@ARTICLE{ :/content/journals/10.1049/ip-syb_20050067, author = {U. Klingmüller}, author = {A. Bauer}, author = {S. Bohl}, author = {P.J. Nickel}, author = {K. Breitkopf}, author = {S. Dooley}, author = {S. Zellmer}, author = {C. Kern}, author = {I. Merfort}, author = {T. Sparna}, author = {J. Donauer}, author = {G. Walz}, author = {M. Geyer}, author = {C. Kreutz}, author = {M. Hermes}, author = {F. Götschel}, author = {A. Hecht}, author = {D. Walter}, author = {L. Egger}, author = {K. Neubert}, author = {C. Borner}, author = {M. Brulport}, author = {W. Schormann}, author = {C. Sauer}, author = {F. Baumann}, author = {R. Preiss}, author = {S. MacNelly}, author = {P. Godoy}, author = {E. Wiercinska}, author = {L. Ciuclan}, author = {J. Edelmann}, author = {K. Zeilinger}, author = {M. Heinrich}, author = {U.M. Zanger}, author = {R. Gebhardt}, author = {T. Maiwald}, author = {R. Heinrich}, author = {J. Timmer}, author = {F. von Weizsäcker}, author = {J.G. Hengstler}, keywords = {critical hepatocellular functions;ERK1/2 phosphorylation;primary mouse hepatocytes;cell regeneration;JAK1 phosphorylation;apoptosis;cell differentiation;STAT3 phosphorylation;Wnt/β-catenin signalling pathways;standardized in vitro system;lactate secretion;signal transduction pathways;NF-κB;transcriptional responses;MAP kinase;cell detoxification;glutamine synthetase;urea secretion;gp130 phosphorylation;luciferase reporter gene constructs;GSK3α inhibition;PI3 kinase;JAK-STAT;systems biology;complex cellular networks;TNF-like cytokine Fas ligand;SMAD;CYP3A4;standardized cell culture;starvation-associated stress;glutathione-S-transferas;TGFβ;hepatocyte growth factor;constant metabolic activities;Akt/PKB phosphorylation;}, ISSN = {1741-2471}, language = {English}, abstract = {Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-κB and Wnt/β-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFβ was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent β-catenin was monitored in response to the inhibition of GSK3β. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFβ-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.}, title = {Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways}, journal = {IEE Proceedings - Systems Biology}, issue = {6}, volume = {153}, year = {2006}, month = {November}, pages = {433-447(14)}, publisher ={}, copyright = {© The Institution of Engineering and Technology}, url = {https://digital-library.theiet.org/;jsessionid=65jhthesbq3mu.x-iet-live-01content/journals/10.1049/ip-syb_20050067} }