Modeling the time resolved transcriptional signature of time sequential stimulation with HGF and IL-6 in hepatocyte proliferation
ABSTRACT: 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: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:Mueller2015 - Hepatocyte proliferation, T160
phosphorylation of CDK2
This model is described in the article:
defines threshold for HGF-dependent proliferation in primary
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
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.
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Project description:c-Met is a receptor tyrosine kinase for hepatocyte growth factor (HGF). Previous work has established that HGF plays a pivotal role in regulating the onset of S phase and DNA replication following partial hepatectomy. In this study, we used c-Met conditional knockout mice (MetLivKO) in which c-met gene is inactivated in postnatal hepatocytes by Alb-Cre recombinase to directly address the net biological outcome of c-Met on liver regeneration. The priming events appear to be intact in MetLivKO livers. Up-regulation of stress response (e.g MAFK, IKBZ, SOCS3) and early growth response (e.g. MYC, DUSP1 and 6) genes as judged by microarray profiling was similar in c-Met deficient regenerating livers as compared to Alb-Cre controls. This was consistent with an early induction of NF-kB, STAT3, and MAPK/ERK. Nevertheless, in the absence of c-Met signaling in the hepatocytes, ERK phosporylation rapidly declined although it remained high in Cre-Ctrl livers, and MetLivKO mice displayed impaired liver regeneration as determined by a decrease in BrdU incorporation and a delay in timely progression into mitosis. Upstream signaling pathways involved in the blockage of G2-M transition included lack of EGR1 transcription factor induction, and inability to up-regulate the levels of cdc2, aurora B and Mad2 followed by defective histone 3 phosphorylation and lag in chromatin condensation. However, after a delayed passage through G2 phase, c-Met deficient cells eventually entered mitosis. In culture, EGF treatment increased proliferation of MetLivKO hepatocytes and restored expression levels of cell cycle regulators aurora B and Mad2 albeit to a lesser degree as compared to Cre-Ctrl hepatocytes. In conclusion, our results assign a novel function for HGF/c-Met signaling in regulation of G2/M transition during liver regeneration and implicate EGR1 as a potential G2/M target of HGF/c-Met pathway. Overall design: Multiple repeats were done of eight timepoint conditions (0, 0.5, 2, 12, 24, 36, 42, and 48 hours) using wild type and knock-out mice. All samples were reverse-fluor labelled. A pooled sample of wild type liver was used as reference.
Project description:Exercise stimulates the release of a plethora of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognising that many circulating proteins might be packaged in extracellular vesicles (EV), we employed quantitative proteomic techniques to characterise the exercise-induced secretion of EV contained proteins. We observed an increase in circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small vesicles, in human arterial blood samples. Pathway analyses revealed significant enrichments in a multitude of biological processes and signalling pathways. Pulse chase and intravital imaging experiments suggest EV-mediated muscle-liver cross talk during exercise. Moreover, by employing arterio-venous balance studies across the contracting human limb, we identified several novel candidate myokines. These data offer a new paradigm by which tissue cross-talk during exercise can exert systemic biological effects
Project description:Arid1a is the subunit of SWI/SNF complex, which was reported to guide SWI/SNF to DNA. Here, we found that loss of Arid1a in the liver and other adult tissues results in improved organ regeneration. Within SWI/SNF complexes, Arid1a physically interacts with C/ebpα, a hepatocyte transcription factor that drives maturation and limits proliferation. Genome-wide analysis showed that loss of Arid1a reduces the recruitment and activity of C/ebpα on target promoters, resulting in expression programs that favor regeneration and cellular fitness during injury. Arid1a binding is enriched in promoters near transcriptional start sites (TSSs), and C/ebpα binds at precisely the same positions, indicating that Arid1a facilitates C/ebpα binding across the genome. Perfuse and isolate primary hepatocytes from mice livers, analysis of genomic occupancy of C/ebpα and H3K4me2 in hepatocytes from Arid1a WT and Arid1a liver specific KO mice by ChIP-seq. Analysis of genomic occupancy of Arid1a in the hepatocytes from V5-Arid1a transgenic mouse by ChIP-seq.
Project description:The circadian clock generates daily rhythms in mammalian liver processes, such as glucose and lipid homeostasis, xenobiotic metabolism, and regeneration. The mechanisms governing these rhythms are not well understood, particularly the distinct contributions of the cell-autonomous clock and central pacemaker to rhythmic liver physiology. Through microarray expression profiling in MMH-D3 hepatocytes, we identified over 1,000 transcripts that exhibit circadian oscillations, demonstrating that many rhythms can be driven by the cell-autonomous clock and that MMH-D3 is a valid circadian model system. The genes represented by these circadian transcripts displayed both co-phasic and anti-phasic organization within a protein-protein interaction network, suggesting the existence of competition for binding sites or partners by genes of disparate transcriptional phases. Multiple pathways displayed enrichment in MMH-D3 circadian transcripts, including the polyamine synthesis module of the glutathione metabolic pathway. The polyamine synthesis module, which is highly associated with cell proliferation and whose products are required for initiation of liver regeneration, includes enzymes whose transcripts exhibit circadian oscillations, such as ornithine decarboxylase (Odc1) and spermidine synthase (Srm). Metabolic profiling revealed that the enzymatic product of SRM, spermidine, cycles as well. Thus, the cell-autonomous hepatocyte clock can drive a significant amount of transcriptional rhythms and orchestrate physiologically relevant modules such as polyamine synthesis. Samples were collected every 2 hours for a duration of 46 hours from differentiated MMH-D3 hepatocytes synchronized via serum shock. Cells were synchronized by serum shock and incubated for 12 hours, and then samples were collected every 2 hours from 12 hours post-serum shock to 58 hours post-serum shock, for a total of 24 samples.
Project description:The HGF/c-Met system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been demonstrated in acute liver regeneration its cell specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined yet. In order to better characterize the role of c-Met in hepatocytes we generated a hepatocyte-specific c-Met knockout mouse (c-Met∆hepa) using the Cre-loxP system and studied its relevance after bile-duct ligation. Two strategies for c-Met deletion in hepatocytes were tested. Early deletion during embryonic development was lethal, while post-natal Cre-expression was successful leading to the generation of viable c-Met∆hepa mice. Bile-duct ligation in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-Met∆hepa mice revealed a significant reduction of anti-apoptotic genes in c-Met deleted hepatocytes. These findings could be functionally tested as c-Met∆hepa mice showed a stronger apoptotic response after bile-duct ligation and Jo-2 stimulation. This phenotype was associated with increased expression of pro-inflammatory cytokines (TNF-a and IL-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger pro-fibrogenic response as evidenced by increased TGF-b1, a-SMA, collagen-1a mRNA expression and enhanced collagen-fiber staining in c-Met∆hepa mice. Overall design: For gene array analysis c-MetDhepa and c-MetloxP/loxP controls were stimulated for 2 hours with 2µg recombinant mouse HGF.Three animals per group were treated in parallel, before and after i.p. injection of recombinant HGF or NaCl.
Project description:Background: Evidence has recently accumulated suggesting that mature hepatocytes have the unique capacity to autonomously decide their replication fate. The molecular mechanism by which hepatocytes switch from an essentially quiescent state to rapidly proliferation after liver injury is not fully understood. To provide molecular insights into the self-renewal of mature hepatocytes, we have applied an ex vivo liver slice culture system. Results: After liver injury, activation of p38 and Erk1/2 began in mature hepatocytes within 5 min. Erk1/2 was activated at the edge of the cut as well as on the surface of liver slices. The number of hepatocytes that contain activated Erk1/2 increased within 1 h and then decreased. Concomitantly, immediate early genes (IEGs), such as Jun, early gene response 1 (Egr1) and Myc were induced and Ki67 positive hepatocytes appeared 24 h after liver injury in the slices. In agreement with in vivo studies, transcriptome analysis using 2 h slice culture reveals that 63% of upregulated genes were downstream of lipopolysaccharide (LPS) stimulation which induces interleukin 6 (IL 6) or tumor necrosis factor (TNF) alpha pathways. Furthermore, circadian clock regulator, such as members of the basic helix-loop-helix family, Bhlhe40 and Bhlhe41, were highly upregulated for 4 h after injury. Although upon injury Jun or Egr1 were induced in liver slices from proliferation-defective liver, no circadian clock regulator genes were upregulated. Moreover, using an in vitro cell culture system we show that Bhlhe40 is required for the G0-G1 transition. Conclusion: Our data suggest that the circadian clock regulator, Bhlhe40, is involved in the G0-G1 transition. An ex vivo system using normal and proliferation defective KO liver is a useful tool for identification of genes that trigger cell proliferation shortly after liver injury. This method may also be applied for measurement of the liver regeneration potential of individual livers at the priming phase. In one dual-color microarray hybridization, mRNA expression changes after 2h ex vivo incubation of liver slices were examined.
Project description:Glucocorticoids are widely used therapeutically to suppress inflammatory/immune responses and most of their effects are produced either by altering transcription of specific genes directly, or by altering the expression of transcription factors that subsequently alter the expression of downstream genes. Relevant data from previous studies indicate that the number of genes regulated by glucocorticoid receptor exceeds 4000 in cells and that 358 different genes are regulated in the liver of adrenalectomized males rats treated with a chronic infusion of methylprednisolone for up to 1 week . However, differences in gene expression between males and females in response to glucocorticoid treatment in isolated hepatocytes are not known. Livers were isolated from adult male and female Sprague-Dawley rats and digested with the collagenase perfusion method developed by Berry and Friend (J Cell Biol 43, 506-520;1969). After collagenase treatment, the liver was excised, minced in balanced salt solution, and centrifuged at 50 g for 3 min. Immediately after isolation, hepatocytes were resuspended in Williams E Medium containing penicillin (100 units/ml), streptomycin (100 μg/ml), 2 mM glutamine pH 7.4, and 10% fetal bovine serum and plated on collagen-coated 94 x 16 mm cell culture dishes and maintained for 24 hours in a 95% air–5% CO2, 37°C incubator. The medium was then changed to free serum Williams E Medium and maintained in culture for an additional 24 hours. Hepatocytes isolated from male and rats were treated with vehicle (0.01% ethanol) or dexamethasone (100 nm) for 6 hours. Total RNA from cells were extracted by using the RNeasy Midi Kit (Qiagen) according to the manufacturer’s instructions. All samples were treated with the RNase-free DNase set (Qiagen) and were kept at -80°C until labeling and hybridization.