasL, UV irradiation 300 J/m² or 600 J/m² or 100nM insulin for the indicated times and caspase-3 activity was measured.

Bid Western Blot in hepatocytes and Jurkat T cells
Hepatocytes were stimulated with TNF-α 25ng/ml, IL-1β 20ng/ml, FasL 50ng/ml, UV irradiation 300J/m² or 600 J/m² and Jurkat T cells with FasL 50ng/ml, respectively, for the indicated times. Total extracts were subjected to western blotting targeting Bid and after stripping actin as a loading control.

Hepatocytes Jurkat T cells
Hepatocytes were stimulated with TNF-α 25ng/ml, IL-1β 20ng/ml, FasL 50ng/ml, UV irradiation 300J/m² or 600 J/m² and Jurkat T cells with FasL 50ng/ml respectively for the indicated times. Total extracts were subjected to western blotting targeting P-JNK and after stripping actin as a loading control.

Vitality [%]
Primary mouse hepatocytes were treated with 25ng/ml TNF-α, 50ng/ml FasL or 20ng/ml IL-1β for the indicated times and vitality was measured by MTT assay and referred to untreated cells. Means of at least 3 independent experiments are shown. Jurkats T cells were treated with 25ng/ml TNF-α, 50ng/ml FasL, UV irradiation 300 J/m² or 600 J/m² or 20ng/ml IL-1β for the indicated times and vitality was measured by MTT assay and referred to untreated cells. Means of at least 3 independent experiments are shown.