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1.  Loss of c-Met Disrupts Gene Expression Program Required for G2/M Progression during Liver Regeneration in Mice 
PLoS ONE  2010;5(9):e12739.
Background
Previous work has established that HGF/c-Met signaling plays a pivotal role in regulating the onset of S phase following partial hepatectomy (PH). In this study, we used Metfl/fl;Alb-Cre+/− conditional knockout mice to determine the effects of c-Met dysfunction in hepatocytes on kinetics of liver regeneration.
Methodology/Principal Finding
The priming events appeared to be intact in Metfl/fl;Alb-Cre+/− livers. Up-regulation of stress response (MAFK, IKBZ, SOCS3) and early growth response (c-Myc, c-Jun, c-Fos, DUSP1 and 6) genes as assessed by RT-qPCR and/or microarray profiling was unchanged. This was consistent with an early induction of MAPK/Erk and STAT3. However, after a successful completion of the first round of DNA replication, c-Met deficient hepatocytes were blocked in early/mid G2 phase as shown by staining with phosphorylated form of histone H3. Furthermore, loss of c-Met in hepatocytes diminished the subsequent G1/S progression and delayed liver recovery after partial hepatectomy. Upstream signaling pathways involved in the blockage of G2/M transition included lack of persistent Erk1/2 activation and inability to up-regulate the levels of Cdk1, Plk1, Aurora A and B, and Mad2 along with a defective histone 3 phosphorylation and lack of chromatin condensation. Continuous supplementation with EGF in vitro increased proliferation of Metfl/fl;Alb-Cre+/− primary hepatocytes and partially restored expression levels of mitotic cell cycle regulators albeit to a lesser degree as compared to control cultures.
Conclusion/Significance
In conclusion, our results assign a novel non-redundant function for HGF/c-Met signaling in regulation of G2/M gene expression program via maintaining a persistent Erk1/2 activation throughout liver regeneration.
doi:10.1371/journal.pone.0012739
PMCID: PMC2940888  PMID: 20862286
2.  c-Myc affects mRNA translation, cell proliferation and progenitor cell function in the mammary gland 
BMC Biology  2009;7:63.
Background
The oncoprotein c-Myc has been intensely studied in breast cancer and mouse mammary tumor models, but relatively little is known about the normal physiological role of c-Myc in the mammary gland. Here we investigated functions of c-Myc during mouse mammary gland development using a conditional knockout approach.
Results
Generation of c-mycfl/fl mice carrying the mammary gland-specific WAPiCre transgene resulted in c-Myc loss in alveolar epithelial cells starting in mid-pregnancy. Three major phenotypes were observed in glands of mutant mice. First, c-Myc-deficient alveolar cells had a slower proliferative response at the start of pregnancy, causing a delay but not a block of alveolar development. Second, while milk composition was comparable between wild type and mutant animals, milk production was reduced in mutant glands, leading to slower pup weight-gain. Electron microscopy and polysome fractionation revealed a general decrease in translational efficiency. Furthermore, analysis of mRNA distribution along the polysome gradient demonstrated that this effect was specific for mRNAs whose protein products are involved in milk synthesis. Moreover, quantitative reverse transcription-polymerase chain reaction analysis revealed decreased levels of ribosomal RNAs and ribosomal protein-encoding mRNAs in mutant glands. Third, using the mammary transplantation technique to functionally identify alveolar progenitor cells, we observed that the mutant epithelium has a reduced ability to repopulate the gland when transplanted into NOD/SCID recipients.
Conclusion
We have demonstrated that c-Myc plays multiple roles in the mouse mammary gland during pregnancy and lactation. c-Myc loss delayed, but did not block proliferation and differentiation in pregnancy. During lactation, lower levels of ribosomal RNAs and proteins were present and translation was generally decreased in mutant glands. Finally, the transplantation studies suggest a role for c-Myc in progenitor cell proliferation and/or survival.
See related minireview by Evan et al:
doi:10.1186/1741-7007-7-63
PMCID: PMC2761394  PMID: 19785743
3.  c-myc, c-fos, and c-jun regulation in the regenerating livers of normal and H-2K/c-myc transgenic mice. 
Molecular and Cellular Biology  1990;10(6):3185-3193.
We investigated the mechanisms of regulation of c-myc, c-fos, and c-jun at the early stages of liver regeneration in mice. We show that the transient increase in steady-state levels of c-myc mRNA at the start of liver regeneration is most probably regulated by posttranscriptional mechanisms. Although there was a marked increase in c-myc transcriptional initiation shortly after partial hepatectomy, a block in elongation prevented the completion of most transcripts. To gain further information on the mechanism of regulation of c-myc expression during liver regeneration, we used transgenic mice harboring the human c-myc gene driven by the H-2K promoter. In these animals, the murine c-myc responded to the growth stimulus generated by partial hepatectomy, whereas the expression of the transgene was constitutive and did not change in the regenerating liver. However, the mRNA from both genes increased markedly after cycloheximide injection, suggesting that the regulation of c-myc mRNA abundance in the regenerating liver differs from that occurring after protein synthesis inhibition. Furthermore, we show that in normal mice c-fos and c-jun mRNA levels and transcriptional rates increase within 30 min after partial hepatectomy. c-fos transcriptional elongation was restricted in nongrowing liver, but the block was partially relieved in the regenerating liver. Nevertheless, for both c-fos and c-jun, changes in steady-state mRNA detected after partial hepatectomy were much greater than the transcriptional increase. In the regenerating liver of H-2K/c-myc mice, c-fos and c-jun expression was diminished, whereas mouse c-myc expression was enhanced in comparison with that in nontransgenic animals.
Images
PMCID: PMC360683  PMID: 2111449
4.  Myc Stimulates Nuclearly Encoded Mitochondrial Genes and Mitochondrial Biogenesis†  
Molecular and Cellular Biology  2005;25(14):6225-6234.
Although several genes involved in mitochondrial function are direct Myc targets, the role of Myc in mitochondrial biogenesis has not been directly established. We determined the effects of ectopic Myc expression or the loss of Myc on mitochondrial biogenesis. Induction of Myc in P493-6 cells resulted in increased oxygen consumption and mitochondrial mass and function. Conversely, compared to wild-type Myc fibroblasts, Myc null rat fibroblasts have diminished mitochondrial mass and decreased number of normal mitochondria. Reconstitution of Myc expression in Myc null fibroblasts partially restored mitochondrial mass and function and normal-appearing mitochondria. Concordantly, we also observed in primary hepatocytes that acute deletion of floxed murine Myc by Cre recombinase resulted in diminished mitochondrial mass in primary hepatocytes. Our microarray analysis of genes responsive to Myc in human P493-6 B lymphocytes supports a role for Myc in mitochondrial biogenesis, since genes involved in mitochondrial structure and function are overrepresented among the Myc-induced genes. In addition to the known direct binding of Myc to many genes involved in mitochondrial structure and function, we found that Myc binds the TFAM gene, which encodes a key transcriptional regulator and mitochondrial DNA replication factor, both in P493-6 lymphocytes with high ectopic MYC expression and in serum-stimulated primary human 2091 fibroblasts with induced endogenous MYC. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis.
doi:10.1128/MCB.25.14.6225-6234.2005
PMCID: PMC1168798  PMID: 15988031
5.  A Novel Role of Nucleostemin in Maintaining the Genome Integrity of Dividing Hepatocytes During Mouse Liver Development and Regeneration 
Hepatology (Baltimore, Md.)  2013;58(6):10.1002/hep.26600.
During liver development and regeneration, hepatocytes undergo rapid cell division and face an increased risk of DNA damage associated with active DNA replication. The mechanism that protects proliferating hepatocytes from replication-induced DNA damage remains unclear. Nucleostemin (NS) is known to be upregulated during liver regeneration, and loss of NS is associated with increased DNA damage in cancer cells. To determine whether NS is involved in protecting the genome integrity of proliferating hepatocytes, we created an albumin promoter-driven NS conditional-null (albNScko) mouse model. Livers of albNScko mice begin to show the loss of NS in developing hepatocytes from the first postnatal week and increased DNA damage and hepatocellular injury at 1-2 weeks old. At 3-4 weeks, albNScko livers develop bile duct hyperplasia and show increased apoptotic cells, necrosis, regenerative nodules, and evidence suggestive of hepatic stem/progenitor cell (HSPC) activation. CCl4 treatment enhances degeneration and DNA damage in NS-deleted hepatocytes and increase biliary hyperplasia and A6+ cells in albNScko livers. Following 70% partial hepatectomy (PHx), albNScko livers show increased DNA damage in parallel with a blunted and prolonged regenerative response. The DNA damage in NS-depleted hepatocytes is explained by the impaired recruitment of a core DNA repair enzyme, RAD51, to replication-induced DNA damage foci. This work reveals a novel genome-protective role of NS in developing and regenerating hepatocytes.
doi:10.1002/hep.26600
PMCID: PMC3844114  PMID: 23813570
biliary hyperplasia; DNA damage; DNA replication; stem cells
6.  Hepatocyte specific deletion of farnesoid X receptor delays, but does not inhibit liver regeneration after partial hepatectomy in mice 
Hepatology (Baltimore, Md.)  2012;56(6):2344-2352.
Farnesoid X Receptor (FXR), the primary bile acid-sensing nuclear receptor, also plays a role in stimulation of liver regeneration. Whole body deletion of FXR results in significant inhibition of liver regeneration after partial hepatectomy (PHX). FXR is expressed in liver and intestine and recent ChIP-seq analysis indicates that FXR regulates distinct set of genes in a tissue-specific manner. These data raise the question about relative contribution of hepatic and intestinal FXR in regulation of liver regeneration. We studied liver regeneration after PHX in hepatocyte-specific FXR knockout (hepFXR-KO) mice over a time course of 0 to 14 days. Whereas the overall kinetics of liver regrowth in hepFXR-KO mice was unaffected, a delay in peak hepatocyte proliferation from day 2 to day 3 after PHX was observed in the hepFXR-KO mice as compared to Cre- control mice. Real Time PCR, Western blot and co-IP studies revealed decreased Cyclin D1 expression and decreased association of Cyclin D1 with CDK4 in hepFXR-KO mice after PHX, correlating with decreased phosphorylation of pRb and delayed cell proliferation in the hepFXR-KO livers. The hepFXR-KO mice also exhibited delay in acute hepatic fat accumulation following PHX, which is associated with regulation of cell cycle. Further, a significant delay in HGF-initiated signaling, including AKT, c-myc and ERK-1/2 pathways, was observed in hepFXR-KO mice. UPLC-mass spectroscopy analysis of hepatic bile acids indicated no difference in levels of bile acids in hepFXR-KO and control mice. In Conclusion, deletion of hepatic FXR did not completely inhibit but delays liver regeneration after PHX secondary to delayed Cyclin D1 activation.
doi:10.1002/hep.25918
PMCID: PMC3469721  PMID: 22730081
HGF; Fat; Proliferation; Cyclin D1; Bile Acids
7.  Liver-specific disruption of PPARγ in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes 
Journal of Clinical Investigation  2003;111(5):737-747.
To elucidate the function of PPARγ in leptin-deficient mouse (ob/ob) liver, a PPARγ liver-null mouse on an ob/ob background, ob/ob-PPARγ(fl/fl)AlbCre+, was produced using a floxed PPARγ allele, PPARγ(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARγ(fl/fl)AlbCre+ mice had a deletion of exon 2 and a corresponding loss of full-length PPARγ mRNA and protein. The PPARγ-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARγ(fl/fl)AlbCre–). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARγ(fl/fl)AlbCre+ mice, and the levels of serum TG and FFA in ob/ob-PPARγ(fl/fl)AlbCre+ mice were significantly higher than in the control ob/ob-PPARγ(fl/fl)AlbCre– mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARγ(fl/fl)AlbCre– mice compared with livers from nonobese Cre– mice; there was no effect of rosiglitazone in ob/ob-PPARγ(fl/fl)AlbCre+ mice. The deficiency of hepatic PPARγ further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARγ plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.
doi:10.1172/JCI200317223
PMCID: PMC151902  PMID: 12618528
8.  Mcl-1 and Bcl-xL Cooperatively Maintain Integrity of Hepatocytes in Developing and Adult Murine Liver 
Hepatology (Baltimore, Md.)  2009;50(4):1217-1226.
Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, especially in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1– deficient mice by crossing mcl-1flox/flox mice and AlbCre mice and further crossed them with bcl-xflox/flox mice, giving Mcl-1/Bcl-xL– deficient mice. The mcl-1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased number of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL)-positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte-specific Bcl-xL– deficient mice. Although mcl-1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-xflox/+ mcl-1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL– deficient or Mcl-1– deficient mice. In contrast, bcl-xflox/flox mcl-1flox/+ AlbCre, bcl-xflox/+ mcl-1flox/flox AlbCre, and bcl-xflox/flox mcl-1flox/flox AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl-1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner.
doi:10.1002/hep.23126
PMCID: PMC3560852  PMID: 19676108
9.  Sequential and Coordinated Actions of c-Myc and N-Myc Control Appendicular Skeletal Development 
PLoS ONE  2011;6(4):e18795.
Background
During limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton.
Methodology/Principal Findings
To investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants.
Conclusions/Significance
Our results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton.
doi:10.1371/journal.pone.0018795
PMCID: PMC3073980  PMID: 21494559
10.  NFAT4 Deficiency Results in Incomplete Liver Regeneration Following Partial Hepatectomy 
The Journal of surgical research  2008;154(2):226-233.
Background
Liver regeneration following partial hepatectomy requires the orchestration of highly regulated molecular pathways; a change in the abundance or activity of a specific gene product has the potential to adversely affect this process. The NFAT (nuclear factor of activated T-cells) transcription factors represent a family of gene transcription signaling intermediates that translate receptor-dependent signaling events into specific transcriptional responses using the Ras/Raf pathway.
Materials/Methods
Eight week old NFAT4 knockout (KO) mice and their wild type counterparts (Balb-c) underwent two-thirds partial hepatectomy. The animals were sacrificed and their livers were harvested at specific timepoints during regeneration. Recovery of liver mass was measured for each timepoint. PCR analysis was used to analyze expression levels of the immediate early genes c-fos, c-jun and c-myc as well as downstream effectors of NFAT4 including FGF-18 and BMP-4.
Results
Hepatocyte proliferation and thus liver regeneration following hepatectomy was suppressed in NFAT4 knockout (KO) mice. Statistical significance was reached at 1 hour, 7 days and 10 days (p < 0.05) with a 22% median reduction in regeneration of liver mass in the NFAT4 KO mice by 10 days, at which time liver regeneration should be complete in mice. The immediate early gene c-fos was elevated in NFAT4 KO mice during early regeneration with a median value at one hour and one day of 1.60E-08 and 1.09E-08 vs 6.10E-09 and 1.55E-09 in the Balb-c mice. C-jun, in contrast, was elevated during late regeneration in the NFAT4 KO mice (3.40E-09 and 5.67E-09 at 7 and 10 days, respectively) in comparison to the Balb-c mice (7.76E-10 and 1.24E-09, respectively.). NFAT2 was also upregulated in the NFAT4 KO mice; however, no changes were detected in its downstream effectors, CCR1 and CCL3.
Conclusions
We demonstrated that NFAT4 deficiency impairs hepatic regeneration in a murine model proving that NFAT4 plays an important yet unclear role in liver regeneration; its absence may be compensated by c-fos, c-jun and NFAT2 expression changes.
doi:10.1016/j.jss.2008.07.023
PMCID: PMC2753792  PMID: 19059606
NFAT4; regeneration; liver; hepatectomy; mouse
11.  Knock-out of Myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes 
Hepatology (Baltimore, Md.)  2009;49(2):627-636.
Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family. It interacts with pro-apoptotic Bcl-2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl-1 is essential for embryonal development and the maintenance of B, T and hematopoietic stem cells. We have recently shown that induction of Mcl-1 by growth factors rescues primary human hepatocytes from CD95-mediated apoptosis. This prompted us to further analyze the relevance of Mcl-1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte-specific Mcl-1 knock-out mouse (Mcl-1flox/flox-AlbCre). Deletion of Mcl-1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl-1flox/flox-AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl-1. Importantly, hepatic pericellular fibrosis occurs in Mcl-1 negative livers in response to chronic liver damage. Furthermore, Mcl-1flox/flox-AlbCre mice are more susceptible towards hepatocellular damage induced by agonistic anti-CD95 antibodies or concanavalin A.
Conclusion
The present study provides in vivo evidence that Mcl-1 is a crucial anti-apoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction.
doi:10.1002/hep.22664
PMCID: PMC2753874  PMID: 19127517
Mcl-1; Bcl-xL; CD95; fibrosis; proliferation
12.  Global IGFBP-1 deletion does not affect prostate cancer development in a c-Myc transgenic mouse model 
The Journal of endocrinology  2011;211(3):297-304.
Circulating IGFBP-1 levels vary in response to nutritional status, and preclinical studies suggest that elevated IGFBP-1 may be protective against the development and progression of prostate cancer. We hypothesized that global deletion of IGFBP-1 would accelerate the development of prostate cancer in a c-Myc transgenic mouse model. To test our hypothesis, c-Myc transgenic mice (Myc/BP-1 WT) were crossed and interbred with the IGFBP-1 knockout mice (Myc/BP-1 KO). The animals were placed on a high protein diet at weaning, weighed every two weeks, and euthanized at 16 weeks of age. Prostate histopathology was assessed and proliferation status was determined by Ki 67 and PCNA analyses. IGF-related serum biomarkers and body composition were measured. No significant difference in the incidence of prostate cancer was observed between the Myc/BP-1 KO and Myc/BP-1 WT mice (65% and 80% respectively, p= 0.48). Proliferation was significantly decreased by 71% in prostate tissue of Myc/BP-1 KO mice compared to Myc/BP-1 WT mice. Myc/BP-1 KO mice exhibited a significant 6.7% increase in body weight relative to the Myc/BP-1 WT mice attributed to an increase in fat mass. Fasting insulin levels were higher in the Myc/BP-1 KO mice without any difference between the groups in fasting glucose concentrations. Thus, contrary to our hypothesis, global deletion of IGFBP-1 in a c-Myc transgenic mouse model did not accelerate the development of prostate cancer. Global IGFBP-1 deletion did result in a significant increase in body weight and body fat mass. Further studies are required to understand the underlying mechanisms for these metabolic effects.
doi:10.1530/JOE-11-0240
PMCID: PMC3271951  PMID: 21903863
Myc; IGFBP-1 knockout; IGF-1; IGFBP; prostate cancer; proliferation; body composition; insulin
13.  Constitutive gray hair in mice induced by melanocyte-specific deletion of c-Myc 
Pigment cell & melanoma research  2012;25(3):312-325.
Summary
c-Myc is involved in the control of diverse cellular processes and implicated in the maintenance of different tissues including the neural crest. Here, we report that c-Myc is particularly important for pigment cell development and homeostasis. Targeting c-Myc specifically in the melanocyte lineage using the floxed allele of c-Myc and Tyr::Cre transgenic mice results in a congenital gray hair phenotype. The gray coat color is associated with a reduced number of functional melanocytes in the hair bulb and melanocyte stem cells in the hair bulge. Importantly, the gray phenotype does not progress with time, suggesting that maintenance of the melanocyte through the hair cycle does not involve c-Myc function. In embryos, at E13.5, c-Myc-deficient melanocyte precursors are affected in proliferation in concordance with a reduction in numbers, showing that c-Myc is required for the proper melanocyte development. Interestingly, melanocytes from c-Myc-deficient mice display elevated levels of the c-Myc paralog N-Myc. Double deletion of c-Myc and N-Myc results in nearly complete loss of the residual pigmentation, indicating that N-Myc is capable of compensating for c-Myc loss of function in melanocytes.
doi:10.1111/j.1755-148X.2012.00998.x
PMCID: PMC3541053  PMID: 22420299
melanocyte; melanoma; c-Myc; N-Myc; conditional knockout; pigment
14.  Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest 
BMC Cancer  2007;7:215.
Background
TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest.
Results
Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity.
Conclusion
The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of these critical cellular proteins can be impaired by common causes of chronic liver disease and HCC, including viral hepatitis B and C proteins, we suggest that disruption of pRb function, and to a lesser extend P21Cip1 and P53 in hepatocytes may represent an additional new mechanism of escape from TGFβ-growth-inhibition in the inflammatory milieu of chronic liver disease and contribute to cancer development.
doi:10.1186/1471-2407-7-215
PMCID: PMC2206047  PMID: 18021445
15.  Contributions of New Hepatocyte Lineages to Liver Growth, Maintenance, and Regeneration in Mice# 
Hepatology (Baltimore, Md.)  2011;54(2):655-663.
The contributions that de novo differentiation of new hepatocyte lineages makes to normal liver physiology are unknown. Here a system that uniquely marks cells during a finite period following primary activation of a serum albumin gene promoter/enhancer-driven Cre transgene (albCre) was used to investigate birthrates of new hepatocyte lineages from Alb-naïve precursors in mice. Elapsed time was measured using a two-color fluorescent marker-gene that converts from expressing tdTomato (tdT, red-fluorescent) to expressing GFP (green-fluorescent) upon exposure to Cre. Accumulation of GFP and decay of tdT each contributed to a regular fluorescence transition, which was calibrated in vivo. In normal adults, this system revealed that a steady-state level of 0.076% hepatocytes had differentiated within the previous four days from cell lineages that had never previously expressed albCre. As compared to resting adult livers, the relative abundance of these newborn hepatocytes was elevated 3.7-fold in normal growing livers of juveniles and 8.6-fold during liver regeneration following partial hepatectomy in normal adults.
Conclusion:
Newborn hepatocyte lineages arising from Alb-naïve cells contribute to liver maintenance under normal conditions. Hepatocyte lineage birthrates can vary in response to the liver’s physiological status.
doi:10.1002/hep.24398
PMCID: PMC3145049  PMID: 21538442
hepatic stem cell; liver development; liver regeneration; liver maintenance; hepatocyte lineage life history
16.  Progressive Effects of N-myc Deficiency on Proliferation, Neurogenesis, and Morphogenesis in the Olfactory Epithelium 
Developmental Neurobiology  2013;74(6):643-656.
N-myc belongs to the myc proto-oncogene family, which is involved in numerous cellular processes such as proliferation, growth, apoptosis, and differentiation. Conditional deletion of N-myc in the mouse nervous system disrupted brain development, indicating that N-myc plays an essential role during neural development. How the development of the olfactory epithelium and neurogenesis within are affected by the loss of N-myc has, however, not been determined. To address these issues, we examined an N-mycFoxg1Cre conditional mouse line, in which N-myc is depleted in the olfactory epithelium. First changes in N-myc mutants were detected at E11.5, with reduced proliferation and neurogenesis in a slightly smaller olfactory epithelium. The phenotype was more pronounced at E13.5, with a complete lack of Hes5-positive progenitor cells, decreased proliferation, and neurogenesis. In addition, stereological analyses revealed reduced cell size of post-mitotic neurons in the olfactory epithelium, which contributed to a smaller olfactory pit. Furthermore, we observed diminished proliferation and neurogenesis also in the vomeronasal organ, which likewise was reduced in size. In addition, the generation of gonadotropin-releasing hormone neurons was severely reduced in N-myc mutants. Thus, diminished neurogenesis and proliferation in combination with smaller neurons might explain the morphological defects in the N-myc depleted olfactory structures. Moreover, our results suggest an important role for N-myc in regulating ongoing neurogenesis, in part by maintaining the Hes5-positive progenitor pool. In summary, our results provide evidence that N-myc deficiency in the olfactory epithelium progressively diminishes proliferation and neurogenesis with negative consequences at structural and cellular levels. © 2013 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc. Develop Neurobiol 74: 643–656, 2014
doi:10.1002/dneu.22162
PMCID: PMC4237195  PMID: 24376126
neurogenesis; N-myc; olfactory epithelium; vomeronasal organ; mouse
17.  The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice 
PLoS ONE  2012;7(10):e46469.
Background
The transcription factor NF-κB consisting of the subunits RelA/p65 and p50 is known to be quickly activated after partial hepatectomy (PH), the functional relevance of which is still a matter of debate. Current concepts suggest that activation of NF-κB is especially critical in non-parenchymal cells to produce cytokines (TNF, IL-6) to adequately prime hepatocytes to proliferate after PH, while NF-κB within hepatocytes mainly bears cytoprotective functions.
Methods
To study the role of the NF-κB pathway in different liver cell compartments, we generated conditional knockout mice in which the transactivating NF-κB subunit RelA/p65 can be inactivated specifically in hepatocytes (RelaF/FAlbCre) or both in hepatocytes plus non-parenchymal cells including Kupffer cells (RelaF/FMxCre). 2/3 and 80% PH were performed in controls (RelaF/F) and conditional knockout mice (RelaF/FAlbCre and RelaF/FMxCre) and analyzed for regeneration.
Results
Hepatocyte-specific deletion of RelA/p65 in RelaF/FAlbCre mice resulted in an accelerated cell cycle progression without altering liver mass regeneration after 2/3 PH. Surprisingly, hepatocyte apoptosis or liver damage were not enhanced in RelaF/FAlbCre mice, even when performing 80% PH. The additional inactivation of RelA/p65 in non-parenchymal cells in RelaF/FMxCre mice reversed the small proliferative advantage observed after hepatocyte-specific deletion of RelA/p65 so that RelaF/FMxCre mice displayed normal cell cycle progression, DNA-synthesis and liver mass regeneration.
Conclusion
The NF-κB subunit RelA/p65 fulfills opposite functions in different liver cell compartments in liver regeneration after PH. However, the effects observed after conditional deletion of RelA/p65 are small and do not alter liver mass regeneration after PH. We therefore do not consider RelA/p65-containing canonical NF-κB signalling to be essential for successful liver regeneration after PH.
doi:10.1371/journal.pone.0046469
PMCID: PMC3462179  PMID: 23049704
18.  N-Myc and L-Myc are Essential for Hair Cell Formation but not Maintenance 
Brain research  2012;1484:1-14.
Sensorineural hearing loss results from damage to the hair cells of the organ of Corti and is irreversible in mammals. While hair cell regeneration may prove to be the ideal therapy after hearing loss, prevention of initial hair cell loss could provide even more benefit at a lower cost. Previous studies have shown that the deletion of Atoh1 results in embryonic loss of hair cells while the absence of Barhl1, Gfi1, and Pou4f3 leads to the progressive loss of hair cells in newborn mice. We recently reported that in the early embryonic absence of N-Myc (using Pax2-Cre), hair cells in the organ of Corti develop and remain until at least seven days after birth, with subsequent progressive loss. Thus, N-Myc plays a role in hair cell viability; however, it is unclear if this is due to its early expression in hair cell precursors and throughout the growing otocyst as it functions through proliferation or its late expression exclusively in differentiated hair cells. Furthermore, the related family member L-Myc is mostly co-expressed in the ear, including in differentiated hair cells, but its function has not been studied and could be partially redundant to N-Myc. To test for a long-term function of the Mycs in differentiated hair cells, we generated nine unique genotypes knocking out N-Myc and/or L-Myc after initial formation of hair cells using the well-characterized Atoh1-Cre. We tested functionality of the auditory and vestibular systems at both P21 and four months of age and under the administration of the ototoxic drug cisplatin. We conclude that neither N-Myc nor L-Myc is likely to play important roles in long-term hair cell maintenance. Therefore, it is likely that the late-onset loss of hair cells resulting from early deletion of the Mycs leads to an unsustainable developmental defect.
doi:10.1016/j.brainres.2012.09.027
PMCID: PMC3482343  PMID: 23022312
L-Myc; N-Myc; Hair Cell; Hearing; Prevention; Regeneration
19.  In Vivo Inhibition of c-MYC in Myeloid Cells Impairs Tumor-Associated Macrophage Maturation and Pro-Tumoral Activities 
PLoS ONE  2012;7(9):e45399.
Although tumor-associated macrophages (TAMs) are involved in tumor growth and metastasis, the mechanisms controlling their pro-tumoral activities remain largely unknown. The transcription factor c-MYC has been recently shown to regulate in vitro human macrophage polarization and be expressed in macrophages infiltrating human tumors. In this study, we exploited the predominant expression of LysM in myeloid cells to generate c-Mycfl/fl LysMcre/+ mice, which lack c-Myc in macrophages, to investigate the role of macrophage c-MYC expression in cancer. Under steady-state conditions, immune system parameters in c-Mycfl/fl LysMcre/+ mice appeared normal, including the abundance of different subsets of bone marrow hematopoietic stem cells, precursors and circulating cells, macrophage density, and immune organ structure. In a model of melanoma, however, TAMs lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions (e.g., reduced expression of VEGF, MMP9, and HIF1α) that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in c-Mycfl/fl LysMcre/+ mice. Macrophage c-Myc deletion also diminished fibrosarcoma growth. These data identify c-Myc as a positive regulator of the pro-tumoral program of TAMs and suggest c-Myc inactivation as an attractive target for anti-cancer therapy.
doi:10.1371/journal.pone.0045399
PMCID: PMC3447925  PMID: 23028984
20.  The Myc 3′ Wnt-Responsive Element Regulates Homeostasis and Regeneration in the Mouse Intestinal Tract 
Molecular and Cellular Biology  2012;32(19):3891-3902.
The Wnt/β-catenin signaling pathway controls cellular proliferation in the intestines. In response to Wnt, β-catenin transits into the nucleus and associates with members of the T-cell factor (TCF) family of transcription factors. β-Catenin/TCF complexes bind Wnt responsive DNA elements (WREs) to activate target gene expression. The c-MYC proto-oncogene (MYC) is a direct target of β-catenin/TCF complexes. We recently identified the MYC 3′ WRE, which maps 1.4-kb downstream from the MYC transcription stop site. To investigate the role of the Myc 3′ WRE in the intestines, we generated a mouse model with a germ line deletion of this element. The intestinal architecture was largely preserved in knockout mice; however, removal of the Myc 3′ WRE compromised the crypt microenvironment. In comparison to wild-type intestines, knockout intestines contained an increased number of proliferative cells and a reduced number of differentiated cells comprising both absorptive and secretory lineages. Using a model of colitis, we found that knockout colons repaired more rapidly during the recovery period of the protocol. These results indicate that regulation of MYC expression through the Myc 3′ WRE contributes to intestinal homeostasis. Furthermore, our study implicates MYC as an important regulator of intestinal regeneration following injury.
doi:10.1128/MCB.00548-12
PMCID: PMC3457533  PMID: 22826434
21.  Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation 
The Journal of Clinical Investigation  2008;118(12):3943-3953.
JNK proteins have been shown to be involved in liver carcinogenesis in mice, but the extent of their involvement in the development of human liver cancers is unknown. Here, we show that activation of JNK1 but not JNK2 was increased in human primary hepatocellular carcinomas (HCCs). Further, JNK1 was required for human HCC cell proliferation in vitro and tumorigenesis after xenotransplantation. Importantly, mice lacking JNK1 displayed decreased tumor cell proliferation in a mouse model of liver carcinogenesis and decreased hepatocyte proliferation in a mouse model of liver regeneration. In both cases, impaired proliferation was caused by increased expression of p21, a cell-cycle inhibitor, and reduced expression of c-Myc, a negative regulator of p21. Genetic inactivation of p21 in JNK1–/– mice restored hepatocyte proliferation in models of both liver carcinogenesis and liver regeneration, and overexpression of c-Myc increased proliferation of JNK1–/– liver cells. Similarly, JNK1 was found to control the proliferation of human HCC cells by affecting p21 and c-Myc expression. Pharmacologic inhibition of JNK reduced the growth of both xenografted human HCC cells and chemically induced mouse liver cancers. These findings provide a mechanistic link between JNK activity and liver cell proliferation via p21 and c-Myc and suggest JNK targeting can be considered as a new therapeutic approach for HCC treatment.
doi:10.1172/JCI37156
PMCID: PMC2579707  PMID: 19033664
22.  Impairment of liver regeneration by the histone deacetylase inhibitor valproic acid in mice*  
Background and objective: Liver regeneration is a complex process regulated by a group of genetic and epigenetic factors. A variety of genetic factors have been reported, whereas few investigations have focused on epigenetic regulation during liver regeneration. In the present study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was used to investigate the effect of HDAC on liver regeneration. Methods: VPA was administered via intraperitoneal injection to 2/3 partially hepatectomized mice to detect hepatocyte proliferation during liver regeneration. The mice were sacrificed, and their liver tissues were harvested at sequential time points from 0 to 168 h after treatment. DNA synthesis was detected via a BrdU assay, and cell proliferation was tested using Ki-67. The expressions of cyclin D1, cyclin E, cyclin dependent kinase 2 (CDK2), and CDK4 were detected by Western blot analysis. Chromatin immunoprecipitation (ChIP) assay was used to examine the recruitment of HDACs to the target promoter regions and the expression of the target gene was detected by Western blot. Results: Immunohistochemical analysis showed that cells positive for BrdU and Ki-67 decreased, and the peak of BrdU was delayed in the VPA-administered mice. Consistently, cyclin D1 expression was also delayed. We identified B-myc as a target gene of HDACs by complementary DNA (cDNA) microarray. The expression of B-myc increased in the VPA-administered mice after hepatectomy (PH). The ChIP assay confirmed the presence of HDACs at the B-myc promoter. Conclusions: HDAC activities are essential for liver regeneration. Inhibiting HDAC activities delays liver regeneration and induces liver cell cycle arrest, thereby causing an anti-proliferative effect on liver regeneration.
doi:10.1631/jzus.B1100362
PMCID: PMC3437367  PMID: 22949360
Liver regeneration; Epigenetic factors; Cell cycle; VPA; B-myc
23.  Akt and mTORC1 Have Different Roles During Liver Tumorigenesis in Mice 
Gastroenterology  2013;144(5):1055-1065.
Background & Aims
Phosphatidylinositide 3-kinase (PI3K) is deregulated in many human tumor types, including primary liver malignancies. The kinase Akt and mammalian target of rapamycin complex (mTORC1) are effectors of PI3K that promote cell growth and survival, but their individual roles in tumorigenesis are not well defined.
Methods
In livers of Alb-Cre mice, we selectively deleted tuberous sclerosis (Tsc)1, a negative regulator of Ras homolog enriched in brain (Rheb) and mTORC1, along with Pten, a negative regulator of PI3K. Tumor tissues were characterized by histologic and biochemical analyses.
Results
The Tsc1fl/fl;AlbCre, Ptenfl/fl;AlbCre, and Tsc1fl/fl;Ptenfl/fl;AlbCre mice developed liver tumors that differed in size, number, and histologic features. Livers of Tsc1fl/fl;AlbCre mice did not develop steatosis; tumors arose later than in the other strains of mice and were predominantly hepatocellular carcinomas (HCCs). Livers of the Ptenfl/fl;AlbCre mice developed steatosis and most of the tumors that formed were intrahepatic cholangiocarcinomas (ICCs). Livers of Tsc1fl/fl;Ptenfl/fl;AlbCre formed large numbers of tumors, of mixed histologies, with the earliest onset of any strain, indicating that loss of Tsc1 and Pten have synergistic effects on tumorigenesis. In these mice, the combination of rapamycin and MK2206 was more effective in reducing liver cell proliferation and inducing cell death than either reagent alone. Tumor differentiation correlated with Akt and mTORC1 activities; the ratio of Akt:mTORC1 activity was high throughout the course of ICC development and low during HCC development. Compared to surrounding non-tumor liver tissue, tumors from all 3 strains had increased activities of Akt, mTORC1, and MAPK and overexpressed fibroblast growth factor receptor (FGFR)1. Inhibition of FGFR1 in Tsc1-null mice suppressed Akt and MAPK activities in tumor cells.
Conclusions
Based on analyses of knockout mice, mTORC1 and Akt have different yet synergistic effects during development of liver tumors in mice.
doi:10.1053/j.gastro.2013.01.053
PMCID: PMC3633657  PMID: 23376645
liver cancer; mouse model; signal transduction; gene disruption
24.  Hepatocyte Growth Factor (HGF)/c-Met Signaling is required for Stem Cell Mediated Liver Regeneration 
Hepatology (Baltimore, Md.)  2012;55(4):1215-1226.
HGF/c-Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c-Met in stem cell-mediated liver regeneration by utilizing mice harboring c-met floxed alleles and Alb-Cre or Mx1-Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3, 5-diethocarbonyl-1,4-dihydrocollidine (DDC). Deletion of c-met in oval cells was confirmed in both models by PCR analysis of FACS- sorted EpCam-positive cells. Loss of c-Met receptor decreased sphere-forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration and decreased hepatocytic differentiation in vivo as demonstrated by double immunofluorescence using oval- (A6 and EpCam) and hepatocyte-specific (HNF-4α) antibodies. Furthermore, lack of c-Met had a profound effect on tissue remodeling and overall composition of HSC niche which was associated with greatly reduced MMP9 activity and decreased expression of SDF1. Using a combination of double immunofluorescence of cell type-specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC-treated livers. The Mx1-Cre-driven c-met deletion caused the greatest phenotypic impact on HSCs response as compared to the selective inactivation in the epithelial cell lineages achieved in c-Metfl/fl; Alb-Cre+/- mice. However, in both models, genetic loss of c-met triggered a similar cascade of events leading to failure of HSCs mobilization and death of the mice. Conclusion: These results establish a direct contribution of c-Met in regulation of HSC response, and support a unique role for HGF/c-Met as an essential growth factor signaling pathway for regeneration of diseased liver.
doi:10.1002/hep.24796
PMCID: PMC3299882  PMID: 22095660
Oval cell; DDC model; MMP9; hepatic stem cell niche; Kupffer cell
25.  Hepatocyte Nuclear Factor 4 alpha Deletion Promotes Diethylnitrosamine-induced Hepatocellular Carcinoma in Mice 
Hepatology (Baltimore, Md.)  2013;57(6):2480-2490.
HNF4α, the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation via unknown mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using a novel TAM-inducible, hepatocyte specific HNF4α knockdown mouse model. Hepatocyte specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation with a significant increase in liver to body weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that, a significant number of up-regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4α knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation.
doi:10.1002/hep.26251
PMCID: PMC3669646  PMID: 23315968

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