Project description:Mueller2015 - Hepatocyte proliferation, T160 phosphorylation of CDK2 This model is described in the article: T160-phosphorylated CDK2 defines threshold for HGF-dependent proliferation in primary hepatocytes. Mueller S, Huard J, Waldow K, Huang X, D'Alessandro LA, Bohl S, Börner K, Grimm D, Klamt S, Klingmüller U, Schilling M. Mol. Syst. Biol. 2015; 11(3): 795 Abstract: Liver regeneration is a tightly controlled process mainly achieved by proliferation of usually quiescent hepatocytes. The specific molecular mechanisms ensuring cell division only in response to proliferative signals such as hepatocyte growth factor (HGF) are not fully understood. Here, we combined quantitative time-resolved analysis of primary mouse hepatocyte proliferation at the single cell and at the population level with mathematical modeling. We showed that numerous G1/S transition components are activated upon hepatocyte isolation whereas DNA replication only occurs upon additional HGF stimulation. In response to HGF, Cyclin:CDK complex formation was increased, p21 rather than p27 was regulated, and Rb expression was enhanced. Quantification of protein levels at the restriction point showed an excess of CDK2 over CDK4 and limiting amounts of the transcription factor E2F-1. Analysis with our mathematical model revealed that T160 phosphorylation of CDK2 correlated best with growth factor-dependent proliferation, which we validated experimentally on both the population and the single cell level. In conclusion, we identified CDK2 phosphorylation as a gate-keeping mechanism to maintain hepatocyte quiescence in the absence of HGF. This model is hosted on BioModels Database and identified by: BIOMD0000000568. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Liver regeneration is characterized by a scheduled sequence of inner and intra-cellular signaling events. It starts with an initial inflammatory phase, followed by a period of rapidly proliferating hepatocytes and stopping abruptly when the liver mass is restored. The cytokines hepatocellular growth factor (HGF) and interleukin 6 (IL-6) play a pivotal role during this process with the former driving proliferation that is enhanced by the latter. While the individual importance of HGF and IL6 has been studied comprahensively the role of cross-talk in control of hepatic proliferation is jet largely unknown. To this end, we performed time-resolved transcriptional profiling of of murine hepatocytes stimulated with HGF and IL-6 indiviually as well as in combination. Thorough systematic investigation performing statistical analysis, mathematical formalization of cross-talk effects on the transcriptional level as well as gene-regulatory network inference revealed the transcriptional program of the cross-talk initiated by HGF and IL-6. Using the proliferation associated Hepcidin (Hamp) and Amphiregulin (Areg) as marker genes for liver regeneration we perform exthensive in-silico experiments with the inferred gene-regulatory network for the identification of the most important players in regulation of the proliferation process. Among other genes, this predicted chemokine (C-X-C motif) ligand 10 (Cxcl10) as an important factor in the temporal regulation of proliferation. These predictions were validated by independent in vitro expression data as well as independent in vivo literature data. While Cxcl10 is known to be involved in liver regeneration, our study extend its role towards its temporal orchestration. Cells were stimulated with either 40 ng/ml rmHGF (all R&D Systems) or 40 ng/ml rhIL-6 alone or in combination. Cells were left untreated as unstimulated control. RNA from three biological triplicates was extracted at 0, 0.5, 2, 4, 8, 16, 24 and 32 hours after stimulation using the RNeasy Mini Plus Kit (Qiagen, Hilden, Germany).

Project description:Liver regeneration is characterized by a scheduled sequence of inner and intra-cellular signaling events. It starts with an initial inflammatory phase, followed by a period of rapidly proliferating hepatocytes and stopping abruptly when the liver mass is restored. The cytokines hepatocellular growth factor (HGF) and interleukin 6 (IL-6) play a pivotal role during this process with the former driving proliferation that is enhanced by the latter. While the individual importance of HGF and IL6 has been studied comprahensively the role of cross-talk in control of hepatic proliferation is jet largely unknown. To this end, we performed time-resolved transcriptional profiling of of murine hepatocytes stimulated with HGF and IL-6 indiviually as well as in combination. Thorough systematic investigation performing statistical analysis, mathematical formalization of cross-talk effects on the transcriptional level as well as gene-regulatory network inference revealed the transcriptional program of the cross-talk initiated by HGF and IL-6. Using the proliferation associated Hepcidin (Hamp) and Amphiregulin (Areg) as marker genes for liver regeneration we perform exthensive in-silico experiments with the inferred gene-regulatory network for the identification of the most important players in regulation of the proliferation process. Among other genes, this predicted chemokine (C-X-C motif) ligand 10 (Cxcl10) as an important factor in the temporal regulation of proliferation. These predictions were validated by independent in vitro expression data as well as independent in vivo literature data. While Cxcl10 is known to be involved in liver regeneration, our study extend its role towards its temporal orchestration. Cells were stimulated with either 40 ng/ml rmHGF (all R&D Systems) and 40 ng/ml rhIL-6 alone or in combination. Cells were left untreated as unstimulated control. RNA was extracted at 1,2,3,4,5,6,7,8,9,10,12,24 hours. Cells were treated with IL6 alone for the first 4 hours and then additionally with HGF from hours 4-24. Controls were taken at -5,0,4,8,12,24 hours.

Project description:During embryogenesis, Hepatocyte Growth Factor (HGF) elicits a distinctive morphogenetic program, the invasive growth, by the activation of MET, whose aberrant activation in cancer drives metastatic progression. Aim of this work is to define and characterize the transcriptional signature of invasive growth, and to verify its activation in human cancers. Global expression profiling was carried out on mouse liver stem/progenitor cells (MLP-29) stimulated for different times, one, six and twenty-four hours, in vitro with HGF to define the invasive growth signature. Meta-analysis of human cancer microarray data was carried out to dissect the transcriptional modules of the invasive growth that are aberrantly activated during carcinogenesis of hepatocellular carcinoma. Differential expression analysis identified 2643 regulated genes by HGF, the invasive growth signature, subdivided in 11 gene expression clusters revealing waves of time coded transcriptional regulation. Those waves have been in-silico associated with the regulative role of the transcriptional unit of Rela/Nfkbia and Fos/Jun and biological features recapitulating the physiological invasive growth phenotype observed in cell line, such as cell motility and scattering, cellular proliferation and protection from apoptosis, cytoskeletal rearangement. Genomic meta-analysis on hepatocellular carcinoma identified of a core genes set (323 gene symbols), consistently regulated between MLP-29 and human tumors and significantly associated with cancer aggressiveness and metastasis p.val < 1*10-6, HR=5.404 CI= 2.570-11.365. The invasive growth signature recapitulates the physiopatological program driven by the stimulation of HGF in normal embryonic liver cells and its activity is observed in HCC as well as in several other tumors. This signature is associated with neoplastic progression and reliably predicts human HCC disease outcome, suggesting the involvement of the invasive growth and cancer in cancer progression. These results prompt the future application of anti-met target therapies in HCC and the application of the signature for both prognostic and predictive purposes.

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