RESUMO
The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.
Assuntos
Fígado/citologia , Fígado/fisiologia , Ribossomos/metabolismo , Animais , Núcleo Celular/metabolismo , Tamanho Celular , Ritmo Circadiano , Exossomos/metabolismo , Hepatócitos/citologia , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fotoperíodo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Ribossomos/químicaRESUMO
It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.
Assuntos
Relógios Circadianos/fisiologia , Hepatócitos/fisiologia , Núcleo Supraquiasmático/fisiologia , Animais , Relógios Circadianos/genética , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Supraquiasmático/cirurgiaRESUMO
Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity.
Assuntos
Células Endoteliais/fisiologia , Células Estreladas do Fígado/fisiologia , Hepatócitos/fisiologia , Células de Kupffer/fisiologia , Fígado/citologia , Macrófagos/fisiologia , Monócitos/fisiologia , Animais , Comunicação Celular , Diferenciação Celular , Células Cultivadas , Microambiente Celular , Feminino , Regulação da Expressão Gênica , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/metabolismoRESUMO
The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.
Assuntos
Diferenciação Celular , Hepatócitos/citologia , Hepatócitos/fisiologia , Fígado/fisiologia , MAP Quinase Quinase 4/genética , Animais , Ciclo Celular , Elementos de DNA Transponíveis , Fibrose , Técnicas de Silenciamento de Genes , Hidrolases/genética , Hidrolases/metabolismo , Fígado/citologia , Fígado/lesões , Fígado/patologia , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Camundongos , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.
Assuntos
Reprogramação Celular/genética , Fibroblastos/fisiologia , Redes Reguladoras de Genes/genética , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Células-Tronco Embrionárias/fisiologia , Feminino , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Defective autophagy and lipotoxicity are the hallmarks of nonalcoholic fatty liver disease. However, the precise molecular mechanism for the defective autophagy in lipotoxic conditions is not fully known. In the current study, we elucidated that activation of the mammalian target of rapamycin complex 1 (mTORC1)-G9a-H3K9me2 axis in fatty acid-induced lipotoxicity blocks autophagy by repressing key autophagy genes. The fatty acid-treated cells show mTORC1 activation, increased histone methyltransferase G9a levels, and suppressed autophagy as indicated by increased accumulation of the key autophagic cargo SQSTM1/p62 and decreased levels of autophagy-related proteins LC3II, Beclin1, and Atg7. Our chromatin immunoprecipitation analysis showed that decrease in autophagy was associated with increased levels of the G9a-mediated repressive H3K9me2 mark and decreased RNA polymerase II occupancy at the promoter regions of Beclin1 and Atg7 in fatty acid-treated cells. Inhibition of mTORC1 in fatty acid-treated cells decreased G9a-mediated H3K9me2 occupancy and increased polymerase II occupancy at Beclin1 and Atg7 promoters. Furthermore, mTORC1 inhibition increased the expression of Beclin1 and Atg7 in fatty acid-treated cells and decreased the accumulation of SQSTM1/p62. Interestingly, the pharmacological inhibition of G9a alone in fatty acid-treated cells decreased the H3K9me2 mark at Atg7 and Beclin1 promoters and restored the expression of Atg7 and Beclin1. Taken together, our findings have identified the mTORC1-G9a-H3K9me2 axis as a negative regulator of the autophagy pathway in hepatocellular lipotoxicity and suggest that the G9a-mediated epigenetic repression is mechanistically a key step during the repression of autophagy in lipotoxic conditions.
Assuntos
Autofagia , Ácidos Graxos , Histona Metiltransferases , Histonas , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Histonas/metabolismo , Ácidos Graxos/toxicidade , Autofagia/fisiologia , Epigênese Genética , Histona Metiltransferases/metabolismo , Hepatócitos/fisiologia , Células Hep G2 , Regulação da Expressão Gênica/efeitos dos fármacos , Palmitatos/toxicidade , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Regiões Promotoras Genéticas , Autofagossomos/genética , Autofagossomos/metabolismo , HumanosRESUMO
BACKGROUND & AIMS: Post-hepatectomy liver failure (PHLF) leads to poor prognosis in patients undergoing hepatectomy, with hepatic vascular reconstitution playing a critical role. However, the regulators of hepatic vascular reconstitution remain unclear. In this study, we aimed to investigate the regulatory mechanisms of hepatic vascular reconstitution and identify biomarkers predicting PHLF in patients undergoing hepatectomy. METHODS: Candidate genes that were associated with hepatic vascular reconstitution were screened using adeno-associated virus vectors in Alb-Cre-CRISPR/Cas9 mice subjected to partial hepatectomy. The biological activities of candidate genes were estimated using endothelial precursor transfusion and associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) models. The level of candidates was detected in biopsies from patients undergoing ALPPS. Risk factors for PHLF were also screened using retrospective data. RESULTS: Downregulation of Gata3 and upregulation of Ramp2 in hepatocytes promoted the proliferation of liver sinusoidal endothelial cells and hepatic revascularization. Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor A (VEGFA) played opposite roles in regulating the migration of endothelial precursors from bone marrow and the formation of new sinusoids after hepatectomy. Gata3 restricted endothelial cell function in patient-derived hepatic organoids, which was abrogated by a Gata3 inhibitor. Moreover, overexpression of Gata3 led to higher mortality in ALPPS mice, which was improved by a PEDF-neutralizing antibody. The expression of Gata3/RAMP and PEDF/VEGFA tended to have a negative correlation in patients undergoing ALPPS. A nomogram incorporating multiple factors, such as serum PEDF/VEGF index, was constructed and could efficiently predict the risk of PHLF. CONCLUSIONS: The balance of Gata3 and Ramp2 in hepatocytes regulates the proliferation of liver sinusoidal endothelial cells and hepatic revascularization via changes in the expression of PEDF and VEGFA, revealing potential targets for the prevention and treatment of PHLF. IMPACT AND IMPLICATIONS: In this study, we show that the balance of Gata3 and Ramp2 in hepatocytes regulates hepatic vascular reconstitution by promoting a shift from pigment epithelium-derived factor (PEDF) to vascular endothelial growth factor A (VEGFA) expression during hepatectomy- or ALLPS (associating liver partition and portal vein ligation for staged hepatectomy)-induced liver regeneration. We also identified serum PEDF/VEGFA index as a potential predictor of post-hepatectomy liver failure in patients who underwent hepatectomy. This study improves our understanding of how hepatocytes contribute to liver regeneration and provides new targets for the prevention and treatment of post-hepatectomy liver failure.
Assuntos
Falência Hepática , Neoplasias Hepáticas , Humanos , Camundongos , Animais , Regeneração Hepática/fisiologia , Fator A de Crescimento do Endotélio Vascular , Estudos Retrospectivos , Células Endoteliais , Fígado/cirurgia , Hepatectomia/efeitos adversos , Hepatócitos/fisiologia , Veia Porta/cirurgia , Falência Hepática/etiologia , Ligadura , Fator de Transcrição GATA3 , Proteína 2 Modificadora da Atividade de ReceptoresRESUMO
BACKGROUND & AIMS: Polyploidy in hepatocytes has been proposed as a genetic mechanism to buffer against transcriptional dysregulation. Here, we aim to demonstrate the role of polyploidy in modulating gene regulatory networks in hepatocytes during ageing. METHODS: We performed single-nucleus RNA sequencing in hepatocyte nuclei of different ploidy levels isolated from young and old wild-type mice. Changes in the gene expression and regulatory network were compared to three independent strains that were haploinsufficient for HNF4A, CEBPA or CTCF, representing non-deleterious perturbations. Phenotypic characteristics of the liver section were additionally evaluated histologically, whereas the genomic allele composition of hepatocytes was analysed by BaseScope. RESULTS: We observed that ageing in wild-type mice results in nuclei polyploidy and a marked increase in steatosis. Haploinsufficiency of liver-specific master regulators (HFN4A or CEBPA) results in the enrichment of hepatocytes with tetraploid nuclei at a young age, affecting the genomic regulatory network, and dramatically suppressing ageing-related steatosis tissue wide. Notably, these phenotypes are not the result of subtle disruption to liver-specific transcriptional networks, since haploinsufficiency in the CTCF insulator protein resulted in the same phenotype. Further quantification of genotypes of tetraploid hepatocytes in young and old HFN4A-haploinsufficient mice revealed that during ageing, tetraploid hepatocytes lead to the selection of wild-type alleles, restoring non-deleterious genetic perturbations. CONCLUSIONS: Our results suggest a model whereby polyploidisation leads to fundamentally different cell states. Polyploid conversion enables pleiotropic buffering against age-related decline via non-random allelic segregation to restore a wild-type genome. IMPACT AND IMPLICATIONS: The functional role of hepatocyte polyploidisation during ageing is poorly understood. Using single-nucleus RNA sequencing and BaseScope approaches, we have studied ploidy dynamics during ageing in murine livers with non-deleterious genetic perturbations. We have identified that hepatocytes present different cellular states and the ability to buffer ageing-associated dysfunctions. Tetraploid nuclei exhibit robust transcriptional networks and are better adapted to genomically overcome perturbations. Novel therapeutic interventions aimed at attenuating age-related changes in tissue function could be exploited by manipulation of ploidy dynamics during chronic liver conditions.
Assuntos
Envelhecimento , Hepatócitos , Poliploidia , Animais , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Camundongos , Envelhecimento/fisiologia , Envelhecimento/genética , Redes Reguladoras de Genes , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Haploinsuficiência , Senescência Celular/genética , Senescência Celular/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/patologiaRESUMO
Spatial metabolomics can reveal intercellular heterogeneity and tissue organization. Here we report on the spatial single nuclear metabolomics (SEAM) method, a flexible platform combining high-spatial-resolution imaging mass spectrometry and a set of computational algorithms that can display multiscale and multicolor tissue tomography together with identification and clustering of single nuclei by their in situ metabolic fingerprints. We first applied SEAM to a range of wild-type mouse tissues, then delineated a consistent pattern of metabolic zonation in mouse liver. We further studied the spatial metabolic profile in the human fibrotic liver. We discovered subpopulations of hepatocytes with special metabolic features associated with their proximity to the fibrotic niche, and validated this finding by spatial transcriptomics with Geo-seq. These demonstrations highlighted SEAM's ability to explore the spatial metabolic profile and tissue histology at the single-cell level, leading to a deeper understanding of tissue metabolic organization.
Assuntos
Microambiente Celular , Biologia Computacional/métodos , Cirrose Hepática/metabolismo , Fígado/citologia , Algoritmos , Animais , Hepatócitos/fisiologia , Humanos , Fígado/fisiologia , Metabolômica/métodos , Camundongos , Reprodutibilidade dos Testes , Imagem Individual de Molécula , TranscriptomaRESUMO
BACKGROUND AND AIMS: The liver has the unique ability of regeneration, which is extremely important for restoring homeostasis after liver injury. Although clinical observations have revealed an association between psychological stress and the liver, whether stress has a causal influence on the liver regeneration remains markedly less defined. APPROACH AND RESULTS: Rearing rodents in an enriched environment (EE) can induce eustress or positive psychological stress. Herein, EE-induced eustress was found to significantly enhance the ability of liver regeneration after partial hepatectomy or carbon tetrachloride-induced liver injury based on the more rapid restoration of liver/body weight ratio and the significantly increased number of proliferating hepatocytes in EE mice. Mechanistically, the cytokine array revealed that IL-22 was markedly increased in the regenerating liver in response to EE. Blockade of IL-22 signaling abrogated the enhanced liver regeneration induced by EE. Group 1 innate lymphoid cells (ILCs), including type 1 ILCs (ILC1s), have been identified as the major sources of IL-22 in the regenerating liver. EE housing led to a rapid accumulation of hepatic ILC1s after partial hepatectomy and the EE-induced enhancement of liver regeneration and elevation of IL-22 was nearly eliminated in ILC1-deficient Tbx21-/- mice. Chemical sympathectomy or blockade of ß-adrenergic signaling also abolished the effect of EE on ILC1s and attenuated the enhanced liver regeneration of EE-housed mice. CONCLUSION: The study findings support the brain-liver axis and suggest that environment-induced eustress promotes liver regeneration through the sympathetic nerve/ILC1/IL-22 axis.
Assuntos
Imunidade Inata , Regeneração Hepática , Camundongos , Animais , Regeneração Hepática/fisiologia , Linfócitos , Interleucinas , Hepatectomia , Fígado , Hepatócitos/fisiologia , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
In cases of end-stage liver diseases, the proliferation of existing hepatocytes is compromised, a feature of human chronic liver disease, in which most hepatocytes are dysfunctional. So far, liver transplantation represents the only curative therapeutic solution for advanced liver diseases, and the shortage of donor organs leads to high morbidity and mortality worldwide. The promising treatment is to prompt the biliary epithelial cells (BECs) transdifferentiation. However, the critical factors governing the initiation of BEC-derived liver regeneration are largely unknown. The zebrafish has advantages in large-scale genetic screens to identify the critical factors involved in liver regeneration. Here, we combined N-ethyl-N-nitrosourea screen, positional cloning, transgenic lines, antibody staining, and in situ hybridization methods and identified a liver regeneration defect mutant ( lrd ) using the zebrafish extensive liver injury model. Through positional cloning and genomic sequencing, we mapped the mutation site to rngtt . Loss of rngtt leads to the defects of BEC dedifferentiation, bipotential progenitor cell activation, and cell proliferation in the initiation stage of liver regeneration. The transdifferentiation from BECs to hepatocytes did not occur even at the late stage of liver regeneration. Mechanically, Rngtt transcriptionally regulates the attachment of mRNA cap to mTOR complex 1 (mTORC1) components and dnmt1 to maintain the activation of mTORC1 and DNA methylation in BECs after severe liver injury and prompt BEC to hepatocyte conversion. Furthermore, rptor and dnmt1 mutants displayed the same liver regeneration defects as rngtt mutation. In conclusion, our results suggest Rngtt is a new factor that initiates BEC-derived liver regeneration.
Assuntos
Regeneração Hepática , Peixe-Zebra , Animais , Humanos , DNA (Citosina-5-)-Metiltransferase 1 , Células Epiteliais , Hepatócitos/fisiologia , Fígado , Regeneração Hepática/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Peixe-Zebra/genéticaRESUMO
Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.
Assuntos
Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/imunologia , Hepatócitos/fisiologia , Fígado/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Viral/imunologia , Animais , Pré-Escolar , Feminino , Células Hep G2 , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Imunidade Inata , Interferons/metabolismo , Fígado/virologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos SCID , Proteínas do Tecido Nervoso/imunologia , RNA Viral/genética , Receptores de Superfície Celular , Transgenes/genética , Quimeras de Transplante , Replicação Viral/genéticaRESUMO
Liver ischemia/reperfusion injury (IRI) is a major complication after partial hepatectomy and liver transplantation and during hypovolemic shock and hypoxia-related diseases. Liver IRI is a current research hotspot. The early stage of liver IRI is characterized by injury and dysfunction of liver sinusoidal endothelial cells (LSECs), which, along with hepatocytes, are the major cells involved in liver injury. In this review, we elaborate on the roles played by LSECs in liver IRI, including the pathological features of LSECs, LSECs exacerbation of the sterile inflammatory response, LSECs interactions with platelets and the promotion of liver regeneration, and the activation of LSECs autophagy. In addition, we discuss the study of LSECs as therapeutic targets for the treatment of liver IRI and the existing problems when applying LSECs in liver IRI research.
Assuntos
Células Endoteliais , Traumatismo por Reperfusão , Humanos , Células Endoteliais/fisiologia , Fígado/patologia , Hepatócitos/fisiologia , Traumatismo por Reperfusão/patologia , Isquemia/patologiaRESUMO
Angiocrine signals derived from endothelial cells are an important component of intercellular communication and have a key role in organ growth, regeneration and disease1-4. These signals have been identified and studied in multiple organs, including the liver, pancreas, lung, heart, bone, bone marrow, central nervous system, retina and some cancers1-4. Here we use the developing liver as a model organ to study angiocrine signals5,6, and show that the growth rate of the liver correlates both spatially and temporally with blood perfusion to this organ. By manipulating blood flow through the liver vasculature, we demonstrate that vessel perfusion activates ß1 integrin and vascular endothelial growth factor receptor 3 (VEGFR3). Notably, both ß1 integrin and VEGFR3 are strictly required for normal production of hepatocyte growth factor, survival of hepatocytes and liver growth. Ex vivo perfusion of adult mouse liver and in vitro mechanical stretching of human hepatic endothelial cells illustrate that mechanotransduction alone is sufficient to turn on angiocrine signals. When the endothelial cells are mechanically stretched, angiocrine signals trigger in vitro proliferation and survival of primary human hepatocytes. Our findings uncover a signalling pathway in vascular endothelial cells that translates blood perfusion and mechanotransduction into organ growth and maintenance.
Assuntos
Comunicação Autócrina , Integrina beta1/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/fisiologia , Mecanotransdução Celular/fisiologia , Transdução de Sinais , Animais , Células Cultivadas , Células Endoteliais/fisiologia , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Fígado/irrigação sanguínea , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND & AIMS: While normal human liver is thought to be generally quiescent, clonal hepatocyte expansions have been observed, though neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. METHODS: Herein, we use in vivo lineage tracing and methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next-generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially distinct areas of laser-captured, microdissected, clones, in tandem with computational modelling in morphologically normal human liver. RESULTS: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mitochondrial DNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mitochondrial DNA variants reveal large numbers of spatially restricted mutations in conjunction with limited numbers of clonal mutations. CONCLUSIONS: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in the normal human liver. IMPACT AND IMPLICATIONS: The liver is mainly composed of hepatocytes, but we know little regarding the source of these cells or how they multiply over time within the disease-free human liver. In this study, we determine a source of new hepatocytes by combining many different lab-based methods and computational predictions to show that hepatocytes share a common cell of origin with bile ducts. Both our experimental and computational data also demonstrate hepatocyte clones are likely to expand in slow waves across the liver in a specific trajectory, but often lie dormant for many years. These data show for the first time the expansion dynamics of hepatocytes in normal liver and their cell of origin enabling the accurate measurment of changes to their dynamics that may lead to liver disease. These findings are important for researchers determining cancer risk in human liver.
Assuntos
Hepatopatias , Nicho de Células-Tronco , Humanos , Teorema de Bayes , Diferenciação Celular , Hepatócitos/fisiologia , Fígado , DNA MitocondrialRESUMO
Cellular proliferation is a basic process during organ development, tissue homeostasis and disease progression. Likewise, after injury typically multiple cell lineages respond to various cues and proliferate to initiate repair and/or remodeling of the injured tissue. Unravelling the specific role of proliferation of one cell type and its lineage in the context of the whole organism during tissue regeneration and/or disease progression would provide valuable information on these processes. Here, we report a new genetic system that allows cell proliferation to be inhibited in a tissue-specific manner. We generated Cre- or Dre-inducible p21-GFP (ip21-GFP) transgenic mice that enable experimentally induced permanent cell cycle arrest of specific cell lineages of interest, while genetically marking these cells. This system allows for the inhibition of pathogenic cell proliferation. We found that cardiac fibroblast proliferation inhibition significantly reduced scar formation, and promoted neovascularization and cardiomyocyte survival. Additionally, we found that inhibition of one type of cell proliferation (namely, hepatocytes) induces the lineage conversion of another type cells (i.e. ductal cells) during tissue regeneration. These results validate the use of ip21-GFP mice as a new genetic tool for cell lineage-specific inhibition of cell proliferation in vivo.
Assuntos
Proliferação de Células , Regulação da Expressão Gênica , Técnicas Genéticas , Alelos , Animais , Linhagem da Célula , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Feminino , Fibroblastos/fisiologia , Proteínas de Fluorescência Verde , Coração/crescimento & desenvolvimento , Coração/fisiologia , Hepatócitos/citologia , Hepatócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/citologiaRESUMO
Single-cell genomics has transformed our ability to examine cell fate choice. Examining cells along a computationally ordered 'pseudotime' offers the potential to unpick subtle changes in variability and covariation among key genes. We describe an approach, scHOT-single-cell higher-order testing-which provides a flexible and statistically robust framework for identifying changes in higher-order interactions among genes. scHOT can be applied for cells along a continuous trajectory or across space and accommodates various higher-order measurements including variability or correlation. We demonstrate the use of scHOT by studying coordinated changes in higher-order interactions during embryonic development of the mouse liver. Additionally, scHOT identifies subtle changes in gene-gene correlations across space using spatially resolved transcriptomics data from the mouse olfactory bulb. scHOT meaningfully adds to first-order differential expression testing and provides a framework for interrogating higher-order interactions using single-cell data.
Assuntos
Fígado/embriologia , Análise de Célula Única/métodos , Animais , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Hepatócitos/fisiologia , Fígado/citologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA , SoftwareRESUMO
Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at â¼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4, leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity.
Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/fisiologia , Vetores Genéticos/genética , Hepatócitos/fisiologia , Inflamação/imunologia , Interleucina-6/metabolismo , Hepatopatias/genética , Animais , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica , Hepatócitos/virologia , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The liver possesses a remarkable regenerative capacity based partly on the ability of hepatocytes to re-enter the cell cycle and divide to replace damaged cells. This capability is substantially reduced upon chronic damage, but it is not clear if this is a cause or consequence of liver disease. Here, we investigate whether blocking hepatocyte division using two different mouse models affects physiology as well as clinical liver manifestations like fibrosis and inflammation. We find that in P14 Cdk1Liv-/- mice, where the division of hepatocytes is abolished, polyploidy, DNA damage, and increased p53 signaling are prevalent. Cdk1Liv-/- mice display classical markers of liver damage two weeks after birth, including elevated ALT, ALP, and bilirubin levels, despite the lack of exogenous liver injury. Inflammation was further studied using cytokine arrays, unveiling elevated levels of CCL2, TIMP1, CXCL10, and IL1-Rn in Cdk1Liv-/- liver, which resulted in increased numbers of monocytes. Ablation of CDK2-dependent DNA re-replication and polyploidy in Cdk1Liv-/- mice reversed most of these phenotypes. Overall, our data indicate that blocking hepatocyte division induces biological processes driving the onset of the disease phenotype. It suggests that the decrease in hepatocyte division observed in liver disease may not only be a consequence of fibrosis and inflammation, but also a pathological cue.
Assuntos
Divisão Celular/fisiologia , Hepatócitos/fisiologia , Cirrose Hepática/metabolismo , Animais , Apoptose/fisiologia , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose/fisiopatologia , Hepatite/metabolismo , Hepatite/fisiopatologia , Hepatócitos/metabolismo , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.