RT Journal Article
A1 U. Klingmüller
A1 A. Bauer
A1 S. Bohl
A1 P.J. Nickel
A1 K. Breitkopf
A1 S. Dooley
A1 S. Zellmer
A1 C. Kern
A1 I. Merfort
A1 T. Sparna
A1 J. Donauer
A1 G. Walz
A1 M. Geyer
A1 C. Kreutz
A1 M. Hermes
A1 F. Götschel
A1 A. Hecht
A1 D. Walter
A1 L. Egger
A1 K. Neubert
A1 C. Borner
A1 M. Brulport
A1 W. Schormann
A1 C. Sauer
A1 F. Baumann
A1 R. Preiss
A1 S. MacNelly
A1 P. Godoy
A1 E. Wiercinska
A1 L. Ciuclan
A1 J. Edelmann
A1 K. Zeilinger
A1 M. Heinrich
A1 U.M. Zanger
A1 R. Gebhardt
A1 T. Maiwald
A1 R. Heinrich
A1 J. Timmer
A1 F. von Weizsäcker
A1 J.G. Hengstler

PB
T1 Primary mouse hepatocytes for systems biology approaches: a standardized in vitro system for modelling of signal transduction pathways
JN IEE Proceedings - Systems Biology
VO 153
IS 6
SP 433
OP 447
AB 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.
K1 critical hepatocellular functions
K1 ERK1/2 phosphorylation
K1 primary mouse hepatocytes
K1 cell regeneration
K1 JAK1 phosphorylation
K1 apoptosis
K1 cell differentiation
K1 STAT3 phosphorylation
K1 Wnt/β-catenin signalling pathways
K1 standardized in vitro system
K1 lactate secretion
K1 signal transduction pathways
K1 NF-κB
K1 transcriptional responses
K1 MAP kinase
K1 cell detoxification
K1 glutamine synthetase
K1 urea secretion
K1 gp130 phosphorylation
K1 luciferase reporter gene constructs
K1 GSK3α inhibition
K1 PI3 kinase
K1 JAK-STAT
K1 systems biology
K1 complex cellular networks
K1 TNF-like cytokine Fas ligand
K1 SMAD
K1 CYP3A4
K1 standardized cell culture
K1 starvation-associated stress
K1 glutathione-S-transferas
K1 TGFβ
K1 hepatocyte growth factor
K1 constant metabolic activities
K1 Akt/PKB phosphorylation
DO https://doi.org/10.1049/ip-syb:20050067
UL https://digital-library.theiet.org/;jsessionid=1bmaqcb4i4ts5.x-iet-live-01content/journals/10.1049/ip-syb_20050067
LA English
SN 1741-2471
YR 2006
OL EN